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Journal Cover   Journal of Cellular Physiology
  [SJR: 1.948]   [H-I: 125]   [4 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  [1597 journals]
  • cAMP and cGMP Play an Essential Role in Galvanotaxis of Cell Fragments
    • Authors: Kan Zhu; Yaohui Sun, Anh Miu, Michael Yen, Bowei Liu, Qunli Zeng, Alex Mogilner, Min Zhao
      Abstract: Cell fragments devoid of the nucleus and major organelles are found in physiology and pathology, for example platelets derived from megakaryocytes, and cell fragments from white blood cells and glioma cells. Platelets exhibit active chemotaxis. Fragments from white blood cells display chemotaxis, phagocytosis and bactericidal functions. Signaling mechanisms underlying migration of cell fragments are poorly understood. Here we used fish keratocyte fragments and demonstrated striking differences in signal transduction in migration of cell fragments and parental cells in a weak electric field. cAMP or cGMP agonists completely abolished directional migration of fragments, but had no effect on parental cells. The inhibition effects were prevented by pre‐incubating with cAMP and cGMP antagonists. Blocking cAMP and cGMP downstream signaling by inhibition of PKA and PKG also recovered fragment galvanotaxis. Both perturbations confirmed that the inhibitory effect was mediated by cAMP or cGMP signaling. Inhibition of cathode signaling with PI3K inhibitor LY294002 also prevented the effects of cAMP or cGMP agonists. Our results suggest that cAMP and cGMP are essential for galvanotaxis of cell fragments, in contrast to the signaling mechanisms in parental cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-30T16:13:41.548036-05:
      DOI: 10.1002/jcp.25229
  • Effects of Sleep Deprivation on Mice Bone Marrow and Spleen B
    • Abstract: B lymphocytes are immune cells crucial for the maintenance and viability of the humoral response. Sleep is an essential event for the maintenance and integrity of all systems, including the immune system (IS). Thus, sleep deprivation (SD) causes problems in metabolism and homeostasis in many cell systems, including the IS. In this study, our goal was to determine changes in B lymphocytes from the bone marrow (BM) and spleen after SD. Three‐month‐old male Swiss mice were used. These mice were sleep deprived through the modified multiple platform method for different periods (24, 48, and 72 h), whereas another group was allowed to sleep for 24 h after 72 h of SD (rebound group) and a third group was allowed to sleep normally during the entire experiment. After this, the spleen and BM were collected, and cell analyses were performed. The numbers of B lymphocytes in the BM and spleen were reduced by SD. Additionally, reductions in the percentage of lymphocyte progenitors and their ability to form colonies were observed. Moreover, an increase in the death of B lymphocytes from the BM and spleen was associated with an increase in oxidative stress indicators, such as DCFH‐DA, CAT and mitochondrial SOD. Rebound was not able to reverse most of the alterations elicited by SD. The reduction in B lymphocytes and their progenitors by cell death, with a concomitant increase in oxidative stress, showed that SD promoted a failure in B lymphopoiesis. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-30T16:12:23.970251-05:
      DOI: 10.1002/jcp.25231
  • Ex Vivo Assay of Electrical Stimulation to Rat Sciatic Nerves: Cell
           Behaviors and Growth Factor Expression
    • Authors: Zhiyong Du; Olexandr Bondarenko, Dingkun Wang, Mahmoud Rouabhia, Ze Zhang
      Abstract: Neurite outgrowth and axon regeneration are known to benefit from electrical stimulation. However, how neuritis and their surroundings react to electrical field is difficult to replicate by monolayer cell culture. In this work freshly harvested rat sciatic nerves were cultured and exposed to two types of electrical field, after which time the nerve tissues were immunohistologically stained and the expression of neurotrophic factors and cytokines were evaluated. ELISA assay was used to confirm the production of specific proteins. All cell populations survived the 48h culture with little necrosis. Electrical stimulation was found to accelerate Wallerian degeneration and help Schwann cells to switch into migratory phenotype. Inductive electrical stimulation was shown to upregulate the secretion of multiple neurotrophic factors. Cellular distribution in nerve tissue was altered upon the application of an electrical field. This work thus presents an ex vivo model to study denervated axon in well controlled electrical field, bridging monolayer cell culture and animal experiment. It also demonstrated the critical role of electrical field distribution in regulating cellular activities. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-30T16:11:54.22287-05:0
      DOI: 10.1002/jcp.25230
  • Research Needs for Understanding the Biology of Overdiagnosis in Cancer
    • Authors: Sudhir Srivastava; Brian J. Reid, Sharmistha Ghosh, Barnett S. Kramer
      Abstract: Many cancers offer an extended window of opportunity for early detection and therapeutic intervention that could lead to a reduction in cause‐specific mortality. The pursuit of early detection in screening settings has resulted in decreased incidence and mortality for some cancers (eg, colon and cervical cancers), and increased incidence with only modest or no effect on cause‐specific mortality in others (eg, breast and prostate). Whereas highly sensitive screening technologies are better at detecting a number of suspected “cancers” that are indolent and likely to remain clinically unimportant in the lifetime of a patient, defined as overdiagnosis, they often miss cancers that are aggressive and tend to present clinically between screenings, known as interval cancers. Unrecognized overdiagnosis leads to overtreatment with its attendant (often long‐lasting) side effects, anxiety, and substantial financial harm. Existing methods often cannot differentiate indolent lesions from aggressive ones or understand the dynamics of neoplastic progression. To correctly identify the population that would benefit the most from screening and identify the lesions that would benefit most from treatment, the evolving genomic and molecular profiles of individual cancers during the clinical course of progression or indolence must be investigated, while taking into account an individual's genetic susceptibility, clinical and environmental risk factors, and the tumor microenvironment. Practical challenges lie not only in the lack of access to tissue specimens that are appropriate for the study of natural history, but also in the absence of targeted research strategies. This commentary summarizes the recommendations from a diverse group of scientists with expertise in basic biology, translational research, clinical research, statistics, and epidemiology and public health professionals convened to discuss research directions. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-27T17:47:33.070998-05:
      DOI: 10.1002/jcp.25227
  • Differential Regulation of Telomerase Reverse Transcriptase Promoter
           Activation and Protein Degradation by Histone Deacetylase Inhibition
    • Authors: Hua Qing; Jun Aono, Hannes M. Findeisen, Karrie L. Jones, Elizabeth B. Heywood, Dennis Bruemmer
      Abstract: Telomerase reverse transcriptase (TERT) maintains telomeres and is rate limiting for replicative life span. While most somatic tissues silence TERT transcription resulting in telomere shortening, cells derived from cancer or cardiovascular diseases express TERT and activate telomerase. In the present study, we demonstrate that histone deacetylase (HDAC) inhibition induces TERT transcription and promoter activation. At the protein level in contrast, HDAC inhibition decreases TERT protein abundance through enhanced degradation, which decreases telomerase activity and induces senescence. Finally, we demonstrate that HDAC inhibition decreases TERT expression during vascular remodeling in vivo. These data illustrate a differential regulation of TERT transcription and protein stability by HDAC inhibition and suggest that TERT may constitute an important target for the anti‐proliferative efficacy of HDAC inhibitors. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-27T17:47:10.176242-05:
      DOI: 10.1002/jcp.25226
  • Mechanical Stimulation and IGF‐1 Enhance mRNA Translation Rate in
           Osteoblasts via Activation of the AKT‐mTOR Pathway
    • Abstract: Insulin‐like growth factor‐1 (IGF‐1) is anabolic for muscle by enhancing the rate of mRNA translation via activation of AKT and subsequent activation of the mammalian target of rapamycin complex 1 (mTOR), thereby increasing cellular protein production. IGF‐1 is also anabolic for bone, but whether the mTOR pathway plays a role in the rate of bone matrix protein production by osteoblasts is unknown. We hypothesized that anabolic stimuli such as mechanical loading and IGF‐1 stimulate protein synthesis in osteoblasts via activation of the AKT‐mTOR pathway. MC3T3‐E1 osteoblasts were either or not subjected for 1 h to mechanical loading by pulsating fluid flow (PFF) or treated with or without human recombinant IGF‐1 (1‐100 ng/ml) for 0.5‐6 h, to determine phosphorylation of AKT and p70S6K (downstream of mTOR) by western blot. After 4 days of culture with or without the mTOR inhibitor rapamycin, total protein, DNA, and gene expression were quantified. IGF‐1 (100 ng/ml) reduced IGF‐1 gene expression, while PFF enhanced IGF‐1 expression. IGF‐1 did not affect collagen‐I gene expression. IGF‐1 dose‐dependently enhanced AKT and p70S6K phosphorylation at 2 and 6 h. PFF enhanced phosphorylation of AKT and p70S6K already within 1 h. Both IGF‐1 and PFF enhanced total protein per cell by ∼30%, but not in the presence of rapamycin. Our results show that IGF‐1 and PFF activate mTOR, thereby stimulating the rate of mRNA translation in osteoblasts. The known anabolic effect of mechanical loading and IGF‐1 on bone may thus be partly explained by mTOR‐mediated enhanced protein synthesis in osteoblasts. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-27T17:46:21.937504-05:
      DOI: 10.1002/jcp.25228
  • Common and Rare Variants Associated with Alzheimer's Disease
    • Authors: Hany Marei; Asmaa Althani, Mohamed El Zowalaty, Mohammad A. Albanna, Carlo Cenciarelli, Tengfei Wang, Thomas Caceci
      Abstract: Alzheimer's disease (AD) is one of the most devastating disorder. Despite the continuing increase of its incidence among aging population, no effective cure has been developed mainly due to difficulties in early diagnosis of the disease before damaging of the brain, and the failure to explore its complex underlying molecular mechanisms. Recent technological advances in genome‐wide association studies (GWAS) and high throughput next generation whole genome, and exome sequencing had deciphered many of AD‐related loci, and discovered single nucleotide polymorphisms (SNPs) that are associated with altered AD molecular pathways. Highlighting altered molecular pathways linked to AD pathogenesis is crucial to identify novel diagnostic and therapeutic AD targets. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-23T17:57:04.715116-05:
      DOI: 10.1002/jcp.25225
  • Coordinated Dynamics of RNA Splicing Speckles in the Nucleus
    • Authors: Qiao Zhang; Krishna Kota Prasad, Samer G. Alam, Jeffrey A. Nickerson, Richard B. Dickinson, Tanmay P. Lele
      Abstract: Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle(Hübner et al., 2013). Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. The movement is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved in a coordinated way along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP‐dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub‐speckles from distal sites of RNA processing to larger splicing speckles a directed ATP‐driven mechanism through interchromatin spaces. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-23T17:56:42.850945-05:
      DOI: 10.1002/jcp.25224
  • TNF‐Alpha/TNFR2 Regulatory Axis Stimulates EphB2‐Mediated
           Neuroregeneration Via Activation of NF‐KappaB
    • Authors: Paul D. Pozniak; Armine Darbinyan, Kamel Khalili
      Abstract: HIV‐1 infected individuals are at high risk of developing HIV‐associated neurocognitive disorders (HAND) as HIV infection leads to neuronal injury and synaptic loss in the central nervous system (CNS). The neurotoxic effects of HIV‐1 are primarily a result of viral replication leading to the production of inflammatory chemokines and cytokines, including TNF‐alpha. Given an important role of TNF‐alpha in regulating synaptic plasticity, we investigated the effects of TNF‐alpha on the development of neuronal processes after mechanical injury, and we showed that TNF‐alpha treatment stimulates the regrowth of neuronal processes. To investigate transcriptional effects of TNF‐alpha on synaptic plasticity, we analyzed both human neurosphere and isolated neuronal cultures for the regulation of genes central to synaptic alterations during learning and memory. TNF‐alpha treatment upregulated Ephrin receptor B2 (EphB2), which is strongly involved in dendritic arborization and synaptic integrity. TNF‐alpha strongly activates the NF‐kappaB pathway, therefore, we propose that TNF‐alpha‐induced neurite regrowth occurs primarily through EphB2 signaling via stimulation of NF‐kappaB. EphB2 promoter activity increased with TNF‐alpha treatment and overexpression of NF‐kappaB. Direct binding of NF‐kappaB to the EphB2 promoter occurred in the ChIP assay, and site‐directed mutagenesis identified binding sites involved in TNF‐alpha‐induced EphB2 activation. TNF‐alpha induction of EphB2 was determined to occur specifically through TNF‐alpha receptor 2 (TNFR2) activation in human primary fetal neurons. Our observations provide a new avenue for the investigation on the impact of TNF‐alpha in the context of HIV‐1 neuronal cell damage as well as providing a potential therapeutic target in TNFR2 activation of EphB2. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-22T17:41:55.486775-05:
      DOI: 10.1002/jcp.25219
  • Different Blood‐Borne Human Osteoclast Precursors Respond in
           Distinct Ways to IL‐17A
    • Authors: Sara Sprangers; Ton Schoenmaker, Yixuan Cao, Vincent Everts, Teun J. de Vries
      Abstract: Osteoclasts are bone‐degrading cells that are formed through fusion of their monocytic precursors. Three distinct subsets of monocytes have been identified in human peripheral blood: classical, intermediate and non‐classical monocytes. They are known to play different roles in physiology and pathology, but their capacity to differentiate into osteoclasts and whether inflammatory cytokines influence this differentiation is unknown. We hypothesized that classical, intermediate and non‐classical monocytes generate functionally different osteoclasts and that they respond in different ways to the inflammatory cytokine interleukin‐17A (IL‐17A). To investigate this, the different monocyte subsets were isolated from human peripheral blood and osteoclastogenesis was induced with the cytokines M‐CSF and RANKL, with or without IL‐17A. We found that all subsets are able to differentiate into osteoclasts in vitro, and that both osteoclastogenesis and subsequent bone resorption was distinctly affected by IL‐17A. Osteoclastogenesis and bone resorption by osteoclasts derived from classical monocytes remained unaffected by IL‐17A, while osteoclast formation from intermediate monocytes was inhibited by the cytokine. Surprisingly, bone resorption by osteoclasts derived from intermediate monocytes remained at similar levels as control cultures, indicating an increased bone resorbing activity by these osteoclasts. Limited numbers of osteoclasts were formed from non‐classical monocytes on bone and no bone resorption was detected, which suggest that these cells belong to a cell lineage different from the osteoclast. By providing more insight into osteoclast formation from human blood monocytes, this study contributes to the possible targeting of specific osteoclast precursors as a therapeutic approach for diseases associated with inflammatory bone loss. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-22T17:41:45.12399-05:0
      DOI: 10.1002/jcp.25220
  • Effects of Sex Hormones on Ocular Surface Epithelia: Lessons Learned from
           Polycystic Ovary Syndrome
    • Abstract: Polycystic ovary syndrome (PCOS) is the most common endocrine abnormality in women of reproductive age. Although its clinical consequences have been known for a long time to extend beyond the reproductive system, with type‐2 diabetes and obesity being the most common, the involvement of the ocular surface in PCOS has been described only more recently. The ocular surface is a morphofunctional unit comprising eyelid margin, tear film, cornea and conjunctiva. Increasing evidence indicates that these structures are under a sex hormone control and relevant diseases such as ocular allergy and dry eye are often caused by alterations in circulating or local steroid hormones levels. Novel treatments targeting sex hormone receptors on ocular surface epithelial cells are also being developed. In this review we aim to describe the current knowledge on the effects of sex hormones at the ocular surface, with a special focus on the effects of androgen imbalance in PCOS. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-22T17:41:41.611322-05:
      DOI: 10.1002/jcp.25221
  • Mitochondrial Dysfunction and Ca2+ Overload Contributes to Hesperidin
           Induced Paraptosis in Hepatoblastoma Cells, HepG2
    • Abstract: Paraptosis is a programmed cell death which is morphologically and biochemically different from apoptosis. In this study, we have investigated the role of Ca2+ in hesperidin‐induced paraptotic cell death in HepG2 cells. Increase in mitochondrial Ca2+ level was observed in hesperidin treated HepG2 cells but not in normal liver cancer cells. Inhibition of inositol‐1,4,5‐triphosphate receptor (IP3) and ryanodine receptor also block the mitochondrial Ca2+ accumulation suggesting that the release of Ca2+ from the endoplasmic reticulum (ER) may probably lead to the increase in mitochondrial Ca2+ level. Pretreatment with ruthenium red (RuRed), a Ca2+ uniporter inhibitor inhibited the hesperidin‐induced mitochondrial Ca2+ overload, swelling of mitochondria and cell death in HepG2 cells. It has also been demonstrated that mitochondrial Ca2+ influxes act upstream of ROS and mitochondrial superoxide production. The increased ROS production further leads to mitochondrial membrane loss in hesperidin treated HepG2 cells. Taken together our results show that IP3R and ryanodine receptor mediated release of Ca2+ from the ER and its subsequent influx through the uniporter into mitochondria contributes to hesperidin‐induced paraptosis in HepG2 cells. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-22T17:41:30.658506-05:
      DOI: 10.1002/jcp.25222
  • Modulation of 17β‐estradiol signaling on cellular proliferation
           by caveolin‐2
    • Authors: Pierangela Totta; Fabio Gionfra, Claudia Busonero, Filippo Acconcia
      Abstract: The sex hormone 17β‐estradiol (E2) exerts pleiotropic effects by binding to the ligand‐activated transcription factor estrogen receptor α (ERα). The E2:ERα complex regulates several physiological processes, including cell survival and proliferation, through transcriptional effects [i.e., estrogen responsive element (ERE)‐based gene transcription] and non‐transcriptional membrane‐initiated effects (i.e., the activation of extra‐nuclear signaling cascades), which derive from the activation of the pool of ERα that is localized to plasma membrane caveolae. Caveolae are ω‐shaped membrane sub‐domains that are composed of scaffold proteins named caveolins (i.e., caveolin‐1, caveolin‐2, and caveolin‐3). While caveolin‐3 is exclusively expressed in muscles, caveolin‐1 and caveolin‐2 are co‐expressed in all human tissues. From a functional point of view, caveolin‐2 can operate both dependently on and independently of caveolin‐1, which is the main coat component of caveolae. Interestingly, while a functional interplay between caveolin‐1 and ERα has been reported in the control of E2‐induced physiological effects, the role of caveolin‐2 in E2:ERα signaling within the cell remains poorly understood. This study shows that siRNA‐mediated caveolin‐2 depletion in breast ductal carcinoma cells (MCF‐7) reduces E2‐induced ERα phosphorylation at serine residue 118 (S118), controls intracellular receptor levels, precludes ERα‐mediated extra‐nuclear activation of signaling pathways, reduces ERα transcriptional activity, and prevents cellular proliferation. Meanwhile, the impact of caveolin‐1 depletion on ERα signaling in MCF‐7 cells is shown to be similar to that elicited by siRNA‐mediated caveolin‐2 depletion. Altogether, these data demonstrate that caveolin‐2 expression is necessary for the control of E2‐dependent cellular proliferation. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-19T09:22:47.26957-05:0
      DOI: 10.1002/jcp.25218
  • Non‐hematopoietic β‐arrestin1 confers protection against
           experimental colitis
    • Authors: Taehyung Lee; Eunhee Lee, David Arrollo, Peter C. Lucas, Narayanan Parameswaran
      Abstract: β‐Arrestins are multifunctional scaffolding proteins that modulate G protein‐coupled receptor (GPCR)‐dependent and ‐independent cell signaling pathways in various types of cells. We recently demonstrated that β‐arrestin1 (β‐arr1) deficiency strikingly attenuates dextran sodium sulfate (DSS)‐induced colitis in mice. Since DSS‐induced colitis is in part dependent on gut epithelial injury, we examined the role of β‐arr1 in intestinal epithelial cells (IECs) using a colon epithelial cell line, SW480 cells. Surprisingly, we found that knockdown of β‐arr1 in SW480 cells enhanced epithelial cell death via a caspase‐3‐dependent process. To understand the in vivo relevance and potential cell type‐specific role of β‐arr1 in colitis development, we generated bone marrow chimeras with β‐arr1 deficiency in either the hematopoietic or non‐hematopoietic compartment. Reconstituted chimeric mice were then subjected to DSS‐induced colitis. Similar to our previous findings, β‐arr1 deficiency in the hematopoietic compartment protected mice from DSS‐induced colitis. However, consistent with the role of β‐arr1 in epithelial apoptosis in vitro, non‐hematopoietic β‐arr1 deficiency led to an exacerbated colitis phenotype. To further understand signaling mechanisms, we examined the effect of β‐arr1 on TNF‐α‐mediated NFκB and MAPK pathways. Our results demonstrate that β‐arr1 has a critical role in modulating ERK, JNK and p38 MAPK pathways mediated by TNF‐α in IECs. Together, our results show that β‐arr1‐dependent signaling in hematopoietic and non‐hematopoietic cells differentially regulates colitis pathogenesis and further demonstrates that β‐arr1 in epithelial cells inhibits TNF‐α‐induced cell death pathways. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-19T08:42:37.610904-05:
      DOI: 10.1002/jcp.25216
  • Effect of docosahexaenoic acid on cell cycle pathways in breast cell lines
           with different transformation degree
    • Authors: Tania Rescigno; Anna Capasso, Mario Felice Tecce
      Abstract: n‐3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), abundant in fish, have been shown to affect development and progression of some types of cancer, including breast cancer. The aim of our study was to further analyze and clarify the effects of these nutrients on the molecular mechanisms underlying breast cancer. Following treatments with DHA we examined cell viability, death, cell cycle and some molecular effects in breast cell lines with different transformation, phenotypic and biochemical characteristics (MCF‐10A, MCF‐7, SK‐BR‐3, ZR‐75‐1). These investigations showed that DHA is able to affect cell viability, proliferation and cell cycle progression in a different way in each assayed breast cell line. The activation of ERK1/2 and STAT3 pathways and the expression and/or activation of molecules involved in cell cycle regulation such as p21Waf1/Cip1 and p53, are very differently regulated by DHA treatments in each cell model. DHA selectively: (i) arrests non tumoral MCF‐10A breast cells in G0/G1 cycle phase, activating p21Waf1/Cip1 and p53, (ii) induces to death highly transformed breast cells SK‐BR‐3, reducing ERK1/2 and STAT3 phosphorylation and (iii) only slightly affects each analyzed process in MCF‐7 breast cell line with transformation degree lower than SK‐BR‐3 cells. These findings suggest a more relevant inhibitory role of DHA within early development and late progression of breast cancer cell transformation and a variable effect in the other phases, depending on individual molecular properties and degree of malignancy of each clinical case. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-19T08:42:00.495249-05:
      DOI: 10.1002/jcp.25217
  • Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing
           Mitophagy through PINK1/Parkin Signal Pathway in H9c2 Cells
    • Abstract: Acetylcholine (ACh) protected against cardiac injury via promoting autophagy and mitochondrial biogenesis, however, the involvement of mitophagy in ACh‐elicited cardioprotection remains unknown. In the present study, H9c2 cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) and ACh treatment during reoxygenation. Mitophagy markers PTEN‐induced kinase 1 (PINK1) and Parkin translocation were examined using western blot and confocal fluorescence microscopy. Mitochondrial membrane potential and reactive oxygen species (ROS) were also detected with fluorescence staining. We found that H/R‐treated cells exhibited reduced levels of PINK1 and Parkin in mitochondria, accompanied with decreased autophagy flux (reduced LC3‐II/LC3‐I and increased p62). Conversely, ACh increased PINK1 and Parkin translocation to mitochondria and enhanced autophagy proteins. Confocal imaging of Parkin and MitoTracker Green‐labeled mitochondria further confirmed ACh‐induced mitochondrial translocation of Parkin, which was reversed by methoctramine and M2 receptor siRNA, suggesting ACh could induce mitophagy by M2 receptor after H/R. Mitophagy inhibitor 3‐methaladenine abolished ACh‐induced mitoprotection, manifesting as aggravated mitochondrial morphology disruption, ATP and membrane potential depletion, increased ROS overproduction and apoptosis. Furthermore, PINK1/Parkin siRNA attenuated the protective effects of ACh against ATP loss and oxidative stress due to mitochondrial‐dependent injury. Taken together, ACh promoted mitochondrial translocation of PINK1/Parkin to stimulate cytoprotective mitophagy via M2 receptor, which may provide beneficial targets in the preservation of cardiac homeostasis against H/R injury. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-14T10:26:29.155661-05:
      DOI: 10.1002/jcp.25215
  • Cathepsin K Deficiency Suppresses Disuse‐Induced Bone Loss
    • Authors: Shuichi Moriya; Yayoi Izu, Smriti Arayal, Makiri Kawasaki, Koki Hata, Chantida Pawaputanon Na Mahasarakhahm, Yuichi Izumi, Paul Saftig, Kazuo Kaneko, Masaki Noda, Yoichi Ezura
      Abstract: Unloading induces bone loss and causes disuse osteoporosis. However, the mechanism underlying disuse osteoporosis is still incompletely understood. Here we examined the effects of cathepsin K (CatK) deficiency on disuse osteoporosis induced by using sciatic neurectomy (Nx) model. After four weeks of surgery, Cat‐K KO and WT mice were sacrificed and subjected to analyses. For cancellous bone rich region, Nx reduced the bone mineral density (BMD) compared to the BMD in the sham operated side in wild type mice. In contrast, CatK deficiency suppressed such Nx‐induced reduction of BMD in cancellous bone. Nx also reduced BMD in the mid shaft cortical bone compared to the BMD in the corresponding region on the sham operated side in wild type mice. In contrast, CatK deficiency suppressed such Nx‐induced reduction of BMD in the mid shaft cortical bone. Bone volume (BV/TV) was reduced by Nx in WT mice. In contrast, Cat‐K deficiency suppressed such reduction in bone volume. Interestingly, CatK deficiency suppressed osteoclast number and osteoclast surface in the Nx side compared to sham side. When bone marrow cells obtained from Nx side femur of CatK‐KO mice were cultured, the levels of the calcified area in culture were increased. Further examination of gene expression indicated that Nx suppressed the expression of genes encoding osteoblast‐phenotype‐related molecules such as Runx2 and alkaline phosphatase in WT mice. In contrast, CatK deficiency suppressed such reduction. These data indicate that CatK is involved in the disuse‐induced bone mass reduction. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-13T11:04:53.470829-05:
      DOI: 10.1002/jcp.25214
  • Body Mass Index and Treatment Outcomes in Metastatic Breast Cancer
           Patients Treated with Eribulin
    • Abstract: Background Eribulin has shown survival advantage and manageable toxicity in heavily pre‐treated metastatic breast cancer (mBC). We assessed whether body mass index (BMI) impacts treatment outcomes in 101 patients treated with eribulin at 6 Italian Oncologic Centers. Methods BMI was addressed as a categorical variable (18.5‐24.9 vs at least 25). Clinical benefit rate (CBR) was assessed overall and in subgroups defined by BMI, line of therapy (LOT) and hormone receptor (HR) status. Analysis of CBR by LOT and HR status were further stratified by BMI. Survival curves were compared using the Kaplan‐Meier method and log‐rank test. Predictors of survival were tested in Cox models. Results Patients treated with eribulin as third line showed greater CBR when their BMI was in the lowest category (77.8 vs 58.1%, p = 0.03). Median progression free survival (PFS) and overall survival (OS) in normal and overweight patients were 4 (95% CI, 3‐5) vs 3 (2.1‐4) months, p = 0.02 and 13 (11‐15) vs 12 (6‐18) months, p = 0.96, respectively. Median PFS and OS in estrogen receptor (ER) positive and negative tumours were 4 (3‐5) vs 3 (2‐4) months, p = 0.005 and 14 (10‐18) vs 7 (4‐10), p = 0.02, respectively. In multivariate analyses, BMI impacted PFS at a nearly significant extent (p = 0.05), while ER expression significantly affected PFS and OS (p = 0.01 and 0.02, respectively). No relevant findings emerged concerning toxicity. Conclusions We found evidence of greater efficacy of eribulin in leaner mBC patients, particularly if given as third line and in ER positive tumours. Further studies are warranted to confirm our findings. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-09T06:41:17.412361-05:
      DOI: 10.1002/jcp.25213
  • Calcineurin‐NFAT Signaling Controls Somatic Cell Reprogramming in a
           Stage‐Dependent Manner
    • Authors: Ming Sun; Bing Liao, Yu Tao, Hao Chen, Feng Xiao, Junjie Gu, Shaorong Gao, Ying Jin
      Abstract: Calcineurin‐NFAT signaling is critical for early lineage specification of mouse embryonic stem cells and early embryos. However, its roles in somatic cell reprogramming remain unknown. Here, we report that calcineurin‐NFAT signaling has a dynamic activity and plays diverse roles at different stages of reprogramming. At the early stage, calcineurin‐NFAT signaling is transiently activated and its activation is required for successful reprogramming. However, at the late stage of reprogramming, activation of calcineurin‐NFAT signaling becomes a barrier for reprogramming and its inactivation is critical for successful induction of pluripotency. Mechanistically, calcineurin‐NFAT signaling contributes to the reprogramming through regulating multiple early events during reprogramming, including mesenchymal to epithelial transition (MET), cell adhesion and emergence of SSEA1+ intermediate cells. Collectively, this study reveals for the first time the important roles of calcineurin‐NFAT signaling during somatic cell reprogramming and provides new insights into the molecular regulation of reprogramming. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-08T08:50:44.374931-05:
      DOI: 10.1002/jcp.25212
  • Appl1 and Appl2 are Expendable for Mouse Development but are Essential for
           HGF‐induced Akt Activation and Migration in Mouse Embryonic
    • Authors: Yinfei Tan; Xiaoban Xin, Francis J. Coffey, David L. Wiest, Lily Q. Dong, Joseph R. Testa
      Abstract: Although Appl1 and Appl2 have been implicated in multiple cellular activities, we and others have found that Appl1 is dispensable for mouse embryonic development, suggesting that Appl2 can substitute for Appl1 during development. To address this possibility, we generated conditionally targeted Appl2 mice. We found that ubiquitous Appl2 knockout (Appl2−/−) mice, much like Appl1−/− mice, are viable and grow normally to adulthood. Intriguingly, when Appl1−/− mice were crossed with Appl2−/− mice, we found that homozygous Appl1;Appl2 double knockout (DKO) animals are also viable and grossly normal with regard to reproductive potential and postnatal growth. Appl2‐null and DKO mice were found to exhibit altered red blood cell physiology, with erythrocytes from these mice generally being larger and having a more irregular shape than erythrocytes from wild type mice. Although Appl1/2 proteins have been previously shown to have a very strong interaction with phosphatidylinositol‐3 kinase (Pi3k) in thymic T cells, Pi3k‐Akt signaling and cellular differentiation was unaltered in thymocytes from Appl1;Appl2 (DKO) mice. However, Appl1/2‐null mouse embryonic fibroblasts exhibited defects in HGF‐induced Akt activation, migration, and invasion. Taken together, these data suggest that Appl1 and Appl2 are required for robust HGF cell signaling but are dispensable for embryonic development and reproduction. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-07T10:43:17.393284-05:
      DOI: 10.1002/jcp.25211
  • The Nox1/4 dual inhibitor GKT137831 or Nox4 knockdown inhibits
           Angiotensin‐II‐induced adult mouse cardiac fibroblast
           proliferation and migration. AT1 physically associates with Nox4
    • Authors: Naveen K. Somanna; Anthony J. Valente, Maike Krenz, William P. Fay, Patrice Delafontaine, Bysani Chandrasekar
      Abstract: Both oxidative stress and inflammation contribute to chronic hypertension‐induced myocardial fibrosis and adverse cardiac remodeling. Here we investigated whether angiotensin (Ang)‐II‐induced fibroblast proliferation and migration are NADPH oxidase (Nox) 4/ROS and IL‐18 dependent. Our results show that the potent induction of mouse cardiac fibroblast (CF) proliferation and migration by Ang‐II is markedly attenuated by Nox4 knockdown and the Nox inhibitor DPI. Further, Nox4 knockdown and DPI pre‐treatment attenuate Ang‐II‐induced IL‐18, IL‐18Rα and collagen expression, and MMP9 activation. While neutralization of IL‐18 blunted Ang‐II‐induced CF proliferation and migration, knockdown of MMP9 attenuated CF migration. The antioxidant NAC and the cell‐permeable SOD mimetics Tempol, MnTBAP, and MnTMPyP attenuated oxidative stress and inhibit CF proliferation and migration. The Nox1/Nox4 dual inhibitor GKT137831 also blunted Ang‐II‐induced H2O2 production and CF proliferation and migration. Further, AT1 binds Nox4, and Ang‐II enhanced their physical association. Notably, GKT137831 attnuated the AT1/Nox4 interaction. These results indicate that Ang‐II induces CF proliferation and migration in part via Nox4/ROS‐dependent IL‐18 induction and MMP9 activation, and may involve AT1/Nox4 physical association. Thus, either (i) neutralizing IL‐18, (ii) blocking AT1/Nox4 interaction or (iii) use of the Nox1/Nox4 inhibitor GKT137831 may have therapeutic potential in chronic hypertension‐induced adverse cardiac remodeling. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-07T10:39:31.576844-05:
      DOI: 10.1002/jcp.25210
  • The IP3R Binding Protein Released with Inositol 1,4,5‐Trisphosphate
           Is Expressed in Rodent Reproductive Tissue and Spermatozoa
    • Authors: Heike Borth; Nele Weber, Dorke Meyer, Andrea Wartenberg, Elisabeth Arlt, Susanna Zierler, Andreas Breit, Gunther Wennemuth, Thomas Gudermann, Ingrid Boekhoff
      Abstract: Besides its capacity to inhibit the 1,4,5‐trisphosphate (IP3) receptor, the regulatory protein IRBIT (IP3 receptor‐binding protein released with IP3) is also able to control the activity of numerous ion channels and electrolyte transporters and thereby creates an optimal electrolyte composition of various biological fluids. Since a reliable execution of spermatogenesis and sperm maturation critically depends on the establishment of an adequate microenvironment, the expression of IRBIT in male reproductive tissue was examined using immunohistochemical approaches combined with biochemical fractionation methods. The present study documents that IRBIT is expressed in Leydig and Sertoli cells. In addition, pronounced IRBIT expression was detected in sperm precursors during early stages of spermatogenesis as well as in spermatozoa. Analyzing tissue sections of rodent epididymides, IRBIT was found to co‐localize with the proton pumping V‐ATPase and the cystic fibrosis transmembrane conductance regulator (CFTR) at the apical surface of narrow and clear cells. A similar co‐localization of IRBIT with CFTR was also observed for Sertoli cells and developing germ cells. Remarkably, assaying caudal sperm in immunogold electron microscopy, IRBIT was found to localize to the acrosomal cap and the flagellum as well as to the sperm nucleus; moreover, a prominent oligomerization was observed for spermatozoa. The pronounced occurrence of IRBIT in the male reproductive system and mature spermatozoa indicates a potential role for IRBIT in establishing the essential luminal environment for a faithful execution of spermatogenesis and epididymal sperm maturation, and suggest a participation of IRBIT during maturations steps after ejaculation and/or the final fertilization process. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-06T06:35:29.112421-05:
      DOI: 10.1002/jcp.25209
  • Reduced dentin matrix protein expression in Camurati‐Engelmann
           disease transgenic mouse model
    • Authors: Angela Gullard; Christina M. Croney, Xiangwei Wu, Olga Mamaeva, Philip Sohn, Xu Cao, Mary MacDougall
      Abstract: Overexpression of transforming growth factor‐beta 1 (TGF‐β1) has been shown to lead to mineralization defects in both the enamel and dentin layers of teeth. A TGFB1 point mutation (H222D), derived from published cases of Camurati‐Engelmann disease (CED), has been shown to constitutively activate TGF‐β1, leading to excess bone matrix production. Although CED has been well documented in clinical case reports, there are no published studies on the effect of CED on the dentition. The objective of this study was to determine the dental manifestations of hyperactivated TGF‐β1 signaling using an established mouse model of CED‐derived TGF‐β1 mutation. Murine dental tissues were studied via radiography, micro‐CT, immunohistochemistry, and qRT‐PCR. Results showed that initial decreased dental mineralized tissue density is resolved Proliferation assays of incisor pulp and alveolar bone cell cultures revealed that cells from transgenic animals displayed a reduced rate of growth compared to alveolar bone cultures from wild‐type mice. TGF‐β family gene expression analysis indicated significant fold changes in the expression of Alpl, Bmp2‐5, Col‐1, ‐2,‐4 and ‐6, Fgf, Mmp, Runx2, Tgfb3, Tfgbr3, and Vdr genes. Assessment of SIBLINGs reveals downregulation of Ibsp, Dmp1, Dspp, Mepe, and Spp1, as well as reduced staining for BMP‐2 and VDR in mesenchymal‐derived pulp tissue in CED animals. Treatment of dental pulp cells with recombinant human TGF‐β1 resulted in increased SIBLING gene expression. Conclusions: Our results provide in vivo evidence suggesting that TFG‐β1 mediates expression of important dentin extracellular matrix components secreted by dental pulp, and when unbalanced, may contribute to abnormal dentin disorders. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-01T11:13:46.627626-05:
      DOI: 10.1002/jcp.25207
  • Smooth muscle cell differentiation: Model systems, regulatory mechanisms,
           and vascular diseases
    • Abstract: Smooth muscle cell (SMC) differentiation is an important process during vascular development. The highly differentiated mature SMCs play critical roles in maintaining structural and functional integrity of blood vessels. However, SMCs are not terminally differentiated, and their phenotype can be modulated between contractile and proliferative states in response to various environmental conditions. Alterations in SMC phenotype contribute to a number of major cardiovascular diseases such as atherosclerosis, hypertension and restenosis following angioplasty, etc. The goal of this review is to provide a brief overview of the recent advancements in our understanding of SMC differentiation and the development of in vitro SMC differentiation models, with a particular emphasis on examination of molecular mechanisms involved in the regulation of SMC differentiation and phenotypic modulation. This article is protected by copyright. All rights reserved
      PubDate: 2015-10-01T11:13:24.410441-05:
      DOI: 10.1002/jcp.25208
  • Involvement of Notch‐1 in Resistance to Regorafenib in Colon Cancer
    • Authors: Giovanna Mirone; Stefania Perna, Arvind Shukla, Gabriella Marfe
      Abstract: Regorafenib, an oral small‐molecule small‐molecule multi kinase inhibitor, is able to block Vascular Endothelial Growth Factor Receptors (VEGFR‐1, 2 and 3), Platelet‐Derived Growth Factor Receptors (PDGF), Fibroblast Growth Factor (FGF) receptor 1, Raf, TIE‐2, and the kinases KIT, RET, and BRAF. Different studies have displayed its antitumor activity in several cancer models (both in vitro and in vivo), particularly in colorectal and gastrointestinal stromal cancers. The mechanism of resistance to regorafenib is largely unknown. In our investigation, we have generated regorafenib‐resistant SW480 cells (Reg‐R‐SW480 cells) by culturing such cells with increasing concentration of regorafenib. Examination of intracellular signaling found that Akt signaling was activated in Reg‐R‐SW480 cells but not in wild‐type SW480 cells, after regorafenib treatment as measured byWestern Blot. The Notch pathway is a fundamental signaling system in the development and homeostasis of tissues since it regulates different cellular process such as proliferation, differentiation, and apoptosis and it can be a potential driver of resistance to a wide array of targeted therapies. In this study, we found that Notch‐1 was significantly up‐regulated in resistant tumor cells as well as HES1 and HEY. Addionally, inhibition of Notch‐1 in resistant cells partially restored sensitivity to regorafenib treatment in vitro. Collectively, these data suggest a key role of Notch‐1 in mediating the resistant effects of regorafenib in colorectal cancer cells, and also provide a rationale to improve the therapeutic efficacy of regorafenib. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-30T05:48:18.10011-05:0
      DOI: 10.1002/jcp.25206
  • Genes in the GABA Pathway Increase in the Lateral Thalamus of Sprague
           Dawley Rats During the Proestrus/Estrus Phase
    • Authors: Mikhail Umorin; Crystal Stinson, Larry L. Bellinger, Phillip R. Kramer
      Abstract: Pain can vary over the estrous cycle as a result of changes in estradiol concentration but the mechanism causing this variation is unclear. Because the thalamus is important in pain control, gene expression in the lateral thalamus (ventral posteromedial, ventral posterolateral, reticular thalamic nuclei) was screened at different phases of the estrous cycle. Gene expression changes in Sprague‐Dawley rats were further analyzed by real‐time PCR and ELISA and plasma estradiol levels were measured by RIAs at different phases of the estrous cycle. Our results indicated that both the RNA and protein expression of glutamate decarboxylase 1 and 2 (GAD1, GAD2), GABA(A) receptor‐associated protein like 1 (GABARAPL1) and vesicular GABA transporter (VGAT) significantly increased in the lateral thalamus when plasma estradiol levels were elevated. Estradiol levels were elevated during the proestrus and estrus phases of the estrous cycle. Estrogen receptor α (ERα) was observed to be co‐localized in thalamic cells and thalamic infusion of an ERα antagonist significantly reduced GAD1 and VGAT transcript. GAD1, GAD2 GABARAPL1 and VGAT have been shown to effect neuronal responses suggesting that modulation of pain during the estrous cycle can be dependent, in part, through estradiol induced changes in thalamic gene expression. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-30T02:18:29.573669-05:
      DOI: 10.1002/jcp.25198
  • Evidences of Biological Functions of Biliverdin Reductase A in the Bovine
    • Abstract: Epididymal sperm binding protein 1 (ELSPBP1) is secreted by the epididymal epithelium via epididymosomes and is specifically transferred to dead spermatozoa during epididymal transit. We identified biliverdin reductase A (BLVRA) as a partner of ELSPBP1 by immunoprecipitation followed by tandem mass spectrometry. Pull down assays showed that these two proteins interact in the presence of zinc ions. The BLVRA enzyme is known to convert biliverdin to bilirubin, both of which possess antioxidant activity. Assessment by real‐time RT‐PCR showed that BLVRA is highly expressed in the caput and the corpus epididymis, but is expressed at lower levels in the testis and the cauda epididymis. It is primarily found in the soluble fraction of the caput epididymal fluid, is barely detectable in the cauda fluid, and is detectable to a lesser extent in the epididymosome fraction of both caput and cauda fluids. Immunocytometry on epididymal sperm showed that BLVRA is found on all sperm recovered from the caput region, whereas it is undetectable on cauda sperm. biliverdin and bilirubin are found in higher concentrations in the caput epididymal fluid, as measured by mass spectrometry. Lipid peroxidation was limited by 1 μM of biliverdin, but not bilirubin when caput spermatozoa were challenged with 500 μM H2O2. Since immature spermatozoa are a source of reactive oxygen species, BLVRA may be involved in the protection of maturing spermatozoa. It is also plausible that BLVRA is implicated in haemic protein catabolism in the epididymal luminal environment. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-30T02:18:00.222198-05:
      DOI: 10.1002/jcp.25200
  • Epigenetic Modulation in Periodontitis: Interaction of Adiponectin and
           JMJD3‐IRF4 Axis in Macrophages
    • Authors: Dongying Xuan; Qianqian Han, Qisheng Tu, Lan Zhang, Liming Yu, Dana Murry, Tianchi Tu, Jane Lian, Gary S. Stein, Jincai Zhang, Jake Chen
      Abstract: Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. JMJD3, a JmjC family histone demethylase necessary for M2 polarization is also required for effective induction of multiple M1 genes by lipopolysaccharide (LPS). However, the effects of JMJD3 to inflammation in the context of obesity remains unknown. To address this deficiency, we firstly examined the expression of JMJD3 in macrophage isolated from bone marrow and adipose tissue of diet induced obesity (DIO) mice. The results indicated that JMJD3 was down‐regulated in obesity. Adiponectin (APN), a factor secreted by adipose tissue which is down‐regulated in obesity, functions to switch macrophage polarization from M1 to M2 (Jenke et al., 2014), thereby attenuating chronic inflammation. Intriguingly, our results indicated that APN contributed to JMJD3 up‐regulation, reduced macrophage infiltration in obese adipose tissue, and abolished the up‐regulation of JMJD3 in peritoneal macrophages isolated from DIO mice when challenged with Porphyromonas gingivalis LPS (pg.lps). To elucidate the interaction of APN and JMJD3 involved in macrophage transformation in the context of inflammation, we designed the loss and gain‐function experiments of APN in vivo with APN‐/‐ mice with experimental periodontitis and in vitro with macrophage isolated from APN ‐/‐ mice. For the first time, we found that APN can help to reduce periodontitis‐related bone loss, modulate JMJD3 and IRF4 expression and macrophage infiltration. Therefore, it can be inferred that APN may contribute to anti‐inflammation macrophage polarization by regulating JMJD3 expression, which provides a basis for macrophage‐centered epigenetic therapeutic strategies. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-24T06:51:20.993116-05:
      DOI: 10.1002/jcp.25201
  • Definitive Hematopoietic Multipotent Progenitor Cells Are Transiently
           Generated from Hemogenic Endothelial Cells in Human Pluripotent Stem Cells
    • Authors: Hao Bai; Yanfeng Liu, Yinliang Xie, Dixie L. Hoyle, Robert A. Brodsky, Linzhao Cheng, Tao Cheng, Zack Z. Wang
      Abstract: Generation of fully functional hematopoietic multipotent progenitor (MPP) cells from human pluripotent stem cells (hPSCs) has a great therapeutic potential to provide an unlimited cell source for treatment of hematological disorders. We previously demonstrated that CD34+CD31+CD144+ population derived from hPSCs contain hemato‐endothelial progenitors (HEPs) that give rise to hematopoietic and endothelial cells. Here, we report a differentiation system to generate definitive hematopoietic MPP cells from HEPs via endothelial monolayer. In the presence of angiogenic factors, HEPs formed an endothelial monolayer, from which hematopoietic clusters emerged through the process of endothelial‐to‐hematopoietic transition (EHT). EHT was significantly enhanced by hematopoietic growth factors. The definitive MPP cells generated from endothelial monolayer were capable of forming multilineage hematopoietic colonies, giving rise to T lymphoid cells, and differentiating into enucleated erythrocytes. Emergence of hematopoietic cells from endothelial monolayer occurred transiently. Hematopoietic potential was lost during prolonged culture of HEPs in endothelial growth conditions. Our study demonstrated that CD34+CD31+CD144+ HEPs gave rise to hematopoietic MPP cells via hemogenic endothelial cells that exist transiently. The established differentiation system provides a platform for future investigation of regulatory factors involved in de novo generation of hematopoietic MPP cells and their applications in transplantation. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-23T04:29:09.590376-05:
      DOI: 10.1002/jcp.25199
  • Downhill Running Excessive Training Inhibits Hypertrophy in Mice Skeletal
           Muscles with Different Fiber Type Composition
    • Abstract: The aim of this study was to verify the effects of running overtraining protocols performed in downhill, uphill and without inclination on the proteins related to hypertrophy signaling pathway in extensor digitorum longus (EDL) and soleus of C57BL/6 mice. We also performed histological and stereological analyses. Rodents were divided into control (CT; sedentary mice), overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up) and overtrained by running without inclination (OTR). The incremental load, exhaustive and grip force tests were used as performance evaluation parameters. 36h after the grip force test, EDL and soleus were removed and immediately used for immunoblotting analysis or stored at ‐80°C for histological and stereological analyses. For EDL, OTR/down decreased the protein kinase B (Akt) and tuberous sclerosis protein 2 (TSC2) phosphorylation (p), and increased myostatin, receptor‐activated Smads (pSMAD2‐3) and insulin receptor substrate‐1 (pIRS‐1; Ser307/636). OTR/down also presented low and high relative proportions of cytoplasm and connective tissue, respectively. OTR/up increased the mammalian target of rapamycin (pmTOR), 70‐kDa ribosomal protein S6 kinase 1 (pS6K1) and pSMAD2‐3, and decreased pTSC2. OTR decreased pTSC2 and increased pIRS‐1 (Ser636). For soleus, OTR/down increased S6 ribosomal protein (pS6RP) and pSMAD2‐3, and decreased pIRS‐1 (Ser639). OTR/up decreased pS6K1, pS6RP and pIRS‐1 (Ser639), and increased pTSC2 (Ser939) and pSMAD2‐3. OTR increased pS6RP, 4E‐binding protein‐1 (p4E‐BP1), pTSC2 (Ser939), and pSMAD2‐3, and decreased pIRS‐1 (Ser639). In summary, OTR/down inhibited the skeletal muscle hypertrophy with concomitant signs of atrophy in EDL. The effects of OTR/up and OTR depended on the analyzed skeletal muscle type. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T06:49:06.822849-05:
      DOI: 10.1002/jcp.25197
  • Mechanical Loading Stimulates Expression of Collagen Cross‐Linking
           Associated Enzymes in Periodontal Ligament
    • Authors: Masaru Kaku; Juan Marcelo Rosales Rocabado, Megumi Kitami, Takako Ida, Yosuke Akiba, Mitsuo Yamauchi, Katsumi Uoshima
      Abstract: Type I collagen, a major extracellular component of the periodontal ligament (PDL), is post‐translationally modified by a series of specific enzymes. Among the collagen‐modifying enzymes, lysyl oxidase (LOX) is essential to initiate collagen cross‐linking and lysyl hydroxylases (LHs) to regulate the cross‐linking pathways that are important for tissue specific mechanical properties. The purpose of this study was to investigate the effects of mechanical loading on the expression of collagen‐modifying enzymes and subsequent tissue changes in PDL. Primary human PDL cells were subjected to mechanical loading in a 3D collagen gel, and gene expression and collagen component were analyzed. Wistar rats were subjected to excessive occlusal loading with or without intraperitoneal injection of a LOX inhibitor, β‐aminopropionitrile (BAPN). Upon mechanical loading, gene expression of LH2 and LOX was significantly elevated, while that of COL1A2 was not affected on hPDL‐derived cells. The mechanical loading also elevated formation of collagen α‐chain dimers in 3D culture. The numbers of LH2 and LOX positive cells in PDL were significantly increased in an excessive occlusal loading model. Notably, an increase of LH2‐positive cells was observed only at the bone‐side of PDL. Intensity of picrosirius red staining was increased by excessive occlusal loading, but significantly diminished by BAPN treatment. These results demonstrated that mechanical loading induced collagen maturation in PDL by up‐regulating collagen‐modifying enzymes and subsequent collagen cross‐linking which are important for PDL tissue maintenance. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T04:03:40.772205-05:
      DOI: 10.1002/jcp.25184
  • Mitotic Inheritance of mRNA Facilitates Translational Activation of the
           Osteogenic‐Lineage Commitment Factor Runx2 in Progeny of
           Osteoblastic Cells
    • Authors: Nelson Varela; Alejandra Aranguiz, Carlos Lizama, Hugo Sepulveda, Marcelo Antonelli, Roman Thaler, Ricardo D. Moreno, Martin Montecino, Gary S. Stein, Andre J. van Wijnen, Mario Galindo
      Abstract: Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype‐committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post‐mitotic re‐activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone‐related runt‐related transcription factor Runx2. Here, we characterized a non‐genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self‐renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non‐chromosomal tubulin‐containing compartment. Subsequently, transcription‐independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast‐specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T04:02:34.052494-05:
      DOI: 10.1002/jcp.25188
  • TRPV4 Channels in Human White Adipocytes: Electrophysiological
           Characterization and Regulation by Insulin
    • Abstract: Intracellular calcium homeostasis in adipocytes is important for the regulation of several functions and is involved in pathological changes in obesity and other associated diseases. TRPV4 (Transient Receptor Potential Vanilloid 4) channels are an important route for calcium entry that operates in a variety of cells and intervenes in a number of functions. In this study, the expression and operation of TRPV4 channels in human cultured adipocytes was evaluated using RT‐PCR, Western blotting, the whole‐cell patch‐clamp technique and fluorescence measurements to characterize these channels and determine intracellular calcium responses. Both the hypoosmolarity and 4αPDD (4alpha‐phorbol‐didecanoate), a specific TRPV4 agonist, induced a similar HC‐067047‐sensitive current, which was predominantly inward, and an intracellular Ca2+ concentration increase, which was exclusively dependent on extracellular calcium, and membrane depolarization. The current had a reverse potential of +31 ± 6 mV and exhibited preferential permeability to Ca2+. Insulin, which regulates metabolic homeostasis in adipocytes, attenuated the TRPV4‐mediated effects. These results confirm the function of TRPV4 in human cultured adipocytes and its regulation by insulin. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T03:56:29.75762-05:0
      DOI: 10.1002/jcp.25187
  • Prolyl Endopeptidase (PREP) is Associated with Male Reproductive Functions
           and Gamete Physiology in Mice
    • Authors: Raffaele Dotolo; Jung Dae Kim, Paolo Pariante, Sergio Minucci, Sabrina Diano
      Abstract: Prolyl endopeptidase (PREP) is a serine protease which has been implicated in many biological processes, such as the maturation and degradation of peptide hormones and neuropeptides, learning and memory, cell proliferation and differentiation, and glucose metabolism. A small number of reports have also suggested PREP participation in both male and female reproduction‐associated processes. In the present work we examined PREP distribution in male germ cells and studied the effects of its knockdown (Prepgt/gt) on testis and sperm in adult mice. The protein is expressed and localized in elongating spermatids and luminal spermatozoa of wild type (wt) mice, as well as Sertoli, Leydig and peritubular cells. PREP is also expressed in the head and midpiece of epididymal spermatozoa, whereas the remaining tail region shows a weaker signal. Furthermore, testis weight, histology of seminiferous tubules, and epididymal sperm parameters were assessed in wt and Prepgt/gt mice: wild type testes have larger average tubule and lumen diameter; in addition, lumenal composition of seminiferous tubules is dissimilar between wt and Prepgt/gt, as the percentage of spermiated tubules is much higher in wt. Finally, total sperm count, sperm motility and normal morphology are also higher in wt than in Prepgt/gt. These results show for the first time that the expression of PREP could be necessary for a correct reproductive function, and suggest that the enzyme may play a role in mouse spermatogenesis and sperm physiology. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-18T02:47:20.832753-05:
      DOI: 10.1002/jcp.25178
  • Alterations of the Plasma Peptidome Profiling in Colorectal Cancer
    • Authors: Chiara Bedin; Sara Crotti, Eugenio Ragazzi, Salvatore Pucciarelli, Lisa Agatea, Ennio Tasciotti, Mauro Ferrari, Pietro Traldi, Flavio Rizzolio, Antonio Giordano, Donato Nitti, Marco Agostini
      Abstract: Early detection of colorectal cancer (CRC) remains a challenge. It has been highlighted that the pathological alterations within an organ and tissues might be reflected in serum or plasma proteomic/peptidic patterns. The aim of the study was to follow the changes in the plasma peptides associated to colorectal cancer progression by mass spectrometry. This study included 27 adenoma, 67 CRC (n = 33 I‐II stage and n = 34 III‐IV stage), 23 liver metastasis from CRC patients and 34 subjects disease‐free as controls. For plasma peptides analysis, samples purification was performed on the Nanoporous Silica Chips technology followed by matrix‐assisted laser desorption/ionisation‐time of flight analysis. Since the high complexity of the obtained dataset, multivariate statistical analysis and discriminant pattern recognition were performed for study groups classification. Forty‐four of 88 ionic species were successfully identified as fragments of peptides and proteins physiologically circulating in the blood and belonging to immune and coagulation systems and inflammatory mediators. Many peptides clustered into sets of overlapping sequences with ladder‐like truncation clearly associated to proteolytic processes of both endo‐ and exoproteases activity. Comparing to controls, a different median ion intensity of the group‐type fragments distribution was observed. Moreover, the degradation pattern obtained by proteolytic cleavage was different into study groups. This pattern was specific and characteristic of each group: controls, colon tumour disease (including adenoma and CRC) and liver metastasis, revealing a role as biomarker in early diagnosis and prognosis. Our findings highlighted peculiar changes in protease activity characteristic of CRC progression from pre‐cancer lesion to metastatic disease. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-17T07:24:37.275826-05:
      DOI: 10.1002/jcp.25196
  • Alpha‐Catulin Co‐Localizes with Vimentin Intermediate
           Filaments and Functions in Pulmonary Vascular Endothelial Cell Migration
           via ROCK
    • Authors: Michael D. Bear; Tiegang Liu, Shereen Abualkhair, Maher A. Ghamloush, Nicholas S. Hill, Ioana Preston, Barry L. Fanburg, Usamah S. Kayyali, Deniz Toksoz
      Abstract: The ubiquitous α‐catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α‐catulin has homology to the cytoskeletal linkers α‐catenin and vinculin, it appears to be functionally divergent. Here we further investigated α‐catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α‐catulin has a unique cytoskeletal role. Examination of endogenous α‐catulin intracellular localization by immunofluorescence revealed a highly organized cytosolic filamentous network suggestive of a cytoskeletal system in a variety of cultured VEC. Double‐immunofluorescence analyses of VEC showed endogenous α‐catulin co‐localization with vimentin intermediate filaments. Similar to vimentin, α‐catulin was found to distribute into detergent‐soluble and ‐insoluble fractions. Treatment of VEC with withaferinA, an agent that targets vimentin filaments, disrupted the α‐catulin network distribution and altered α‐catulin solubility. Vimentin participates in cell migration, and withaferinA was found to inhibit VEC migration in vitro; similarly, α‐catulin knock‐down reduced VEC migration. Based on previous reports showing that ROCK modulates vimentin, we found that ROCK depletion attenuated VEC migration; furthermore, α‐catulin depletion was shown to reduce ROCK‐induced signaling. These findings indicate that α‐catulin has a unique function in co‐localization with vimentin filaments that contributes to VEC migration via a pathway that may involve ROCK signaling. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-17T04:29:20.726411-05:
      DOI: 10.1002/jcp.25185
  • HhAntag, a Hedgehog Signaling Antagonist, Suppresses Chondrogenesis and
           Modulates Canonical and Non‐Canonical BMP Signaling
    • Authors: Christina Mundy; Adebayo Bello, Federica Sgariglia, Eiki Koyama, Maurizio Pacifici
      Abstract: Chondrogenesis subtends the development of most skeletal elements and involves mesenchymal cell condensations differentiating into growth plate chondrocytes that proliferate, undergo hypertrophy and are replaced by bone. In the pediatric disorder Hereditary Multiple Exostoses, however, chondrogenesis occurs also at ectopic sites and causes formation of benign cartilaginous tumors –exostoses‐ near the growth plates. No treatment is currently available to prevent or reverse exostosis formation. Here, we asked whether chondrogenesis could be stopped by targeting the hedgehog pathway, one of its major regulators. Micromass cultures of limb mesenchymal cells were treated with increasing amounts of the hedgehog inhibitor HhAntag or vehicle. The drug effectively blocked chondrogenesis and did so in a dose‐dependent manner as monitored by: alcian blue‐positive cartilage nodule formation; gene expression of cartilage marker genes; and reporter activity in Gli1‐LacZ cell cultures. HhAntag blocked chondrogenesis even when the cultures were co‐treated with bone morphogenetic protein 2 (rhBMP‐2), a strong pro‐chondrogenic factor. Immunoblots showed that HhAntag action included modulation of canonical (pSmad1/5/8) and non‐canonical (pp38) BMP signaling. In cultures co‐treated with HhAntag plus rhBMP‐2, there was a surprising strong up‐regulation of pp38 levels. Implantation of rhBMP‐2‐coated beads near metacarpal elements in cultured forelimb explants induced formation of ectopic cartilage that however, was counteracted by HhAntag co‐treatment. Collectively, our data indicate that HhAntag inhibits not only hedgehog signaling, but also modulates canonical and non‐canonical BMP signaling and blocks basal and rhBMP2‐stimulated chondrogenesis, thus representing a potentially powerful drug‐based strategy to counter ectopic cartilage growth or induce its involution. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-12T09:39:16.573423-05:
      DOI: 10.1002/jcp.25192
  • Lipidomics of Mesenchymal Stromal Cells: Understanding the Adaptation of
           Phospholipid Profile in Response to Pro‐Inflammatory Cytokines
    • Abstract: Mesenchymal stromal cells (MSCs) present anti‐inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. In clinical applications MSCs are subjected to a strong pro‐inflammatory environment, essential to their immunosuppressive action. Despite the wide clinical use of these cells, how MSCs exert their effect remains unclear. Several lipids are known to be involved in cell's signaling and modulation of cellular functions. The aim of this paper is to examine the variation in lipid profile of MSCs under pro‐inflammatory environment, induced by the presence of tumor necrosis factor alpha (TNF‐α) and interferon gamma (IFN‐γ), using the most modern lipidomic approach. Major changes in lipid molecular profile of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysoPC (LPC) and sphingomyelin (SM) classes were found. No changes were observed in the phosphatidylinositol (PI) profile. The levels of PC species with shorter fatty acids (FAs) acyl chains, mainly C16:0, decreased under pro‐inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of LPC(18:0), which is known to be an anti‐inflammatory LPC, observed in MSCs subjected to TNF‐α and IFN‐γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF‐α and IFN‐γ also enhanced the levels of PE(40:6) and decreased the levels of PE(O‐38:6). Higher expression of PS(36:1) and SM(34:0) along with a decrease in PS(38:6) levels were observed. These results indicate that lipid metabolism and signaling are modulated during MSCs activation, which suggests that lipids may be involved in MSCs functional and anti‐inflammatory activities. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-12T09:38:24.556302-05:
      DOI: 10.1002/jcp.25191
  • Review: Tumor Microenvironment Versus Cancer Stem Cells in
           Cholangiocarcinoma: Synergistic Effects'
    • Authors: Maurizio Romano; Francesco De Francesco, Enrico Gringeri, Antonio Giordano, Giuseppe A. Ferraro, Marina Di Domenico, Umberto Cillo
      Abstract: Cholangiocarcinoma (CCAs) may be defined as tumors that derived from the biliary tree with the differentiation in the biliary epithelial cells. This tumor is malignant, extremely aggressive with a poor prognosis. It can be treated surgically and its pathogenesis is poorly understood. The tumor microenvironment (TME) is a very important factor in the regulation of tumor angiogenesis, invasion, and metastasis. Besides cancer stem cells (CSCs) can modulate tumor growth, stroma formation and migratory capability. The initial stage of tumorigenesis is characterized by genetic mutations and epigenetic alterations due to intrinsic factors which lead to the generation of oncogenes thus inducing tumorigenesis. CSCs may result from precancerous stem cells, cell de‐differentiation, normal stem cells or an epithelial‐mesenchymal transition (EMT). CSCs have been found in the cancer niche, and EMT may occur early within the tumor microenvironment. Previous studies have demonstrated evidence of cholangiocarcinoma stem cells (CD133, CD24, EpCAM, CD44, and others) and the presence of these markers has been associated with malignant potential. The interaction between TME and cholangiocarcinoma stem cells via signaling mediators may create an environment that accommodates tumor growth, yielding resistance to cytotoxic insults (chemotherarapeutic). While progress has been made in the understanding of the mechanisms, the interactions in the tumorigenic process still remain a major challenge. Our review, addresses recent concepts of TME‐CSCs interaction and will emphasize the importance of early detection with the use of novel diagnostic mechanisms such as CCA‐CSC biomarkers and the importance of tumor stroma to define new treatments. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-11T04:31:25.258196-05:
      DOI: 10.1002/jcp.25190
  • Glucose and Inflammatory Cells Decrease Adiponectin in Epicardial Adipose
           Tissue Cells: Paracrine Consequences on Vascular Endothelium
    • Abstract: Epicardial adipose tissue (EAT) is a source of energy for heart that expresses the insulin‐sensitizer, anti‐inflammatory and anti‐atherogenic protein, adiponectin. But, in coronary artery disease, adiponectin production declines. Our objective was to determine its regulation by glucose and inflammation in stromal cells from EAT and subcutaneous adipose tissue (SAT) and its paracrine effect on endothelial cells. Stromal cells of EAT and SAT were obtained from patients who underwent cardiac surgery. Adipogenesis was induced at 117, 200 or 295 mg/dL glucose, with or without macrophage‐conditioned medium (MCM). Expression of adiponectin, GLUT‐4 and the insulin receptor was analyzed by real‐time PCR. The paracrine effect of stromal cells was determined in co‐cultures with endothelial cells, by exposing them to high glucose and/or MCM, and, additionally, to leukocyte‐conditioned medium from patients with myocardial infarction. The endothelial response was determined by analyzing vascular adhesion molecule expression. Our results showed a U‐shaped dose‐response curve of glucose on adiponectin in EAT, but not in SAT stromal cells. Conversely, MCM reduced the adipogenesis‐induced adiponectin expression of EAT stromal cells. The presence of EAT stromal increased the inflammatory molecules of endothelial cells. This deleterious effect was emphasized in the presence of inflammatory cell‐conditioned medium from patients with myocardial infarction. Thus, high glucose and inflammatory cells reduced adipogenesis‐induced adiponectin expression of EAT stromal cells, which induced an inflammatory paracrine process in endothelial cells. This inflammatory effect was lower in presence of mature adipocytes, producers of adiponectin. These results contribute to understanding the role of EAT dysfunction on coronary atherosclerosis progression. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-10T06:38:31.719517-05:
      DOI: 10.1002/jcp.25189
  • Neurotransmitters and Neuropeptides in the Endocrine Pancreas: New Players
           in the Control of Islet Cells Mass and Function
    • Authors: Eliana S. Di Cairano; Stefania Moretti, Paola Marciani, Vellea Franca Sacchi, Michela Castagna, Alberto Davalli, Franco Folli, Carla Perego
      Abstract: Islets of Langerhans control whole body glucose homeostasis, as they respond, releasing hormones, to changes in nutrient concentrations in the blood stream. The regulation of hormone secretion has been the focus of attention for a long time because it is related to many metabolic disorders, including diabetes mellitus. Endocrine cells of the islet use a sophisticate system of paracrine and autocrine signals to synchronize their activities. These signals provide a fast and accurate control not only for hormone release but also for cell differentiation and survival, key aspects in islet physiology and pathology. Among the different categories of paracrine/autocrine signals, this review highlights the role of neurotransmitters and neuropeptides. In a manner similar to neurons, endocrine cells synthesize, accumulate, release neurotransmitters in the islet milieu, and possess receptors able to decode these signals. In this review, we provide a comprehensive description of neurotransmitter/neuropetide signaling pathways present within the islet. Then, we focus on evidence supporting the concept that neurotransmitters/neuropeptides and their receptors are interesting new targets to preserve β‐cell function and mass. A greater understanding of how this network of signals works in physiological and pathological conditions would advance our knowledge of islet biology and physiology and uncover potentially useful areas of pharmacological intervention. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-10T02:41:38.974466-05:
      DOI: 10.1002/jcp.25176
  • Osteosarcoma Stem Cells Have Active Wnt/β‐Catenin and
           Overexpress SOX2 and KLF4
    • Abstract: Osteosarcoma is a bone tumor displaying significant cellular and histological heterogeneity and a complex genetic phenotype. Although multiple studies strongly suggest the presence of cancer stem cells in osteosarcoma a consensus on their characterization is still missing. We used a combination of functional assays (sphere‐forming, Aldefluor and side‐population) for identification of cancer stem cell populations in osteosarcoma cell lines. Expression of stemness‐related transcription factors, quiescent nature, in vivo tumorigenicity and Wnt/β‐catenin activation were evaluated. We show that different cancer stem cell populations may co‐exist in osteosarcoma cell lines exhibiting distinct functional properties. Osteosarcoma spheres are slowly‐proliferating populations, overexpress SOX2 and KLF4 stemness‐related genes and have enhanced tumorigenic potential. Additionally, spheres show specific activation of Wnt/β‐catenin signaling as evidenced by increased nuclear β‐catenin, TCF/LEF activity and AXIN2 expression, in a subset of the cell lines. Aldefluor‐positive populations were detected in all osteosarcoma cell lines and overexpress SOX2, but not KLF4. The side‐population phenotype is correlated with ABCG2 drug‐efflux transporter expression. Distinct functional methods seem to identify cancer stem cells with dissimilar characteristics. Intrinsic heterogeneity may exist within osteosarcoma cancer stem cells and can have implications on the design of targeted therapies aiming to eradicate these cells within tumors. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:44:02.134755-05:
      DOI: 10.1002/jcp.25179
  • Kidney Injury Molecule‐1 Enhances Endocytosis of Albumin in Renal
           Proximal Tubular Cells
    • Authors: Xueying Zhao; Chen Jiang, Rebecca Olufade, Dong Liu, Nerimiah Emmett
      Abstract: Receptor‐mediated endocytosis plays an important role in albumin reabsorption by renal proximal tubule epithelial cells. Kidney injury molecule‐1 (KIM‐1) is a scavenger receptor that is upregulated on the apical membrane of proximal tubules in proteinuric kidney disease. In this study, we examined the cellular localization and functional role of KIM‐1 in cultured renal tubule epithelial cells (TECs). Confocal immunofluorescence microscopy reveals intracellular and cell surface localization of KIM‐1 in primary renal TECs. Albumin stimulation resulted in a redistribution of KIM‐1 and tight junction protein zonula occludens‐1 in primary TEC monolayer. An increase in albumin internalization was observed in both primary TECs expressing endogenous KIM‐1 and rat kidney cell line (NRK‐52E) overexpressing exogenous KIM‐1. KIM‐1‐induced albumin accumulation was abolished by its specific antibody. Moreover, endocytosed KIM‐1 and its cargo proteins were delivered from endosomes to lysosomes for degradation in a clathrin‐dependent pathway. Supportive evidence includes a) detection of KIM‐1 in Rab5‐positive early endosomes, Rab7‐positive late endosomes/multivesicular bodies, and LAMP1‐positive lysosomes, b) colocalization of KIM‐1 and clathrin in the intracellular vesicles, and c) blockade of KIM‐1‐mediated albumin internalization by chlorpromazine, an inhibitor of clathrin‐dependent endocytosis. KIM‐1 expression was upregulated by albumin but downregulated by transforming growth factor‐β1. Taken together, our data indicate that KIM‐1 increases albumin endocytosis in renal tubule epithelial cells, at least partially via a clathrin‐dependent mechanism. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:39:37.965251-05:
      DOI: 10.1002/jcp.25181
  • Modulating Intrafollicular Hormonal Milieu in Controlled Ovarian
           Stimulation: Insights from PPAR Expression in Human Granulosa Cells
    • Abstract: Controlled ovarian stimulation (COS) leading to ovulation of multiple follicles is a crucial aspect of biomedical infertility care. Nevertheless, biomarkers useful for COS management are still lacking. Peroxisome proliferator‐activated receptors (PPARs) are nuclear hormone receptors relevant to steroid metabolism in granulosa cells (GCs). We investigated whether PPARs and their steroidogenic targets were differentially expressed in GCs differentiated under different recombinant or urinary gonadotropin preparations. GCs from women subjected to COS with r‐hFSH, r‐hFSH/r‐hLH or hMG‐HP were processed to assess expression of PPARα, PPARβ/δ, PPARγ and steroidogenic enzymes under PPAR modulation. As an evidence of their activation, all PPAR isotypes with their coactivators, the retinoic‐X‐receptors (RXRs), localized in the nucleus. When GCs from r‐hFSH/r‐hLH group were compared with r‐hFSH, a significant reduction of PPARα protein was observed. By contrast, an increase of PPARβ/δ at both protein and mRNA levels along with that of PPARγ protein were detected. The steroidogenic enzymes 17βHSD IV, 3βHSD II and HMG‐CoA red were downregulated in the r‐hFSH/r‐hLH group in comparison to r‐hFSH unlike CYP19A1 that remained unchanged. In GCs from urinary FSH‐LH stimulation (hMG‐HP), PPARα was more expressed in comparison with r‐hFSH/r‐hLH group. Likewise, 3βHSD II and 17βHSD IV were increased suggesting that hMG‐HP partially mimicked r‐hFSH/r‐hLH effects. In summary, transcript analysis associated to protein investigation revealed differential effects of COS protocols on PPARs and their steroidogenic targets in relation to LH and gonadotropin source. These observations candidate PPARs as new biomarkers of follicle competence opening new hypotheses on COS effects on ovarian physiology. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:35:04.594288-05:
      DOI: 10.1002/jcp.25182
  • Downregulation of Long Noncoding RNA Meg3 Affects Insulin Synthesis and
           Secretion in Mouse Pancreatic Beta Cells
    • Authors: LiangHui You; Ning Wang, DanDan Yin, LinTao Wang, FeiYan Jin, YaNan Zhu, QingXin Yuan, Wei De
      Abstract: Increasing evidence indicates that long noncoding RNAs (lncRNAs) are involved in diverse biological process. Mouse maternal expressed gene 3 (Meg3), is an imprinted gene and essential for development. Here, we explored the relationship between Meg3 and the function of mouse beta cells in vitro and vivo. Real‐time PCR analyses revealed Meg3 was more abundantly expressed in Balb/c mouse islets than exocrine glands. Moreover, the expression of Meg3 in islets was decreased in T1DM (NOD female mice) and T2DM (db/db mice) models. Meg3 expression was modulated dynamically by glucose in Min6 cells and isolated mouse islets. The function role of Meg3 was investigated in Min6 cells and normal mouse by knockdown of Meg3 using small interfering RNA. After suppression of Meg3 expression in vitro, insulin synthesis and secretion were impaired and the rate of beta cells apoptosis was increased. Moreover, knockdown of Meg3 in vivo led to the impaired glucose tolerance and decreased insulin secretion, consisted with the reduction of insulin positive cells areas by immunochemistry assays. Notably, islets from Meg3 interference groups showed significant decrease of Pdx‐1 and MafA expression in mRNA and protein levels. These results indicate that Meg3 may function as a new regulator of maintaining beta cells identity via affecting insulin production and cell apoptosis. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T04:04:54.515521-05:
      DOI: 10.1002/jcp.25175
  • Circulating MicroRNA and Long Noncoding RNA as Biomarkers of
           Cardiovascular Diseases
    • Authors: Qiang Shi; Xi Yang
      Abstract: Although >90 of the human genome is transcribed, only  200 nucleotides). It is now clear that these RNAs fulfil critical roles as transcriptional and post‐transcriptional regulators and as guides of chromatin‐modifying complexes. Although not translated into protein, noncoding RNAs can regulate cardiac function through diverse mechanisms and their dysregulation is increasingly linked with cardiovascular pathophysiology. Furthermore, a series of recent studies have discovered that noncoding RNAs can be found in the bloodstream and some species are remarkably stable. This has raised the possibility that such noncoding RNAs may be measured in body fluids and serve as novel diagnostic biomarkers. Here, we summarize the current knowledge of noncoding RNAs' function and biomarker potential in cardiac diseases, concentrating mainly on circulating miRNAs and lncRNAs. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T03:49:20.492542-05:
      DOI: 10.1002/jcp.25174
  • Peripheral Blood Mononuclear Cells Spontaneous Osteoclastogenesis:
    • Abstract: In vitro peripheral blood mononuclear cells differentiate into osteoclasts under the influence of osteoclast‐stimulating factors. However, accumulating evidence suggests spontaneous osteoclasts formation and activity in patients affected by local or systemic bone remodeling diseases in comparison with healthy controls. Therefore, within this review, we summarize the studies where spontaneous osteoclastogenesis of peripheral blood mononuclear cells was observed in pathological conditions of the skeletal system. We indicate a linkage between immunoregulation by T cells and spontaneous osteoclasts formation with increased levels of tumor necrosis factors‐α, receptor activator of nuclear factor kappa‐B ligand and inteleukin‐7 production. In the light of these results, it would be of crucial importance to deepen the correlation between systemic bone remodeling diseases and spontaneous osteoclastogenesis as well as to investigate in detail the mechanisms underlying this phenomenon and the clinical relevance in bone remodeling disease diagnosis and monitoring. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-09T03:32:50.397314-05:
      DOI: 10.1002/jcp.25134
  • Stress‐Induced Activation of Apoptosis Signal‐Regulating
           Kinase 1 Promotes Osteoarthritis
    • Abstract: Apoptosis signal‐regulated kinase 1 (ASK1) has been shown to affect a wide range of cellular processes including stress‐related responses, cytokine and growth factor signaling, cell cycle and cell death. Recently, we reported that lack of ASK1 slowed chondrocyte hypertrophy, terminal differentiation and apoptosis resulting in an increase in trabecular bone formation. Herein, we investigated the role of ASK1 in the pathogenesis of osteoarthritis (OA). Immunohistochemistry performed on articular cartilage samples from patients with OA showed ASK1 expression increased with OA severity. In vitro analysis of chondrocyte hypertrophy, maturation and ASK1 signaling in embryonic fibroblasts from ASK1 knockout (KO) and wild type (WT) mice was examined. Western analysis demonstrated an increase in ASK1 signaling commensurate with chondrogenic maturation during differentiation or in response to stress by the cytokines, tumor necrosis factor alpha or interleukin 1 beta in WT, but not in ASK1 KO embryonic fibroblasts. Surgically induced moderate or severe OA or OA due to natural aging in WT and ASK1 KO mice was assessed by microCT of subchondral bone, immunohistochemistry, histology and OARSI scoring. Immunohistochemistry, microCT and OARSI scoring all indicated that the lack of ASK1 protected against OA joint degeneration, both in surgically induced OA and in aging mice. We propose that the ASK1 MAP kinase signaling cascade is an important regulator of chondrocyte terminal differentiation and inhibitors of this pathway could be useful for slowing chondrocyte maturation and cell death observed with OA progression. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-07T05:44:12.455325-05:
      DOI: 10.1002/jcp.25186
  • Epigenetic Priming Confers Direct Cell Trans‐Differentiation from
           Adipocyte to Osteoblast in a Transgene‐Free State
    • Abstract: The bone marrow of healthy individuals is primarily composed of osteoblasts and hematopoietic cells, while that of osteoporosis patients has a larger portion of adipocytes. There is evidence that the epigenetic landscape can strongly influence cell differentiation. We have shown that it is possible to direct the trans‐differentiation of adipocytes to osteoblasts by modifying the epigenetic landscape with a DNA methyltransferase inhibitor (DNMTi), 5′‐aza‐dC, followed by Wnt3a treatment to signal osteogenesis. Treating 3T3‐L1 adipocytes with 5′‐aza‐dC induced demethylation in the hypermethylated CpG regions of bone marker genes; subsequent Wnt3a treatment drove the cells to osteogenic differentiation. When old mice with predominantly adipose marrow were treated with both 5′‐aza‐dC and Wnt3a, decreased fatty tissue and increased bone volume were observed. Together, our results indicate that epigenetic modification permits direct programming of adipocytes into osteoblasts in a mouse model of osteoporosis, suggesting that this approach could be useful in bone tissue‐engineering applications. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-03T17:22:44.577997-05:
      DOI: 10.1002/jcp.25183
  • BMP‐2 Enhances Lgr4 Gene Expression in Osteoblastic Cells
    • Authors: Chantida Pawaputanon Na Mahasarakham; Yoichi Ezura, Makiri Kawasaki, Arayal Smriti, Shuichi Moriya, Takayuki Yamada, Yayoi Izu, Katsuhiko Nishimori, Yuichi Izumi, Masaki Noda
      Abstract: Osteoporosis is one of the most prevalent diseases and the number of patients suffering from this disease is soaring due to the increase in the aged population in the world. The severity of bone loss in osteoporosis is based on the levels of impairment in the balance between bone formation and bone resorption, two arms of the bone metabolism and bone remodeling. However, determination of bone formation levels is under many layers of control that are as yet fully defined. Bone morphogenetic protein (BMP) plays a key role in regulation of bone formation while its downstream targets are still incompletely understood. Lgr4 gene encodes an orphan receptor and has been identified as a genetic determinant for bone mass in osteoporotic patients. Here, we examine the effects of BMP on the expression of Lgr4 in osteoblastic cells. Lgr4 gene is expressed in an osteoblastic cell line, MC3T3E1 in a time dependent manner during the culture. BMP treatment enhances Lgr4 mRNA expression at least in part via transcriptional event. When Lgr4 mRNA is knocked down, the levels of BMP‐induced increase in alkaline phosphatase (Alp) activity and Alp mRNA are suppressed. BMP enhancement of Lgr4 gene expression is suppressed by FGF and reversed by dexamethasone. BMP also enhances Lgr4 expression in primary cultures of calvarial osteoblasts. These data indicate that Lgr4 gene is regulated by BMP and is required for BMP effects on osteoblastic differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-02T17:08:43.507094-05:
      DOI: 10.1002/jcp.25180
  • Mesenchymal Stromal Cells Differentiating to Adipocytes Accumulate
           Autophagic Vesicles Instead of Functional Lipid Droplets
    • Abstract: Adult bone marrow mesenchymal stromal cells (BMSCs) can easily be differentiated into a variety of cells. In vivo transplantation of BMSCs‐differentiated cells has had limited success, suggesting that these cells may not be fully compatible with the cells they are intended to replace in vivo. We investigated the structural and functional features of BMSCs‐derived adipocytes as compared with adipocytes from adipose tissue, and the structure and functionality of lipid vesicles formed during BMSCs differentiation to adipocytes. Gas chromatography‐mass spectrometry showed fatty acid composition of BMSCs‐derived adipocytes and adipocytes from the adipose tissue to be very different, as is the lipid rafts composition, caveolin‐1 expression, caveolae distribution in their membranes, and the pattern of expression of fatty acid elongases. Confocal microscopy confirmed the absence from BMSCs‐derived adipocytes of markers of lipid droplets. BMSCs‐derived adipocytes cannot convert deuterated glucose into deuterated species of fatty acids and cannot uptake the deuterated fatty acid‐bovine serum albumin complexes from the culture medium, suggesting that intra‐cellular accumulation of lipids does not occur by lipogenesis. We noted that BMSCs differentiation to adipocytes is accompanied by an increase in autophagy. Autophagic vesicles accumulate in the cytoplasm of BMSCs‐derived adipocytes and their size and distribution resembles that of Nile Red‐stained lipid vesicles. Stimulation of autophagy in BMSCs triggers the intra‐cellular accumulation of lipids while inhibition of autophagy prevents this accumulation. In conclusion, differentiation of BMSCs‐derived adipocytes leads to intra‐cellular accumulation of autophagic vesicles rather than functional lipid droplets, suggesting that these cells are not authentic adipocytes. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T18:31:06.575644-05:
      DOI: 10.1002/jcp.25177
  • Toll‐Like Receptors and Tissue Remodeling: The Pro/Cons Recent
    • Authors: Alessandra Micera; Bijorn Omar Balzamino, Antonio Di Zazzo, Filippo Biamonte, Gigliola Sica, Stefano Bonini
      Abstract: The Toll‐like Receptor (TLR) family ensures prompt response towards pathogens, protecting the host against infections, and guarantees a realistic balance between protective and detrimental activities. Multiple regulating mechanisms characterize TLR activity that is not limited to innate and adaptive antimicrobial immune responses, as observed in the inflammatory (either infective, allergic or autoimmune) responses associated with tissue remodeling. Following the insult and the arise of inflammatory response, tissue remodeling takes place and might develop in fibrosis, depending on microenvironment as a result of imbalanced fibroblasts (FBs) and myofibroblasts (myoFBs) activation/survival. The process is driven by an epithelial‐fibroblast‐immune cell cross‐talk. While the main FB function is the matrix metabolism for tissue homeostasis or repair, the myoFB differentiation represents a crucial step in attempting repair of injury. FBs/myoFBs provide more than structural support at site of injury, synthesizing and/or reacting to different cytokines, growth factors, neuromediators and soluble/lipid mediators. TLR‐bearing FBs/myoFBs might contribute at the innate immune level, providing a second line of protection/defense as well as being a target/effector cell of tissue remodeling. TLRs might also interfere with acute inflammation as well as with established fibrosis, triggering structural/functional changes in agreement with the genetic background, the site of lesion, the entity of associated infection, the poor blood circulation or the pharmacological treatments, all together strictly influencing tissue repair/remodeling process. This review will focus on the recent findings on TLRs at launch and long‐lasting tissue remodeling process, that strongly suggest TLRs as optional targets for future therapies. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T03:50:56.588812-05:
      DOI: 10.1002/jcp.25124
  • G Protein‐Coupled Receptor 120 Signaling Negatively Regulates
           Osteoclast Differentiation, Survival, and Function
    • Abstract: G protein‐coupled receptor 120 (GPR120) plays an important role in the regulation of inflammation and lipid metabolism. In this study, we investigated the role of GPR120 in osteoclast development and found that GPR120 regulates osteoclast differentiation, survival and function. We observed that GPR120 was highly expressed in osteoclasts compared to their precursors, bone marrow‐derived macrophages (BMMs). Activation of GPR120 by its ligand GW9508 suppressed receptor activator of NF‐κB ligand (RANKL)‐induced osteoclast differentiation and the expression of nuclear factor of activated T cells c1 (NFATc1), a key modulator of osteoclastogenesis. GPR120 activation further inhibited the RANKL‐stimulated phosphorylation of IκBα and JNK. In addition to osteoclast differentiation, GPR120 activation increased the apoptosis of mature osteoclasts by inducing caspase‐3 and Bim expression. Activation of GPR120 also interfered with cell spreading and actin cytoskeletal organization mediated by M‐CSF but not by RANKL. Coincident with the impaired cytoskeletal organization, GPR120 activation blocked osteoclast bone resorbing activity. Furthermore, knockdown of GPR120 using small hairpin RNA abrogated all these inhibitory effects on osteoclast differentiation, survival, and function. Together, our findings identify GPR120 as a negative modulator of osteoclast development that may be an attractive therapeutic target for bone‐destructive diseases. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T02:47:54.011233-05:
      DOI: 10.1002/jcp.25133
  • Vitamin D Induces Cyclooxygenase 2 Dependent Prostaglandin E2 Synthesis in
           HaCaT keratinocytes
    • Abstract: The active metabolite of vitamin D calcitriol and its analogs are well‐known for their anti‐inflammatory action in the skin, while their main side effect associated with topical treatment of inflammatory disorders is irritant contact dermatitis. Prostaglandin E2 (PGE2) is pro‐inflammatory at the onset of inflammation and anti‐inflammatory at its resolution. We hypothesized that induction of PGE2 synthesis by calcitriol in epidermal keratinocytes may contribute both to its pro‐inflammatory and anti‐inflammatory effects on the skin. Treatment of human immortalized HaCaT keratinocytes with calcitriol (3–100 nM, 2–24 h) increased PGE2 production due to increased mRNA and protein expression of COX‐2, but not to increase of COX‐1 or release of arachidonic acid. The effect of calcitriol on COX‐2 mRNA was observed also in primary human keratinocytes. The increase in COX‐2 mRNA is associated with COX‐2 transcript stabilization. Calcitriol exerts this effect by a rapid (2 h) and protein synthesis independent mode of action that is dependent on PKC and Src kinase activities. Treatment with a COX‐2 inhibitor partially prevented the attenuation of the keratinocyte inflammatory response by calcitriol. We conclude that up‐regulation of COX‐2 expression with the consequent increase in PGE2 synthesis may be one of the mechanisms explaining the Janus face of calcitriol as both a promoter and attenuator of cutaneous inflammation. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T01:48:46.324798-05:
      DOI: 10.1002/jcp.25132
  • Comparison of Dasatinib, Nilotinib and Imatinib in the Treatment of
           Chronic Myeloid Leukemia
    • Authors: Roberto Ciarcia; Sara Damiano, Maria Valeria Puzio, Serena Montagnaro, Francesco Pagnini, Carmen Pacilio, Giuseppe Caparrotti, Cristiana Bellan, Tiziana Garofano, Maria Sole Polito, Antonio Giordano, Salvatore Florio
      Abstract: To overcome the drug resistance phenomenon induced by Imatibib (IM), in clinical practice, are often used second generation of tyrosine kinase inhibitors as Nilotinib (NIL) a such potent inhibitor of the BCR/ABL kinase and Dasatinib (DAS) a inhibitor of BCR/ABL kinase and inhibitor SrC family kinase. In this study we evaluated the in vivo effect of DAS, NIL and IM on intracellular calcium concentration, oxidative stress and apoptosis in peripheral blood leukocytes of 45 newly diagnosed patients with chronic myeloid leukaemia (CML‐PBM). Our data demonstrated that treatment with DAS and NIL showed an higher modulating potential than IM on intracellular calcium concentration by inhibiting the thapsigargin, a sarcoplasmic/endoplasmic reticulum Ca2+‐ATPase (SERCA) inhibitor and Lithium (Li) an inositol 1,4,5‐triphosphate (InsP3) receptor inhibitor activities. Moreover our data demonstrated that NIL and DAS have significantly increased apoptosis more than IM by involving both intracellular calcium signaling as well as oxidative stress. The acquisition of the oxidative stress and calcium channels receptors values data could help the hematologist to modulate and improve the treatment of chronic myeloid leukaemia (CML) pathology. This article is protected by copyright. All rights reserved
      PubDate: 2015-09-01T01:42:17.468069-05:
      DOI: 10.1002/jcp.25118
  • Cell Surface Human Airway Trypsin‐Like Protease Is Lost During
           Squamous Cell Carcinogenesis
    • Authors: Michael J. Duhaime; Khaliph O. Page, Fausto A. Varela, Andrew S. Murray, Michael E. Silverman, Gina L. Zoratti, Karin List
      Abstract: Cancer progression is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix, as well as cleaving and activating growth factors and receptors that are involved in pro‐cancerous signaling pathways. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression, however, the expression or function of the TTSP Human Airway Trypsin‐like protease (HAT) in carcinogenesis has not been examined. In the present study we aimed to determine the expression of HAT during squamous cell carcinogenesis. HAT transcript is present in several tissues containing stratified squamous epithelium and decreased expression is observed in carcinomas. We determined that HAT protein is consistently expressed on the cell surface in suprabasal/apical layers of squamous cells in healthy cervical and esophageal epithelia. To assess whether HAT protein is differentially expressed in normal tissue versus tissue in different stages of carcinogenesis, we performed a comprehensive immunohistochemical analysis of HAT protein expression levels and localization in arrays of paraffin embedded human cervical and esophageal carcinomas compared to the corresponding normal tissue. We found that HAT protein is expressed in the non‐proliferating, differentiated cellular strata and is lost during the dedifferentiation of epithelial cells, a hallmark of squamous cell carcinogenesis. Thus, HAT expression may potentially be useful as a marker for clinical grading and assessment of patient prognosis in squamous cell carcinomas. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-22T17:23:43.72242-05:0
      DOI: 10.1002/jcp.25173
  • MDA‐9/Syntenin Control
    • Authors: Julie Philley; Anbarasu Kannan, Santanu Dasgupta
      Abstract: MDA‐9/Syntenin is a small PDZ domain containing scaffolding protein with diverse array of function regulating membrane trafficking, cell adhesion, neural and synaptic development, ubiquitination and exosome biogenesis. An appreciable number of studies also established a pivotal role of MDA‐9/Syntenin in cancer development and progression. In this review, we will discuss the dynamic role of MDA‐9/Syntenin in regulating normal and abnormal fate of various cellular processes. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-20T17:45:36.201263-05:
      DOI: 10.1002/jcp.25136
  • Defining Plasma MicroRNAs Associated With Cognitive Impairment in
           Hiv‐Infected Patients
    • Abstract: Human Immunodeficiency Virus (HIV)‐infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age‐matched, HIV‐infected (HIV+) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV+ patients with cognitive impairment from those without cognitive impairment. These results justify follow‐on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV+ patients with neurocognitive impairment. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-18T14:15:44.419205-05:
      DOI: 10.1002/jcp.25131
  • Autophagy‐Induced Apoptosis in Lung Cancer Cells By a Novel
           Digitoxin Analog
    • Authors: Yogesh M. Kulkarni; Vivek Kaushik, Neelam Azad, Clayton Wright, Yon Rojanasakul, George O'Doherty, Anand Krishnan V. Iyer
      Abstract: We have synthesized a novel derivative of Digitoxin, termed “MonoD”, which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within one hour of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles and increased expression of pro‐autophagic markers Beclin‐1 and LC3‐II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3‐II and Beclin‐1. Prolonged treatment with MonoD induced apoptosis in lung cancer cells. We investigated MonoD‐dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3‐II and Beclin‐1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD‐dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-11T17:32:01.320926-05:
      DOI: 10.1002/jcp.25129
  • The Histone‐Deacetylase‐Inhibitor Suberoylanilide Hydroxamic
           Acid Promotes Dental Pulp Repair Mechanisms Through Modulation of Matrix
           Metalloproteinase‐13 Activity
    • Authors: Henry F. Duncan; Anthony J. Smith, Garry J. P. Fleming, Nicola C. Partridge, Emi Shimizu, Gary P. Moran, Paul R. Cooper
      Abstract: Direct application of histone‐deacetylase‐inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular‐reparative events. We have previously demonstrated that HDACis (Valproic acid, Trichostatin A) increase mineralization in dental papillae‐derived cell‐lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 μM); while low concentrations (1 μM) SAHA did not increase apoptosis. HDACi‐exposure for 24 h induced mineralization‐per‐cell dose‐dependently after 2 weeks; however, constant 14d SAHA‐exposure inhibited mineralization. Microarray analysis (24 h and 14d) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0‐fold change at 24 h, which reduced to 36 genes at 14d. 59% of genes were down‐regulated at 24 h and 36% at 14d, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA‐supplementation increased MMP‐13 protein expression (7d, 14 d) and enzyme activity (48 h, 14d). Selective MMP‐13‐inhibition (MMP‐13i) dose‐dependently accelerated mineralization in both SAHA‐treated and non‐treated cultures. MMP‐13i‐supplementation promoted expression of several mineralization‐associated markers, however, HDACi‐induced cell migration and wound healing were impaired. Data demonstrate that short‐term low‐dose SAHA‐exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP‐13i further increased mineralization‐associated events, but decreased HDACi cell migration indicating a specific role for MMP‐13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-11T17:31:15.884875-05:
      DOI: 10.1002/jcp.25128
  • Nuclear Translocation of p65 is Controlled by Sec6 Via the Degradation of
    • Authors: Toshiaki Tanaka; Mitsuyoshi Iino
      Abstract: Nuclear factor‐κB (NF‐κB) is an inducible transcription factor that mediates immune and inflammatory responses. NF‐κB pathways are also involved in cell adhesion, differentiation, proliferation, autophagy, senescence, and protection against apoptosis. The deregulation of NF‐κB activity is found in a number of disease states, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease. The 90 kDa ribosomal S6 kinase (p90RSK) family, which is serine/threonine kinases, is phosphorylated by extracellular signal‐regulated kinase1/2 (ERK1/2) and is related to NF‐κB pathways. Our previous studies revealed that Sec6, a component of the exocyst complex, plays specific roles in cell‐cell adhesion and cell cycle arrest. However, the mechanism by which Sec6 regulates the NF‐κB signaling pathway is unknown. We demonstrated that Sec6 knockdown inhibited the degradation of IκBα and delayed the nucleus‐cytoplasm translocation of p65 in HeLa cells transfected with Sec6 siRNAs after treatment with tumor necrosis factor alpha (TNF‐α). Furthermore, the binding of p65 and cAMP response element binding protein (CREB) binding protein (CBP) or p300 decreased and NF‐κB related genes which were inhibitors of NF‐κB alpha (IκBα), A20, B cell lymphoma protein 2 (Bcl‐2), and monocyte chemoattractant protein‐1 (MCP‐1) were low in cells transfected with Sec6 siRNAs in response to TNF‐α stimulation. Sec6 knockdown decreased the expression of p90RSKs and the phosphorylation of ERK or p90RSK1 at Ser380 or IκBα at Ser32. The present study suggests that Sec6 regulates NF‐κB transcriptional activity via the control of the phosphorylation of IκBα, p90RSK1, and ERK. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-06T07:01:22.740635-05:
      DOI: 10.1002/jcp.25122
  • CD44 Influences Fibroblast Behaviors Via Modulation of Cell‐Cell and
           Cell‐Matrix Interactions, Affecting Survivin and Hippo Pathways
    • Authors: Masayuki Tsuneki; Joseph A. Madri
      Abstract: CD44 has been studied in a wide variety of cell types, in a diverse array of cell behaviors and in a diverse range of signaling pathways. We now document a role for CD44 in mediating fibroblast behaviors via regulation of N‐cadherin, extracellular matrix expression, Survivin and the Hippo pathway. Here we report our findings on the roles of CD44 in modulating proliferation, apoptosis, migration and invasion of murine wild‐type (WT‐FB) and CD44 knockout dermal fibroblasts (CD44KO‐FB). As we have documented in microvascular endothelial cells lacking CD44, we found persistent increased proliferation, reduced activation of cleaved caspase 3, increased initial attachment, but decreased strength of cell attachment in high cell density, post confluent CD44KO‐FB cultures. Additionally, we found that siRNA knock‐down of CD44 mimicked the behaviors of CD44KO‐FB, restoring the decreases in N‐cadherin, collagen type I, fibronectin, Survivin, nuclear fractions of YAP and phospho‐YAP and decreased levels of cleaved caspase 3 to the levels observed in CD44KO‐FB. Interestingly, plating CD44KO‐FB on collagen type I or fibronectin resulted in significant decreases in secondary proliferation rates compared to plating cells on non‐coated dishes, consistent with increased cell adhesion compared to their effects on WT‐FB. Lastly, siRNA knockdown of CD44 in WT‐FB resulted in increased fibroblast migration compared to WT‐FB, albeit at reduced rates compared to CD44KO‐FB. These results are consistent with CD44's pivotal role in modulating several diverse behaviors important for adhesion, proliferation, apoptosis, migration and invasion during development, growth, repair, maintenance and regression of a wide variety of mesenchymal tissues. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-06T07:00:50.701007-05:
      DOI: 10.1002/jcp.25123
  • Retinoblastoma Protein Knockdown Favors Oxidative Metabolism and Glucose
           and Fatty Acid Disposal in Muscle Cells
    • Abstract: Deficiency in the retinoblastoma protein (Rb) favors leanness and a healthy metabolic profile in mice largely attributed to activation of oxidative metabolism in white and brown adipose tissues. Less is known about Rb modulation of skeletal muscle metabolism. This was studied here by transiently knocking down Rb expression in differentiated C2C12 myotubes using small interfering RNAs. Compared with control cells transfected with non‐targeting RNAs, myotubes silenced for Rb (by 80–90%) had increased expression of genes related to fatty acid uptake and oxidation such as Cd36 and Cpt1b (by 61% and 42%, respectively), increased Mitofusin 2 protein content (∼2.5‐fold increase), increased mitochondrial to nuclear DNA ratio (by 48%), increased oxygen consumption (by 65%) and decreased intracellular lipid accumulation. Rb silenced myotubes also displayed up‐regulated levels of glucose transporter type 4 expression (∼5‐fold increase), increased basal glucose uptake, and enhanced insulin‐induced Akt phosphorylation. Interestingly, exercise in mice led to increased Rb phosphorylation (inactivation) in skeletal muscle as evidenced by immunohistochemistry analysis. In conclusion, the silencing of Rb enhances mitochondrial oxidative metabolism and fatty acid and glucose disposal in skeletal myotubes, and changes in Rb status may contribute to muscle physiological adaptation to exercise. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-04T06:58:00.065611-05:
      DOI: 10.1002/jcp.25121
  • Impaired Hematopoiesis and Disrupted Monocyte/Macrophage Homeostasis in
           Mucopolysaccharidosis Type I Mice
    • Abstract: Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disease caused by alpha‐L‐iduronidase deficiency in which heparan and dermatan sulfate degradation is compromised. Besides primary lysosomal glycosaminoglycan accumulation, further changes in cellular functions have also been described in several murine MPS models. Herein, we evaluated alterations in hematopoiesis and its implications on the production of mature progeny in a MPS I murine model. Despite the significant increase in hematopoietic stem cells, a reduction in common myeloid progenitors and granulocyte‐macrophage progenitor cells was observed in Idua ‐/‐ mice bone marrow. Furthermore, no alterations in number, viability nor activation of cell death mechanisms were observed in Idua ‐/‐ mice mature macrophages but they presented higher sensitivity to apoptotic induction after staurosporine treatment. In addition, changes in Ca2+ signaling and a reduction in phagocytosis ability were also found. In summary, our results revealed significant intracellular changes in mature Idua ‐/‐ macrophages related to alterations in Idua ‐/‐ mice hematopoiesis, revealing a disruption in cell homeostasis. These results provide new insights into physiopathology of MPS I. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-03T06:59:30.534856-05:
      DOI: 10.1002/jcp.25120
  • Fucoidan Suppresses the Growth of Human Acute Promyelocytic Leukemia Cells
           In Vitro and In Vivo
    • Authors: Farzaneh Atashrazm; Ray M Lowenthal, Gregory M Woods, Adele F Holloway, Samuel S Karpiniec, Joanne L Dickinson
      Abstract: Fucoidan, a natural component of seaweeds, is reported to have immunomodulatory and anti‐tumor effects. The mechanisms underpinning these activities remain poorly understood. In this study, the cytotoxicity and anti‐tumor activities of fucoidan were investigated in acute myeloid leukemia (AML) cells. The human AML cell lines NB4, KG1a, HL60 and K562 were treated with fucoidan and cell cycle, cell proliferation and expression of apoptotic pathways molecules were analyzed. Fucoidan suppressed the proliferation and induced apoptosis through the intrinsic and extrinsic pathways in the acute promyelocytic leukemia (APL) cell lines NB4 and HL60, but not in KG1a and K562 cells. In NB4 cells, apoptosis was caspase‐dependent as it was significantly attenuated by pre‐treatment with a pan‐caspase inhibitor. P21/WAF1/CIP1 was significantly up‐regulated leading to cell cycle arrest. Fucoidan decreased the activation of ERK1/2 and down‐regulated the activation of AKT through hypo‐phosphorylation of Thr(308) residue but not Ser(473). In vivo, a xenograft model using the NB4 cells was employed. Mice were fed with fucoidan and tumor growth was measured following inoculation with NB4 cells. Subsequently, splenic natural killer (NK) cell cytotoxic activity was also examined. Oral doses of fucoidan significantly delayed tumor growth in the xenograft model and increased cytolytic activity of NK cells. Taken together, these data suggest that the selective inhibitory effect of fucoidan on APL cells and its protective effect against APL development in mice warrant further investigation of fucoidan as a useful agent in treatment of certain types of leukemia. This article is protected by copyright. All rights reserved
      PubDate: 2015-08-03T06:58:16.202878-05:
      DOI: 10.1002/jcp.25119
  • Hypoxia Promotes the Inflammatory Response and Stemness Features in
           Visceral Fat Stem Cells from Obese Subjects
    • Authors: Elisa Petrangeli; Giuseppe Coroniti, Anna T. Brini, Laura de Girolamo, Deborah Stanco, Stefania Niada, Gianfranco Silecchia, Emanuela Morgante, Carla Lubrano, Matteo A. Russo, Luisa Salvatori
      Abstract: Low‐grade chronic inflammation is a salient feature of obesity and many associated disorders. This condition frequently occurs in central obesity and is connected to alterations of the visceral adipose tissue (AT) microenvironment. Understanding how obesity is related to inflammation may allow the development of therapeutics aimed at improving metabolic parameters in obese patients. To achieve this aim, we compared the features of 2 subpopulations of adipose‐derived stem cells (ASC) isolated from both subcutaneous and visceral AT of obese patients with the features of 2 subpopulations of ASC from the same isolation sites of non‐obese individuals. In particular, the behavior of ASC of obese vs non‐obese subjects during hypoxia, which occurs in obese AT and is an inducer of the inflammatory response, was evaluated. Obesity deeply influenced ASC from visceral AT (obV‐ASC); these cells appeared to exhibit clearly distinguishable morphology and ultrastructure as well as reduced proliferation, clonogenicity and expression of stemness, differentiation and inflammation‐related genes. These cells also exhibited a deregulated response to hypoxia, which induced strong tissue‐specific NF‐kB activation and an NF‐kB‐mediated increase in inflammatory and fibrogenic responses. Moreover, obV‐ASC, which showed a less stem‐like phenotype, recovered stemness features after hypoxia. Our findings demonstrated the peculiar behavior of obV‐ASC, their influence on the obese visceral AT microenvironment and the therapeutic potential of NF‐kB inhibitors. These novel findings suggest that the deregulated hyper‐responsiveness to hypoxic stimulus of ASC from visceral AT of obese subjects may contribute via paracrine mechanisms to low‐grade chronic inflammation, which has been implicated in obesity‐related morbidity. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-30T05:15:07.745025-05:
      DOI: 10.1002/jcp.25113
  • Regulation of Spermatogenic Cell T‐Type Ca2+ Currents by Zn2+:
           Implications in Male Reproductive Physiology
    • Abstract: Zn2+ is a trace metal which is important for spermatogenesis progression; its deficiency causes atrophy or malignant growth of the testis. Although testis, epididymis and prostate contain high Zn2+ concentrations, the molecular entities which are modulated by this metal are still under study. Interestingly, spermatogenic cells mainly express CaV3.2‐encoded T‐type Ca2+ currents (ICaT) which are positively or negatively modulated by Zn2+ in other tissues. To explore whether ICaT could be regulated by Zn2+ and albumin, its main physiological carrier, we performed whole cell electrophysiological recordings of spermatogenic cell ICaT in the absence or presence of different Zn2+ concentrations. Zn2+ decreased ICaT in a concentration‐dependent manner (IC50= 2 μM) and this inhibition could only be completely removed in presence of albumin. Differently to previous reports, ICaT did not show a tonic inhibition by Zn2+. Further analysis showed that Zn2+ did not affect the voltage dependency or the kinetics of current activation, but right shifted the steady‐state inactivation curve and slowed inactivation and deactivation kinetics. Recovery from inactivation was also altered. However, these apparent alterations in gating properties are not enough to explain the strong ICaT reduction. Using non‐stationary fluctuation analysis, we found that Zn2+ mainly reduced the number of available Ca2+ channels without changing the single channel current amplitude. ICaT modulation by Zn2+ could be relevant for spontaneous Ca2+ oscillations during spermatogenesis and in pathophysiological conditions such as diabetes. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-29T09:24:14.760195-05:
      DOI: 10.1002/jcp.25112
  • BMP‐2 Induced Expression of PLCβ1 That Is a Positive Regulator
           of Osteoblast Differentiation
    • Authors: Giulia Ramazzotti; Alberto Bavelloni, William Blalock, Manuela Piazzi, Lucio Cocco, Irene Faenza
      Abstract: Bone morphogenetic protein 2 (BMP‐2) is a critical growth factor that directs osteoblast differentiation and bone formation. Phosphoinositide‐phospholipase Cβ 1 (PLCβ1) plays a crucial role in the initiation of the genetic program responsible for muscle differentiation. Differentiation of C2C12 mouse myoblasts in response to insulin stimulation is characterized by a marked increase in nuclear PLCβ1. Here, the function of PLCβ1 in the osteogenic differentiation was investigated. Briefly, in C2C12 cells treated with BMP‐2 we assist to a remarkable increase in PLCβ1 protein and mRNA expression. The data regarding the influence on differentiation demonstrated that PLCβ1 promotes osteogenic differentiation by up‐regulating alkaline phosphatase (ALP). Moreover, PLCβ1 is present in the nuclear compartment of these cells and overexpression of a cytosolic‐PLCβ1mutant (cyt‐PLCβ1), which lacks a nuclear localization sequence, prevented the differentiation of C2C12 cells into osteocytes. Recent evidence indicates that miRNAs act as important post transcriptional regulators in a large number of processes, including osteoblast differentiation. Since miR‐214 is a regulator of Osterix (Osx) which is an osteoblast‐specific transcription factor that is needful for osteoblast differentiation and bone formation, we further investigated whether PLCβ1 could be a potential target of miR‐214 in the control of osteogenic differentiation by gain‐ and loss‐ of function experiment. The results indicated that inhibition of miR‐214 in C2C12 cells significantly enhances the protein level of PLCβ1 and promotes C2C12 BMP‐2‐induced osteogenesis by targeting PLCβ1. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:43:25.727826-05:
      DOI: 10.1002/jcp.25107
  • Leptin Effect on Acetylation and Phosphorylation of PGC1α in Muscle
           Cells Associated With AMPK and Akt Activation in High‐Glucose Medium
    • Abstract: Leptin is crucial in energy metabolism, including muscle regulation. Peroxisome proliferator activated receptor gamma co‐activator 1α (PGC1α) orchestrates energy metabolism and is tightly controlled by post‐translational covalent modifications such as phosphorylation and acetylation. We aimed to further the knowledge of PGC1α control by leptin (at physiological levels) in muscle cells by time‐sequentially analysing the activation of AMP activated protein kinase (AMPK), p38 mitogen‐activated protein kinase (p38 MAPK) and Akt (Protein kinase B) – all known to phosphorylate PGC1α and to be involved in the regulation of its acetylation status – in C2C12 myotubes placed in a high‐glucose serum‐free medium. We also studied the protein levels of PGC1α, Sirtuin 1, adiponectin, COX IV, mitofusin 2 (Mfn2) and pyruvate dehydrogenase kinase 4 (PDK4). Our main findings suggest an important role of leptin regulating AMPK and Akt phosphorylation, mitofusin 2 induction and PGC1α acetylation status, with the novelty that the latter in transitorily increased in response to leptin, an effect dependent, at least in part, on AMPK regulation. These post‐translational reversible changes in PGC1α in response to leptin, especially the increase in acetylation status, may be related to the physiological role of the hormone in modulating muscle cell response to the physiological/nutritional status. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:41:43.604498-05:
      DOI: 10.1002/jcp.25109
  • Sustained Inhibition of Proliferative Response After Transient FGF
           Stimulation is Mediated by Interleukin 1 Signaling
    • Authors: Ashleigh Poole; Doreen Kacer, Emily Cooper, Francesca Tarantini, Igor Prudovsky
      Abstract: Transient FGF stimulation of various cell types results in FGF memory – a sustained blockage of efficient proliferative response to FGF and other growth factors. FGF memory establishment requires HDAC activity, indicating its epigenetic character. FGF treatment stimulates proinflammatory NFκB signaling, which is also critical for FGF memory formation. The search for FGF‐induced mediators of FGF memory revealed that FGF stimulates HDAC‐dependent expression of the inflammatory cytokine IL1α. Similarly to FGF, transient cell treatment with recombinant IL1α inhibits the proliferative response to further FGF and EGF stimulation, but does not prevent FGF receptor‐mediated signaling. Interestingly, like cells pretreated with FGF1, cells pretreated with IL1α exhibit enhanced restructuring of actin cytoskeleton and increased migration in response to FGF stimulation. IRAP, a specific inhibitor of IL1 receptor, and a neutralizing anti‐IL1α antibody prevent the formation of FGF memory and rescue an efficient proliferative response to FGF restimulation. A similar effect results following treatment with the anti‐inflammatory agents aspirin and dexamethasone. Thus, FGF memory is mediated by proinflammatory IL1 signaling. It may play a role in the limitation of proliferative response to tissue damage and prevention of wound‐induced hyperplasia. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:41:20.046371-05:
      DOI: 10.1002/jcp.25111
  • Failure to Target RANKL Signaling Through p38‐MAPK Results in
           Defective Osteoclastogenesis in the Microphthalmia Cloudy‐eyed
    • Abstract: The microphthalmia‐associated transcription factor (MITF) is a basic helix‐loop‐helix leucine zipper family factor that is essential for terminal osteoclast differentiation. Previous work demonstrates that phosphorylation of MITF by p38 MAPK downstream of receptor activator of NFκB ligand (RANKL) signaling is necessary for MITF activation in osteoclasts. The spontaneous Mitf cloudy eyed (ce) allele results in production of a truncated MITF protein that lacks the leucine zipper and C‐terminal end. Here we show that the Mitfce allele leads to a dense bone phenotype in neonatal mice due to defective osteoclast differentiation. In response to RANKL stimulation, in vitro osteoclast differentiation was impaired in myeloid precursors derived from neonatal or adult Mitfce/ce mice. The loss of the leucine zipper domain in Mitfce/ce mice does not interfere with the recruitment of MITF/PU.1 complexes to target promoters. Further, we have mapped the p38 MAPK docking site within the region deleted in Mitfce. This interaction is necessary for the phosphorylation of MITF by p38 MAPK. Site‐directed mutations in the docking site interfered with the interaction between MITF and its co‐factors FUS and BRG1. MITF‐ce fails to recruit FUS and BRG1 to target genes, resulting in decreased expression of target genes and impaired osteoclast function. These results highlight the crucial role of signaling dependent MITF/ p38 MAPK interactions in osteoclast differentiation. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-27T05:40:54.04586-05:0
      DOI: 10.1002/jcp.25108
  • IL‐6 Activates PI3K and PKCζ Signaling and Determines Cardiac
           Differentiation in Rat Embryonic H9c2 Cells
    • Authors: Maria Angela D'amico; Barbara Ghinassi, Pascal Izzicupo, Annalisa Di Ruscio, Angela Di Baldassarre
      Abstract: Introduction IL‐6 influences several biological processes, including cardiac stem cell and cardiomyocyte physiology. Although JAK‐STAT3 activation is the defining feature of IL‐6 signaling, signaling molecules such as PI3K, PKCs and ERK1/2 are also activated and elicit different responses. Moreover, most studies on the specific role of these signaling molecules focus on the adult heart, and few studies are available on the biological effects evoked by IL‐6 in embryonic cardiomyocytes. Aim The aim of this study was to clarify the biological response of embryonic heart derived cells to IL‐6 by analyzing the morphological modifications and the signaling cascades evoked by the cytokine in H9c2 cells. Results IL‐6 stimulation determined the terminal differentiation of H9c2 cells, as evidenced by the increased expression of cardiac transcription factors (NKX2.5 and GATA4), structural proteins (α‐myosin heavy chain and cardiac Troponin T) and the gap junction protein Connexin 43. This process was mediated by the rapid modulation of PI3K, Akt, PTEN and PKCζ phosphorylation levels. PI3K recruitment was an upstream event in the signaling cascade and when PI3K was inhibited, IL‐6 failed to modify PKCζ, PTEN and Akt phosphorylation. Blocking PKCζ activity affected only PTEN and Akt. Finally, the overexpression of a constitutively active form of PKCζ in H9c2 cells largely mimicked the morphological and molecular effects evoked by IL‐6. Conclusion This study demonstrated that IL‐6 induces the cardiac differentiation of H9c2 embryonic cells though a signaling cascade that involves PI3K, PTEN and PKCζ activities. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:37:46.759391-05:
      DOI: 10.1002/jcp.25101
  • RhoA‐Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate
    • Abstract: Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho‐family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M) and nanotopography (N) to evaluate the effect of surface topography on RhoA‐mediated functions (cell spreading, adhesion, migration and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S< M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness‐dependent (S 
      PubDate: 2015-07-23T18:37:20.245806-05:
      DOI: 10.1002/jcp.25100
  • The PPARβ/δ Agonist GW0742 Induces Early Neuronal Maturation of
           Cortical Post‐Mitotic Neurons: Role of PPARβ/δ in Neuronal
    • Authors: E. Benedetti; S. Di Loreto, B. D'Angelo, L. Cristiano, M. d'Angelo, A. Antonosante, A. Fidoamore, Golini Raffaella, B. Cinque, MG Cifone, R Ippoliti, A. Giordano, A. Cimini
      Abstract: Increasing evidences support that signalling lipids participate in synaptic plasticity and cell survival, and that the lipid signalling is closely associated with neuronal differentiation, learning and memory and with pathologic events such as epilepsy and Alzheimer's disease. The Peroxisome Proliferator‐Activated Receptors (PPAR) are strongly involved in the fatty acid cell signalling, as many of the natural lypophylic compounds are PPAR ligands. We have previously shown that PPARβ/δ is the main isotype present in cortical neuron primary cultures and that during neuronal maturation, PPARβ/δ is gradually increased and activated. To get more insight into the molecular mechanism by which PPARβ/δ may be involved in neuronal maturation processes, in this work a specific PPARβ/δ agonist, GW0742 was used administered alone or in association with a specific PPARβ/δ antagonist, the GSK0660 and the parameters involved in neuronal differentiation and maturation were assayed. The data obtained demonstrated the strong involvement of PPARβ/δ in neuronal maturation, triggering the agonist an anticipation of neuronal differentiation and the antagonist abolishing the observed effects. These effects appear to be mediated by the activation of both BDNF canonical (BDNF7TrkB) pathway. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:36:55.606691-05:
      DOI: 10.1002/jcp.25103
  • CyclinD1 Down Regulation and Increased Apoptosis Are Common Features of
    • Abstract: Genetic variants within components of the cohesin complex (NIPBL, SMC1A, SMC3, RAD21, PDS5, ESCO2, HDAC8) are believed to be responsible for a spectrum of human syndromes known as “cohesinopathies” that includes Cornelia de Lange Syndrome (CdLS). CdLS is a multiple malformation syndrome affecting almost any organ and causing severe developmental delay. Cohesinopathies seem to be caused by dysregulation of specific developmental pathways downstream of mutations in cohesin components. However, it is still unclear how mutations in different components of the cohesin complex affect the output of gene regulation. In this study, zebrafish embryos and SMC1A‐mutated patient‐derived fibroblasts were used to analyze abnormalities induced by SMC1A loss of function. We show that the knockdown of smc1a in zebrafish impairs neural development, increases apoptosis and specifically down‐regulates Ccnd1 levels. The same down‐regulation of cohesin targets is observed in SMC1A‐mutated patient fibroblasts. Previously, we have demonstrated that haploinsufficiency of NIPBL produces similar effects in zebrafish and in patients fibroblasts indicating a possible common feature for neurological defects and mental retardation in cohesinopathies. Interestingly, expression analysis of Smc1a and Nipbl in developing mouse embryos reveals a specific pattern in the hindbrain, suggesting a role for cohesin in neural development in vertebrates. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:36:04.714796-05:
      DOI: 10.1002/jcp.25106
  • Osteogenic Potential of Human Oral‐Periosteal Cells (PCs) Isolated
           from Different Oral Origin: An In Vitro Study
    • Authors: Gabriele Ceccarelli; Antonio Graziano, Laura Benedetti, Marcello Imbriani, Federica Romano, Francesco Ferrarotti, Mario Aimetti, Gabriella M. Cusella De Angelis
      Abstract: The periosteum is a specialized connective tissue containing multipotent stem cells capable of bone formation. In this study, we aimed at demonstrating that human oral periosteal cells derived from three different oral sites (upper vestibule, lower vestibule and hard palate) represent an innovative cell source for maxillo‐facial tissue engineering applications in terms of accessibility and self‐commitment towards osteogenic lineage. Periosteal cells (PCs) were isolated from patients with different ages (20‐30 yy, 40‐50 yy, 50‐60 yy); we then analyzed the in vitro proliferation capacity and the bone self‐commitment of cell clones culturing them without any osteogenic supplement to support their differentiation. We found that oral PCs, independently of their origin and age of patients, are mesenchymal stem cells with stem cell characteristics (clonogenical and proliferative activity) and that, even in absence of any osteogenic induction, they undertake the osteoblast lineage after 45 days of culture. These results suggest that oral periosteal cells could replace mesenchymal cells from bone marrow in oral tissue‐engineering applications. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:35:38.582213-05:
      DOI: 10.1002/jcp.25104
  • Reciprocal Control of Osteogenic and Adipogenic Differentiation by ERK/MAP
           Kinase Phosphorylation of Runx2 and PPARγ Transcription Factors
    • Authors: Chunxi Ge; William P. Cawthorn, Yan Li, Guisheng Zhao, Ormond A. MacDougald, Renny T. Franceschi
      Abstract: In many skeletal diseases, including osteoporosis and disuse osteopenia, defective osteoblast differentiation is associated with increased marrow adipogenesis. The relative activity of two transcription factors, RUNX2 and PPARγ, controls whether a mesenchymal cell will differentiate into an osteoblast or adipocyte. Herein we show that the ERK/MAP kinase pathway, an important mediator of mechanical and hormonal signals in bone, stimulates osteoblastogenesis and inhibits adipogenesis via phosphorylation of RUNX2 and PPARγ. Induction of osteoblastogenesis in ST2 mesenchymal cells was associated with increased MAPK activity and RUNX2 phosphorylation. Under these conditions PPARγ phosphorylation also increased, but adipogenesis was inhibited. In contrast, during adipogenesis MAPK activity and phosphorylation of both transcription factors was reduced. RUNX2 phosphorylation and transcriptional activity were directly stimulated by MAPK, a response requiring phosphorylation at S301 and S319. MAPK also inhibited PPARγ‐dependent transcription via S112 phosphorylation. Stimulation of MAPK increased osteoblastogenesis and inhibited adipogenesis, while dominant‐negative suppression of activity had the opposite effect. In rescue experiments using Runx2‐/‐ mouse embryo fibroblasts (MEFs), wild type or, to a greater extent, phosphomimetic mutant RUNX2 (S301E,S319E) stimulated osteoblastogenesis while suppressing adipogenesis. In contrast, a phosphorylation‐deficient RUNX2 mutant (S301A,S319A) had reduced activity. Conversely, wild type or, to a greater extent, phosphorylation‐resistant S112A mutant PPARγ strongly stimulated adipogenesis and inhibited osteoblastogenesis in Pparg ‐/‐ MEFs, while S112E mutant PPARγ was less active. Competition between RUNX2 and PPARγ was also observed at the transcriptional level. Together, these studies highlight the importance of MAP kinase signaling and RUNX2/PPARγ phosphorylation in the control of osteoblast and adipocyte lineages. This article is protected by copyright. All rights reserved
      PubDate: 2015-07-23T18:35:14.838053-05:
      DOI: 10.1002/jcp.25102
  • Agents Which Inhibit NF‐κB Signaling Block Spontaneous
           Contractile Activity and Negatively Influence Survival of Developing
    • Authors: C. George Carlson; Lauren Stein, Elizabeth Dole, Ross M. Potter, David Bayless
      Abstract: Inhibiting the NF‐κB signaling pathway provides morphological and functional benefits for the mdx mouse, a model for Duchenne muscular dystrophy characterized by chronic elevations in the nuclear expression of p65, the transactivating component of the NF‐κB complex. The purpose of this study was to examine p65 expression in nondystrophic and mdx myotubes using confocal immunofluorescence, and determine whether inhibitors of the NF‐κB pathway alter myotube development. Primary cultures of nondystrophic and mdx myotubes had identical levels of nuclear and cytosolic p65 expression and exhibited equivalent responses to TNF‐α, thus excluding the hypothesis that the lack of dystrophin is sufficient to induce increases in NF‐κB signaling. The NF‐κB inhibitors pyrrolidine dithiocarbamate (PDTC) and sulfasalazine decreased spontaneous contractile activity and reduced myotube viability in a dose‐ and time‐dependent manner. Similarly, a vivo‐morpholino designed to block translation of murine p65 (m‐p65tb‐vivomorph1) rapidly abolished spontaneous contractile activity, reduced p65 expression measured by confocal immunofluorescence, and induced cell death in primary cultures of nondystrophic and mdx myotubes. Similar effects on p65 immunofluorescence and cell viability were observed following m‐p65tb‐vivomorph1 exposure to spontaneously inactive C2C12 myotubes, while exposure to a control scrambled vivo morpholino had no effect. These results indicate a direct role of the NF‐κB pathway in myotube development and identify a potential therapeutic limitation to the use of NF‐κB inhibitors in treating Duchenne and related muscular dystrophies. This article is protected by copyright. All rights reserved
      PubDate: 2015-06-30T17:53:25.163707-05:
      DOI: 10.1002/jcp.25085
  • Electrospun Type 1 Collagen Matrices Using a Novel Benign Solvent for
           Cardiac Tissue Engineering
    • Authors: Alan M. Punnoose; Anuradha Elamparithi, Sarah Kuruvilla
      Abstract: Electrospinning is a well‐established technique that uses a high electric field to fabricate ultra fine fibrous scaffolds from both natural and synthetic polymers to mimic the cellular microenvironment. Collagen is one of the most preferred biopolymers due to its biocompatibility and widespread occurrence in nature. Electrospinning of Collagen alone has been reported with fluoroalcohols such as Hexafluoroisopropanol (HFIP) and Trifluoroethanol (TFE), which are toxic to the environment. In this study we describe the use of a novel benign binary solvent to generate nanofibers of Collagen type 1, which is non‐toxic and economical. Transmission electron microscopy (TEM) analysis revealed the characteristic feature of native collagen namely the 67 nm banding pattern, confirming that the triple helical structure was maintained. Scanning Electron Microscopy (SEM) analysis showed the fiber diameters to be in the 200‐800 nm range. Biocompatibility of the three dimensional (3D) scaffolds was established by MTT assays using skeletal myoblasts and Confocal Microscopic analysis of immunofluorescent stained sections for muscle specific markers such as Desmin and Actin. Primary neonatal rat ventricular cardiomyocytes seeded onto the scaffolds were able to maintain their contractile function for a period of 17 days. Our work provides evidence that Collagen 1 can be electrospun without combining with other polymers using a novel benign solvent and we are currently exploring the potential of this approach for cardiac and skeletal muscle tissue engineering. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-12T13:48:51.922438-05:
      DOI: 10.1002/jcp.25035
  • Protein Kinase C Is Involved in the Induction of ATP‐Binding
           Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor
           Agonist in Human Macrophages
    • Authors: Etimad A. Huwait; Nishi N. Singh, Daryn R. Michael, Thomas S. Davies, Joe W. E. Moss, Dipak P. Ramji
      Abstract: The transcription of the ATP‐binding cassette transporter A1 (ABCA1) gene, which plays a key anti‐atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c‐Jun N‐terminal kinase (JNK) and phosphoinositide 3‐kinase (PI3K) in the LXRs‐mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have therefore investigated a potential role for PKC in the action of LXR/RXR agonist 22‐(R)‐hydroxycholesterol (22‐(R)‐HC)/9‐cis‐retinoic acid (9cRA) in THP‐1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein‐1 (AP‐1) DNA binding activity in macrophages treated with 22‐(R)‐HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype‐specific inhibitors. These studies therefore reveal a potentially important role for PKC in the action of 22‐(R)‐HC and 9cRA in human macrophages. This article is protected by copyright. All rights reserved
      PubDate: 2015-05-07T18:01:38.140641-05:
      DOI: 10.1002/jcp.25157
  • Rab7 Regulates CDH1 Endocytosis, Circular Dorsal Ruffles Genesis and
           Thyroglobulin Internalization in a Thyroid Cell Line
    • Abstract: Rab7 regulates the biogenesis of late endosomes, lysosomes and autophagosomes. It has been proposed that a functional and physical interaction exists between Rab7 and Rac1 GTPases in CDH1 endocytosis and ruffled border formation. In FRT cells over‐expressing Rab7, increased expression and activity of Rac1 was observed, whereas a reduction of Rab7 expression by RNAi resulted in reduced Rac1 activity, as measured by PAK1 phosphorylation. We found that CDH1 endocytosis was extremely reduced only in Rab7 over‐expressing cells but was unchanged in Rab7 silenced cells. In Rab7 under or over‐expressing cells, Rab7 and LC3B‐II co‐localized and co‐localization in large circular structures occurred only in Rab7 over‐expressing cells. These large circular structures occurred in about 10% of the cell population; some of them (61%) showed co‐localization of Rab7 with cortactin and f‐actin and were identified as circular dorsal ruffles (CDRs), the others as mature autophagosomes. We propose that the over‐expression of Rab7 is sufficient to induce CDRs. Furthermore, in FRT cells, we found that the expression of the insoluble/active form of Rab7, rather than Rab5 or Rab8, was inducible by cAMP and that cAMP‐stimulated FRT cells showed increased PAK1 phosphorylation and were no longer able to endocytose CDH1. Finally, we demonstrated that Rab7 over‐expressing cells are able to endocytose exogenous thyroglobulin via pinocytosis/CDRs more efficiently than control cells. We propose that the major thyroglobulin endocytosis described in thyroid autonomous adenomas due to Rab7 increased expression, occurs via CDRs. This article is protected by copyright. All rights reserved
  • Molecular Mechanism of Transcriptional Regulation of Matrix
           Metalloproteinase‐9 in Diabetic Retinopathy
    • Abstract: Increase in matrix metalloproteinase‐9 (MMP‐9) is implicated in retinal capillary cell apoptosis, a phenomenon which precedes the development of diabetic retinopathy. MMP‐9 promoter has multiple sites for binding the transcriptional factors, including two for activator protein 1 (AP‐1). The binding of AP‐1, a heterodimer of c‐Jun and c‐Fos, is regulated by posttranslational modifications, and in diabetes, deacetylating enzyme, Sirt1, is inhibited. Our aim is to investigate the molecular mechanism of MMP‐9 transcriptional regulation in diabetes. Binding of AP‐1 (c‐Jun, c‐Fos) at the MMP‐9 promoter, and AP‐1 acetylation were analyzed in retinal endothelial cells incubated in normal or high glucose by chromatin‐immunoprecipitation and co‐immunoprecipitation respectively. Role of AP‐1 in MMP‐9 regulation was confirmed by c‐Jun or c‐Fos siRNAs, and that of its acetylation, by Sirt1 overexpression. In vitro results were validated in the retina from diabetic mice overexpressing Sirt1, and in the retinal microvessels from human donors with diabetic retinopathy. In experimental models, AP‐1 binding was increased at the proximal and distal sites of the MMP‐9 promoter, and similar phenomenon was confirmed in the retinal microvessels from human donors with diabetic retinopathy. Silencing of AP‐1, or overexpression of Sirt1 ameliorated glucose‐induced increase in MMP‐9 expression and cell apoptosis. Thus, in diabetes, due to Sirt1 inhibition, AP‐1 is hyperacetylated, which increases its binding at MMP‐9 promoter, and hence, activation of Sirt1 could inhibit the development of diabetic retinopathy by impeding MMP‐9‐mediated mitochondrial damage. This article is protected by copyright. All rights reserved
  • Nuclear Factor κB1/RelA Mediates Inflammation in Human Lung
           Epithelial Cells at Atmospheric Oxygen Levels
    • Abstract: Oxygen levels range from 2‐9% in vivo. Atmospheric O2 levels (21%) are known to induce cell proliferation defects and cellular senescence in primary cell cultures. However, the mechanistic basis of the deleterious effects of higher O2 levels is not fully understood. On the other hand, immortalized cells including cancer cell lines, which evade cellular senescence are normally cultured at 21% O2 and the effects of higher O2 on these cells are understudied. Here we addressed this problem by culturing immortalized human bronchial epithelial (BEAS‐2B) cells at ambient atmospheric, 21% O2 and lower, 10% O2. Our results show increased inflammatory response at 21% O2 but not at 10% O2. We found higher RelA binding at the NF‐κB1/RelA target gene promoters as well as upregulation of several pro‐inflammatory cytokines in cells cultured at 21% O2. RelA knockdown prevented the upregulation of the pro‐inflammatory cytokines at 21% O2, suggesting NF‐κB1/RelA as a major mediator of inflammatory response in cells cultured at 21% O2. Interestingly, unlike the 21% O2 cultured cells, exposure of 10% O2 cultured cells to H2O2 did not elicit inflammatory response, suggesting increased ability to tolerate oxidative stress in cells cultured at lower O2 levels. This article is protected by copyright. All rights reserved
  • Rab, Arf and Arl‐Regulated Membrane Traffic in Cortical Neuron
    • Abstract: The migration of projection neurons from its birthplace in the subventricular zone to their final destination in the cortical plate is a complex process that requires a series of highly coordinated cellular events. Amongst the key factors involved in the processes would include modulators of cytoskeletal dynamics, as well as cellular membrane traffic. Members of the small GTPases family responsible for the latter process, the Rabs and Arfs, have been recently implicated in cortical neuron migration. Rab5 and Rab11, which are key modulators of endocytosis and endocytic recycling respectively, ensure proper surface expression and distribution of N‐cadherin, a key adhesion protein that tethers migrating neurons to the radial glia fiber tracts during pia‐directed migration. Rab7, which is associated with lysosomal biogenesis and function, is important for the final step of terminal translocation when N‐cadherin is downregulated by lysosomal degradation. Arf6 activity, which is known to be important in neuronal processes outgrowth, may negatively impact the multipolar‐bipolar transition of cortical neurons undergoing radial migration, but the downstream effector of Arf6 in this regard is not yet known. In addition to the above, members of the Arl family which have been recently shown to be important in radial glia scaffold formation, would also be important for cortical neuron migration. In this short review, we discuss recent advances in our understanding in the importance of membrane traffic regulated by the Rab, Arf and Arl family members in cortical neuron migration. This article is protected by copyright. All rights reserved
  • The LPV Motif is Essential for the Efficient Export of Secretory DMP1 from
           the Endoplasmic Reticulum
    • Abstract: Dentin matrix protein 1 (DMP1) is found abundantly in the extracellular matrices of bone and dentin. Secretory DMP1 begins with a tripeptide of leucine‐proline‐valine (LPV) after the endoplasmic reticulum (ER)‐entry signal peptide is cleaved. The goal of this study was to determine the role of the LPV motif in the secretion of DMP1. A series of DNA constructs was generated to express various forms of DMP1 with or without the LPV motif. These constructs were transfected into a preosteoblast cell line, the MC3T3‐E1 cells, and the subcellular localization and secretion of various forms of DMP1 were examined by immunofluorescent staining and Western‐blotting analyses. Immunofluorescent staining showed that the LPV‐containing DMP1 variants were primarily localized in the Golgi complex, whereas the LPV‐lacking DMP1 variants were found abundantly within the ER. Western‐blotting analyses demonstrated that the LPV‐containing DMP1 variants were rapidly secreted from the transfected cells, as they did not accumulate within the cells, and the amounts increased in the conditioned media over time. In contrast, the LPV‐lacking DMP1 variants were predominantly retained within the cells, and only small amounts were secreted out of the cells over time. These results suggest that the LPV motif is essential for the efficient export of secretory DMP1 from the ER to the Golgi complex. This article is protected by copyright. All rights reserved
  • Establishment of Immortalized BMP2/4 Double Knock‐Out Osteoblastic
           Cells is Essential for Study of Osteoblast Growth, Differentiation and
    • Abstract: Bone morphogenetic proteins 2 and 4 (BMP2/4) are essential for osteoblast differentiation and osteogenesis. Generation of a BMP2/4 dual knock out (ko/ko) osteoblastic cell line is a valuable asset for studying effects of BMP2/4 on skeletal development. In this study, our goal was to create immortalized mouse deleted BMP2/4 osteoblasts by infecting adenoviruses with Cre recombinase and green fluorescent protein genes into immortalized murine floxed BMP2/4 osteoblasts. Transduced BMP2/4ko/ko cells were verified by green immunofluorescence and PCR. BMP2/4ko/ko osteoblasts exhibited small size, slow cell proliferation rate and cell growth was arrested in G1 and G2 phases. Expression of bone‐relate genes was reduced in the BMP2/4ko/ko cells, resulting in delay of cell differentiation and mineralization. Importantly, extracellular matrix remodeling was impaired in the BMP2/4ko/ko osteoblasts as reflected by decreased Mmp‐2 and Mmp‐9 expressions. Cell differentiation and mineralization were rescued by exogenous BMP2 and/or BMP4. Therefore, we for the first time described establishment of an immortalized deleted BMP2/4 osteoblast line useful for study of mechanisms in regulating osteoblast lineages. This article is protected by copyright. All rights reserved
  • Stretching Impacts Inflammation Resolution in Connective Tissue
    • Abstract: Acute inflammation is accompanied from its outset by the release of specialized pro‐resolving mediators (SPMs), including resolvins, that orchestrate the resolution of local inflammation. We showed earlier that, in rats with subcutaneous inflammation of the back induced by carrageenan, stretching for 10 minutes twice daily reduced inflammation and improved pain, two weeks after carrageenan injection. In this study, we hypothesized that stretching of connective tissue activates local pro‐resolving mechanisms within the tissue in the acute phase of inflammation. In rats injected with carrageenan and randomized to stretch vs. no stretch for 48 hours, stretching reduced inflammatory lesion thickness and neutrophil count, and increased resolvin (RvD1) concentrations within lesions. Furthermore, subcutaneous resolvin injection mimicked the effect of stretching. In ex vivo experiments, stretching of connective tissue reduced the migration of neutrophils and increased tissue RvD1 concentration. These results demonstrate a direct mechanical impact of stretching on inflammation‐regulation mechanisms within connective tissue. This article is protected by copyright. All rights reserved
  • Table of Contents: Volume 231, Number 3
  • Editor's Choice
  • Highlights: Volume 231, Number 3
  • Announcement
  • Punnoose, A.M., Elamparithi, A., and Kuruvilla, S. (2015), Electrospun
           Type 1 Collagen Matrices Using a Novel Benign Solvent for Cardiac Tissue
           Engineering. J. Cell. Physiol. doi: 10.1002/jcp.25035
  • Autophagy as a Potential Target for Sarcopenia
    • Abstract: Sarcopenia is an aging‐related disease with a significant reduction in mass and strength of skeletal muscle due to the imbalance between protein synthesis and protein degradation. The loss of skeletal muscle is an inevitable event during aging process, which can result in the significant impact on the quality of life, and also can increase the risk for other aging‐associated diseases in the elderly. However, the underlying molecular mechanism of aging‐related skeletal muscle loss is still poorly understood. Autophagy is a degradation pathway for the clearance of dysfunctional organelles and damaged macromolecules during aging process. Appropriate induction or accurate regulation of autophagic process and improved quality control of mitochondria through autophagy or other strategies are required for the maintenance of skeletal muscle mass. In this article, we have summarized the current understanding of autophagic pathways in sarcopenia, and discussed the functional status of autophagy and autophagy‐associated quality control of mitochondria in the pathogenesis of sarcopenia. Moreover, this article will provide some theoretical references for the exploration of scientific and optimal intervention strategies such as exercise and caloric restriction for the prevention and treatment of sarcopenia through the regulation of autophagic pathways. This article is protected by copyright. All rights reserved
  • Fusion Pore Size Limits 5‐HT Release from Single Enterochromaffin
           Cell Vesicles
    • Abstract: Enterochromaffin cells are the major site of serotonin (5‐HT) synthesis and secretion providing ∼95% of the body's total 5‐HT. 5‐HT can act as a neurotransmitter or hormone and has several important endocrine and paracrine roles. We have previously demonstrated that EC cells release small amounts of 5‐HT per exocytosis event compared to other endocrine cells. We utilised a recently developed method to purify EC cells to demonstrate the mechanisms underlying 5‐HT packaging and release. Using the fluorescent probe FFN511 we demonstrate that EC cells express VMAT and that VMAT plays a functional role in 5‐HT loading into vesicles. Carbon fibre amperometry studies illustrate that the amount of 5‐HT released per exocytosis event from EC cells is dependent on both VMAT and the H+‐ATPase pump, as demonstrated with reserpine or bafilomycin, respectively. We also demonstrate that increasing the amount of 5‐HT loaded into EC cell vesicles does not result in an increase in quantal release. As this indicates that fusion pore size may be a limiting factor involved, we compared pore diameter in EC and chromaffin cells by assessing the vesicle capture of different‐sized fluorescent probes to measure the extent of fusion pore dilation. This identified that EC cells have a reduced fusion pore expansion that does not exceed 9 nm in diameter. These results demonstrate that the small amounts of 5‐HT released per fusion event in EC cells can be explained by a smaller fusion pore that limits 5‐HT release capacity from individual vesicles. This article is protected by copyright. All rights reserved
  • Alternative RUNX1 Promoter Regulation by Wnt/β‐Catenin
           Signaling in Leukemia Cells and Human Hematopoietic Progenitors
    • Abstract: Two distantly located promoter regions regulate the dynamic expression of RUNX genes during development: distal P1 and proximal P2 promoters. We have recently described that β‐catenin increases total Runx1 mRNA levels in human CD34+ hematopoietic progenitors and enhances spatial proximity with its translocation partner ETO. Here we report that induction of Wnt/β‐catenin signaling in HL60 and Jurkat leukemia‐derived cell lines and CD34+ progenitors selectively activate the production of the longer distal P1‐Runx1 mRNA isoform. Gain‐ and loss‐of‐function experiments revealed that the differential increase in P1‐Runx1 expression is accomplished through a minimal β‐catenin responsive region that includes a highly conserved TCF/LEF‐binding element, located ‐20/‐16 bp upstream of the canonical distal P1‐Runx1 transcription start site. We conclude that the distal P1‐Runx1 promoter is a direct transcriptional target of Wnt/β‐catenin signaling that may be important in normal hematopoiesis or its transition into malignant stem cells during the onset or progression of leukemia. This article is protected by copyright. All rights reserved
  • Endothelial and Epithelial Cell Transition to a Mesenchymal Phenotype Was
           Delineated by Nestin Expression
    • Abstract: Endothelial and epithelial cell transition to a mesenchymal phenotype was identified as cellular paradigms implicated in the appearance of fibroblasts and development of reactive fibrosis in interstitial lung disease. The intermediate filament protein nestin was highly expressed in fibrotic tissue, detected in fibroblasts and participated in proliferation and migration. The present study tested the hypothesis that the transition of endothelial and epithelial cells to a mesenchymal phenotype was delineated by nestin expression. Three weeks following hypobaric hypoxia, adult male Sprague‐Dawley rats characterized by alveolar and perivascular lung fibrosis were associated with increased nestin protein and mRNA levels and marked appearance of nestin/collagen type I(+)‐fibroblasts. In the perivascular region of hypobaric hypoxic rats, displaced CD31(+)‐endothelial cells were detected, exhibited a mesenchymal phenotype and co‐expressed nestin. Likewise, epithelial cells in the lungs of hypobaric hypoxic rats transitioned to a mesenchymal phenotype distinguished by the co‐expression of E‐cadherin and collagen. Following the removal of FBS from primary passage rat alveolar epithelial cells, TGF‐β1 was detected in the media and a subpopulation acquired a mesenchymal phenotype characterized by E‐cadherin downregulation and concomitant induction of collagen and nestin. Bone morphogenic protein‐7 treatment of alveolar epithelial cells prevented E‐cadherin downregulation, suppressed collagen induction but partially inhibited nestin expression. These data support the premise that the transition of endothelial and epithelial cells to a mesenchymal cell may have contributed in part to the appearance nestin/collagen type I(+)‐fibroblasts and the reactive fibrotic response in the lungs of hypobaric hypoxic rats. This article is protected by copyright. All rights reserved
  • The Role of Kv1.2 Channel in Electrotaxis Cell Migration
    • Abstract: Voltage‐gated potassium Kv1.2 channels play pivotal role in maintaining of resting membrane potential and, consequently, regulation of cellular excitability of neurons. Endogenously generated electric field (EF) have been proven as an important regulator for cell migration and tissue repair. The mechanisms of ion channel involvement in EF‐induced cell responses are extensively studied but largely are poorly understood. In this study we generated three COS‐7 clones with different expression levels of Kv1.2 channel, and confirmed their functional variations with patch clamp analysis. Time‐lapse imaging analysis showed that EF‐induced cell migration response was Kv1.2 channel expression level depended. Inhibition of Kv1.2 channels with charybdotoxin (ChTX) constrained the sensitivity of COS‐7 cells to EF stimulation more than their motility. Immunocytochemistry and pull‐down analyses demonstrated association of Kv1.2 channels with actin‐binding protein cortactin and its re‐localization to the cathode‐facing membrane at EF stimulation, which confirms the mechanism of EF‐induced directional migration. This study displays that Kv1.2 channels represent an important physiological link in EF‐induced cell migration. The described mechanism suggests a potential application of EF which may improve therapeutic performance in curing injuries of neuronal and/or cardiac tissue repair, post operational therapy and various degenerative syndromes. This article is protected by copyright. All rights reserved
  • Mitogen‐Dependent Regulation of DUSP1 Governs ERK and p38 Signaling
           During Early 3T3‐L1 Adipocyte Differentiation
    • Abstract: Knowledge concerning mechanisms that control proliferation and differentiation of preadipocytes is essential to our understanding of adipocyte hyperplasia and the development of obesity. Evidence has shown that temporal regulation of mitogen‐activated protein kinase (MAPK) phosphorylation and dephosphorylation is critical for coupling extracellular stimuli to cellular growth and differentiation. Using differentiating 3T3‐L1 preadipocytes as a model of adipocyte hyperplasia, we examined a role for dual‐specificity phosphatase 1 (DUSP1) on the timely modulation of MAPK signaling during states of growth arrest, proliferation, and differentiation. Using real‐time reverse transcription PCR (qRT‐PCR), we report that DUSP1 is induced during early preadipocyte proliferation concomitant with ERK and p38 dephosphorylation. As deactivation of ERK and p38 is essential for the progression of adipocyte differentiation, we further showed that de novo mRNA synthesis was required for ERK and p38 dephosphorylation, suggesting a role for ‘inducible’ phosphatases in regulating MAPK signaling. Pharmacological and genetic inhibition of DUSP1 markedly increased ERK and p38 phosphorylation during early adipocyte differentiation. Based on these findings, we postulated that loss of DUSP1 would block adipocyte hyperplasia. However, genetic loss of DUSP1 was not sufficient to prevent preadipocyte proliferation or differentiation, suggesting a role for other phosphatases in the regulation of adipogenesis. In support of this, qRT‐PCR identified several MAPK‐specific DUSPs induced during early (DUSP2, ‐4, ‐5, & ‐6), mid (DUSP4 & ‐16) and late (DUSP9) stages of adipocyte differentiation. Collectively, these data suggest an important role for DUSPs in regulating MAPK dephosphorylation, with an emphasis on DUSP1, during early adipogenesis. This article is protected by copyright. All rights reserved
  • High Methionine Diet Poses Cardiac Threat: A Molecular Insight
    • Abstract: High methionine diet (HMD) for example red meat which includes lamb, beef, pork can pose cardiac threat and vascular dysfunction but the mechanisms are unclear. We hypothesize that a diet rich in methionine can malfunction the cardiovascular system in three ways: 1) by augmenting oxidative stress; 2) by inflammatory manifestations; and 3) by matrix/vascular remodeling. To test this hypothesis we used four groups of mice 1) WT; 2) WT + methionine; 3) CBS+/−; 4) CBS+/− + methionine. We observed high oxidative stress in mice fed with methionine which was even higher in CBS+/− and CBS+/− + methionine. Higher oxidative stress was indicated by high levels of SOD‐1 in methionine fed mouse hearts while IL‐1, IL‐6, TNFα and TLR4 showed high inflammatory manifestations. The upregulated levels of eNOS/iNOS and upregulated levels of MMP2/MMP9 along with high collagen deposition indicated vascular and matrix remodeling in methionine fed mouse. We evaluated the cardiac function which was dysregulated in the mice fed with HMD. These mice had decreased ejection fraction and left ventricular dysfunction which subsequently leads to adverse cardiac remodeling. In conclusion our study clearly shows that HMD poses a cardiac threat by increasing oxidative stress, inflammatory manifestations, matrix/vascular remodeling and decreased cardiac function. This article is protected by copyright. All rights reserved
  • MSCs: The Sentinel and Safe‐Guards of Injury
    • Abstract: Mesenchymal Stem Cells (MSCs) were originally named because they could differentiate in a variety of mesenchymal phenotypes in culture. Evidence indicates that MSCs arise from perivascular cells, pericytes, when the blood vessels are broken or inflamed. These pericyte/MSCs are situated on every blood vessel in the body. The MSCs sense the microenvironment of the injury site and secrete a site‐specific factor that serve several important reparative functions: First, a curtain of molecules from the front of the MSCs provide a barrier from the interrogation of the over‐aggressive immune system. Second, from the back of the MSCs, a different set of bioactive agents inhibit scar formation and establish a regenerative microenvironment. Third, if bacteria are sensed by the MSCs, they produce powerful protein antibiotics that kill the bacteria on contact. Last, the MSCs surround and encyst intruding solid objects like a piece of wood (a “splinter”) or other foreign objects. The MSCs act as a combination paramedic and emergency room (ER) staff to survey the damage, isolate foreign components, stabilize the injured tissues, provide antibiotics and encysting protection before a slower, medicinal sequence can be initiated to regenerate the damaged tissue. The MSCs thus act as sentinels to safeguard the individual from intrusion and chronic injury. A societal treatment system has evolved, paramedics and ER procedures, which mirror in a macro‐ sense what MSCs orchestrate in a micro‐ sense. Key to this new understanding is that MSCs are not “stem cells”, but rather as Medicinal Signaling Cells as the therapeutic agents. This article is protected by copyright. All rights reserved
  • The Spectrum of E2F in Liver Disease – Mediated Regulation in
           Biology and Cancer
    • Abstract: Uncoordinated cell growth is one of the fundamental concepts in carcinogenesis and occurs secondary to dysregulation of the cell cycle. The E2Fs are a large family of transcription factors and are key regulators of the cell cycle. The activation of E2Fs is intimately regulated by retinoblastoma 1 (RB1). The RB pathway has been implicated in almost every human malignancy. Recently there have been exciting developments in the E2F field using animal models to better understand the role of E2Fs in vivo. Genetic mouse models have proven essential in implicating E2Fs in hepatocellular carcinoma (HCC) and liver disease. In this review, the general structure and function of E2Fs as well as the role for E2Fs in the development of HCC and liver disease is evaluated. Specifically what is known about E2Fs in human disease is explored in depth and future directions are discussed. This article is protected by copyright. All rights reserved
  • Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial
           Cells to Galectin‐1
    • Abstract: Galectins play key roles in the inflammatory cascade. In this study we aimed to analyze the effect of galectin‐1 (Gal‐1) on function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro‐inflammatory cytokines, and Gal‐1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal‐1 binding and modulation of IECs. We found an enhanced binding of Gal‐1 to: a) murine IECs exposed to IL‐1β, TNF, and IL‐13, b) IECs from inflamed areas in intestinal tissue from IBD patients, c) small bowel of allergic mice and d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal‐1 favored a tolerogenic microenvironment, while high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase‐9‐ and modulation of Bcl‐2 protein family members. Our results showed that, when added in the presence of diverse pro‐inflammatory cytokines (TNF, IL‐13 and IL‐5), Gal‐1 differentially promoted the secretion of growth factors including TSLP, EGF, IL‐10, IL‐25, and TGF‐β1. In conclusion, we found an augmented binding of Gal‐1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal‐1 concentration. These findings highlight the importance of the inflammatory microenvironment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal‐1 and its role in epithelial cell homeostasis. This article is protected by copyright. All rights reserved
  • Sex Steroids Influence Brain‐Derived Neurotropic Factor Secretion
           from Human Airway Smooth Muscle Cells
    • Abstract: Brain derived neurotropic factor (BDNF) is emerging as an important player in airway inflammation, remodeling, and hyperreactivity. Separately, there is increasing evidence that sex hormones contribute to pathophysiology in the lung. BDNF and sex steroid signaling are thought to be intricately linked in the brain. There is currently little information on BDNF and sex steroid interactions in the airway, but is relevant to understanding growth factor signaling in the context of asthma in men vs. women. In this study, we assessed the effect of sex steroids on BDNF expression and secretion in human airway smooth muscle (ASM). Human ASM was treated with estrogen (E2) or testosterone (T, 10nM each) and intracellular BDNF and secreted BDNF measured. E2 and T significantly reduced secretion of BDNF; effects prevented by estrogen and androgen receptor inhibitor, ICI 182,780 (1uM) and flutamide (10uM), respectively. Interestingly, no significant changes were observed in intracellular BDNF mRNA or protein expression. High affinity BDNF receptor, TrkB, was not altered by E2 or T. E2 (but not T) significantly increased intracellular cyclic AMP levels. Notably, Epac1 and Epac2 expression were significantly reduced by E2 and T. Furthermore, SNARE complex protein SNAP25 was decreased. Overall, these novel data suggest that physiologically relevant concentrations of E2 or T inhibit BDNF secretion in human ASM, suggesting a potential interaction of sex steroids with BDNF in the airway that is different from brain. The relevance of sex steroid‐BDNF interactions may lie in their overall contribution to airway diseases such as asthma. This article is protected by copyright. All rights reserved
  • Galectin‐1 Controls the Proliferation and Migration of Liver
           Sinusoidal Endothelial Cells and Their Interaction with Hepatocarcinoma
    • Abstract: Galectin‐1 (Gal1), a β‐galactoside‐binding protein elevated in hepatocellular carcinoma (HCC), promotes epithelial‐mesenchymal transition (EMT) and its expression correlates with HCC growth, invasiveness and metastasis. During the early stages of HCC, transforming growth factor β1 (TGF‐β1) acts as a tumor suppressor; however in advanced stages, HCC cells lose their cytostatic response to TGF‐β1 and undergo EMT. Here, we investigated the role of Gal1 on liver endothelial cell biology, and the interplay between Gal1 and TGF‐β1 in HCC progression. By Western blot and immunofluorescence, we analyzed Gal1 expression, secretion and localization in HepG2 and HuH‐7 human HCC cells, and in SK‐HEP‐1 human liver sinusoidal endothelial cells (SECs). We used loss‐of‐function and gain‐of‐function experiments to down‐ or up‐regulate Gal1 expression, respectively, in HepG2 cells. We cultured SK‐HEP‐1 cells with conditioned media from HCC cells secreting different levels of Gal1, and demonstrated that Gal1 derived from tumor hepatocytes induced its own expression in SECs. Colorimetric and scratch‐wound assays revealed that secretion of Gal1 by HCC cells induced SEC proliferation and migration. Moreover, by fluorescence microscopy we demonstrated that Gal1 promoted glycan‐dependent heterotypic adhesion of HepG2 cells to SK‐HEP‐1 SECs. Furthermore, TGF‐β1 induced Gal1 expression and secretion by HCC cells, and promoted HepG2 cell adhesion to SK‐HEP‐1 SECs through a Gal1‐dependent mechanism. Finally, Gal1 modulated HepG2 cell proliferation and sensitivity to TGF‐β1‐induced growth inhibition. Our results suggest that Gal1 and TGF‐β1 might function coordinately within the HCC microenvironment to regulate tumor growth, invasion, metastasis and angiogenesis. This article is protected by copyright. All rights reserved
  • Morphine‐Induced MOR‐1X and ASF/SF2 Expression Is Independent
           of Transcriptional Regulation: Implications for MOR‐1X Signaling
    • Abstract: Recently, multiple μ‐opioid receptor (MOR) isoforms have been identified that originate from a single gene, OPRM1; however, both their regulation and their functional significance are poorly characterized. The objectives of this study were to decipher, first, the regulation of alternatively spliced μ‐opioid receptor isoforms and the spliceosome components that determine splicing specificity and, second, the signaling pathways utilized by particular isoforms both constitutively and following agonist binding. Our studies demonstrated that the expression of a particular splice variant, MOR‐1X, was up‐regulated by morphine, and this coincided with an increase in the essential splicing factor ASF/SF2. Structural comparison of this isoform to the prototypical variant MOR‐1 revealed that the unique distal portion of the C‐terminal domain contains additional phosphorylation sites, while functional comparison found distinct signaling differences, particularly in the ERK and p90 RSK pathways. Additionally, MOR‐1X expression significantly reduced Bax expression and mitochondrial dehydrogenase activity, suggesting a unique functional consequence for MOR‐1X specific signaling. Collectively, these findings suggest that alternative splicing of the MOR is altered by exogenous opioids, such as morphine, and that individual isoforms, such as MOR‐1X, mediate unique signal transduction with distinct functional consequence. Furthermore, we have identified for the first time a potential mechanism that involves the essential splicing factor ASF/SF2 through which morphine regulates splicing specificity of the MOR encoding gene, OPRM1. This article is protected by copyright. All rights reserved
  • Carbonic anhydrase 9 mRNA/microRNA34a interplay in hypoxic human
    • Abstract: The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up‐regulates the expression of Carbonic Anhydrase iso‐enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down‐regulates miR34a expression in hypoxic MCF7 cell‐derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over‐expression of cloned CA9‐mRNA‐3'UTR increases the mRNA half‐life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2‐dependent down‐regulation of miR34a substantially contributes to the post‐transcriptional up‐regulation of CA9, and that CA9‐mRNA‐3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore may play a relevant role in the hypoxic breast cancer stem cell niche. This article is protected by copyright. All rights reserved
  • Therapeutic Approaches in Mitochondrial Dysfunction, Proteolysis, and
           Structural Alterations of Diaphragm and Gastrocnemius in Rats With Chronic
           Heart Failure
    • Abstract: Patients with chronic heart failure (CHF) experience exercise intolerance, fatigue and muscle wasting, which negatively influence their survival. We hypothesized that treatment with either the antioxidant N‐acetyl cysteine (NAC) or the proteasome inhibitor bortezomib of rats with monocrotaline‐induced CHF may restore inspiratory and limb muscle mass, function, and structure through several molecular mechanisms involved in protein breakdown and metabolism in the diaphragm and gastrocnemius. In these muscles of CHF‐cachectic rats with and without treatment with NAC or bortezomib (n = 10/group) and non‐cachectic controls, proteolysis (tyrosine release, proteasome activities, ubiquitin‐proteasome markers), oxidative stress, inflammation, mitochondrial function, myosin, NF‐κB transcriptional activity, muscle structural abnormalities and fiber morphometry were analyzed together with muscle and cardiac functions. In diaphragm and gastrocnemius of CHF‐cachectic rats, tyrosine release, proteasome activity, protein ubiquitination, atrogin‐1, MURF‐1, NF‐κB activity, oxidative stress, inflammation and structural abnormalities were increased, while muscle and cardiac functions, myosin content, slow‐ and fast‐twitch fiber sizes, and mitochondrial activity were decreased. Concomitant treatment of CHF‐cachectic rats with NAC or bortezomib improved protein catabolism, oxidative stress, inflammation, muscle fiber sizes, function and damage, superoxide dismutase and myosin levels, mitochondrial function (complex I, gastrocnemius), cardiac function and decreased NF‐κB transcriptional activity in both muscles. Treatment of CHF‐cachectic animals with NAC or bortezomib attenuated the functional (heart, muscles), biological, and structural alterations in muscles. Nonetheless, future studies conducted in actual clinical settings are warranted in order to assess the potential beneficial effects and safety concerns of these pharmacological agents on muscle mass loss and wasting in CHF‐cachectic patients. This article is protected by copyright. All rights reserved
  • Complex‐I alteration and enhanced mitochondrial fusion are
           associated with prostate cancer progression
    • Abstract: Mitochondria (mt) encoded respiratory complex‐I (RCI) mutations and their pathogenicity remain largely unknown in prostate cancer (PCa). Little is known about the role of mtDNA loss on mt integrity in PCa. We determined mtDNA mutation in human and mice PCa and assessed the impact of mtDNA depletion on mt integrity. We also examined whether the circulating exosomes from PCa patients are transported to mt and carry mtDNA or mt proteins. We have employed next generation sequencing of the whole mt genome in human and Hi‐myc PCa. The impact of mtDNA depletion on mt integrity, presence of mtDNA and protein in sera exosomes was determined. A co‐culture of human PCa cells and the circulating exosomes followed by confocal imaging determined co‐localization of exosomes and mt. We observed frequent RCI mutations in human and Hi‐myc PCa which disrupted corresponding complex protein expression. Depletion of mtDNA in PCa cells influenced mt integrity, increased expression of MFN1, MFN2, PINK1 and decreased expression of MT‐TFA. Increased mt fusion and expression of PINK1 and DNM1L were also evident in the Hi‐myc tumors. RCI‐mtDNA, MFN2 and IMMT proteins were detected in the circulating exosomes of men with benign prostate hyperplasia (BPH) and progressive PCa. Circulating exosomes and mt co‐localized in PCa cells. Our study identified new pathogenic RCI mutations in PCa and defined the impact of mtDNA loss on mt integrity. Presence of mtDNA and mt proteins in the circulating exosomes implicated their usefulness for biomarker development. This article is protected by copyright. All rights reserved
  • Vascular endothelial growth factor A regulates the secretion of different
           angiogenic factors in lung cancer cells
    • Abstract: Vascular endothelial growth factor A (VEGFA) is one of the main mediators of angiogenesis in non‐small cell lung cancer (NSCLC). Recently, it has been described an autocrine feed‐forward loop in NSCLC cells in which tumor‐derived VEGFA promoted the secretion of VEGFA itself, amplifying the proangiogenic signal. In order to investigate the role of VEGFA in lung cancer progression, we assessed the effects of recombinant VEGFA on proliferation, migration and secretion of other angiogenic factors in A549, H1975 and HCC827 NSCLC cell lines. We found that VEGFA did not affect NSCLC cell proliferation and migration. On the other hand, we demonstrated that VEGFA not only produced a strong and persistent increase of VEGFA itself but also significantly induced the secretion of a variety of angiogenic factors, including follistatin (FST), hepatocyte growth factor (HGF), angiopoietin‐2 (ANGPT2), granulocyte‐colony stimulating factor (G‐CSF), interleukin (IL)‐8, leptin (LEP), platelet/endothelial cell adhesion molecule 1 (PECAM‐1) and platelet‐derived growth factor bb (PDGF‐BB). PI3K/AKT, RAS/ERK and STAT3 signalling pathways were found to mediate the effects of VEGFA in NSCLC cell lines. We also observed that VEGFA regulation mainly occurred at post‐transcriptional level and that NSCLC cells expressed different isoforms of VEGFA. Collectively, our data suggested that VEGFA contributes to lung cancer progression by inducing a network of angiogenic factors, which might offer potential for therapeutic intervention. This article is protected by copyright. All rights reserved
  • The Transcription Factor EB (TFEB) Regulates Osteoblast Differentiation
           through ATF4/CHOP‐Dependent Pathway
    • Abstract: Osteoblasts are bone‐forming cells that produce large amounts of collagen type I and various bone matrix proteins. Although osteoblast differentiation is highly regulated by various factors, it remains unknown whether lysosomes are directly involved in osteoblast differentiation. Here we demonstrate the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, modulates osteoblast differentiation. The expression levels of TFEB as well as those of endosomal/lysosomal proteins were up‐regulated during osteoblast differentiation using mouse osteoblastic MC3T3‐E1 cells. By gene knockdown (KD) experiments with small interfering RNA (siRNA), TFEB depletion caused markedly reduced osteoblast differentiation as compared with the control cells. Conversely, overexpression (OE) of TFEB resulted in strikingly enhanced osteoblastogenesis compared to the control cells. By analysis of down‐stream effector molecules, TFEB KD was found to cause marked up‐regulation of activating transcription factor4 (ATF4) and CCAAT/enhancer‐binding protein homologous protein (CHOP), both of which are essential factors for osteoblastogenesis. In contrast, TFEB OE promoted osteoblast differentiation through reduced expression of ATF4 and CHOP without differentiation agents. Given the importance of ATF4 and CHOP in osteoblastogenesis, it is clear that the TFEB‐regulated signaling pathway for osteoblast differentiation is involved in ATF4/CHOP‐dependent signaling pathway. This article is protected by copyright. All rights reserved
  • Inhibition of polyamine uptake potentiates the anti‐proliferative
           effect of polyamine synthesis inhibition and preserves the contractile
           phenotype of vascular smooth muscle cells
    • Abstract: Increased vascular smooth muscle cell (VSMC) proliferation is a factor in atherosclerosis and injury‐induced arterial (re)stenosis. Inhibition of polyamine synthesis by α‐difluoro‐methylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, attenuates VSMC proliferation with high sensitivity and specificity. However, cells can escape polyamine synthesis blockade by importing polyamines from the environment. To address this issue, polyamine transport inhibitors (PTIs) have been developed. We investigated the effects of the novel trimer44NMe (PTI‐1) alone and in combination with DFMO on VSMC polyamine uptake, proliferation and phenotype regulation. PTI‐1 efficiently inhibited polyamine uptake in primary mouse aortic and human coronary VSMCs in the absence as well as in the presence of DFMO. Interestingly, culture with DFMO for 2 days substantially (>95%) reduced putrescine (Put) and spermidine (Spd) contents without any effect on proliferation. Culture with PTI‐1 alone had no effect on either polyamine levels or proliferation rate, but the combination of both treatments reduced Put and Spd levels below the detection limit and inhibited proliferation. Treatment with DFMO for a longer time period (4 days) reduced Put and Spd below their detection limits and reduced proliferation, showing that only a small pool of polyamines is needed to sustain VSMC proliferation. Inhibited proliferation by polyamine depletion was associated with maintained expression of contractile smooth marker genes. In cultured intact mouse aorta, PTI‐1 potentiated the DFMO‐induced inhibition of cell proliferation. The combination of endogenous polyamine synthesis inhibition with uptake blockade is thus a viable approach for targeting unwanted vascular cell proliferation in vivo, including vascular restenosis. This article is protected by copyright. All rights reserved
  • Identification of a hematopoietic cell dedifferentiation‐inducing
    • Abstract: It has long been realized that hematopoietic cells may have the capacity to trans‐differentiate into non‐lymphohematopoietic cells under specific conditions. However, the mechanisms and the factors for hematopoietic cell trans‐differentiation remain unknown. In an in vitro culture system, we found that using a conditioned medium from proliferating fibroblasts can induce a subset of hematopoietic cells to become adherent fibroblast‐like cells (FLCs). FLCs are not fibroblasts nor other mesenchymal stromal cells, based on their expression of type‐1 collagen and other stromal cell marker genes. To identify the active factors in the conditioned medium, we cultured fibroblasts in a serum‐free medium and collected it for further purification. Using the fractions from filter devices of different molecular weight cut‐offs, and ammonium sulfate precipitation collected from the medium, we found the active fraction is a protein. We then purified this fraction by using fast protein liquid chromatography (FPLC) and identified it by mass spectrometer as macrophage colony‐stimulating factor (M‐CSF). The mechanisms of M‐CSF‐inducing trans‐differentiation of hematopoietic cells seem to involve a tyrosine kinase signalling pathway and its known receptor. The FLCs express a number of stem cell markers including SSEA‐1 and ‐3, OCT3/4, NANOG and SOX2. Spontaneous and induced differentiation experiments confirmed that FLCs can be further differentiated into cell types of three germ layers. These data indicate that hematopoietic cells can be induced by M‐CSF to dedifferentiate to multipotent stem cells. This study also provides a simple method to generate multipotent stem cells for clinical applications. This article is protected by copyright. All rights reserved
  • Interaction between Human Polyomavirus BK and Hypoxia inducible
           factor‐1 alpha
    • Abstract: BK polyomavirus (BKV) has a worldwide seroprevalence of approximately 90%. After primary infection, BKV establishes a life‐long latency within the urogenital tract. The severe immunological impairment occurring in renal transplant recipients leads to BKV reactivation, which may result in Polyomavirus Associated Nephropathy (PVAN). While the transplanted kidney is transiently unperfused, Hypoxia Inducible Factors (HIFs) mediate the cellular response to hypoxia. The α‐subunit of HIF isoform 1 (HIF‐1α) may interact with several viruses, but until now, there has been no information regarding the interaction between BKV and HIF‐1α. The aim of this study is to investigate the possible interaction between HIF‐1α and BKV and its potential effect on the pathogenesis of PVAN. Screening of 17 kidney tissue samples revealed that HIF‐1α expression was 13.6‐fold higher in PVAN tissues compared to control tissues. A luminometric assay in co‐transfected African green monkey kidney cells (VERO) demonstrated BKV promoter activation ranging from 2‐ to 6‐fold (p 
  • Regulation and Functional Implications of Opioid Receptor Splicing in
           Opioid Pharmacology and HIV Pathogenesis
    • Abstract: Despite the identification and characterization of four opioid receptor subtypes and the genes from which they are encoded, pharmacological data does not conform to the predications of a four opioid receptor model. Instead, current studies of opioid pharmacology suggest the existence of additional receptor subtypes; however, no additional opioid receptor subtype has been identified to date. It is now understood that this discrepancy is due to the generation of multiple isoforms of opioid receptor subtypes. While several mechanisms are utilized to generate these isoforms, the primary mechanism involves alternative splicing of the pre‐mRNA transcript. Extensive alternative splicing patterns for opioid receptors have since been identified and discrepancies in opioid pharmacology are now partially attributed to variable expression of these isoforms. Recent studies have been successful in characterizing the localization of these isoforms as well as their specificity in ligand binding; however, the regulation of opioid receptor splicing specificity is poorly characterized. Furthermore, the functional significance of individual receptor isoforms and the extent to which opioid‐ and/or HIV‐mediated changes in the opioid receptor isoform profile contributes to altered opioid pharmacology or the well‐known physiological role of opioids in the exacerbation of HIV neurocognitive dysfunction is unknown. As such, the current review details constitutive splicing mechanisms as well as the specific architecture of opioid receptor genes, transcripts, and receptors in order to highlight the current understanding of opioid receptor isoforms, potential mechanisms of their regulation and signaling, and their functional significance in both opioid pharmacology and HIV‐associated neuropathology. This article is protected by copyright. All rights reserved
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