p130Cas Is Required for Mammary Tumor Growth and Transforming Growth Factor-��-mediated Metastasis through Regulation of Smad2/3 Activity

During breast cancer progression, transforming growth factor-β (TGF-β) switches from a tumor suppressor to a pro-metastatic molecule. Several recent studies suggest that this conversion in TGF-β function depends upon fundamental changes in the TGF-β signaling system. We show here that these changes in TGF-β signaling are concomitant with aberrant expression of the focal adhesion protein, p130Cas. Indeed, elevating expression of either the full-length (FL) or just the carboxyl terminus (CT) of p130Cas in mammary epithelial cells (MECs) diminished the ability of TGF-β1 to activate Smad2/3, but increased its coupling to p38 MAPK. This shift in TGF-β signaling evoked (i) resistance to TGF-β-induced growth arrest, and (ii) acinar filling upon three-dimensional organotypic cultures of p130Cas-FL or -CT expressing MECs. Furthermore, rendering metastatic MECs deficient in p130Cas enhanced TGF-β-stimulated Smad2/3 activity, which restored TGF-β-induced growth inhibition both in vitro and in mammary tumors produced in mice. Additionally, whereas elevating TβR-II expression in metastatic MECs had no affect on their phosphorylation of Smad2/3, this event markedly enhanced their activation of p38 MAPK, leading to increased MEC invasion and metastasis. Importantly, depleting p130Cas expression in TβR-II-expressing metastatic MECs significantly increased their activation of Smad2/3, which (i) reestablished the physiologic balance between canonical and noncanonical TGF-β signaling, and (ii) reversed cellular invasion and early mammary tumor cell dissemination stimulated by TGF-β. Collectively, our findings identify p130Cas as a molecular rheostat that regulates the delicate balance between canonical and noncanonical TGF-β signaling, a balance that is critical to maintaining the tumor suppressor function of TGF-β during breast cancer progression.     

Pin1 Promotes Transforming Growth Factor-��-induced Migration and Invasion

Transforming growth factor-β (TGF-β) regulates a wide variety of biological activities. It induces potent growth-inhibitory responses in normal cells but promotes migration and invasion of cancer cells. Smads mediate the TGF-β responses. TGF-β binding to the cell surface receptors leads to the phosphorylation of Smad2/3 in their C terminus as well as in the proline-rich linker region. The serine/threonine phosphorylation sites in the linker region are followed by the proline residue. Pin1, a peptidyl-prolyl cis/trans isomerase, recognizes phosphorylated serine/threonine-proline motifs. Here we show that Smad2/3 interacts with Pin1 in a TGF-β-dependent manner. We further show that the phosphorylated threonine 179-proline motif in the Smad3 linker region is the major binding site for Pin1. Although epidermal growth factor also induces phosphorylation of threonine 179 and other residues in the Smad3 linker region the same as TGF-β, Pin1 is unable to bind to the epidermal growth factor-stimulated Smad3. Further analysis suggests that phosphorylation of Smad3 in the C terminus is necessary for the interaction with Pin1. Depletion of Pin1 by small hairpin RNA does not significantly affect TGF-β-induced growth-inhibitory responses and a number of TGF-β/Smad target genes analyzed. In contrast, knockdown of Pin1 in human PC3 prostate cancer cells strongly inhibited TGF-β-mediated migration and invasion. Accordingly, TGF-β induction of N-cadherin, which plays an important role in migration and invasion, is markedly reduced when Pin1 is depleted in PC3 cells. Because Pin1 is overexpressed in many cancers, our findings highlight the importance of Pin1 in TGF-β-induced migration and invasion of cancer cells.     

Hepatic deletion of Smad7 in mouse leads to spontaneous liver dysfunction and aggravates alcoholic liver injury

Background

TGF-β has been known to play an important role in various liver diseases including fibrosis and alcohol-induced fatty liver. Smad7 is an intracellular negative regulator of TGF-β signaling. It is currently unclear whether endogenous Smad7 has an effect on liver function and alcoholic liver damage.

Methodology/Principal Findings

We used Cre/loxP system by crossing Alb-Cre mice with Smad7^loxP/loxP mice to generate liver-specific deletion of Smad7 with loss of the indispensable MH2 domain. Alcoholic liver injury was achieved by feeding mice with a liquid diet containing 5% ethanol for 6 weeks, followed by a single dose of ethanol gavage. Deletion of Smad7 in the liver was associated with increased Smad2/3 phosphorylation in the liver or upon TGF-β treatment in primary hepatocytes. The majority of mice with liver specific deletion of Smad7 (Smad7^liver-KO) were viable and phenotypically normal, accompanied by only slight or no reduction of Smad7 expression in the liver. However, about 30% of Smad7^liver-KO mice with high efficiency of Smad7 deletion had spontaneous liver dysfunction, demonstrated as low body weight, overall deterioration, and increased serum levels of AST and ALT. Degeneration and elevated apoptosis of liver cells were observed with these mice. TGF-β-induced epithelial to mesenchymal transition (EMT) was accelerated in Smad7-deleted primary hepatocytes. In addition, alcohol-induced liver injury and steatosis were profoundly aggravated in Smad7 deficient mice, associated with upregulation of critical genes involved in lipogenesis and inflammation. Furthermore, alcohol-induced ADH1 expression was significantly abrogated by Smad7 deletion in hepatocytes.

Conclusion/Significance

In this study, we provided in vivo evidence revealing that endogenous Smad7 plays an important role in liver function and alcohol-induced liver injury.     

Endoglin in liver fibrosis

Liver fibrosis occurs in most types of chronic liver diseases and is characterized by excessive accumulation of extracellular matrix proteins, leading to disruption of tissue function and eventually organ failure. Transforming growth factor (TGF)-β represents an important pro-fibrogenic factor and aberrant TGF-β action has been implicated in many disease processes of the liver. Endoglin is a TGF-β co-receptor expressed mainly in endothelial cells that has been shown to differentially regulates TGF-β signal transduction by inhibiting ALK5-Smad2/3 signalling and augmenting ALK1-Smad1/5 signalling. Recent reports demonstrating upregulation of endoglin expression in pro-fibrogenic cell types such as scleroderma fibroblasts and hepatic stellate cells have led to studies exploring the potential involvement of this TGF-β co-receptor in organ fibrosis. A recent article by Meurer and colleagues now shows that endoglin expression is increased in transdifferentiating hepatic stellate cells in vitro and in two different models (carbon tetrachloride intoxication and bile duct ligation) of liver fibrosis in vivo. Moreover, they show that endoglin overexpression in hepatic stellate cells is associated with enhanced TGF-β-driven Smad1/5 phosphorylation and α-smooth muscle actin production without altering Smad2/3 signaling. These findings suggest that endoglin may play an important role in hepatic fibrosis by altering the balance of TGF-β signaling via the ALK1-Smad1/5 and ALK-Smad2/3 pathways and raise the possibility that targeting endoglin expression in transdifferentiating hepatic stellate cells may represent a novel therapeutic strategy for the treatment of liver fibrosis.     

Transforming Growth Factor-�� Promotes Recruitment of Bone Marrow Cells and Bone Marrow-derived Mesenchymal Stem Cells through Stimulation of MCP-1 Production in Vascular Smooth Muscle Cells

Bone marrow-derived progenitor cells have recently been shown to be involved in the development of intimal hyperplasia after vascular injury. Transforming growth factor-β (TGF-β) has profound stimulatory effects on intimal hyperplasia, but it is unknown whether these effects involve progenitor cell recruitment. In this study we found that although TGF-β had no direct effect on progenitor cell recruitment, conditioned media derived from vascular smooth muscle cells (VSMC) stimulated with TGF-β induced migration of both total bone marrow (BM) cells and BM-mesenchymal stem cells (MSC) and also induced MSC differentiation into smooth muscle like cells. Furthermore, overexpression of the signaling molecule Smad3 in VSMC via adenovirus-mediated gene transfer (AdSmad3) enhanced the TGF-β''s chemotactic effect. Microarray analysis of VSMC stimulated by TGF-β/AdSmad3 revealed monocyte chemoattractant protein-1 (MCP-1) as a likely factor responsible for progenitor cell recruitment. We then demonstrated that TGF-β through Smad3 phosphorylation induced a robust expression of MCP-1 in VSMC. Recombinant MCP-1 mimicked the stimulatory effect of conditioned media on BM and MSC migration. In the rat carotid injury model, Smad3 overexpression significantly increased MCP-1 expression after vascular injury, consistent with our in vitro results. Interestingly, TGF-β/Smad3-induced MCP-1 was completely blocked by both Ro-32-0432 and rotterlin, suggesting protein kinase C-δ (PKCδ) may play a role in TGF-β/Smad3-induced MCP-1 expression. In summary, our data demonstrate that TGF-β, through Smad3 and PKCδ, stimulates VSMC production of MCP-1, which is a chemoattractant for bone marrow-derived cells, specifically MSC. Manipulation of this signaling system may provide a novel approach to inhibition of intimal hyperplasia.     

Smad3 Prevents ��-Catenin Degradation and Facilitates ��-Catenin Nuclear Translocation in Chondrocytes

Our previous study demonstrated that transforming growth factor (TGF)-β activates β-catenin signaling through Smad3 interaction with β-catenin in chondrocytes. In the present studies, we further investigated the detailed molecular mechanism of the cross-talk between TGF-β/Smad3 and Wnt/β-catenin signaling pathways. We found that C-terminal Smad3 interacted with both the N-terminal region and the middle region of β-catenin protein in a TGF-β-dependent manner. Both Smad3 and Smad4 were required for the interaction with β-catenin and protected β-catenin from an ubiquitin-proteasome-dependent degradation. In addition, the formation of the Smad3-Smad4-β-catenin protein complex also mediated β-catenin nuclear translocation. This Smad3-mediated regulatory mechanism of β-catenin protein stability enhanced the activity of β-catenin to activate downstream target genes during chondrogenesis. Our findings demonstrate a novel mechanism between TGF-β and Wnt/β-catenin signaling pathways during chondrocyte development.     

Toxicarioside A Inhibits Tumor Growth and Angiogenesis: Involvement of TGF-β/Endoglin Signaling

Toxicarioside A is a cardenolide isolated mainly from plants and animals. Emerging evidence demonstrate that cardenolides not only have cardiac effects but also anticancer effects. In this study, we used in vivo models to investigate the antitumor activities of toxicarioside A and the potential mechanisms behind them. Murine colorectal carcinoma (CT26) and Lewis lung carcinoma (LL/2) models were established in syngeneic BALB/c and C57BL/6 mice, respectively. We found that the optimum effective dose of toxicarioside A treatment significantly suppressed tumor growth and angiogenesis in CT and LL/2 tumor models in vivo. Northern and Western blot analysis showed significant inhibition of endoglin expression in toxicarioside A-treated human umbilical vein endothelial cells (HUVECs) in vitro and tumor tissues in vivo. Toxicarioside A treatment significantly inhibited cell proliferation, migration and invasion, but did not cause significant cell apoptosis and affected other membrane protein (such as CD31 and MHC I) expression. In addition, TGF-β expression was also significantly inhibited in CT26 and LL/2 tumor cells treated with toxicarioside A. Western blot analysis indicated that Smad1 and phosphorylated Smad1 but not Smad2/3 and phosphorylated Smad2/3 were attenuated in HUVECs treated with toxicarioside A. Smad1 and Smad2/3 signaling remained unchanged in CT26 and LL/2 tumor cells treated with toxicarioside A. Endoglin knockout by small interfering RNA against endoglin induced alternations in Smad1 and Smad2/3 signaling in HUVECs. Our results indicate that toxicarioside A suppresses tumor growth through inhibition of endoglin-related tumor angiogenesis, which involves in the endoglin/TGF-β signal pathway.     

Smurf2 Participates in Human Trophoblast Cell Invasion by Inhibiting TGF-�� Type I Receptor

Successful embryo implantation depends on the ability of the trophoblast cells to invade the endometrium and the receptivity of the endometrium. Unlike tumor invasion, trophoblast invasion is spatio-temporaly restricted. Transforming growth factor (TGF)-β is a key inhibitory factor in the invasion of early trophoblast cells. Smad ubiquitination regulatory factor 2 (Smurf2), a HECT type E3 ubiquitin ligase, is an important regulator of the TGF-β signaling pathway, targeting TGF-β receptors and various Smads for proteasome-mediated degradation. In this context, we wished to determine whether Smurf2 has a physiological role during embryo implantation, especially in trophoblast invasion. We examined the spatio-temporal expression of Smurf2 in human placental villi and the function of Smurf2 in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, HTR-8/SVneo. Results from RT-PCR and immunohistochemical studies showed that expression of Smurf2 in placental villi was the highest during the first trimester and decreased as the pregnancy progressed. Overexpression of Smurf2 in HTR-8/SVneo cells reduced TGF-β type I receptor levels, and enhanced cell migration and invasion. Conversely, RNA interference–mediated downregulation of Smurf2 resulted in a significant increase in TGF-β type I receptor protein levels. However, the levels of Smad2, another potential target of Smurf2, remained unchanged. In conclusion, the present study suggests that Smurf2 promotes trophoblast cell migration and invasion, and this function may involve downregulation of TGF-β type I receptor. (J Histochem Cytochem 57:605–612, 2009)     

Endoglin promotes transforming growth factor beta-mediated Smad 1/5/8 signaling and inhibits endothelial cell migration through its association with GIPC

Transforming growth factor beta (TGF-beta) signals through two distinct pathways to regulate endothelial cell proliferation, migration, and angiogenesis, the ALK-1/Smad 1/5/8 and ALK-5/Smad2/3 pathways. Endoglin is a co-receptor predominantly expressed in endothelial cells that participates in TGFbeta-mediated signaling with ALK-1 and ALK-5 and regulates critical aspects of cellular and biological responses. The embryonic lethal phenotype of knock-out mice because of defects in angiogenesis and disease-causing mutations resulting in human vascular diseases both support essential roles for endoglin, ALK-1, and ALK-5 in the vasculature. However, the mechanism by which endoglin mediates TGF-beta signaling through ALK-1 and ALK-5 has remained elusive. Here we describe a novel interaction between endoglin and GIPC, a scaffolding protein known to regulate cell surface receptor expression and trafficking. Co-immunoprecipitation and immunofluorescence confocal studies both demonstrate a specific interaction between endoglin and GIPC in endothelial cells, mediated by a class I PDZ binding motif in the cytoplasmic domain of endoglin. Subcellular distribution studies demonstrate that endoglin recruits GIPC to the plasma membrane and co-localizes with GIPC in a TGFbeta-independent manner, with GIPC-promoting cell surface retention of endoglin. Endoglin specifically enhanced TGF-beta1-induced phosphorylation of Smad 1/5/8, increased a Smad 1/5/8 responsive promoter, and inhibited endothelial cell migration in a manner dependent on the ability of endoglin to interact with GIPC. These studies define a novel mechanism for the regulation of endoglin signaling and function in endothelial cells and demonstrate a new role for GIPC in TGF-beta signaling.     

Identification of retinoic acid in a high content screen for agents that overcome the anti-myogenic effect of TGF-beta-1

Background

Transforming growth factor beta 1 (TGF-β1) is an inhibitor of muscle cell differentiation that is associated with fibrosis, poor regeneration and poor function in some diseases of muscle. When neutralizing antibodies to TGF-β1 or the angiotensin II inhibitor losartan were used to reduce TGF-β1 signaling, muscle morphology and function were restored in mouse models of Marfan Syndrome and muscular dystrophy. The goal of our studies was to identify additional agents that overcome the anti-myogenic effect of TGF-β1.

Methodology/Principal Findings

A high-content cell-based assay was developed in a 96-well plate format that detects the expression of myosin heavy chain (MHC) in C2C12 cells. The assay was used to quantify the dose-dependent responses of C2C12 cell differentiation to TGF-β1 and to the TGF-β1 Type 1 receptor kinase inhibitor, SB431542. Thirteen agents previously described as promoting C2C12 differentiation in the absence of TGF-β1 were screened in the presence of TGF-β1. Only all-trans retinoic acid and 9-cis retinoic acid allowed a maximal level of C2C12 cell differentiation in the presence of TGF-β1; the angiotensin-converting enzyme inhibitor captopril and 10 nM estrogen provided partial rescue. Vitamin D was a potent inhibitor of retinoic acid-induced myogenesis in the presence of TGF-β1. TGF-β1 inhibits myoblast differentiation through activation of Smad3; however, retinoic acid did not inhibit TGF-β1-induced activation of a Smad3-dependent reporter gene in C2C12 cells.

Conclusions/Significance

Retinoic acid alleviated the anti-myogenic effect of TGF-β1 by a Smad3-independent mechanism. With regard to the goal of improving muscle regeneration and function in individuals with muscle disease, the identification of retinoic acid is intriguing in that some retinoids are already approved for human therapy. However, retinoids also have well-described adverse effects. The quantitative, high-content assay will be useful to screen for less-toxic retinoids or combinations of agents that promote myoblast differentiation in the presence of TGF-β1.     

Poly(ADP-ribose) polymerase 1 is indispensable for transforming growth factor-β Induced Smad3 activation in vascular smooth muscle cell

Background

Transforming growth factor type-β (TGF-β)/Smad pathway plays an essential role in vascular fibrosis. Reactive oxygen species (ROS) generation also mediates TGF-β signaling-induced vascular fibrosis, suggesting that some sort of interaction exists between Smad and redox pathways. However, the underlying molecular mechanism is largely unknown. This study aims to investigate the influence of poly(ADP-ribose) polymerase 1 (PARP1), a downstream effector of ROS, on TGF-β signaling transduction through Smad3 pathway in rat vascular smooth muscle cells (VSMCs).

Methods and Results

TGF-β1 treatment promoted PARP1 activation through induction of ROS generation in rat VSMCs. TGF-β1-induced phosphorylation and nuclear accumulation of Smad3 was prevented by treatment of cells with PARP inhibitor, 3-aminobenzamide (3AB) or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)-2-(N,N-dimethylamino)acetami (PJ34), or PARP1 siRNA. TGF-β1 treatment promoted poly(ADP-ribosy)lation of Smad3 via activation of PARP1 in the nucleus. Poly(ADP-ribosy)lation enhanced Smad-Smad binding element (SBE) complex formation in nuclear extracts and increased DNA binding activity of Smad3. Pretreatment with 3AB, PJ34, or PARP1 siRNA prevented TGF-β1-induced Smad3 transactivation and expression of Smad3 target genes, including collagen Iα1, collagen IIIα1 and tissue inhibitor of metalloproteinase 1, in rat VSMCs.

Conclusions

PARP1 is indispensable for TGF-β1 induced Smad3 activation in rat VSMCs. Targeting PARP1 may be a promising therapeutic approach against vascular diseases induced by dysregulation of TGF-β/Smad3 pathway.     

Src-mediated Post-translational Regulation of Endoglin Stability and Function Is Critical for Angiogenesis

Endoglin is a transforming growth factor β (TGF-β) co-receptor essential for angiogenesis and tumor vascularization. Endoglin modulates the crucial balance between pro- and anti-angiogenic signaling by activin receptor-like kinase (ALK) 1, 5, and TGF-β type II (TβRII) receptors. Despite its established role in physiology and disease, the mechanism of endoglin down-regulation remains unknown. Here we report that the conserved juxtamembrane cytoplasmic tyrosine motif (⁶¹²YIY⁶¹⁴) is a critical determinant of angiogenesis. Src directly phosphorylates this motif to induce endoglin internalization and degradation via the lysosome. We identified epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) as Src-activators that induce endoglin turnover following ⁶¹²YIY⁶¹⁴ phosphorylation. Interestingly, Src phosphorylation of endoglin-⁶¹²YIY⁶¹⁴ was also an important process for receptor down-regulation by TRACON105 (TRC105), an endoglin-targeting antibody currently in clinical trials. The regulation of ⁶¹²YIY⁶¹⁴ phosphorylation was critical for angiogenesis, as both the phosphomimetic and unphosphorylatable mutants impaired endothelial functions including proliferation, migration, and capillary tube formation. Collectively, these findings establish Src and pro-angiogenic mitogens as critical mediators of endoglin stability and function.     

Regulation of TGF-β receptor hetero-oligomerization and signaling by endoglin

Complex formation among transforming growth factor-β (TGF-β) receptors and its modulation by coreceptors represent an important level of regulation for TGF-β signaling. Oligomerization of ALK5 and the type II TGF-β receptor (TβRII) has been thoroughly investigated, both in vitro and in intact cells. However, such studies, especially in live cells, are missing for the endothelial cell coreceptor endoglin and for the ALK1 type I receptor, which enables endothelial cells to respond to TGF-β by activation of both Smad2/3 and Smad1/5/8. Here we combined immunoglobulin G–mediated immobilization of one cell-surface receptor with lateral mobility studies of a coexpressed receptor by fluorescence recovery after photobleaching (FRAP) to demonstrate that endoglin forms stable homodimers that function as a scaffold for binding TβRII, ALK5, and ALK1. ALK1 and ALK5 bind to endoglin with differential dependence on TβRII, which plays a major role in recruiting ALK5 to the complex. Signaling data indicate a role for the quaternary receptor complex in regulating the balance between TGF-β signaling to Smad1/5/8 and to Smad2/3.