Journal of Cellular Physiology 
[4 followers] Follow
Subscription journal
ISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
Published by John Wiley and Sons
[1587 journals]
[4 followers] Follow Subscription journalISSN (Print) 0021-9541 - ISSN (Online) 1097-4652
Published by John Wiley and Sons
[1587 journals]- Oncogenic cooperation between PI3K/Akt signaling and transcription factor Runx2 promotes the invasive properties of metastatic breast cancer cells
- Authors: Sandhya Pande; Gillian Browne, Srivatsan Padmanabhan, Sayyed K. Zaidi, Jane B. Lian, Andre J van Wijnen, Janet L. Stein, Gary S. Stein
Pages: n/a - n/a
Abstract: The serine/threonine kinase Akt/PKB promotes cancer cell growth and invasion through several downstream targets. Identification of novel substrates may provide new avenues for therapeutic intervention. Our study shows that Akt phosphorylates the cancer related transcription factor Runx2 resulting in stimulated DNA binding of the purified recombinant protein in vitro. Pharmacological inhibition of the PI3K/Akt pathway in breast cancer cells reduces DNA binding activity of Runx2 with concomitant reduction in the expression of metastasis related Runx2 target genes. Akt phosphorylates Runx2 at three critical residues within the runt DNA binding domain to enhance its in vivo genomic interactions with a target gene promoter, MMP13. Mutation of these three phosphorylation sites reduces Runx2 DNA binding activity, but does not interefere with CBFβ‐Runx2 interactions. Consequently, expression of multiple metastasis‐related genes is decreased and Runx2 mediated cell invasion is supressed. Thus, our work identifies Runx2 as a novel and important downstream mediator of the PI3K/Akt pathway that is linked to metastatic properties of breast cancer cells. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-02-06T07:57:46.334325-05:
DOI: 10.1002/jcp.24339
- Authors: Sandhya Pande; Gillian Browne, Srivatsan Padmanabhan, Sayyed K. Zaidi, Jane B. Lian, Andre J van Wijnen, Janet L. Stein, Gary S. Stein
- Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice
- Authors: Laura R. McCabe; Regina Irwin, Laura Schaefer, Robert A. Britton
Pages: n/a - n/a
Abstract: Osteoporosis can result from intestinal inflammation, as is seen with inflammatory bowel disease. Probiotics, microorganisms that provide a health benefit to the host when ingested in adequate amounts, can have anti‐inflammatory properties and are currently being examined to treat inflammatory bowel disease. Here, we examined if treating healthy male mice with Lactobacillus reuteri ATCC PTA 6475 (a candidate probiotic with anti‐TNFα activity) could affect intestinal TNFα levels and enhance bone density. Adult male mice were given L. reuteri 6475 orally by gavage for 3X/week for four weeks. Examination of jejunal and ileal RNA profiles indicates that L. reuteri suppressed basal TNFα mRNA levels in the jejunum and ileum in male mice, but surprisingly not in female mice. Next, we examined bone responses. Micro‐computed tomography demonstrated that L. reuteri 6475 treatment increased male trabecular bone parameters (mineral density, bone volume fraction, trabecular number and trabecular thickness) in the distal femur metaphyseal region as well as in the lumbar vertebrae. Cortical bone parameters were unaffected. Dynamic and static histomorphometry and serum remodeling parameters indicate that L. reuteri ingestion increases osteoblast serum markers and dynamic measures of bone formation in male mice. In contrast to male mice, L. reuteri had no effect on bone parameters in female mice. Taken together our studies indicate that femoral and vertebral bone formation increases in response to oral probiotic use, leading to increased trabecular bone volume in male mice. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-02-06T07:57:44.336942-05:
DOI: 10.1002/jcp.24340
- Authors: Laura R. McCabe; Regina Irwin, Laura Schaefer, Robert A. Britton
- Myocardin and MicroRNA‐1 modulate bladder activity through connexin 43 expression during post‐natal development
- Authors: Masaaki Imamura; Yoshio Sugino, Xiaochun Long, Orazio J. Slivano, Nobuyuki Nishikawa, Naoki Yoshimura, Joseph M. Miano
Pages: n/a - n/a
Abstract: Overactive bladder (OAB) is a pervasive clinical problem involving alterations in both neurogenic and myogenic activity. While there has been some progress in understanding neurogenic inputs to OAB, the mechanisms controlling myogenic bladder activity are unclear. We report the involvement of myocardin (MYOCD) and microRNA‐1 (miR‐1) in the regulation of connexin 43 (GJA1), a major gap junction in bladder smooth muscle, and the collective role of these molecules during post‐natal bladder development. Wild type mouse bladders showed normal development from early postnatal to adult including increases in bladder capacity and maintenance of normal sensitivity to cholinergic agents concurrent with down‐regulation of MYOCD and several smooth muscle cell (SMC) contractile genes. Myocardin heterozygous‐knockout mice exhibited reduced expression of Myocd mRNA and several SMC contractile genes concurrent with bladder SMC hypersensitivity that was mediated by gap junctions. In both cultured rat bladder SMC and in vivo bladders, MYOCD down‐regulated GJA1 expression through miR‐1 up‐regulation. Interestingly, adult myocardin heterozygous‐knockout mice showed normal increases in bladder and body weight but lower bladder capacity compared to wild type mice. These results suggest that MYOCD down‐regulates GJA1 expression via miR‐1 up‐regulation, thereby contributing to maintenance of normal sensitivity and development of bladder capacity. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:47:12.457749-05:
DOI: 10.1002/jcp.24333
- Authors: Masaaki Imamura; Yoshio Sugino, Xiaochun Long, Orazio J. Slivano, Nobuyuki Nishikawa, Naoki Yoshimura, Joseph M. Miano
- SIRT1 silencing confers neuroprotection through IGF‐1 pathway activation
- Authors: Luigi Sansone; Valentina Reali, Laura Pellegrini, Lidia Villanova, Michele Aventaggiato, Gabriella Marfe, Roberta Rosa, Marcella Nebbioso, Marco Tafani, Massimo Fini, Matteo A. Russo, Bruna Pucci
Pages: n/a - n/a
Abstract: The following study demonstrated that, in in vitro differentiated neurons, SIRT1 silencing induced an increase of IGF‐1 protein expression and secretion and of IGF‐1R protein levels which, in turn prolonged neuronal cell survival in presence of an apoptotic insult. On the contrary SIRT1 overexpression increased cell death. In particular, IGF‐1 and IGF‐1R expression levels were negatively regulated by SIRT1. In SIRT1 silenced cells, the increase in IGF‐1 and IGF‐1R expression was associated to an increase in AKT and ERK1/2 phosphorylation. Moreover, neuronal differentiation was reduced in SIRT1 overexpressing cells and increased in SIRT1 silenced cells. We conclude that SIRT1 silenced neurons appear more committed to differentiation and more resistant to cell death through the activation of IGF‐1 survival pathway. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:47:10.70884-05:0
DOI: 10.1002/jcp.24334
- Authors: Luigi Sansone; Valentina Reali, Laura Pellegrini, Lidia Villanova, Michele Aventaggiato, Gabriella Marfe, Roberta Rosa, Marcella Nebbioso, Marco Tafani, Massimo Fini, Matteo A. Russo, Bruna Pucci
- RKIP: Much more than Raf kinase inhibitory protein
- Authors: Fahd Al‐Mulla; Milad S. Bitar, Zainab Taqi, Kam Yeung
Pages: n/a - n/a
Abstract: From its discovery as a phosphatidylethanolamine binding protein in bovine brain to its designation as a physiological inhibitor of Raf kinase protein, RKIP has emerged as a critical molecule for maintaining subdued, well‐orchestrated cellular responses to stimuli. The disruption of RKIP in a wide range of pathologies, including cancer, Alzheimer's disease, and pancreatitis, makes it an exciting target for individualized therapy and disease‐specific interventions. This review attempts to highlight recent advances in the RKIP field underscoring its potential role as a master modulator of many pivotal intracellular signaling cascades that control cellular growth, motility, apoptosis, genomic integrity and therapeutic resistance. Specific biological and functional niches are highlighted to focus future research towards an enhanced understanding of the multiple roles of RKIP in health and disease. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:47:09.861267-05:
DOI: 10.1002/jcp.24335
- Authors: Fahd Al‐Mulla; Milad S. Bitar, Zainab Taqi, Kam Yeung
- Human NG2+ adipose stem cells loaded in vivo on a new crosslinked hyaluronic acid‐lys scaffold fabricate a skeletal muscle tissue
- Authors: Vincenzo Desiderio; Francesco De Francesco, Chiara Schiraldi, Alfredo De Rosa, Annalisa La Gatta, Francesca Paino, Riccardo d'Aquino, Giuseppe Andrea Ferraro, Virginia Tirino, Gianpaolo Papaccio
Pages: n/a - n/a
Abstract: Mesenchymal Stem Cell (MSC) therapy holds promise for treating diseases and tissue repair. Regeneration of skeletal muscle tissue that is lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. Human Adipose Stem Cells (ASCs) have been reported to regenerate muscle fibers and reconstitute the pericytic cell pool after myogenic differentiation in vitro. Our aim was to evaluate the differentiation potential of constructs made from a new cross‐linked hyaluronic acid (XHA) scaffold on which different sorted subpopulations of ASCs were loaded. 30 days after engraftment in mice, we found that NG2+ ASCs underwent a complete myogenic differentiation, fabricating a human skeletal muscle tissue, while NG2− ASCs merely formed a human adipose tissue. Myogenic differentiation was confirmed by the expression of MyoD, MF20, laminin, and lamin A/C by immunofluorescence and/or RT‐PCR. In contrast, adipose differentiation was confirmed by the expression of adiponectin, Glut‐4, and PPAR‐γ. Both tissues formed expressed Class I HLA, confirming their human origin and excluding any contamination by murine cells. In conclusion, our study provides novel evidence that NG2+ ASCs loaded on XHA scaffolds are able to fabricate a human skeletal muscle tissue in vivo without the need of a myogenic pre‐differentiation step in vitro. We emphasize the translational significance of our findings for human skeletal muscle regeneration. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:47:07.438268-05:
DOI: 10.1002/jcp.24336
- Authors: Vincenzo Desiderio; Francesco De Francesco, Chiara Schiraldi, Alfredo De Rosa, Annalisa La Gatta, Francesca Paino, Riccardo d'Aquino, Giuseppe Andrea Ferraro, Virginia Tirino, Gianpaolo Papaccio
- Interferon‐γ promotes vascular remodeling in human microvascular endothelial cells by upregulating endothelin (ET)‐1 and transforming growth factor (TGF) β2
- Authors: Izabela Chrobak; Stefania Lenna, Lukasz Stawski, Maria Trojanowska
Pages: n/a - n/a
Abstract: Systemic sclerosis (SSc) is a complex disease characterized by vascular alterations, activation of the immune system and tissue fibrosis. Previous studies have implicated activation of the interferon pathways in the pathogenesis of SSc. The goal of this study was to determine whether interferon type I and/or type II could play a pathogenic role in SSc vasculopathy. Human dermal microvascular endothelial cells (HDMVECs) and fibroblasts were obtained from foreskins of healthy newborns. The RT Profiler PCR Array System was utilized to screen for EndoMT genes. Treatment with IFN‐α or IFN‐γ downregulated Fli1 and VE‐cadherin. In contrast, IFN‐α and IFN‐γ exerted opposite effects on the expression of α‐SMA, CTGF, ET‐1 and TGFβ2, with IFN‐α downregulating and IFN‐γ upregulating this set of genes. Blockade of TGFβ signaling normalized IFN‐γ‐mediated changes in Fli1, VE‐cadherin, CTGF and ET‐1 levels, whereas upregulation of α‐SMA and TGFβ2 was not affected. Bosentan treatment was more effective than TGFβ blockade in reversing the actions of IFN‐γ, including downregulation of α‐SMA and TGFβ2, suggesting that activation of the ET‐1 pathway plays a main role in the IFN‐γ responses in HDMECs. IFN‐γ induced expression of selected genes related to endothelial‐to‐mesenchymal transition (EndoMT), including Snail1, FN1, PAI1, TWIST1, STAT3, RGS2, and components of the WNT pathway. The effect of IFN‐γ on EndoMT was mediated via TGFβ2 and ET‐1 signaling pathways. This study demonstrates distinct effects of IFN‐α and IFN‐γ on the biology of vascular endothelial cells. IFN‐γ may contribute to abnormal vascular remodeling and fibrogenesis in SSc, partially via induction of EndoMT. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:47:06.028803-05:
DOI: 10.1002/jcp.24337
- Authors: Izabela Chrobak; Stefania Lenna, Lukasz Stawski, Maria Trojanowska
- Sestrins: Novel antioxidant and AMPK‐modulating functions regulated by exercise'
- Authors: Fabian Sanchis‐Gomar
Pages: n/a - n/a
Abstract: Oxidative stress results from damage to tissues caused by free radicals and is increased by exercise. Peroxiredoxins (PRXs) maintain the cellular reducing environment by scavenging intracellular hydrogen peroxide. It has been recently noted that physical exercise has a positive effect on the PRX system, exerting a protective effect against oxidative stress‐induced damage. However, other compounds, such as sestrins (SESNs), a stress‐inducible protein family with antioxidant properties, should also be considered in the function of PRXs. SESNs are clearly involved in the regeneration process of PRXs and therefore may also be modulated by physical exercise. In addition, SESNs are clearly involved in TOR, AMPK, p53, FoxO, and PRXs signaling pathways. The aforementioned pathways are implicated in aging processes by inducing an increased resistance to subsequent stress, thus delaying age‐related changes, such as sarcopenia and frailty, and consequently promoting longevity. Likewise, exercise also modulates these pathways. In fact, exercise is one of the most important recommended strategies to prevent sarcopenia and frailty, increase longevity, and improve health in the elderly. Loss of SESNs can cause several chronic pathologies, such as fat accumulation, mitochondrial dysfunction, cardiac arrhythmia, and/or muscle degeneration. Accordingly, physical inactivity leads to accumulation of visceral fat and consequently the activation of a network of inflammatory pathways, which promote development of insulin resistance, atherosclerosis, neurodegeneration, and tumor growth. To date, the SESNs‐exercise relationship has not been explored. However, this emerging family of stress proteins may be part of the redox‐based adaptive response to exercise. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:47:03.760865-05:
DOI: 10.1002/jcp.24338
- Authors: Fabian Sanchis‐Gomar
- Activation and promotion of adipose tissue‐derived mesenchymal stem cells by tumour necrosis factor‐alpha preconditioning for bone tissue engineering
- Authors: ZuFu Lu; GuoCheng Wang, Colin R Dunstan, YongJun Chen, William Yenn‐Ru Lu, Ben Davies, Hala Zreiqat
Pages: n/a - n/a
Abstract: There is a major medical need for developing novel and effective approaches for repairing non‐union and critical‐sized bone defects. Although the mechanisms remain to be determined, it is known that inflammation plays a crucial role in initiating bone repair and regeneration. This study investigated the effect of short‐term (three days) preconditioning with tumour necrosis factor‐alpha (TNF‐α) on proliferation, mobilization and differentiation of adipose tissue‐derived mesenchymal stem cells (ASCs). We demonstrated that TNF‐α pre‐conditioning increased proliferation, mobilization, and osteogenic differentiation of ASCs and up‐regulated bone morphogenetic protein‐2 (BMP‐2) protein levels. BMP‐2 silencing by siRNA partially inhibited osteogenic differentiation of ASCs induced by TNF‐α; BMP‐2 pre‐conditioning also significantly increased osteogenic differentiation of ASCs but the effects were significantly smaller than those observed for TNF‐α preconditioning. Furthermore, TNF‐α treatment promoted extracellular‐signal‐regulated kinases(Erk)1/2 and p38 mitogen‐activated protein kinase (MAPK) signaling pathways, but only Erk1/2 inhibition reduced the BMP‐2 levels and osteogenic differentiation induced by TNF‐α preconditioning. Together, these results support the hypothesis that inflammation contributes to bone regeneration by promoting proliferation, mobilization, and osteogenic differentiation of ASCs; three days of TNF‐α preconditioning, mimicking the short boost of inflammation normally occurring after bone injury, might serve as a feasible approach for directing stem cells into osteogenic differentiation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:47:00.205299-05:
DOI: 10.1002/jcp.24330
- Authors: ZuFu Lu; GuoCheng Wang, Colin R Dunstan, YongJun Chen, William Yenn‐Ru Lu, Ben Davies, Hala Zreiqat
- TNFα induces expression of HIF‐1α mRNA and protein but inhibits hypoxic stimulation of HIF‐1 transcriptional activity in airway smooth muscle cells
- Authors: Stamatina Tsapournioti; Ilias Mylonis, Apostolia Hatziefhimiou, Maria G. Ioannou, Rodopi Stamatiou, Georgios K. Koukoulis, George Simos, Paschalis‐Adam Molyvdas, Efrosyni Paraskeva
Pages: n/a - n/a
Abstract: Airway smooth muscle cells (ASMCs) participate in tissue remodeling characteristic of airway inflammatory diseases like asthma. Inflammation and hypoxia pathways are often interconnected and the regulatory subunit of the Hypoxia Inducible Factor, HIF‐1α, has been recently shown to be induced by cytokines. Here we investigate the effect of individual or combined treatment of ASMCs with the inflammatory mediator TNFα and/or hypoxia on the expression of HIF‐1α, HIF‐1 targets and inflammation markers. TNFα enhances HIF‐1α protein and mRNA levels, under both normoxia and hypoxia. TNFα‐mediated induction of HIF‐1α gene transcription is repressed by inhibition of the NF‐κB pathway. Despite the up‐regulation of HIF‐1α protein, the transcription of HIF‐1 target genes remains low in the presence of TNFα at normoxia and is even reduced at hypoxia. We show that the reduction in HIF‐1 transcriptional activity by TNFα is due to inhibition of the interaction of HIF‐1α with ARNT and subsequent blocking of its binding to HREs. Comparison between hypoxia and TNFα for their effects on the expression of inflammatory markers shows significant differences: hypoxia up‐regulates the expression of IL‐6, but not RANTES or ICAM, and reduces the induction of VCAM by TNFα. Finally, ex vivo treatment of rabbit trachea strips with TNFα increases HIF‐1α protein levels, but reduces the expression of HIF‐1 targets under hypoxia. Overall, TNFα induces HIF‐1α mRNA synthesis via an NF‐κB dependent pathway but inhibits binding of HIF‐1α to ARNT and DNA, while hypoxia and TNFα have distinct effects on ASMC inflammatory gene expression. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:46:50.484583-05:
DOI: 10.1002/jcp.24331
- Authors: Stamatina Tsapournioti; Ilias Mylonis, Apostolia Hatziefhimiou, Maria G. Ioannou, Rodopi Stamatiou, Georgios K. Koukoulis, George Simos, Paschalis‐Adam Molyvdas, Efrosyni Paraskeva
- First evidence of Prothymosin alpha localization in the acrosome of mammalian male gametes
- Authors: Diana Ferrara; Paolo Pariante, Loredana Di Matteo, Ismene Serino, Richard Oko, Sergio Minucci
Pages: n/a - n/a
Abstract: Prothymosin α (PTMA) is a highly acidic intrinsically unstructured protein. Its expression in male gonads is evolutionary conserved; in rat testis it is specifically localized in the cytoplasm of post‐meiotic germ cells, in association with the developing acrosome system. In the present paper we investigated on PTMA localization inside the head of mammalian spermatozoa (SPZ). We chose a confocal approach to ascertain whether PTMA is expressed in the acrosome or in the perinuclear theca, two regions that are tightly linked and partially overlapped in the mature haploid cells. The obtained results showed that PTMA is specifically localized in the acrosome of rat epididymal SPZ; the same experimental approach evidenced, for the first time, PTMA presence in human ejaculated SPZ. A Western blot analysis on protein extracts from human sperm head fractions confirmed the confocal data and demonstrated that the peptide is specifically associated with the inner acrosomal membrane fraction. Finally, when the acrosome reaction was induced in vitro by progesterone treatment on both rat and human sperm, PTMA signal was retained in the apical region of reacted SPZ. In conclusion, this study confirms the conservation of PTMA distribution in vertebrate male gametes and strongly supports a role for this polypeptide in their physiology. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:46:48.968105-05:
DOI: 10.1002/jcp.24332
- Authors: Diana Ferrara; Paolo Pariante, Loredana Di Matteo, Ismene Serino, Richard Oko, Sergio Minucci
- “RB1 in cancer: Different mechanisms of RB1 inactivation and alterations of pRb pathway in tumorigenesis”
- Authors: Riccardo Di Fiore; Antonella D'Anneo, Giovanni Tesoriere, Renza Vento
Pages: n/a - n/a
Abstract: Loss of RB1 gene is considered either a causal or an accelerating event in retinoblastoma. A variety of mechanisms inactivates RB1 gene, including intragenic mutations, loss of expression by methylation and chromosomal deletions, with effects which are species‐ and cell type‐ specific. RB1 deletion can even lead to aneuploidy thus greatly increasing cancer risk. The RB1gene is part of a larger gene family that includes RBL1 and RBL2, each of the three encoding structurally related proteins indicated as pRb, p107 and p130, respectively. The great interest in these genes and proteins springs from their ability to slow down neoplastic growth. pRb can associate with various proteins by which it can regulate a great number of cellular activities. In particular, its association with the E2F transcription factor family allows the control of the main pRb functions, while the loss of these interactions greatly enhances cancer development. As RB1 gene, also pRb can be functionally inactivated through disparate mechanisms which are often tissue specific and dependent on the scenario of the involved tumor suppressors and oncogenes. The critical role of the context is complicated by the different functions played by the RB proteins and the E2F family members. In this review, we want to emphasize the importance of the mechanisms of RB1/pRb inactivation in inducing cancer cell development. The review is divided in three chapters describing in succession the mechanisms of RB1 inactivation in cancer cells, the alterations of pRb pathway in tumorigenesis and the RB protein and E2F family in cancer. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:46:46.37659-05:0
DOI: 10.1002/jcp.24329
- Authors: Riccardo Di Fiore; Antonella D'Anneo, Giovanni Tesoriere, Renza Vento
- Molecular biomarkers as potential targets for therapeutic strategies in human testicular germ cell tumours: An overview
- Authors: Paolo Chieffi; Sergio Chieffi
Pages: n/a - n/a
Abstract: Testicular germ cell tumors (TGCTs), the most common malignancy in males between 15 and 34 years of age and the most frequent cause of death from solid tumors in this age group. TGCTs can be subdivided into seminoma and non‐seminoma germ cell tumors (NSGCTs), including embryonal cell carcinoma, choriocarcinoma, yolk sac tumor, and teratoma. Seminomas and NSGCTs do not only present distinctive clinical features, but they also show significant differences as far as therapy and prognosis are concerned. Seminomas are highly sensitive to both radiation and chemotherapy, with a good prognosis, non‐seminomas are sensitive to platinum‐based combination chemotherapy and are less susceptible to radiation, with the exception of teratomas. The different therapeutic outcome might be explained by inherent properties of the cells from which testicular neoplasia originate. The unique treatment sensitivity of TGCTs is unexplained so far, but it is likely to be related to intrinsic molecular characteristics of the PGCs/gonocytes, from which these tumours originate. Many discovered bio‐markers including OCT3/4, SOX2, SOX17, HMGA1, HMGA2, PATZ1, GPR30, Aurora B, estrogen receptor β, and others have given further advantages to discriminate between histological subgroups. In addition, therapeutic approaches for the treatment of TGCTs have been proposed: humanized antibodies against receptors/surface molecules on cancer cells, inhibitors of serine‐threonine, and tyrosine kinases, and others. The mini‐review will be an overview on the molecular alterations identified in TGCTs and on novel targeted antineoplastic strategies that might help to treat chemotherapy resistant TGCTs. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
PubDate: 2013-01-28T08:46:46.243432-05:
DOI: 10.1002/jcp.24328
- Authors: Paolo Chieffi; Sergio Chieffi
- Pin1 and nuclear receptors: A new language'
- Authors: Raffaele La Montagna; Isabella Caligiuri, Antonio Giordano, Flavio Rizzolio
Pages: n/a - n/a
Abstract: Pin1 is a unique enzyme that can isomerize specific phospho‐Ser/Thr‐Pro peptide bonds, inducing a conformational change in the target protein. Such activity represents a novel and tightly controlled signaling mechanism regulating a spectrum of protein functions during the normal physiology of the cell and in pathological conditions. Our last study demonstrated that Pin1 interacts with the androgen receptor protein and that this interaction is important for its transcriptional activity. Here, we consider the activity that Pin1 plays on the N‐terminal domain of different nuclear receptors and provide an interpretation of this phenomenon. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.
PubDate: 2012-12-31T10:52:06.881898-05:
DOI: 10.1002/jcp.24316
- Authors: Raffaele La Montagna; Isabella Caligiuri, Antonio Giordano, Flavio Rizzolio
- Glycated collagen and altered glucose increase endothelial cell adhesion strength
- Authors: Steven Frank Kemeny; Stephanie Cicalese, Dannielle Solomon Figueroa, Alisa Morss Clyne
Pages: n/a - n/a
Abstract: Cell adhesion strength is important to cell survival, proliferation, migration, and mechanotransduction, yet changes in endothelial cell adhesion strength have not yet been examined in diseases such as diabetes with high rates of cardiovascular complications. We therefore investigated porcine aortic endothelial cell adhesion strength on native and glycated collagen‐coated substrates and in low, normal, and high glucose culture using a spinning disc apparatus. Adhesion strength increased by 30 dynes/cm2 in cells on glycated collagen as compared to native collagen. Attachment studies revealed that cells use higher adhesion strength αvβ3 integrins to bind to glycated collagen instead of the typical α2β1 integrins used to bind to native collagen. Similarly, endothelial cells cultured in low and high glucose had 15 dynes/cm2 higher adhesion strength than cells in normal glucose after two days. Increased adhesion strength was due to elevated VEGF release and intracellular PKC in low and high glucose cells, respectively. Thus glucose increased endothelial cell adhesion strength via different underlying mechanisms. These adhesion strength changes could contribute to diabetic vascular disease, including accelerated atherosclerosis and disordered angiogenesis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.
PubDate: 2012-12-31T10:51:56.383398-05:
DOI: 10.1002/jcp.24313
- Authors: Steven Frank Kemeny; Stephanie Cicalese, Dannielle Solomon Figueroa, Alisa Morss Clyne
- EMT markers in lung adenocarcinoma pleural effusion spheroid cells
- Authors: Enrico Giarnieri; Claudia De Vitis, Alessia Noto, Giuseppe Roscilli, Gerardo Salerno, Salvatore Mariotta, Alberto Ricci, Bruno Pierdonato, Giuseppe Russo, Andrea Laurenzi, Maria Rosaria Giovagnoli, Gennaro Ciliberto, Rita Mancini
Pages: n/a - n/a
Abstract: Epithelial‐to‐Mesenchymal Transition (EMT) is a process in which cells undergo a developmental switch from epithelial to mesenchymal phenotype. This process has been related to embryologic morphogenesis but also to cancer progression and metastasis. The aim of the current study was to investigate the expression of EMT related markers in adherent and spheroid cell cultures derived from Malignant Pleural Effusions (MPEs) of patients affected by lung adenocarcinoma. On the basis of efficient in vitro propagation, six cases of MPEs were selected and analyzed by immunocytochemistry staining for EMT markers and by RT‐PCR for transcription factors known to orchestrate EMT. EMT markers immunostaining showed in spheroids a statistically significant correlation between the loss of E‐cadherin immunoreactivity and overexpression of N‐cadherin (P
PubDate: 2012-12-18T08:40:57.74587-05:0
DOI: 10.1002/jcp.24300
- Authors: Enrico Giarnieri; Claudia De Vitis, Alessia Noto, Giuseppe Roscilli, Gerardo Salerno, Salvatore Mariotta, Alberto Ricci, Bruno Pierdonato, Giuseppe Russo, Andrea Laurenzi, Maria Rosaria Giovagnoli, Gennaro Ciliberto, Rita Mancini
- Alpha‐2‐macroglobulin: A physiological guardian
- Authors: Ahmed Abdur Rehman; Haseeb Ahsan, Fahim Halim Khan
Pages: n/a - n/a
Abstract: Alpha macroglobulins are large glycoproteins which are present in the body fluids of both invertebrates and vertebrates. Alpha‐2‐macroglobulin (α2M), a key member of alpha macroglobulin superfamily, is a high molecular weight homotetrameric glycoprotein. α2M has many diversified and complex functions, but it is primarily known by its ability to inhibit a broad spectrum of proteases without the direct blockage of the protease active site. α2M is also known to be involved in the regulation, transport, and a host of other functions. For example, apart from inhibiting proteinases, it regulates binding of transferrin to its surface receptor, binds defensin and myelin basic protein, etc, binds several important cytokines, including basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), interleukin‐1β (IL‐1β) and interleukin‐6 (IL‐6), and modify their biological activity. α2M also binds a number of hormones and regulate their activity. α2M is said to protect the body against various infections, and hence, can be used as a biomarker for the diagnosis and prognosis of a number of diseases. However, this multipurpose antiproteinse is not “fail safe” and could be damaged by reactive species generated endogenously or exogenously, leading to various pathophysiological conditions. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.
PubDate: 2012-10-19T07:08:48.043798-05:
DOI: 10.1002/jcp.24266
- Authors: Ahmed Abdur Rehman; Haseeb Ahsan, Fahim Halim Khan
- Sorafenib and HDAC inhibitors synergize with TRAIL to kill tumor cells
- Abstract: The present studies were designed to compare and contrast the abilities of TRAIL (death receptor agonist) and obatoclax (BCL‐2 family inhibitor) to enhance sorafenib + HDAC inhibitor toxicity in GI tumor cells. Sorafenib and HDAC inhibitor treatment required expression of CD95 to kill GI tumor cells in vitro and in vivo. In cells lacking CD95 expression, TRAIL treatment, and to a lesser extent obatoclax, enhanced the lethal effects of sorafenib + HDAC inhibitor exposure. In hepatoma cells expressing CD95 a similar data pattern emerged with respect to the actions of TRAIL. Downstream of the death receptor the ability of TRAIL to enhance cell killing correlated with reduced AKT, ERK1/2, p70 S6K and mTOR activity and enhanced cleavage of pro‐caspase 3 and reduced expression of MCL‐1 and BCL‐XL. Over‐expression of BCL‐XL or MCL‐1 or expression of dominant negative pro‐caspase 9 protected cells from drug toxicity. Expression of activated AKT, p70 S6K, mTOR and to a lesser extent MEK1EE also protected cells that correlated with maintained c‐FLIP‐s expression, reduced BIM expression and increased BAD phosphorylation. In vivo sorafenib + HDAC inhibitor toxicity against tumors was increased in a greater than additive fashion by TRAIL. Collectively, our data argue that TRAIL, rather than obatoclax, is the most efficacious agent at promoting sorafenib + HDAC inhibitor lethality. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The present studies were designed to compare and contrast the abilities of TRAIL (death receptor agonist) and obatoclax (BCL‐2 family inhibitor) to enhance sorafenib + HDAC inhibitor toxicity in GI tumor cells. Sorafenib and HDAC inhibitor treatment required expression of CD95 to kill GI tumor cells in vitro and in vivo. In cells lacking CD95 expression, TRAIL treatment, and to a lesser extent obatoclax, enhanced the lethal effects of sorafenib + HDAC inhibitor exposure. In hepatoma cells expressing CD95 a similar data pattern emerged with respect to the actions of TRAIL. Downstream of the death receptor the ability of TRAIL to enhance cell killing correlated with reduced AKT, ERK1/2, p70 S6K and mTOR activity and enhanced cleavage of pro‐caspase 3 and reduced expression of MCL‐1 and BCL‐XL. Over‐expression of BCL‐XL or MCL‐1 or expression of dominant negative pro‐caspase 9 protected cells from drug toxicity. Expression of activated AKT, p70 S6K, mTOR and to a lesser extent MEK1EE also protected cells that correlated with maintained c‐FLIP‐s expression, reduced BIM expression and increased BAD phosphorylation. In vivo sorafenib + HDAC inhibitor toxicity against tumors was increased in a greater than additive fashion by TRAIL. Collectively, our data argue that TRAIL, rather than obatoclax, is the most efficacious agent at promoting sorafenib + HDAC inhibitor lethality. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Functional and morphological impact of ER stress on mitochondria
- Abstract: Over the past years, knowledge and evidence about the existence of crosstalks between cellular organelles and their potential effects on survival or cell death have been constantly growing. More recently, evidence accumulated showing an intimate relationship between endoplasmic reticulum and mitochondria. These close contacts not only establish extensive physical links allowing exchange of lipids and calcium but they can also coordinate pathways involved in cell life and death. It is now obvious that ER dysfunction/stress and Unfolded Protein Response (UPR) as well as mitochondria play major roles in apoptosis. However, while the effects of major ER stress on cell death have been largely studied and reviewed, it becomes more and more evident that cells might regularly deal with sublethal ER stress, a condition that does not necessarily lead to cell death but might affect the function/activity of other organelles such as mitochondria. In this review, we will particularly focus on these new, interesting and intriguing metabolic and morphological events that occur during the early adaptative phase of the ER stress, before the onset of cell death, and that remain largely unknown. Relevance and implication of these mitochondrial changes in response to ER stress conditions for human diseases such as type II diabetes and Alzheimer's disease will also be considered. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Over the past years, knowledge and evidence about the existence of crosstalks between cellular organelles and their potential effects on survival or cell death have been constantly growing. More recently, evidence accumulated showing an intimate relationship between endoplasmic reticulum and mitochondria. These close contacts not only establish extensive physical links allowing exchange of lipids and calcium but they can also coordinate pathways involved in cell life and death. It is now obvious that ER dysfunction/stress and Unfolded Protein Response (UPR) as well as mitochondria play major roles in apoptosis. However, while the effects of major ER stress on cell death have been largely studied and reviewed, it becomes more and more evident that cells might regularly deal with sublethal ER stress, a condition that does not necessarily lead to cell death but might affect the function/activity of other organelles such as mitochondria. In this review, we will particularly focus on these new, interesting and intriguing metabolic and morphological events that occur during the early adaptative phase of the ER stress, before the onset of cell death, and that remain largely unknown. Relevance and implication of these mitochondrial changes in response to ER stress conditions for human diseases such as type II diabetes and Alzheimer's disease will also be considered. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Cell Proliferation is promoted by Compressive Stress during early stage of chondrogenic differentiation of rat BMSCs
- Abstract: The presence of an appropriate number of viable cells is prerequisite for successive differentiation during chondrogenesis. Chondrogenic differentiation has been reported to be influenced by mechanical stimuli. This research aimed to study the effects of cyclic compressive stress on cell viability of rat bone marrow‐derived MSCs (BMSCs) during chondrogenesis as well as its underlying mechanisms. The results showed that dynamic compression increased cell quantity and viability remarkably in the early stage of chondrogenesis, during which the expression of Ihh, Cyclin D1, CDK4 and Col2α1 were enhanced significantly. Possible signal pathways implicated in the process were explored in our study. MEK/ERK and p38 MAPK were not found to function in this process while BMP signaling seemed to play an important role in the mechanotransduction during chondrogenic proliferation. In conclusion, dynamic compressive stress could enhance cell viability during chondrogenesis, which might be achieved by activating BMP signaling. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The presence of an appropriate number of viable cells is prerequisite for successive differentiation during chondrogenesis. Chondrogenic differentiation has been reported to be influenced by mechanical stimuli. This research aimed to study the effects of cyclic compressive stress on cell viability of rat bone marrow‐derived MSCs (BMSCs) during chondrogenesis as well as its underlying mechanisms. The results showed that dynamic compression increased cell quantity and viability remarkably in the early stage of chondrogenesis, during which the expression of Ihh, Cyclin D1, CDK4 and Col2α1 were enhanced significantly. Possible signal pathways implicated in the process were explored in our study. MEK/ERK and p38 MAPK were not found to function in this process while BMP signaling seemed to play an important role in the mechanotransduction during chondrogenic proliferation. In conclusion, dynamic compressive stress could enhance cell viability during chondrogenesis, which might be achieved by activating BMP signaling. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Akt inhibition promotes hexokinase 2 redistribution and glucose uptake in cancer cells
- Abstract: Hexokinase II (HK2), the enzyme that catalyzes the first committed step of glycolysis, is overexpressed in many cancers, as is the central signaling kinase Akt. Akt activity promotes HK2 association with the mitochondria, as well as glucose uptake by cancer cells. In HeLa cervical cancer cells, Akt inhibitor IV (Ai4) increased nuclear HK2 localization, while in MDA‐MB‐231 breast cancer cells, Ai4 merely induced cytoplasmic redistribution without increased nuclear accumulation. Small interfering RNA (siRNA) directed against Akt confirmed the effect in HeLa cells. Next, we treated the cells with clotrimazole (CTZ), which detaches HK2 from the mitochondria, or leptomycin B (LMB), which promotes HK2 nuclear accumulation, and determined the effect on HK2 subcellular distribution. In both cell lines, CTZ detached HK2 from the mitochondria, without substantially increasing nuclear HK2, while LMB increased nuclear HK2, without redistributing cytoplasmic HK2. Contrary to expectations, Akt inhibition promoted glucose uptake in both cell lines, suggesting that Akt inhibition may increase glucose uptake by detaching HK2 from the mitochondria. In both cell lines, CTZ and LMB increased glucose uptake. However, the results in the HeLa cells showed greater effects: CTZ increased glucose uptake to a similar degree to Ai4, while LMB was far more effective than either. These data suggest that both detachment of HK2 from the mitochondria and increased nuclear HK2 are important for Ai4‐induced increased glucose uptake. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Hexokinase II (HK2), the enzyme that catalyzes the first committed step of glycolysis, is overexpressed in many cancers, as is the central signaling kinase Akt. Akt activity promotes HK2 association with the mitochondria, as well as glucose uptake by cancer cells. In HeLa cervical cancer cells, Akt inhibitor IV (Ai4) increased nuclear HK2 localization, while in MDA‐MB‐231 breast cancer cells, Ai4 merely induced cytoplasmic redistribution without increased nuclear accumulation. Small interfering RNA (siRNA) directed against Akt confirmed the effect in HeLa cells. Next, we treated the cells with clotrimazole (CTZ), which detaches HK2 from the mitochondria, or leptomycin B (LMB), which promotes HK2 nuclear accumulation, and determined the effect on HK2 subcellular distribution. In both cell lines, CTZ detached HK2 from the mitochondria, without substantially increasing nuclear HK2, while LMB increased nuclear HK2, without redistributing cytoplasmic HK2. Contrary to expectations, Akt inhibition promoted glucose uptake in both cell lines, suggesting that Akt inhibition may increase glucose uptake by detaching HK2 from the mitochondria. In both cell lines, CTZ and LMB increased glucose uptake. However, the results in the HeLa cells showed greater effects: CTZ increased glucose uptake to a similar degree to Ai4, while LMB was far more effective than either. These data suggest that both detachment of HK2 from the mitochondria and increased nuclear HK2 are important for Ai4‐induced increased glucose uptake. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- FUS/TLS assembles into stress granules and is a prosurvival factor during hyperosmolar stress
- Abstract: FUsed in Sarcoma/Translocated in LipoSarcoma (FUS/TLS or FUS) has been linked to several biological processes involving DNA and RNA processing, and has been associated with multiple diseases, including myxoid liposarcoma and amyotrophic lateral sclerosis (ALS). ALS‐associated mutations cause FUS to associate with stalled translational complexes called stress granules under conditions of stress. However, little is known regarding the normal role of endogenous (non‐disease linked) FUS in cellular stress response. Here, we demonstrate that endogenous FUS exerts a robust response to hyperosmolar stress induced by sorbitol. Hyperosmolar stress causes an immediate re‐distribution of nuclear FUS to the cytoplasm, where it incorporates into stress granules. The redistribution of FUS to the cytoplasm is modulated by methyltransferase activity, whereas the inhibition of methyltransferase activity does not affect the incorporation of FUS into stress granules. The response to hyperosmolar stress is specific, since endogenous FUS does not redistribute to the cytoplasm in response to sodium arsenite, hydrogen peroxide, thapsigargin, or heat shock, all of which induce stress granule assembly. Intriguingly, cells with reduced expression of FUS exhibit a loss of cell viability in response to sorbitol, indicating a prosurvival role for endogenous FUS in the cellular response to hyperosmolar stress. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: FUsed in Sarcoma/Translocated in LipoSarcoma (FUS/TLS or FUS) has been linked to several biological processes involving DNA and RNA processing, and has been associated with multiple diseases, including myxoid liposarcoma and amyotrophic lateral sclerosis (ALS). ALS‐associated mutations cause FUS to associate with stalled translational complexes called stress granules under conditions of stress. However, little is known regarding the normal role of endogenous (non‐disease linked) FUS in cellular stress response. Here, we demonstrate that endogenous FUS exerts a robust response to hyperosmolar stress induced by sorbitol. Hyperosmolar stress causes an immediate re‐distribution of nuclear FUS to the cytoplasm, where it incorporates into stress granules. The redistribution of FUS to the cytoplasm is modulated by methyltransferase activity, whereas the inhibition of methyltransferase activity does not affect the incorporation of FUS into stress granules. The response to hyperosmolar stress is specific, since endogenous FUS does not redistribute to the cytoplasm in response to sodium arsenite, hydrogen peroxide, thapsigargin, or heat shock, all of which induce stress granule assembly. Intriguingly, cells with reduced expression of FUS exhibit a loss of cell viability in response to sorbitol, indicating a prosurvival role for endogenous FUS in the cellular response to hyperosmolar stress. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Blood type B antigen modulates cell migration through regulating cdc42 expression and activity in HaCaT cells
- Abstract: ABO blood group is determined by carbohydrate antigens, called ABH antigens. It has been known that the change of carbohydrate antigen expression, including ABH antigens, has correlation with the tumor metastasis and survival; however, the exact mechanism remains to be elucidated. ABH antigens are expressed not only in blood cells but also in several tissues. In epidermis, ABH antigen is expressed in the uppermost spinous and granular layer. We investigated the role of ABH antigens on the cell migration of HaCaT keratinocytes, which express B antigen. Knock‐down of B antigen expression by small interference RNA of FUT1 inhibited HaCaT cell migration. At that time, we found that lamellipodia and actin fiber were also reduced by knock‐down of B antigen expression. The transcription of cdc42, a kind of Rho GTPase which plays a key role in actin polymerization, was reduced by down‐regulated B antigen expression. Furthermore, the reduced B antigen expression also inhibited the interaction of cdc42 and N‐WASP. Collectively, our data provide a clue how ABH antigens regulate the cell migration mechanism. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: ABO blood group is determined by carbohydrate antigens, called ABH antigens. It has been known that the change of carbohydrate antigen expression, including ABH antigens, has correlation with the tumor metastasis and survival; however, the exact mechanism remains to be elucidated. ABH antigens are expressed not only in blood cells but also in several tissues. In epidermis, ABH antigen is expressed in the uppermost spinous and granular layer. We investigated the role of ABH antigens on the cell migration of HaCaT keratinocytes, which express B antigen. Knock‐down of B antigen expression by small interference RNA of FUT1 inhibited HaCaT cell migration. At that time, we found that lamellipodia and actin fiber were also reduced by knock‐down of B antigen expression. The transcription of cdc42, a kind of Rho GTPase which plays a key role in actin polymerization, was reduced by down‐regulated B antigen expression. Furthermore, the reduced B antigen expression also inhibited the interaction of cdc42 and N‐WASP. Collectively, our data provide a clue how ABH antigens regulate the cell migration mechanism. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- MicroRNA‐302a sensitizes testicular embryonal carcinoma cells to cisplatin‐induced cell death
- Abstract: Cisplatin is a commonly used chemotherapeutic agent for the treatment of several human malignancies, such as testicular germ cell tumors (TGCT). The toxic effects persist and those that are present long after chemotherapy affect the overall quality of life of patients. MicroRNAs (miRNAs) play important roles in the responses of cancer cells to chemotherapy and have been shown to modulate cell sensitivity to chemotherapeutic drugs. However, the relationship between miRNA expression and cisplatin sensitivity of TGCT has not been fully explored. In this study, the effects of miR‐302a on cisplatin cytotoxicity in TGCT‐derived cell line NTERA‐2 (NT2) were evaluated. We found that expression levels of miR‐302a were increased in cisplatin‐treated NT2 cells. Up‐regulation of miR‐302a significantly increased the sensitivity of NT2 cells to cisplatin by enhancing cisplatin‐induced G2/M phase arrest and the subsequent progression to apoptosis. MiR‐302a also increased the killing effects of cisplatin by lowering the apoptotic threshold; the same result was also observed in another TGCT‐derived cell line, NCCIT. Furthermore, miR‐302a‐enhanced cisplatin sensitivity was partially mediated through the down‐regulation of p21 in NT2 cells. MiR‐302a induced apoptosis was further enhanced by silencing of p53 in NT2 cells. p53 levels were inversely associated with the expression of Oct4, Sox2 and Nanog in response to cisplatin. Thus, targeting miR‐302a may offer new therapeutic interventions in TGCT. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Cisplatin is a commonly used chemotherapeutic agent for the treatment of several human malignancies, such as testicular germ cell tumors (TGCT). The toxic effects persist and those that are present long after chemotherapy affect the overall quality of life of patients. MicroRNAs (miRNAs) play important roles in the responses of cancer cells to chemotherapy and have been shown to modulate cell sensitivity to chemotherapeutic drugs. However, the relationship between miRNA expression and cisplatin sensitivity of TGCT has not been fully explored. In this study, the effects of miR‐302a on cisplatin cytotoxicity in TGCT‐derived cell line NTERA‐2 (NT2) were evaluated. We found that expression levels of miR‐302a were increased in cisplatin‐treated NT2 cells. Up‐regulation of miR‐302a significantly increased the sensitivity of NT2 cells to cisplatin by enhancing cisplatin‐induced G2/M phase arrest and the subsequent progression to apoptosis. MiR‐302a also increased the killing effects of cisplatin by lowering the apoptotic threshold; the same result was also observed in another TGCT‐derived cell line, NCCIT. Furthermore, miR‐302a‐enhanced cisplatin sensitivity was partially mediated through the down‐regulation of p21 in NT2 cells. MiR‐302a induced apoptosis was further enhanced by silencing of p53 in NT2 cells. p53 levels were inversely associated with the expression of Oct4, Sox2 and Nanog in response to cisplatin. Thus, targeting miR‐302a may offer new therapeutic interventions in TGCT. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Functional role of Runx3 in the regulation of aggrecan expression during cartilage development
- Abstract: Runx2 and Runx3 are known to be expressed in the growth plate during endochondral bone formation. Here we addressed the functional role of Runx3 as distinct from Runx2 by using two models of post natal bone repair: fracture healing that proceeds by an endochondral process and marrow ablation that proceeds by only an intramembraneous process. Both Runx2 and Runx3 mRNAs were differentially up regulated during fracture healing. In contrast, only Runx2 showed increased expression after marrow ablation. During fracture healing, Runx3 was expressed earlier than Runx2, was concurrent with the period of chondrogenesis, and coincident with maximal aggrecan expression a protein associated with proliferating and permanent cartilage. Immunohistological analysis showed Runx3 protein was also expressed by chondrocytes in vivo. In contrast, Runx2 was expressed later during chondrocyte hypertrophy, and primary bone formation. The functional activities of Runx3 during chondrocyte differentiation were assessed by examining its regulatory actions on aggrecan gene expression. Aggrecan mRNA levels and aggrecan promoter activity were enhanced in response to the over‐expression of either Runx2 and Runx3 in ATDC5 chondrogenic cell line, while sh‐RNA knocked down of each Runx protein showed that only Runx3 knock down specifically suppressed aggrecan mRNA expression and promoter activity. ChIP assay demonstrated that Runx3 interactions were selective to sites within the aggrecan promoter and were only observed during early periods of chondrogenesis before hypertrophy. Our studies suggest that Runx3 positively regulates aggrecan expression and suggest that its function is more limited to cartilage development than to bone. In aggregate these data further suggest that the various members of the Runx transcription factors are involved in the coordination of chondrocyte development, maturation and hypertrophy during endochondral bone formation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Runx2 and Runx3 are known to be expressed in the growth plate during endochondral bone formation. Here we addressed the functional role of Runx3 as distinct from Runx2 by using two models of post natal bone repair: fracture healing that proceeds by an endochondral process and marrow ablation that proceeds by only an intramembraneous process. Both Runx2 and Runx3 mRNAs were differentially up regulated during fracture healing. In contrast, only Runx2 showed increased expression after marrow ablation. During fracture healing, Runx3 was expressed earlier than Runx2, was concurrent with the period of chondrogenesis, and coincident with maximal aggrecan expression a protein associated with proliferating and permanent cartilage. Immunohistological analysis showed Runx3 protein was also expressed by chondrocytes in vivo. In contrast, Runx2 was expressed later during chondrocyte hypertrophy, and primary bone formation. The functional activities of Runx3 during chondrocyte differentiation were assessed by examining its regulatory actions on aggrecan gene expression. Aggrecan mRNA levels and aggrecan promoter activity were enhanced in response to the over‐expression of either Runx2 and Runx3 in ATDC5 chondrogenic cell line, while sh‐RNA knocked down of each Runx protein showed that only Runx3 knock down specifically suppressed aggrecan mRNA expression and promoter activity. ChIP assay demonstrated that Runx3 interactions were selective to sites within the aggrecan promoter and were only observed during early periods of chondrogenesis before hypertrophy. Our studies suggest that Runx3 positively regulates aggrecan expression and suggest that its function is more limited to cartilage development than to bone. In aggregate these data further suggest that the various members of the Runx transcription factors are involved in the coordination of chondrocyte development, maturation and hypertrophy during endochondral bone formation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Journal of Cellular Physiology: Volume 228, Number 8, August 2013
- Abstract: Cover: Different transporters for miRNA transport. Please see article by Xu et al., pages 1713–1719. Cover designed by Lucía Sagredo Sánchez.
- Abstract: Cover: Different transporters for miRNA transport. Please see article by Xu et al., pages 1713–1719. Cover designed by Lucía Sagredo Sánchez.
- Table of Contents: Volume 228, Number 8
- Highlights: Volume 228, Number 8
- Altered bone development and turnover in transgenic mice over‐expressing lipocalin‐2 in bone
- Abstract: Lipocalin‐2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over‐expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed‐down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate.
In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional Receptor Activator of Nuclear factor‐κB Ligand (RANKL) and of the IL‐6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Lipocalin‐2 (LCN2) is a protein largely expressed in many tissues, associated with different biological phenomena such as cellular differentiation, inflammation and cancer acting as a survival/apoptotic signal. We found that LCN2 was expressed during osteoblast differentiation and we generated transgenic (Tg) mice over‐expressing LCN2 in bone. Tg mice were smaller and presented bone microarchitectural changes in both endochondral and intramembranous bones. In particular, Tg bones displayed a thinner layer of cortical bone and a decreased trabecular number. Osteoblast bone matrix deposition was reduced and osteoblast differentiation was slowed‐down. Differences were also observed in the growth plate of young transgenic mice where chondrocyte displayed a more immature phenotype and a lower proliferation rate.
In bone marrow cell cultures from transgenic mice, the number of osteoclast progenitors was increased whereas in vivo it was increased the number of mature osteoclasts expressing tartrate resistant acid phosphatase (TRAP). Finally, while osteoprotegerin (OPG) levels remained unchanged, the expression of the conventional Receptor Activator of Nuclear factor‐κB Ligand (RANKL) and of the IL‐6 was enhanced in Tg mice. In conclusion, we found that LCN2 plays a role in bone development and turnover having both a negative effect on bone formation, by affecting growth plate development and interfering with osteoblast differentiation, and a positive effect on bone resorption by enhancing osteoclast compartment. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Culture under low physiological oxygen conditions improves the stemness and quality of induced pluripotent stem cells
- Abstract: The ex vivo expansion of stem cells under low physiological oxygen (O2) conditions has been demonstrated to improve the stemness and genomic stability of the cells. We investigated whether low‐oxygen culture would be beneficial for the culture of induced pluripotent stem (iPS) cells. Two human iPS cell lines (201B7 and 253G1) were used for the experiments. Cells expanded from a single colony of each cell line were initiated for culture in 2.5% O2, 5% O2, or 20% O2 and maintained for 2 months in parallel. The levels of intracellular and mitochondrial reactive oxygen species did not differ between the cells cultured under different conditions. More colonies of uniformly smaller size were observed at 2.5% and 5% O2 than at 20% O2. All of these iPS colonies that expanded under the various oxygen conditions stained positively for Oct3/4, Nanog, SSEA‐4, and ALP. However, Western blot analysis showed that the iPS cells cultured at 2.5% and 5% O2 expressed significantly more Nanog but less 53BP1 than those cultured at 20% O2. Data from an array CGH showed no significant chromosomal abnormalities, although some genes involved in cellular and metabolic processes were amplified in the low oxygen culture, particularly at 2.5% O2. Our data suggest that low physiological oxygen culture could improve the stemness and quality of iPS cells, a result that might be associated with the amplification of genes involved in metabolic and cellular processes. Long‐term culture will be necessary to confirm whether low physiological oxygen levels also improve genomic stability. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The ex vivo expansion of stem cells under low physiological oxygen (O2) conditions has been demonstrated to improve the stemness and genomic stability of the cells. We investigated whether low‐oxygen culture would be beneficial for the culture of induced pluripotent stem (iPS) cells. Two human iPS cell lines (201B7 and 253G1) were used for the experiments. Cells expanded from a single colony of each cell line were initiated for culture in 2.5% O2, 5% O2, or 20% O2 and maintained for 2 months in parallel. The levels of intracellular and mitochondrial reactive oxygen species did not differ between the cells cultured under different conditions. More colonies of uniformly smaller size were observed at 2.5% and 5% O2 than at 20% O2. All of these iPS colonies that expanded under the various oxygen conditions stained positively for Oct3/4, Nanog, SSEA‐4, and ALP. However, Western blot analysis showed that the iPS cells cultured at 2.5% and 5% O2 expressed significantly more Nanog but less 53BP1 than those cultured at 20% O2. Data from an array CGH showed no significant chromosomal abnormalities, although some genes involved in cellular and metabolic processes were amplified in the low oxygen culture, particularly at 2.5% O2. Our data suggest that low physiological oxygen culture could improve the stemness and quality of iPS cells, a result that might be associated with the amplification of genes involved in metabolic and cellular processes. Long‐term culture will be necessary to confirm whether low physiological oxygen levels also improve genomic stability. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Regulation of the Delta and Alpha Epithelial Sodium Channel (ENaC) by Ubiquitination and Nedd8
- Abstract: The δ epithelial sodium channel (δENaC1) is a proton‐activated sodium‐selective, amiloride‐sensitive ion channel in the ENaC/degenerin family of ion channels involved in blood pressure regulation and mechanosensation. Other ENaC family members are subject to ubiquitin modification leading to internalization from the cell surface, and degradation of the channel. Here we show that δENaC is also modified by ubiquitin on three intracellular lysine residues. Absence of these lysines abolished ubiquitin modification of δENaC and increased cell surface levels of δENaC. Although the HECT‐domain ubiquitin ligase Nedd4‐2 reduced amiloride‐sensitive current generated by δβγENaC‐containing channels, δENaC does not contain a binding site for Nedd4‐2; therefore, this effect is probably mediated by the βγENaC subunits. Nedd8, a ubiquitin‐like protein that regulates RING‐domain E3 ubiquitin ligases, promoted δENaC ubiquitination, decreased both the intracellular and cell surface δENaC populations, and decreased δβγENaC amiloride‐sensitive short circuit current (Isc‐amiloride) in a mammalian epithelium. Nedd8 also promoted α− and γENaC ubiquitination; decreased the cell surface pools, and decreased αβγENaC Isc‐amiloride. Conversely, XIAP, a single subunit RING E3 ligase, decreased ubiquitinated δENaC, increased the δENaC cell surface pool and increased δβγENaC Isc‐amiloride. Therefore δ− and α − βγENaC channel function may be influenced by RING‐domain E3 ubiquitin ligases. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The δ epithelial sodium channel (δENaC1) is a proton‐activated sodium‐selective, amiloride‐sensitive ion channel in the ENaC/degenerin family of ion channels involved in blood pressure regulation and mechanosensation. Other ENaC family members are subject to ubiquitin modification leading to internalization from the cell surface, and degradation of the channel. Here we show that δENaC is also modified by ubiquitin on three intracellular lysine residues. Absence of these lysines abolished ubiquitin modification of δENaC and increased cell surface levels of δENaC. Although the HECT‐domain ubiquitin ligase Nedd4‐2 reduced amiloride‐sensitive current generated by δβγENaC‐containing channels, δENaC does not contain a binding site for Nedd4‐2; therefore, this effect is probably mediated by the βγENaC subunits. Nedd8, a ubiquitin‐like protein that regulates RING‐domain E3 ubiquitin ligases, promoted δENaC ubiquitination, decreased both the intracellular and cell surface δENaC populations, and decreased δβγENaC amiloride‐sensitive short circuit current (Isc‐amiloride) in a mammalian epithelium. Nedd8 also promoted α− and γENaC ubiquitination; decreased the cell surface pools, and decreased αβγENaC Isc‐amiloride. Conversely, XIAP, a single subunit RING E3 ligase, decreased ubiquitinated δENaC, increased the δENaC cell surface pool and increased δβγENaC Isc‐amiloride. Therefore δ− and α − βγENaC channel function may be influenced by RING‐domain E3 ubiquitin ligases. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Olfactory Ensheathing Cells (OEC)‐ and Neuroblastoma B104 Cell Line‐Conditioned Medium Promotes Differentiation of Human Mesenchymal Stem Cells from Adipose Tissue toward a Neural Phenotype
- Abstract: Olfactory ensheathing cells (OECs) are known to be capable of continuous neurogenesis throughout lifetime and are a source of multiple trophic factors important in central nervous system regeneration. B104 neuroblastoma cells are recognized to induce differentiation of neural stem cells into oligodendrocyte precursor cells. Therefore, the aim of this study was to verify if conditioned medium (CM) obtained from OECs or B104 cells was capable of inducing differentiation of adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) to a neuronal phenotype. In order to, immunocytochemical procedures and flow cytometry analysis were used and some neural markers, as nestin, protein gene product 9.5 (PGP 9.5), microtubule‐associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and neuron cell surface antigen (A2B5) were examined 24 hours and 7 days after the treatment. The results showed that both OECs‐ or B104‐CM treated AT‐MSCs express markers of progenitor and mature neurons (nestin, PGP 9.5 and MAP2) in time‐dependent manner, display morphological features resembling neuronal cells, and result negative for GFAP and A2B5, astrocyte and oligodendrocyte markers, respectively. This study demonstrated that AT‐MSCs can be influenced by the environment, indicating that these cells can respond to environmental cues also versus a neuronal phenotype. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Olfactory ensheathing cells (OECs) are known to be capable of continuous neurogenesis throughout lifetime and are a source of multiple trophic factors important in central nervous system regeneration. B104 neuroblastoma cells are recognized to induce differentiation of neural stem cells into oligodendrocyte precursor cells. Therefore, the aim of this study was to verify if conditioned medium (CM) obtained from OECs or B104 cells was capable of inducing differentiation of adipose tissue‐derived mesenchymal stem cells (AT‐MSCs) to a neuronal phenotype. In order to, immunocytochemical procedures and flow cytometry analysis were used and some neural markers, as nestin, protein gene product 9.5 (PGP 9.5), microtubule‐associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), and neuron cell surface antigen (A2B5) were examined 24 hours and 7 days after the treatment. The results showed that both OECs‐ or B104‐CM treated AT‐MSCs express markers of progenitor and mature neurons (nestin, PGP 9.5 and MAP2) in time‐dependent manner, display morphological features resembling neuronal cells, and result negative for GFAP and A2B5, astrocyte and oligodendrocyte markers, respectively. This study demonstrated that AT‐MSCs can be influenced by the environment, indicating that these cells can respond to environmental cues also versus a neuronal phenotype. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- 1α,25‐dihydroxyvitamin D3 stimulates activin A production to fine‐tune osteoblast‐induced mineralization
- Abstract: In healthy bones mineralization has to be tightly controlled to avoid pathological phenotypes. In this study we investigated interactions between 1α,25(OH)2D3 (1,25D3) and activin A in the regulation of osteoblast induced mineralization. In human osteoblast cultures we demonstrated that besides stimulation of mineralization, 1,25D3 also induced activin A, a strong inhibitor of mineralization. Simultaneously, follistatin (FST), the natural antagonist of activin A, was down‐regulated by1,25D3. This resulted in an increase in activin A activity during 1,25D3 treatment. We also showed that in 1,25D3‐treated osteoblasts, mineralization can be further increased when activin A activity was abrogated by adding exogenous FST. This observation implies that, besides stimulation of mineralization, 1,25D3 also controls activin A‐mediated inhibition of mineralization. Besides activin A, 1,25D3 also induces osteocalcin (BGLAP), another inhibitor of mineralization. Warfarin, which has been shown to inactivate osteocalcin, increased 1,25D3‐induced mineralization. Interaction between these two systems became evident from the synergistic increase in BGLAP expression upon blocking activin activity in 1,25D3‐treated cultures. In conclusion, we demonstrate that 1,25D3 stimulation of mineralization by human osteoblasts is suppressed by concomitant induction of inhibitors of mineralization. Mineralization induction by 1,25D3 may actually be controlled via interplay with activin A and osteocalcin. Finally, this complex regulation of mineralization substantiates the significance of tight control of mineralization to prevent excessive mineralization and consequently reduction in bone quality and strength. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: In healthy bones mineralization has to be tightly controlled to avoid pathological phenotypes. In this study we investigated interactions between 1α,25(OH)2D3 (1,25D3) and activin A in the regulation of osteoblast induced mineralization. In human osteoblast cultures we demonstrated that besides stimulation of mineralization, 1,25D3 also induced activin A, a strong inhibitor of mineralization. Simultaneously, follistatin (FST), the natural antagonist of activin A, was down‐regulated by1,25D3. This resulted in an increase in activin A activity during 1,25D3 treatment. We also showed that in 1,25D3‐treated osteoblasts, mineralization can be further increased when activin A activity was abrogated by adding exogenous FST. This observation implies that, besides stimulation of mineralization, 1,25D3 also controls activin A‐mediated inhibition of mineralization. Besides activin A, 1,25D3 also induces osteocalcin (BGLAP), another inhibitor of mineralization. Warfarin, which has been shown to inactivate osteocalcin, increased 1,25D3‐induced mineralization. Interaction between these two systems became evident from the synergistic increase in BGLAP expression upon blocking activin activity in 1,25D3‐treated cultures. In conclusion, we demonstrate that 1,25D3 stimulation of mineralization by human osteoblasts is suppressed by concomitant induction of inhibitors of mineralization. Mineralization induction by 1,25D3 may actually be controlled via interplay with activin A and osteocalcin. Finally, this complex regulation of mineralization substantiates the significance of tight control of mineralization to prevent excessive mineralization and consequently reduction in bone quality and strength. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Targeting Deregulated Epigenetic Control in Cancer: Cancer Epigenomics as a Platform for Risk Assessment, Early Detection, Targeted Therapy and Potential for Relapse
- Abstract: Cancer is a multifaceted disease that involves acquisition of genetic mutations, deletions, and amplifications as well as deregulation of epigenetic mechanisms that fine‐tune gene regulation. Key epigenetic mechanisms that include histone modifications, DNA methylation, and non‐coding RNA‐mediated gene silencing are often deregulated in a variety of cancers. Subnuclear localization of key proteins in the interphase nucleus and bookmarking of genes by lineage commitment factors in mitosis – a new dimension to epigenetic control of fundamental biological processes – is also modified in cancer. In this review, we discuss the various aspects of epigenetic control that are operative in a variety of cancers and their potential for risk assessment, early detection, targeted therapy and personalized medicine. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Cancer is a multifaceted disease that involves acquisition of genetic mutations, deletions, and amplifications as well as deregulation of epigenetic mechanisms that fine‐tune gene regulation. Key epigenetic mechanisms that include histone modifications, DNA methylation, and non‐coding RNA‐mediated gene silencing are often deregulated in a variety of cancers. Subnuclear localization of key proteins in the interphase nucleus and bookmarking of genes by lineage commitment factors in mitosis – a new dimension to epigenetic control of fundamental biological processes – is also modified in cancer. In this review, we discuss the various aspects of epigenetic control that are operative in a variety of cancers and their potential for risk assessment, early detection, targeted therapy and personalized medicine. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- PHLP2 is essential and plays a role in ciliogenesis and microtubule assembly in the ciliate Tetrahymena thermophila
- Abstract: Recent studies have implicated the phosducin‐like protein‐2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 hr. Overexpressed Phlp2p‐HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP‐Phlp2p caused a dominant‐negative effect. Cells overproducing GFP‐Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non‐tagged Phlp2p. Growing GFP‐Phlp2p‐overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel‐treated GFP‐Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP‐Phlp2p‐overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule‐dependent functions including cilia assembly. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Recent studies have implicated the phosducin‐like protein‐2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 hr. Overexpressed Phlp2p‐HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP‐Phlp2p caused a dominant‐negative effect. Cells overproducing GFP‐Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non‐tagged Phlp2p. Growing GFP‐Phlp2p‐overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel‐treated GFP‐Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP‐Phlp2p‐overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule‐dependent functions including cilia assembly. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Therapeutic potential of a pyridoxal‐based vanadium(IV) complex showing selective cytotoxicity for cancer vs. healthy cells
- Abstract: Vanadium compounds can exert anticancer effects, partly due to inhibition of tyrosine phosphatases. Here we report the effect of N,N'‐ethylenebis (pyridoxylideneiminato) vanadium (IV) complex [Pyr2enV(IV)], that induced 93% and 57% of cell mortality in A375 (human melanoma) and A549 (human lung carcinoma) cells, respectively; the mortality was
- Abstract: Vanadium compounds can exert anticancer effects, partly due to inhibition of tyrosine phosphatases. Here we report the effect of N,N'‐ethylenebis (pyridoxylideneiminato) vanadium (IV) complex [Pyr2enV(IV)], that induced 93% and 57% of cell mortality in A375 (human melanoma) and A549 (human lung carcinoma) cells, respectively; the mortality was
- Small GTPase Rap1 regulates cell migration through regulation of small GTPase RhoA activity in response to transforming growth factor‐β1
- Abstract: Transforming growth factor (TGF)‐β1 regulates diverse cellular functions. Particularly, TGF‐β1 induces monocyte migration to sites of injury or inflammation in early period, whereas TGF‐β1 inhibits cell migration in late phase. In this study, we attempted to understand how TGF‐β1 suppresses cell migration in late phase. We found that TGF‐β1 of short exposure induces the production of chemokines, such as macrophage inflammatory protein (MIP)‐1α, by Raw 264.7 cells. However, knock‐down of small GTPase RhoA by sh‐RhoA inhibited the production of MIP‐1α and macrophage migration, suggesting that RhoA is essential for expression of this chemokine. An activator of Epac (exchange proteins directly activated by cAMP; a guanine nucleotide exchange factor of Rap1), 8CPT‐2Me‐cAMP which leads to Rap1 activation abrogated MIP‐1α expression and macrophage migration. Indeed, GTP‐RhoA and GTP‐Rap1 levels were reciprocally regulated in a time‐dependent manner following TGF‐β1 stimulation. 8CPT‐2Me‐cAMP suppressed GTP‐RhoA levels, whereas si‐Rap1 augmented GTP‐RhoA levels and cell migration. TGF‐β1 produced cAMP in late period and si‐RNAs of Epac1 (exchange protein directly activated by cAMP 1) and Epac2 reduced GTP‐Rap1 levels leading to promotion of GTP‐RhoA levels. Furthermore, si‐RNA of ARAP3 (Rap‐dependent RhoGAP) increased GTP‐RhoA level and cell migration. Therefore, we propose the mechanism that prolonged TGF‐β1 treatment produce cAMP, which activates sequentially Epac, Rap1 and ARAP3, resulting in suppression of RhoA, chemokine expression, and macrophage migration. Contrary to the general concept that Rap1 stimulates cell migration, we demonstrated in this study that Rap1 inhibits cell migration by suppression of RhoA activity in response to TGF‐β1. © 2013 Wiley Periodicals, Inc.
- Abstract: Transforming growth factor (TGF)‐β1 regulates diverse cellular functions. Particularly, TGF‐β1 induces monocyte migration to sites of injury or inflammation in early period, whereas TGF‐β1 inhibits cell migration in late phase. In this study, we attempted to understand how TGF‐β1 suppresses cell migration in late phase. We found that TGF‐β1 of short exposure induces the production of chemokines, such as macrophage inflammatory protein (MIP)‐1α, by Raw 264.7 cells. However, knock‐down of small GTPase RhoA by sh‐RhoA inhibited the production of MIP‐1α and macrophage migration, suggesting that RhoA is essential for expression of this chemokine. An activator of Epac (exchange proteins directly activated by cAMP; a guanine nucleotide exchange factor of Rap1), 8CPT‐2Me‐cAMP which leads to Rap1 activation abrogated MIP‐1α expression and macrophage migration. Indeed, GTP‐RhoA and GTP‐Rap1 levels were reciprocally regulated in a time‐dependent manner following TGF‐β1 stimulation. 8CPT‐2Me‐cAMP suppressed GTP‐RhoA levels, whereas si‐Rap1 augmented GTP‐RhoA levels and cell migration. TGF‐β1 produced cAMP in late period and si‐RNAs of Epac1 (exchange protein directly activated by cAMP 1) and Epac2 reduced GTP‐Rap1 levels leading to promotion of GTP‐RhoA levels. Furthermore, si‐RNA of ARAP3 (Rap‐dependent RhoGAP) increased GTP‐RhoA level and cell migration. Therefore, we propose the mechanism that prolonged TGF‐β1 treatment produce cAMP, which activates sequentially Epac, Rap1 and ARAP3, resulting in suppression of RhoA, chemokine expression, and macrophage migration. Contrary to the general concept that Rap1 stimulates cell migration, we demonstrated in this study that Rap1 inhibits cell migration by suppression of RhoA activity in response to TGF‐β1. © 2013 Wiley Periodicals, Inc.
- Regulation of intracellular pH by glycosaminoglycans
- Abstract: The intracellular pH is regulated by a delicate balance of ion distribution across the plasma membrane and the physico‐chemical properties of intra‐ and extracellular components. We analysed the effects of glycosaminoglycans on the intracellular pH of fibroblasts by using the fluorescent pH indicator BCECF‐AM. Addition of hyaluronan, hyaluronan oligosaccharides, chondroitin sulfate or heparin to the culture medium of fibroblasts caused intracellular acidification from pH 7.2 to pH 6.7 in a concentration dependent manner. High molecular weight hyaluronan acidified more than hyaluronan oligosaccharides at the same concentrations. Hyaluronidase treatment or inhibition of hyaluronan export with xanthohumol led to intracellular alkalization. These observations indicated that extracellular glycosaminoglycans participate in intracellular pH regulation. The mechanism was explained by Donnan effects and molecular crowding. © 2013 Wiley Periodicals, Inc.
- Abstract: The intracellular pH is regulated by a delicate balance of ion distribution across the plasma membrane and the physico‐chemical properties of intra‐ and extracellular components. We analysed the effects of glycosaminoglycans on the intracellular pH of fibroblasts by using the fluorescent pH indicator BCECF‐AM. Addition of hyaluronan, hyaluronan oligosaccharides, chondroitin sulfate or heparin to the culture medium of fibroblasts caused intracellular acidification from pH 7.2 to pH 6.7 in a concentration dependent manner. High molecular weight hyaluronan acidified more than hyaluronan oligosaccharides at the same concentrations. Hyaluronidase treatment or inhibition of hyaluronan export with xanthohumol led to intracellular alkalization. These observations indicated that extracellular glycosaminoglycans participate in intracellular pH regulation. The mechanism was explained by Donnan effects and molecular crowding. © 2013 Wiley Periodicals, Inc.
- KLF4 promoted odontoblastic differentiation of mouse dental papilla cells via regulation of DMP1
- Abstract: Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix‐secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of odontoblasts. We have recently found that Krüppel‐like factor 4 (Klf4) was expressed in the polarizing and elongating odontoblasts, but the function of Klf4 in the differentiation of odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of odontoblasts by up‐regulating some odontoblast‐related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line‐mDPC6T. Overexpression of Klf4 significantly up‐regulated odontoblast‐related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock‐down of Klf4 down‐regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4—Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock‐down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of odontoblasts via the up‐regulation of Dmp1. © 2013 Wiley Periodicals, Inc.
- Abstract: Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix‐secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of odontoblasts. We have recently found that Krüppel‐like factor 4 (Klf4) was expressed in the polarizing and elongating odontoblasts, but the function of Klf4 in the differentiation of odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of odontoblasts by up‐regulating some odontoblast‐related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line‐mDPC6T. Overexpression of Klf4 significantly up‐regulated odontoblast‐related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock‐down of Klf4 down‐regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4—Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock‐down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of odontoblasts via the up‐regulation of Dmp1. © 2013 Wiley Periodicals, Inc.
- Suppression of interactions between prostate tumor cell integrin αvβ3 and endothelial ICAM‐1 by simvastatin inhibits prostate cancer micrometastasis
- Abstract: Cancer micrometastasis relies on the ability of cancer cells to secrete angiogenic modulators, to interact with the vascular endothelium, and to overcome the resistance offered by the endothelial‐barrier. Being an essential step prior to metastasis, blockage of micrometastasis can have potential applications in cancer therapy and metastasis prevention. Due to poorly known molecular mechanisms leading to micrometastasis, developing therapeutic strategies to target prostate cancer utilizing drugs that block micrometastasis is far from reality. Here we demonstrate the potential benefits of simvastatin in the inhibition of prostate cancer micrometastasis and reveal the novel molecular mechanisms underlying this process. First, we showed that simvastatin inhibited the ability of human PC3 prostate cancer cells for transendothelial migration in vitro. Second, our data indicated that simvastatin modulates the expression of tumor derived factors such as angiopoietins and VEGF‐A at the mRNA and protein levels by the PC3 cells, thus preventing endothelial‐barrier disruption. Third, simvastatin directly activated endothelial cells and enhances endothelial‐barrier resistance. Apart from this, our study revealed that simvastatin‐mediated effect on PC3 micrometastasis was mediated through inhibition of integrin αvβ3 activity and suppression of interaction between prostate cancer cell integrin αvβ3 with endothelial ICAM‐1. © 2013 Wiley Periodicals, Inc.
- Abstract: Cancer micrometastasis relies on the ability of cancer cells to secrete angiogenic modulators, to interact with the vascular endothelium, and to overcome the resistance offered by the endothelial‐barrier. Being an essential step prior to metastasis, blockage of micrometastasis can have potential applications in cancer therapy and metastasis prevention. Due to poorly known molecular mechanisms leading to micrometastasis, developing therapeutic strategies to target prostate cancer utilizing drugs that block micrometastasis is far from reality. Here we demonstrate the potential benefits of simvastatin in the inhibition of prostate cancer micrometastasis and reveal the novel molecular mechanisms underlying this process. First, we showed that simvastatin inhibited the ability of human PC3 prostate cancer cells for transendothelial migration in vitro. Second, our data indicated that simvastatin modulates the expression of tumor derived factors such as angiopoietins and VEGF‐A at the mRNA and protein levels by the PC3 cells, thus preventing endothelial‐barrier disruption. Third, simvastatin directly activated endothelial cells and enhances endothelial‐barrier resistance. Apart from this, our study revealed that simvastatin‐mediated effect on PC3 micrometastasis was mediated through inhibition of integrin αvβ3 activity and suppression of interaction between prostate cancer cell integrin αvβ3 with endothelial ICAM‐1. © 2013 Wiley Periodicals, Inc.
- Polycyclic aromatic hydrocarbons ‐ induced ROS accumulation enhances mutagenic potential of T‐antigen from human polyomavirus JC
- Abstract: Polycyclic Aromatic Hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep‐fried food, and in natural crude oil. Since PAH‐metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T‐antigen (JCV T‐antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil‐PAHs), and detected several carcinogenic PAHs. The oil‐PAHs were tested in exponentially growing cultures on normal mouse fibroblasts (R508), and in R508 stably expressing JCV T‐antigen (R508/T). The oil‐PAHs were cytotoxic only at relatively high doses (1:50–1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non‐toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil‐PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T‐antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low‐fidelity repair by non‐homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil‐PAHs. Our results indicate for the first time carcinogenic synergy in which oil‐PAHs trigger oxidative DNA damage and JCV T‐antigen compromises DNA repair fidelity. © 2013 Wiley Periodicals, Inc.
- Abstract: Polycyclic Aromatic Hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep‐fried food, and in natural crude oil. Since PAH‐metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T‐antigen (JCV T‐antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil‐PAHs), and detected several carcinogenic PAHs. The oil‐PAHs were tested in exponentially growing cultures on normal mouse fibroblasts (R508), and in R508 stably expressing JCV T‐antigen (R508/T). The oil‐PAHs were cytotoxic only at relatively high doses (1:50–1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non‐toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil‐PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T‐antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low‐fidelity repair by non‐homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil‐PAHs. Our results indicate for the first time carcinogenic synergy in which oil‐PAHs trigger oxidative DNA damage and JCV T‐antigen compromises DNA repair fidelity. © 2013 Wiley Periodicals, Inc.
- The zebrafish as a model for nociception studies
- Abstract: Nociception is the sensory mechanism used to detect cues that can harm an organism. The understanding of the neural networks and molecular controls of the reception of pain remains an ongoing challenge for biologists. While we have made significant progress in identifying a number of molecules and pathways that are involved in transduction of noxious stimuli, from the skin through the sensory receptor cell and from this to the spinal cord on into the central nervous system, we still lack a clear understanding of the perceptual processes, the responses to pain and the regulation of pain perception. Mice and rat animal models have been extensively used for nociception studies. However, the study of pain and noiception in these organisms can be rather laborious, costly and time consuming. Conversely the use of Drosophila and C. elegans may be affected by the large evolutionary distance between these animals and humans. We outline here the reasons why zebrafish presents a new and attractive model for studying pain reception and responses and the most interesting findings in the study of nociception that have been obtained using the zebrafish model. © 2013 Wiley Periodicals, Inc.
- Abstract: Nociception is the sensory mechanism used to detect cues that can harm an organism. The understanding of the neural networks and molecular controls of the reception of pain remains an ongoing challenge for biologists. While we have made significant progress in identifying a number of molecules and pathways that are involved in transduction of noxious stimuli, from the skin through the sensory receptor cell and from this to the spinal cord on into the central nervous system, we still lack a clear understanding of the perceptual processes, the responses to pain and the regulation of pain perception. Mice and rat animal models have been extensively used for nociception studies. However, the study of pain and noiception in these organisms can be rather laborious, costly and time consuming. Conversely the use of Drosophila and C. elegans may be affected by the large evolutionary distance between these animals and humans. We outline here the reasons why zebrafish presents a new and attractive model for studying pain reception and responses and the most interesting findings in the study of nociception that have been obtained using the zebrafish model. © 2013 Wiley Periodicals, Inc.
- FGF‐2 enhances Runx‐2/Smads nuclear localization in BMP‐2 canonical signaling in osteoblasts
- Abstract: Bone morphogenetic protein 2 (BMP‐2) is one of the most potent regulators of osteoblast differentiation and bone formation. R‐Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they are translocated in the nucleus regulating transcription target genes by interacting with various transcription factors. Runx‐2 proteins have been shown to interact through their C‐terminal segment with Smads and this interaction is required for in vivo osteogenesis. In particular, recruitment of Smads to intranuclear sites is Runx‐2 dependent, and Runx‐2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites. Previously, we have shown, by in vitro and in vivo experiments, that BMP‐2 up‐regulated FGF‐2 which is important for the maximal responses of BMP‐2 in bone. In this study, we found that endogenous FGF2 is necessary for BMP‐2 induced nuclear accumulation and co‐localization of Runx‐2 and phospho‐Smads1/5/8, while Runx/Smads nuclear accumulation and co‐localization was reduced in Fgf2‐/‐ osteoblasts. Based on these novel data, we conclude that the impaired nuclear accumulation of Runx‐2 in Fgf2‐/‐ osteoblasts reduces R‐Smads sub‐nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice. © 2013 Wiley Periodicals, Inc.
- Abstract: Bone morphogenetic protein 2 (BMP‐2) is one of the most potent regulators of osteoblast differentiation and bone formation. R‐Smads (Smads 1/5/8) are the major transducers for BMPs receptors and, once activated, they are translocated in the nucleus regulating transcription target genes by interacting with various transcription factors. Runx‐2 proteins have been shown to interact through their C‐terminal segment with Smads and this interaction is required for in vivo osteogenesis. In particular, recruitment of Smads to intranuclear sites is Runx‐2 dependent, and Runx‐2 factor may accommodate the dynamic targeting of signal transducer to active transcription sites. Previously, we have shown, by in vitro and in vivo experiments, that BMP‐2 up‐regulated FGF‐2 which is important for the maximal responses of BMP‐2 in bone. In this study, we found that endogenous FGF2 is necessary for BMP‐2 induced nuclear accumulation and co‐localization of Runx‐2 and phospho‐Smads1/5/8, while Runx/Smads nuclear accumulation and co‐localization was reduced in Fgf2‐/‐ osteoblasts. Based on these novel data, we conclude that the impaired nuclear accumulation of Runx‐2 in Fgf2‐/‐ osteoblasts reduces R‐Smads sub‐nuclear targeting with a consequent decreased expression of differentiating markers and impaired bone formation in Fgf2 null mice. © 2013 Wiley Periodicals, Inc.
- Caveolin and TGF‐β entanglements
- Abstract: Transforming growth factor (TGF)‐β is a multifunctional cytokine acting during development, tissue homeostasis, regeneration processes and disease progression. Due to its pleiotropic effects, tight regulation of the induced signaling cascades is mandatory. Caveolin proteins regulate a specific endocytic pathway and modulate diverse signaling pathways and thus have been related to severe disorders, e.g. cancer and fibrosis. Caveolin affects TGF‐β/‐Smad and non‐Smad signaling in many ways and thus can determine the cellular outcome upon TGF‐β challenge. Reciprocal regulation of caveolin and TGF‐β is also evident, ranging from gene expression to miRNA regulation. Finally, there is in vivo evidence that this crosstalk influences disease development and progression. This review gives an overview about the multifaceted relations of caveolin and TGF‐β. © 2013 Wiley Periodicals, Inc.
- Abstract: Transforming growth factor (TGF)‐β is a multifunctional cytokine acting during development, tissue homeostasis, regeneration processes and disease progression. Due to its pleiotropic effects, tight regulation of the induced signaling cascades is mandatory. Caveolin proteins regulate a specific endocytic pathway and modulate diverse signaling pathways and thus have been related to severe disorders, e.g. cancer and fibrosis. Caveolin affects TGF‐β/‐Smad and non‐Smad signaling in many ways and thus can determine the cellular outcome upon TGF‐β challenge. Reciprocal regulation of caveolin and TGF‐β is also evident, ranging from gene expression to miRNA regulation. Finally, there is in vivo evidence that this crosstalk influences disease development and progression. This review gives an overview about the multifaceted relations of caveolin and TGF‐β. © 2013 Wiley Periodicals, Inc.
- WEIGHTED REGISTRATION OF 123I‐FP‐CIT SPECT IMAGES IMPROVES ACCURACY OF BINDING POTENTIAL ESTIMATES IN PATHOLOGICALLY LOW STRIATAL UPTAKE
- Abstract: Purpose
To test whether the use of a striatum weighted image may improve registration accuracy and diagnostic outcome in patients with parkinsonian syndromes (PS).
Methods
Weighted images were generated by increasing signal intensity of striatal voxels and used as intermediate dataset for co‐registering the brain image onto template. Experimental validation was performed using an anthropomorphic striatal phantom. 123I‐FP‐CIT SPECT binding ratios were manually determined in 67 PS subjects an and compared to those obtained using unsupervised standard (UWR) and weighted registered (WR) approach. Normalized cost function was used to evaluate the accuracy of phantom and subjects registered images to the template. Reproducibility between unsupervised and manual ratios was assessed by using intra‐class correlation coefficient (ICC) and Bland and Altman analysis. Correlation coefficient was used to assess the dependence of semi‐quantitative ratios on clinical findings.
Results
Weighted method improves accuracy of brain registration onto template as determined by cost function in phantom (0.86 ± 0.06vs.0.98 ± 0.02; Student's t test, P = 0.04) and in subject scans (0.69 ± 0.06vs.0.53 ± 0.06; Student's t test, P
- Abstract: Purpose
To test whether the use of a striatum weighted image may improve registration accuracy and diagnostic outcome in patients with parkinsonian syndromes (PS).
Methods
Weighted images were generated by increasing signal intensity of striatal voxels and used as intermediate dataset for co‐registering the brain image onto template. Experimental validation was performed using an anthropomorphic striatal phantom. 123I‐FP‐CIT SPECT binding ratios were manually determined in 67 PS subjects an and compared to those obtained using unsupervised standard (UWR) and weighted registered (WR) approach. Normalized cost function was used to evaluate the accuracy of phantom and subjects registered images to the template. Reproducibility between unsupervised and manual ratios was assessed by using intra‐class correlation coefficient (ICC) and Bland and Altman analysis. Correlation coefficient was used to assess the dependence of semi‐quantitative ratios on clinical findings.
Results
Weighted method improves accuracy of brain registration onto template as determined by cost function in phantom (0.86 ± 0.06vs.0.98 ± 0.02; Student's t test, P = 0.04) and in subject scans (0.69 ± 0.06vs.0.53 ± 0.06; Student's t test, P
- Induction of STEAP4 correlates with 1,25‐dihydroxyvitamin D3 stimulation of adipogenesis in mesenchymal progenitor cells derived from human adipose tissue
- Abstract: The vitamin D receptor (VDR) is expressed in human adipocytes and is transiently induced during early adipogenesis in mesenchymal progenitor cell models. VDR null mice exhibit enhanced energy expenditure and reduced adiposity even when fed high fat diets. Adipocyte‐specific transgenic‐expression of human VDR in mice enhances adipose tissue mass, indicating that VDR activation in adipocytes enhances lipid storage in vivo. In these studies we conducted genomic profiling and differentiation assays in primary cultures of human adipose‐derived mesenchymal progenitor cells to define the role of the VDR and its ligand 1,25‐dihydroxyvitamin D3 (1,25D) in adipogenesis. In the presence of adipogenic media, 1,25D promoted lipid accumulation and enhanced the expression of FABP4, FASN and PPARγ. Mesenchymal cells derived from 6‐month old VDR null mice exhibited impaired adipogenesis ex vivo but differentiation was restored by stable expression of human VDR. STEAP4, a gene that encodes a metalloreductase linked to obesity, insulin sensitivity, metabolic homeostasis and inflammation, was highly induced in human adipose cells differentiated in the presence of 1,25D but was minimally affected by 1,25D in undifferentiated precursors. These studies provide a molecular basis for recent epidemiological associations between vitamin D status, body weight and insulin resistance which may have relevance for prevention or treatment of metabolic syndrome and obesity. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The vitamin D receptor (VDR) is expressed in human adipocytes and is transiently induced during early adipogenesis in mesenchymal progenitor cell models. VDR null mice exhibit enhanced energy expenditure and reduced adiposity even when fed high fat diets. Adipocyte‐specific transgenic‐expression of human VDR in mice enhances adipose tissue mass, indicating that VDR activation in adipocytes enhances lipid storage in vivo. In these studies we conducted genomic profiling and differentiation assays in primary cultures of human adipose‐derived mesenchymal progenitor cells to define the role of the VDR and its ligand 1,25‐dihydroxyvitamin D3 (1,25D) in adipogenesis. In the presence of adipogenic media, 1,25D promoted lipid accumulation and enhanced the expression of FABP4, FASN and PPARγ. Mesenchymal cells derived from 6‐month old VDR null mice exhibited impaired adipogenesis ex vivo but differentiation was restored by stable expression of human VDR. STEAP4, a gene that encodes a metalloreductase linked to obesity, insulin sensitivity, metabolic homeostasis and inflammation, was highly induced in human adipose cells differentiated in the presence of 1,25D but was minimally affected by 1,25D in undifferentiated precursors. These studies provide a molecular basis for recent epidemiological associations between vitamin D status, body weight and insulin resistance which may have relevance for prevention or treatment of metabolic syndrome and obesity. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Frequency‐dependent Cell Death by Optical Tweezers Manipulation
- Abstract: Optical tweezers were used to scan individual Chronic Myelogenous Leukemia cells to determine if the cell death depends on the scanning conditions. Although increasing the scanning frequency or amplitude means greater force applied to the cells, their effects on cell death are not a simple increasing trend, as observed in the optical microscopy. Indeed, cell death sharply increased at particular screening frequencies and amplitudes, whereas other frequencies or amplitudes were less detrimental. These results suggest that cell damage was more sensitive to certain scanning conditions, rather than simply high applied forces. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Optical tweezers were used to scan individual Chronic Myelogenous Leukemia cells to determine if the cell death depends on the scanning conditions. Although increasing the scanning frequency or amplitude means greater force applied to the cells, their effects on cell death are not a simple increasing trend, as observed in the optical microscopy. Indeed, cell death sharply increased at particular screening frequencies and amplitudes, whereas other frequencies or amplitudes were less detrimental. These results suggest that cell damage was more sensitive to certain scanning conditions, rather than simply high applied forces. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Na+/H+ exchange is inactivated during mouse oocyte meiosis, facilitating glycine accumulation that maintains cell volume in embryos
- Abstract: The coupled action of the Na+/H+ exchanger NHE1 and the HCO3‐/Cl‐ exchanger AE2 constitutes the principal mechanism for acute correction of decreased cell volume in mammalian somatic cells, while, when acting separately, they regulate intracellular pH. It was previously found that AE2 becomes inactivated during meiosis in mouse oocytes. Similarly, NHE1 activity stimulated by intracellular acidosis was present in preovulatory germinal vesicle stage (GV) mouse oocytes and then decreased during meiotic maturation. In contrast, NHE1 activity stimulated by decreased cell volume was low in GV oocytes but became active during meiotic maturation as the oocyte detached from the zona pellucida. It then decreased again in mature eggs similar to activity stimulated by acidosis. The subcellular localization of NHE1 was investigated with YFP‐tagged NHE1. Exogenous NHE1 expressed in GV oocytes localized to the plasma membrane and resulted in increased Na+/H+ exchanger activity, but only when co‐expressed with Calcineurin Homologous Protein 1 (CHP1). When oocytes expressing functional NHE1 were matured to eggs, however, membrane localization of NHE1 and Na+/H+ exchanger activity were lost. It was unknown why NHE1 and AE2 activities are suppressed during meiotic maturation. Maintenance of cell volume in preimplantation embryos requires glycine accumulation via the GLYT1 transporter, a process unique to eggs and early embryos that is initiated during meiotic maturation. When NHE1 and AE2 activities were maintained in GV oocytes by exogenous expression, glycine accumulation was inhibited. We propose that NHE1‐mediated acute cell volume regulation is inactivated during meiotic maturation to allow preferential accumulation of glycine in eggs. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The coupled action of the Na+/H+ exchanger NHE1 and the HCO3‐/Cl‐ exchanger AE2 constitutes the principal mechanism for acute correction of decreased cell volume in mammalian somatic cells, while, when acting separately, they regulate intracellular pH. It was previously found that AE2 becomes inactivated during meiosis in mouse oocytes. Similarly, NHE1 activity stimulated by intracellular acidosis was present in preovulatory germinal vesicle stage (GV) mouse oocytes and then decreased during meiotic maturation. In contrast, NHE1 activity stimulated by decreased cell volume was low in GV oocytes but became active during meiotic maturation as the oocyte detached from the zona pellucida. It then decreased again in mature eggs similar to activity stimulated by acidosis. The subcellular localization of NHE1 was investigated with YFP‐tagged NHE1. Exogenous NHE1 expressed in GV oocytes localized to the plasma membrane and resulted in increased Na+/H+ exchanger activity, but only when co‐expressed with Calcineurin Homologous Protein 1 (CHP1). When oocytes expressing functional NHE1 were matured to eggs, however, membrane localization of NHE1 and Na+/H+ exchanger activity were lost. It was unknown why NHE1 and AE2 activities are suppressed during meiotic maturation. Maintenance of cell volume in preimplantation embryos requires glycine accumulation via the GLYT1 transporter, a process unique to eggs and early embryos that is initiated during meiotic maturation. When NHE1 and AE2 activities were maintained in GV oocytes by exogenous expression, glycine accumulation was inhibited. We propose that NHE1‐mediated acute cell volume regulation is inactivated during meiotic maturation to allow preferential accumulation of glycine in eggs. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Epigallocatechin gallate inhibits growth and Epithelial‐to‐Mesenchymal Transition in human thyroid carcinoma cell lines
- Abstract: Well‐differentiated papillary and follicular thyroid carcinoma are the most frequent types of thyroid cancer and the prognosis is generally favorable however, a number of patients develops recurrences. Epigallocatechin‐3‐gallate (EGCG), a major catechin in green tea, was shown to possess remarkable therapeutic potential against various types of human cancers, although data on thyroid cancer cells are still lacking. The aim of this study was to investigate the effect of EGCG on the proliferation and motility of human thyroid papillary (FB‐2) and follicular (WRO) carcinoma cell lines.
Our results demonstrate that EGCG (10, 40, 60 μM) treatment inhibited the growth of FB‐2 and WRO cells in a dose‐dependent manner. These changes were associated with reduced cyclin D1, increased p21 and p53 expression. Furthermore, EGCG suppressed phosphorylation of AKT and ERK1/2. In addition EGCG treatment results in reduction of cell motility and migration. Changes in motility and migration in FB‐2 were associated with modulation in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. After 24h EGCG caused an increase of the E‐cadherin expression and a concomitant decrease of SNAIL, ZEB and the basic helix–loop–helix transcription factor TWIST. Besides expression of Vimentin, N‐cadherin and α5‐integrin was down‐regulated. These data well correlate with a reduction of MMP9 activity as evidenced by gelatin zymography.
Our findings support the inhibitory role of EGCG on thyroid cancer cell proliferation and motility with concomitant loss of epithelial‐to‐mesenchymal cell transition markers. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Well‐differentiated papillary and follicular thyroid carcinoma are the most frequent types of thyroid cancer and the prognosis is generally favorable however, a number of patients develops recurrences. Epigallocatechin‐3‐gallate (EGCG), a major catechin in green tea, was shown to possess remarkable therapeutic potential against various types of human cancers, although data on thyroid cancer cells are still lacking. The aim of this study was to investigate the effect of EGCG on the proliferation and motility of human thyroid papillary (FB‐2) and follicular (WRO) carcinoma cell lines.
Our results demonstrate that EGCG (10, 40, 60 μM) treatment inhibited the growth of FB‐2 and WRO cells in a dose‐dependent manner. These changes were associated with reduced cyclin D1, increased p21 and p53 expression. Furthermore, EGCG suppressed phosphorylation of AKT and ERK1/2. In addition EGCG treatment results in reduction of cell motility and migration. Changes in motility and migration in FB‐2 were associated with modulation in the expression of several proteins involved in cell adhesion and reorganization of actin cytoskeleton. After 24h EGCG caused an increase of the E‐cadherin expression and a concomitant decrease of SNAIL, ZEB and the basic helix–loop–helix transcription factor TWIST. Besides expression of Vimentin, N‐cadherin and α5‐integrin was down‐regulated. These data well correlate with a reduction of MMP9 activity as evidenced by gelatin zymography.
Our findings support the inhibitory role of EGCG on thyroid cancer cell proliferation and motility with concomitant loss of epithelial‐to‐mesenchymal cell transition markers. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Reversal of Cardiac Dysfunction and Subcellular Alterations by Metoprolol in Heart Failure Due to Myocardial Infarction
- Abstract: In order to examine the reversibility of heart failure due to myocardial infarction (MI) by β‐adrenoceptor blockade, 12 wks infarcted rats were treated with or without metoprolol (50 mg/kg/day) for 8 wks. The depressed left ventricular (LV) systolic pressure, positive and negative rates of changes in pressure development, ejection fraction, fractional shortening and cardiac output, as well as increased LV end‐diastolic pressure in 20 wks MI animals were partially reversed by metoprolol. MI‐induced decreases in septum (systolic) thickness as well as increase in LV posterior wall thickness and LV internal diameter were partially or fully reversible by metoprolol. Treatment of MI animals with metoprolol partially reversed the elevated levels of plasma norepinephrine and dopamine without affecting the elevated levels of epinephrine. Although sarcoplasmic reticular (SR) Ca2+‐uptake, as well as protein content for SR Ca2+‐pump and phospholamban, were reduced in the infarcted hearts; these changes were partially reversible with metoprolol. Depressed myofibrillar Ca2+‐stimulated ATPase activity, as well as mRNA levels for SR Ca2+‐pump, phospholamban and α‐myosin heavy chain, were unaffected whereas increased mRNA level for β‐myosin heavy chain was partially reversed by metoprolol. The results suggest that partial improvement of cardiac performance by β‐adrenoceptor blockade at advanced stages of heart failure may be due to partial reversal of changes in SR Ca2+‐pump function whereas partial to complete reverse cardiac remodeling may be due to partial reduction in the elevated levels of plasma catecholamines. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: In order to examine the reversibility of heart failure due to myocardial infarction (MI) by β‐adrenoceptor blockade, 12 wks infarcted rats were treated with or without metoprolol (50 mg/kg/day) for 8 wks. The depressed left ventricular (LV) systolic pressure, positive and negative rates of changes in pressure development, ejection fraction, fractional shortening and cardiac output, as well as increased LV end‐diastolic pressure in 20 wks MI animals were partially reversed by metoprolol. MI‐induced decreases in septum (systolic) thickness as well as increase in LV posterior wall thickness and LV internal diameter were partially or fully reversible by metoprolol. Treatment of MI animals with metoprolol partially reversed the elevated levels of plasma norepinephrine and dopamine without affecting the elevated levels of epinephrine. Although sarcoplasmic reticular (SR) Ca2+‐uptake, as well as protein content for SR Ca2+‐pump and phospholamban, were reduced in the infarcted hearts; these changes were partially reversible with metoprolol. Depressed myofibrillar Ca2+‐stimulated ATPase activity, as well as mRNA levels for SR Ca2+‐pump, phospholamban and α‐myosin heavy chain, were unaffected whereas increased mRNA level for β‐myosin heavy chain was partially reversed by metoprolol. The results suggest that partial improvement of cardiac performance by β‐adrenoceptor blockade at advanced stages of heart failure may be due to partial reversal of changes in SR Ca2+‐pump function whereas partial to complete reverse cardiac remodeling may be due to partial reduction in the elevated levels of plasma catecholamines. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- MicroRNA miR‐24 promotes cell proliferation by targeting the CDKs inhibitors p27Kip1 and p16INK4a
- Abstract: Cell cycle progression is controlled by numerous mechanisms ensuring correct cell division. The transition from one cell cycle phase to another occurs in an orderly fashion and is regulated by different cellular proteins. Therefore an alteration of the regulatory mechanisms of the cell cycle results in uncontrolled cell proliferation, which is a distinctive feature of human cancers.
Recent evidences suggest that microRNAs (miRs) may also control the levels of multiple cell cycle regulators and therefore control cell proliferation. In fact miRs are a class of small non‐coding RNAs, which modulate gene expression. They are involved in numerous physiological cellular processes and most importantly accumulating evidence indicates that many miRs are aberrantly expressed in human cancers.
In this report we describe that miR‐24 directly targets p27Kip1 and p16Ink4a in primary keratinocyte and in different cancer derived cell lines promoting their proliferation, suggesting that miR‐24 is involved in cyclin‐dependent kinase inhibitors posttranscriptional regulation and that upregulation of miR‐24 may play a role in carcinogenesis. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Cell cycle progression is controlled by numerous mechanisms ensuring correct cell division. The transition from one cell cycle phase to another occurs in an orderly fashion and is regulated by different cellular proteins. Therefore an alteration of the regulatory mechanisms of the cell cycle results in uncontrolled cell proliferation, which is a distinctive feature of human cancers.
Recent evidences suggest that microRNAs (miRs) may also control the levels of multiple cell cycle regulators and therefore control cell proliferation. In fact miRs are a class of small non‐coding RNAs, which modulate gene expression. They are involved in numerous physiological cellular processes and most importantly accumulating evidence indicates that many miRs are aberrantly expressed in human cancers.
In this report we describe that miR‐24 directly targets p27Kip1 and p16Ink4a in primary keratinocyte and in different cancer derived cell lines promoting their proliferation, suggesting that miR‐24 is involved in cyclin‐dependent kinase inhibitors posttranscriptional regulation and that upregulation of miR‐24 may play a role in carcinogenesis. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Hemophagocytosis‐mediated keratinization in oral carcinoma in‐situ and squamous cell carcinoma: a possible histopathogenesis of keratin pearls
- Abstract: Although the histopathogenetic process of keratin pearls is still poorly understood, acceleration of keratinization in squamous cell carcinoma (SCC) cells may represent one possible therapeutic avenue. Based on our histopathological observations, we have hypothesized that SCC cells are keratinized by phagocytosis of extravasated erythrocytes. To confirm this hypothesis, we firstly examined immature keratin pearls in oral carcinoma in‐situ (CIS) and mature ones in SCC by immunohistochemistry. Concentric dyskeratotic cells in CIS keratin pearls became positive for keratin (K) 10, K17, heme oxygenase‐1 (HO‐1), or protease activated receptor‐2 (PAR‐2), a candidate regulator for hemophagocytosis. When ZK‐1 cells, a SCC cell system, were incubated with human peripheral blood erythrocytes, or with crude and purified hemoglobins (Hbs), their erythro‐hemophagocytotic activities were confirmed by immunofluorescence. Immunofluorescence signals for K10, K17 and HO‐1 were enhanced due to hemophagocytosis in time‐dependent manners. mRNA expression levels for the three molecules were most enhanced by purified Hb, followed by crude Hb and erythrocytes. K17/K10 mRNA expression levels were more elevated when PAR‐2 was activated in ZK‐1 cells. The results indicated that immature and mature keratin pearls in CIS and SCC were generated by oxidative stresses derived from erythro‐hemophagocytosis, which might mediate HO‐1 expression and be regulated by PAR‐2. Thus, hemorrhage from the rupture of blood vessels can be one of the triggers for keratin pearl formation in oral CIS and SCC. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Although the histopathogenetic process of keratin pearls is still poorly understood, acceleration of keratinization in squamous cell carcinoma (SCC) cells may represent one possible therapeutic avenue. Based on our histopathological observations, we have hypothesized that SCC cells are keratinized by phagocytosis of extravasated erythrocytes. To confirm this hypothesis, we firstly examined immature keratin pearls in oral carcinoma in‐situ (CIS) and mature ones in SCC by immunohistochemistry. Concentric dyskeratotic cells in CIS keratin pearls became positive for keratin (K) 10, K17, heme oxygenase‐1 (HO‐1), or protease activated receptor‐2 (PAR‐2), a candidate regulator for hemophagocytosis. When ZK‐1 cells, a SCC cell system, were incubated with human peripheral blood erythrocytes, or with crude and purified hemoglobins (Hbs), their erythro‐hemophagocytotic activities were confirmed by immunofluorescence. Immunofluorescence signals for K10, K17 and HO‐1 were enhanced due to hemophagocytosis in time‐dependent manners. mRNA expression levels for the three molecules were most enhanced by purified Hb, followed by crude Hb and erythrocytes. K17/K10 mRNA expression levels were more elevated when PAR‐2 was activated in ZK‐1 cells. The results indicated that immature and mature keratin pearls in CIS and SCC were generated by oxidative stresses derived from erythro‐hemophagocytosis, which might mediate HO‐1 expression and be regulated by PAR‐2. Thus, hemorrhage from the rupture of blood vessels can be one of the triggers for keratin pearl formation in oral CIS and SCC. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Opposite roles of myocardin and atrogin‐1 in L6 myoblast differentiation
- Abstract: L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low‐concentration of serum, but the underlying mechanism is not well understood. The role of atrogin‐1, an E3 ligase with well‐characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin‐1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up‐regulation of atrogin‐1 and down‐regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, overexpression of atrogin‐1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co‐expression of myocardin with atrogin‐1 inhibited atrogin‐1‐induced myogenin expression. Overexpression of atrogin‐1 decreased myocardin protein level, albeit without affecting its mRNA level. Small‐interfering RNA‐mediated knockdown of atrogin‐1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin‐1 without involving Foxo1. Taken together, our results have demonstrated that atrogin‐1 plays a positive role in skeletal muscle differentiation through down‐regulation of myocardin. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: L6 rat myoblasts undergo differentiation and myotube formation when cultured in medium containing a low‐concentration of serum, but the underlying mechanism is not well understood. The role of atrogin‐1, an E3 ligase with well‐characterized roles in muscle atrophy, has not been defined in muscle differentiation. Myocardin is a coactivator of serum response factor (SRF), which together promotes smooth muscle differentiation. Myocardin is transiently expressed in skeletal muscle progenitor cells with inhibitory effects on the expression of myogenin and muscle differentiation. It remains unknown whether myocardin, which undergoes ubiquitination degradation, plays a role in L6 cell differentiation. The current study aimed to investigate the potential roles of myocardin and atrogin‐1 in differentiation of L6 cells. As reported by many others, shifting to medium containing 2% serum induced myotube formation of L6 cells. Differentiation was accompanied by up‐regulation of atrogin‐1 and down‐regulation of myocardin, suggesting that both may be involved in muscle differentiation. As expected, overexpression of atrogin‐1 stimulated the expression of troponin T and myogenin and differentiation of the L6 myoblasts. Co‐expression of myocardin with atrogin‐1 inhibited atrogin‐1‐induced myogenin expression. Overexpression of atrogin‐1 decreased myocardin protein level, albeit without affecting its mRNA level. Small‐interfering RNA‐mediated knockdown of atrogin‐1 increased myocardin protein. Consistently, ectopic expression of myocardin inhibited myogenic differentiation. Unexpectedly, myocardin decreased the expression of atrogin‐1 without involving Foxo1. Taken together, our results have demonstrated that atrogin‐1 plays a positive role in skeletal muscle differentiation through down‐regulation of myocardin. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- MicroRNA‐195 chemosensitizes colon cancer cells to the chemotherapeutic drug doxorubicin by targeting the first binding site of BCL2L2 mRNA
- Abstract: The mechanisms underlying doxorubicin (Dox) resistance in colon cancer cells are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the relationship between miRNA and doxorubicin resistance in colon cancer cells has not been previously explored. In this study, we utilized microRNA array and real‐time PCR to verify that some microRNAs including miR‐127, miR‐195, miR‐22, miR‐137 were significantly down‐regulated, while miR‐21, miR‐592 were up‐regulated in both HT29/DOX and LOVO/DOX cell lines. In vitro cell viability assay showed that knockdown of miR‐195 in HT29 and LOVO cells caused a marked inhibition of Dox‐induced cytotoxicity. Moreover, we explored that miR‐195 is involved in repression of BCL2L2 expression through targeting its 3'‐untranslated region, especially the first binding site within its mRNA. Furthermore, down‐regulation of miR‐195 conferred DOX resistance in parental cells and reduced cell apoptosis activity, while over‐expression of miR‐195 sensitized resistant cells to DOX and enhanced cell apoptosis activity, all of which can be partly rescued by BCL2L2 siRNA and cDNA expression. These results may have implications for therapeutic strategies aiming to overcome colon cancer cells resistance to doxorubicin. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The mechanisms underlying doxorubicin (Dox) resistance in colon cancer cells are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the relationship between miRNA and doxorubicin resistance in colon cancer cells has not been previously explored. In this study, we utilized microRNA array and real‐time PCR to verify that some microRNAs including miR‐127, miR‐195, miR‐22, miR‐137 were significantly down‐regulated, while miR‐21, miR‐592 were up‐regulated in both HT29/DOX and LOVO/DOX cell lines. In vitro cell viability assay showed that knockdown of miR‐195 in HT29 and LOVO cells caused a marked inhibition of Dox‐induced cytotoxicity. Moreover, we explored that miR‐195 is involved in repression of BCL2L2 expression through targeting its 3'‐untranslated region, especially the first binding site within its mRNA. Furthermore, down‐regulation of miR‐195 conferred DOX resistance in parental cells and reduced cell apoptosis activity, while over‐expression of miR‐195 sensitized resistant cells to DOX and enhanced cell apoptosis activity, all of which can be partly rescued by BCL2L2 siRNA and cDNA expression. These results may have implications for therapeutic strategies aiming to overcome colon cancer cells resistance to doxorubicin. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Atomic Force Microscopy: High Resolution Dynamic Imaging of Cellular and Molecular Structure in Health and Disease
- Abstract: The atomic force microscope (AFM), invented in 1986, and a member of the scanning probe family of microscopes, offers the unprecedented ability to image biological samples unfixed and in a hydrated environment at high resolution. This opens the possibility to investigate biological mechanisms temporally in a heretofore unattainable resolution. We have used AFM to investigate: (1) fundamental issues in cell biology (secretion) and, (2) the pathological basis of a human thrombotic disease, the antiphospholipid syndrome (APS). These studies have incorporated the imaging of live cells at nanometer resolution, leading to discovery of the “porosome”, the universal secretory portal in cells, and a molecular understanding of membrane fusion from imaging the interaction and assembly of proteins between opposing lipid membranes. Similarly, the development of an in vitro simulacrum for investigating the molecular interactions between proteins and lipids has helped define an etiological explanation for APS. The prime importance of AFM in the success of these investigations will be presented in this manuscript, as well as a discussion of the limitations of this technique for the study of biomedical samples. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The atomic force microscope (AFM), invented in 1986, and a member of the scanning probe family of microscopes, offers the unprecedented ability to image biological samples unfixed and in a hydrated environment at high resolution. This opens the possibility to investigate biological mechanisms temporally in a heretofore unattainable resolution. We have used AFM to investigate: (1) fundamental issues in cell biology (secretion) and, (2) the pathological basis of a human thrombotic disease, the antiphospholipid syndrome (APS). These studies have incorporated the imaging of live cells at nanometer resolution, leading to discovery of the “porosome”, the universal secretory portal in cells, and a molecular understanding of membrane fusion from imaging the interaction and assembly of proteins between opposing lipid membranes. Similarly, the development of an in vitro simulacrum for investigating the molecular interactions between proteins and lipids has helped define an etiological explanation for APS. The prime importance of AFM in the success of these investigations will be presented in this manuscript, as well as a discussion of the limitations of this technique for the study of biomedical samples. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Acetaminophen attenuates doxorubicin‐induced cardiac fibrosis via osteopontin and GATA4 regulation: Reduction of oxidant levels
- Abstract: It is well documented in animal and human studies that therapy with the anti‐cancer drug doxorubicin (DOX) induces fibrosis, cardiac dysfunction, and cell death. The most widely accepted mechanism of cardiac injury is through production of reactive oxygen species (ROS), which cause mitochondrial damage, sarcomere structural alterations, and altered gene expression in myocytes and fibroblasts. Here we investigated the effects of acetaminophen (APAP, N‐acetyl‐para‐aminophenol) on DOX‐induced cardiac injury and fibrosis in the presence or absence of osteopontin (OPN). H9c2 rat heart‐derived embryonic myoblasts were exposed to increasing concentrations of DOX ± APAP; cell viability, oxidative stress, and OPN transcript levels were analyzed. We found a dose‐dependent decrease in cell viability and a corresponding increase in intracellular oxidants at the tested concentrations of DOX. These effects were attenuated in the presence of APAP. RT‐PCR analysis revealed a small increase in OPN transcript levels in response to DOX, which was suppressed by APAP. When male 10–12‐week‐old mice (OPN+/+ or OPN‐/‐) were given weekly injections of DOX ± APAP for 4 weeks there was substantial cardiac fibrosis in OPN+/+ and, to a lesser extent, in OPN‐/‐ mice. In both groups, APAP decreased fibrosis to near baseline levels. Activity of the pro‐survival GATA4 transcription factor was diminished by DOX in both mouse genotypes, but retained baseline activity in the presence of APAP. These effects were mediated, in part, by the ability of APAP, acting as an anti‐inflammatory agent, to decrease intracellular ROS levels, consequently diminishing the injury‐induced increase in OPN levels. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: It is well documented in animal and human studies that therapy with the anti‐cancer drug doxorubicin (DOX) induces fibrosis, cardiac dysfunction, and cell death. The most widely accepted mechanism of cardiac injury is through production of reactive oxygen species (ROS), which cause mitochondrial damage, sarcomere structural alterations, and altered gene expression in myocytes and fibroblasts. Here we investigated the effects of acetaminophen (APAP, N‐acetyl‐para‐aminophenol) on DOX‐induced cardiac injury and fibrosis in the presence or absence of osteopontin (OPN). H9c2 rat heart‐derived embryonic myoblasts were exposed to increasing concentrations of DOX ± APAP; cell viability, oxidative stress, and OPN transcript levels were analyzed. We found a dose‐dependent decrease in cell viability and a corresponding increase in intracellular oxidants at the tested concentrations of DOX. These effects were attenuated in the presence of APAP. RT‐PCR analysis revealed a small increase in OPN transcript levels in response to DOX, which was suppressed by APAP. When male 10–12‐week‐old mice (OPN+/+ or OPN‐/‐) were given weekly injections of DOX ± APAP for 4 weeks there was substantial cardiac fibrosis in OPN+/+ and, to a lesser extent, in OPN‐/‐ mice. In both groups, APAP decreased fibrosis to near baseline levels. Activity of the pro‐survival GATA4 transcription factor was diminished by DOX in both mouse genotypes, but retained baseline activity in the presence of APAP. These effects were mediated, in part, by the ability of APAP, acting as an anti‐inflammatory agent, to decrease intracellular ROS levels, consequently diminishing the injury‐induced increase in OPN levels. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Linking membrane dynamics and trafficking to autophagy and the unfolded protein response
- Abstract: Cellular stressors typically induce two protective counter‐responses – autophagy and the unfolded protein response (UPR). It is conceivable that these two endoplasmic reticulum (ER) membrane‐based processes would intersect/interact somehow with the constitutive housekeeping process of exocytic membrane traffic from the ER. How exactly might this occur' Recent evidence indicates that a conserved Rab protein, Rab1/Ypt1p, has functional roles in UPR and autophagy. This molecular switch and its associated effectors may therefore serve to link up a network of cellular responses to stress through changes in membrane dynamics and protein turnover. The notion provides further explanations as to why elevation of Rab1/Ypt1p levels could counter the cytotoxicity of α‐synuclein, and a similar mode of protection may well be at work against other stresses. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Cellular stressors typically induce two protective counter‐responses – autophagy and the unfolded protein response (UPR). It is conceivable that these two endoplasmic reticulum (ER) membrane‐based processes would intersect/interact somehow with the constitutive housekeeping process of exocytic membrane traffic from the ER. How exactly might this occur' Recent evidence indicates that a conserved Rab protein, Rab1/Ypt1p, has functional roles in UPR and autophagy. This molecular switch and its associated effectors may therefore serve to link up a network of cellular responses to stress through changes in membrane dynamics and protein turnover. The notion provides further explanations as to why elevation of Rab1/Ypt1p levels could counter the cytotoxicity of α‐synuclein, and a similar mode of protection may well be at work against other stresses. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- MicroRNA transport: A new way in cell communication
- Abstract: MicroRNAs (miRNAs) can efficiently regulate gene expression by targeting mRNA to cause mRNA cleavage or translational repression. Growing evidence indicates that miRNAs exist not only in cells but also in a variety of body fluids, which stimulates substantial interest in the transport mechanism and regulating process of extracellular miRNAs. This article reviews the basic biogenesis of miRNAs in detail to explore the origin of extracellular miRNAs. Different miRNA transporters have been summarized (e.g. exosomes, microvesicles, apoptosis bodies and RNA‐binding proteins). In addition, we discuss the regulators affecting miRNA transport (e.g. ATP and ceramide) and the selection mechanism for different miRNA transporters. Studies about miRNA transporters and the transport mechanism are new and developing. With the progress of the research, new functions of extracellular miRNAs may be uncovered in the future. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: MicroRNAs (miRNAs) can efficiently regulate gene expression by targeting mRNA to cause mRNA cleavage or translational repression. Growing evidence indicates that miRNAs exist not only in cells but also in a variety of body fluids, which stimulates substantial interest in the transport mechanism and regulating process of extracellular miRNAs. This article reviews the basic biogenesis of miRNAs in detail to explore the origin of extracellular miRNAs. Different miRNA transporters have been summarized (e.g. exosomes, microvesicles, apoptosis bodies and RNA‐binding proteins). In addition, we discuss the regulators affecting miRNA transport (e.g. ATP and ceramide) and the selection mechanism for different miRNA transporters. Studies about miRNA transporters and the transport mechanism are new and developing. With the progress of the research, new functions of extracellular miRNAs may be uncovered in the future. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Hypoxia promotes tumor cell survival in acidic conditions by preserving ATP levels
- Abstract: The efficacy of targeting pH disruption to induce cell death in the acidic and hypoxic tumor microenvironment continues to be assessed. Here we analyzed the impact of varying levels of hypoxia in acidic conditions on fibroblasts and tumor cell survival. Across all cell lines tested, hypoxia (1% O2) provided protection against acidosis induced cell death compared to normoxia. Meanwhile severe hypoxia (0.1% O2) removed this protection and in some cases exacerbated acidosis‐induced cell death. Differential survival between cell types during external acidosis correlated with their respective intracellular pH regulating capabilities. Cellular ATP measurements were conducted to determine their contribution to cell survival under these combined stresses. In general, hypoxia (1% O2) maintained elevated ATP levels in acidic conditions while severe hypoxia did not. To further explore this interaction we combined acidosis with ATP depletion using 2‐Deoxyglucose and observed an enhanced rate of cell mortality. Striking results were also observed with hypoxia providing protection against cell death in spite of a severe metabolic stress induced by a combination of acidosis and oligomycin. Finally, we demonstrated that both HIF1α and HIF2α expression were drastically reduced in hypoxic and acidic conditions indicating a sensitivity of this protein to cellular pH conditions. This knockdown of HIF expression by acidosis has implications for the development of therapies targeting the disruption of cellular pH regulation. Our results reinforce the proof of concept that acidosis and metabolic disruption affecting ATP levels could be exploited as a tumor cell killing strategy. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The efficacy of targeting pH disruption to induce cell death in the acidic and hypoxic tumor microenvironment continues to be assessed. Here we analyzed the impact of varying levels of hypoxia in acidic conditions on fibroblasts and tumor cell survival. Across all cell lines tested, hypoxia (1% O2) provided protection against acidosis induced cell death compared to normoxia. Meanwhile severe hypoxia (0.1% O2) removed this protection and in some cases exacerbated acidosis‐induced cell death. Differential survival between cell types during external acidosis correlated with their respective intracellular pH regulating capabilities. Cellular ATP measurements were conducted to determine their contribution to cell survival under these combined stresses. In general, hypoxia (1% O2) maintained elevated ATP levels in acidic conditions while severe hypoxia did not. To further explore this interaction we combined acidosis with ATP depletion using 2‐Deoxyglucose and observed an enhanced rate of cell mortality. Striking results were also observed with hypoxia providing protection against cell death in spite of a severe metabolic stress induced by a combination of acidosis and oligomycin. Finally, we demonstrated that both HIF1α and HIF2α expression were drastically reduced in hypoxic and acidic conditions indicating a sensitivity of this protein to cellular pH conditions. This knockdown of HIF expression by acidosis has implications for the development of therapies targeting the disruption of cellular pH regulation. Our results reinforce the proof of concept that acidosis and metabolic disruption affecting ATP levels could be exploited as a tumor cell killing strategy. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Snail mediates PDGF‐BB‐induced invasion of rat bone marrow mesenchymal stem cells in 3‐D collagen and chick chorioallantoic membrane
- Abstract: We previously reported that membrane type‐1 matrix metalloproteinase (MT1‐MMP) enables mesenchymal stem cells (MSCs) to move through both three‐dimensional (3D) type I collagen and basement membrane barriers; however, its upstream regulating factors were unidentified. Here, we report that PDGF‐BB upregulates both mRNA and protein expression of snail in rat bone marrow MSCs (rBMMSCs). PDGF‐BB enhances rBMMSC invasion in 3D collagen, which is blocked by snail specific siRNA transfection. Snail overexpression induced by plasmid transfection results in increased rBMMSC invasion in 3D collagen. Snail expression induced by PDGF‐BB in MSCs is inhibited by LY294002 and PD98059, which are inhibitors of the PI3K/AKT and MAPK1/2/ERK1/2 signaling pathways, respectively. MT1‐MMP expression in rBMMSCs, both as mRNA and protein, is decreased by snail siRNA transfection, but increased by snail overexpression, indicating that they are controlled by snail. Finally, snail controls MSC transmigration through chorioallantoic membrane of 11‐day‐old chick embryos. Taken together, these in vitro and in vivo data identify snail as a critical mediator for rBMMSC invasion induced by PDGF‐BB. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: We previously reported that membrane type‐1 matrix metalloproteinase (MT1‐MMP) enables mesenchymal stem cells (MSCs) to move through both three‐dimensional (3D) type I collagen and basement membrane barriers; however, its upstream regulating factors were unidentified. Here, we report that PDGF‐BB upregulates both mRNA and protein expression of snail in rat bone marrow MSCs (rBMMSCs). PDGF‐BB enhances rBMMSC invasion in 3D collagen, which is blocked by snail specific siRNA transfection. Snail overexpression induced by plasmid transfection results in increased rBMMSC invasion in 3D collagen. Snail expression induced by PDGF‐BB in MSCs is inhibited by LY294002 and PD98059, which are inhibitors of the PI3K/AKT and MAPK1/2/ERK1/2 signaling pathways, respectively. MT1‐MMP expression in rBMMSCs, both as mRNA and protein, is decreased by snail siRNA transfection, but increased by snail overexpression, indicating that they are controlled by snail. Finally, snail controls MSC transmigration through chorioallantoic membrane of 11‐day‐old chick embryos. Taken together, these in vitro and in vivo data identify snail as a critical mediator for rBMMSC invasion induced by PDGF‐BB. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Novel role of VMP1 as modifier of the pancreatic tumor cell response to chemotherapeutic drugs
- Abstract: We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress‐induced autophagy‐associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over‐expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi‐based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1‐binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: We hypothesized that inhibiting molecules that mediate the adaptation response to cellular stress can antagonize the resistance of pancreatic cancer cells to chemotherapeutic drugs. Toward this end, here, we investigated how VMP1, a stress‐induced autophagy‐associated protein, modulate stress responses triggered by chemotherapeutic agents in PDAC. We find that VMP1 is particularly over‐expressed in poorly differentiated human pancreatic cancer. Pharmacological studies show that drugs that work, in part, via the endoplasmic reticulum stress response, induce VMP1 expression. Similarly, VMP1 is induced by known endoplasmic reticulum stress activators. Genetic inactivation of VMP1 using RNAi‐based antagonize the pancreatic cancer stress response to antitumoral agents. Functionally, we find that VMP1 regulates both autophagy and chemotherapeutic resistance even in the presence of chloroquin, ATG5 or Beclin 1 siRNAs, or a Beclin 1‐binding VMP1 mutant. In addition, VMP1 modulates endoplasmic reticulum stress independently of its coupling to the molecular and cellular autophagy machinery. Preclinical studies demonstrate that xenografts expressing an inducible and tractable form of VMP1 show increased resistance to the gemcitabine treatment. These results underscore a novel role for VMP1 as a potential therapeutic target for combinatorial therapies aimed at sensitizing pancreatic cancer cells to chemotherapeutic agents as well as provide novel molecular mechanisms to better understand this phenomenon. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- GRK2 negatively regulates IGF‐1R signaling pathway and cyclins' expression in HepG2 cells
- Abstract: G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin‐like growth factor 1 receptor (IGF‐1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF‐1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF‐1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF‐1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF‐1R signaling pathway and cyclins' expression. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: G protein coupled receptor kinase 2 (GRK2) plays a central role in the regulation of a variety of important signaling pathways. Alternation of GRK2 protein level and activity casts profound effects on cell physiological functions and causes diseases such as heart failure, rheumatoid arthritis, and obesity. We have previously reported that overexpression of GRK2 has an inhibitory role in cancer cell growth. To further examine the role of GRK2 in cancer, in this study, we investigated the effects of reduced protein level of GRK2 on insulin‐like growth factor 1 receptor (IGF‐1R) signaling pathway in human hepatocellular carcinoma (HCC) HepG2 cells. We created a GRK2 knockdown cell line using a lentiviral vector mediated expression of GRK2 specific short hairpin RNA (shRNA). Under IGF‐1 stimulation, HepG2 cells with reduced level of GRK2 showed elevated total IGF‐1R protein expression as well as tyrosine phosphorylation of receptor. In addition, HepG2 cells with reduced level of GRK2 also demonstrated increased tyrosine phosphorylation of IRS1 at the residue 612 and increased phosphorylation of Akt, indicating a stronger activation of IGF‐1R signaling pathway. However, HepG2 cells with reduced level of GRK2 did not display any growth advantage in culture as compared with the scramble control cells. We further detected that reduced level of GRK2 induced a small cell cycle arrest at G2/M phase by enhancing the expression of cyclin A, B1, and E. Our results indicate that GRK2 has contrasting roles on HepG2 cell growth by negatively regulating the IGF‐1R signaling pathway and cyclins' expression. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Oxygen‐induced transcriptional dynamics in human osteoblasts are most prominent at the onset of mineralization
- Abstract: Oxygen tension plays an important role in the regulation of cellular processes. During hematopoietic stem cell (HSC) differentiation, HSCs migrate from one stem cell niche to the next, each with a different oxygen tension that determines which signalling pathways are on and off, determining the differentiation stage of the cell. Oxygen tension influences osteoblast differentiation and mineralization. Low oxygen levels inhibit matrix formation and mineralization. We were interested in the regulatory mechanisms that underlie this inhibition and wondered whether a switch in oxygen tension could have varying effects depending on the differentiation phase of the osteoblasts. We performed an oxygen tension switch phase study in which we switched osteoblasts from high to low oxygen tension during their three week differentiation and mineralization process. We performed microarray expression profiling on samples collected during this three week period and analyzed biochemical and histo‐chemical endpoint parameters to determine the effect of a switch in oxygen levels on mineralization. We found that low oxygen tension has the most profound impact on mineralization when administered during the period of matrix maturation. Additionally, a large set of genes was regulated by oxygen, independent of the differentiation phase. These genes were involved in cell metabolisms and matrix formation. Our study demonstrates that variation in oxygen tension strongly affects gene expression in differentiating osteoblasts. The magnitude of this change for either expression levels or the number of regulated probes, depends on the osteoblast differentiation stage, with the phase prior to the onset of mineralization being most sensitive. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Oxygen tension plays an important role in the regulation of cellular processes. During hematopoietic stem cell (HSC) differentiation, HSCs migrate from one stem cell niche to the next, each with a different oxygen tension that determines which signalling pathways are on and off, determining the differentiation stage of the cell. Oxygen tension influences osteoblast differentiation and mineralization. Low oxygen levels inhibit matrix formation and mineralization. We were interested in the regulatory mechanisms that underlie this inhibition and wondered whether a switch in oxygen tension could have varying effects depending on the differentiation phase of the osteoblasts. We performed an oxygen tension switch phase study in which we switched osteoblasts from high to low oxygen tension during their three week differentiation and mineralization process. We performed microarray expression profiling on samples collected during this three week period and analyzed biochemical and histo‐chemical endpoint parameters to determine the effect of a switch in oxygen levels on mineralization. We found that low oxygen tension has the most profound impact on mineralization when administered during the period of matrix maturation. Additionally, a large set of genes was regulated by oxygen, independent of the differentiation phase. These genes were involved in cell metabolisms and matrix formation. Our study demonstrates that variation in oxygen tension strongly affects gene expression in differentiating osteoblasts. The magnitude of this change for either expression levels or the number of regulated probes, depends on the osteoblast differentiation stage, with the phase prior to the onset of mineralization being most sensitive. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- mTOR signaling for biological control and cancer
- Abstract: Mammalian target of rapamycin (mTOR) is a major intersection that connects signals from the extracellular milieu to corresponding changes in intracellular processes. When abnormally regulated, the mTOR signaling pathway is implicated in a wide spectrum of cancers, neurological diseases and proliferative disorders. Therefore, pharmacological agents that restore the regulatory balance of the mTOR pathway could be beneficial for a great number of diseases. This review summarizes current understanding of mTOR signaling and some unanswered questions in the field. We describe the composition of the mTOR complexes, upstream signals that activate mTOR, and physiological processes that mTOR regulates. We also discuss the role of mTOR and its downstream effectors in cancer, obesity and diabetes, and autism. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Mammalian target of rapamycin (mTOR) is a major intersection that connects signals from the extracellular milieu to corresponding changes in intracellular processes. When abnormally regulated, the mTOR signaling pathway is implicated in a wide spectrum of cancers, neurological diseases and proliferative disorders. Therefore, pharmacological agents that restore the regulatory balance of the mTOR pathway could be beneficial for a great number of diseases. This review summarizes current understanding of mTOR signaling and some unanswered questions in the field. We describe the composition of the mTOR complexes, upstream signals that activate mTOR, and physiological processes that mTOR regulates. We also discuss the role of mTOR and its downstream effectors in cancer, obesity and diabetes, and autism. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Adenosine, adenosine receptors and their role in glucose homeostasis and lipid metabolism
- Abstract: Adenosine is an endogenous metabolite that is released from all tissues and cells including liver, pancreas, muscle and fat, particularly under stress, intense exercise, or during cell damage. The role of adenosine in glucose homeostasis has been attributed to its ability to regulate, through its membrane receptors, processes such as insulin secretion, glucose release and clearance, glycogenolysis, and glycogenesis. Additionally, adenosine and its multiple receptors have been connected to lipid metabolism by augmenting insulin‐mediated inhibition of lipolysis, and the subsequent increase in free fatty acids and glycerol levels. Furthermore, adenosine was reported to control liver cholesterol synthesis, consequently affecting plasma levels of cholesterol and triglycerides, and the amount of fat tissue. Alterations in the balance of glucose and lipid homeostasis have implications in both cardiovascular disease and diabetes. The ability of different adenosine receptors to activate and inhibit the same signaling cascades has made it challenging to study the influence of adenosine, adenosine analogs and their receptors in health and disease. This review focuses on the role and significance of different adenosine receptors in mediating the effect of adenosine on glucose and lipid homeostasis. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Adenosine is an endogenous metabolite that is released from all tissues and cells including liver, pancreas, muscle and fat, particularly under stress, intense exercise, or during cell damage. The role of adenosine in glucose homeostasis has been attributed to its ability to regulate, through its membrane receptors, processes such as insulin secretion, glucose release and clearance, glycogenolysis, and glycogenesis. Additionally, adenosine and its multiple receptors have been connected to lipid metabolism by augmenting insulin‐mediated inhibition of lipolysis, and the subsequent increase in free fatty acids and glycerol levels. Furthermore, adenosine was reported to control liver cholesterol synthesis, consequently affecting plasma levels of cholesterol and triglycerides, and the amount of fat tissue. Alterations in the balance of glucose and lipid homeostasis have implications in both cardiovascular disease and diabetes. The ability of different adenosine receptors to activate and inhibit the same signaling cascades has made it challenging to study the influence of adenosine, adenosine analogs and their receptors in health and disease. This review focuses on the role and significance of different adenosine receptors in mediating the effect of adenosine on glucose and lipid homeostasis. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Lactoferricin mediates anti‐inflammatory and anti‐catabolic effects via inhibition of IL‐1 and LPS activity in the intervertebral disc
- Abstract: The catabolic cytokine interleukin‐1 (IL‐1) and endotoxin lipopolysaccharide (LPS) are well‐known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL‐1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti‐catabolic and anti‐inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL‐1 and LPS‐mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL‐1 and LPS‐mediated proteoglycan (PG) depletion, matrix‐degrading enzyme production and enzyme activity in long‐term (alginate beads) and short‐term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL‐1 and LPS‐mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage‐degrading enzymes, including MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, and ADAMTS‐5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor‐induced stimulation of oxidative and inflammatory factors such as iNOS, IL‐6, and toll‐like receptor‐2 (TLR‐2) and TLR‐4. Finally, the ability of LfcinB to antagonize IL‐1 and LPS‐mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The catabolic cytokine interleukin‐1 (IL‐1) and endotoxin lipopolysaccharide (LPS) are well‐known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL‐1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti‐catabolic and anti‐inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL‐1 and LPS‐mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL‐1 and LPS‐mediated proteoglycan (PG) depletion, matrix‐degrading enzyme production and enzyme activity in long‐term (alginate beads) and short‐term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL‐1 and LPS‐mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage‐degrading enzymes, including MMP‐1, MMP‐3, MMP‐13, ADAMTS‐4, and ADAMTS‐5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor‐induced stimulation of oxidative and inflammatory factors such as iNOS, IL‐6, and toll‐like receptor‐2 (TLR‐2) and TLR‐4. Finally, the ability of LfcinB to antagonize IL‐1 and LPS‐mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Fibroblast cytoskeletal remodeling induced by tissue stretch involves ATP signaling
- Abstract: Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 minutes, returning to baseline at 60 min. No increase in ATP was observed in tissue incubated without stretch, or tissue stretched in the presence of the Rho kinase inhibitor Y27632. The increase in fibroblast cross sectional area in response to tissue stretch was blocked by both suramin (a purinergic receptor blocker) and apyrase (an enzyme that selectively degrades extracellular ATP). Furthermore, connexin channel blockers (octanol and carbenoxolone), but not VRAC (fluoxetine) or pannexin (probenecid) channel blockers, inhibited fibroblast expansion. Together, these results support a mechanism in which extracellular ATP signaling via connexin hemichannels mediated the active change in fibroblast shape that occurs in response to a static increase in tissue length. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Fibroblasts in whole areolar connective tissue respond to static stretching of the tissue by expanding and remodeling their cytoskeleton within minutes both ex vivo and in vivo. This study tested the hypothesis that the mechanism of fibroblast expansion in response to tissue stretch involves extracellular ATP signaling. In response to tissue stretch ex vivo, ATP levels in the bath solution increased significantly, and this increase was sustained for 20 minutes, returning to baseline at 60 min. No increase in ATP was observed in tissue incubated without stretch, or tissue stretched in the presence of the Rho kinase inhibitor Y27632. The increase in fibroblast cross sectional area in response to tissue stretch was blocked by both suramin (a purinergic receptor blocker) and apyrase (an enzyme that selectively degrades extracellular ATP). Furthermore, connexin channel blockers (octanol and carbenoxolone), but not VRAC (fluoxetine) or pannexin (probenecid) channel blockers, inhibited fibroblast expansion. Together, these results support a mechanism in which extracellular ATP signaling via connexin hemichannels mediated the active change in fibroblast shape that occurs in response to a static increase in tissue length. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- CCL20 induces migration and proliferation on breast epithelial cells
- Abstract: The communication between the tumour cells and the surrounding cells helps drive the process of tumour progression. Since the microenvironment of breast cancer includes CCL20 chemokine, the purpose of this study was to determine whether CCL20 modulates the physiology of healthy breast epithelial cells in areas adjacent to the tumour. Therefore, primary cultures of mammary cells taken from normal peritumoral areas were used. We assessed that breast cells expressed CCR6 CCL20 receptor. Using molecular (siRNA) and pharmacological (inhibitors) techniques we found multiple signalling kinases to be activated by CCR6 and involved in CCL20‐induced breast cell proliferation and migration. The binding of 10 ng/ml CCL20 to CCR6 induced cell migration whilst higher concentrations (from 15 to 25 ng/ml) led to cell proliferation. CCL20 controlled cell migration and MMP‐9 expression by PKC‐alpha that activated Src, which caused the activation of downstream Akt, JNK and NF‐kB pathways. Furthermore, higher CCL20 concentrations increased cycE and decreased p27Kip expression ending in enhanced cell proliferation. Cell proliferation occurred through PKC‐epsilon activation that transactivated EGFR and ERK1/2/MAPK pathway. Although activated by different CCL20 concentrations, these pathways function in parallel and crosstalk to some extent, inasmuch as Akt activation was responsible for ERK1/2 nuclear translocation and enhanced the transcription of of c‐fos and c‐myc, involved in cell proliferation. In summary, tumour cells exchange signals with the surrounding healthy cells modifying the extracellular matrix through enzyme secretion; thus, CCL20 might be a factor involved in the ontogeny of breast carcinoma. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The communication between the tumour cells and the surrounding cells helps drive the process of tumour progression. Since the microenvironment of breast cancer includes CCL20 chemokine, the purpose of this study was to determine whether CCL20 modulates the physiology of healthy breast epithelial cells in areas adjacent to the tumour. Therefore, primary cultures of mammary cells taken from normal peritumoral areas were used. We assessed that breast cells expressed CCR6 CCL20 receptor. Using molecular (siRNA) and pharmacological (inhibitors) techniques we found multiple signalling kinases to be activated by CCR6 and involved in CCL20‐induced breast cell proliferation and migration. The binding of 10 ng/ml CCL20 to CCR6 induced cell migration whilst higher concentrations (from 15 to 25 ng/ml) led to cell proliferation. CCL20 controlled cell migration and MMP‐9 expression by PKC‐alpha that activated Src, which caused the activation of downstream Akt, JNK and NF‐kB pathways. Furthermore, higher CCL20 concentrations increased cycE and decreased p27Kip expression ending in enhanced cell proliferation. Cell proliferation occurred through PKC‐epsilon activation that transactivated EGFR and ERK1/2/MAPK pathway. Although activated by different CCL20 concentrations, these pathways function in parallel and crosstalk to some extent, inasmuch as Akt activation was responsible for ERK1/2 nuclear translocation and enhanced the transcription of of c‐fos and c‐myc, involved in cell proliferation. In summary, tumour cells exchange signals with the surrounding healthy cells modifying the extracellular matrix through enzyme secretion; thus, CCL20 might be a factor involved in the ontogeny of breast carcinoma. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- C6orf89 encodes three distinct HDAC enhancers that function in the nucleolus, the Golgi and the midbody
- Abstract: We report here that C6orf89, which encodes a protein that interacts with bombesin receptor subtype‐3 and accelerates cell cycle progression and wound repair in human bronchial epithelial cells (Liu et al. PLoS One 2011), encodes one soluble and two type II membrane proteins that function as histone deacetylases (HDAC) enhancers. Soluble 34/64sp is selectively targeted to the nucleolus and is retained in nucleolar organiser regions (NORs) in mitotic cells. Nucleolar 34/64sp is integrated into the ribosomal gene transcription machinery, colocalises and coimmunoprecipitates with the Pol I transcription factor UBF, and undergoes a dramatic relocalisation to the nucleolus upon the arrest of rDNA transcription, protein synthesis and PI3K/mTORC2 signalling. Membrane 42/116mp localises to the Golgi and the midbody, and its controlled ectopic expression provokes the disruption of the Golgi cisternae and hinders the separation of daughter cells and the completion of mitosis. The latter effect is also produced by the microinjection of an affinity‐purified amfion antibody. The identification of C60rf89 as a gene that encodes three distinct proteins with the capacity to enhance the activity of histone deacetylases (HDACs) in the nucleolus, the Golgi and the midbody provides new information regarding the components of the acetylome and their capacity to interact with different functional groups in the cell. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: We report here that C6orf89, which encodes a protein that interacts with bombesin receptor subtype‐3 and accelerates cell cycle progression and wound repair in human bronchial epithelial cells (Liu et al. PLoS One 2011), encodes one soluble and two type II membrane proteins that function as histone deacetylases (HDAC) enhancers. Soluble 34/64sp is selectively targeted to the nucleolus and is retained in nucleolar organiser regions (NORs) in mitotic cells. Nucleolar 34/64sp is integrated into the ribosomal gene transcription machinery, colocalises and coimmunoprecipitates with the Pol I transcription factor UBF, and undergoes a dramatic relocalisation to the nucleolus upon the arrest of rDNA transcription, protein synthesis and PI3K/mTORC2 signalling. Membrane 42/116mp localises to the Golgi and the midbody, and its controlled ectopic expression provokes the disruption of the Golgi cisternae and hinders the separation of daughter cells and the completion of mitosis. The latter effect is also produced by the microinjection of an affinity‐purified amfion antibody. The identification of C60rf89 as a gene that encodes three distinct proteins with the capacity to enhance the activity of histone deacetylases (HDACs) in the nucleolus, the Golgi and the midbody provides new information regarding the components of the acetylome and their capacity to interact with different functional groups in the cell. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Cardiac resident nestin+ cells participate in reparative vascularisation
- Abstract: The rodent heart contains a population of nestin(+) cells derived from the embryonic neural crest and migrate to the scar after myocardial infarction. The present study tested the hypothesis that intron 2 of the nestin gene drives expression and a subpopulation of nestin(+) cells participate in reparative vascularisation. The directed expression of the green fluorescent protein (GFP) by the second intron of the nestin gene identified GFP/nestin(+) cells intercalated among ventricular myocytes in the heart of normal transgenic mice. Ischemic injury led to the migration of GFP(+) cells to the scar and a subpopulation was detected in CD31/nestin(+) endothelial cells of newly formed blood vessels. The direct contribution to reparative vascularisation provided the impetus to test the hypothesis that increasing the population of nestin(+) cells in the infarcted heart will improve scar healing. Skin‐derived cells isolated from E18 Sprague‐Dawley rats grew as spheres, expressed nestin, sox2, neural crest‐related transcriptional genes and a panel of peptide growth factors. Skin‐derived cells transplanted in the non‐infarcted left ventricle of 3‐day post‐MI rats migrated to the peri‐infarct/infarct region and remained engrafted for 21 days. A significantly smaller infarct, increased number of small calibre blood vessels and improved ventricular function were observed in engrafted infarcted rat hearts. Thus, the second intron of the nestin gene drives expression in the mouse heart and a subpopulation of GFP/nestin(+) cells directly participate in reparative vascularisation. Increasing the population of nestin(+) cells via the transplantation of skin‐derived cells represents a potential approach to limit ischemic damage to the heart. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: The rodent heart contains a population of nestin(+) cells derived from the embryonic neural crest and migrate to the scar after myocardial infarction. The present study tested the hypothesis that intron 2 of the nestin gene drives expression and a subpopulation of nestin(+) cells participate in reparative vascularisation. The directed expression of the green fluorescent protein (GFP) by the second intron of the nestin gene identified GFP/nestin(+) cells intercalated among ventricular myocytes in the heart of normal transgenic mice. Ischemic injury led to the migration of GFP(+) cells to the scar and a subpopulation was detected in CD31/nestin(+) endothelial cells of newly formed blood vessels. The direct contribution to reparative vascularisation provided the impetus to test the hypothesis that increasing the population of nestin(+) cells in the infarcted heart will improve scar healing. Skin‐derived cells isolated from E18 Sprague‐Dawley rats grew as spheres, expressed nestin, sox2, neural crest‐related transcriptional genes and a panel of peptide growth factors. Skin‐derived cells transplanted in the non‐infarcted left ventricle of 3‐day post‐MI rats migrated to the peri‐infarct/infarct region and remained engrafted for 21 days. A significantly smaller infarct, increased number of small calibre blood vessels and improved ventricular function were observed in engrafted infarcted rat hearts. Thus, the second intron of the nestin gene drives expression in the mouse heart and a subpopulation of GFP/nestin(+) cells directly participate in reparative vascularisation. Increasing the population of nestin(+) cells via the transplantation of skin‐derived cells represents a potential approach to limit ischemic damage to the heart. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Combined activity of post‐exercise concentrations of NA and eHsp72 on human neutrophil function: Role of cAMP
- Abstract: Extracellular heat shock proteins of 72 kDa (eHsp72) and noradrenaline (NA) can act as “danger signals” during exercise‐induced stress by activating neutrophil function (chemotaxis, phagocytosis, and fungicidal capacity). In addition, post‐exercise concentrations of NA increase the expression and release of Hsp72 by human neutrophils, and adrenoreceptors and cAMP are involved in the stimulation of neutrophils by eHsp72. This suggests an interaction between the two molecules in the modulation of neutrophils during exercise‐induced stress. Given this context, the aim of the present investigation was to study the combined activity of post‐exercise circulating concentrations of NA and eHsp72 on the neutrophil phagocytic process, and to evaluate the role of cAMP as intracellular signal in these effects.
Results showed an accumulative stimulation of chemotaxis induced by NA and eHsp72. However, while NA and eHsp72, separately, stimulate the phagocytosis and fungicidal activity of neutrophils, when they act together they do not modify these capacities of neutrophils. Similarly, post‐exercise concentrations of NA and eHsp72 separately increased the intracellular level of cAMP, but NA and eHsp72 acting together did not modify the intracellular concentration of cAMP.
These results confirm that cAMP can be involved in the autocrine/paracrine physiological regulation of phagocytosis and fungicidal capacity of human neutrophils mediated by NA and eHsp72 in the context of exercise‐induced stress. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Extracellular heat shock proteins of 72 kDa (eHsp72) and noradrenaline (NA) can act as “danger signals” during exercise‐induced stress by activating neutrophil function (chemotaxis, phagocytosis, and fungicidal capacity). In addition, post‐exercise concentrations of NA increase the expression and release of Hsp72 by human neutrophils, and adrenoreceptors and cAMP are involved in the stimulation of neutrophils by eHsp72. This suggests an interaction between the two molecules in the modulation of neutrophils during exercise‐induced stress. Given this context, the aim of the present investigation was to study the combined activity of post‐exercise circulating concentrations of NA and eHsp72 on the neutrophil phagocytic process, and to evaluate the role of cAMP as intracellular signal in these effects.
Results showed an accumulative stimulation of chemotaxis induced by NA and eHsp72. However, while NA and eHsp72, separately, stimulate the phagocytosis and fungicidal activity of neutrophils, when they act together they do not modify these capacities of neutrophils. Similarly, post‐exercise concentrations of NA and eHsp72 separately increased the intracellular level of cAMP, but NA and eHsp72 acting together did not modify the intracellular concentration of cAMP.
These results confirm that cAMP can be involved in the autocrine/paracrine physiological regulation of phagocytosis and fungicidal capacity of human neutrophils mediated by NA and eHsp72 in the context of exercise‐induced stress. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Carcinoma‐associated fibroblasts: Non‐neoplastic tumour‐promoting mesenchymal cells
- Abstract: Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non‐transformed fibroblasts expressing α‐smooth muscle actin (α‐SMA). MFs together with non‐MF cells present in the tumour‐associated stroma are collectively referred to as carcinoma‐associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour‐promoting interactions with other tumour‐constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour‐promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Cancerous stroma coevolves alongside tumour progression, thereby promoting the malignant conversion of epithelial carcinoma cells. To date, an abundance of data have supported crucial roles of the tumour microenvironment (TME) in providing cancer cells with proliferative, migratory, survival and invasive propensities favouring the processes of tumourigenesis. The cancerous reactive stroma is frequently populated by a large number of myofibroblasts (MFs), which are activated, non‐transformed fibroblasts expressing α‐smooth muscle actin (α‐SMA). MFs together with non‐MF cells present in the tumour‐associated stroma are collectively referred to as carcinoma‐associated fibroblasts (CAFs), one of the major stromal cell types recognised in various human carcinomas. Recruitment of fibroblasts and/or their progenitors to a tumour mass and their subsequent transdifferentiation into MFs, as well as ongoing maintenance of their activated state, are believed to be essential processes facilitating tumour progression. However, the complex networks of signalling pathways mediating the phenotypic conversion into CAFs, as well as those underlying their tumour‐promoting interactions with other tumour‐constituting cells, have yet to be fully explored. Histopathological confirmation of the presence of large numbers of CAF MFs within TME and their altered gene expression profiles are known to be associated with disease progression and to serve as independent negative prognostic factors for a wide range of tumour types. In this review, we examine the current evidence shedding light on the emerging roles of tumour‐promoting CAFs, cells that are pivotal for epithelial cancer development and progression, and discuss the therapeutic potential of targeting these cells. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Transcriptional profiling of endometriosis tissues identifies genes related to organogenesis defects
- Abstract: Endometriosis is a common benign pathology, characterised by the presence of endometrial tissue outside the endometrial cavity with a prevalence of 10–15% in reproductive‐aged women. The pathogenesis is not completely understood, and several theories have been proposed to explain the etiology. Our group has recently described the presence of ectopic endometrium in a consistent number of human female foetuses analysed by autopsy, reinforcing the hypothesis that endometriosis may be generated by defects during the organogenesis of the female reproductive trait. Herein, in order to identify, at molecular level, changes involved in the disease, we compared the transcriptional profiling of ectopic endometrium with the corresponding eutopic one. Statistical analyses lead us to identify some genes specifically deregulated in the ectopic endometrium, that are involved in gonad developmental process or in wound healing process. Among them, we identified BMP4 and GREM1. BMP4 was never associated before to endometriosis and is involved in the mesoderm‐Müllerian duct differentiation. GREM1 is needed for the initial step of the ureter growth and perhaps could possibly be involved in Müller ducts differentiation. These molecules might be related to the endometriosis etiology since we showed that their expression is not related to the menstrual cycle phase both at RNA and at protein levels. These data support the theory that embryological defects could be responsible of the endometriosis generation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Endometriosis is a common benign pathology, characterised by the presence of endometrial tissue outside the endometrial cavity with a prevalence of 10–15% in reproductive‐aged women. The pathogenesis is not completely understood, and several theories have been proposed to explain the etiology. Our group has recently described the presence of ectopic endometrium in a consistent number of human female foetuses analysed by autopsy, reinforcing the hypothesis that endometriosis may be generated by defects during the organogenesis of the female reproductive trait. Herein, in order to identify, at molecular level, changes involved in the disease, we compared the transcriptional profiling of ectopic endometrium with the corresponding eutopic one. Statistical analyses lead us to identify some genes specifically deregulated in the ectopic endometrium, that are involved in gonad developmental process or in wound healing process. Among them, we identified BMP4 and GREM1. BMP4 was never associated before to endometriosis and is involved in the mesoderm‐Müllerian duct differentiation. GREM1 is needed for the initial step of the ureter growth and perhaps could possibly be involved in Müller ducts differentiation. These molecules might be related to the endometriosis etiology since we showed that their expression is not related to the menstrual cycle phase both at RNA and at protein levels. These data support the theory that embryological defects could be responsible of the endometriosis generation. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Inactivation of p27kip1 promotes carcinogens induced liver hepatocarcinogenesis through enhancing inflammatory cytokine secretion and STAT3 signaling activation
- Abstract: Although the expression of p27 has been regarded as a prognostic parameter in human liver cancer since the implication of decreased p27 expression levels in the genesis and progression of hepatocellular carcinoma (HCC), the molecular mechanism linking p27 deficiency and HCC development is still unclear. Here, we report an increase in tumorigenesis and progression as well as an enhanced inflammatory response in p27 deficient mice (p27‐/‐) and hypothesize the possible mechanism. We show that p27‐/‐ mice display increased proliferation and decreased apoptosis of tumor cells, accompanied by an increase in the serum inflammatory cytokines IL‐6 and TNF‐α. Furthermore, our data indicated that the increased number and signal transducers and activator of transcription 3 (STAT3) phosphorylation status of infiltrated inflammatory cells was accompanied by increased IL‐6 and TNF‐α mRNA levels in tumor and normal liver tissue in the p27‐/‐ mice. Moreover, using tumor cell and splenocytes co‐culture and tumor homologous transplantation, we validated our hypothesis in vitro and in vivo. Collectively, these data demonstrate that the loss of p27 promotes carcinogens‐induced HCC genesis and progression via the elevation of inflammatory cytokines and the augmented activation of STAT3 signaling in tumor cells and infiltrated inflammatory cells. Altogether, the loss of the cyclin kinase inhibitor p27, traditionally regarded as a consequence of DNA damage, can in turn promote HCC progression through enhancing the inflammatory response, potentially representing a promising therapeutic target in the prevention of HCC genesis and progression. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
- Abstract: Although the expression of p27 has been regarded as a prognostic parameter in human liver cancer since the implication of decreased p27 expression levels in the genesis and progression of hepatocellular carcinoma (HCC), the molecular mechanism linking p27 deficiency and HCC development is still unclear. Here, we report an increase in tumorigenesis and progression as well as an enhanced inflammatory response in p27 deficient mice (p27‐/‐) and hypothesize the possible mechanism. We show that p27‐/‐ mice display increased proliferation and decreased apoptosis of tumor cells, accompanied by an increase in the serum inflammatory cytokines IL‐6 and TNF‐α. Furthermore, our data indicated that the increased number and signal transducers and activator of transcription 3 (STAT3) phosphorylation status of infiltrated inflammatory cells was accompanied by increased IL‐6 and TNF‐α mRNA levels in tumor and normal liver tissue in the p27‐/‐ mice. Moreover, using tumor cell and splenocytes co‐culture and tumor homologous transplantation, we validated our hypothesis in vitro and in vivo. Collectively, these data demonstrate that the loss of p27 promotes carcinogens‐induced HCC genesis and progression via the elevation of inflammatory cytokines and the augmented activation of STAT3 signaling in tumor cells and infiltrated inflammatory cells. Altogether, the loss of the cyclin kinase inhibitor p27, traditionally regarded as a consequence of DNA damage, can in turn promote HCC progression through enhancing the inflammatory response, potentially representing a promising therapeutic target in the prevention of HCC genesis and progression. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.
