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Journal Cover Journal of Cellular Physiology
  [SJR: 1.842]   [H-I: 139]   [5 followers]  Follow
    
   Hybrid Journal Hybrid journal (It can contain Open Access articles)
   ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
   Published by John Wiley and Sons Homepage  [1609 journals]
  • Interfering of the Reelin/ApoER2/PSD95 Signaling Axis Reactivates
           Dendritogenesis of Mature Hippocampal Neurons
    • Abstract: Reelin, an extracellular glycoprotein secreted in embryonic and adult brains, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity‐dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2‐tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippococampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re‐insertion of NR2B‐NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-21T12:25:21.899058-05:
      DOI: 10.1002/jcp.25605
       
  • Heat Stress Modulates both Anabolic and Catabolic Signaling Pathways
           Preventing Dexamethasone‐Induced Muscle Atrophy In Vitro
    • Authors: Wakako Tsuchida; Masahiro Iwata, Takayuki Akimoto, Shingo Matsuo, Yuji Asai, Shigeyuki Suzuki
      Abstract: It is generally recognized that synthetic glucocorticoids induce skeletal muscle weakness, and endogenous glucocorticoid levels increase in patients with muscle atrophy. It is reported that heat stress attenuates glucocorticoid‐induced muscle atrophy; however, the mechanisms involved are unknown. Therefore, we examined the mechanisms underlying the effects of heat stress against glucocorticoid‐induced muscle atrophy using C2C12 myotubes in vitro, focusing on expression of key molecules and signaling pathways involved in regulating protein synthesis and degradation. The synthetic glucocorticoid dexamethasone decreased myotube diameter and protein content, and heat stress prevented the morphological and biochemical glucocorticoid effects. Heat stress also attenuated increases in mRNAs of regulated in development and DNA damage responses 1 (REDD1) and Kruppel‐like factor 15 (KLF15). Heat stress recovered the dexamethasone‐induced inhibition of PI3K/Akt signaling. These data suggest that changes in anabolic and catabolic signals are involved in heat stress‐induced protection against glucocorticoid‐induced muscle atrophy. These results have a potentially broad clinical impact because elevated glucocorticoid levels are implicated in a wide range of diseases associated with muscle wasting. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-20T14:20:23.775655-05:
      DOI: 10.1002/jcp.25609
       
  • MnTBAP Inhibits Bleomycin‐Induced Pulmonary Fibrosis by Regulating
           VEGF and Wnt Signaling
    • Authors: Rajkumar Venkatadri; Anand Krishnan V. Iyer, Vani Ramesh, Clayton Wright, Carlos A. Castro, Juan S. Yakisich, Neelam Azad
      Abstract: Cellular oxidative stress is implicated not only in lung injury but also in contributing to the development of pulmonary fibrosis. We demonstrate that a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger, Manganese (III) tetrakis (4‐benzoic acid) porphyrin chloride (MnTBAP) significantly inhibited bleomycin‐induced fibrogenic effects both in vitro and in vivo. Further investigation into the underlying mechanisms revealed that MnTBAP targets canonical Wnt and non‐canonical Wnt/Ca2+ signaling pathways, both of which were upregulated by bleomycin treatment. The effect of MnTBAP on canonical Wnt signaling was significant in vivo but inconclusive in vitro and the non‐canonical Wnt/Ca2+ signaling pathway was observed to be the predominant pathway regulated by MnTBAP in bleomycin‐induced pulmonary fibrosis. Furthermore, we show that the inhibitory effects of MnTBAP involve regulation of VEGF which is upstream of the Wnt signaling pathway. Overall, the data shows that the superoxide scavenger MnTBAP attenuates bleomycin‐induced pulmonary fibrosis by targeting VEGF and Wnt signaling pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-20T14:15:23.908239-05:
      DOI: 10.1002/jcp.25608
       
  • Human Telomerase Reverse Transcriptase (hTERT) Positively Regulates 26S
           Proteasome Activity
    • Abstract: Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, an RNA‐dependent DNA polymerase that elongates telomeric DNA. hTERT displays several extra‐telomeric functions that are independent of its telomere‐regulatory function, including tumor progression and neuronal cell death regulation. In this study, we evaluated these additional hTERT non‐telomeric functions. We determined that hTERT interacts with several 19S and 20S proteasome subunits. The 19S regulatory particle and 20S core particle are part of 26S proteasome complex, which plays a central role in ubiquitin‐dependent proteolysis. In addition, hTERT positively regulated 26S proteasome activity independent of its enzymatic activity. Moreover, hTERT enhanced subunit interactions, which may underlie hTERT's ability of hTERT to stimulate the 26S proteasome. Furthermore, hTERT displayed cytoprotective effect against ER stress via the activation of 26S proteasome in acute myeloid leukemia cells. Our data suggest that hTERT acts as a novel chaperone to promote 26S proteasome assembly and maintenance. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-20T14:15:22.299963-05:
      DOI: 10.1002/jcp.25607
       
  • Cover Image, Volume 232, Number 1, January 2017
    • Authors: Areg Zingiryan; Nicholas H. Farina, Kristiaan H. Finstad, Janet L. Stein, Jane B. Lian, Gary S. Stein
      Abstract: Cover: The cover image, by Nicholas H Farina et al., is based on the From the Bench Paper Dissection of Individual Prostate Lobes in Mouse Models of Prostate Cancer to Obtain High Quality RNA,
      DOI : 10.1002/jcp.25384.
      PubDate: 2016-09-19T14:38:07.232144-05:
       
  • Pulsed Electromagnetic Field Exposure Reduces Hypoxia and Inflammation
           Damage in Neuron‐Like and Microglial Cells
    • Authors: Fabrizio Vincenzi; Annalisa Ravani, Silvia Pasquini, Stefania Merighi, Stefania Gessi, Stefania Setti, Ruggero Cadossi, Pier Andrea Borea, Katia Varani
      Abstract: In the present study the effect of low‐frequency, low‐energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron‐like cells and microglial cells. In particular, the primary aim was to evaluate the effect of PEMF exposure in inflammation‐ and hypoxia‐induced injury in two different neuronal cell models, the human neuroblastoma‐derived SH‐SY5Y cells and rat pheochromocytoma PC12 cells and in N9 microglial cells. In neuron‐like cells, live/dead and apoptosis assays were performed in hypoxia conditions from 2 to 48 h. Interestingly, PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis. In the same cell lines, PEMFs inhibited the activation of the hypoxia‐inducible factor 1α (HIF‐1α), the master transcriptional regulator of cellular response to hypoxia. The effect of PEMF exposure on reactive oxygen species (ROS) production in both neuron‐like and microglial cells was investigated considering their key role in ischemic injury. PEMFs significantly decreased hypoxia‐induced ROS generation in PC12, SH‐SY5Y and N9 cells after 24 or 48 h of incubation. Moreover, PEMFs were able to reduce some of the most well‐known pro‐inflammatory cytokines such as tumor necrosis factor ‐α (TNF‐α), interleukin (IL)‐1β, IL‐6 and IL‐8 release in N9 microglial cells stimulated with different concentrations of LPS for 24 or 48 h of incubation time. These results show a protective effect of PEMFs on hypoxia damage in neuron‐like cells and an anti‐inflammatory effect in microglial cells suggesting that PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:30:51.474977-05:
      DOI: 10.1002/jcp.25606
       
  • Valproic Acid and Lithium Meditate Anti‐Inflammatory Effects by
           Differentially Modulating Dendritic Cell Differentiation and Function
    • Abstract: Valproic acid (VPA), with inhibition activity mainly towards histone deacetylase (HDAC) and Glycogen Synthase Kinase (GSK)‐3, and lithium, with inhibition activity mainly toward GSK‐3, are both prescribed in clinical as mood‐stabilizers and anticonvulsants for the control of bipolar disorder. This study aims to compare the immuno‐modulation activities of VPA and lithium, especially on the differentiation and functions of dendritic cells (DC). Our data show that treatment with VPA or lithium effectively alleviated the severity of collagen‐induced arthritis triggered by LPS in mice. Both agents reduced the serum level of IL‐6 and IL‐10 after LPS challenge in mice. VPA and lithium both induce significant down‐regulation of group I CD1 expression and secretion of IL‐6 during differentiation of human monocyte‐derived immature DC, while they differ in the induction of CD83 and CD86 expression, secretion of IL‐8, IL‐10, and TNF‐α. Upon stimulation of immature DC with LPS, VPA and lithium both reduced the secretion of IL‐6 and TNF‐α. However, only lithium significantly increased the production of IL‐10, while VPA increased the production of IL‐8 but substantially reduce the secretion of IL‐10 and IL‐23. Treatment with VPA resulted in a reduced capacity of LPS‐stimulated DC to promote the differentiation of T helper 17 cells that are critical in the promotion of inflammatory responses. Taken together, our results suggest that VPA and lithium may differentially modulate inflammation through regulating the capacity of DC to mediate distinct T cell responses, and they may provide a complementary immunomodulatory effects for the treatment of inflammation‐related diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:30:50.294937-05:
      DOI: 10.1002/jcp.25604
       
  • Human Brain Malignant Glioma (BMG‐1) 3D Aggregate Morphology and
           Screening for Cytotoxicity and Anti‐Proliferative Effects
    • Authors: Maddaly Ravi; Aarthi Ramesh, Aishwarya Pattabhi
      Abstract: Two interesting aspects of cell lines grown in 3D conditions are their distinct morphology and production of extracellular matrix (ECM). Also, it is known that 3D aggregates have different susceptibilities to damage‐inducing agents compared to their 2D monolayer counterparts. We describe the effect of ECM on 3D aggregate morphology, the effect of cisplatin, bleomycin and UV on the 3D aggregates and 2D monolayers of the BMG‐1 cell line. We also present a rapid method for analyzing cytotoxicity and anti‐proliferative effects of 3D aggregates in 96‐well plates. We utilized a single‐step protocol using the dye resazurin.BMG‐1 cells formed floating aggregates on 1% agarose hydrogels. The extent of ECM formed by them was dependent on number of cells seeded irrespective of the seeding density, which in turn directed the 3D aggregates compactness. The 3D aggregates were less susceptible to cisplatin and UV‐induced cytotoxicity compared to 2D counterparts. The IC 50 value of cisplatin was elevated at 210 µg/ml for the aggregates compared to 170µg/ml for the monolayers. Exposure to UV for 10, 20 and 30 minutes gave inhibition values of 2.98%, 8%, 22.99% and 31.8% for the aggregates as compared to 3.06%, 7.5%, 39.4% and 46.7% for the monolayers. While bleomycin‐induced effects were unapparent when analyzed by vital staining for the doses used, the rapid, single‐step method in 96‐well plates was able to provide a dose‐response for cytotoxicity and anti‐proliferative effects. Also, comparative analysis of results obtained from vital staining and the single‐step method demonstrates the reliability of the assay described. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-17T14:26:20.762476-05:
      DOI: 10.1002/jcp.25603
       
  • Early Subchondral Bone Loss at Arthritis Onset Predicted Late Arthritis
           Severity in a Rat Arthritis Model
    • Abstract: Synovitis is usually observed before loss of articular function in rheumatoid arthritis (RA). In addition to the synovium and according to the “Inside‐Outside” theory, bone compartment is also involved in RA pathogenesis. Then, we investigated time dependent articular bone loss and prediction of early bone loss to late arthritis severity on the rat adjuvant‐induced arthritis (AIA) model. Lewis female rats were longitudinally monitored from arthritis induction (day 0), with early (day 10) and late (day 17) steps. Trabecular and cortical microarchitecture parameters of 4 ankle bones were assessed by microcomputed tomography. Gene expression was determined at sacrifice. Arthritis occurred at day 10 in AIA rats. At this time, bone erosions were detected on 4 ankle bones, with cortical porosity increase (+67%) and trabecular alterations including bone volume fraction (BV/TV: ‐13%) and trabecular thickness decrease. Navicular bone assessment was the most reproducible and sensitive. Furthermore, strong correlations were observed between bone alterations at day 10 and arthritis severity or bone loss at day 17, including predictability of day 10 BV/TV to day 17 articular index (R2 = 0.76). Finally, gene expression at day 17 confirmed massive osteoclast activation and interestingly provided insights on strong activation of bone formation inhibitor markers at the joint level. In rat AIA, bone loss was already observed at synovitis onset and was predicted late arthritis severity. Our results reinforced the key role of subchondral bone in arthritis pathogenesis, in favour to the “Inside‐Outside” theory. Mechanisms of bone loss in rat AIA involved resorption activation and formation inhibition changes. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:05:52.041277-05:
      DOI: 10.1002/jcp.25601
       
  • Differential Expression of miR‐4520a Associated with Pyrin Mutations
           Suggesting a Role of Autophagy in Familial Mediterranean Fever (FMF)
    • Abstract: Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent, acute and self‐limiting attacks of fever. Mutations in MEFV gene encoding pyrin account for FMF but the high number of heterozygote patients with typical symptoms of the disease has driven a number of alternative aetiopathogenic hypotheses. The MEFV gene was knocked down in human myelomonocytic cells that express endogenous pyrin to identify deregulated microRNAs (miRNAs). Microarray analyses revealed 29 significantly differentially expressed miRNAs implicated in pathways associated with cellular integrity and survival. Implementation of in silico gene network prediction algorithms and bioinformatics analyses showed that miR‐4520a is predicted to target genes implicated in autophagy through regulation of RHEB/mTOR signaling. Differential expression levels of RHEB were confirmed by luciferase reporter gene assays providing further evidence that is directly targeted by miR‐4520a. Although the relative expression levels of miR‐4520a were variable among FMF patients, the statistical expression of miR‐4520a was different between FMF mutation carriers and controls (p = 0.032), indicating an association between miR‐4520a expression and MEFV mutations. Comparison between FMF patients bearing the M694V mutation, associated with severe disease, and healthy controls showed a significant increase in miR‐4520a expression levels (p = 0.00548). These data suggest that RHEB, the main activator of mTOR signaling, is a valid target of miR‐4520a with the relative expression levels of the latter being significantly deregulated in FMF patients and highly dependent on the presence of pyrin mutations, especially of the M694V type. These results support a role of deregulated autophagy in the pathogenesis of FMF. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:05:47.865817-05:
      DOI: 10.1002/jcp.25602
       
  • A MicroRNA Cluster miR‐23∼24∼27 Is Upregulated by Aldosterone in the
           Distal Kidney Nephron where It Alters Sodium Transport
    • Authors: X. Liu; R.S. Edinger, C.A. Klemens, Y.L. Phua, A.J. Bodnar, W.A. LaFramboise, J. Ho, M.B. Butterworth
      Abstract: The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu‐miR‐23∼24∼27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR‐27a/b was verified to bind to the 3'‐untranslated region of intersectin‐2, a multi‐domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC‐mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR‐27 in aldosterone's action via a novel target. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:01:00.182963-05:
      DOI: 10.1002/jcp.25599
       
  • Inflammatory Events and Oxidant Production in the Diaphragm,
           Gastrocnemius, and Blood of Rats Exposed to Chronic Intermittent Hypoxia:
           Therapeutic Strategies
    • Abstract: We hypothesized that inflammatory events and reactive oxygen species (ROS) production may be differentially expressed in respiratory and limb muscles, and blood of a chronic intermittent hypoxia (CIH) experimental model and that antioxidants and TNF‐alpha blockade may influence those events. In blood, diaphragm, and gastrocnemius of rats non‐invasively exposed to CIH (10% hypoxia, two hours/day, 14 consecutive days) with/without concomitant treatment with either anti‐TNF‐alpha antibody (infliximab) or N‐acetyl cysteine (NAC), inflammatory cytokines, superoxide anion production, muscle structural abnormalities, and fiber‐type composition were assessed. Compared to non‐exposed controls, in CIH‐exposed rats, body weight gain was reduced, TNF‐alpha, IL‐1beta, IL‐6, and interferon‐gamma levels were increased in diaphragm, TNF‐alpha and IL‐1 beta plasma levels were greater, systemic and muscle superoxide anion production was higher, diaphragm and gastrocnemius inflammatory cells and internal nuclei were higher, and muscle fiber‐type and morphometry remained unmodified. CIH rats treated with infliximab further increased TNF‐alpha, IL‐1beta, IL‐6, and interferon‐gamma diaphragm levels, whereas NAC induced a reduction only in TNF‐alpha and IL‐1beta levels in diaphragm and plasma. Infliximab and NAC elicited a significant decline in superoxide anion production in diaphragm, gastrocnemius, and plasma, while inducing a further increase in inflammatory cells and internal nuclei in both muscles. Proinflammatory cytokines are differentially expressed in respiratory and limb muscles and plasma of CIH‐exposed rats, while superoxide anion production increased in both muscle types and blood. Infliximab and NAC exerted different effects. These findings may help understand the biology underlying CIH in skeletal muscles and blood of patients with chronic respiratory diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-16T17:00:51.109077-05:
      DOI: 10.1002/jcp.25600
       
  • RNA‐Generated and Gene‐Edited Induced Pluripotent Stem Cells for
           Disease Modeling and Therapy
    • Authors: James Kehler; Marianna Greco, Valentina Martino, Manickam Pachiappan, Hiroko Yokoe, Alice Chen, Miranda Yang, Joel Jessee, Martin Gotte, Luciano Milanesi, Alberto Albertini, Gianfranco Bellipanni, Ileana Zucchi, Rolland A. Reinbold, Antonio Giordano
      Abstract: Cellular reprogramming by epigenomic remodeling of chromatin holds great promise in the field of human regenerative medicine. As an example, human induced Pluripotent Stem Cells (iPSCs) obtained by reprograming of patient somatic cells are sufficiently similar to embryonic stem cells (ESCs) and can generate all cell types of the human body. Clinical use of iPSCs is dependent on methods that do not utilize genome altering transgenic technologies that are potentially unsafe and ethically unacceptable. Transient delivery of exogenous RNA into cells provides a safer reprogramming system to transgenic approaches that rely on exogenous DNA or viral vectors. RNA reprogrammed iPSCs lines may prove to be more suitable for clinical applications and provide stable starting cell lines for gene‐editing, isolation and characterization of patient iPSC lines. The introduction and rapid evolution of CRISPR/Cas9 gene‐editing systems has provided a readily accessible research tool to perform functional human genetic experiments. Similar to RNA reprogramming, transient delivery of mRNA encoding Cas9 in combination with guide RNA sequences to target specific points in the genome eliminates the risk of potential integration of Cas9 plasmid constructs. We present optimized RNA based laboratory procedure for making and editing iPSCs. In the near‐term these two powerful technologies are being harnessed to dissect mechanisms of human development and disease in vitro, supporting both basic and translational research. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-15T11:05:27.599142-05:
      DOI: 10.1002/jcp.25597
       
  • Nephroprotective Effects of Metformin in Diabetic Nephropathy
    • Authors: Sreenithya Ravindran; Vinitha Kuruvilla, Kerry Wilbur, Shankar Munusamy
      Abstract: Metformin, a well‐known anti‐diabetic agent, is very effective in lowering blood glucose in patients with type 2 diabetes with minimal side‐effects. Metformin is also being recommended in the treatment of obesity and polycystic ovary syndrome. Metformin elicits its therapeutic effects mainly via activation of AMP‐activated kinase (AMPK) pathway. Renal cells under hyperglycemic or proteinuric conditions exhibit inactivation of cell defense mechanisms such as AMPK and autophagy, and activation of pathologic pathways such as mammalian target of rapamycin (mTOR), endoplasmic reticulum (ER) stress, epithelial‐to‐mesenchymal transition (EMT), oxidative stress and hypoxia. As these pathologic pathways are intertwined with AMPK signaling, the potential benefits of metformin therapy in patients with type 2 diabetes would extend beyond its anti‐hyperglycemic effects. However, since metformin is eliminated unchanged through the kidneys and some studies have shown the incidence of lactic acidosis with its use during severe renal dysfunction, the use of metformin was contraindicated in patients with renal disease until recently. With more studies indicating the relatively low incidence of lactic acidosis and revealing the additional benefits with metformin therapy, the US FDA has now approved metformin to be administered in patients with established renal disease based on their renal function. The purpose of this review is to highlight the various mechanisms by which metformin protects renal cells that have lost its functionality in a diabetic or non‐diabetic setting and to enlighten the advantages and therapeutic potential of metformin as a nephroprotectant for patients with diabetic nephropathy and other non‐diabetic forms of chronic kidney disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-14T18:00:30.414624-05:
      DOI: 10.1002/jcp.25598
       
  • A Novel Skin Splint for Accurately Mapping Dermal Remodeling and
           Epithelialization During Wound Healing
    • Authors: Shiro Jimi; Francesco De Francesco, Giuseppe A. Ferraro, Michele Riccio, Shuuji Hara
      Abstract: The mouse excisional dorsal full‐thickness wound model with a silicon splint fixed on the skin has been widely used to mimic human wound healing. However, the method cannot accurately quantify dermal remodeling, since the initial point of epithelialization on the wound surface is unclear. To overcome this limitation, we have developed a novel mouse excisional wound model to assess the degree of epithelial extension and regeneration, using a plastic ring‐shaped splint fixed beneath the surrounding epidermal tissue. At the end of the experiment, tissue samples were fixed in formalin, the splint was excised, and paraffin sections were prepared. Splint holes, corresponding to the prior location of the splint, were evident on the tissue cross‐sections, and the epidermis above the holes was considered the initial excision site. The epidermal contraction and epithelial regeneration, as independent essential tissue alterations in wound healing, could be distinguishable and quantified. Compared with previous splint models, this method provides an accurate evaluation of epidermal processes in wound healing, and can be a platform to assess the effects of various wound healing factors. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-14T17:55:47.612886-05:
      DOI: 10.1002/jcp.25595
       
  • Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal
           Epithelial Cells
    • Abstract: Intestinal epithelial cell culture is important for biological, functional and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered‐cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio‐membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL‐25 and TSLP), inhibitory (IL‐10 and TGF‐β) or pro‐inflammatory mediators (IL‐1β and TNF) were induced. In conclusion, this novel protocol using cow's collagen‐ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell‐microorganism interaction studies and stem cell expansion. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-14T17:55:38.907459-05:
      DOI: 10.1002/jcp.25594
       
  • Identifying Nuclear Matrix–Attached DNA Across the Genome
    • Authors: Jason R. Dobson; Deli Hong, A. Rasim Barutcu, Hai Wu, Anthony N. Imbalzano, Jane B. Lian, Janet L. Stein, Andre J. van Wijnen, Jeffrey A. Nickerson, Gary S. Stein
      Abstract: Experimental approaches to define the relationship between gene expression and nuclear matrix attachment regions (MARs) have given contrasting and method‐specific results. We have developed a next generation sequencing strategy to identify MARs across the human genome (MAR‐Seq). The method is based on crosslinking chromatin to its nuclear matrix attachment sites to minimize changes during biochemical processing. We used this method to compare nuclear matrix organization in MCF‐10A mammary epithelial‐like cells and MDA‐MB‐231 breast cancer cells and evaluated the results in the context of global gene expression (array analysis) and positional enrichment of gene‐regulatory histone modifications (ChIP‐Seq). In the normal‐like cells, nuclear matrix–attached DNA was enriched in expressed genes, while in the breast cancer cells, it was enriched in non‐expressed genes. In both cell lines, the chromatin modifications that mark transcriptional activation or repression were appropriately associated with gene expression. Using this new MAR‐Seq approach, we provide the first genome‐wide characterization of nuclear matrix attachment in mammalian cells and reveal that the nuclear matrix–associated genome is highly cell‐context dependent. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-14T17:55:32.395329-05:
      DOI: 10.1002/jcp.25596
       
  • Cyclic Tensile Strain Reduces TNF‐α Induced Expression of MMP‐13 by
           Condylar Temporomandibular Joint Cells
    • Authors: Hessam Tabeian; Astrid D. Bakker, Beatriz F. Betti, Frank Lobbezoo, Vincent Everts, Teun J de Vries
      Abstract: ObjectiveTo investigate whether the disproportionate degradation of mandibular condyle cartilage in arthritic juvenile temporomandibular joint (TMJ) is related to distinctive responses of TMJ‐derived cells to tumor necrosis factor‐α (TNF‐α), and whether mechanical loading affects this response.MethodsThe effect of TNF‐α (0.1‐10ng/ml) was tested on juvenile porcine TMJ cells isolated from the condyle, fossa, and disc, grown in 3D agarose gels. Expression of anabolic and catabolic factors was quantified by RT‐qPCR and/or immunohistochemistry. Condylar cells were stimulated for 12h with TNF‐α (10ng/ml), followed by 8h of 6% cyclic tensile strain, and gene expression of MMPs was quantified.ResultsTNF‐α (10ng/ml) reduced the expression of the matrix proteins collagen type I and type II after 6h of incubation. Aggrecan gene expression was increased in the presence of 0.1ng/ml TNF‐α. The fossa and disc cells responded to TNF‐α with an increased expression of the aggrecanase ADAMTS4. TNF‐α enhanced MMP‐13 gene and protein expression only by condylar cells. Mechanical loading reduced this effect.ConclusionCells isolated from the different cartilaginous structures reacted differently to TNF‐α. Since the disc and fossa contain a very low level of proteoglycans in comparison to the condyle, the role played by ADAMTS4 in degradation of the fossa and disc might be limited. TNF‐α induced MMP‐13 expression by condylar cells might be involved in the degradation of the juvenile condyle. Since this expression was reduced by mechanical loading, functional loading with oral physiotherapy or orthodontic activators may help to reduce the catabolic effect of TNF‐α. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-12T17:50:41.79315-05:0
      DOI: 10.1002/jcp.25593
       
  • Tight Junction Protein Abundance and Apoptosis During Involution of Rat
           Mammary Glands
    • Authors: Claire V. C. Phyn; Kerst Stelwagen, Stephen R. Davis, Christopher D. Mcmahon, Joanne M. Dobson, Kuljeet Singh
      Abstract: To examine tight junction protein abundance and apoptosis of epithelial cells at the onset of involution in rodent mammary glands, milk accumulation and mammary engorgement was induced by teat‐sealing with an adhesive for 0, 6, 12, 18, 24, and 36 h (n = 6 per group) at peak lactation. In non‐sealed control glands, histological analysis confirmed a lactating phenotype, indicating suckling by pups throughout the experiment. In contrast, alveoli of teat‐sealed glands were distended within 6 h, with maximal luminal size observed by 12 h of non‐suckling. By 18 h following teat‐sealing, an involuting phenotype was observed, indicated by alveolar lumina engorged with milk vesicles and increased leukocytes. Relative to non‐sealed glands, mammary apoptosis was increased in engorged glands 18 h following teat‐sealing. The abundance of ZO‐1 and occludin proteins was decreased in engorged glands by 12 and 18 h, respectively, following teat‐sealing. In contrast, the claudin‐1 22 kDa band was increased by 6 h and peaked at 12‐18 h, whereas the 28 kDa band declined by 36 h, relative to controls. There were no temporal changes in ZO‐1, occludin, and claudin‐1 22 kDa proteins within control glands, although there were minor differences in claudin‐1 28 kDa. These data indicate that intramammary milk accumulation due to cessation of milk removal is associated with mammary apoptosis. The apoptotic event is preceded by a rapid loss of abundance of ZO‐1, occludin and an initial increase in claudin‐1. The loss of cell‐cell communication may initiate involution and apoptosis of mammary epithelial cells and is a localized intramammary event, occurring only in non‐suckled glands. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T16:35:38.400884-05:
      DOI: 10.1002/jcp.25591
       
  • Overexpression of Large‐Conductance Calcium‐Activated Potassium
           Channels in Human Glioblastoma Stem‐Like Cells and Their Role in Cell
           Migration
    • Authors: Paolo Rosa; Luigi Sforna, Silvia Carlomagno, Giorgio Mangino, Massimo Miscusi, Mauro Pessia, Fabio Franciolini, Antonella Calogero, Luigi Catacuzzeno
      Abstract: Glioblastomas (GBMs) are brain tumors characterized by diffuse invasion of cancer cells into the healthy brain parenchyma, and establishment of secondary foci. GBM cells abundantly express large‐conductance, calcium‐activated potassium (BK) channels that are thought to promote cell invasion. Recent evidence suggests that the GBM high invasive potential mainly originates from a pool of stem‐like cells, but the expression and function of BK channels in this cell subpopulation have not been studied. We investigated the expression of BK channels in GBM stem‐like cells using electrophysiological and immunochemical techniques, and assessed their involvement in the migratory process of this important cell subpopulation. In U87‐MG cells, BK channel expression and function were markedly upregulated by growth conditions that enriched the culture in GBM stem‐like cells (U87‐NS). Cytofluorimetric analysis further confirmed the appearance of a cell subpopulation that co‐expressed high levels of BK channels and CD133, as well as other stem cell markers. A similar association was also found in cells derived from freshly resected GBM biopsies. Finally, transwell migration tests showed that U87‐NS cells migration was much more sensitive to BK channel block than U87‐MG cells. Our data show that BK channels are highly expressed in GBM stem‐like cells, and participate to their high migratory activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T16:35:32.146807-05:
      DOI: 10.1002/jcp.25592
       
  • Cardiotrophin‐1 Regulates Adipokine Production in 3T3‐L1 Adipocytes
           and Adipose Tissue from Obese Mice
    • Abstract: Cardiotrophin‐1 (CT‐1) belongs to the IL‐6 family of cytokines. Previous studies of our group revealed that CT‐1 is a key regulator of glucose and lipid metabolism. The aim of the present study was to analyze the in vitro and in vivo effects of CT‐1 on the production of several adipokines involved in body weight regulation, nutrient metabolism and inflammation. For this purpose, 3T3‐L1 adipocytes were incubated with recombinant protein CT‐1 (rCT‐1) (1‐ 40 ng/mL) for 1 and 18 h. Moreover, the acute effects of rCT‐1 administration (0.2 mg/kg, i.v.) for 30 min and 3 h on adipokines levels were also evaluated in high‐fat fed obese mice. In 3T3‐L1 adipocytes, rCT‐1 treatment downregulated the expression and secretion of leptin, resistin and visfatin. However, rCT‐1 significantly stimulated apelin mRNA and secretion. rCT‐ 1 (18 h) also promoted the activation by phosphorylation of AKT, ERK 1/2 and STAT3. Interestingly, pre‐treatment with the PI3K inhibitor LY294002 reversed the stimulatory effects of rCT‐1 on apelin expression, suggesting that this pathway could be mediating the effects of rCT‐1 on apelin production. In contrast, acute administration of rCT‐1 (30 min and 3 h) to diet‐ induced obese mice downregulated leptin and resistin, without significantly modifying apelin or visfatin mRNA in adipose tissue. Furthermore, CT‐1 null mice exhibited altered expression of adipokines in adipose tissue. The present study demonstrates that rCT‐1 modulates the production of adipokines in vitro and in vivo, suggesting that the regulation of the secretory function of adipocytes could be involved in the metabolic actions of this cytokine. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T16:30:37.064707-05:
      DOI: 10.1002/jcp.25590
       
  • Live cell imaging: Assessing the phototoxicity of 488 nm and 546 nm light
           and methods to alleviate it
    • Authors: Stephen Douthwright; Greenfield Sluder
      Abstract: Inlive cellimaging of fluorescent proteins, phototoxicity of the excitation light can be problematical. Cell death is obvious, but reduced cell viability can make the interpretation of observations error prone. We characterized the phototoxic consequences of 488 and 546nm light on untransformed human cells and tested methodsthat have or could be used to alleviate photodamage.Unlabeled RPE1 cells were given single 0.5 to 2.5 minute irradiations in early G1 from a mercury arc lamp on a fluorescence microscope. 488nm lightproduced a dose dependent decrease in the percentage of cells that progressed to mitosis, slowing of the cell cycle for some of those entering mitosis, and a ∼12% incidence of cell deathfor the highest dose. For 546nm light we found a 10‐15% reduction in the percentage of cells entering mitosis, no strong dose dependency, and a ∼2% incidence of cell deathfor the longest irradiations. For cells expressing GFP‐centrin1 or mCherry‐centrin1, fewer entered mitosis for each dose than unlabeled cells. For constant total dose 488nm light irradiations of unlabeled cells, reducing the intensity tenfold or spreading the exposures out as a series of 10 second pulses at 1 minute intervals produced a minor and not consistent improvement in the percentage of cells entering mitosis. Reducing oxidative processes, by culturing at ∼3% oxygen or adding the reducing agent Troloxnoticeably increased the fraction of cells entering mitosis. Thus, for long term imaging there can be value to using RFP constructs and for GFP tagged proteins reducing oxidative processes. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:45:36.075005-05:
      DOI: 10.1002/jcp.25588
       
  • Hyaluronic acid nanohydrogel loaded with Quercetin alone or in combination
           to a macrolide derivative of rapamycin RAD001 (Everolimus) as a new
           treatment for hormone‐responsive human breast cancer
    • Authors: V Quagliariello; RV Iaffaioli, E Armenia, O Clemente, M Barbarisi, G Nasti, M Berretta, A Ottaiano, A Barbarisi
      Abstract: The aim of this study is based on the evaluation of anticancer, anti‐inflammatory activities and cellular uptake of hyaluronic acid nanohydrogel of quercetin tested alone and in combination to a macrolide derivative of rapamycin RAD001 (everolimus) on hormone‐responsive breast cancer cell line MCF‐7. Biological investigations were focused on the receptor mediated cellular internalization of the nanohydrogel and its abilities to reduce secretion of several cytokines (IL‐8, IL‐6, IL‐19), VEGF and metalloproteases (MMP‐2, MMP‐9) under pro‐inflammatory conditions. Nanohydrogel show a CD44 dependent endocytosis with evident time dependent cytoplasmatic accumulation with abilities to reduce secretion of all cytokines of ∼60% compared to untreated cells. Combination of formulated quercetin and everolimus leads to a synergistic cytotoxic effects with a Combination Index of 0.38. These results highlights the importance of synergistic effect of the hyaluronic acid nanohydrogel of quercetin with everolimus in the regulation of human breast cancer cell proliferation and emphasize the antitumor and anti‐inflammatory properties of the nanocarrier. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:35:42.566155-05:
      DOI: 10.1002/jcp.25587
       
  • Acute ethanol increases IGF‐I‐induced phosphorylation of ERKs by
           enhancing recruitment of p52‐Shc to the Grb2/Shc complex
    • Authors: Matthew Dean; Adam Lassak, Anna Wilk, Adriana Zapata, Luis Marrero, Patricia Molina, Krzysztof Reiss
      Abstract: Ethanol plays a detrimental role in the development of the brain. Multiple studies have shown that ethanol inhibits insulin‐like growth factor I receptor (IGF‐IR) function. Because the IGF‐IR contributes to brain development by supporting neural growth, survival, and differentiation, we sought to determine the molecular mechanism(s) involved in ethanol's effects on this membrane‐associated tyrosine kinase. Using multiple neuronal cell types, we performed Western blot, immunoprecipitation, and GST‐pulldowns following acute (1 ‐ 24 hours) or chronic (3 weeks) treatment with ethanol. Surprisingly, exposure of multiple neuronal cell types to acute (up to 24 hours) ethanol (50mM) enhanced IGF‐I‐induced phosphorylation of ERKs, without affecting IGF‐IR tyrosine phosphorylation itself, or Akt phosphorylation. This acute increase in ERKs phosphorylation was followed by the expected inhibition of the IGF‐IR signaling following 3‐week ethanol exposure. We then expressed a GFP‐tagged IGF‐IR construct in PC12 cells and used them to perform fluorescence recovery after photobleaching (FRAP) analysis. Using these fluorescently‐labeled cells, we determined that 50mM ethanol decreased the half‐time of the IGF‐IR‐associated FRAP, which implied that cell membrane‐associated signaling events could be affected. Indeed, co‐immunoprecipitation and GST‐pulldown studies demonstrated that the acute ethanol exposure increased the recruitment of p52‐Shc to the Grb2‐Shc complex, which is known to engage the Ras‐Raf‐ERKs pathway following IGF‐1 stimulation. These experiments indicate that even a short and low‐dose exposure to ethanol may dysregulate function of the receptor, which plays a critical role in brain development. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:31:17.661918-05:
      DOI: 10.1002/jcp.25586
       
  • Protective role of GPER agonist g‐1 on cardiotoxicity induced by
           doxorubicin
    • Authors: Ernestina M. De Francesco; Carmine Rocca, Francesco Scavello, Daniela Amelio, Teresa Pasqua, Damiano C. Rigiracciolo, Andrea Scarpelli, Silvia Avino, Francesca Cirillo, Nicola Amodio, Maria C. Cerra, Marcello Maggiolini, Tommaso Angelone
      Abstract: The use of Doxorubicin (Dox), a frontline drug for many cancers, is often complicated by dose‐limiting cardiotoxicity in approximately 20% of patients. The G‐protein estrogen receptor GPER/GPR30 mediates estrogen action as the cardioprotection under certain stressful conditions. For instance, GPER activation by the selective agonist G‐1 reduced myocardial inflammation, improved immunosuppression, triggered pro‐survival signaling cascades, improved myocardial mechanical performance and reduced infarct size after ischemia/reperfusion (I/R) injury. Hence, we evaluated whether ligand‐activated GPER may exert cardioprotection in male rats chronically treated with Dox. 1 week of G‐1 (50 µg/kg/day) intraperitoneal administration mitigated Dox (3 mg/kg/day) adverse effects, as revealed by reduced TNF‐α, IL‐1β, LDH and ROS levels. Western blotting analysis of cardiac homogenates indicated that G‐1 prevents the increase in p‐c‐jun, BAX, CTGF, iNOS and COX2 expression induced by Dox. Moreover, the activation of GPER rescued the inhibitory action elicited by Dox on the expression of BCL2, pERK and pAKT. TUNEL assay indicated that GPER activation may also attenuate the cardiomyocyte apoptosis upon Dox exposure. Using ex vivo Langendorff perfused heart technique, we also found an increased systolic recovery and a reduction of both infarct size and LDH levels in rats treated with G‐1 in combination with Dox respect to animals treated with Dox alone. Accordingly, the beneficial effects induced by G‐1 were abrogated in the presence of the GPER selective antagonist G15. These data suggest that GPER activation mitigates Dox‐induced cardiotoxicity, thus proposing GPER as a novel pharmacological target to limit the detrimental cardiac effects of Dox treatment. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-08T07:31:15.491629-05:
      DOI: 10.1002/jcp.25585
       
  • Erratum: All‐Trans Retinoic Acid Attenuates Ultraviolet
           Radiation‐Induced Down‐Regulation of Aquaporin‐3 and Water
           Permeability in Human Keratinocytes by Cong Cao, Shu Wan, Qin Jiang,
           Ashley Amaral, Shan Lu, Gang Hu, Zhigang Bi, Nicola Kouttab, Wenming Chu
           and Yinsheng Wan
    • PubDate: 2016-09-07T14:30:25.15386-05:0
      DOI: 10.1002/jcp.25479
       
  • Store Operated Ca2+ Entry in Oocytes Modulates the Dynamics of
           IP3‐Dependent Ca2+ Release from Oscillatory to Tonic
    • Abstract: Ca2+ signaling is ubiquitous and mediates various cellular functions encoded in its spatial, temporal and amplitude features. Here we investigate the role of Store‐Operated Ca2+ Entry (SOCE) in regulating the temporal dynamics of Ca2+ signals in Xenopus oocytes, which can be either oscillatory or tonic. Oscillatory Ca2+ release from intracellular stores is typically observed at physiological agonist concentration. When Ca2+ release is coupled to Ca2+ store depletion, this triggers the activation of SOCE that translates into a low amplitude tonic Ca2+ signal. SOCE has also been implicated in fueling Ca2+ oscillations when activated at low levels. Here we show that sustained SOCE activation in the presence of IP3 to gate IP3 receptors (IP3R) results in a pump‐leak steady state across the endoplasmic reticulum (ER) membrane that inhibits Ca2+ oscillations and produces a tonic Ca2+ signal. Tonic signaling downstream of SOCE activation relies on focal Ca2+ entry through SOCE ER‐plasma membrane (PM) junctions, Ca2+ uptake into the ER, followed by release through open IP3Rs at distant sites, a process we refer to as ‘Ca2+ teleporting’. Therefore, sustained SOCE activation in the presence of an IP3‐dependent ‘leak’ pathway at the ER membrane results in a switch from oscillatory to tonic Ca2+ signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-07T14:25:44.356793-05:
      DOI: 10.1002/jcp.25513
       
  • Vertebrate Lonesome Kinase Regulated Extracellular Matrix Protein
           Phosphorylation, Cell Shape and Adhesion in Trabecular Meshwork Cells
    • Authors: Rupalatha Maddala; Nikolai P. Skiba, Ponugoti Vasantha Rao
      Abstract: Glaucoma, a leading cause of irreversible blindness is commonly associated with elevated intraocular pressure (IOP) due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although dysregulated production and organization of extracellular matrix (ECM) is presumed to increase resistance to AH outflow and elevate IOP by altering TM cell contractile and adhesive properties, it is not known whether regulation of ECM protein phosphorylation via the secretory vertebrate lonesome kinase (VLK) influences TM cellular characteristics. Here we tested this possibility. Experiments carried out in this study reveal that the 32 kDa protein is a prominent VLK isoform detectable in lysates and conditioned media (CM) of human TM cells. Increased levels of VLK were observed in CM of TM cells subjected to cyclic mechanical stretch, or treated with dexamethasone, TGF‐β2 and TM cells expressing constitutively active RhoA GTPase. Downregulation of VLK expression in TM cells using siRNA decreased tyrosine phosphorylation (TyrP) of ECM proteins and focal adhesions, and induced changes in cell shape in association with reduced levels of actin stress fibers and phospho‐paxillin. VLK was also demonstrated to regulate TGF‐β2‐induced TyrP of ECM proteins. Taken together, these results suggest that VLK secretion can be regulated by external cues, intracellular signal proteins and mechanical stretch, and VLK can in turn regulate TyrP of ECM proteins secreted by TM cells and control cell shape, actin stress fibers and focal adhesions. These observations indicate a potential role for VLK in homeostasis of AH outflow and IOP, and in the pathobiology of glaucoma. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-03T16:00:28.92223-05:0
      DOI: 10.1002/jcp.25582
       
  • NOD Mice Having a Lyn Tyrosine Kinase Mutation Exhibit Abnormal Neutrophil
           Chemotaxis
    • Abstract: Neutrophils from NOD (Non‐Obese Diabetic) mice exhibited reduced migration speed, decreased frequency of directional changes and loss of directionality during chemotaxis (compared to wild type (WT) C57BL/6 mice). Additionally, F‐actin of chemotaxing NOD neutrophils failed to orient toward the chemoattractant gradient and NOD neutrophil adhesion was impaired. A point mutation near the autophosphorylation site of Lyn in NOD mice was identified. Point mutations of G to A (G1412 in LynA and G1199 in LynB) cause a change of amino acid E393 (glutamic acid) to K (lysine) in LynA (E393→K) (E372 of LynB), affecting fMLP‐induced tyrosine phosphorylation. These data indicate that the Lyn mutation in NOD neutrophils is likely responsible for dysregulation of neutrophil adhesion and directed migration, implying the role of Lyn in modulating diabetic patient's susceptibility to bacterial and fungal infections. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-03T16:00:25.524895-05:
      DOI: 10.1002/jcp.25583
       
  • TNT‐Induced Phagocytosis: Tunneling Nanotubes Mediate the Transfer of
           Pro‐Phagocytic Signals from Apoptotic to Viable Cells
    • Authors: Margarethe Bittins; Xiang Wang
      Abstract: The exposure of phosphatidylserine (PS) on the surface membrane of apoptotic cells triggers the recruitment of phagocytic receptors and subsequently results in uptake by phagocytes. Here we describe how apoptotic cells can use intercellular membrane nanotubes to transfer exposed PS to neighboring viable cells, and thus deposit an “eat‐me” tag on the viable cells. Tunneling nanotubes (TNTs) connected UV‐treated apoptotic rat pheochromocytoma PC12 cells with neighboring untreated cells. These TNTs were composed of PS‐exposed plasma membrane and facilitated the transfer of the membrane from apoptotic to viable cells. Other pro‐phagocytic signals, such as oxidized phospholipids and calreticulin, were also transferred to viable cells. In addition, anti‐phagocytic signal CD47 presenting on the plasma membrane of viable cells was masked by the transferred PS‐membrane. Confocal imaging revealed an increase of phagocytosis of viable PC12 cells by murine RAW264.7 macrophages when the viable PC12 cells were cocultured with UV‐treated PC12 cells. Treatment with 50 nM cytochalasin D would abolish TNTs and correspondingly inhibit this phagocytosis of the viable cells. Our study indicates that exposed‐PS membrane is delivered from apoptotic to viable cells through TNTs. This transferred membrane may act as a pro‐phagocytic signal for macrophages to induce phagocytosis of viable cells in a situation where they are in the vicinity of apoptotic cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-03T16:00:23.18093-05:0
      DOI: 10.1002/jcp.25584
       
  • Mouse Oocytes Acquire Mechanisms that Permit Independent Cell Volume
           Regulation at the End of Oogenesis
    • Authors: Samantha Richard; Alina P. Tartia, Detlev Boison, Jay M. Baltz
      Abstract: Mouse embryos employ a unique mechanism of cell volume regulation in which glycine is imported via the GLYT1 transporter to regulate intracellular osmotic pressure. Independent cell volume regulation normally becomes active in the oocyte after ovulation is triggered. This involves two steps: the first is the release of the strong adhesion between the oocyte and zona pellucida (ZP) while the second is the activation of GLYT1. In fully‐grown oocytes, release of adhesion and GLYT1 activation also occur spontaneously in oocytes removed from the follicle. It is unknown, however, whether the capacity to release oocyte‐ZP adhesion or activate GLYT1 first arises in the oocyte after ovulation is triggered or instead growing oocytes already possess these capabilities but they are suppressed in the follicle. Here, we assessed when during oogenesis oocyte‐ZP adhesion can be released and when GLYT1 can be activated, with adhesion assessed by an osmotic assay and GLYT1 activity determined by [3H]‐glycine uptake. Oocyte‐ZP adhesion could not be released by growing oocytes until they were nearly fully grown. Similarly, the amount of GLYT1 activity that can be elicited in oocytes increased sharply at the end of oogenesis. The SLC6A9 protein that is responsible for GLYT1 activity and Slc6a9 transcripts are present in growing oocytes and increased over the course of oogenesis. Furthermore, SLC6A9 becomes localized to the oocyte plasma membrane as the oocyte grows. Thus, oocytes acquire the ability to regulate their cell volume by releasing adhesion to the ZP and activating GLYT1 as they approach the end of oogenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-09-02T14:05:28.234774-05:
      DOI: 10.1002/jcp.25581
       
  • Involvement of CX3CL1 in the Migration of Osteoclast Precursors Across
           Osteoblast Layer Stimulated by Interleukin‐1ß
    • Abstract: The trigger for bone remodeling is bone resorption by osteoclasts. Osteoclast differentiation only occurs on the old bone, which needs to be repaired under physiological conditions. However, uncontrolled bone resorption is often observed in pro‐inflammatory bone diseases, such as rheumatoid arthritis. Mature osteoclasts are multinuclear cells that differentiate from monocyte/macrophage lineage cells by cell fusion. Although Osteoclast precursors should migrate across osteoblast layer to reach bone matrix before maturation, the underlying mechanisms have not yet been elucidated in detail. We herein found that osteoclast precursors utilize two routes to migrate across osteoblast layer by confocal‐ and electro‐microscopic observations. The osteoclast supporting activity of osteoblasts inversely correlated with osteoblast density and was positively related to the number of osteoclast precursors under the osteoblast layer. Osteoclast differentiation was induced by IL‐1ß, but not by PGE2 in high‐density osteoblasts. Osteoblasts and osteoclast precursors expressed CX3CL1 and CX3CR1, respectively, and the expression of CX3CL1 increased in response to interleukin‐1ß. An anti‐CX3CL1‐neutralizing antibody inhibited the migration of osteoclast precursors and osteoclast differentiation. These results strongly suggest the involvement of CX3CL1 in the migration of osteoclast precursors and osteoclastogenesis, and will contribute to the development of new therapies for bone diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T16:55:26.96921-05:0
      DOI: 10.1002/jcp.25577
       
  • HMGB1 Inhibits Apoptosis Following MI and Induces Autophagy via mTORC1
           Inhibition
    • Authors: Eleonora Foglio; Giovanni Puddighinu, Antonia Germani, Matteo A Russo, Federica Limana
      Abstract: Exogenous High Mobility Group Box‐1 protein (HMGB1) has been reported to protect the infarcted heart but the underlying mechanism is quite complex. In particular, its effect on ischemic cardiomyocytes has been poorly investigated. Aim of the present study was to verify whether and how autophagy and apoptosis were involved in HMGB1‐induced heart repair following myocardial infarction (MI). HMGB1 (200 ng) or denatured HMGB1 were injected in the peri‐infarcted region of mouse hearts following acute MI. Three days after treatment, an upregulation of autophagy was detected in infarcted HMGB1 treated hearts compared to controls. Specifically, HMGB1 induced autophagy by significantly upregulating the protein expression of LC3, Beclin‐1 and Atg7 in the border zone. To gain further insights into the molecular mechanism of HMGB1‐mediated autophagy, WB analysis were performed in cardiomyocytes isolated from three day infarcted hearts in the presence and in the absence of HMGB1 treatment. Results showed that upregulation of autophagy by HMGB1 treatment was potentially related to activation of AMP‐activated protein kinase (AMPK) and inhibition of the mammalian target of rapamycin complex 1 (mTORC1). Accordingly, in these hearts, phospho‐Akt signalling pathway was inhibited. The induction of autophagy was accompanied by reduced cardiomyocyte apoptotic rate and decreased expression levels of Bax/Bcl‐2 and active caspase‐3 in the border zone of three day infarcted mice following HMGB1 treatment. We report the first in vivo evidence that HMGB1 treatment in a murine model of acute MI might induce cardiomyocyte survival through attenuation of apoptosis and AMP‐activated protein kinase‐dependent autophagy. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T16:55:22.425714-05:
      DOI: 10.1002/jcp.25576
       
  • Risk Differences between Prediabetes and Diabetes According to Breast
           Cancer Molecular Subtypes
    • Authors: A Crispo; LSA Augustin, M Grimaldi, F Nocerino, A Giudice, E Cavalcanti, M Di Bonito, G Botti, M De Laurentiis, M Rinaldo, E Esposito, A Amore, M Libra, G Ciliberto, G Riccardi, DJA Jenkins, M Montella
      Abstract: Hyperglycemia and hyperinsulinemia may play a role in breast carcinogenesis and prediabetes and diabetes have been associated with increased breast cancer (BC) risk. However, whether BC molecular subtypes may modify these associations is less clear. We therefore investigated these associations in all cases and by BC molecular subtypes among women living in Southern Italy.Cases were 557 patients with non‐metastatic incident BC and controls were 592 outpatients enrolled during the same period as cases and in the same hospital for skin‐related non‐malignant conditions. Adjusted multivariate logistic regression models were built to assess the risks of developing BC in the presence of prediabetes or diabetes. The analyses were repeated by strata of BC molecular subtypes: Luminal A, Luminal B, HER2+ and Triple Negative (TN).Prediabetes and diabetes were significantly associated with higher BC incidence after controlling for known risk factors (OR= 1.94, 95% CI 1.32‐2.87 and OR = 2.46, 95% CI 1.38‐4.37, respectively). Similar results were seen in Luminal A and B while in the TN subtype only prediabetes was associated with BC (OR = 2.43, 95% CI 1.11‐5.32). Among HER2+ patients, only diabetes was significantly associated with BC risk (OR = 3.04, 95% CI 1.24‐7.47). Furthermore, when post‐menopausal HER2+ was split into hormone receptor positive versus negative, the association with diabetes remained significant only in the former (OR = 5.13, 95% CI 1.53‐17.22).These results suggest that prediabetes and diabetes are strongly associated with BC incidence and that these metabolic conditions may be more relevant in the presence of breast cancer molecular subtypes with positive hormone receptors. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T16:50:25.696429-05:
      DOI: 10.1002/jcp.25579
       
  • Meta‐Analysis of Transcriptome Regulation During Induction to Cardiac
           Myocyte Fate from Mouse and Human Fibroblasts
    • Abstract: Ectopic expression of a defined set of transcription factors (TFs) can directly convert fibroblasts into a cardiac myocyte cell fate. Beside inefficiency in generating induced cardiomyocytes (iCMs), the molecular mechanisms that regulate this process remained to be well defined. The main purpose of this study was to provide better insight on the transcriptome regulation and to introduce a new strategy for candidating TFs for the transdifferentiation process. Eight mouse and three human high quality microarray data sets were analyzed to find differentially expressed genes (DEGs), which we integrated with TF‐binding sites and protein‐protein interactions to construct gene regulatory and protein‐protein interaction networks. Topological and biological analyses of constructed gene networks revealed the main regulators and most affected biological processes. The DEGs could be categorized into two distinct groups, first, up‐regulated genes that are mainly involved in cardiac‐specific processes and second, down‐regulated genes that are mainly involved in fibroblast‐specific functions. Gata4, Mef2a, Tbx5, Tead4 TFs were identified as main regulators of cardiac‐specific gene expression program; and Trp53, E2f1, Myc, Sfpi1, Lmo2, and Meis1 were identified as TFs which mainly regulate the expression of fibroblast‐specific genes. Furthermore, we compared gene expression profiles and identified TFs between mouse and human to find the similarities and differences. In summary, our strategy of meta‐analyzing the data of high‐throughput techniques by computational approaches, besides revealing the mechanisms involved in the regulation of the gene expression program, also suggests a new approach for increasing the efficiency of the direct reprogramming of fibroblasts into iCMs. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T16:50:24.851247-05:
      DOI: 10.1002/jcp.25580
       
  • Overexpression of Gremlin1 in Mesenchymal Stem Cells Improves Hindlimb
           Ischemia in Mice by Enhancing Cell Survival
    • Authors: Qiuling Xiang; Dongxi Hong, Yan Liao, Yong Cao, Muyun Liu, Jun Pang, Junjie Zhou, Guang Wang, Renhao Yang, Maosheng Wang, Andy Peng Xiang
      Abstract: Mesenchymal stem cells (MSCs) are a promising cell resource for the treatment of ischemic diseases, partially through paracrine effects. One of the major obstacles of MSC treatment is the poor survival rate and low efficiency of transplanted stem cells due to ischemic or inflammatory environments. Gremlin1 (GREM1), a regulator of growth, differentiation and development, has been identified as a novel proangiogenic factor. However, the role and mechanism of GREM1 in MSCs remains unclear. Therefore, we assessed the putative beneficial effects of GREM1 on MSC‐based therapy for hindlimb ischemia. The lentiviral vector, EF1a‐GREM1, was constructed using the Multisite Gateway System and used to transduce MSCs. In vitro studies demonstrated increased survival of GREM1‐MSCs exposed to H2O2, which is consistent with the activation of caspase‐3. Conditional medium from GREM1‐MSCs (GREM1‐MSC‐CM) increased the anti‐apoptotic effects of human umbilical vein endothelial cells (HUVECs), and this effect was attenuated by treatment with the PI3K/Akt pathway inhibitor LY294002. MSCs modified with GREM1 could significantly increase blood perfusion of the ischemic hindlimb in vivo in a mouse model, which was correlated to improved MSC survival. This study demonstrates that overexpression of GREM1 in MSCs have greater therapeutic effects against ischemia compared with wild‐type MSCs by enhancing the survival of MSCs and ECs, which may provide new tools for studies investigating the treatment of ischemic diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-31T13:45:25.493412-05:
      DOI: 10.1002/jcp.25578
       
  • Endothelial STAT3 Activation Increases Vascular Leakage Through
           Downregulating Tight Junction Proteins: Implications for Diabetic
           Retinopathy
    • Abstract: Vascular inflammation is characteristic feature of diabetic retinopathy. In diabetic retina, a variety of the pro‐inflammatory cytokines are elevated and involved in endothelial dysfunction. STAT3 transcription factor has been implicated in mediating cytokine signaling during vascular inflammation. However, whether and how STAT3 is involved in the direct regulation of the endothelial permeability is currently undefined. Our studies revealed that IL‐6‐induced STAT3 activation increases retinal endothelial permeability and vascular leakage in retinas of mice through the reduced expression of the tight junction proteins ZO‐1 and occludin. In a co‐culture model with microglia and endothelial cells under a high glucose condition, the microglia‐derived IL‐6 induced STAT3 activation in the retinal endothelial cells, leading to increasing endothelial permeability. In addition, IL‐6‐induced STAT3 activation was independent of ROS generation in the retinal endothelial cells. Moreover, we demonstrated that STAT3 activation downregulates the ZO‐1 and occludin levels and increases the endothelial permeability through the induction of VEGF production in retinal endothelial cells. These results suggest the potential importance of IL‐6/STAT3 signaling in regulating endothelial permeability and provide a therapeutic target to prevent the pathology of diabetic retinopathy.
      PubDate: 2016-08-31T13:45:21.833116-05:
      DOI: 10.1002/jcp.25575
       
  • Mild Aerobic Exercise Training Hardly Affects the Diaphragm of mdx Mice
    • Abstract: In the mdx mice model of Duchenne Muscular Dystrophy (DMD), mild endurance exercise training positively affected limb skeletal muscles, whereas few and controversial data exist on the effects of training on the diaphragm. The diaphragm was examined in mdx (C57BL/10ScSn‐Dmdmdx) and wild type (WT, C57BL/10ScSc) mice under sedentary conditions (mdx‐SD, WT‐SD) and during mild exercise training (mdx‐EX, WT‐EX). At baseline, and after 30 and 45 days (training: 5 d/wk for 6 weeks), diaphragm muscle morphology and Cx39 protein were assessed. In addition, tissue levels of the chaperonins Hsp60 and Hsp70 and the p65 subunit of nuclear factor‐kB (NF‐kB) were measured in diaphragm, gastrocnemius, and quadriceps in each experimental group at all time points. Although morphological analysis showed unchanged total area of necrosis/regeneration in the diaphragm after training, there was a trend for larger areas of regeneration than necrosis in the diaphragm of mdx‐EX compared to mdx‐SD mice. However, the levels of Cx39, a protein associated with active regeneration in damaged muscle, were similar in the diaphragm of mdx‐EX and mdx‐SD mice. Hsp60 significantly decreased at 45 days in the diaphragm, but not in limb muscles, in both trained and sedentary mdx compared to WT mice. In limb muscles, but not in the diaphragm, Hsp70 and NF‐kB p65 levels were increased in mdx mice irrespective of training at 30 and 45 days. Therefore, the diaphragm of mdx mice showed little inflammatory and stress responses over time, and appeared hardly affected by mild endurance training. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-30T17:45:40.562165-05:
      DOI: 10.1002/jcp.25573
       
  • Early Growth Response 3 (Egr3) Contributes a Maintenance of C2C12 Myoblast
           Proliferation
    • Authors: Mitsutoshi Kurosaka; Yuji Ogura, Toshiya Funabashi, Tatsuo Akema
      Abstract: Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc‐finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF‐кB transcriptional activity in Egr3‐inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V+ cell fraction and apoptotic protein activity including caspase‐3 and caspase‐7, and Poly‐ADP ribose polymerase. By contrast, the reduction of cellular proliferation by inhibition of Egr3 was partially recovered by treatment of pan‐caspase inhibitor Z‐VAD‐FMK. Collectively, these results suggest that Egr3 is involved in myoblast proliferation by interaction with survival signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-30T17:45:37.199268-05:
      DOI: 10.1002/jcp.25574
       
  • Wnt3A Induces GSK‐3β Phosphorylation and β‐Catenin Accumulation
           through RhoA/ROCK
    • Abstract: In canonical pathway, Wnt3A has been known to stabilize β‐catenin through the dissociation between β‐catenin and glycogen synthase kinase‐3β (GSK‐3β) that suppresses the phosphorylation and degradation of β‐catenin. In non‐canonical signaling pathway, Wnt was known to activate Rho GTPases and to induce cell migration. The cross‐talk between canonical and non‐canonical pathways by Wnt signaling, however, has not been fully elucidated. Here, we revealed that Wnt3A induces not only the phosphorylation of GSK‐3β and accumulation of β‐catenin but also RhoA activation in RAW264.7 and HEK293 cells. Notably, sh‐RhoA and Tat‐C3 abolished both the phosphorylation of GSK‐3β and accumulation of β‐catenin. Y27632, an inhibitor of Rho‐associated coiled coil kinase (ROCK) and si‐ROCK inhibited both GSK‐3β phosphorylation and β‐catenin accumulation. Furthermore, active domain of ROCK directly phosphorylated the purified recombinant GSK‐3β in vitro. In addition, Wnt3A induced cell proliferation and migration, which were inhibited by Tat‐C3 and Y27632. Taken together, we propose the cross‐talk between canonical and non‐canonical signaling pathways of Wnt3A, which induces GSK‐3β phosphorylation and β‐catenin accumulation through RhoA and ROCK activation. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-30T17:45:33.804796-05:
      DOI: 10.1002/jcp.25572
       
  • Unique Regulatory Mechanisms for the Human Embryonic Stem Cell Cycle
    • Authors: Jennifer J. VanOudenhove; Rodrigo A. Grandy, Prachi N. Ghule, Jane B. Lian, Janet L. Stein, Sayyed K. Zaidi, Gary S. Stein
      Abstract: The cell cycle in pluripotent human embryonic stem cells is governed by unique mechanisms that support unrestricted proliferation and competency for endodermal, mesodermal and ectodermal differentiation. The abbreviated G1 period with retention of uncompromised fidelity for genetic and epigenetic mechanisms operative in control of proliferation support competency for expansion of the pluripotent cell population that is fundamental for initial stages of development. Regulatory events during the G1 period of the pluripotent cell cycle are decisive for the transition from pluripotency to lineage commitment. Recent findings indicate that a G2 cell cycle pause is present in both endodermal and mesodermal lineage cells, and is obligatory for differentiation to endoderm. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-17T17:35:41.441477-05:
      DOI: 10.1002/jcp.25567
       
  • MAP Kinase‐Dependent RUNX2 Phosphorylation Is Necessary for Epigenetic
           Modification of Chromatin During Osteoblast Differentiation
    • Authors: Yan Li; Chunxi Ge, Renny T. Franceschi
      Abstract: RUNX2, an essential transcription factor for osteoblast differentiation and bone formation, is activated by ERK/MAP kinase‐dependent phosphorylation. However, relationship between these early events and specific epigenetic modifications of chromatin during osteoblast differentiation have not been previously examined. Here, we explore these relationships using chromatin immunoprecipitation (ChIP) to detect chromatin modifications in RUNX2‐binding regions of Bglap2 and Ibsp. Growth of MC3T3‐E1c4 preosteoblast cells in differentiation conditions rapidly induced Bglap2 and lbsp mRNAs. For both genes, osteogenic stimulation increased chromatin‐bound P‐ERK, P‐RUNX2, p300 and RNA polymerase II as well as histone H3K9 and H4K5 acetylation. The level of H3K4 di‐methylation, another gene activation‐associated histone mark, also increased. In contrast, levels of the gene repressive marks, H3K9 mono‐, di‐, and tri‐methylation, in the same regions were reduced. Inhibition of MAP kinase signaling blocked differentiation‐dependent chromatin modifications and Bglap2 and Ibsp expression. To evaluate the role of RUNX2 phosphorylation in these responses, RUNX2‐deficient C3H10T1/2 cells were transduced with adenovirus encoding wild type or phosphorylation site mutant RUNX2 (RUNX2 S301A/S319A). Wild type RUNX2, but not the non‐phosphorylated mutant, increased H3K9 and H4K5 acetylation as well as chromatin‐associated P‐ERK, p300 and polymerase II. Thus, RUNX2 phosphorylation is necessary for subsequent epigenetic changes required for osteoblast gene expression. Taken together, this study reveals a molecular mechanism through which osteogenic genes are controlled by a MAPK and P‐RUNX2‐dependent process involving epigenetic modifications of specific promoter regions. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-11T15:55:33.213599-05:
      DOI: 10.1002/jcp.25517
       
  • Neurotransmitters: The Critical Modulators Regulating Gut‐Brain Axis
    • Authors: Rahul Mittal; Luca H. Debs, Amit P. Patel, Desiree Nguyen, Kunal Patel, Gregory O'Connor, M'hamed Grati, Jeenu Mittal, Denise Yan, Adrien A. Eshraghi, Sapna K. Deo, Sylvia Daunert, Xue Zhong Liu
      Abstract: Neurotransmitters including catecholamines and serotonin play a crucial role in maintaining homeostasis in the human body. Studies on these neurotransmitters mainly revolved around their role in the “fight or flight” response, transmitting signals across a chemical synapse and modulating blood flow throughout the body. However, recent research has demonstrated that neurotransmitters can play a significant role in the gastrointestinal (GI) physiology. Norepinephrine (NE), epinephrine (E), dopamine (DA), and serotonin have recently been a topic of interest because of their roles in the gut physiology and their potential roles in gastrointestinal and central nervous system pathophysiology. These neurotransmitters are able to regulate and control not only blood flow, but also affect gut motility, nutrient absorption, gastrointestinal innate immune system, and the microbiome. Furthermore, in pathological states such as inflammatory bowel disease (IBD) and Parkinson's disease, the levels of these neurotransmitters are dysregulated, therefore causing a variety of gastrointestinal symptoms. Research in this field has shown that exogenous manipulation of catecholamine serum concentrations can help in decreasing symptomology and/or disease progression. In this review article, we discuss the current state‐of‐the‐art research and literature regarding the role of neurotransmitters in regulation of normal gastrointestinal physiology, their impact on several disease processes, and novel work focused on the use of exogenous hormones and/or psychotropic medications to improve disease symptomology. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-11T15:55:30.576165-05:
      DOI: 10.1002/jcp.25518
       
  • DJ‐1/Park7 Sensitive Na+/H+ Exchanger 1 (NHE1) in CD4+ T Cells
    • Abstract: DJ‐1/Park7 is a redox‐sensitive chaperone protein counteracting oxidation and presumably contributing to the control of oxidative stress responses and thus inflammation. DJ‐1 gene deletion exacerbates the progression of Parkinson's disease presumably by augmenting oxidative stress. Formation of reactive oxygen species (ROS) is paralleled by activation of the Na+/H+ exchanger 1 (NHE1). ROS formation in CD4+ T cells plays a decisive role in regulating inflammatory responses. In the present study we explored whether DJ‐1 is expressed in CD4+ T cells and affects ROS production as well as NHE1 in those cells. To this end, DJ‐1 and NHE1 transcript and protein levels were quantified by qRT‐PCR and Western blotting respectively, intracellular pH (pHi) utilizing bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF) fluorescence, NHE activity from realkalinization after an ammonium pulse, and ROS production utilizing 2',7' –dichlorofluorescin diacetate (DCFDA) fluorescence. As a result DJ‐1 was expressed in CD4+ T cells. ROS formation, NHE1 transcript levels, NHE1 protein, and NHE activity were higher in CD4+ T cells from DJ‐1 deficient mice than in CD4+ T cells from wild type mice. Antioxidant N‐acetyl‐cysteine (NAC) and protein tyrosine kinase (PTK) inhibitor staurosporine decreased the NHE activity in DJ‐1 deficient CD4+ T cells, and blunted the difference between DJ‐1‐/‐ and DJ‐1+/+ CD4+ T cells, an observation pointing to a role of ROS in the up‐regulation of NHE1 in DJ‐1‐/‐ CD4+ T cells. In conclusion, DJ‐1 is a powerful regulator of ROS production as well as NHE1 expression and activity in CD4+ T cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-10T18:30:46.184168-05:
      DOI: 10.1002/jcp.25516
       
  • Dedicator of Cytokinesis 2 in Cell Signaling Regulation and Disease
           Development
    • Abstract: Dedicator of cytokinesis 2 (DOCK2) is a CDM family protein containing DOCK homology region (DHR) ‐1 and DHR‐2, Src‐homology 3 (SH3) domain, and C‐terminal polybasic amino acid cluster. The CDM family consists of 11 mammalian members and is classified into four subfamilies, the DOCK‐A, ‐B, ‐C, and ‐D. DOCK2 is a member of DOCK‐A subfamily and an atypical guanine exchange factor regulating the loading of GTP to activate Rac. It is primarily found in peripheral blood, spleen, and thymus and mainly expressed in lymphocytes and macrophages of various organs. DOCK2 is also expressed in microglial in brain and is induced in neointima smooth muscle following vascular injury. Functionally, DOCK2 is involved in cell motility, polarity, adhesion, proliferation, and apoptosis. It is essential for lymphocyte migration and activation as well as neutrophil chemotaxis. DOCK2 also regulates the differentiation of natural killer T cells, type 2 T helper cells, and plasmacytoid dendritic cells. In addition, it is important for the growth of B cell lymphoma and prostate cancer cells. Deletion of DOCK2 enables long‐term cardiac allograft survival. Moreover, DOCK2 is associated with the Alzheimer Disease, HIV development, and the early‐onset of invasive infections. Recently, we found that DOCK2 plays a critical role in SMC phenotypic modulation and vascular remodeling. In this review, we will briefly summarize recent advancement of DOCK2 function. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-09T17:51:08.979659-05:
      DOI: 10.1002/jcp.25512
       
  • Pathophysiology of the Desmo‐Adhesome
    • Authors: Antonio Celentano; Michele Davide Mignogna, Michael McCullough, Nicola Cirillo
      Abstract: Advances in our understanding of desmosomal diseases have provided a clear demonstration of the key role played by desmosomes in tissue and organ physiology, highlighting the importance of their dynamic and finely regulated structure. In this context, non‐desmosomal regulatory molecules have acquired increasing relevance in the study of this organelle resulting in extending the desmosomal interactome, named the “desmo‐adhesome”. Spatiotemporal changes in the expression and regulation of the desmo‐adhesome underlie a number of genetic, infectious, autoimmune, and malignant conditions. The aim of the present article was to examine the structural and functional relationship of the desmosome, by providing a comprehensive, yet focused overview of the constituents targeted in human disease. The inclusion of the novel regulatory network in the desmo‐adhesome pathophysiology opens new avenues to a deeper understanding of desmosomal diseases, potentially unveiling pathogenic mechanisms waiting to be explored. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-09T17:47:27.213856-05:
      DOI: 10.1002/jcp.25515
       
  • Chemoresistance of Lung and Breast Cancer Cells Growing Under Prolonged
           Periods of Serum Starvation
    • Authors: Juan Sebastian Yakisich; Rajkumar Venkatadri, Neelam Azad, Anand Krishnan V. Iyer
      Abstract: The efficacy of chemotherapy is hindered by both tumor heterogeneity and acquired or intrinsic multi‐ drug resistance caused by the contribution of multidrug resistance proteins and stemness‐associated prosurvival markers. Therefore, targeting multi‐drug resistant cells would be much more effective against cancer. In this study, we characterized the chemoresistance properties of adherent (anchorage‐ dependent) lung H460 and breast MCF‐7 cancer cells growing under prolonged periods of serum starvation (PPSS). We found that under PPSS, both cell lines were highly resistant to Paclitaxel, Colchicine, Hydroxyurea, Obatoclax, Wortmannin and LY294002. Levels of several proteins associated with increased stemness such as Sox2, MDR1, ABCG2 and Bcl‐2 were found to be elevated in H460 cells but not in MCF‐7 cells. While pharmacological inhibition of either MDR1, ABCG2, Bcl‐2 with Verapamil, Sorafenib or Obatoclax respectively decreased the levels of their target proteins under routine culture conditions as expected, such inhibition did not reverse PX resistance in PPSS conditions. Paradoxically, treatment with inhibitors in serum‐starved conditions produced an elevation of their respective target proteins. In addition, we found that Digitoxin, an FDA approved drug that decrease the viability of cancer cells growing under PPSS, downregulates the expression of Sox2, MDR1, phospho‐ AKT, Wnt5a/b and β‐catenin. Our data suggests that PPSS‐induced chemoresistance is the result of extensive rewiring of intracellular signaling networks and that multi‐resistance can be effectively overcome by simultaneously targeting multiple targets of the rewired network. Furthermore, our PPSS model provides a simple and useful tool to screen drugs for their ability to target multiple pathways of cancer resistance. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-09T17:47:17.98153-05:0
      DOI: 10.1002/jcp.25514
       
  • Effects of Conjugated Linoleic Acid Associated with Endurance Exercise on
           Muscle Fibres and Peroxisome Proliferator‐Activated Receptor γ
           Coactivator 1 α Isoforms
    • Authors: Rosario Barone; Claudia Sangiorgi, Antonella Marino Gammazza, Daniela D'Amico, Monica Salerno, Francesco Cappello, Cristoforo Pomara, Giovanni Zummo, Felicia Farina, Valentina Di Felice, Filippo Macaluso
      Abstract: Conjugated linoleic acid (CLA) has been reported to improve muscle hypertrophy, steroidogenesis, physical activity, and endurance capacity in mice, although the molecular mechanisms of its actions are not completely understood. The aim of the present study was to identify whether CLA alters the expression of any of the peroxisome proliferator‐activated receptor γ coactivator 1α (PGC1α) isoforms, and to evaluate the possible existence of fibre‐type‐specific hypertrophy in the gastrocnemius and plantaris muscles. Mice were randomly assigned to one of four groups: placebo sedentary, CLA sedentary, placebo trained, or CLA trained. The CLA groups were gavaged with 35 µl per day of Tonalin® FFA 80 food supplement containing CLA throughout the 6‐week experimental period, whereas the placebo groups were gavaged with 35 µl sunflower oil each day. Each administered dose of CLA corresponded to approximately 0.7 g/kg or 0.5%, of the dietary daily intake. Trained groups ran 5 days per week on a Rota‐Rod for 6 weeks at increasing speeds and durations. Mice were sacrificed by cervical dislocation and hind limb posterior muscle groups were dissected and used for histological and molecular analyses. Endurance training stimulated mitochondrial biogenesis by PGC1α isoforms (tot, α1, α2 and α3) but CLA supplementation did not stimulate PGC1α isoforms or mitochondrial biogenesis in trained or sedentary mice. In the plantaris muscle, CLA supplementation induced a fibre‐type‐specific hypertrophy of type IIx muscle fibres, which was associated with increased capillary density and was different from the fibre‐type‐specific hypertrophy induced by endurance exercise (of type I and IIb muscle fibres). This article is protected by copyright. All rights reserved
      PubDate: 2016-08-02T17:06:00.484007-05:
      DOI: 10.1002/jcp.25511
       
  • VIP Family Members Prevent Outer Blood Retinal Barrier Damage in a Model
           of Diabetic Macular Edema
    • Authors: Grazia Maugeri; Agata Grazia D'Amico, Caterina Gagliano, Salvatore Saccone, Concetta Federico, Sebastiano Cavallaro, Velia D'Agata
      Abstract: Diabetic macular edema (DME), characterized by an increase of thickness in the eye macular area, is due to breakdown of the blood‐retinal barrier (BRB). Hypoxia plays a key role in the progression of this pathology by activating the hypoxia‐inducible factors. In the last years, various studies have put their attention on the role of pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) in retinal dysfunction. However, until now, no study has investigated their protective role against the harmful combined effect of both hyperglycemia and hypoxia on outer BRB. Therefore, in the present study, we have analyzed the role of these peptides on permeability, restoration of tight junctions expression and inhibition of hyperglycemia/hypoxia‐induced apoptosis, in an experimental in vitro model of outer BRB. Our results have demonstrated that the peptides' treatment have restored the integrity of outer BRB induced by cell exposure to hyperglycemia/hypoxia. Their effect is mediated through the activation of phosphoinositide 3 kinase (PI3K)/Akt and mammalian mitogen activated protein kinase/Erk kinase (MAPK/ERK) signaling pathways.In conclusion, our study further clarifies the mechanism through which PACAP and VIP perform the beneficial effect on retinal damage induced by hyperglycemic/hypoxic insult, responsible of DME progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-08-02T17:05:58.282499-05:
      DOI: 10.1002/jcp.25510
       
  • Functional Effects of Cigarette Smoke‐Induced Changes in Airway Smooth
           Muscle Mitochondrial Morphology
    • Authors: Bharathi Aravamudan; Michael Thompson, Gary C. Sieck, Robert Vassallo, Christina M. Pabelick, Y.S. Prakash
      Abstract: Long‐term exposure to cigarette smoke (CS) triggers airway hyperreactivity and remodeling, effects that involve airway smooth muscle (ASM). We previously showed that CS destabilizes the networked morphology of mitochondria in human ASM by regulating the expression of mitochondrial fission and fusion proteins via multiple signaling mechanisms. Emerging data link regulation of mitochondrial morphology to cellular structure and function. We hypothesized that CS‐induced changes in ASM mitochondrial morphology detrimentally affect mitochondrial function, leading to CS effects on contractility and remodeling. Here, ASM cells were exposed to 1% cigarette smoke extract (CSE) for 48 hours to alter mitochondrial fission/fusion, or by inhibiting the fission protein Drp1 or the fusion protein Mfn2. Mitochondrial function was assessed via changes in bioenergetics or altered rates of proliferation and apoptosis. Our results indicate that both exposure to CS and inhibition of mitochondrial fission/ fusion proteins affect mitochondrial function (i.e., energy metabolism, proliferation and apoptosis) in ASM cells. In vivo, the airways in mice chronically exposed to CS are thickened and fibrotic, and the expression of proteins involved in mitochondrial function is dramatically altered in the ASM of these mice. We conclude that CS‐induced changes in mitochondrial morphology (fission/ fusion balance) correlate with mitochondrial function, and thus may control ASM proliferation, which plays a central role in airway health. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-30T15:00:30.808461-05:
      DOI: 10.1002/jcp.25508
       
  • Uric Acid Amplifies Aβ Amyloid Effects Involved in the Cognitive
           Dysfunction/Dementia: Evidences from an Experimental Model In Vitro
    • Authors: G Desideri; R Gentile, A Antonosante, E Benedetti, D Grassi, L Cristiano, A Manocchio, S Selli, R Ippoliti, C Ferri, Claudio Borghi, A Giordano, Annamaria Cimini
      Abstract: There is still a considerable debate concerning whether uric acid is neuroprotective or neurotoxic agent. To clarify this topic we tested the effects of uric acid on neuronal cells biology by using differentiated SHSY5Y neuroblastoma cells incubated with amyloid ß to reproduce an in vitro model of Alzheimer's disease. The incubation of cells with uric acid at the dose of 40 µM or higher significantly reduced cell viability and potentiated the proapoptotic effect of amyloid ß. Finally, uric acid enhanced the generation of 4‐hydroxynonenal and the expression of PPARβ/δ promoted by amyloid β, indicating a prooxidant effects. In conclusion, uric acid could exert a detrimental influence on neuronal cell biology being this influence further potentiated by concomitant exposure to neurotoxic stimuli. This effect is evident for uric acid concentrations close to those achievable in cerebrospinal fluid in presence of mild hyperuricemia thus suggesting a potential role of uric acid in pathophysiology of cognitive dysfunction. These effects are influenced by the concentrations of uric acid and by the presence of favouring conditions that commonly occur in neurodegenerative disorders and well as in the aging brain, including increased oxidative stress and exposure to amyloid β. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-30T14:55:25.58167-05:0
      DOI: 10.1002/jcp.25509
       
  • Chondrocyte Morphology in Stiff and Soft Agarose Gels and the Influence of
           Foetal Calf Serum
    • Authors: A. Karim; A. C. Hall
      Abstract: Changes to chondrocyte volume/morphology may have deleterious effects on extracellular matrix (ECM) metabolism potentially leading to cartilage deterioration and osteoarthritis (OA). The factors controlling chondrocyte properties are poorly understood however pericellular matrix (PCM) weakening may be involved. We have studied the density, volume, morphology and clustering of cultured bovine articular chondrocytes within stiff (2%w/v) and soft (0.2%w/v) 3‐dimensional agarose gels. Gels with encapsulated chondrocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM; foetal calf serum (FCS) 1‐10%;380mOsm) for up to 7d. Chondrocytes were fluorescently‐labelled after 1, 3 and 7d with 5‐chloromethylfluorescein‐diacetate (CMFDA) and propidium iodide (PI) or 1,5‐bis{[2‐(di‐methylamino)ethyl]amino}‐4,8‐dihydroxyanthracene‐9,10‐dione (DRAQ5) to identify cytoplasmic space or DNA and imaged by confocal laser scanning microscopy (CLSM). Chondrocyte density, volume, morphology and clustering were quantified using VolocityTM software. In stiff gels after 7d with 10% FCS, chondrocyte density remained unaffected and morphology was relatively normal with occasional cytoplasmic processes. However in soft gels by day 1, chondrocyte volume increased (P = 0.0058) and by day 7, density increased (P = 0.0080), along with the percentage of chondrocytes of abnormal morphology (P 
      PubDate: 2016-07-29T16:35:23.286636-05:
      DOI: 10.1002/jcp.25507
       
  • Emerging Roles of Transforming Growth Factor β Signaling in Diabetic
           Retinopathy
    • Authors: Sarah E. Wheeler; Nam Y. Lee
      Abstract: Diabetic retinopathy (DR) is a serious complication of diabetes mellitus affecting about one third of diabetic adults. Despite its prevalence, treatment options are limited and often implemented only in the later stages of the disease. To date, the pathogenesis of DR has been extensively characterized in the context of elevated glucose, insulin, and VEGF signaling, although a growing number of other growth factors and molecules, including transforming growth factor‐β (TGF‐β) are being recognized as important contributors and/or therapeutic targets. Here, we review the complex roles of TGF‐β signaling in DR pathogenesis and progression. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-29T16:30:27.799001-05:
      DOI: 10.1002/jcp.25506
       
  • An Overview on Predictive Biomarkers of Testicular Germ Cell Tumors
    • Authors: Paolo Chieffi
      Abstract: Testicular germ cell tumors (TGCTs) are frequent solid malignant tumors and cause of death in men between 20–40 years of age. Genetic and environmental factors play an important role in the origin and development of TGCTs. Although the majority of TGCTs are responsive to chemotherapy, about 20% of patient presents incomplete response or tumors relapse. In addition, the current treatments cause acute toxicity and several chronic collateral effects, including sterility.The present mini‐review collectively summarize the most recent findings on the new discovered molecular biomarkers such as tyrosine kinases, HMGAs, Aurora B kinase, and GPR30 receptor predictive of TGCTs and as emerging new possible molecular targets for therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-29T10:10:23.798428-05:
      DOI: 10.1002/jcp.25482
       
  • Novel Instruments for the Implementation of Electrochemotherapy Protocols:
           From Bench Side to Veterinary Clinic
    • Authors: Enrico P. Spugnini; Stefano Fais, Tommaso Azzarito, Alfonso Baldi
      Abstract: Electrochemotherapy (ECT) is a medical strategy that allows an increased efficacy of chemotherapy agents after the application of permeabilizing electric pulses having appropriate characteristics (form, voltage, frequency). In the past ten years, the clinical efficacy of this therapeutic approach in several spontaneous models of tumors in animals has been shown. Moreover, some of the molecular and cellular mechanisms responsible for this phenomenon have been elucidated. Our group has been deeply involved in the development of new ECT protocols for companion animals, implementing the use of the technique as first line treatment, and evaluating different chemotherapy agents in laboratory animals as well as pets. This article summarizes the most important advances in veterinary ECT, including the development of novel equipment, therapeutic protocols and their translation to humans. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-28T06:35:31.771816-05:
      DOI: 10.1002/jcp.25505
       
  • Coumestrol Inhibits Proliferation and Migration of Prostate Cancer Cells
           by Regulating AKT, ERK1/2 and JNK MAPK Cell Signaling Cascades
    • Authors: Whasun Lim; Muhah Jeong, Fuller W. Bazer, Gwonhwa Song
      Abstract: Coumestrol is the one of the major phytoestrogens which is abundant in soybeans, legumes, brussel sprouts and spinach. The beneficial effects of coumestrol are well known in various biological processes including; neuroprotective effects on the nervous system, function of the female reproductive system, anti‐bacterial properties and anti‐cancer effects. Although the anti‐tumor activity of coumestrol has been demonstrated for ovarian, breast, lung and cervical cancers, little is known of its effects on prostate cancer. Therefore, in the present study, we investigated the chemotherapeutic effects of coumestrol on two prostate cancer cell lines, PC3 and LNCaP. Our results showed that coumestrol decreased proliferation and migration and induced apoptosis in both PC3 and LNCaP cells. Moreover, effects of coumestrol on cell signaling pathways were investigated and it increased phosphorylation of ERK1/2, JNK, P90RSK and P53 proteins in a dose‐ and time‐dependent manner whereas phosphorylation of AKT was reduced by coumestrol under the same conditions for culture of PC3 and LNCaP cells. In addition, mitochondrial dysfunction was induced by coumestrol as evidenced by a significant loss of mitochondrial membrane potential. Furthermore, cleavage of caspase‐3 and caspase‐9, the apoptotic proteins associated with mitochondria, also changed in response to coumestrol. Coumestrol also caused mitochondrial dysfunction resulting in an increase in ROS production in PC3 and LNCaP cells. These results suggest that coumestrol can inhibit progression of prostate cancer and may be a novel chemotherapeutic agent for treatment of prostate cancer via effects mediated via the PI3K/AKT and ERK1/2 and JNK MAPK cell signaling pathways. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-28T05:51:13.440339-05:
      DOI: 10.1002/jcp.25494
       
  • Tissue Inhibitor of Metalloproteinase 1 Influences Vascular Adaptations to
           Chronic Alterations in Blood Flow
    • Authors: Erin R. Mandel; Cassandra Uchida, Emmanuel Nwadozi, Armin Makki, Tara L. Haas
      Abstract: Remodeling of the skeletal muscle microvasculature involves the coordinated actions of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs). We hypothesized that the loss of TIMP1 would enhance both ischemia and flow‐induced vascular remodeling by increasing MMP activity. TIMP1 deficient (Timp1−/−) and wild‐type (WT) C57BL/6 mice underwent unilateral femoral artery (FA) ligation or were treated with prazosin, an alpha‐1 adrenergic receptor antagonist, in order to investigate vascular remodeling to altered flow. Under basal conditions, Timp1−/− mice had reduced microvascular content as compared to WT mice. Furthermore, vascular remodeling was impaired in Timp1−/− mice. Timp1−/− mice displayed reduced blood flow recovery in response to FA ligation and no arteriogenic response to prazosin treatment. Timp1−/− mice failed to undergo angiogenesis in response to ischemia or prazosin, despite maintaining the capacity to increase VEGF‐A and eNOS mRNA. Vascular permeability was increased in muscles of Timp1−/− mice in response to both prazosin treatment and FA ligation, but this was not accompanied by greater MMP activity. This study highlights a previously undescribed integral role for TIMP1 in both vascular network maturation and adaptations to ischemia or alterations in flow. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-28T05:45:36.851462-05:
      DOI: 10.1002/jcp.25491
       
  • Aging Selectively Modulates Vitamin C Transporter Expression Patterns in
           the Kidney
    • Abstract: In the kidney, vitamin C is reabsorbed from the glomerular ultrafiltrate by sodium‐vitamin C cotransporter isoform 1 (SVCT1) located in the brush border membrane of the proximal tubules. Although we know that vitamin C levels decrease with age, the adaptive physiological mechanisms used by the kidney for vitamin C reabsorption during aging remain unknown. In this study, we used an animal model of accelerated senescence (SAMP8 mice) to define the morphological alterations and aging‐induced changes in the expression of vitamin C transporters in renal tissue. Aging induced significant morphological changes, such as periglomerular lymphocytic infiltrate and glomerular congestion, in the kidneys of SAMP8 mice, although no increase in collagen deposits was observed using 2‐photon microscopy analysis and second harmonic generation. The most characteristic histological alteration was the dilation of intracellular spaces in the basolateral region of proximal tubule epithelial cells. Furthermore, a combination of laser microdissection, qRT‐PCR and immunohistochemical analyses allowed us to determine that SVCT1 expression specifically increased in the proximal tubules from the outer strip of the outer medulla (segment S3) and cortex (segment S2) during aging and that these tubules also express GLUT1. We conclude that aging modulates vitamin C transporter expression and that renal over‐expression of SVCT1 enhances vitamin C reabsorption in aged animals that may synthesize less vitamin C. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T17:00:23.819469-05:
      DOI: 10.1002/jcp.25504
       
  • Late Endosomal Recycling of Open MHC‐I Conformers
    • Abstract: With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty Ld (eLd) molecules, an open form of murine MHC‐I allele, in fibroblast‐like cells. Pulse labeling of cell surface eLd with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC‐I conformers. Kinetic modeling, using in‐house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eLd demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eLd distributed into pre‐degradative multivesicular bodies (MVBs), these LE subsets were not a source for eLd recycling. The LE recycling of eLd did not require Rab7 membrane domains, as demonstrated by Rab7‐silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eLd in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:50:44.218757-05:
      DOI: 10.1002/jcp.25495
       
  • Nestin Expressed by Pre‐Existing Cardiomyocytes Recapitulated in Part an
           Embryonic Phenotype; Suppressive Role of p38 MAPK
    • Abstract: Nestin(+)‐cardiomyocytes were identified in the ischemically damaged human/rodent heart, albeit the cellular source and signaling events implicated in the appearance of the intermediate filament protein remained undefined. Expression of the enhanced green fluorescent protein (EGFP) driven by the second intron of the nestin gene identified a subpopulation of EGFP/nestin(+) cells that differentiated to a vascular phenotype in the peri‐infarct/infarct region of post‐MI mice albeit the transgene was not detected in nestin(+)‐cardiomyocytes. α‐MHC‐driven expression of the reporter mCherry was detected in troponin‐T(+)‐ and nestin(+)‐cardiomyocytes in the peri‐infarct/infarct region of post‐MI mice. However, the cell cycle re‐entry of nestin/mCherry(+)‐cardiomyocytes was not observed. Nestin staining was identified in a paucity of neonatal rat ventricular cardiomyocytes (NNVM). Exposure to phorbol 12, 13‐dibutyrate (PDBu) induced NNVM hypertrophy but did not promote nestin expression or Brdu incorporation. PDBu treatment of NNVMs phosphorylated p38 MAPK and HSP27 and HSP27 phosphorylation was abrogated by the p38 MAPK inhibitor SB203580. PDBu/SB203580 co‐treatment significantly increased the percentage of NNVMs that expressed nestin and incorporated Brdu. In the heart of embryonic 10.5 day mice, nestin was detected in cycling troponin‐T(+)‐cardiomyocytes. Nestin was also detected in embryonic rat ventricular cardiomyocytes and depletion of the intermediate filament protein attenuated cell cycle re‐entry. Thus, nestin expressed by pre‐existing cardiomyocytes following ischemic damage recapitulated in part an embryonic phenotype and may provide the requisite phenotype to initiate cell cycle re‐entry. However, the overt activation of the p38 MAPK pathway post‐MI may in part limit the appearance and inhibit the cell cycle re‐entry of nestin(+)‐cardiomyocytes. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:50:36.765274-05:
      DOI: 10.1002/jcp.25496
       
  • Neuronatin Protein in Health and Disease
    • Authors: Priyamvada M. Pitale; Wayne Howse, Marina Gorbatyuk
      Abstract: Neuronatin (NNAT) was first identified as a brain‐specific gene crucial for brain development. Over the years, NNAT has been studied in different developing and post‐developed tissues and organs. While NNAT manifests functional and structural similarities to the phospholamban gene, its physiological and pathological roles in healthy and diseased tissues have not been precisely identified. Ca2+ signaling, glucose transport, insulin secretion, and inflammation modulated at different pathological conditions have been proposed to be governed by NNAT. This review describes the current findings of cellular molecular pathways known to be modified concomitantly with an alteration in NNAT expression, and it highlights the need to conduct extensive investigation regarding the role of NNAT in health and disease. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:50:23.162398-05:
      DOI: 10.1002/jcp.25498
       
  • Tyrosine Residues Regulate Multiple Nuclear Functions of p54nrb
    • Authors: Ahn R. Lee; Wayne Hung, Ning Xie, Liangliang Liu, Leye He, Xuesen Dong
      Abstract: The non‐POU‐domain‐containing octamer binding protein (NONO; also known as p54nrb) has various nuclear functions ranging from transcription, RNA splicing, DNA synthesis and repair. Although tyrosine phosphorylation has been proposed to account for the multi‐functional properties of p54nrb, direct evidence on p54nrb as a phosphotyrosine protein remains unclear. To investigate the tyrosine phosphorylation status of p54nrb, we performed site‐directed mutagenesis on the five tyrosine residues of p54nrb, replacing the tyrosine residues with phenylalanine or alanine, and immunoblotted for tyrosine phosphorylation. We then preceded with luciferase reporter assays, RNA splicing minigene assays, co‐immunoprecipitation, and confocal microscopy to study the function of p54nrb tyrosine residues on transcription, RNA splicing, protein‐protein interaction, and cellular localization. We found that p54nrb was not phosphorylated at tyrosine residues. Rather, it has non‐specific binding affinity to anti‐phosphotyrosine antibodies. However, replacement of tyrosine with phenylalanine altered p54nrb activities in transcription co‐repression and RNA splicing in gene context‐dependent fashions by means of differential regulation of p54nrb protein association with its interacting partners and co‐regulators of transcription and splicing. These results demonstrate that tyrosine residues, regardless of phosphorylation status, are important for p54nrb function. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-27T10:45:26.08248-05:0
      DOI: 10.1002/jcp.25493
       
  • Glioblastoma Multiforme and Adult Neurogenesis in the
           Ventricular‐Subventricular Zone: A Review
    • Abstract: Brain cancers account for less than 1,5% of all new cancer cases reported in the United States each year. Due to their invasive and heterogeneous nature, in addition to their resistance to multimodal treatments, these tumors are usually fatal. Gliomas, and in particular high‐grade astrocytomas such as glioblastoma multiforme (GBM), are the most common and lethal primary tumors of the central nervous system. The median survival of most patients is less than one year after application of multimodal therapies. The question is why are these cancers so injurious' And above all, how is it possible for a so carefully orchestrated area like the brain to develop such tumors' This brings us to the study of glioma stem cells, their specialized niches (perivascular and hypoxic), and the neurogenic phenomena that takes place within the adult ventricular‐subventricular zone: a structure that lies at the intersection between brain development and gliomagenesis. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-26T15:30:24.649733-05:
      DOI: 10.1002/jcp.25502
       
  • Role of Thrombin in the Pathogenesis of Central Nervous System
           Inflammatory Diseases
    • Authors: Safieh Ebrahimi; Najme Jaberi, Amir Avan, Mikhail Ryzhikov, Mohammad Reza Keramati, Mohammad Reza Parizadeh, Seyed Mahdi Hassanian
      Abstract: Thrombin initiates proinflammatory signaling responses through activation of protease‐activated receptors (PARs) in in vitro and in vivo systems. Proinflammatory signaling function of thrombin increases secretion of proinflammatory cytokines and chemokines, triggers vascular permeability, promotes leukocyte migration, and induces adhesion molecule expression. Thrombin as a potent signaling molecule is strongly implicated in a number of proinflammatory disorders including severe sepsis, cancer, cardiovascular disease, and of special interest in this review neurodegenerative disorders. This review summarizes the role of thrombin in the pathogenesis of central nervous system (CNS) inflammatory diseases, including Alzheimer's disease (AD) and Parkinson's disease (PD), promoting greater understanding and clinical management of these diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-26T15:30:21.373423-05:
      DOI: 10.1002/jcp.25501
       
  • Quantitative Analysis of Endocytic Recycling of Membrane Proteins by
           Monoclonal Antibody‐Based Recycling Assays
    • Abstract: In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin‐dependent endocytosis, represented by the transferrin receptor, and by clathrin‐independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin‐dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC‐conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti‐Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody‐capture protocols. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-26T15:25:23.719938-05:
      DOI: 10.1002/jcp.25503
       
  • Cytoskeleton Aberrations in Alkaptonuric Chondrocytes
    • Authors: Michela Geminiani; Silvia Gambassi, Lia Millucci, Pietro Lupetti, Giulia Collodel, Lucia Mazzi, Bruno Frediani, Daniela Braconi, Barbara Mazzocchi, Marcella Laschi, Giulia Bernardini, Annalisa Santucci
      Abstract: Alkaptonuria (AKU) is an ultra‐rare autosomal genetic disorder caused by a defect in the activity of the enzyme homogentisate 1,2‐dioxygenase (HGD) that leads to the accumulation of homogentisic acid (HGA) and its oxidized product, benzoquinone acetic acid (BQA), in the connective tissues causing a pigmentation called “ochronosis”. The consequent progressive formation of ochronotic aggregates generate a severe condition of oxidative stress and inflammation in all the affected areas. Experimental evidences have also proved the presence of serum amyloid A (SAA) in several AKU tissues and it allowed classifying AKU as a secondary amyloidosis. Although AKU is a multisystemic disease, the most affected system is the osteoarticular one and articular cartilage is the most damaged tissue. In this work, we have analyzed for the first time the cytoskeleton of AKU chondrocytes by means of immunofluorescence staining. We have shown the presence of SAA within AKU chondrocytes and finally we have demonstrated the co‐localization of SAA with three cytoskeletal proteins: actin, vimentin and β‐tubulin. Furthermore, in order to observe the ultrastructural features of AKU chondrocytes we have performed TEM analysis, focusing on the Golgi apparatus structure and, to demonstrate that pigmented areas in AKU cartilage are correspondent to areas of oxidation, 4‐HNE presence has been evaluated by means of immunofluorescence. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-25T14:55:30.2404-05:00
      DOI: 10.1002/jcp.25500
       
  • MiR‐145 Regulates Lipogenesis in Goat Mammary Cells via Targeting INSIG1
           and Epigenetic Regulation of Lipid‐Related Genes
    • Authors: Hui Wang; Huaiping Shi, Jun Luo, Yongqing Yi, Dawei Yao, Xueying Zhang, Gongzhen Ma, Juan J. Loor
      Abstract: MicroRNAs (miRNAs) are noncoding RNA molecules that regulate gene expression at the post‐transcriptional level to cause translational repression or degradation of targets. The profiles of miRNAs across stages of lactation in small ruminant species such as dairy goats is unknown. A small RNA library was constructed using tissue samples from mammary gland of Saanen dairy goats harvested at mid‐lactation followed by sequencing via Solexa technology. A total of 796 conserved miRNAs, 263 new miRNAs and 821 pre‐miRNAs were uncovered. After comparative analyses of our sequence data with published mammary gland transcriptome data across different stages of lactation, a total of 37 miRNAs (including miR‐145) had significant differences in expression over the lactation cycle. Further studies revealed that miR‐145 regulates metabolism of fatty acids in goat mammary gland epithelial cells (GMEC). Compared with nonlactating mammary tissue, lactating mammary gland had a marked increase in expression of miR‐145. Overexpression of miR‐145 increased transcription of genes associated with milk fat synthesis resulting in greater fat droplet formation, triacylglycerol accumulation, and proportion of unsaturated fatty acids. In contrast, silencing of miR‐145 impaired fatty acid synthesis. Inhibition of miR‐145 increased methylation levels of fatty acid synthase (FASN), stearoyl‐CoA desaturase 1 (SCD1), peroxisome proliferator‐activated receptor gamma (PPARG), and sterol regulatory element binding transcription factor 1 (SREBF1). Luciferase reporter assays confirmed that insulin induced gene 1 (INSIG1) is a direct target of miR‐145. These findings underscore the need for further studies to evaluate the potential for targeting miR‐145 for improving beneficial milk components in ruminant milk. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-22T16:10:27.305623-05:
      DOI: 10.1002/jcp.25499
       
  • BAG3 Protein Is Over‐ Expressed in Endometrioid Endometrial
           Adenocarcinomas
    • Authors: Veronica Esposito; Carlo Baldi, Pio Zeppa, Michelina Festa, Luana Guerriero, Morena d'Avenia, Massimiliano Chetta, Fulvio Zullo, Vincenzo De Laurenzi, Maria Caterina Turco, Alessandra Rosati, Maurizio Guida
      Abstract: Endometrioid adenocarcinomas represent 80% of endometrial carcinomas1. Molecular features, including microsatellite instability, mutations of the PTEN, PIK3CA, K‐Ras and β‐catenin genes1 and dysregulations in sncRNAs (small non coding RNAs) are described for this disease. However, mechanisms and molecules that determine cell survival and response/resistance to therapy in different subtypes of this tumour are not fully clarified1. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-22T05:50:31.02862-05:0
      DOI: 10.1002/jcp.25489
       
  • 3‐Methylcholanthrene/Aryl‐Hydrocarbon Receptor–Mediated Hypertension
           through eNOS Inactivation
    • Abstract: Endothelial nitric oxide synthase (eNOS) modulates vascular blood pressure and is predominantly expressed in endothelial cells and activated through the protein kinase B (Akt/PKB)‐dependent pathway. We previously reported that 3‐methylcholanthrene (3MC) activates the aryl hydrocarbon receptor (AhR) and reduces PI3K/Akt phosphorylation. This study investigated the mechanism underlying the downregulatory effects of 3‐MC on nitric oxide (NO) production occurring through the AhR/RhoA/Akt‐mediated mechanism. The mechanism underlying the effects of 3‐MC on eNOS activity and blood pressure was examined in vitro and in vivo through genetic and pharmacological approaches. Results indicated that 3‐MC modified heat shock protein 90 (HSP90), caveolin‐1, dynein, and eNOS mRNA and protein expression through the AhR/RhoA‐dependent mechanism in mouse cerebral vascular endothelial cells (MCVECs) and that 3‐MC reduced eNOS phosphorylation through the AhR/RhoA‐mediated inactivation of Akt1. The upregulation of dynein expression was associated with decreased eNOS dimer formation (eNOS dimer; an activated form of the enzyme). Coimmunoprecipitation assay results indicated that 3‐MC significantly reduced the interaction between eNOS and its regulatory proteins, including Akt1 and HSP90, but increased the interaction between eNOS and caveolin‐1. Immunofluorescence and western blot analysis revealed that 3‐MC reduced the amount of membrane‐bound activated eNOS, and a modified Griess assay revealed that 3‐MC concomitantly reduced NO production. However, simvastatin reduced 3‐MC–mediated murine hypertension. Our study results indicate that AhR, RhoA, and eNOS have major roles in blood pressure regulation. Statin intervention might provide a potential therapeutic approach for reducing hypertension caused by 3‐MC. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-21T11:30:22.668827-05:
      DOI: 10.1002/jcp.25497
       
  • Atrogin‐1 Increases Smooth Muscle Contractility through Myocardin
           Degradation
    • Abstract: Atrogin‐1, an E3 ligase present in skeletal, cardiac and smooth muscle, down‐regulates myocardin protein during skeletal muscle differentiation. Myocardin, the master regulator of smooth muscle cell (SMC) differentiation, induces expression of smooth muscle marker genes through its association with serum response factor (SRF), which binds to the CArG box in the promoter. Myocardin undergoes ubiquitylation and proteasomal degradation. Evidence suggests that proteasomal degradation of myocardin is critical for myocardin to exert its transcriptional activity, but there is no report about the E3 ligase responsible for myocardin ubiquitylation and subsequent transactivation. Here, we showed that overexpression of atrogin‐1 increased contractility of cultured SMCs and mouse aortic tissues in organ culture. Overexpression of dominant‐negative myocardin attenuated the increase in SMC contractility induced by atrogin‐1. Atrogin‐1 overexpression increased expression of the SM contractile markers while downregulated expression of myocardin protein but not mRNA. Atrogin‐1 also ubiquitylated myocardin for proteasomal degradation in vascular SMCs. Deletion studies showed that atrogin‐1 directly interacted with myocardin through its amino acids 284‐345. Immunostaining studies showed nuclear localization of atrogin‐1, myocardin and the Rpt6 subunit of the 26S proteasome. Atrogin‐1 overexpression not only resulted in degradation of myocardin but also increased recruitment of RNA Polymerase II onto the promoters of myocardin target genes. In summary, our results have revealed the roles for atrogin‐1 in the regulation of smooth muscle contractility through enhancement of myocardin ubiquitylation/degradation and its transcriptional activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-21T11:20:36.573109-05:
      DOI: 10.1002/jcp.25485
       
  • Can Cholesterol Metabolism Modulation Affect Brain Function and
           Behavior'
    • Authors: Veronica Cartocci; Michela Servadio, Viviana Trezza, Valentina Pallottini
      Abstract: Cholesterol is an important component for cell physiology. It regulates the fluidity of cell membranes and determines the physical and biochemical properties of proteins. In the central nervous system, cholesterol controls synapse formation and function and supports the saltatory conduction of action potential. In recent years, the role of cholesterol in the brain has caught the attention of several research groups since a breakdown of cholesterol metabolism has been associated with different neurodevelopmental and neurodegenerative diseases, and interestingly also with psychiatric conditions. The aim of this review is to summarize the current knowledge about the connection between cholesterol dysregulation and various neurologic and psychiatric disorders based on clinical and preclinical studies. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T12:06:03.33063-05:0
      DOI: 10.1002/jcp.25488
       
  • Vimentin as a Marker of Early Differentiating, Highly Motile Corneal
           Epithelial Cells
    • Abstract: Vimentin (Vim), a cytoskeletal intermediate filament, is part of a naturally occurring reversible program, the Epithelial‐Mesenchymal Transition (EMT), which converts epithelial cells into mesenchymal‐like derivatives. Based on previous results showing that epithelial cells co‐express Vim and keratin (Krt) as part of a cytoskeletal network which confers them a highly motile phenotype, we explored the role of Vim in rabbit corneal epithelial cells or RCE1(5T5) cells, an established model of corneal epithelial differentiation. Vim and keratin filaments were co‐expressed in cells localized at the proliferative/migratory rim of the growing colonies, but not in basal cells from the center of the colonies nor at suprabasal cell layers. Flow cytometry and qPCR demonstrated that there was a decrease in Krt+/Vim+ cell number and ΔNp63α expression when cells reached confluence and formed a 4‐5 layered epithelium, while there was a concomitant increase of both Pax‐6 expression and Krt+/Vim‐ cells. Inhibition of cell proliferation with mitomycin C did not modify cell motility nor the expression of Vim. We studied the distribution and expression of α6 integrin, a protein also involved in cell migration. The results demonstrated that α6 integrin had a distribution which was, in part, co‐linear with Vim at the proliferative/migratory rim of cell colonies, suggesting an indirect interaction between these proteins. Immunoprecipitation and immunostaining assays indicated that plectin might be mediating such interaction. These data suggest that Vim expression in corneal epithelium is found in a cell population composed of highly motile cells with a Vim+/Krt+/ΔNp63α+/Pax‐6low/α6 integrin+ phenotype. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T12:01:04.868486-05:
      DOI: 10.1002/jcp.25487
       
  • Expression of P2 Purinergic Receptors in Mesenchymal Stem Cells and their
           Roles in Extracellular Nucleotide Regulation of Cell Functions
    • Abstract: Extracellular ATP and other nucleotides induce autocrine and/or paracrine purinergic signalling via activation of the P2 receptors on the cell surface, which represents one of the most common signalling mechanisms. Mesenchymal stem cells (MSC) are a type of multipotent adult stem cells that have many promising applications in regenerative medicine. There is increasing evidence to show that extracellular nucleotides regulate MSC functions and P2 receptor‐mediated purinergic signalling plays an important role in such functional regulation. P2 receptors comprise ligand‐gated ion channel P2X receptors and G‐protein‐coupled P2Y receptors. In this review, we provide an overview of the current understanding with respect to expression of the P2X and P2Y receptors in MSC and their roles in mediating extracellular nucleotide regulation of MSC proliferation, migration and differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T12:00:41.476616-05:
      DOI: 10.1002/jcp.25484
       
  • The β2‐Adrenoceptor Agonist Terbutaline Stimulates Angiogenesis
           via Akt and ERK Signaling
    • Authors: Stefanie Lemmens; Lauren Kusters, Annelies Bronckaers, Nathalie Geurts, Sven Hendrix
      Abstract: Angiogenesis is associated with changes in endothelial cell (EC) proliferation and tube formation, controlled by extracellular receptor‐activated kinase (ERK)/mitogen activated protein kinase (MAPK) and Akt signaling. Important regulators of these systems include hormones acting on G‐protein‐coupled receptors, such as beta 2‐adrenoceptors (β2‐ARs).In central nervous system (CNS) trauma, the importance of β2‐AR modulation has been highlighted, although the effects on revascularization remain unclear. Vascular protection and revascularization are, however, key to support regeneration.We have investigated the angiogenic capacity of the specific β2‐AR agonist terbutaline on ECs derived from the CNS, namely bEnd.3‐cells. As angiogenesis is a multistep process involving increased proliferation and tube formation of ECs, we investigated the effects of terbutaline on these processes. We show that terbutaline significantly induced bEnd.3 tube formation in a matrigel in vitro assay. Moreover, administration of specific inhibitors of ERK and Akt signaling both inhibited terbutaline‐induced tube formation. The proliferation rate of the ECs was not affected. In order to investigate the general effects of terbutaline in an organotypic system, we have used the chick chorioallantoic membrane (CAM)‐assay. Most importantly, terbutaline increased the number of blood vessels in this in ovo setting. Although we observed a positive trend, the systemic administration of terbutaline did not significantly improve the functional outcome, nor did it affect revascularization in our spinal cord injury model.In conclusion, these data indicate that terbutaline is promising to stimulate blood vessel formation, underscoring the importance of further research into the angiotherapeutic relevance of terbutaline and β2‐AR signaling after CNS‐trauma. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-20T11:50:35.977902-05:
      DOI: 10.1002/jcp.25483
       
  • Extracellular Proteasomes are Deficient in 19S Subunits as Revealed by
           iTRAQ Quantitative Proteomics
    • Authors: Anna S. Tsimokha; Julia J. Zaykova, Andrew Bottrill, Nikolai A. Barlev
      Abstract: Proteasome‐mediated proteolysis is critical for regulation of vast majority of cellular processes. In addition to their well‐documented functions in the nucleus and cytoplasm proteasomes have also been found in extracellular space. The origin and functions of these proteasomes, dubbed as circulating/plasmatic or extracellular proteasomes, are unclear. To gain insights into the molecular and functional differences between extracellular (EPs) and cellular proteasomes (CPs) we compared their subunit composition using iTRAQ‐based quantitative proteomics (iTRAQ LC/MS‐MS). Our analysis of purified from K562 cells or conditioned medium intact proteasome complexes led to an identification and quantification of 114 proteins, out of which 19 were 26S proteasome proteins (all subunits of the 20S proteasome and a small number of the 19S regulatory particle proteins), and 3 belonged to the ubiquitin system. 62 of proteasome interacting proteins (PIPs) were differentially represented in CP versus EP, with folds difference ranging from 1.5 to 4.8. The bioinformatics analysis revealed that functionally most of EP‐PIPs were associated with protein biosynthesis and, unlike CP‐PIPs, were under represented by chaperon/ATP‐binding proteins. Identities of some of the proteasome proteins and PIPs were verified by Western blotting. Importantly, we uncovered that the stoichiometry of the 20S versus 19S complexes in the extracellular proteasomes was different compared to the one calculated for the intracellular proteasomes. Specifically, the EP prep contained only three 19S subunits versus at least 18 in the CP one, suggesting that the extracellular proteasomes are deficient in 19S complexes, which may imply that they have special biological functions. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-19T05:05:44.685377-05:
      DOI: 10.1002/jcp.25492
       
  • DNA Methylation Profiling in Chondrocyte Dedifferentiation In Vitro
    • Authors: Li Duan; Yujie Liang, Bin Ma, Daming Wang, Wei Liu, Jianghong Huang, Jianyi Xiong, Liangquan Peng, Jielin Chen, Weimin Zhu, Daping Wang
      Abstract: DNA methylation has emerged as a crucial regulator of chondrocyte dedifferentiation, which severely compromises the outcome of autologous chondrocyte implantation (ACI) treatment for cartilage defects. However, the full ‐scale DNA methylation profiling in chondrocyte dedifferentiation remains to be determined. Here, we performed a genome‐wide DNA methylation profiling of dedifferentiated chondrocytes in monolayer culture and chondrocytes treated with DNA methylation inhibitor 5‐azacytidine (5‐AzaC). This research revealed that the general methylation level of CpG was increased while the COL‐1A1 promoter methylation level was decreased during the chondrocyte dedifferentiation. 5‐AzaC could reduce general methylation levels and reverse the chondrocyte dedifferentiation. Surprisingly, the DNA methylation level of COL‐1A1 promoter was increased after 5‐AzaC treatment. The COL‐1A1 expression level was increased while that of SOX‐9 was decreased during the chondrocyte dedifferentiation. 5‐AzaC treatment up‐regulated the SOX‐9 expression while down‐regulated the COL‐1A1 promoter activity and gene expression. Taken together, these results suggested that differential regulation of the DNA methylation level of cartilage‐specific genes might contribute to the chondrocyte dedifferentiation. Thus, the epigenetic manipulation of these genes could be a potential strategy to counteract the chondrocyte dedifferentiation accompanying in vitro propagation. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-12T16:26:00.893453-05:
      DOI: 10.1002/jcp.25486
       
  • Combined Multidimensional Microscopy as a Histopathology Imaging Tool
    • Authors: Gerald J. Shami; Delfine Cheng, Filip Braet
      Abstract: Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X‐ray micro‐computed tomography, bright‐field light microscopy and backscattered scanning electron microscopy, thus facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterise various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high‐resolution backscattered scanning electron microscopy, we were able to further characterise the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-09T09:15:35.773428-05:
      DOI: 10.1002/jcp.25470
       
  • Evidence for the Role of BAG3 in Mitochondrial Quality Control in
           Cardiomyocytes
    • Authors: Farzaneh G. Tahrir; Tijana Knezevic, Manish K. Gupta, Jennifer Gordon, Joseph Y. Cheung, Arthur M. Feldman, Kamel Khalili
      Abstract: Mitochondrial abnormalities impact the development of myofibrillar myopathies. Therefore, understanding the mechanisms underlying the removal of dysfunctional mitochondria from cells is of great importance toward understanding the molecular events involved in the genesis of cardiomyopathy. Earlier studies have ascribed a role for BAG3 in the development of cardiomyopathy in experimental animals leading to the identification of BAG3 mutations in patients with heart failure which may play a part in the onset of disease development and progression. BAG3 is co‐chaperone of heat shock protein 70 (HSP70), which has been shown to modulate apoptosis and autophagy, in several cell models. In this study, we explore the potential role of BAG3 in mitochondrial quality control. We demonstrate that siRNA mediated suppression of BAG3 production in neonatal rat ventricular cardiomyocytes (NRVCs) significantly elevates the level of Parkin, a key component of mitophagy. We found that both BAG3 and Parkin are recruited to depolarized mitochondria and promote mitophagy. Suppression of BAG3 in NRVCs significantly reduces autophagy flux and eliminates expression of Tom20, an essential import receptor for mitochondria proteins, after induction of mitophagy. These observations suggest that BAG3 is critical for the maintenance of mitochondrial homeostasis under stress conditions, and disruptions in BAG3 expression impact cardiomyocyte function. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-06T06:50:22.924349-05:
      DOI: 10.1002/jcp.25476
       
  • Biochemical and Pathophysiological Premises to Positron Emission
           Tomography with Choline Radiotracers
    • Authors: Vincenzo Cuccurullo; Giuseppe Danilo Di Stasio, Laura Evangelista, Gabriella Castoria, Luigi Mansi
      Abstract: Choline is a quaternary ammonium base that represents an essential component of phospholipids and cell membranes. Malignant transformation is associated with an abnormal choline metabolism at a higher levels with respect to those exclusively due to cell multiplication. The use of Positron Emission Tomography/Computed Tomography (PET/CT) with radiocholine (RCH), labeled with 11C or 18F, is widely diffuse in oncology, with main reference to restaging of patients with prostate cancer. The enhanced concentration in neoplasm is based not only on the increasing growing rate, but also on more specific issues, such as the augmented uptake in malignant cells due to the up‐regulation of choline kinase. Furthermore the role of hypoxia in decreasing choline's uptake determine an in vivo concentration only in well oxygenated tumors, with a lower uptake when malignancy increases, i.e. in tumors positive at 18F‐Fluoro‐deoxyglucose. In this paper we have analyzed the most important issues related to the possible utilization of RCH in diagnostic imaging of human cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-06T06:46:59.668408-05:
      DOI: 10.1002/jcp.25478
       
  • The Effects of Acute and Chronic Exercise on Skeletal Muscle Proteome
    • Abstract: Skeletal muscle plasticity and its adaptation to exercise is a topic that is widely discussed and investigated due to its primary role in the field of exercise performance and health promotion. Repetitive muscle contraction through exercise stimuli leads to improved cardiovascular output and the regulation of endothelial dysfunction and metabolic disorders such as insulin resistance and obesity. Considerable improvements in proteomic tools and data analysis have broth some new perspectives in the study of the molecular mechanisms underlying skeletal muscle adaptation in response to physical activity. In this sense, this review updates the main relevant studies concerning muscle proteome adaptation to acute and chronic exercise, from aerobic to resistance training, as well as the proteomic profile of natural inbred high running capacity animal models. Also, some promising prospects in the muscle secretome field are presented, in order to better understand the role of physical activity in the release of extracellular microvesicles and myokines activity. Thus, the present review aims to update the fast‐growing exercise‐proteomic scenario, leading to some new perspectives about the molecular events under skeletal muscle plasticity in response to physical activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-06T06:46:56.937592-05:
      DOI: 10.1002/jcp.25477
       
  • An Innovative Assay for the Analysis of In Vitro Endothelial Remodeling:
           Experimental and Computational Evidence
    • Authors: Marco Scianna; Eleonora Bassino, Luca Munaron
      Abstract: The molecular and cellular mechanisms underlying vascular remodeling are currently investigated several experimental strategies which aim to mimic the complex environmental conditions found in vivo. Some of them focus on the tubulogenic activity of dispersed endothelial cell populations, while others evaluate vascular sprouting. Here we propose a new method to assess matrigel invasion starting from confluent or subconfluent monolayers of human microvascular ECs (HMVEC) seeded on different substrates. The experimental setting is also validated by an improved hybrid multiscale mathematical approach, which integrates a mesoscopic grid‐based cellular Potts model, that describes HMVEC phenomenology, with a continuous one, accounting for the kinetics of diffusing growth factors. Both experimental and theoretical approaches show that the endothelial potential to invade, migrate and organize in tubule structures is a function of selected environmental parameters. The present methodology is intended to be simple to use, standardized for rapid screening and suitable for mechanistic studies. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-30T10:30:30.870964-05:
      DOI: 10.1002/jcp.25468
       
  • microRNA‐30b Suppresses Epithelial‐Mesenchymal Transition and
           Metastasis of Hepatoma Cells
    • Authors: Xiaolin Sun; Shuhua Zhao, Huanan Li, Hanwen Chang, Zhen Huang, Zhi Ding, Lei Dong, Jiangning Chen, Yuhui Zang, Junfeng Zhang
      Abstract: Epithelial–mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis in HCC. Growing evidence indicates that upregulation of Snail, the major EMT inducer, significantly correlates with the metastasis and poor prognosis of HCC. Here, we investigate the underlying mechanism of miR‐30b in suppressing metastasis of hepatoma cells by targeting Snail. In this study, we found that miR‐30b was significantly downregulated and negatively associated with Snai1 production in HCC cell lines with higher metastatic potentials. Gain‐ and loss‐of‐function studies revealed that miR‐30b could dramatically inhibit in vitro HCC cell migration and invasion. In vivo orthotopic liver xenograft model further demonstrated that stable over‐expression of miR‐30b significantly repressed the local invasion and lung metastasis of hepatoma cells. Meanwhile, the restoration of miR‐30b expression suppressed the distant colonization of hepatoma cells. Both gain‐ and loss‐of‐function studies showed that miR‐30b suppressed the EMT of hepatoma cells as indicated by the morphology changes and deregulation of epithelial and mesenchymal markers. Using RNAi, we further investigated the role of Snai1 in HCC cell EMT and demonstrated that knockdown of Snai1 significantly inhibited the EMT and cancer cell metastasis. Additionally, miR‐30b exhibited inhibitory effects on HCC cell proliferation in vitro and in vivo. In conclusion, our findings highlight the significance of miR‐30b downregulation in HCC tumor metastasis and invasiveness, and implicate a new potential therapeutic target for HCC metastasis. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-30T10:25:27.648539-05:
      DOI: 10.1002/jcp.25466
       
  • Parathyroid Hormone‐Related Protein Protects Osteoblastic Cells from
           Oxidative Stress by Activation of MKP1 Phosphatase
    • Abstract: Oxidative damage is an important contributor to the morphological and functional changes in osteoporotic bone. Aging increases the levels of reactive oxygen species (ROS) that cause oxidative stress and induce osteoblast apoptosis. ROS modify several signaling responses, including mitogen‐activated protein kinase (MAPK) activation, related to cell survival. Both parathyroid hormone (PTH) and its bone counterpart, PTH‐related protein (PTHrP), can regulate MAPK activation by modulating MAPK phosphatase‐1 (MKP1). Thus, we hypothesized that PTHrP might protect osteoblasts from ROS‐induced apoptosis by targeting MKP1. In osteoblastic MC3T3‐E1 and MG‐63 cells, H2O2 triggered p38, JNK, ERK and p66Shc phosphorylation and cell apoptosis. Meanwhile, PTHrP (1‐37) rapidly but transiently increased ERK and Akt phosphorylation without affecting p38, JNK or p66Shc activation. H2O2‐induced p38 and ERK phosphorylation and apoptosis were both decreased by pre‐treatment with specific kinase inhibitors or PTHrP (1‐37) in both osteoblastic cell types. These dephosphorylating and prosurvival actions of PTHrP (1‐37) were prevented by a phosphatase inhibitor cocktail, the phosphatase MKP1 inhibitor sanguinarine or a MKP1 siRNA. PTHrP (1‐37) promptly enhanced MKP1 protein and gene expression and MKP1‐dependent catalase activity in osteoblastic cells. Furthermore, exposure to PTHrP (1‐37) adsorbed in an implanted hydroxyapatite‐based ceramic into a tibial defect in aging rats increased MKP1 and catalase gene expression in the healing bone area. Our findings demonstrate that PTHrP counteracts the pro‐apoptotic actions of ROS by a mechanism dependent on MKP1‐induced dephosphorylation of MAPKs in osteoblasts. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-30T06:25:27.24047-05:0
      DOI: 10.1002/jcp.25473
       
  • Transcriptomic Analyses of Adipocyte Differentiation from Human
           Mesenchymal Stromal‐Cells (MSC)
    • Abstract: Adipogenesis is a physiological process required for fat‐tissue development, mainly involved in regulating the organism energetic‐state. Abnormal distribution‐changes and dysfunctions in such tissue are associated to different pathologies. Adipocytes are generated from progenitor cells, via a complex differentiating process not yet well understood. Therefore, we investigated differential mRNA and miRNA expression patterns of human mesenchymal stromal‐cells (MSC) induced and not induced to differentiate into adipocytes by next (second)‐generation sequencing. A total of 2,866 differentially‐expressed genes (101 encoding miRNA) were identified, with 705 (46 encoding miRNA) being upregulated in adipogenesis. They were related to different pathways, including PPARG, lipid, carbohydrate and energy metabolism, redox, membrane‐organelle biosynthesis and endocrine system. Downregulated genes were related to extracellular matrix and cell migration, proliferation and differentiation. Analyses of mRNA‐miRNA interaction showed that repressed miRNA‐encoding genes can act downregulating PPARG‐related genes; mostly the PPARG activator (PPARGC1A). Induced miRNA‐encoding genes regulate downregulated genes related to TGFB1. These results shed new light to understand adipose‐tissue differentiation and physiology, increasing our knowledge about pathologies like obesity, type‐2 diabetes and osteoporosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-28T04:17:05.839447-05:
      DOI: 10.1002/jcp.25472
       
  • Integrated Transcriptome Map Highlights Structural and Functional Aspects
           of the Normal Human Heart
    • Authors: Maria Caracausi; Allison Piovesan, Lorenza Vitale, Maria Chiara Pelleri
      Abstract: A systematic meta‐analysis of the available gene expression profiling datasets for the whole normal human heart generated a quantitative transcriptome reference map of this organ. TRAM (Transcriptome Mapper) software integrated 32 gene expression profile datasets from different sources returning a reference value of expression for each of the 43,360 known, mapped transcripts assayed by any of the experimental platforms used in this regard. Main findings include the visualization at the gene and chromosomal levels of the classical description of the basic histology and physiology of the heart, the identification of suitable housekeeping reference genes, the analysis of stoichiometry of gene products and the focusing on chromosome 21 genes, which are present in one excess copy in Down syndrome subjects, presenting cardiovascular defects in 30‐40% of cases. Independent in vitro validation showed an excellent correlation coefficient (r = 0.98) with the in silico data. Remarkably, heart/non‐cardiac tissues expression ratio may also be used to anticipate that effects of mutations will most probably affect or not the heart. The quantitative reference global portrait of gene expression in the whole normal human heart illustrates the structural and functional aspects of the whole organ and is a general model to understand the mechanisms underlying heart pathophysiology. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-27T06:55:22.777055-05:
      DOI: 10.1002/jcp.25471
       
  • SCD1 Alters Long‐Chain Fatty Acid (LCFA) Composition and its Expression
           is Directly Regulated by SREBP‐1 and PPARγ 1 in Dairy Goat Mammary
           Cells
    • Authors: Dawei Yao; Jun luo, Qiuya He, Hengbo Shi, Jun Li, Hui Wang, Huifen Xu, Zhi Chen, Yongqing Yi, Juan J. Loor
      Abstract: Stearoyl‐CoA desaturase 1 (SCD1) is a key enzyme for the synthesis of the monounsaturated fatty acids (MUFA) palmitoleic acid and oleic acid. In non‐ruminant species, SCD1 expression is known to be tightly regulated by a variety of transcription factors. Although the role of SCD1 and the transcriptional regulatory mechanism by SREBP‐1 and PPARs in other species is clear, changes in lipid metabolism related to SCD1 and via the regulation of SREBP‐1 or PPARG1 in ruminant mammary tissue remain largely unknown. Here, we demonstrated that SCD1 expression in goat mammary tissue is higher during lactation than the dry period. Overexpression of SCD1 increased the intracellular MUFA content and lipid accumulation, whereas SCD1 silencing resulted in a significant decrease in oleic acid concentration and triacylglycerol (TAG) accumulation. The overexpression of SREBF1 in goat mammary epithelial cells (GMEC) enhanced SCD1 expression and its promoter activity, but that effect was abolished when SREBF1 was silenced. Furthermore, deletion of sterol regulatory element (SRE) and the nuclear factor (NF‐Y) binding sites within a ‐1713∼ + 65‐base pair region of the SCD1 promoter completely abolished SREBP‐1‐induced SCD1 transcription. Otherwise, PPARG1 overexpression also stimulated the expression of SCD1 and its transcriptional activity directly via a PPAR response element (PPRE) in the SCD1 promoter. Together, these results indicate that SCD1 could markedly affect the fatty acid composition and rate of TAG synthesis through direct regulation via SREBP‐1 and PPARG1, hence, underscoring an important role of the enzyme and this transcription regulator in controlling mammary gland lipid synthesis in the goat. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-24T08:10:24.186014-05:
      DOI: 10.1002/jcp.25469
       
  • HDAC1, HDAC4 and HDAC9 Bind to PC3/Tis21/Btg2 and Are Required for its
           Inhibition of Cell Cycle Progression and Cyclin D1 Expression
    • Authors: Laura Micheli; Giorgio D'Andrea, Luca Leonardi, Felice Tirone
      Abstract: PC3/Tis21 is a transcriptional cofactor that inhibits proliferation in several cell types, including neural progenitors. Here we report that PC3/Tis21 associates with HDAC1, HDAC4 and HDAC9 in vivo, in fibroblast cells. Furthermore, when HDAC1, HDAC4 or HDAC9 are silenced in fibroblasts or in a line of cerebellar progenitor cells, the ability of PC3/Tis21 to inhibit proliferation is significantly reduced. Overexpression of HDAC1, HDAC4 or HDAC9 in fibroblasts and in cerebellar precursor cells synergizes with PC3/Tis21 in inhibiting the expression of cyclin D1, a cyclin selectively inhibited by PC3/Tis21. Conversely, the depletion of HDAC1 or HDAC4 (but not HDAC9) in fibroblasts and in cerebellar precursor cells significantly impairs the ability of PC3/Tis21 to inhibit cyclin D1 expression. An analysis of HDAC4 deletion mutants shows that both the amino‐ terminal moiety and the catalytic domain of HDAC4 associate to PC3/Tis21, but neither alone is sufficient to potentiate the inhibition of cyclin D1 by PC3/Tis21. As a whole, our findings indicate that PC3/Tis21 inhibits cell proliferation in a way dependent on the presence of HDACs, in fibroblasts as well as in neural cells.Considering that several reports have demonstrated that HDACs can act as transcriptional corepressors on the cyclin D1 promoter, our data suggest that the association of PC3/Tis21 to HDACs is functional to recruit them to target genes, such as cyclin D1, for repression of their expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-23T06:55:33.829497-05:
      DOI: 10.1002/jcp.25467
       
  • Further Support for ECM Control of Receptor Trafficking and Signaling
    • Authors: Lindsay Clegg; Feilim Mac Gabhann
      Abstract: Recently, Sack et al. presented an interesting, novel data set in JCP examining the effect of substrate stiffness on VEGF processing and signaling. The data represents a clear contribution to the field. However, the authors' conclusion that “extracellular matrix binding is essential for VEGF internalization” conflicts with other knowledge in the field, and is not supported by their data. Instead, their data demonstrates the effect of heparin addition and changing ECM stiffness on both VEGF binding to fibronectin and VEGF binding to endothelial receptors. This is consistent with other work showing that matrix‐ binding reduces VEGF‐VEGFR internalization, shifting downstream signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-20T09:45:37.567799-05:
      DOI: 10.1002/jcp.25464
       
  • DGKζ Downregulation Enhances Osteoclast Differentiation and Bone
           Resorption Activity Under Inflammatory Conditions
    • Authors: Kiyoshi Iwazaki; Toshiaki Tanaka, Yasukazu Hozumi, Masashi Okada, Rieko Tsuchiya, Ken Iseki, Matthew K. Topham, Kaneyuki Kawamae, Michiaki Takagi, Kaoru Goto
      Abstract: Bone homeostasis is maintained by a balance between resorption of the bone matrix and its replacement by new bone. Osteoclasts play a crucially important role in bone metabolism. They are responsible for bone resorption under pathophysiological conditions. Differentiation of these cells, which are derived from bone marrow cells, depends on receptor activator of NF‐κB ligand (RANKL). RANKL‐induced osteoclastogenesis is regulated by the phosphoinositide (PI) signaling pathway, in which diacylglycerol (DG) serves as a second messenger in signal transduction. In this study, we examined the functional implications of DG kinase (DGK), an enzyme family responsible for DG metabolism, for osteoclast differentiation and activity. Of DGKs, DGKζ is most abundantly expressed in osteoclast precursors such as bone marrow‐derived monocytes/macrophages. During osteoclast differentiation from precursor cells, DGKζ is downregulated at the protein level. In this regard, we found that DGKζ deletion enhances osteoclast differentiation and bone resorption activity under inflammatory conditions in an animal model of osteolysis. Furthermore, DGKζ deficiency upregulates RANKL expression in response to TNFα stimulation. Collectively, results suggest that DGKζ is silent under normal conditions, but it serves as a negative regulator in osteoclast function under inflammatory conditions. Downregulation of DGKζ might be one factor predisposing a person to osteolytic bone destruction in pathological conditions. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-17T05:50:26.946779-05:
      DOI: 10.1002/jcp.25461
       
  • Pancreatic Endoderm‐Derived from Diabetic Patient‐Specific Induced
           Pluripotent Stem Cell Generates Glucose‐Responsive Insulin‐Secreting
           Cells
    • Authors: Bahareh Rajaei; Mehdi Shamsara, Masume Fakhr Taha, Mohammad Massumi, Mohammad Hossein Sanati
      Abstract: Human induced pluripotent stem cells (hiPSCs) can potentially serve as an invaluable source for cell replacement therapy and allow the creation of patient‐ and disease‐specific stem cells without the controversial use of embryos and avoids any immunological incompatibility. The generation of insulin‐producing pancreatic β‐cells from pluripotent stem cells in vitro provides an unprecedented cell source for personal drug discovery and cell transplantation therapy in diabetes. A new five‐step protocol was introduced in this study, effectively induced hiPSCs to differentiate into glucose‐responsive insulin‐producing cells. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, primitive gut‐tube endoderm, posterior foregut, pancreatic endoderm and endocrine precursor. Each stage of differentiation were characterized by stage‐specific markers. The produced cells exhibited many properties of functional β‐cells, including expression of critical β‐cells transcription factors, the potency to secrete C‐peptide in response to high levels of glucose and the presence of mature endocrine secretory granules. This high efficient differentiation protocol, established in this study, yielded 79.18% insulin‐secreting cells which were responsive to glucose five times higher than the basal level. These hiPSCs‐derived glucose‐responsive insulin‐secreting cells might provide a promising approach for the treatment of type I diabetes mellitus. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-16T06:20:33.930205-05:
      DOI: 10.1002/jcp.25459
       
  • FGF23 Neutralizing Antibody Ameliorates Hypophosphatemia and Impaired FGF
           Receptor Signaling in Kidneys of HMWFGF2 Transgenic Mice
    • Authors: E. Du; L. Xiao, M. M. Hurley
      Abstract: High molecular weight FGF2 transgenic mice (HMWTg) phenocopy the Hyp mouse, homolog of human X‐linked hypophosphatemic rickets with phosphate wasting and abnormal fibroblast growth factor (FGF23), fibroblast growth factor receptor (FGFR), Klotho and mitogen activated protein kinases (MAPK) signaling in kidney. In this study, we assessed whether short term (24 hour) in vivo administration of FGF23 neutralizing antibody (FGF23Ab) could rescue hypophosphatemia and impaired FGFR signaling in kidneys of HMWTg male mice. Bone mineral density and bone mineral content in one‐month‐old HMWTg mice were significantly reduced compared with Control/VectorTg mice. Serum FGF23 was significantly increased in HMWTg compared with VectorTg. Serum phosphate was significantly reduced in HMWTg and was rescued by FGF23Ab. Serum parathyroid hormone (PTH) was significantly increased in HMWTg but was not reduced by FGF23Ab. 1, 25(OH)2 D was inappropriately normal in serum of HMWTg and was significantly increased in both Vector and HMWTg by FGF23Ab. Analysis of HMWTg kidneys revealed significantly increased mRNA expression of the FGF23 co‐receptor Klotho, transcription factor mRNAs for early growth response‐1 transcription factor (Egr‐1) and c‐fos were all significantly decreased by FGF23Ab. A significant reduction in the phosphate transporter Npt2a mRNA was also observed in HMWTg kidneys, which was increased by FGF23Ab. FGF23Ab reduced p‐FGFR1, p‐FGFR3, KLOTHO, p‐ERK1/2, C‐FOS and increased NPT2A protein in HMWTg kidneys. We conclude that FGF23 blockade rescued hypophosphatemia by regulating FGF23/FGFR downstream signaling in HMWTg kidneys. Furthermore, HMWFGF2 isoforms regulate PTH expression independent of FGF23/FGFR signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-16T06:15:25.784878-05:
      DOI: 10.1002/jcp.25458
       
  • Ethanol Extract of Cissus quadrangularis Enhances Osteoblast
           Differentiation and Mineralization of Murine Pre‐Osteoblastic MC3T3‐E1
           Cells
    • Abstract: Traditional medicinal literature and previous studies have reported the possible role of Cissus quadrangularis (CQ) as an anti‐osteoporotic agent. This study examines the effectiveness of CQ in promoting osteoblast differentiation of the murine pre‐osteoblast cell line, MC3T3‐E1. Ethanolic extract of CQ (CQ‐E) was found to affect growth kinetics of MC3T3‐E1 cells in a dosage dependent manner. High concentrations of CQ‐E (more than 10 µg/ml) have particularly adverse effects, while lower concentrations of 0.1 and 1 µg/ml were non‐toxic and did not affect cell viability. Notably, cell proliferation was significantly increased at the lower concentrations of CQ‐E. CQ‐E treatment also augmented osteoblast differentiation, as reflected by a substantial increase in expression of the early osteoblast marker ALP activity, and at later stage, by mineralization of extracellular matrix compared to the control group. These findings suggest dose‐dependent effect of CQ‐E with lower concentrations exhibiting anabolic and osteogenic properties. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:47.466708-05:
      DOI: 10.1002/jcp.25449
       
  • MicroRNA‐590‐5p stabilizes Runx2 by targeting Smad7 during
           osteoblast differentiation
    • Authors: M. Vishal; S. Vimalraj, R. Ajeetha, M. Gokulnath, R. Keerthana, Z. He, N.C. Partridge, N. Selvamurugan
      Abstract: Mesenchymal stem cells (MSCs) are multipotent cells and their differentiation into the osteoblastic lineage is strictly controlled by several regulators, including microRNAs (miRNAs). Runx2 is a bone transcription factor required for osteoblast differentiation. Here, we used in silico analysis to identify a number of miRNAs that putatively target Runx2 and its co‐factors to mediate both positive and negative regulation of osteoblast differentiation. Among these miRNAs, miR‐590‐5p was selected and its expression was found to be increased during osteoblast differentiation. When mouse MSCs (mMSCs) were transiently transfected with a miR‐590‐5p mimic, we detected an increase in both calcium deposition and the mRNA expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP) and type I collagen genes. Smad7 was found to be among the putative target genes of miR‐590‐5p and its mRNA and protein expression decreased after miR‐590‐5p mimic transfection in human osteoblast‐like cells (MG63). Our analysis indicated that Runx2 was not a putative target of miR‐590‐5p. However, Runx2 protein, but not mRNA expression, increased after miR‐590‐5p mimic transfection in MG63 cells. Runx2 protein expression was increased with knockdown of Smad7 expression by Smad7 siRNA in these cells. We further identified that the 3′‐untranslated region of Smad7 was directly targeted by miR‐590‐5p; this was done using the luciferase reporter gene system. It is known that Smad7 inhibits osteoblast differentiation via Smurf2‐mediated Runx2 degradation. Hence, based on our results, we suggest that miR‐590‐5p promotes osteoblast differentiation by indirectly protecting and stabilizing the Runx2 protein by targeting Smad7 gene expression. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:34.725034-05:
      DOI: 10.1002/jcp.25434
       
  • Inhibition of mTOR Signaling Pathway Delays Follicle Formation in Mice
    • Authors: Jing Zhang; Wenwen Liu, Xinhui Sun, Feifei Kong, Ye Zhu, Yue Lei, Youqiang Su, Yiping Su, Jing Li
      Abstract: In mammalian ovaries, follicle assembly requires proper germ cell cyst breakdown and the invasion of somatic cells to encapsulate individual oocytes. Abnormalities in this process lead to a number of pathologies such as premature ovarian failure and infertility. As a conserved pathway regulating cell growth and metabolism in response to growth factors and nutrients, the roles of mTOR signaling in follicular development have been extensively studied in recent years. However, its functions during follicle formation remain unknown. In this study, the expression of p‐rpS6 (phospho‐ribosomal proteinS6), a downstream marker of mTORC1, showed dynamic changes in perinatal ovaries. When E18.5 ovaries, which mainly contained germ cell nests, were incubated with the mTOR inhibitors Rapamycin and Torin1 for 24 h, follicle assembly was delayed with differential somatic cell invasion into germ cell cyst among the groups. After transplanting treated or untreated ovaries into kidney capsules of recipient ovariectomized mice, follicular development was blocked in treated ovaries, as shown by fewer antral follicles and a higher proportion of primordial follicles. Further studies showed a significant decrease in somatic cell proliferation and the expression of marker genes related to follicular development (Kitl, Kit, Gdf9, Bmp15, Zp3, and Amhr2) in treated ovaries. Moreover, the addition of KITL, a growth factor that is mainly produced by pregranulosa cells during germ cell nest breakdown, rescued the extension of follicle formation induced by mTOR inhibitors. These results suggest that KITL functions downstream of mTOR in somatic cells to regulate their communication with oocytes during follicle formation. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T07:15:34.10707-05:0
      DOI: 10.1002/jcp.25456
       
  • Cholesterol and its Derivatives that Are Components of an Immune
           Stimulatory Drug Reversibly Inhibit Proteinase K
    • Authors: Namrata Singh; Debasish Bhattacharyya
      Abstract: Microorganisms express a variety of proteases that degrade many proteins of the host body and subvert host immune response. While elucidating the mechanism/s of an immune stimulatory drug that contains bile lipid, regulation of proteolytic activity was investigated. The drug and bile lipids both stabilize Proteinase K, an aggressive protease of fungal origin against auto‐digestion. Among the components of bile lipids, only cholesterol and its derivatives stabilize the enzyme. Biophysical evidences such as scattering of light, intrinsic and extrinsic fluorescence emission spectra, circular dichroism spectra, atomic force microscopy and transmission electron microscopy images indicated that cholesterol and its derivatives interact with Proteinase K. Inhibition kinetics using esterolysis of ATEE revealed non‐competitive inhibition by cholesterol. Surface Plasmon Resonance and mass spectrometric analysis indicated 1:1 stoichiometry of binding and with dissociation constant in the µM range. Further, the presence of four cholesterol recognition amino acid consensus motifs (CRAC motifs I‐IV) was identified in Proteinase K. Bioinformatics analysis revealed that the whole stretch of cholesterol interacts very well with the hydrophobic groove of motif II only among the four CRAC motifs. Variation of cholesterol content of HepG2 human liver carcinoma cells showed positive correlation with binding of fluorescence tagged Proteinase K. Under these conditions, binding of Proteinase K to the cells did not affect their morphology, viability and growth kinetics. Cell bound Proteinase K could be released by an excess of its substrate, thereby restoring reversibly its proteolytic activity. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T07:15:28.51616-05:0
      DOI: 10.1002/jcp.25457
       
  • Post‐Transcriptional Regulation of the Human Mu‐Opioid Receptor (MOR)
           by Morphine‐Induced RNA Binding Proteins hnRNP K and PCBP1
    • Abstract: Expression of the mu‐opioid receptor (MOR) protein is controlled by extensive transcriptional and post‐transcriptional processing. MOR gene expression has previously been shown to be altered by a post‐transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)‐binding protein (hnRNP) K and poly(C)‐binding protein 1 (PCBP1) as post‐transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5‘‐untranslated region of human MOR mRNA. In addition, we also found that morphine‐induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine‐induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). This article is protected by copyright. All rights reserved
      PubDate: 2016-06-13T06:56:20.948968-05:
      DOI: 10.1002/jcp.25455
       
  • Affinity Selection of FGF2‐Binding Heparan Sulfates for Ex Vivo
           Expansion of Human Mesenchymal Stem Cells
    • Authors: Sampath Jeewantha Wijesinghe; Ling Ling, Sadasivam Murali, Hui Qing Yeong, Simon F.R. Hinkley, Susan M. Carnachan, Tracey J. Bell, Kunchithapadam Swaminathan, James H. Hui, Andre J. van Wijnen, Victor Nurcombe, Simon M. Cool
      Abstract: The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture‐expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand‐receptor complex formation. We previously reported on an FGF2‐interacting HS variant (termed HS2) isolated in microgram amounts from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here we detail the isolation of gram amounts of an FGF2 affinity‐purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP‐2 or VEGF165. This process used a peptide sequence derived from the heparin‐binding domain of FGF2 as a substrate to affinity‐isolate HS8 from a commercially available source of porcine mucosal HS. Our data show that HS8 binds to FGF2 with higher affinity than to FGF1, FGF7, BMP2, PDGF‐BB, or VEGF165. Also, HS8 protects FGF2 from thermal destabilization and increases FGF signaling and hMSC proliferation through FGF receptor 1. Long‐term supplementation of cultures with HS8 increased both hMSC numbers and their colony‐forming efficiency without adversely affecting the expression of hMSC‐related surface antigens. This strategy further exemplifies the utility of affinity‐purifying HS variants against particular ligands important to the stem cell microenvironment and advocates for their addition as adjuvants for the culture‐expansion of hMSCs destined for cellular therapy. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-13T06:56:16.469175-05:
      DOI: 10.1002/jcp.25454
       
  • Combination of Rapamycin and Resveratrol for Treatment of Bladder Cancer
    • Authors: Anya Alayev; Rachel S. Salamon, Naomi S. Schwartz, Adi Y. Berman, Sara L. Wiener, Marina K. Holz
      Abstract: Loss of TSC1 function, a crucial negative regulator of mTOR signaling, is a common alteration in bladder cancer. Mutations in other members of the PI3K pathway, leading to mTOR activation, are also found in bladder cancer. This provides rationale for targeting mTOR for treatment of bladder cancer characterized by TSC1 mutations and/or mTOR activation. In this study, we asked whether combination treatment with rapamycin and resveratrol could be effective in concurrently inhibiting mTOR and PI3K signaling and inducing cell death in bladder cancer cells. In combination with rapamycin, resveratrol was able to block rapamycin‐induced Akt activation, while maintaining mTOR pathway inhibition. In addition, combination treatment with rapamycin and resveratrol induced cell death specifically in TSC1‐/‐ MEF cells, and not in wild‐type MEFs. Similarly, resveratrol alone or in combination with rapamycin induced cell death in human bladder cancer cell lines. These data indicate that administration of resveratrol together with rapamycin may be a promising therapeutic option for treatment of bladder cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T10:01:48.493314-05:
      DOI: 10.1002/jcp.25443
       
  • Non‐Metabolic Role of PKM2 in Regulation of the HIV‐1 LTR
    • Authors: Satarupa Sen; Satish L. Deshmane, Rafal Kaminski, Shohreh Amini, Prasun K. Datta
      Abstract: Identification of cellular proteins, in addition to already known transcription factors such as NF‐κB, Sp1, C‐EBPβ, NFAT, ATF/CREB, and LEF‐1, which interact with the HIV‐1 LTR is critical in understanding the mechanism of HIV‐1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells, a macrophage model of latency. We observed that HIV‐1SF162 infection of monocyte/macrophages and reactivation of HIV‐1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA‐induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA‐induced U1 cells in comparison to PMA‐induced U937 cells. We focused on understanding the potential role of PKM2 in HIV‐1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA‐activated U1 and TZM‐ bl cells demonstrated the interaction of PKM2 with the HIV‐1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV‐1 LTR‐luciferase reporter in U937, U‐ 87 MG, and TZM‐bl cells. Using various truncated constructs of the HIV‐1 LTR, we mapped the region spanning ‐120 bp to ‐80 bp to be essential for PKM2‐mediated transactivation. This region contains the NF‐κB binding site and deletion of this site attenuated PKM2 mediated activation of HIV‐1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF‐κB. These observations demonstrate for the first time that PKM2 is a transcriptional co‐activator of HIV‐1 LTR. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T10:01:42.21665-05:0
      DOI: 10.1002/jcp.25445
       
  • Leucine‐Rich Amelogenin Peptide (LRAP) Uptake by Cementoblast Requires
           Flotillin‐1 Mediated Endocytosis
    • Abstract: Basic, pre‐clinical and clinical studies have documented the potential of amelogenin, and its variants, to affect cell response and tissue regeneration. However, the mechanisms are unclear. Thus, the aim of the present study was to identify, in cementoblasts, novel binding partners for an alternatively spliced amelogenin form (Leucine‐Rich Amelogenin Peptide ‐ LRAP), which is supposed to act as a signaling molecule in epithelial‐mesenchymal interactions. LRAP‐binding protein complexes from immortalized murine cementoblasts (OCCM‐30) were achieved by capture affinity assay (GST pull down) and proteins present in these complexes were identified by mass spectrometry and immunoblotting. Flotillin‐1, which functions as a platform for signal transduction, vesicle trafficking, endocytosis, and exocytosis, was identified and confirmed by co‐precipitation and co‐localization assays as a protein‐binding partner for LRAP in OCCM‐30 cells. In addition, we found that exogenously added GST‐LRAP recombinant protein was internalized by OCCM‐30 cells, predominantly localized in the perinuclear region and, that inhibition of flotillin1‐dependent functions by small interference RNA (siRNA) methodology significantly affected LRAP uptake and its biological properties on OCCM‐30 cells, including LRAP effect on the expression of genes encoding osteocalcin (Ocn), bone sialoprotein (Bsp) and runt‐related transcription factor 2 (RunX2). In conclusion, LRAP uptake by cementoblast involves flotillin‐assisted endocytosis, which suggests an involvement of LRAP in lipid‐raft‐dependent signaling pathways which are mediated by flotillin‐1. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:25:20.31246-05:0
      DOI: 10.1002/jcp.25453
       
  • In Vitro and In Vivo Differentiation of Progenitor Stem Cells Obtained
           after Mechanical Digestion of Human Dental Pulp
    • Authors: Manuela Monti; Antonio Graziano, Silvana Rizzo, Cesare Perotti, Claudia Del Fante, Riccardo d'Aquino, CarloAlberto Redi, Ruggero Rodriguez y Baena
      Abstract: Human population is facing a revolutionary change in the demographic structure with an increasing number of elderly people requiring an unmet need to ensure a smooth aging process and dental care is certainly an important aspect that has to be considered. To date, dentistry has been conservative and the need of transferring the scientific models of regenerative dentistry into clinical practice is becoming a necessity.The aim of this study was to characterize the differentiation commitment (in vitro) and the clinical grafting ability (in vivo) of a population of progenitor stem cells obtained after mechanical digestion of dental pulp with an innovative system recently developed. This approach was successfully used in previous studies to obtain a clinical‐grade ready to use dental pulp fragments that could be grafted in autologous tissues to obtain bone. We are thus showing that micro grafts resulting from mechanical digestion contain stem cells with a mesenchymal phenotype, able to differentiate towards different cell types and to generate new bone in patients.We are providing data for the establishment of standardized and routinely oral surgery approaches, having outlined the cellular properties of human stem cells obtained from the dental pulp. This method can represent a valid tool for both regenerative medicine and tissue engineering purposes not only applicable to the cranio‐maxillofacial region but, likely, to different bone pathologies for a fastening and healing recovering of patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:23:35.810575-05:
      DOI: 10.1002/jcp.25452
       
  • Effects of sex and notch signaling on the osteocyte cell pool
    • Authors: Ernesto Canalis; Lauren Schilling, Stefano Zanotti
      Abstract: Osteocytes play a fundamental role in mechanotransduction and skeletal remodeling. Sex is a determinant of skeletal structure, and female C57BL/6J mice have increased osteoblast number in cancellous bone when compared to male mice. Activation of Notch in the skeleton causes profound cell‐context dependent changes in skeletal physiology. To determine the impact of sex and of Notch signaling on the osteocyte cell pool, we analyzed cancellous and cortical bone of 1 to 6 month old C57BL/6J or 129SvJ/C57BL/6J mice and determined the osteocyte number/area. There was an age‐dependent decline in osteocyte number in cancellous bone of male but not female mice, so that 6 month old female mice had a greater number of osteocytes than male littermates. Although differences between male and female mice were modest, female mice had ∼10‐15% greater number of osteocytes/area. RNA sequence analysis of osteocyte‐rich preparations did not reveal differences between sexes in the expression of genes known to influence bone homeostasis. Neither the activation of Notch1 nor the concomitant inactivation of Notch1 and Notch2 in Osterix (Sp7) or Dentin matrix protein 1 (Dmp1) expressing cells had a pronounced and consistent effect on cancellous or cortical bone osteocyte number in either sex. Moreover, inactivation of Notch1 and Notch2 in Dmp1 expressing cells did not influence the bone loss in a muscle immobilization model of skeletal unloading. In conclusion, cancellous bone osteocytes decline with age in male mice, cortical osteocytes are influenced by sex in younger mice, but osteocyte cell density is not affected substantially by Notch signaling. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-07T11:50:40.061135-05:
      DOI: 10.1002/jcp.25433
       
  • Early Treatment with Enalapril and Later Renal Injury in Programmed Obese
           Adult Rats
    • Authors: Hyung Eun Yim; Kee Hwan Yoo, In Sun Bae, Young Sook Hong
      Abstract: Obesity‐related kidney disease should be prevented or retarded. We aimed to investigate whether early treatment with enalapril ameliorates later renal injury induced by early postnatal overnutrition. Three or ten male pups per mother were assigned to either the Obese or Lean group during the first 21 days of life. These pups were treated with enalapril (Obese enalapril, OE; Lean enalapril, LE) or vehicle (Obese control, OC; Lean control, LC) for 15–28 days. Body weight, blood pressure (BP), and renal alterations were determined at 3 months. Enalapril decreased body weight only in the Lean group at 3 months (P 
      PubDate: 2016-06-06T10:15:29.747504-05:
      DOI: 10.1002/jcp.25444
       
  • Gating Modulation of the Tumor‐Related Kv10.1 Channel by Mibefradil
    • Abstract: Several reports credit mibefradil with tumor suppressing properties arising from its known inhibition of Ca2+ currents. Given that mibefradil (Mb) is also known to inhibit K+ channels, we decided to study the interaction between this organic compound and the tumor‐related Kv10.1 channel. Here we report that Mb modulates the gating of Kv10.1. Mb induces an apparent inactivation from both open and early closed states where the channels dwell at hyperpolarized potentials. Additionally Mb accelerates the kinetics of current activation, in a manner that depends on initial conditions. Our observations suggest that Mb binds to the voltage sensor domain of Kv10.1 channels, thereby modifying the gating of the channels in a way that in some, but not all, aspects opposes to the gating effects exerted by divalent cations. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-03T06:58:46.738119-05:
      DOI: 10.1002/jcp.25448
       
  • T11TS Treatment Augments Apoptosis of Glioma Associated Brain Endothelial
           Cells, Hint towards Anti‐Angiogenic Action in Glioma
    • Authors: Debanjan Bhattacharya; Manoj Kumar Singh, Suhnrita Chaudhuri, Ankur Datta, Swapna Chaudhuri
      Abstract: Malignant glioma continues to be a clinical challenge with an urgent need for developing curative therapeutic intervention. Apoptosis induction in tumor associated endothelial cells represent a central mechanism that counteracts angiogenesis in glioma and other solid tumors. We previously demonstrated that intraperitoneal administration of sheep erythrocyte membrane glycopeptide T11‐target structure (T11TS) in rodent glioma model inhibits PI3K/Akt pathway and Raf/MEK/ERK signaling in glioma associated brain endothelial cells. In the present study we investigated whether T11TS treatment influence apoptosis signaling in vivo in glioma associated brain endothelial cells. Annexin‐V/PI staining showed that T11TS treatment in glioma induced rats increases apoptosis of glioma associated endothelial cells within glioma milieu compared to brain endothelial cells in glioma induced and control groups. Flowcytometric JC‐1 assay revealed that T11TS administration triggers loss of mitochondrial membrane potential in glioma associated brain endothelial cells. Flowcytometry, immunoblotting and in‐situ immunofluoresecnt imaging were employed to investigate the effect of T11TS on apoptotic regulatory proteins in brain endothelial cells. T11TS treatment upmodulated expression of p53, Bax, Fas, FasL and FADD in glioma associated endothelial cells and downregulated Bcl‐2 protein. T11TS therapy induced cytochrome‐c release into cytosol, activated caspase ‐9, 8, 3, and cleaved Bid in glioma associated brain endothelial cells. The study demonstrates that T11TS induces apoptosis in glioma associated brain endothelial cells via p53 accumulation and activation of intrinsic as well as Fas dependent extrinsic pathway. The pro‐apoptotic action of T11TS on glioma associated endothelial cells provides crucial insight into how T11TS exerts its anti‐angiogenic function in glioma. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-03T06:51:42.586441-05:
      DOI: 10.1002/jcp.25447
       
  • Generation of neural crest‐like cells from human periodontal ligament
           cell‐derived induced pluripotent stem cells
    • Authors: Atsushi Tomokiyo; Kim Hynes, Jia Ng, Danijela Menicanin, Esther Camp, Agnes Arthur, Stan Gronthos, Peter Mark Bartold
      Abstract: Neural crest cells (NCC) hold great promise for tissue engineering, however the inability to easily obtain large numbers of NCC is a major factor limiting their use in studies of regenerative medicine. Induced pluripotent stem cells (iPSC) are emerging as a novel candidate that could provide an unlimited source of NCC. In the present study, we examined the potential of neural crest tissue‐derived periodontal ligament (PDL) iPSC to differentiate into neural crest‐like cells (NCLC) relative to iPSC generated from a non‐neural crest derived tissue, foreskin fibroblasts (FF). We detected high HNK1 expression during the differentiation of PDL and FF iPSC into NCLC as a marker for enriching for a population of cells with NCC characteristics. We isolated PDL iPSC‐ and FF iPSC‐derived NCLC, which highly expressed HNK1. A high proportion of the HNK1‐positive cell populations generated, expressed the MSC markers, whilst very few cells expressed the pluripotency markers or the hematopoietic markers. The PDL and FF HNK1‐positive populations gave rise to smooth muscle, neural, glial, osteoblastic and adipocytic like cells and exhibited higher expression of smooth muscle, neural, and glial cell‐associated markers than the PDL and FF HNK1‐negative populations. Interestingly, the HNK1‐positive cells derived from the PDL‐iPSC exhibited a greater ability to differentiate into smooth muscle, neural, glial cells and adipocytes, than the HNK1‐positive cells derived from the FF‐iPSC. Our work suggests that HNK1‐enriched NCLC from neural crest tissue‐derived iPSC more closely resemble the phenotypic and functional hallmarks of NCC compared to the HNK1‐low population and non‐neural crest iPSC‐derived NCLC. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T10:43:14.571204-05:
      DOI: 10.1002/jcp.25437
       
  • Anti‐Diabetic Agent Sodium Tungstate Induces the Secretion of Pro‐ and
           Anti‐Inflammatory Cytokines by Human Kidney Cells
    • Abstract: Diabetic kidney disease (DKD) is the major cause of end stage renal disease. Sodium tungstate (NaW) exerts anti‐diabetic and immunomodulatory activities in diabetic animal models. Here, we used primary cultures of renal proximal tubule epithelial cells derived from type‐2‐diabetic (D‐RPTEC) and non‐diabetic (N‐RPTEC) subjects as in vitro models to study the effects of NaW on cytokine secretion, as these factors participate in intercellular regulation of inflammation, cell growth and death, differentiation, angiogenesis, development, and repair, all processes that are dysregulated during DKD. In basal conditions, D‐RPTEC cells secreted higher levels of prototypical pro‐inflammatory IL‐6, IL‐8, and MCP‐1 than N‐RPTEC cells, in agreement with their diabetic phenotype. Unexpectedly, NaW further induced IL‐6, IL‐8, and MCP‐1 secretion in both N‐ and D‐RPTEC, together with lower levels of IL‐1 RA, IL‐4, IL‐10, and GM‐CSF, suggesting that it may contribute to the extent of renal damage/repair during DKD. Besides, NaW induced the accumulation of IκBα, the main inhibitor protein of one major pathway involved in cytokine production, suggesting further anti‐inflammatory effect in the long‐term. A better understanding of the mechanisms involved in the interplay between the anti‐diabetic and immunomodulatory properties of NaW will facilitate future studies about its clinical relevance. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.Sodium tungstate (NaW) exerts anti‐diabetic activities. We further show that NaW induces IL‐6, IL‐8, and MCP‐1 secretion, together with lower levels of IL1‐RA, IL‐4, IL‐10, GM‐CSF in renal proximal tubule epithelial cells from diabetic and non‐diabetic subjects, suggesting that it may contribute to the extent of renal damage/repair of diabetic kidney.
      PubDate: 2016-06-02T10:25:19.149981-05:
      DOI: 10.1002/jcp.25429
       
  • Ginkgolic Acid Inhibits Invasion and Migration and TGF‐β‐Induced EMT
           of Lung Cancer Cells Through PI3K/Akt/mTOR Inactivation
    • Abstract: Epithelial‐to‐mesenchymal transition (EMT) is a critical cellular phenomenon regulating tumor metastases. In the present study, we investigated whether ginkgolic acid can affect EMT in lung cancer cells and the related underlying mechanism(s) of its actions. We found that ginkgolic acid C15:1 (GA C15:1) inhibited cell proliferation, invasion, and migration in both A549 and H1299 lung cancer cells. GA C15:1 also suppressed the expression of EMT related genes (Fibronectin, Vimentin, N‐cadherin, MMP‐9, MMP‐2, Twist and Snail) and suppressed TGF‐β‐induced EMT as assessed by reduced expression of mesenchymal markers (Fibronectin, Vimentin, N‐cadherin), MMP‐9, MMP‐2, Twist and Snail. However, GA C15:1 did not affect the expression of various epithelial marker proteins (Occludin and E‐cadherin) in both A549 and H1299 cells. TGF‐β‐induced morphologic changes from epithelial to mesenchymal cells and induction of invasion and migration were reversed by GA C15:1. Finally, GA C15:1 not only abrogated basal PI3K/Akt/mTOR signaling cascade, but also reduced TGF‐β‐induced phosphorylation of PI3K/Akt/mTOR pathway in lung cancer cells. Overall, these findings suggest that GA C15:1 suppresses lung cancer invasion and migration through the inhibition of PI3K/Akt/mTOR signaling pathway and provide a source of potential therapeutic compounds to control the metastatic dissemination of tumor cells. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.GA C15:1 also suppressed the expression of EMT related genes. GA C15:1 suppressed TGF‐beta‐induced mesenchymal markers, MMP‐9, MMP‐2, Twist and Snail. GA C15:1 suppresses metastatic lung cancer through inhibition of PI3K/Akt/mTOR activation.
      PubDate: 2016-06-02T10:13:27.95026-05:0
      DOI: 10.1002/jcp.25426
       
  • Sex Differences in Estrogen Receptor α and β Levels and Activation
           Status in LPS‐Stimulated Human Macrophages
    • Authors: Ilaria Campesi; Maria Marino, Andrea Montella, Sara Pais, Flavia Franconi
      Abstract: Immune function, inflammation, and atherosclerosis display sex differences and are influenced by 17β‐estradiol through estrogen receptors subtypes ERα and ERβ. Male tissues express active ERs, but their possible involvement in inflammation in males has never been assessed. Macrophages express both ERα and ERβ and offer the opportunity to evaluate the role of ER levels and activation in inflammation. We assessed the ability of lipopolysaccharide (LPS) to modulate, in a sex‐specific way, the expression and the activation status of ERα and ERβ in blood monocytes‐derived macrophages (MDMs) from men and women. MDMs were incubated with 100 ng/ml LPS for 24 h and used to evaluate ERα, ERβ, P‐ERα, p38, and P‐p38 expression by Western Blotting. In basal conditions, ERα and ERβ were significantly higher in female MDMs than in male MDMs. LPS up‐regulated ERα and ERα phosphorylation in both sexes, with a significantly higher effect observed in male MDMs, and down‐regulated ERβ level only in female MDMs. p38 and P‐p38 proteins, indicative of ERβ activity, did not show sex differences both in basal conditions and after LPS treatment. Finally, ERα/ERβ and P‐ERα/ERα ratios were significantly higher in male MDMs than in female ones. Our data indicate, for the first time, that LPS affects ERα but not ERβ activation status. We identify a significant role of ERα in LPS‐mediated inflammatory responses in MDMs, which represents an initial step in understanding the influence of sex in the relationship between LPS and ERα. J. Cell. Physiol. 9999: 1–6, 2016. © 2016 Wiley Periodicals, Inc.
      PubDate: 2016-06-02T10:06:28.352424-05:
      DOI: 10.1002/jcp.25425
       
  • Chrysophanol Induces Apoptosis of Choriocarcinoma Through Regulation of
           ROS and the AKT and ERK1/2 Pathways
    • Authors: Whasun Lim; Changwon Yang, Fuller W. Bazer, Gwonhwa Song
      Abstract: Chrysophanol is an anthraquinone compound, mainly isolated from rhubarb, with anti‐cancer effects on some types of cancer cells. However, effects of chrysophanol on human choriocarcinoma cells are not known. Therefore, the objective of this study was to determine effects of chrysophanol on choriocarcinoma cells (JAR and JEG‐3) and identify signal transduction cascades activated by chrysophanol. Results of present study, showed that chrysophanol decreased cell viability and induced apoptosis of JEG‐3, but not JAR cells, in a dose‐dependent manner. Chrysophanol also increased oxidative stress in JEG‐3 cells by inducing ROS generation followed by mitochondrial dysfunction including depolarization of mitochondrial inner membrane potential. Western blot analysis revealed that ERK1/2, P90RSK, AKT, and P70S6K were increased significantly in JEG‐3 cells by chrysophanol. Next, we investigated chrysophanol‐mediated effects on proliferation of JEG‐3 cells using pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126). Inhibition of AKT and ERK1/2 prevented chrysophanol‐induced stimulation of proliferation of JEG‐3 cells. In addition, the phosphorylation of AKT and ERK1/2 was suppressed by LY294002 and U0126 in JEG‐3 cells treated with chrysophanol, whereas, the AKT protein was activated by pre‐treatment of JEG‐3 cells with U0126. Furthermore, we compared therapeutic effects of chrysophanol with cisplatin and paclitaxel which are conventional salvage regimens for choriocarcinoma. Our results verified that chrysophanol has synergistic effects with traditional therapy to increase apoptosis of JEG‐3 cells. Collectively, these results indicate that chrysophanol is a potential effective chometherapeutic agent for treatment of choriocarcinoma therapy, and minimizing side effects of conventional treatment regimens. J. Cell. Physiol. 9999: 1–9, 2016. © 2016 Wiley Periodicals, Inc.Results of the present study indicated that chrysophanol has anti‐cancer effects by reducing cell viability and inducing apoptosis by increasing ROS production and decreasing mitochondrial membrane potential through the ERK1/2 and AKT signaling cascades. Moreover, chrysophanol is a potential effective chometherapeutic agent for treatment of choriocarcinoma therapy when combined with cisplatin and paclitaxel and minimizing side effects of conventional treatment regimens.
      PubDate: 2016-06-02T10:03:58.124871-05:
      DOI: 10.1002/jcp.25423
       
  • Repression of Primitive Erythroid Program Is Critical for the Initiation
           of Multi‐Lineage Hematopoiesis in Mouse Development
    • Authors: Toshiyuki Yamane; Chie Ito, Aya Washino, Kana Isono, Hidetoshi Yamazaki
      Abstract: Formation of the hematopoietic cells occurs in multiple steps. The first hematopoietic cells observed during ontogeny are primitive erythrocytes, which are produced in the early yolk sac within a limited temporal window. Multi‐lineage hematopoiesis, which supplies almost the entire repertoire of blood cell lineages, lags behind primitive erythropoiesis in the tissue. However, molecular mechanisms regulating sequential generation of primitive erythrocytes and multipotent hematopoietic progenitors in the yolk sac are largely unknown. In this study, the transcription factors involved in the development of hematopoietic cells were examined in purified progenitor cell populations from pluripotent stem cell cultures and from the yolk sac of developing embryos. We found that the earliest committed hematopoietic progenitors highly expressed Gata1, Scl/tal1, and Klf1 genes. Expression of these transcription factors, which is known to form a core erythroid transcriptional network, explained the prompt generation of primitive erythrocytes from these earliest progenitors. Importantly, the multipotent hematopoietic cells, which lack the differentiation potential into primitive erythroid cells, down‐regulated these genes during a transition from the earliest committed progenitors. In addition, we showed that Pu.1 is involved in the multipotent cell differentiation through the suppression of erythroid transcription program. We propose that these molecular mechanisms governed by transcription factors form sequential waves of primitive erythropoiesis and multi‐lineage hematopoiesis in the early yolk sac of developing embryos. J. Cell. Physiol. 9999: 1–8, 2016. © 2016 Wiley Periodicals, Inc.The first blood cell progenitors highly expressed red blood cell‐specific transcription factors. This expression of transcription factors was considered to enable the immediate production of primitive red blood cells. Importantly, the multipotent blood cell progenitors derived from the earliest progenitors shut‐off the expression of red blood cell‐specific transcription factors, which was partly mediated through the upregulation of Pu.1 expression.
      PubDate: 2016-06-02T09:57:07.656034-05:
      DOI: 10.1002/jcp.25422
       
  • Flavopiridol: An Old Drug With New Perspectives? Implication for
           Development of New Drugs
    • Authors: Annamaria Cimini; Michele d'Angelo, Elisabetta Benedetti, Barbara D'Angelo, Giulio Laurenti, Andrea Antonosante, Loredana Cristiano, Antonella Di Mambro, Marcella Barbarino, Vanessa Castelli, Benedetta Cinque, Maria Grazia Cifone, Rodolfo Ippoliti, Francesca Pentimalli, Antonio Giordano
      Abstract: Glioblastoma, the most common brain tumor, is characterized by high proliferation rate, invasion, angiogenesis, and chemo‐ and radio‐resistance. One of most remarkable feature of glioblastoma is the switch toward a glycolytic energetic metabolism that leads to high glucose uptake and consumption and a strong production of lactate. Activation of several oncogene pathways like Akt, c‐myc, and ras induces glycolysis and angiogenesis and acts to assure glycolysis prosecution, tumor proliferation, and resistance to therapy. Therefore, the high glycolytic flux depends on the overexpression of glycolysis‐related genes resulting in an overproduction of pyruvate and lactate. Metabolism of glioblastoma thus represents a key issue for cancer research. Flavopiridol is a synthetic flavonoid that inhibits a wide range of Cyclin‐dependent kinase, that has been demonstrate to inactivate glycogen phosphorylase, decreasing glucose availability for glycolysis. In this work the study of glucose metabolism upon flavopiridol treatment in the two different glioblastoma cell lines. The results obtained point towards an effect of flavopiridol in glycolytic cells, thus suggesting a possible new use of this compound or flavopiridol‐derived formulations in combination with anti‐proliferative agents in glioblastoma patients. J. Cell. Physiol. 9999: 1–11, 2016. © 2016 Wiley Periodicals, Inc.FLAP treatment affects glucose utilization pathways in two glioblastoma cell lines, bearing different PTEN status. The treatment strongly decreases the glycolytic enzymes with concomitant increase of cell death.
      PubDate: 2016-06-02T09:52:49.630745-05:
      DOI: 10.1002/jcp.25421
       
  • Gene and microRNA Expression Are Predictive of Tumor Response in Rectal
           Adenocarcinoma Patients Treated with Preoperative Chemoradiotherapy
    • Authors: Caterina Millino; Isacco Maretto, Beniamina Pacchioni, Maura Digito, Antonino De Paoli, Vincenzo Canzonieri, Edoardo D'Angelo, Marco Agostini, Flavio Rizzolio, Antonio Giordano, Andrea Barina, Senthilkumar Rajendran, Giovanni Esposito, Gerolamo Lanfranchi, Donato Nitti, Salvatore Pucciarelli
      Abstract: Preoperative chemoradiotherapy (pCRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). However, tumor response to pCRT is not uniform, and there are no effective predictive methods. This study investigated whether specific gene and miRNA expression are associated with tumor response to pCRT.Tissue biopsies were obtained from patients before pCRT and resection. Gene and miRNA expression were analyzed using a one‐color microarray technique that compares signatures between responders (R) and non‐responders (NR), as measured based on tumor regression grade.Two groups composed of 38 “exploration cohort” and 21 “validation cohort” LARC patients were considered for a total of 32 NR and 27 R patients. In the first cohort, using SAM Two Class analysis, 256 genes and 29 miRNAs that were differentially expressed between the NR and R patients were identified. The anti‐correlation analysis showed that the same 8 miRNA interacted with different networks of transcripts. The miR‐630 appeared only with the NR patients and was anti‐correlated with a single transcript: RAB5B. After PAM, the following 8 transcripts were strong predictors of tumor response: TMEM188, ITGA2, NRG, TRAM1, BCL2L13, MYO1B, KLF7 and GTSE1. Using this gene set, an unsupervised cluster analysis was applied to the validation cohort and correctly assigned the patients to the NR or R group with 85.7% accuracy, 90% sensitivity and 82% specificity. All three parameters reached 100% when both cohorts were considered together.In conclusion, gene and miRNA expression profiles may be helpful for predicting response to pCRT in LARC patients. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-25T17:40:42.055244-05:
      DOI: 10.1002/jcp.25441
       
  • Pin1, the Master Orchestrator of Bone Cell Differentiation
    • Abstract: Pin1 is an enzyme that specifically recognizes the peptide bond between phosphorylated serine or threonine (pS/pT‐P) and proline. This recognition causes a conformational change of its substrate, which further regulates downstream signaling. Pin1‐/‐ mice show developmental bone defects and reduced mineralization. Pin1 targets RUNX2 (Runt‐Related Transcription Factor 2), SMAD1/5, and β‐catenin in the FGF, BMP, and WNT pathways, respectively. Pin1 has multiple roles in the crosstalk between different anabolic bone signaling pathways. For example, it controls different aspects of osteoblastogenesis and increases the transcriptional activity of Runx2, both directly and indirectly. Pin1 also influences osteoclastogenesis at different stages by targeting PU.1 (Purine‐rich nucleic acid binding protein 1), C‐FOS, and DC‐STAMP. The phenotype of Pin1‐/‐ mice has led to the recent identification of multiple roles of Pin1 in different molecular pathways in bone cells. These roles suggest that Pin1 can be utilized as an efficient drug target in congenital and acquired bone diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-25T17:40:37.258658-05:
      DOI: 10.1002/jcp.25442
       
  • Ascorbic acid induces necrosis in human laryngeal squamous cell carcinoma
           via ROS, PKC, and calcium signaling
    • Abstract: Ascorbic acid induces apoptosis, autophagy, and necrotic cell death in cancer cells. We investigated the mechanisms by which ascorbic acid induces death in laryngeal squamous cell carcinoma Hep2 cells. Ascorbic acid markedly reduced cell viability and induced death without caspase activation and an increase in cytochrome c. Hep2 cells exposed to ascorbic acid exhibited membrane rupture and swelling, the morphological characteristics of necrotic cell death. The generation of reactive oxygen species (ROS) was increased in Hep2 cells treated with ascorbic acid, and pretreatment with N‐acetylcysteine blocked ascorbic acid–induced cell death. Ascorbic acid also stimulated protein kinase C (PKC) signaling, especially PKC α/β activation, and subsequently increased cytosolic calcium levels. However, ascorbic acid–induced necrotic cell death was inhibited by Ro‐31‐8425 (PKC inhibitor) and BAPTA‐AM (cytosolic calcium–selective chelator). ROS scavenger NAC inhibited PKC activation induced by ascorbic acid and Ro‐31‐8425 suppressed the level of cytosolic calcium increased by ascorbic acid, indicating that ROS is represented as an upstream signal of PKC pathway and PKC activation leads to the release of calcium into the cytosol, which ultimately regulates the induction of necrosis in ascorbic acid‐treated Hep2 cells. These data demonstrate that ascorbic acid induces necrotic cell death through ROS generation, PKC activation, and cytosolic calcium signaling in Hep2 cells. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-22T23:40:54.779976-05:
      DOI: 10.1002/jcp.25438
       
  • Hypoxia Modulates the Swelling‐Activated Cl Current in Human
           Glioblastoma Cells: Role in Volume Regulation and Cell Survival
    • Authors: Luigi Sforna; Marta Cenciarini, Silvia Belia, Antonio Michelucci, Mauro Pessia, Fabio Franciolini, Luigi Catacuzzeno
      Abstract: The malignancy of glioblastoma multiform (GBM), the most common human brain tumor, correlates with the presence of hypoxic areas, but the underlying mechanisms are unclear. GBM cells express abundant Cl channels whose activity supports cell volume and membrane potential changes, ultimately leading to cell proliferation, migration, and escaping death. In non‐tumor tissues Cl channels are modulated by hypoxia, which prompted us to verify whether hypoxia would also modulate Cl channels in GBM cells. Our results show that in GBM cell lines, acute application of a hypoxic solution activates a Cl current displaying the biophysical and pharmacological features of the swelling‐activated Cl current (ICl,swell). We also found that acute hypoxia increased the cell volume by about 20%, and a 30% hypertonic solution partially inhibited the hypoxia‐activated Cl current, suggesting that cell swelling and the activation of the Cl current are sequential events. Notably the hypoxia‐induced cell swelling was followed by a regulatory volume decrease (RVD) mediated mainly by ICl,swell. Since a hypoxia‐induced prolonged cell swelling is usually regarded as a death insult, we hypothesized that the hypoxia‐activated Cl current could limit cell swelling and prevent necrotic death of GBM cells under hypoxic conditions. In accordance, we found that the ICl,swell inhibitor DCPIB hampered the RVD process, and more importantly it sensibly increased the hypoxia‐induced necrotic death in these cells. Taken together, these results suggest that Cl channels are strongly involved in the survival of GBM cells in a hypoxic environment, and may thus represent a new therapeutic target for this malignant tumor. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-29T03:36:23.324328-05:
      DOI: 10.1002/jcp.25393
       
  • Table of Contents, Editor's Choice, Highlights
    • Pages: 1 - 6
      PubDate: 2016-09-19T14:38:00.707127-05:
      DOI: 10.1002/jcp.25571
       
  • Stiffness of Intact Endothelial Cells from Fresh Aortic Bifurcations of
           Atherosclerotic Rabbits ‐ Atomic Force Microscopic Study
    • Authors: Kozaburo Hayashi; Michitaka Higaki
      First page: 7
      Abstract: Stiffness of intact endothelial cells (ECs) in the abdominal aorta (AA) and in the medial and lateral wall of the common iliac artery (CIA(Medial) and CIA(Lateral), respectively), which were freshly obtained from cholesterol‐fed rabbits, were measured with an atomic force microscopic indentation method. In the areas away from atherosclerotic plaques (Off‐plaque), ECs were significantly stiffer in CIA(Medial) than in the other two locations; this result was similar to that from normal diet‐fed animals. On the other hand, there were no significant differences in the stiffness of ECs located on atherosclerotic plaques (On‐plaque) among the three sites; the stiffness was equal to those in “Off‐plaque” wall of CIA(Lateral) and AA. Moreover, the stiffness of ECs covering plaques decreased with the progression of atherosclerosis. The precise quantification of the stiffness of vascular ECs would provide a better understanding of cellular remodeling and adaptation in atherosclerosis. This article is protected by copyright. All rights reserved
      PubDate: 2016-03-17T09:43:15.558698-05:
      DOI: 10.1002/jcp.25379
       
  • Dissection of Individual Prostate Lobes in Mouse Models of Prostate Cancer
           to Obtain High Quality RNA
    • Authors: Areg Zingiryan; Nicholas H. Farina, Kristiaan H. Finstad, Janet L. Stein, Jane B. Lian, Gary S. Stein
      Pages: 14 - 18
      Abstract: Genetically engineered mouse models of prostate cancer allow for study of disease progression from localized tumor formation through distal metastasis. The anatomy of the mouse prostate differs dramatically from the human prostate, being composed of four lobe pairs (anterior, dorsal, lateral, and ventral), making the identification and dissection technically challenging. Although the entire murine prostate and surrounding tissue, including urethra, bladder, seminal vesicles, and associated adipose tissue, can be quickly dissected for en bloc analysis, it is necessary to isolate individual prostate lobes for gene expression studies elucidating the molecular mechanisms of prostate cancer. The procedure as described here includes full color images, allowing the researcher to appreciate the unique prostate morphology and tissue manipulation required to harvest individual prostate lobes. Along with removing all extraneous tissue, the procedure allows for direct comparison of the different prostate lobes by established downstream techniques. Importantly, high quality RNA required for next‐generation gene expression analysis can only consistently be obtained from ventral and lateral lobes. Finally, preclinical studies using prostate targeted therapies can be monitored specifically in individual prostate lobes for histological and gene expression studies. J. Cell. Physiol. 232: 14–18, 2017. © 2016 Wiley Periodicals, Inc.Here, we present a procedure with full color images to isolate individual murine prostate lobes, free of adjacent tissue, for use in downstream gene expression studies. All eight murine prostate lobes can be harvested within an hour and processed for histologic analysis and molecular component isolation (RNA, DNA, protein, etc.). Only the lateral and ventral prostate lobes yield non‐degraded RNA.
      PubDate: 2016-04-01T03:22:20.741891-05:
      DOI: 10.1002/jcp.25384
       
  • Probing Micromechanical Properties of the Extracellular Matrix of Soft
           Tissues by Atomic Force Microscopy
    • Pages: 19 - 26
      Abstract: The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy (AFM) has unique capabilities to measure ECM mechanics at the scale at which cells probe the mechanical features of their microenvironment. Moreover, AFM measurements can be readily combined with bright field and fluorescence microscopy. Performing reliable mechanical measurements with AFM requires accurate calibration of the device and correct computation of the mechanical parameters. A suitable approach to isolate ECM mechanics from cell contribution is removing the cells by means of an effective decellularization process that preserves the composition, structure and mechanical properties of the ECM. AFM measurement of ECM micromechanics provides important insights into organ biofabrication, cell‐matrix mechanical crosstalk and disease‐induced tissue stiffness alterations. J. Cell. Physiol. 232: 19–26, 2017. © 2016 Wiley Periodicals, Inc.The extracellular matrix (ECM) determines 3D tissue architecture and provides structural support and chemical and mechanical cues to the cells. Atomic force microscopy allows the measurement of ECM mechanics at the microscale at which cells probe the mechanical features of their microenvironment.
      PubDate: 2016-06-02T06:06:07.36028-05:0
      DOI: 10.1002/jcp.25420
       
  • Light Sheet Microscopy to Measure Protein Dynamics
    • Authors: Matthias Rieckher
      First page: 27
      Abstract: Visualizing protein dynamics is the key to a quantitative understanding of molecular mechanisms in biological systems. Recent developments in fluorescent microscopy techniques allow for novel experimental approaches to study stochastic localization, distribution and movement of fluorescently labeled molecules in high resolution as they occur over time in the context of living cells and whole organisms. Particularly suitable for such studies is the application of light sheet microscopy, as it enables rapid in vivo imaging of a wide range and size of specimen, from whole organisms and organs, down to the dynamics of subcellular structures and single molecules. This article summarizes the principles of light sheet microscopy and its advantages over other optical imaging techniques, such as light microscopy and confocal microscopy, and highlights recent innovations that significantly enhance spatio‐temporal resolution. Also, this manuscript contains basic guidelines for the implementation of light sheet microscopy in the laboratory and presents thus far unpublished light sheet microscopy applications demonstrating the ability of this technology to measure protein dynamics in a whole‐organism context. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-10T01:31:54.123735-05:
      DOI: 10.1002/jcp.25451
       
  • Animal Models of Congenital Cardiomyopathies Associated With Mutations in
           Z‐Line Proteins
    • Pages: 38 - 52
      Abstract: The cardiac Z‐line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. The Z‐line is important for efficient force generation and transmission as well as the maintenance of structural stability and integrity. Furthermore, it is a nodal point for intracellular signaling, in particular mechanosensing and mechanotransduction. Mutations in various genes encoding Z‐line proteins have been associated with different cardiomyopathies, including dilated cardiomyopathy, hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, restrictive cardiomyopathy, and left ventricular noncompaction, and mutations even within the same gene can cause widely different pathologies. Animal models have contributed to a great advancement in the understanding of the physiological function of Z‐line proteins and the pathways leading from mutations in Z‐line proteins to cardiomyopathy, although genotype–phenotype prediction remains a great challenge. This review presents an overview of the currently available animal models for Z‐line and Z‐line associated proteins involved in human cardiomyopathies with special emphasis on knock‐in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z‐line proteins. Pros and cons of mouse models will be discussed and a future outlook will be given. J. Cell. Physiol. 232: 38–52, 2017. © 2016 Wiley Periodicals, Inc.The cardiac Z‐line at the boundary between sarcomeres is a multiprotein complex connecting the contractile apparatus with the cytoskeleton and the extracellular matrix. This review presents an overview of the currently available animal models for Z‐line and Z‐line associated proteins involved in human cardiomyopathies with special emphasis on knock‐in and transgenic mouse models recapitulating the clinical phenotypes of human cardiomyopathy patients carrying mutations in Z‐line proteins.
      PubDate: 2016-06-02T10:06:33.346872-05:
      DOI: 10.1002/jcp.25424
       
  • Targeting the Breast Cancer Kinome
    • Authors: Samantha M. Miller; Daniel R. Goulet, Gary L. Johnson
      Pages: 53 - 60
      Abstract: Protein kinases are highly tractable targets for the treatment of many cancers including breast cancer, due to their essential role in tumor cell proliferation and survival. Sequencing of the breast cancer genome and transcriptome has defined breast cancer as a heterogeneous disease that is classified into five molecular subtypes: luminal A, luminal B, HER2‐enriched, basal‐like, and claudin‐low. Each subtype displays a unique expression profile of protein kinases that can be targeted by small molecule kinase inhibitors or biologics. An understanding of genomic changes, including mutations or copy number variations, for specific protein kinases and dependencies on kinases across breast cancer subtypes is allowing for a more rational design of targeted breast cancer therapies. While specific kinase inhibitors have had success in the clinic, including the CDK4/6 inhibitor palbociclib in combination with aromatase inhibitors in luminal breast cancer, patients often become resistant to treatment. An understanding of the mechanisms allowing cells to bypass targeted kinase inhibition has led to the development of combination therapies that are more durable in pre‐clinical studies. However, the heterogeneity of resistance mechanisms and rapid adaptability of the kinome through feedback regulation greatly inhibit the long‐term efficacy of combination kinase inhibitor therapies. It is becoming apparent that epigenetic inhibitors, such as HDAC and BET bromodomain inhibitors can block the transcriptional adaptability of tumor cells to kinase inhibitors and prevent the onset of resistance. Such novel combination therapies are currently showing promise in preclinical studies to markedly increase the durability of kinase inhibitors in breast cancer. J. Cell. Physiol. 232: 53–60, 2017. © 2016 Wiley Periodicals, Inc.Sequencing of the breast cancer genome and transcriptome has identified specific protein kinases as promising drug targets across the five molecular subtypes. While some kinase inhibitors have had success in the clinic, resistance to treatment often occurs. An understanding of the molecular mechanisms of resistance has allowed for the design of combination therapies which have the potential to block resistance in the clinic.
      PubDate: 2016-05-31T07:45:26.494401-05:
      DOI: 10.1002/jcp.25427
       
  • Browning of White Fat: Novel Insight into Factors, Mechanisms and
           Therapeutics
    • Authors: Nevena Jeremic; Pankaj Chatuverdi, Suresh C. Tyagi
      First page: 61
      Abstract: What is more interesting about brown adipose tissue (BAT) is its ability to provide thermogenesis, protection against obesity by clearing triglycerides and mitigating insulin resistance. White adipose tissue (WAT) on the other hand stores excess energy and secretes some endocrine factors like leptin for regulating satiety. For the last decade there has been an increasing interest in the browning of fat keeping in view its beneficial effects on metabolic disorders and protection in the form of perivascular fat. Obesity is one such metabolic disorder that leads to significant morbidity and mortality from obesity‐related disorders such as type 2 diabetes mellitus (T2D) and cardiovascular disease risk. Browning of white fat paves the way to restrict obesity and obesity related disorders. Although exercise has been the most common factor for fat browning, however there are other factors that involve: 1) beta aminoisobutyric acid (BAIBA); 2) gamma amino butyric acid (GABA); 3) PPARɣ agonists; 4) JAK inhibition; 5) IRISIN. In this review we propose two novel factors musclin and TFAM for fat browning. Musclin a myokine released from muscles during exercise activates PPARɣ which induces browning of WAT that has beneficial metabolic and cardiac effects. TFAM is a transcription factor that induces mitochondrial biogenesis. Since BAT is rich in mitochondria, higher expression of TFAM in WAT or TFAM treatment in WAT cells can induce browning of WAT. We propose that fat browning can be used as a therapeutic tool for metabolic disorders and cardiovascular diseases. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-09T08:23:39.343408-05:
      DOI: 10.1002/jcp.25450
       
  • Multifaceted Breast Cancer: The Molecular Connection with Obesity
    • Authors: Feola Antonia; Ricci Serena, Kouidhi Soumaya, Rizzo Antonietta, Penon Antonella, Formisano Pietro, Giordano Antonio, Di Carlo Angelina, Di Domenico Marina
      First page: 69
      Abstract: Obesity is characterized by a disruption in energy balance regulation that results in an excess accumulation of body fat. Its increasing prevalence poses a major public health concern because it is a risk factor for a host of additional chronic conditions, including type 2 diabetes, hypertension, and cardiovascular disease. Obesity is increasingly recognized as a growing cause of cancer risk. In particular excessive adipose expansion during obesity causes adipose dysfunction and inflammation that can regulate tumor growth. In obesity, dysregulated systemic metabolism and inflammation induce hyperinsulinemia, hyperglycemia, dyslipidemia and enhance sex hormone production with increased secretion of proinflammatory adipokine that impact breast cancer development and progression. This review describes how adipose inflammation that characterizes obesity is responsible of microenvironment to promote cancer, and discuss how steroid hormones, that are essential for the maintenance of the normal development, growth and differentiation of the cells, influence the induction and progression of breast cancer. This article is protected by copyright. All rights reserved
      PubDate: 2016-07-01T05:36:16.572172-05:
      DOI: 10.1002/jcp.25475
       
  • Cartilage‐specific and Cre‐dependent Nkx3.2 overexpression in vivo
           causes skeletal dwarfism by delaying cartilage hypertrophy
    • First page: 78
      Abstract: Nkx3.2, the vertebrate homologue of Drosophila bagpipe, has been implicated as playing a role in chondrogenic differentiation. In brief, Nkx3.2 is initially expressed in chondrocyte precursor cells and, later during cartilage maturation, its expression is diminished in hypertrophic chondrocytes. In addition to Nkx3.2 expression analyses, previous studies using ex vivo chick embryo cultures and in vitro cell cultures have suggested that Nkx3.2 can suppress chondrocyte hypertrophy. However, it has never been demonstrated that Nkx3.2 functions in regulating chondrocyte hypertrophy during cartilage development in vivo. Here, we show that cartilage‐ specific and Cre‐dependent Nkx3.2 overexpression in mice results in significant postnatal dwarfism in endochondral skeletons, while intramembranous bones remain unaltered. Further, we observed significant delays in cartilage hypertrophy in conditional transgenic ciTg‐Nkx3.2 mice. Together, these findings confirm that Nkx3.2 is capable of controlling hypertrophic maturation of cartilage in vivo, and this regulation plays a significant role in endochondral ossification and longitudinal bone growth. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-02T12:28:17.938636-05:
      DOI: 10.1002/jcp.25446
       
  • Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in
           Murine Primary Splenocytes
    • Pages: 101 - 109
      Abstract: Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation, and survival. Leptin receptor is integrated inside cholesterol‐rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl‐β‐cyclodextrin (β‐MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/ml). Unlike β‐MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. J. Cell. Physiol. 232: 101–109, 2017. © 2016 Wiley Periodicals, Inc.This article suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling.
      PubDate: 2016-04-07T03:41:38.328614-05:
      DOI: 10.1002/jcp.25394
       
  • Cannabinoid Receptor 1 Mediates Homing of Bone Marrow‐Derived
           Mesenchymal Stem Cells Triggered by Chronic Liver Injury
    • Authors: Lin Wang; Le Yang, Lei Tian, Ping Mai, Shuangshuang Jia, Lin Yang, Liying Li
      Pages: 110 - 121
      Abstract: Cannabinoid receptors (CBs) have been implicated in the pathogenesis of various liver diseases, including liver fibrosis. Our previous studies have demonstrated that after liver injury, mouse bone marrow‐derived mesenchymal stem cells (BMSCs) can migrate to the injured liver and differentiate to myofibroblasts, contributing to hepatic fibrogenesis. However, the role of CBs in the homing of BMSCs in liver injury is yet unclear. In this study, we found that both CB1 and CB2 were expressed in BMSCs. Migration assays were performed by transwell chambers. CB1 agonist ACEA promoted the migration of BMSCs, but CB2 agonist JWH133 had no effect. Pharmacological or genetic ablation of CB1 reduced ACEA‐induced migration, whereas CB2 did not. Moreover, activation of CB1 increased active GTP‐bound Rac1, RhoA, and Cdc42 protein levels. The elevated GTP‐bound Rac1 and RhoA protein levels were decreased by CB1 antagonist AM281 treatment, but not Cdc42. In addition, ACEA‐induced migration was suppressed by NSC23766 (Rac1 inhibitor) or C3 transferase (RhoA inhibitor), whereas MLS‐573151 (Cdc42 inhibitor) had no effect. Consistent with these data, Rac1 or RhoA knock‐down significantly blocked CB1‐mediated migration. Meanwhile, CB1‐mediated migration was associated with cytoskeletal remodeling. In vivo, administration of CB1 antagonist AM281 markedly inhibited the recruitment of BMSCs to the injured liver using fluorescence‐activated cell sorting. Furthermore, blockade of CB1 significantly attenuated liver fibrosis. In conclusion, our results suggest that CB1 plays a crucial role in liver fibrosis through mediating the homing of BMSCs to damaged liver, which may provide new insight into the pathogenesis and treatment of liver fibrosis. J. Cell. Physiol. 232: 110–121, 2017. © 2016 Wiley Periodicals, Inc.The present study aims to evaluate the impact of Cannabinoid receptors (CBs) on the migration of BMSCs in liver injury and investigate the mechanisms involved. We found that BMSCs expressed CB1 and CB2. CB1 rather than CB2 mediated the migration of BMSCs via the Rac1 and RhoA pathways. And CB1‐mediated migration was associated with cytoskeletal remodeling. Furthermore, pharmacological blockade of CB1 markedly reduced the recruitment of BMSCs to injured liver and attenuated liver fibrosis. These results may contribute to the better understanding of the involvement of CB1 in liver injury and open new perspectives for development of potential therapeutics for liver disease.
      PubDate: 2016-04-08T00:12:05.373199-05:
      DOI: 10.1002/jcp.25395
       
  • MG132 Induces Expression of Monocyte Chemotactic Protein‐Induced Protein
           1 in Vascular Smooth Muscle Cells
    • Pages: 122 - 128
      Abstract: Monocyte chemoattractant protein‐1 (MCP‐1) has been reported to induce the expression of monocyte chemotactic protein‐induced protein 1 (MCPIP1), which undergoes ubiquitination degradation. Therefore, we predict that in vascular smooth muscle (VSMCs), MCPIP1 may be induced by MCP‐1 and undergo degradation, which can be inhibited by the proteasome inhibitor, MG132. Our results showed that treatment of human VSMCs with MCP‐1 did not increase the expression of MCPIP1. Treatment with MG132, however, elevated MCPIP1 protein levels through stimulation of the gene transcription, but not through increasing protein stability. MCPIP1 expression induced by MG132 was inhibited by α‐amanitin inhibition of gene transcription or cycloheximide inhibition of protein synthesis. Our further studies showed that MCPIP1 expression induced by MG132 was inhibited by the inhibitors of AKT and p38 kinase, suggesting a role of the AKT‐p38 pathway in MG132 effects. We also found that treatment with MG132 induces apoptosis, but overexpression of MCPIP1 inhibited bromodeoxyuridine (BrdU) incorporation of human VSMCs without induction of significant apoptosis. In summary, MCPIP1 expression is induced by MG132 likely through activation of the AKT‐p38 pathway. MCPIP1 inhibits SMC proliferation without induction of apoptosis. J. Cell. Physiol. 232: 122–128, 2017. © 2016 Wiley Periodicals, Inc.Monocyte chemoattractant protein‐1 (MCP‐1) has been reported to induce the expression of monocyte chemotactic protein‐induced protein 1 (MCPIP1), which undergoes ubiquitination degradation. In cultured human vascular smooth muscle, MCPIP1 expression is induced by MG132, a proteasome inhibitor, through signal transduction and gene transcription. Overexpression of MCPIP1 results in inhibition of smooth muscle cell proliferation.
      PubDate: 2016-04-14T02:00:50.27102-05:0
      DOI: 10.1002/jcp.25396
       
  • SRC Family Kinase Inhibition in Ewing Sarcoma Cells Induces p38 MAP
           Kinase‐Mediated Cytotoxicity and Reduces Cell Migration
    • Authors: Paola Indovina; Nadia Casini, Iris Maria Forte, Tiziana Garofano, Daniele Cesari, Carmelina Antonella Iannuzzi, Leonardo Del Porro, Francesca Pentimalli, Luca Napoliello, Silvia Boffo, Silvia Schenone, Maurizio Botta, Antonio Giordano
      Pages: 129 - 135
      Abstract: Ewing sarcoma (ES) is a highly aggressive bone and soft tissue cancer, representing the second most common primary malignant bone tumor in children and adolescents. Although the development of a multimodal therapy, including both local control (surgery and/or radiation) and systemic multidrug chemotherapy, has determined a significant improvement in survival, patients with metastatic and recurrent disease still face a poor prognosis. Moreover, considering that ES primarily affects young patients, there are concerns about long‐term adverse effects of the therapy. Therefore, more rational strategies, targeting specific molecular alterations underlying ES, are required. Recent studies suggest that SRC family kinases (SFKs), which are aberrantly activated in most cancer types, could represent key therapeutic targets also for ES. Here, we challenged ES cell lines with a recently developed selective SFK inhibitor (a pyrazolo[3,4‐d]pyrimidine derivative, called SI221), which was previously shown to be a valuable proapoptotic agent in other tumor types while not affecting normal cells. We observed that SI221 significantly reduced ES cell viability and proved to be more effective than the well‐known SFK inhibitor PP2. SI221 was able to induce apoptosis in ES cells and also reduced ES cell clonogenic potential. Furthermore, SI221 was also able to reduce ES cell migration. At the molecular level, our data suggest that SFK inhibition through SI221 could reduce ES cell viability at least in part by hindering an SFK‐NOTCH1 receptor‐p38 mitogen‐activated protein kinase (MAPK) axis. Overall, our study suggests a potential application of specific SFK inhibition in ES therapy. J. Cell. Physiol. 232: 129–135, 2017. © 2016 Wiley Periodicals, Inc.In this study, we challenged Ewing sarcoma (ES) cell lines with SI221, a recently developed selective SRC family kinase (SFK) inhibitor. We observed that this compound significantly reduced ES cell viability, clonogenic potential and migration. At the molecular level, our data suggest that SI221 could reduce ES cell viability at least in part by hindering an SFK‐NOTCH1 receptor‐p38 mitogen‐activated protein kinase (MAPK) axis.
      PubDate: 2016-04-19T03:41:36.652493-05:
      DOI: 10.1002/jcp.25397
       
  • Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and
           Epithelial Factors
    • Pages: 136 - 144
      Abstract: Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long‐term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre‐culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co‐culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136–144, 2017. © 2016 Wiley Periodicals, Inc.In a human stromal mammary cell line, glucose pre‐culture and soluble epithelial factors constitute a stimulus for lactate production. In a co‐culture setting, a vectorial flux of lactate from stroma to epithelial cells were stablished. The uptake of lactate by epithelial cells correlates with the degree of malignancy.
      PubDate: 2016-04-28T02:20:45.897269-05:
      DOI: 10.1002/jcp.25398
       
  • Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of
           Osteoblast Growth and Differentiation
    • Pages: 145 - 153
      Abstract: Inhibition of Runx2 is one of many mechanisms that suppress bone formation in glucocorticoid (GC)‐induced osteoporosis (GIO). We profiled mRNA expression in ST2/Rx2dox cells after treatment with doxycycline (dox; to induce Runx2) and/or the synthetic GC dexamethasone (dex). As expected, dex typically antagonized Runx2‐driven transcription. Select genes, however, were synergistic stimulated and this was confirmed by RT‐qPCR. Among the genes synergistically stimulated by GCs and Runx2 was Wnt inhibitory Factor 1 (Wif1), and Wif1 protein was readily detectable in medium conditioned by cultures co‐treated with dox and dex, but neither alone. Cooperation between Runx2 and GCs in stimulating Wif1 was also observed in primary preosteoblast cultures. GCs strongly inhibited dox‐driven alkaline phosphatase (ALP) activity in control ST2/Rx2dox cells, but not in cells in which Wif1 was silenced. Unlike its anti‐mitogenic activity in committed osteoblasts, induction of Runx2 transiently increased the percentage of cells in S‐phase and accelerated proliferation in the ST2 mesenchymal pluripotent cell culture model. Furthermore, like the inhibition of Runx2‐driven ALP activity, dex antagonized the transient mitogenic effect of Runx2 in ST2/Rx2dox cultures, and this inhibition eased upon Wif1 silencing. Plausibly, homeostatic feedback loops that rely on Runx2 activation to compensate for bone loss in GIO are thwarted, exacerbating disease progression through stimulation of Wif1. J. Cell. Physiol. 232: 145–153, 2017. © 2016 Wiley Periodicals, Inc.The work by Morimoto et al. demonstrates target gene‐dependent outcomes of the interaction between Runx2 and glucocorticoids (GCs) in preosteoblasts. While Runx2‐driven gene expression is generally antagonized by GCs, a few genes are synergistically stimulated by Runx2 and GCs. One of them, Wif1, potentially thwarts a homeostatic feedback loop and exacerbates GC‐induced osteoporosis.
      PubDate: 2016-04-26T03:35:45.489811-05:
      DOI: 10.1002/jcp.25399
       
  • Differentiation of Keratinocytes Modulates Expression of Epidermal
           Elements of the Cutaneous Analog of Hypothalamus‐Pituitary‐Adrenal
           Axis
    • First page: 154
      Abstract: It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus‐pituitary‐adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA).Expression of CRF, UCN1‐3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies.Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from Day 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium.Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. This article is protected by copyright. All rights reserved
      PubDate: 2016-04-06T17:02:14.046921-05:
      DOI: 10.1002/jcp.25400
       
  • Metabolic Adaptation of the Small Intestine to Short‐ and Medium‐Term
           High‐Fat Diet Exposure
    • Pages: 167 - 175
      Abstract: The small intestine is the main organ involved in the digestion and absorption of nutrients. It is in an ideal position to sense the availability of energy in the lumen in addition to its absorptive function. Consumption of a high‐fat diet (HFD) influences the metabolic characteristics of the small intestine. Therefore, to better understand the metabolic features of the small intestine and their changes in response to dietary fat, we characterized the metabolism of duodenal, jejunal, and hepatic cell lines and assessed the metabolic changes in the enterocytes and the liver after short‐term (3 days) or medium‐term (14 days) HFD feeding in mice. Experiments with immortalized enterocytes indicated a higher glycolytic capacity in the duodenal cell line compared to the other two cell lines, whereas the jejunal cell line exhibited a high oxidative metabolism. Short‐term HFD feeding induced changes in the expression of glucose and lipid metabolism‐related genes in the duodenum and the jejunum of mice, but not in the liver. When focusing on fatty acid oxidation both, short‐ and medium‐term HFD feeding induced an upregulation of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A, the key enzyme of ketogenesis, at the protein level in the intestinal epithelial cells, but not in the liver. These results suggest that HFD feeding induces an early adaptation of the small intestine rather than the liver in response to a substantial fat load. This highlights the importance of the small intestine in the adaptation of the body to the metabolic changes induced by HFD exposure. J. Cell. Physiol. 232: 167–175, 2017. © 2016 Wiley Periodicals, Inc.Characterization of intestinal cell lines from the proximal and distal part of the small intestine, showed a differential metabolism in response to metabolic modulators. Short‐ and medium‐term access to high‐fat‐diet induced the expression of HMG‐CoAS2, the ketogenesis key‐enzyme, specifically in the jejunum but not in the duodenum or in the liver of mice.
      PubDate: 2016-04-28T02:21:10.59553-05:0
      DOI: 10.1002/jcp.25402
       
  • Antibodies Against Mimotopes of Simian Virus 40 Large T Antigen, the
           Oncoprotein, in Serum Samples From Elderly Healthy Subjects
    • Authors: Elisa Mazzoni; Giovanni Guerra, Maria Vittoria Casali, Silvia Pietrobon, Ilaria Bononi, Andrea Puozzo, Andrea Tagliapietra, Pier Francesco Nocini, Mauro Tognon, Fernanda Martini
      Pages: 176 - 181
      Abstract: Simian Virus 40 (SV40), a monkey polyomavirus, was administered to human populations by early anti‐poliomylitis vaccines contaminated by this small DNA tumor virus. Data on SV40 infection in humans remain controversial. Elderly subjects represent an interesting cohort to investigate, because they were not immunized with SV40‐contaminated vaccines. Taking advantage of the Italian population, the second oldest worldwide, elderly subjects (n = 237) up to 100 years old were enrolled in this study. Their sera were analyzed, by ELISA tests with synthetic peptides mimicking the viral epitopes, for IgG antibodies reacting with SV40 large Tumor antigen (Tag), the viral oncoprotein. An overall seroprevalence of 22% was revealed in subjects aged 66–100 years, ranging from 19% in individuals 66–74 years old, to 24% in subjects 82–100 years old, with a lower SV40 titer detected in the oldest group. Our data show that: (i) SV40 infection is not frequent in old individuals; (ii) the infection rate increases in elderly with the age; (iii) the antibody titer of SV40 Tag decreases with the age. In conclusion, SV40 infection seems to spread in old subjects independently from SV40‐contaminated vaccines. This study seems to confirm that SV40 is also a human virus. J. Cell. Physiol. 232: 176–181, 2017. © 2016 Wiley Periodicals, Inc.Specific antibodies reacting against Simian Virus 40 large T antigen mimotopes were detected in serum samples from elderly healthy subjects, up to 100 years old, with a prevalence of 22%. Immunological data were obtained with a novel indirect ELISA with synthetic peptides, which mimic the immunogenic epitopes of the viral oncoprotein of SV40. Our data suggests that the exposure to Simian Virus 40 infection is not frequent in old individuals. The immunological results showed that (i) the infection rate increases in elderly with the age; (ii) the proportion of elderly subjects with high Simian Virus 40 large T antigen antibody levels decreases with age; (iii) Simian Virus 40 infection spreads in old subjects independently from SV40‐contaminated vaccines; (iv) this monkey polyomavirus is also a viral agent in humans.
      PubDate: 2016-04-29T05:20:52.781399-05:
      DOI: 10.1002/jcp.25405
       
  • Runx2/DICER/miRNA Pathway in Regulating Osteogenesis
    • Authors: Leilei Zheng; Qisheng Tu, Shu Meng, Lan Zhang, Liming Yu, Jinlin Song, Yun Hu, Lei Sui, Jin Zhang, Michel Dard, Jessica Cheng, Dana Murray, Yin Tang, Jane B. Lian, Gary S. Stein, Jake Chen
      Pages: 182 - 191
      Abstract: DICER is the central enzyme that cleaves precursor microRNAs (miRNAs) into 21–25 nucleotide duplex in cell lineage differentiation, identity, and survival. In the current study, we characterized the specific bone metabolism genes and corresponding miRNAs and found that DICER and Runt‐related transcription factor 2 (Runx2) expressions increased simultaneously during osteogenic differentiation. Luciferase assay showed that Runx2 significantly increased the expression levels of DICER luciferase promoter reporter. Our analysis also revealed weaker DICER expression in embryos of Runx2 knock out mice (Runx2 −/−) compared with that of Runx2 +/− and Runx2 +/+ mice. We further established the calvarial bone critical‐size defect (CSD) mouse model. The bone marrow stromal cells (BMSCs) transfected with siRNA targeting DICER were combined with silk scaffolds and transplanted into calvarial bone CSDs. Five weeks post‐surgery, micro‐CT analysis revealed impaired bone formation, and repairing in calvarial defects with the siRNA targeting DICER group. In conclusion, our results suggest that DICER is specifically regulated by osteogenic master gene Runx2 that binds to the DICER promoter. Consequently, DICER cleaves precursors of miR‐335‐5p and miR‐17‐92 cluster to form mature miRNAs, which target and decrease the Dickkopf‐related protein 1 (DKK1), and proapoptotic factor BIM levels, respectively, leading to an enhanced Wnt/β‐catenin signaling pathway. These intriguing results reveal a central mechanism underlying lineage‐specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. Our study, also suggests a potential application of modulating DICER expression for bone tissue repair and regeneration. J. Cell. Physiol. 232: 182–191, 2017. © 2016 Wiley Periodicals, Inc.These intriguing results reveal a central mechanism underlying lineage‐specific regulation by a Runx2/DICER/miRNAs cascade during osteogenic differentiation and bone development. This cell‐ and development‐specific regulation is essential and mandatory for the initiation and progression of osteogenic differentiation. Furthermore, our study also suggests a potential application of specifically regulating DICER for bone tissue repair and regeneration.
      PubDate: 2016-04-26T03:36:35.324038-05:
      DOI: 10.1002/jcp.25406
       
  • Low‐Dose Farnesyltransferase Inhibitor Suppresses HIF‐1α and Snail
           Expression in Triple‐Negative Breast Cancer MDA‐MB‐231 Cells In
           Vitro
    • First page: 192
      Abstract: The aggressiveness of triple‐negative breast cancer (TNBC), which lacks estrogen receptor, progesterone receptor and epidermal growth factor receptor 2 (HER2), represents a major challenge in breast cancer. Migratory and self‐renewal capabilities are integral components of invasion, metastasis and recurrence of TNBC. Elevated hypoxia‐inducible factor‐1α (HIF‐1α) expression is associated with aggressiveness of cancer. Nonetheless, how HIF‐1α expression is regulated and how HIF‐1α induces aggressive phenotype are not completely understood in TNBC. The cytotoxic effects of farnesyltransferase (FTase) inhibitors (FTIs) have been studied in cancer and leukemia cells. In contrast, the effect of FTIs on HIF‐1α expression has not yet been studied. Here, we show that clinically relevant low‐dose FTI, tipifarnib (300 nM), decreased HIF‐1α expression, migration and tumorsphere formation in human MDA‐MB‐231 TNBC cells under a normoxic condition. In contrast, the low‐dose FTIs did not inhibit cell growth and activity of the Ras pathway in MDA‐MB 231 cells. Tipifarnib‐induced decrease in HIF‐1α expression was associated with amelioration of the Warburg effect, hypermetabolic state, increases in Snail expression and ATP release, and suppressed E‐cadherin expression, major contributors to invasion, metastasis and recurrence of TBNC. These data suggest that FTIs may be capable of ameliorating the aggressive phenotype of TNBC by suppressing the HIF‐1α‐Snail pathway. This article is protected by copyright. All rights reserved
      PubDate: 2016-06-15T11:55:28.989913-05:
      DOI: 10.1002/jcp.25411
       
  • Prostaglandin E2‐Dependent Phosphorylation of RAS Inhibition 1 (RIN1) at
           Ser 291 and 292 Inhibits Transforming Growth Factor‐β‐Induced RAS
           Activation Pathway in Human Synovial Fibroblasts: Role in Cell Migration
    • Authors: Casimiro Gerarduzzi; QingWen He, Beibei Zhai, John Antoniou, John A. Di Battista
      Pages: 202 - 215
      Abstract: Prostaglandin E2 (PGE2)‐stimulated G‐protein–coupled receptor (GPCR) activation inhibits pro‐fibrotic TGFβ‐dependent stimulation of human fibroblast to myofibroblast transition (FMT), though the precise molecular mechanisms are not fully understood. In the present study, we describe the PGE2‐dependent suppression and reversal of TGFβ‐induced events such as α‐sma expression, stress fiber formation, and Ras/Raf/ERK/MAPK pathway‐dependent activation of myofibroblast migration. In order to elucidate post‐ligand‐receptor signaling pathways, we identified a predominant PKA phosphorylation motif profile in human primary fibroblasts after treatment with exogenous PGE2 (EC50 30 nM, Vmax 100 nM), mimicked by the adenyl cyclase activator forskolin (EC50 5 μM, Vmax 10 μM). We used a global phosphoproteomic approach to identify a 2.5‐fold difference in PGE2‐induced phosphorylation of proteins containing the PKA motif. Deducing the signaling pathway of our migration data, we identified Ras inhibitor 1 (RIN1) as a substrate, whereby PGE2 induced its phosphorylation at Ser291 and at Ser292 by a 5.4‐ and 4.8‐fold increase, respectively. In a series of transient and stable over expression studies in HEK293T and HeLa cells using wild‐type (wt) and mutant RIN1 (Ser291/292Ala) or Ras constructs and siRNA knock‐down experiments, we showed that PGE2‐dependent phosphorylation of RIN1 resulted in the abrogation of TGFβ‐induced Ras/Raf signaling activation and subsequent downstream blockade of cellular migration, emphasizing the importance of such phosphosites in PGE2 suppression of wound closure. Overexpression experiments in tandem with pull‐down assays indicated that specific Ser291/292 phosphorylation of RIN1 favored binding to activated Ras. In principal, understanding PGE2‐GPCR activated signaling pathways mitigating TGFβ‐induced fibrosis may lead to more evidence‐based treatments against the disease. J. Cell. Physiol. 232: 202–215, 2017. © 2016 Wiley Periodicals, Inc.Background: PGE2 is an antifibrotic agent with a limited molecular mechanism of action against myofibroblasts. Results: PGE2 induces the phosphorylation of RIN1 at Ser291/292 to inhibit the TGFβ‐activated Ras/Raf pathway of myofibroblast migration. Conclusion: An antifibrotic mechanism of PGE2 blocks myofibroblast migration through RIN1. Significance: Understanding the endogenous mechanisms of antifibrosis could be extrapolated for therapeutic means.
      PubDate: 2016-05-26T08:46:51.819046-05:
      DOI: 10.1002/jcp.25412
       
  • Targeted Modification of Mitochondrial ROS Production Converts High
           Glucose‐Induced Cytotoxicity to Cytoprotection: Effects on Anesthetic
           Preconditioning
    • Authors: Filip Sedlic; Maria Muravyeva, Ana Sepac, Marija Sedlic, Anna Marie Williams, Meiying Yang, Xiaowen Bai, Zeljko J. Bosnjak
      First page: 216
      Abstract: Contradictory reports on the effects of diabetes and hyperglycemia on myocardial infarction range from cytotoxicity to cytoprotection. The study was designed to investigate acute effects of high glucose‐driven changes in mitochondrial metabolism and osmolarity on adaptive mechanisms and resistance to oxidative stress of isolated rat cardiomyocytes.We examined the effects of high glucose on several parameters of mitochondrial bioenergetics, including changes in oxygen consumption, mitochondrial membrane potential and NAD(P)H fluorometry. Effects of high glucose on the endogenous cytoprotective mechanisms elicited by anesthetic preconditioning (APC) and the mediators of cell injury were also tested. These experiments included real‐time measurements of reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) opening in single cells by laser scanning fluorescence confocal microscopy, and cell survival assay.High glucose rapidly enhanced mitochondrial energy metabolism, observed by increase in NAD(P)H fluorescence intensity, oxygen consumption and mitochondrial membrane potential. This substantially elevated production of ROS, accelerated opening of the mPTP and decreased survival of cells exposed to oxidative stress. Abrogation of high glucose‐induced mitochondrial hyperpolarization with 2,4 dinitrophenol (DNP) significantly, but not completely, attenuated ROS production to a level similar to hyperosmotic mannitol control. DNP treatment reversed high glucose‐induced cytotoxicity to cytoprotection. Hyperosmotic mannitol treatment also induced cytoprotection. High glucose abrogated APC‐induced mitochondrial depolarization, delay in mPTP opening and cytoprotection.In conclusion, high glucose‐induced mitochondrial hyperpolarization abolishes APC and augments cell injury. Attenuation of high glucose‐induced ROS production by eliminating mitochondrial hyperpolarization protects cardiomyocytes. This article is protected by copyright. All rights reserved
      PubDate: 2016-05-02T13:22:40.120836-05:
      DOI: 10.1002/jcp.25413
       
  • Milk‐Derived Nanoparticle Fraction Promotes the Formation of Small
           Osteoclasts But Reduces Bone Resorption
    • Authors: Marina C. Oliveira; Irene Di Ceglie, Onno J. Arntz, Wim B. van den Berg, Frank H.J. van den Hoogen, Adaliene V.M. Ferreira, Peter L.E.M. van Lent, Fons A.J. van de Loo
      Pages: 225 - 233
      Abstract: The general consensus is that milk promotes bone growth and density because is a source of calcium and contains components that enhance intestinal calcium uptake or directly affect bone metabolism. In this study, we investigated the effect of bovine‐derived milk 100,000 g pellet (P100), which contains nanoparticles (
      PubDate: 2016-06-02T05:51:51.12926-05:0
      DOI: 10.1002/jcp.25414
       
  • Erratum: ATP‐Sensitive Potassium Channel: A Novel Target for Protection
           Against UV‐Induced Human Skin Cell Damage by Cong Cao, Sarah Healey,
           Ashley Amaral, Avery Lee‐Couture, Shu Wan, Nicola Kouttab, Wenming Chu
           and Yinsheng Wan
    • First page: 236
      PubDate: 2016-07-14T10:30:44.777224-05:
      DOI: 10.1002/jcp.25480
       
  • MED28 Regulates Epithelial–Mesenchymal Transition through NFκB in Human
           Breast Cancer Cells
    • Abstract: MED28, a mammalian Mediator subunit, was found highly expressed in several types of malignancy, including breast cancer. Recently, we have identified a role of MED28 in regulating both cell growth and migration in human breast cancer cells. In epithelium‐derived solid tumor, migration and invasion are preceded by the progression of epithelial–mesenchymal transition (EMT) which calls for downregulation of epithelial markers as well as upregulation of mesenchymal markers, among other features. The objective of this study was to investigate a putative role of MED28 in the progression of EMT in human breast cancer cells. In fibroblast‐like MDA‐MB‐231 cells, suppression of MED28 attenuated the mesenchymal morphology, concomitantly with a reduction of several mesenchymal biomarkers and Snail, a transcriptional repressor of E‐cadherin. The suppression effect was also accompanied by downregulation of p‐NFκB/p65. However, overexpression of MED28 exhibited in an opposite manner. In epithelial MCF7 cells, administration of Adriamycin®, an experimental EMT induction system, led to a mesenchyme‐like appearance correlated with increased expression of MED28, p‐p65, and Snail, and a reciprocal change of epithelial and mesenchymal markers. Furthermore, suppression of MED28 attenuated the experimental EMT effect and restored the original expression status of E‐cadherin and MMP9 in MCF7 cell. Our data indicate that MED28 modulates the development of EMT through NFκB in human breast cancer cells, further reinforcing the significance of MED28 in the progression of breast cancer on top of its role in cell growth and migration. This article is protected by copyright. All rights reserved
       
  • Anti‐Tumor Effects of Cardiac Glycosides on Human Lung Cancer Cells
           and Lung Tumorspheres
    • Abstract: Lung cancer is a leading cause of cancer‐related death in the United States. While several drugs have been developed that target individual biomarkers, their success has been limited due to intrinsic or acquired resistance for the specific targets of such drugs. A more effective approach is to target multiple pathways that dictate cancer progression. Cardiac glycosides demonstrate such multimodal effects on cancer cell survival, and our aim was to evaluate the effect of two naturally occurring monosaccaridic cardiac glycosides – Convallatoxin and Peruvoside on lung cancer cells. While both drugs had significant anti‐proliferative effects on H460 and Calu‐3 lung cancer cells, Convallatoxin demonstrated two‐fold higher activity as compared to Peruvoside using both viability and colony forming assays, suggesting a role for the aglycone region in dictating drug potency. The tumor suppressor p53 was found to be important for action of both drugs – p53‐underexpressing cells were less sensitive as compared to p53‐positive H460 cells. Further assessment of p53‐underexpressing H460 cells showed that drugs were able to arrest cells in the G0/G1 phase of the cell cycle in a dose‐dependent manner. Both drugs significantly inhibited migration and invasion of cancer cells and decreased the viability of floating tumorspheres. An assessment of intracellular pathways indicated that both drugs were able to modulate proteins that are involved in apoptosis, autophagy, cell cycle, proliferation and EMT. Our data suggests a promising role for cardiac glycosides in lung cancer treatment, and provides impetus for further investigation of the anti‐cancer potential of this class of drugs. This article is protected by copyright. All rights reserved
       
  • Hypoglycemia Enhances Epithelial‐Mesenchymal Transition and
           Invasiveness, and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell
           Cultures and Microspheroids
    • Abstract: The accelerated growth of solid tumors leads to episodes of both hypoxia and hypoglycemia (HH) affecting their intermediary metabolism, signal transduction and transcriptional activity. A previous study showed that normoxia (20% O2) plus 24 h hypoglycemia (2.5 mM glucose) increased glycolytic flux whereas oxidative phosphorylation (OxPhos) was unchanged vs. normoglycemia in HeLa cells. However, the simultaneous effect of HH on energy metabolism has not been yet examined. Therefore, the effect of hypoxia (0.1‐1% O2) plus hypoglycemia on the energy metabolism of HeLa cells was analyzed by evaluating protein content and activity, along with fluxes of both glycolysis and OxPhos. Under hypoxia, in which cell growth ceased and OxPhos enzyme activities, ΔΨm and flux were depressed, hypoglycemia did not stimulate glycolytic flux despite increasing H‐RAS, p‐AMPK, GLUT1, GLUT3 and HKI levels, and further decreasing mitochondrial enzyme content. The impaired mitochondrial function in HH cells correlated with mitophagy activation. The depressed OxPhos and unchanged glycolysis pattern was also observed in quiescent cells from mature multicellular tumor spheroids, suggesting that these inner cell layers are similarly subjected to HH. The principal ATP supplier was glycolysis for HH 2D monolayer and 3D quiescent spheroid cells. Accordingly, the glycolytic inhibitors iodoacetate and gossypol were more effective than mitochondrial inhibitors in decreasing HH‐cancer cell viability. Under HH, stem cell‐, angiogenic‐, and EMT‐biomarkers, as well as glycoprotein‐P content and invasiveness, were also enhanced. These observations indicate that HH cancer cells develop an attenuated Warburg and pronounced EMT‐ and invasive‐phenotype. This article is protected by copyright. All rights reserved
       
  • The Role of Calcium‐Sensing Receptors in Endothelin‐1‐Dependent
           Effects on Adult Rat Ventricular Cardiomyocytes: Possible Contribution to
           Adaptive Myocardial Hypertrophy
    • Abstract: Nitric oxide (NO)‐deficiency as it occurs during endothelial dysfunction activates the endothelin‐1 system and increases the expression of receptor activity modifying protein (RAMP)‐1 that acts as a chaperon for calcium‐sensing receptors (CaR) that have recently been identified to improve cardiac function. Here we hypothesized that endothelin‐1 increases the cardiac expression of CaR and thereby induces an adaptive type of hypertrophy. Expressions of RAMP‐1, endothelin receptors, and CaR were analyzed by RT‐PCR in left ventricular tissues of L‐NAME‐treated rats. Effects of ET‐1 on CaR expression and cell function (load free cell shortening) were analyzed in adult rat ventricular cardiomyocytes. siRNA directed against CaR and RAMP‐1 was used to investigate a causal relationship. PD142893 and BQ788 were used to dissect the contribution of ETB1, ETB2, and ETA receptors. Non‐specific NO synthase inhibition with L‐Nitro arginine methyl ester (L‐NAME) caused a cardiac upregulation of ETB receptors and CaR suggesting a paracrine effect of endothelin‐1 on cardiomyocytes. Indeed, endothelin‐1 induced the expression of CaR in cultured cardiomyocytes. Under these conditions cardiomyocytes increased cell size (hypertrophy) but maintained normal function. Down‐regulation of RAMP‐1 or selective inhibition of ETA and ETB1 receptors led endothelin‐1 reducing cell shortening and attenuates up‐regulation of CaR. In conclusion, endothelin‐1 causes an adaptive type of hypertrophy by up‐regulation of CaR in cardiomyocytes via ETA receptors. This article is protected by copyright. All rights reserved
       
  • PACAP and VIP Inhibit HIF‐1α‐Mediated VEGF Expression in a Model of
           Diabetic Macular Edema
    • Abstract: Pituitary adenylate cyclase‐activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) exert a protective role against retinal injuries, including diabetic macular edema (DME). The macular damage is induced by hyperglycemia, which damages vessels supplying blood to the retina and induces hypoxia. The microenvironmental changes stimulate the expression of hypoxia‐inducible factors (HIFs), which promote the choroidal endothelial cell transmigration across the retinal pigmented epithelium (RPE) into neurosensory retina, where they proliferate into new vessels under stimulation of the vascular endothelial growth factor (VEGF). In the present study, we have investigated whether PACAP and VIP prevent retinal damage by modulating the expression of HIFs, VEGF and its receptors. In accord to our hypothesis, we have shown that both peptides are able to significantly reduce HIF‐1α and increase HIF‐3α expression in ARPE‐19 cells exposed to hyperglycemic/hypoxic insult. This effect is also related to a reduction of VEGF and its receptors expression. Moreover, both peptides also reduce the activation of p38 mitogen‐activated protein kinase (MAPK), a pro‐apoptotic signaling pathway, which is activated by VEGFR‐1 and 2 receptors.In conclusion, our study has further elucidated the protective role performed by PACAP and VIP, against the harmful combined effect of hyperglycemia/hypoxia characterizing the DME microenvironment. This article is protected by copyright. All rights reserved
       
  • Role of Protein Kinase G and Reactive Oxygen Species in the Regulation of
           Podocyte Function in Health and Disease
    • Abstract: Podocytes and their foot processes form an important cellular layer of the glomerular barrier involved in the regulation of glomerular permeability. Disturbing the function of podocytes plays a central role in the development of proteinuria in diabetic nephropathy. Retraction of the podocyte foot processes that form slit diaphragms is a common feature of proteinuria; although, the correlation between these events in not well understood. Notably, it is unclear whether podocyte foot processes are able to regulate slit diaphragm permeability and glomerular ultrafiltration. The occurrence of reactive oxygen species generation, insulin resistance, and hyperglycemia characterizes early stages of type 2 diabetes.Protein kinase G type I alpha (PKGIα) is an intracellular target for vasorelaxant factors. It is activated in both cGMP‐dependent and cGMP‐independent manners. Recently, we demonstrated a relationship between oxidative stress, PKGIα activation, actin reorganization, and changes in the permeability of the filtration barrier. This review discusses how redox imbalance affects both the activity of PKGIα and PKGI‐dependent signaling pathways in podocytes. This article is protected by copyright. All rights reserved
       
  • Differences in Activation of HIV‐1 Replication by Superinfection with
           HIV‐1 and HIV‐2 in U1 Cells
    • Abstract: Macrophages contribute to HIV‐1 pathogenesis by forming a viral reservoir that serve as a viral source for the infection of CD4 T cells. The relationship between HIV‐1 latent infection and superinfection in macrophages has not been well studied. Using susceptible U1 cells chronically infected with HIV‐1, we studied the effects of HIV superinfection on latency and differences in superinfection with HIV‐1 and HIV‐2 in macrophages. We found that HIV‐1 (MN) superinfection displayed increased HIV‐1 replication in a time‐dependent manner; while cells infected with HIV‐2 (Rod) initially showed increased HIV‐1 replication, followed by a decrease in HIV‐1 RNA production. HIV‐1 superinfection upregulated/activated NF‐κB, NFAT, AP‐1, SP‐1, and MAPK Erk through expression/activation of molecules, CD4, CD3, TCRβ, Zap‐70, PLCγ1 and PKCΘ in T cell receptor‐related signaling pathways; while HIV‐2 superinfection initially increased expression/activation of these molecules followed by decreased protein expression/activation. HIV superinfection initially downregulated HDAC1 and upregulated Acetyl‐histone H3 and Histone H3 (K4), while HIV‐2 superinfection demonstrated an increase in HDAC1 and a decrease in Acetyl‐histone H3 and Histone H3 (K4) relative to HIV‐1 superinfection. U1 cells superinfected with HIV‐1 or HIV‐2 showed differential expression of proteins, IL‐2, PARP‐1, YB‐1, and LysRS. These findings indicate that superinfection with HIV‐1 or HIV‐2 has different effects on reactivation of HIV‐1 replication. HIV‐1 superinfection with high load of viral replication may result in high levels of cytotoxicity relative to HIV‐2 superinfection. Cells infected with HIV‐2 showed lower level of HIV‐1 replication, suggesting that co‐infection with HIV‐2 may result in slower progression towards AIDS. This article is protected by copyright. All rights reserved
       
  • G Protein‐Coupled Receptor Endosomal Signaling and Regulation of
           Neuronal Excitability and Stress Responses: Signaling Options and Lessons
           from the PAC1 Receptor
    • Abstract: Our understanding of G protein coupled receptor (GPCR) mechanisms and functions have evolved considerably. Among the many conceptual realignments, GPCRs can exist in an ensemble of active microstates that have the potential to differentially engage specific downstream signaling events. Furthermore, among GPCR dynamics, GPCR internalization and vesicular trafficking are no longer solely mechanisms for desensitization, but now appreciated to form intricate endosomal signaling complexes that can potentially target second messengers to intracellular compartments with high temporal and spatial resolution. The PACAPergic system is important in the maintenance of physiological homeostasis in the central and peripheral nervous systems and activation of the PACAP‐selective PAC1 receptor can generate differential but coordinate plasma membrane and endosomal signals for cellular responses. The integration of these signals can modulate PACAP‐induced changes in ionic conductances that gate neuronal excitability. PACAP/PAC1 receptor generation of endosomal ERK signals participate in chronic pain and anxiety‐like responses which can be attenuated with endocytosis inhibitors. From the abilities of ligands to stabilize the different GPCR microstates for biased downstream signaling, the development of biased PAC1 receptor agonists and antagonists may provide opportunities to dissociate the homeostatic regulatory signals of PACAP from the maladaptive effects. In particular, the development of biased antagonists to PAC1 receptor‐mediated endosomal signaling may offer therapeutic options for chronic pain and stress‐related disorders. This article is protected by copyright. All rights reserved
       
 
 
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