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Hui Y. Lan - One of the best experts on this subject based on the ideXlab platform.
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tgf β SMAD signaling in renal fibrosis
Frontiers in Physiology, 2015Co-Authors: Xiaoming Meng, Patrick Mingkuen Tang, Hui Y. LanAbstract:TGF-β (transforming growth factor-β) is well identified as a central mediator in renal fibrosis. TGF-β initiates canonical and non-canonical pathways to exert multiple biological effects. Among them, SMAD signaling is recognized as a major pathway of TGF-β signaling in progressive renal fibrosis. During fibrogenesis, SMAD3 is highly activated, which is associated with the down-regulation of an inhibitory SMAD7 via an ubiquitin E3-ligases-dependent degradation mechanism. The equilibrium shift between SMAD3 and SMAD7 leads to accumulation and activation of myofibroblasts, overproduction of ECM (extracellular matrix), and reduction in ECM degradation in the diseased kidney. Therefore, overexpression of SMAD7 has been shown to be a therapeutic agent for renal fibrosis in various models of kidney diseases. In contrast, another downstream effecter of TGF-β/SMAD signaling pathway, SMAD2, exerts its renal protective role by counter-regulating the SMAD3. Furthermore, recent studies demonstrated that SMAD3 mediates renal fibrosis by down-regulating miR-29 and miR-200 but up-regulating miR-21 and miR-192. Thus, overexpression of miR-29 and miR-200 or down-regulation of miR-21 and miR-192 is capable of attenuating SMAD3-mediated renal fibrosis in various mouse models of chronic kidney diseases (CKD). Taken together, TGF-β/SMAD signaling plays an important role in renal fibrosis. Targeting TGF-β/SMAD3 signaling may represent a specific and effective therapy for CKD associated with renal fibrosis.
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Opposing roles for SMAD2 and SMAD3 in peritoneal fibrosis in vivo and in vitro.
The American journal of pathology, 2014Co-Authors: Wen-juan Duan, Xiao-ru Huang, Hui Y. LanAbstract:Peritoneal fibrosis is a major cause of ultrafiltration failure in patients receiving continuous ambulatory peritoneal dialysis. Transforming growth factor (TGF)-β1 is an important mediator in this process; however, its signaling mechanisms had not been explored. Thus, we examined TGF-β1/SMAD signaling in human peritoneal biopsy specimens associated with continuous ambulatory peritoneal dialysis. We found that TGF-β/SMAD2/3 signaling was highly activated in patients with increased collagen deposition and thickening of the peritoneal membrane who were receiving continuous ambulatory peritoneal dialysis. Long-term exposure of wild-type mice to 4.25% peritoneal dialysis solution for 30 days induced significant peritoneal fibrosis with impaired peritoneal equilibrium, which was prevented in SMAD3 knockout mice. In contrast, conditional SMAD2 gene deletion in the peritoneum exacerbated peritoneal fibrosis and dysfunction. The contrasting roles of SMAD2 and SMAD3 in peritoneal fibrosis were also examined in vitro . Cultured mesothelial cells from SMAD3 knockout mice were resistant to TGF-β1–induced collagen I production and the transition toward a myofibroblast phenotype as seen in wild-type cells, whereas SMAD2 deficiency in mesothelial cells failed to modulate the profibrotic response to TGF-β1. In conclusion, this study found activation of TGF-β/SMAD signaling in peritoneal fibrosis in patients receiving continuous ambulatory peritoneal dialysis and identifies opposing roles for SMAD2 and SMAD3 in peritoneal dialysis–associated peritoneal fibrosis. These findings provide a mechanistic basis for future therapies targeting TGF-β/SMAD signaling in peritoneal fibrosis.
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role of the tgf β bmp 7 SMAD pathways in renal diseases
Clinical Science, 2013Co-Authors: Xiaoming Meng, Arthur C K Chung, Hui Y. LanAbstract:TGF-β (transforming growth factor-β) and BMP-7 (bone morphogenetic protein-7), two key members in the TGF-β superfamily, play important but diverse roles in CKDs (chronic kidney diseases). Both TGF-β and BMP-7 share similar downstream SMAD signalling pathways, but counter-regulate each other to maintain the balance of their biological activities. During renal injury in CKDs, this balance is significantly altered because TGF-β signalling is up-regulated by inducing TGF-β1 and activating SMAD3, whereas BMP-7 and its downstream SMAD1/5/8 are down-regulated. In the context of renal fibrosis, SMAD3 is pathogenic, whereas SMAD2 and SMAD7 are renoprotective. However, this counter-balancing mechanism is also altered because TGF-β1 induces Smurf2, a ubiquitin E3-ligase, to target SMAD7 as well as SMAD2 for degradation. Thus overexpression of renal SMAD7 restores the balance of TGF-β/SMAD signalling and has therapeutic effect on CKDs. Recent studies also found that SMAD3 mediated renal fibrosis by up-regulating miR-21 (where miR represents microRNA) and miR-192, but down-regulating miR-29 and miR-200 families. Therefore restoring miR-29/miR-200 or suppressing miR-21/miR-192 is able to treat progressive renal fibrosis. Furthermore, activation of TGF-β/SMAD signalling inhibits renal BMP-7 expression and BMP/SMAD signalling. On the other hand, overexpression of renal BMP-7 is capable of inhibiting TGF-β/SMAD3 signalling and protects the kidney from TGF-β-mediated renal injury. This counter-regulation not only expands our understanding of the causes of renal injury, but also suggests the therapeutic potential by targeting TGF-β/SMAD signalling or restoring BMP-7 in CKDs. Taken together, the current understanding of the distinct roles and mechanisms of TGF-β and BMP-7 in CKDs implies that targeting the TGF-β/SMAD pathway or restoring BMP-7 signalling may represent novel and effective therapies for CKDs.
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tgf β SMAD signaling in kidney disease
Seminars in Nephrology, 2012Co-Authors: Hui Y. Lan, Arthur C K ChungAbstract:Chronic progressive kidney diseases typically are characterized by active renal fibrosis and inflammation. Transforming growth factor-β1 (TGF-β1) is a key mediator in the development of renal fibrosis and inflammation. TGF-β1 exerts its biological effects by activating SMAD2 and SMAD3, which is regulated negatively by an inhibitory SMAD7. In the context of fibrosis, although SMAD3 is pathogenic, SMAD2 and SMAD7 are protective. Under disease conditions, SMADs also interact with other signaling pathways, such as the mitogen-activated protein kinase and nuclear factor-κB pathways. In contrast to the pathogenic role of active TGF-β1, latent TGF-β1 plays a protective role in renal fibrosis and inflammation. Furthermore, recent studies have shown that TGF-β/SMAD signaling plays a regulating role in microRNA-mediated renal injury. Thus, targeting TGF-β signaling by gene transfer of either SMAD7 or microRNAs into diseased kidneys has been shown to retard progressive renal injury in a number of experimental models. In conclusion, TGF-β/SMAD signaling plays a critical role in renal fibrosis and inflammation. Advances in understanding of the mechanisms of TGF-β/SMAD signaling in renal fibrosis and inflammation during chronic kidney diseases should provide a better therapeutic strategy to combat kidney diseases.
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SMAD7 Transgene Attenuates Peritoneal Fibrosis in Uremic Rats Treated with Peritoneal Dialysis
Journal of the American Society of Nephrology : JASN, 2007Co-Authors: Hong Guo, Hui Y. Lan, Joseph Leung, Man Fai Lam, Loretta Y.y. Chan, Anita W. L. Tsang, Kar Neng LaiAbstract:Transforming growth factor β (TGF-β) plays a critical role in the pathogenesis of the peritoneal fibrosis that complicates long-term peritoneal dialysis (PD). We studied the TGF-β/SMAD signaling pathway in peritoneal fibrosis induced in uremic rats treated with PD and explored the therapeutic potential of SMAD7 to prevent fibrogenesis. After subtotal nephrectomy, uremic rats were treated with peritoneal dialysis using 4.25% dextrose-containing fluid. The peritoneum of uremic rats treated with PD demonstrated fibrosis, increased TGF-β expression, increased SMAD2/3 activation, decreased SMAD7 expression, and increased expression of fibrogenic and angiogenic factors. In addition, peritoneal function was impaired and its structure was altered, including a thickened submesothelial layer. In rats transfected with a SMAD7 transgene using an ultrasound-microbubble–mediated system, peritoneal fibrosis was attenuated, peritoneal function was improved, and SMAD2/3 activation was inhibited. We suggest that administration of SMAD7 inhibits peritoneal fibrogenesis in uremic rats treated with PD by correcting the imbalance between downregulated SMAD7 and activated SMAD2/3. Blockade of the TGF-β/SMAD signaling pathway may represent a novel therapeutic approach to prevent peritoneal fibrosis in patients treated with PD.
Peter Ten Dijke - One of the best experts on this subject based on the ideXlab platform.
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SMAD protein and TGF-beta signaling in vascular smooth muscle cells.
International journal of molecular medicine, 2003Co-Authors: Hirofumi Ikedo, Peter Ten Dijke, Kiyoshi Tamaki, Seiji Ueda, Seiya Kato, Makiko Fujii, Seiya OkudaAbstract:Transforming growth factor-beta1 (TGF-beta1) plays a role in vascular remodeling by stimulating vascular smooth muscle cell (SMC) growth and matrix-protein synthesis at sites of vascular injury. SMAD proteins have been shown to mediate intracellular signaling of this growth factor. We investigated the expression and phosphorylation of SMADs in cultured rat aortic smooth muscle cells. In addition, we evaluated the effects of overexpression of SMAD proteins on TGF-beta signal transduction by adenovirus-mediated gene transfer. In rat SMC, SMAD1, SMAD2, SMAD3, SMAD4 and SMAD5 were detected by immunoprecipitation. Using antisera against phosphorylated SMAD2, we showed that TGF-beta1-induced SMAD2 phosphorylation in a concentration- and time-dependent manner. Using adenovirus-mediated transfection method, we demonstrated that overexpression of SMAD2 or SMAD4 was associated with an increased production of TGF-beta1-induced plasminogen activator inhibitor-1 (PAI-1). However, the most prominent expression of PAI-1 was observed upon cotransfection of both SMAD2 and SMAD4. Both the proliferative effect of TGF-beta1 under serum-free conditions and its anti-proliferative effect under serum-rich conditions were suppressed by the adenovirus-mediated overexpression of SMAD7. These results indicated that SMADs proteins were expressed in vascular SMC and that they mediated TGF-beta signaling in those cells.
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The FYVE domain in SMAD anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-beta/SMAD signalling.
Genes to cells : devoted to molecular & cellular mechanisms, 2002Co-Authors: Fumiko Itoh, Susumu Itoh, Nullin Divecha, Lenny Brocks, L. C. J. M. Oomen, Hans Janssen, Jero Calafat, Peter Ten DijkeAbstract:Transforming growth factor-β (TGF-β) superfamily members are multi-functional regulators of cell fate. These factors signal by binding to a limited number of highly conserved transmembrane type I and type II serine/threonine kinase receptors. These receptors initiate signals into the cell via the SMAD proteins. Up to date, 8 different mammalian SMADs are reported and are divided into three subgroups; receptor regulated SMADs (R-SMADs), common mediator SMADs (Co-SMADs) and inhibitory SMADs (I-SMADs). This thesis investigates the function and regulation of TGF-β/SMAD signaling through identification and characterization of SMAD interacting proteins.I-SMADs, i.e. SMAD6 and SMAD7, are potent antagonists of the TGF-β superfamily signaling. We found that SMAD7, but not SMAD6, inhibits TGF-β1-induced growth inhibition and expression of immediate early response genes. Interestingly, in the absence of ligand, SMAD7 was found to be predominantly localized in the nucleus, whereas SMAD7 accumulated in the cytoplasm upon TGF-β receptor activation. Moreover, we found that the MH2 domain is important for nuclear export.To investigate further the role of inhibitory SMADs, we have identified AMSH as a SMAD6 interacting protein using a yeast two-hybrid screening method. AMSH was previously discovered as the associated molecule with the SH3 domain of STAM. AMSH interacts with I-SMADs, but not with R- and Co-SMADs upon receptor activation and potentiates BMP-induced activation of transcriptional reporter activity, growth arrest and apoptosis. AMSH was found to prevent SMAD6 from binding to activated type I receptors and/or activated R-SMADs. SMAD anchor for receptor activation (SARA) is critical for SMAD2 and SMAD3 activation by TGF-β receptors. The present studies show that the localization of SARA in early endosomes is regulated through its FYVE domain. We have found that the FYVE domain of SARA is sufficient and necessary for the early endosomal localization, probably through its interaction with PtdIns(3)P. Moreover, the localization of SARA in early endosomes is required for efficient TGF-β/SMAD signaling.Both Notch and BMP signaling pathways are important for vascular development. We have found that Herp2, which is originally known as one of the Notch target genes, is synergistically induced upon activation of Notch and BMP signaling pathways in endothelial cells (ECs). The critical elements for synergistical activation of Herp2 gene by BMP and Notch pathway were identified. Furthermore, the Notch intracellular domain interacts with SMAD5 upon BMP receptor and this interaction becomes stronger in the presence of pCAF. Interestingly, Herp2 was found to antagonize BMP receptor- or Id-mediated EC migration.
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deficient SMAD7 expression a putative molecular defect in scleroderma
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Chunming Dong, Frederick M Wigley, Woohyun Yoon, Rene Alvarez, Barbara White, Peter Ten Dijke, Zhiru Li, Tao Wang, Pascal J GoldschmidtclermontAbstract:Scleroderma is a chronic systemic disease that leads to fibrosis of affected organs. Transforming growth factor (TGF) β has been implicated in the pathogenesis of scleroderma. SMAD proteins are signaling transducers downstream from TGF-β receptors. Three families of SMADs have been identified: (i) receptor-regulated SMAD2 and -3 (R-SMADs); (ii) common partner SMAD4 (Co-SMAD); and (iii) inhibitory SMAD6 and -7 (I-SMADs, part of a negative feedback loop). We have investigated the signaling components for the TGF-β pathway and TGF-β activity in scleroderma lesions in vivo and in scleroderma fibroblasts in vitro. Basal level and TGF-β-inducible expression of SMAD7 are selectively decreased, whereas SMAD3 expression is increased both in scleroderma skin and in explanted scleroderma fibroblasts in culture. TGF-β signaling events, including phosphorylation of SMAD2 and -3, and transcription of the PAI-1 gene are increased in scleroderma fibroblasts, relative to normal fibroblasts. In vitro adenoviral gene transfer with SMAD7 restores normal TGF-β signaling in scleroderma fibroblasts. These results suggest that alterations in the SMAD pathway, including marked SMAD7 deficiency and SMAD3 up-regulation, may be responsible for TGF-β hyperresponsiveness observed in scleroderma.
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signaling of transforming growth factor β family members through SMAD proteins
FEBS Journal, 2000Co-Authors: Susumu Itoh, Fumiko Itoh, Mariejose Goumans, Peter Ten DijkeAbstract:SMADs are pivotal intracellular nuclear effectors of transforming growth factor-beta (TGF-beta) family members. Ligand-induced activation of TGF-beta family receptors with intrinsic serine/threonine kinase activity trigger phosphorylation of receptor-regulated SMADs (R-SMADs), whereas SMAD2 and SMAD3 are phosphorylated by TGF-beta, and activin type I receptors, SMAD1, SMAD5 and SMAD8, act downstream of BMP type I receptors. Activated R-SMADs form heteromeric complexes with common-partner SMADs (Co-SMADs), e.g. SMAD4, which translocate efficiently to the nucleus, where they regulate, in co-operation with other transcription factors, coactivators and corepressors, the transcription of target genes. Inhibitory SMADs act in most cases in an opposite manner from R- and Co-SMADs. Like other components in the TGF-beta family signaling cascade, SMAD activity is intricately regulated. The multifunctional and context dependency of TGF-beta family responses are reflected in the function of SMADs as signal integrators. Certain SMADs are somatically mutated at high frequency in particular types of human cancers. Gene ablation of SMADs in the mouse has revealed their critical roles during embryonic development. Here we review the latest advances in our understanding of the SMAD mechanism of action and their in vivo functions.
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role of SMAD proteins and transcription factor sp1 in p21waf1 cip1 regulation by transforming growth factor β
Journal of Biological Chemistry, 2000Co-Authors: Katerina Pardali, Peter Ten Dijke, Akira Kurisaki, Anita Moren, Dimitris Kardassis, Aristidis MoustakasAbstract:Abstract Transforming growth factor-β (TGF-β) inhibits cell cycle progression, in part through up-regulation of gene expression of the p21WAF1/Cip1(p21) cell cycle inhibitor. Previously we have reported that the intracellular effectors of TGF-β, SMAD3 and SMAD4, functionally cooperate with Sp1 to activate the human p21 promoter in hepatoma HepG2 cells. In this study we show that SMAD3 and SMAD4 when overexpressed in HaCaT keratinocytes lead to activation of the p21 promoter. Activation requires the binding sites for the ubiquitous transcription factor Sp1 on the proximal promoter. Induction of the endogenous HaCaTp21 gene by TGF-β1 is further enhanced after overexpression of SMAD3 and SMAD4, whereas dominant negative mutants of SMAD3 and SMAD4 and the inhibitory SMAD7 all inhibit p21induction by TGF-β1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficient Drosophila SL-2 cells binds to the proximal p21 promoter sequences, whereas SMAD proteins do not. In support of this finding, we show that DNA-binding domain mutants of SMAD3 and SMAD4 are capable of transactivating the p21 promoter as efficiently as wild type SMADs. Co-expression of SMAD3 with SMAD4 and Sp1 in SL-2 cells or co-incubation of phosphorylated SMAD3, SMAD4, and Sp1 in vitro results in enhanced binding of Sp1 to the p21 proximal promoter sequences. We demonstrate that Sp1 physically and directly interacts with SMAD2, SMAD3, and weakly with SMAD4 via their amino-terminal (Mad-Homology 1) domain. Finally, by using GAL4 fusion proteins we show that the glutamine-rich sequences in the transactivation domain of Sp1 contribute to the cooperativity with SMAD proteins. In conclusion, SMAD proteins play important roles in regulation of the p21 gene by TGF-β, and the functional cooperation of SMAD proteins with Sp1 involves the physical interaction of these two types of transcription factors.
Aristidis Moustakas - One of the best experts on this subject based on the ideXlab platform.
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PARP-1 Attenuates SMAD-Mediated Transcription
Molecular cell, 2010Co-Authors: Peter Lönn, Carl-henrik Heldin, Lars P. Van Der Heide, Markus Dahl, Ulf Hellman, Aristidis MoustakasAbstract:The versatile cytokine transforming growth factor β (TGF-β) regulates cellular growth, differentiation, and migration during embryonic development and adult tissue homeostasis. Activation of TGF-β receptors leads to phosphorylation of SMAD2 and SMAD3, which oligomerize with SMAD4 and accumulate in the nucleus where they recognize gene regulatory regions and orchestrate transcription. Termination of SMAD-activated transcription involves SMAD dephosphorylation, nuclear export, or ubiquitin-mediated degradation. In an unbiased proteomic screen, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a SMAD-interacting partner. PARP-1 dissociates SMAD complexes from DNA by ADP-ribosylating SMAD3 and SMAD4, which attenuates SMAD-specific gene responses and TGF-β-induced epithelial-mesenchymal transition. Thus, our results identify ADP-ribosylation of SMAD proteins by PARP-1 as a key step in controlling the strength and duration of SMAD-mediated transcription.
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tgf β and the SMAD signaling pathway support transcriptomic reprogramming during epithelial mesenchymal cell transition
Molecular Biology of the Cell, 2005Co-Authors: Ulrich Valcourt, Carl-henrik Heldin, M. Kowanetz, H. Niimi, Aristidis MoustakasAbstract:Epithelial-mesenchymal transition (EMT) contributes to normal tissue patterning and carcinoma invasiveness. We show that transforming growth factor (TGF)-β/activin members, but not bone morphogenetic protein (BMP) members, can induce EMT in normal human and mouse epithelial cells. EMT correlates with the ability of these ligands to induce growth arrest. Ectopic expression of all type I receptors of the TGF-β superfamily establishes that TGF-β but not BMP pathways can elicit EMT. Ectopic SMAD2 or SMAD3 together with SMAD4 enhanced, whereas dominant-negative forms of SMAD2, SMAD3, or SMAD4, and wild-type inhibitory SMAD7, blocked TGF-β–induced EMT. Transcriptomic analysis of EMT kinetics identified novel TGF-β target genes with ligand-specific responses. Using a TGF-β type I receptor that cannot activate SMADs nor induce EMT, we found that SMAD signaling is critical for regulation of all tested gene targets during EMT. One such gene, Id2, whose expression is repressed by TGF-β1 but induced by BMP-7 is critical for regulation of at least one important myoepithelial marker, α-smooth muscle actin, during EMT. Thus, based on ligand-specific responsiveness and evolutionary conservation of the gene expression patterns, we begin deciphering a genetic network downstream of TGF-β and predict functional links to the control of cell proliferation and EMT.
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role of SMAD proteins and transcription factor sp1 in p21waf1 cip1 regulation by transforming growth factor β
Journal of Biological Chemistry, 2000Co-Authors: Katerina Pardali, Peter Ten Dijke, Akira Kurisaki, Anita Moren, Dimitris Kardassis, Aristidis MoustakasAbstract:Abstract Transforming growth factor-β (TGF-β) inhibits cell cycle progression, in part through up-regulation of gene expression of the p21WAF1/Cip1(p21) cell cycle inhibitor. Previously we have reported that the intracellular effectors of TGF-β, SMAD3 and SMAD4, functionally cooperate with Sp1 to activate the human p21 promoter in hepatoma HepG2 cells. In this study we show that SMAD3 and SMAD4 when overexpressed in HaCaT keratinocytes lead to activation of the p21 promoter. Activation requires the binding sites for the ubiquitous transcription factor Sp1 on the proximal promoter. Induction of the endogenous HaCaTp21 gene by TGF-β1 is further enhanced after overexpression of SMAD3 and SMAD4, whereas dominant negative mutants of SMAD3 and SMAD4 and the inhibitory SMAD7 all inhibit p21induction by TGF-β1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficient Drosophila SL-2 cells binds to the proximal p21 promoter sequences, whereas SMAD proteins do not. In support of this finding, we show that DNA-binding domain mutants of SMAD3 and SMAD4 are capable of transactivating the p21 promoter as efficiently as wild type SMADs. Co-expression of SMAD3 with SMAD4 and Sp1 in SL-2 cells or co-incubation of phosphorylated SMAD3, SMAD4, and Sp1 in vitro results in enhanced binding of Sp1 to the p21 proximal promoter sequences. We demonstrate that Sp1 physically and directly interacts with SMAD2, SMAD3, and weakly with SMAD4 via their amino-terminal (Mad-Homology 1) domain. Finally, by using GAL4 fusion proteins we show that the glutamine-rich sequences in the transactivation domain of Sp1 contribute to the cooperativity with SMAD proteins. In conclusion, SMAD proteins play important roles in regulation of the p21 gene by TGF-β, and the functional cooperation of SMAD proteins with Sp1 involves the physical interaction of these two types of transcription factors.
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regulation of the human p21 waf1 cip1 promoter in hepatic cells by functional interactions between sp1 and SMAD family members
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Aristidis Moustakas, Dimitris KardassisAbstract:The cell cycle inhibitor p21/WAF1/Cip1 is expressed in many cell types and is regulated by p53-dependent and p53-independent mechanisms. p21 is an important regulator of hepatocyte cell cycle, differentiation, and liver development, but little is known about the regulation of its synthesis in hepatocytes. We report herein that the p21 gene is constitutively expressed in human hepatoma HepG2 cells. Deletion analysis of the p21 promoter showed that it contains a distal (positions −2,300/−210) and a proximal (positions −124 to −61) region that act synergistically to achieve high levels of constitutive expression. The proximal region that consists of multiple Sp1 binding sites is essential for constitutive p21 promoter activity in hepatocytes. This region also mediates the transcriptional activation of the p21 promoter by members of the SMAD family of proteins, which play important role in the transduction of extracellular signals such as transforming growth factor β, activin, etc. Constitutive expression of p21 was severely reduced by a C-terminally truncated form of SMAD4 that was shown previously to block signaling through SMADs. SMAD3/4 and to a much lesser extent SMAD2/4 caused high levels of transcriptional activation of the p21 promoter. Transactivation was compromised by N- or C-terminally truncated forms of SMAD3. By using Gal4-Sp1 fusion proteins, we show that SMAD proteins can activate gene transcription via functional interactions with the ubiquitous factor Sp1. These data demonstrate that SMAD proteins and Sp1 participate in the constitutive or inducible expression of the p21 gene in hepatic cells.
Joan Massagué - One of the best experts on this subject based on the ideXlab platform.
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Distinct Domain Utilization by SMAD3 and SMAD4 for Nucleoporin Interaction and Nuclear Import
Journal of Biological Chemistry, 2003Co-Authors: Lan Xu, Claudio Alarcón, Joan MassaguéAbstract:Abstract SMAD proteins undergo rapid nuclear translocation upon stimulation by transforming growth factor-β (TGFβ) and in so doing transduce the signal into the nucleus. In this report we unraveled nuclear import mechanisms of SMAD3 and SMAD4 that are dependent on their interaction with FG-repeat-containing nucleoporins such as CAN/Nup214, without the involvement of importin molecules that are responsible for most of the known nuclear import events. A surface hydrophobic corridor within the MH2 domain of SMAD3 is critical for association with CAN/Nup214 and nuclear import, whereas SMAD4 interaction with CAN/Nup214, and nuclear import requires structural elements present only in the full-length SMAD4. As exemplified by the different susceptibility to inhibition of import by cytoplasmic retention factor SARA (SMAD anchor for receptor activation), such utilization of distinct domains for nuclear import of SMAD3 and SMAD4 suggests that nuclear transport of SMAD3 and SMAD4 is subject to control by different retention factors.
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A SMAD Transcriptional Corepressor
Cell, 1999Co-Authors: David Wotton, Susan Lee, Joan MassaguéAbstract:Following TGFbeta receptor-mediated phosphorylation and association with SMAD4, SMAD2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a SMAD2-binding protein and a repressor of transcription. A TGFbeta-activated SMAD complex can recruit TGIF and histone deacetylases (HDACs) to a SMAD target promoter, repressing transcription. Thus, upon entering the nucleus, a SMAD2-SMAD4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of SMAD corepressors and coactivators within the cell.
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a SMAD transcriptional corepressor
Cell, 1999Co-Authors: David Wotton, Susan Lee, Joan MassaguéAbstract:Abstract Following TGFβ receptor–mediated phosphorylation and association with SMAD4, SMAD2 moves into the nucleus, binds to target promoters in association with DNA-binding cofactors, and recruits coactivators such as p300/CBP to activate transcription. We identified the homeodomain protein TGIF as a SMAD2-binding protein and a repressor of transcription. A TGFβ-activated SMAD complex can recruit TGIF and histone deacetylases (HDACs) to a SMAD target promoter, repressing transcription. Thus, upon entering the nucleus, a SMAD2–SMAD4 complex may interact with coactivators, forming a transcriptional activation complex, or with TGIF and HDACs, forming a transcriptional repressor complex. Formation of one of these two mutually exclusive complexes is determined by the relative levels of SMAD corepressors and coactivators within the cell.
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a mechanism of repression of tgfbeta SMAD signaling by oncogenic ras
Genes & Development, 1999Co-Authors: Marcus Kretzschmar, Jacqueline Doody, Inna Timokhina, Joan MassaguéAbstract:TGFβ can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFβ antimitogenic responses. Here we report that oncogenic Ras inhibits TGFβ signaling in mammary and lung epithelial cells by negatively regulating the TGFβ mediators SMAD2 and SMAD3. Oncogenically activated Ras inhibits the TGFβ-induced nuclear accumulation of SMAD2 and SMAD3 and SMAD-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of SMAD2 and SMAD3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFβ receptor phosphorylation sites that activate SMAD nuclear translocation. Mutation of these MAP kinase sites in SMAD3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFβ in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of SMAD2 and SMAD3. Our results suggest a mechanism for the counterbalanced regulation of SMAD2/SMAD3 by TGFβ and Ras signals in normal cells, and for the silencing of antimitogenic TGFβ functions by hyperactive Ras in cancer cells.
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inhibition of transforming growth factor β SMAD signalling by the interferon γ stat pathway
Nature, 1999Co-Authors: Luis Ulloa, Jacqueline Doody, Joan MassaguéAbstract:Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) have opposite effects on diverse cellular functions, but the basis for this antagonism is not known. TGF-beta signals through a receptor serine kinase that phosphorylates and activates the transcription factors SMADs 2 and 3, whereas the IFN-gamma receptor and its associated protein tyrosine kinase Jak1 mediate phosphorylation and activation of the transcription factor Stat1. Here we present a basis for the integration of TGF-beta and IFN-gamma signals. IFN-gamma inhibits the TGF beta-induced phosphorylation of SMAD3 and its attendant events, namely, the association of SMAD3 with SMAD4, the accumulation of SMAD3 in the nucleus, and the activation of TGFbeta-responsive genes. Acting through Jak1 and Stat1, IFN-gamma induces the expression of SMAD7, an antagonistic SMAD, which prevents the interaction of SMAD3 with the TGF-beta receptor. The results indicate a mechanism of transmodulation between the STAT and SMAD signal-transduction pathways.
Alain Mauviel - One of the best experts on this subject based on the ideXlab platform.
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Stable overexpression of SMAD7 in human melanoma cells inhibits their tumorigenicity in vitro and in vivo.
Oncogene, 2005Co-Authors: Delphine Javelaud, Véronique Delmas, Maria Möller, Peggy Sextius, Jocelyne André, Suzanne Menashi, Lionel Larue, Alain MauvielAbstract:We previously identified constitutive SMAD signaling in human melanoma cells despite resistance to transforming growth factor-beta (TGF-beta) control of cell proliferation. This led us to investigate the effect of inhibitory SMAD7 overexpression on melanoma cell behavior. Using the highly metastatic cell line, 1205-Lu, we thus generated melanoma cell clones constitutively expressing SMAD7, and their mock-transfected counterparts. Stable expression of SMAD7 resulted in an inhibition of constitutive SMAD2/3 phosphorylation, and in a reduced TGF-beta response of SMAD3/SMAD4-driven gene transactivation, as measured using transfected SMAD3/4-specific reporter gene constructs. SMAD7 overexpression, however, did not alter their proliferative capacity and resistance to TGF-beta-driven growth inhibition. On the other hand, expression of SMAD7 efficiently reduced the capacity of human melanoma cells to invade Matrigel in Boyden migration chambers, while not affecting their motility and adhesion to collagen and laminin. Gelatin zymography identified reduced MMP-2 and MMP-9 secretion by SMAD7-expressing melanoma cells as compared with their control counterparts. SMAD7-expressing melanoma cells exhibited a dramatically reduced capacity to form colonies under anchorage-independent culture conditions, and, when injected subcutaneously into nude mice, were largely delayed in their ability to form tumors. These results suggest that TGF-beta production by melanoma cells not only affects the tumor environment but also directly contributes to tumor cell aggressiveness through autocrine activation of SMAD signaling.
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Cytoplasmic SnoN in normal tissues and nonmalignant cells antagonizes TGF-beta signaling by sequestration of the SMAD proteins.
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Ariel Krakowski, Alain Mauviel, Julien Laboureau, Minna Bissell, Kunxin LuoAbstract:TGF-beta is a ubiquitously expressed cytokine that signals through the SMAD proteins to regulate many diverse cellular processes. SnoN is an important negative regulator of SMAD signaling. It has been described as a nuclear protein, based on studies of ectopically expressed SnoN and endogenous SnoN in cancer cell lines. In the nucleus, SnoN binds to SMAD2, SMAD3, and SMAD4 and represses their ability to activate transcription of TGF-beta target genes through multiple mechanisms. Here, we show that, whereas SnoN is localized exclusively in the nucleus in cancer tissues or cells, in normal tissues and nontumorigenic or primary epithelial cells, SnoN is predominantly cytoplasmic. Upon morphological differentiation or cell-cycle arrest, SnoN translocates into the nucleus. In contrast to nuclear SnoN that represses the transcriptional activity of the SMAD complexes, cytoplasmic SnoN antagonizes TGF-beta signaling by sequestering the SMAD proteins in the cytoplasm. Interestingly, cytoplasmic SnoN is resistant to TGF-beta-induced degradation and therefore is more potent than nuclear SnoN in repressing TGF-beta signaling. Thus, we have identified a mechanism of regulation of TGF-beta signaling via differential subcellular localization of SnoN that is likely to produce different patterns of downstream TGF-beta responses and may influence the proliferation or differentiation states of epithelial cells.
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Retinoic acid receptors interfere with the TGF-beta/SMAD signaling pathway in a ligand-specific manner.
Oncogene, 2003Co-Authors: Valérie Pendaries, Franck Verrecchia, Serge Michel, Alain MauvielAbstract:Transforming growth factor-beta (TGF-beta) and retinoic acid (RA) are important regulators of cell growth and differentiation. The TGF-beta receptors utilize SMAD proteins to transduce signals intracellularly and regulate transcription of target genes, either directly or in combination with other sequence-specific transcription factors. Two classes of nuclear receptors, the retinoic acid receptors (RARs) and the retinoic X receptors, are involved in mediating transcriptional responses to RA. Given the known interactions between the TGF-beta and RAR pathways, we have investigated the role played by RAR ligands in modulating functional interactions between SMAD3 and RARs. Using transient cell transfection experiments with an artificial SMAD3/SMAD4-dependent reporter construct, we demonstrate that RAR overexpression enhances SMAD-driven transactivation, an effect that requires both SMAD3 and SMAD4. We provide evidence that RAR effect on SMAD3/SMAD4-driven transcription is prevented by natural and synthetic RAR agonists, and potentiated by synthetic RAR antagonists. The activity of two TGF-beta-responsive human gene promoter constructs was regulated in a parallel fashion. Using both mammalian two-hybrid and immunoprecipitation/Western methods, we demonstrate a direct interaction between the region DEF of RARgamma and the MH2 domain of SMAD3, inhibited by RAR agonists and enhanced by their antagonists. We propose that RARs may function as coactivators of the SMAD pathway in the absence of RAR agonists or in the presence of their antagonists, a phenomenon that contrasts with their known role as agonist-activated transcriptional regulators of RA-dependent genes.
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Tumor necrosis factor-alpha inhibits transforming growth factor-beta /SMAD signaling in human dermal fibroblasts via AP-1 activation.
Journal of Biological Chemistry, 2000Co-Authors: Franck Verrecchia, Marcia Pessah, Azeddine Atfi, Alain MauvielAbstract:Understanding the molecular mechanisms underlying the antagonistic activities of tumor necrosis factor-alpha (TNF-alpha) against transforming growth factor-beta (TGF-beta) is of utmost importance given the physiopathological implications of these cytokines. In this report, we demonstrate that TNF-alpha prevents TGF-beta-induced SMAD-specific gene transactivation without inducing detectable levels of inhibitory SMAD7 in human dermal fibroblasts. On the other hand, c-Jun and JunB, both induced by TNF-alpha, block SMAD3-mediated transcription. Expression of antisense c-Jun mRNA prevents TNF-alpha inhibition of TGF-beta/SMAD signaling whereas that of dominant-negative Ikappa-B kinase-alpha or antisense SMAD7 does not. We provide evidence for off-DNA interactions between SMAD3 and both c-Jun and JunB accompanied with reduced SMAD3-DNA interactions. Finally, we show that overexpression of the transcriptional co-activator p300 prevents TNF-alpha/AP-1 inhibition of TGF-beta/SMAD signaling. These data suggest that TNF-alpha interferes with SMAD signaling through the induction of AP-1 components, the latter forming off-DNA complexes with SMAD3 and preventing its binding to specific cis-element(s). In addition, Jun members compete with SMAD3 for the common transcription co-activator p300. These two mechanisms are likely to act in concert to decrease SMAD-specific transcription.
