TGF-ß induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways.

Transforming Growth Factor-ß (TGF-ß) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-ß is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-ß regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-ß1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-ß1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-ß1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-ß1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-ß action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention.

Nodal promotes glioblastoma cell growth.

Nodal is a member of the transforming growth factor-ß (TGF-ß) superfamily that plays critical roles during embryogenesis. Recent studies in ovarian, breast, prostate, and skin cancer cells suggest that Nodal also regulates cell proliferation, apoptosis, and invasion in cancer cells. However, it appears to exert both tumor-suppressing and tumor-promoting effects, depending on the cell type. To further understand the role of Nodal in tumorigenesis, we examined the effect of Nodal in glioblastoma cell growth and spheroid formation using U87 cell line. Treatment of U87 with recombinant Nodal significantly increased U87 cell growth. In U87 cells stably transfected with the plasmid encoding Nodal, Smad2 phosphorylation was strongly induced and cell growth was significantly enhanced. Overexpression of Nodal also resulted in tight spheroid formation. On the other hand, the cells stably transfected with Nodal siRNA formed loose spheroids. Nodal is known to signal through activin receptor-like kinase 4 (ALK4) and ALK7 and the Smad2/3 pathway. To determine which receptor and Smad mediate the growth promoting effect of Nodal, we transfected siRNAs targeting ALK4, ALK7, Smad2, or Smad3 into Nodal-overexpressing cells and observed that cell growth was significantly inhibited by ALK4, ALK7, and Smad3 siRNAs. Taken together, these findings suggest that Nodal may have tumor-promoting effects on glioblastoma cells and these effects are mediated by ALK4, ALK7, and Smad3.

Coupling of dephosphorylation and nuclear export of Smads in TGF-beta signaling.

In eukaryotes, regulation of signaling mediators/effectors in the nucleus is one of the principal mechanisms that govern duration and strength of signaling. Smads are a family of structurally related intracellular proteins that serve as signaling effectors for transforming growth factor beta (TGF-beta) and TGF-beta-related proteins. Accumulating evidence demonstrates that Smads possess intrinsic nucleocytoplasmic shuttling capacity, which enables them to transmit TGF-beta signals from cell membrane to nucleus. We recently identified two important steps in the termination of nuclear Smad signaling. The first step is initiated by a serine/threonine phosphatase PPM1A that dephosphorylates Smad2/3 in the nucleus, thereby shutting down signaling capacity of phosphorylated Smad2/3. The second step involves nuclear export of dephosphorylated Smad2/3 with the aid of nuclear protein RanBP3 to terminate Smad signaling. This chapter introduces methods for examining nuclear export of Smad2/3 in TGF-beta signaling.

TGFBR2 mutations alter smooth muscle cell phenotype and predispose to thoracic aortic aneurysms and dissections.

AIMS: Transforming growth factor-ß (TGF-ß) signaling is critical for the differentiation of smooth muscle cells (SMCs) into quiescent cells expressing a full repertoire of contractile proteins. Heterozygous mutations in TGF-ß receptor type II (TGFBR2) disrupt TGF-ß signaling and lead to genetic conditions that predispose to thoracic aortic aneurysms and dissections (TAADs). The aim of this study is to determine the molecular mechanism by which TGFBR2 mutations cause TAADs. METHODS AND RESULTS: Using aortic SMCs explanted from patients with TGFBR2 mutations, we show decreased expression of SMC contractile proteins compared with controls. Exposure to TGF-ß1 fails to increase expression of contractile genes in mutant SMCs, whereas control cells further increase expression of these genes. Analysis of fixed and frozen aortas from patients with TGFBR2 mutations confirms decreased in vivo expression of contractile proteins relative to unaffected aortas. Fibroblasts explanted from patients with TGFBR2 mutations fail to transform into mature myofibroblasts with TGF-ß1 stimulation as assessed by expression of contractile proteins. CONCLUSIONS: These data support the conclusion that heterozygous TGFBR2 mutations lead to decreased expression of SMC contractile protein in both SMCs and myofibroblasts. The failure of TGFBR2-mutant SMCs to fully express SMC contractile proteins predicts defective contractile function in these cells and aligns with a hypothesis that defective SMC contractile function contributes to the pathogenesis of TAAD.

Smad3 mediates immediate early induction of Id1 by TGF-beta.

Id1 is a member of the inhibitor of differentiation (Id) protein family that regulates a wide range of cell functions. Previous studies have shown that expression of the Id1 gene is down-regulated by TGF-beta in epithelial cells, whereas it is up-regulated by BMP in a variety of cell types. During our study of the biological function of TGF-beta1, we found that Id1 can be strongly up-regulated by TGF-beta1 in the human mammary gland epithelial cell line MCF10A. Quantitative real-time RT-PCR has revealed as high as 7.5-fold induction of Id1 mRNA by TGF-beta1 in MCF10A cells after 1 h of TGF-beta1 stimulation, and this induction does not require de novo protein synthesis. Using Smad knockdown and knockout approaches, we have identified Smad3 as the responsible R-Smad for mediating transcriptional activation of the Id1 gene. Chromatin immunoprecipitation assay confirms that Smad3 and Smad4 bind to the upstream region of the Id1 gene. Our results demonstrate that Smad3, but not Smad2, mediates TGF-beta1-dependent early transcriptional induction of Id1.

Phosphatase PPM1A regulates phosphorylation of Thr-186 in the Cdk9 T-loop.

Cdk9 is the catalytic subunit of a general RNA polymerase II elongation factor known as positive transcription elongation factor b (P-TEFb). The kinase function of P-TEFb requires phosphorylation of Thr-186 in the T-loop of Cdk9 to allow substrates to access the catalytic core of the enzyme. To identify human phosphatases that dephosphorylate the T-loop of Cdk9, we used a Thr-186-phosphospecific antiserum to screen a phosphatase expression library. Overexpression of PPM1A and the related PPM1B greatly reduced Cdk9 T-loop phosphorylation in vivo. PPM1A and Cdk9 appear to associate in vivo as the proteins could be co-immunoprecipitated. The short hairpin RNA depletion of PPM1A resulted in an increase in Cdk9 T-loop phosphorylation. In phosphatase reactions in vitro, purified PPM1A could dephosphorylate Thr-186 both with and without the association of 7SK RNA, a small nuclear RNA that is bound to approximately 50% of total cellular P-TEFb. PPM1B only efficiently dephosphorylated Cdk9 Thr-186 in vitro when 7SK RNA was depleted from P-TEFb. Taken together, our data indicate that PPM1A and to some extent PPM1B are important negative regulators of P-TEFb function.

Essential phosphatases and a phospho-degron are critical for regulation of SRC-3/AIB1 coactivator function and turnover.

SRC-3/AIB1 is a master growth coactivator and oncogene, and phosphorylation activates it into a powerful coregulator. Dephosphorylation is a potential regulatory mechanism for SRC-3 function, but the identity of such phosphatases remains unexplored. Herein, we report that, using functional genomic screening of human Ser/Thr phosphatases targeting SRC-3's known phosphorylation sites, the phosphatases PDXP, PP1, and PP2A were identified to be key negative regulators of SRC-3 transcriptional coregulatory activity in steroid receptor signalings. PDXP and PP2A dephosphorylate SRC-3 and inhibit its ligand-dependent association with estrogen receptor. PP1 stabilizes SRC-3 protein by blocking its proteasome-dependent turnover through dephosphorylation of two previously unidentified phosphorylation sites (Ser101 and S102) required for activity. These two sites are located within a degron of SRC-3 and are primary determinants of SRC-3 turnover. Moreover, PP1 regulates the oncogenic cell proliferation and invasion functions of SRC-3 in breast cancer cells.

Smad7-induced beta-catenin degradation alters epidermal appendage development.

To assess whether Smad signaling affects skin development, we generated transgenic mice in which a Smad antagonist, Smad7, was induced in keratinocytes, including epidermal stem cells. Smad7 transgene induction perturbed hair follicle morphogenesis and differentiation, but accelerated sebaceous gland morphogenesis. Further analysis revealed that independent of its role in anti-Smad signaling, Smad7 bound beta-catenin and induced beta-catenin degradation by recruiting an E3 ligase, Smurf2, to the Smad7/beta-catenin complex. Consequently, Wnt/beta-catenin signaling was suppressed in Smad7 transgenic hair follicles. Coexpression of the Smurf2 and Smad7 transgenes exacerbated Smad7-induced abnormalities in hair follicles and sebaceous glands. Conversely, when endogenous Smad7 was knocked down, keratinocytes exhibited increased beta-catenin protein and enhanced Wnt signaling. Our data reveal a mechanism for Smad7 in antagonizing Wnt/beta-catenin signaling, thereby shifting the skin differentiation program from forming hair follicles to sebaceous glands.

Protein serine/threonine phosphatase PPM1A dephosphorylates Smad1 in the bone morphogenetic protein signaling pathway.

Bone morphogenetic proteins (BMPs) are secreted polypeptides belonging to the transforming growth factor-beta (TGF-beta) superfamily that activates a broad range of biological responses in the metazoan organism. The BMP-initiated signaling pathway is under tight control by processes including regulation of the ligands, the receptors, and the key downstream intracellular effector Smads. A critical point of control in BMP signaling is the phosphorylation of Smad1, Smad5, and Smad8 in their C-terminal SXS motif. Although such phosphorylation, which is mediated by the type I BMP receptor kinases in response to BMP stimulation, is well characterized, biochemical mechanisms underlying Smad dephosphorylation remain to be elucidated. In this study, we have found that PPM1A, a metal ion-dependent protein serine/threonine phosphatase, physically interacts with and dephosphorylates Smad1 both in vitro and in vivo. Functionally, overexpression of PPM1A abolishes BMP-induced transcriptional responses, whereas RNA interference-mediated knockdown of PPM1A enhances BMP signaling. Collectively, our study suggests that PPM1A plays an important role in controlling BMP signaling through catalyzing Smad dephosphorylation.

PPM1A functions as a Smad phosphatase to terminate TGFbeta signaling.

TGFbeta signaling controls diverse normal developmental processes and pathogenesis of diseases including cancer and autoimmune and fibrotic diseases. TGFbeta responses are generally mediated through transcriptional functions of Smads. A key step in TGFbeta signaling is ligand-induced phosphorylation of receptor-activated Smads (R-Smads) catalyzed by the TGFbeta type I receptor kinase. However, the potential of Smad dephosphorylation as a regulatory mechanism of TGFbeta signaling and the identity of Smad-specific phosphatases remain elusive. Using a functional genomic approach, we have identified PPM1A/PP2Calpha as a bona fide Smad phosphatase. PPM1A dephosphorylates and promotes nuclear export of TGFbeta-activated Smad2/3. Ectopic expression of PPM1A abolishes TGFbeta-induced antiproliferative and transcriptional responses, whereas depletion of PPM1A enhances TGFbeta signaling in mammalian cells. Smad-antagonizing activity of PPM1A is also observed during Nodal-dependent early embryogenesis in zebrafish. This work demonstrates that PPM1A/PP2Calpha, through dephosphorylation of Smad2/3, plays a critical role in terminating TGFbeta signaling.

Repression of bone morphogenetic protein and activin-inducible transcription by Evi-1.

Smads, key effectors of transforming growth factor (TGF)-beta, activin, and bone morphogenetic protein (BMP) signaling, regulate gene expression and interact with coactivators and corepressors that modulate Smad activity. The corepressor Evi-1 exerts its oncogenic effects by repressing TGF-beta/Smad3-mediated transcription, thereby blocking TGF-beta-induced growth arrest. Because Evi-1 interacts with the highly conserved MH2 domain of Smad3, we investigated the physical and functional interaction of Evi-1 with Smad1 and Smad2, downstream targets of BMP and activin signaling, respectively. Evi-1 interacted with and repressed the receptor-activated transcription through Smad1 and Smad2, similarly to Smad3. In addition, Evi-1 repressed BMP/Smad1- and activin/Smad2-mediated induction of endogenous Xenopus gene expression, suggesting a role of repression of BMP and activin signals by Evi-1 in vertebrate embryogenesis. Evi-1 also repressed the induction of endogenous Smad7 expression by TGF-beta family ligands. In the course of these studies, we observed Evi-1 repression of Smad transactivation even when Smad binding to DNA was kept constant. We therefore explored the mechanism of Evi-1 repression of TGF-beta family-inducible transcription. Evi-1 repression did not result from displacement of Smad binding to DNA or to CREB-binding protein but from the recruitment of Evi-1 by Smad3 and CREB-binding protein to DNA. Following TGF-beta stimulation, Evi-1 and the associated corepressor CtBP were recruited to the endogenous Smad7 promoter. Evi-1 recruitment to the promoter decreased TGF-beta-induced histone acetylation, coincident with its repression of Smad7 gene expression. In this way, Evi-1 acts as a general Smad corepressor to inhibit TGF-beta-, activin-, and BMP-inducible transcription.

PG-M/versican binds to P-selectin glycoprotein ligand-1 and mediates leukocyte aggregation.

P-selectin glycoprotein ligand-1 (PSGL-1), a glycoprotein expressed on the cell surface of leukocytes, binds to selectins and mediates leukocyte rolling on the vascular endothelium. Here we report that PSGL-1 binds to the C-terminal (G3 domain) of the extracellular proteoglycan PG-M/versican. Cells transfected with PSGL-1 or a shorter form containing the binding site, or cells expressing endogenous PSGL-1 aggregate in the presence of versican or G3 product. The aggregation appears to be induced by G3 multimers that bind to PSGL-1 and form a network. Endogenous versican and/or G3-containing fragments also bind to PSGL-1 in human plasma. Removal of the endogenous G3-containing fragments reduces the effect of plasma on leukocyte aggregation. Finally, the roles of G3-containing fragments in leukocyte aggregation were confirmed in a mouse model. Taken together, our results strongly support a physiologically relevant role for PSGL-1/versican binding and may have implications in the immunoresponse.

Ubiquitination and proteolysis of cancer-derived Smad4 mutants by SCFSkp2.

Smad4/DPC4, a common signal transducer in transforming growth factor beta (TGF-beta) signaling, is frequently inactivated in human cancer. Although the ubiquitin-proteasome pathway has been established as one mechanism of inactivating Smad4 in cancer, the specific ubiquitin E3 ligase for ubiquitination-mediated proteolysis of Smad4 cancer mutants remains unclear. In this report, we identified the SCFSkp2 complex as candidate Smad4-interacting proteins in an antibody array-based screen and further elucidated the functions of SCFSkp2 in mediating the metabolic instability of cancer-derived Smad4 mutants. We found that Skp2, the F-box component of SCFSkp2, physically interacted with Smad4 at the physiological levels. Several cancer-derived unstable mutants exhibited significantly increased binding to Skp2, which led to their increased ubiquitination and accelerated proteolysis. These results suggest an important role for the SCFSkp2 complex in switching cancer mutants of Smad4 to undergo polyubiquitination-dependent degradation.

Smad6 recruits transcription corepressor CtBP to repress bone morphogenetic protein-induced transcription.

Smad6 and Smad7 are inhibitory Smads induced by transforming growth factor beta-Smad signal transduction pathways in a negative-feedback mechanism. Previously it has been thought that inhibitory Smads bind to the type I receptor and block the phosphorylation of receptor-activated Smads, thereby inhibiting the initiation of Smad signaling. Conversely, few studies have suggested the possible nuclear functions of inhibitory Smads. Here, we present compelling evidence demonstrating that Smad6 repressed bone morphogenetic protein-induced Id1 transcription through recruiting transcriptional corepressor C-terminal binding protein (CtBP). A consensus CtBP-binding motif, PLDLS, was identified in the linker region of Smad6. Our findings show that mutation in the motif abolished the Smad6 binding to CtBP and subsequently its repressor activity of transcription. We conclude that the nuclear functions and physical interaction of Smad6 and CtBP provide a novel mechanism for the transcriptional regulation by inhibitory Smads.

SUMO-1/Ubc9 promotes nuclear accumulation and metabolic stability of tumor suppressor Smad4.

Tumor suppressor Smad4/DPC4 is a central intracellular signal transducer for transforming growth factor-beta (TGF-beta) signaling. We recently reported that transcriptional potential of Smad4 was regulated by SUMOylation in transfected HeLa cells (1), but the precise mechanism and function of Smad4 SUMOylation in TGF-beta signaling remain to be elucidated. Here, we describe the regulation of TGF-beta signaling by SUMOylation through the control of Smad4 metabolic stability and subcellular localization. We found that SUMO-1 overexpression strongly increases Smad4 levels, while inhibition of SUMOylation by small interfering RNA (siRNA)-mediated knockdown of the E2 enzyme Ubc9 reduces endogenous Smad4 levels. Concomitantly, SUMO-1 overexpression enhances and Ubc9 knockdown reduces levels of intranuclear Smad4, growth inhibitory response, as well as transcriptional responses to TGF-beta. Comparison of wild type and mutant forms of Smad4 for SUMOylation, ubiquitination, and half-life allows the conclusion that SUMO-1 modification serves to protect Smad4 from ubiquitin-dependent degradation and consequently enhances the growth inhibitory and transcriptional responses of Smad4.

dSmurf selectively degrades decapentaplegic-activated MAD, and its overexpression disrupts imaginal disc development.

MAD plays an important role in decapentaplegic (DPP) signaling throughout Drosophila development. Despite a recent study describing the restriction of DPP signaling via putative ubiquitin E3 ligase dSmurf (1), the molecular mechanisms of how dSmurf affects DPP signaling remain unexplored. Toward this goal we demonstrated the degradation of phosphorylated MAD by dSmurf. dSmurf selectively interacted with MAD, but not Medea and Dad, and the MAD-dSmurf interaction was induced by constitutively active DPP type I receptor thickveins. Wild type dSmurf, but not its C1029A mutant, mediated ubiquitination-dependent degradation of MAD. Silencing of dSmurf using RNA interference stabilized MAD protein in Drosophila S2 cells. Targeted expression of dSmurf in various tissues abolished phosphorylated MAD and disrupted patterning and growth. In contrast, similar overexpression of inactive dSmurf(C1029A) showed no significant effects on development. We conclude that dSmurf specifically targets phosphorylated MAD to proteasome-dependent degradation and regulates DPP signaling during development.