Non-canonical TGF-β Pathway Research Articles

GW25-e0832 TNF receptor-associated factor 6 (TRAF6) mediates the angiotensin-induced non-canonical TGFβ pathway activation and differentiation of c-kit+ cardiac stem cells

GW25-e0832 TNF receptor-associated factor 6 (TRAF6) mediates the angiotensin-induced non-canonical TGFβ pathway activation and differentiation of c-kit+ cardiac stem cells

An integrative modeling framework reveals plasticity of TGF-β signaling

BackgroundThe TGF-β transforming growth factor is the most pleiotropic cytokine controlling a broad range of cellular responses that include proliferation, differentiation and apoptosis. The context-dependent multifunctional nature of TGF-β is associated with complex signaling pathways. Differential models describe the dynamics of the TGF-β canonical pathway, but modeling the non-canonical networks constitutes a major challenge. Here, we propose a qualitative approach to explore all TGF-β-dependent signaling pathways.ResultsUsing a new formalism, CADBIOM, which is based on guarded transitions and includes temporal parameters, we have built the first discrete model of TGF-β signaling networks by automatically integrating the 137 human signaling maps from the Pathway Interaction Database into a single unified dynamic model. Temporal property-checking analyses of 15934 trajectories that regulate 145 TGF-β target genes reveal the association of specific pathways with distinct biological processes. We identify 31 different combinations of TGF-β with other extracellular stimuli involved in non-canonical TGF-β pathways that regulate specific gene networks. Extensive analysis of gene expression data further demonstrates that genes sharing CADBIOM trajectories tend to be co-regulated.ConclusionsAs applied here to TGF-β signaling, CADBIOM allows, for the first time, a full integration of highly complex signaling pathways into dynamic models that permit to explore cell responses to complex microenvironment stimuli.

Stimulation of the soluble guanylate cyclase (sGC) inhibits fibrosis by blocking non-canonical TGFβ signalling

ObjectivesWe have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between...

Smad6 inhibits non-canonical TGF-β1 signalling by recruiting the deubiquitinase A20 to TRAF6

Transforming growth factor (TGF)-β, a pivotal cytokine involved in a variety of cellular functions, transmits signals through Smad-dependent canonical and Smad-independent noncanonical pathways. In contrast to the canonical TGF-β pathway, it is unknown how noncanonical TGF-β pathways are negatively regulated. Here we demonstrate that the inhibitory Smad Smad6, but not Smad7, negatively regulates TGF-β1-induced activation of the TRAF6-TAK1-p38 MAPK/JNK pathway, a noncanonical TGF-β pathway. TGF-β1-induced Smad6 abolishes K63-linked polyubiquitination of TRAF6 by recruiting the A20 deubiquitinating enzyme in AML-12 mouse liver cells and primary hepatocytes. In addition, the knockdown of Smad6 or A20 in an animal model or cell culture system maintains TAK1 and p38 MAPK/JNK phosphorylation and increases apoptosis, emphasizing the crucial role of the Smad6-A20 axis in negative regulation of the TGF-β1-TRAF6-TAK1-p38 MAPK/JNK pathway. Therefore, our findings provide insight into the molecular mechanisms underlying negative regulation of noncanonical TGF-β pathways.

P21‐activated kinase‐2 is a critical mediator of transforming growth factor‐β‐induced hepatoma cell migration

Transforming growth factor-β (TGF-β) has been shown to play a central role in the promotion of cell motility, but its functional mechanism has remained unclear. With the aim of investigating the diagnostic and treatment modalities for patients with hepatocellular carcinoma (HCC), the signaling pathway that may contribute to TGF-β-mediated cell invasion in hepatoma cells was evaluated. Three hepatoma cell lines, HepG2, PLC/PRF/5, and HLF, were treated with TGF-β, and the involvement of the non-canonical TGF-β pathway was analyzed by cell migration assays. HepG2 cells were treated with a p21-activated kinase-2 (PAK2)-targeting small interfering RNA and analyzed for their cell motility. The relationships between the PAK2 status and the clinicopathological characteristics of 62 HCC patients were also analyzed. The cell migration assays showed that Akt is a critical regulator of TGF-β-mediated cell migration. Western blotting analyses showed that TGF-β stimulated Akt and PAK2 in all three hepatoma cell lines, and phosphorylated PAK2 was blocked by Akt inhibitor. Suppression of PAK2 expression by small interfering RNA resulted in increased focal adhesions with significantly repressed cell migration in the presence of TGF-β. Clinicopathological analyses showed that the phosphorylation level of PAK2 was closely associated with tumor progression, metastasis, and early recurrence of HCC. PAK2 may be a critical mediator of TGF-β-mediated hepatoma cell migration, and may represent a potential target for the treatment of HCC.

Abstract 3103: Overexpression of homeoprotein six1 activates non-canonical TGF-beta signaling to induce epithelial-mesenchymal transition in human papillomavirus type 16-immortalized human keratinocytes.

Abstract Inappropriate expression of embryonic genes, particularly homeodomain transcription factors, contributes to tumorigenesis and tumor progression. The overexpression of Six1, a member of the Six family of homeodomain transcription factors, has been found in various human cancers, and is associated with increased tumor progression and metastasis. Previous studies in our laboratory discovered that the expression of Six1 mRNA and protein increased during in vitro progression of human papillomavirus type 16 (HPV16)-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. However, the precise mechanisms of how Six1 promotes tumor onset at early and late stages of HPV16-mediated transformation remain unknown. In this study, we found that HKc/HPV16 and HKc/DR overexpressing Six1 exhibited a more mesenchymal phenotype compared to vector controls, as characterized by a fibroblastic morphology, and increased migration and invasion. We utilized Whole Human Genome Microarrays (4x44K from Agilent Technologies) to explore the gene expression changes associated with Six1 overexpression in HKc/HPV16 and HKc/DR. We found that overexpression of Six1 down-regulated epithelial-related genes (ex., E-cadherin and occludin) and up-regulated mesenchymal-related genes (ex., N-cadherin, vimentin, and snail), which strongly suggests that Six1 overexpression induces epithelial-mesenchymal transition (EMT). Gene expression changes determined by microarray analysis were confirmed by real time PCR and western blotting. Increased expression of TGF-beta receptors in response to overexpression of Six1 in HKc/DR did not lead to increased Smad-dependent signaling but did activate the Mitogen-activated protein kinase (MAPK) pathway, suggesting that MAPK non-Smad TGF-β signaling may be involved in Six1-mediated EMT. In conclusion, our study demonstrates that Six1 overexpression induces EMT, possibly by activation of the MAPK non-canonical (non-Smad) TGF-β pathway, during HPV16-mediated transformation of human keratinocytes. Citation Format: Hanwen Xu, Lucia Pirisi, Kim E. Creek. Overexpression of homeoprotein six1 activates non-canonical TGF-beta signaling to induce epithelial-mesenchymal transition in human papillomavirus type 16-immortalized human keratinocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3103. doi:10.1158/1538-7445.AM2013-3103

MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway

Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN.

TTRAP Is a Novel Component of the Non-Canonical TRAF6-TAK1 TGF-β Signaling Pathway

Transforming growth factor-β (TGF-β) principally relays its effects through the Smad pathway however, accumulating evidence indicate that alternative signaling routes are also employed by this pleiotropic cytokine. For instance recently, we have demonstrated that ligand occupied TGF-β receptors can directly trigger the TRAF6-TAK1 signaling module, resulting in MAP kinase activation. Here we report identification of the adaptor molecule TTRAP as a novel component of this non-canonical TGF-β pathway. We show that the protein associates with TGF-β receptors and components of the TRAF6-TAK1 signaling module, resulting in differential regulation of TGF-β activated p38 and NF-κB responses. Modulation of cellular TTRAP level affects cell viability in the presence of TGF-β, suggesting that the protein is an important component of the TGF-β induced apoptotic process.

Excessive Dpp signaling induces cardial apoptosis through dTAK1 and dJNK during late embryogenesis of Drosophila

BackgroundTo identify genes involved in the heart development of Drosophila, we found that embryos lacking raw function exhibited cardial phenotypes. raw was initially identified as a dorsal open group gene. The dorsal open phenotype was demonstrated to be resulted from the aberrant expression of decapentaplegic (dpp), a member of the tumor growth factor beta (TGF-β), signaling pathway. Despite the role of dpp in pattering cardioblasts during early embryogenesis of Drosophila have been demonstrated, how mutation in raw and/or excessive dpp signaling involves in the differentiating heart of Drosophila has not been fully elaborated at late stages.ResultsWe show that raw mutation produced a mild overspecification of cardial cells at stage 14, but these overproduced cells were mostly eliminated in late mutant embryos due to apoptosis. Aberrant dpp signaling is likely to contribute to the cardial phenotype found in raw mutants, because expression of dpp or constitutively activated thickven (tkvCA), the type I receptor of Dpp, induced a raw-like phenotype. Additionally, we show that dpp induced non-autonomous apoptosis through TGFβ activated kinase 1 (TAK1), because mis-expression of a dominant negative form of Drosophila TAK1 (dTAK1DN) was able to suppress cell death in raw mutants or embryos overexpressing dpp. Importantly, we demonstrated that dpp induce its own expression through dTAK1, which also leads to the hyperactivation of Drosophila JNK (DJNK). The hyperactivated DJNK was attributed to be the cause of Dpp/DTAK1-induced apoptosis because overexpression of a dominant negative DJNK, basket (bskDN), suppressed cell death induced by Dpp or DTAK1. Moreover, targeted overexpression of the anti-apoptotic P35 protein, or a dominant negative proapoptotic P53 (P53DN) protein blocked Dpp/DTAK1-induced apoptosis, and rescued heart cells under the raw mutation background.ConclusionsWe find that ectopic Dpp led to DJNK-dependent cardial apoptosis through the non-canonical TGF-β pathway during late embryogenesis of Drosophila. This certainly will increase our understanding of the pathogenesis of cardiomyopathy, because haemodynamic overload can up-regulate TGF-β and death of cardiomyocytes is observed in virtually every myocardial disease. Thus, our study may provide possible medical intervention for human cardiomyopathy.

Noncanonical TGF-β pathways, mTORC1 and Abl, in renal interstitial fibrogenesis

Renal interstitial fibrosis is a major determinant of renal failure in the majority of chronic renal diseases. Transforming growth factor-beta (TGF-beta) is the single most important cytokine promoting renal fibrogenesis. Recent in vitro studies identified novel non-smad TGF-beta targets including p21-activated kinase-2 (PAK2), the abelson nonreceptor tyrosine kinase (c-Abl), and the mammalian target of rapamycin (mTOR) that are activated by TGF-beta in mesenchymal cells, specifically in fibroblasts but less in epithelial cells. In the present studies, we show that non-smad effectors of TGF-beta including PAK2, c-Abl, Akt, tuberin (TSC2), and mTOR are activated in experimental unilateral obstructive nephropathy in rats. Treatment with c-Abl or mTOR inhibitors, imatinib mesylate and rapamycin, respectively, each blocks noncanonical (non-smad) TGF-beta pathways in the kidney in vivo and diminishes the number of interstitial fibroblasts and myofibroblasts as well as the interstitial accumulation of extracellular matrix proteins. These findings indicate that noncanonical TGF-beta pathways are activated during the early and rapid renal fibrogenesis of obstructive nephropathy. Moreover, the current findings suggest that combined inhibition of key regulators of these non-smad TGF-beta pathways even in dose-sparing protocols are effective treatments in renal fibrogenesis.

Revealing Differences Between Fibroblasts and Epithelial Cells

Transforming growth factor-β (TGF-β) stimulates different responses depending on cell type; for example, epithelial cells undergo growth arrest in response to TGF-β, whereas fibroblasts undergo morphological changes and proliferate. Wilkes et al. determined that p21-activated kinase 2 (PAK2) is one downstream signaling component that is selectively activated in fibroblasts. The kinase activity of PAK2 was stimulated by TGF-β in all cultured fibroblast cells tested but was not stimulated in any of the epithelial cells treated with TGF-β. Surprisingly, neither Smad2 nor Smad 3, the mediators of the canonical TGF-β pathway, were required for PAK2 activation in fibroblasts. Full stimulation of PAK2 by TGF-β required guanosine triphosphate (GTP) loading of the GTPases Cdc42 and Rac1, which interacted directly with PAK2 in TGF-β-stimulated fibroblasts. This interaction was not observed in epithelial cells. Finally, the TGF-β-stimulated morphological changes and proliferative response of fibroblasts were blocked by transfection of dominant-negative PAK2 or antisense treatments to decrease PAK2 protein. Thus, cell-specific responses can arise from a noncanonical TGF-β pathway that does not involve Smads and that regulates the cytoskeleton through the stimulation of PAK2. M. C. Wilkes, S. J. Murphy, N. Garamszegi, E. B. Leof, Cell-type-specific activation of PAK2 by transforming growth factor β independent of Smad2 and Smad3. Mol. Cell. Biol. 23 , 8878-8889 (2003). [Abstract] [Full Text]