Smad3-Smad4 and AP-1 complexes synergize in transcriptional activation of the c-Jun promoter by transforming growth factor beta. (1/1304)

Transcriptional regulation by transforming growth factor beta (TGF-beta) is a complex process which is likely to involve cross talk between different DNA responsive elements and transcription factors to achieve maximal promoter activation and specificity. Here, we describe a concurrent requirement for two discrete responsive elements in the regulation of the c-Jun promoter, one a binding site for a Smad3-Smad4 complex and the other an AP-1 binding site. The two elements are located 120 bp apart in the proximal c-Jun promoter, and each was able to independently bind its corresponding transcription factor complex. The effects of independently mutating each of these elements were nonadditive; disruption of either sequence resulted in complete or severe reductions in TGF-beta responsiveness. This simultaneous requirement for two distinct and independent DNA binding elements suggests that Smad and AP-1 complexes function synergistically to mediate TGF-beta-induced transcriptional activation of the c-Jun promoter.  (+info)

Transforming growth factor-beta induces formation of a dithiothreitol-resistant type I/Type II receptor complex in live cells. (2/1304)

Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, the type I (TbetaRI) and type II (TbetaRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TbetaRI and TbetaRII. Velocity centrifugation of endogenous receptors suggests that ligand-bound TbetaRI and TbetaRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope-tagged receptors provides the first evidence in live cells that TbetaRI. TbetaRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-beta binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-beta to TbetaRI, does not prevent formation of the TbetaRI.TbetaRII complex, but increases its sensitivity to detergent and prevents TGF-beta-activated TbetaRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TbetaRI. TbetaRII complex, disrupted by dithiothreitol, or direct binding of TGF-beta to TbetaRI is required for signaling.  (+info)

Convergence of transforming growth factor-beta and vitamin D signaling pathways on SMAD transcriptional coactivators. (3/1304)

Cell proliferation and differentiation are regulated by growth regulatory factors such as transforming growth factor-beta (TGF-beta) and the liphophilic hormone vitamin D. TGF-beta causes activation of SMAD proteins acting as coactivators or transcription factors in the nucleus. Vitamin D controls transcription of target genes through the vitamin D receptor (VDR). Smad3, one of the SMAD proteins downstream in the TGF-beta signaling pathway, was found in mammalian cells to act as a coactivator specific for ligand-induced transactivation of VDR by forming a complex with a member of the steroid receptor coactivator-1 protein family in the nucleus. Thus, Smad3 may mediate cross-talk between vitamin D and TGF-beta signaling pathways.  (+info)

Targeted disruption of SMAD3 results in impaired mucosal immunity and diminished T cell responsiveness to TGF-beta. (4/1304)

SMAD3 is one of the intracellular mediators that transduces signals from transforming growth factor-beta (TGF-beta) and activin receptors. We show that SMAD3 mutant mice generated by gene targeting die between 1 and 8 months due to a primary defect in immune function. Symptomatic mice exhibit thymic involution, enlarged lymph nodes, and formation of bacterial abscesses adjacent to mucosal surfaces. Mutant T cells exhibit an activated phenotype in vivo, and are not inhibited by TGF-beta1 in vitro. Mutant neutrophils are also impaired in their chemotactic response toward TGF-beta. Chronic intestinal inflammation is infrequently associated with colonic adenocarcinoma in mice older than 6 months of age. These data suggest that SMAD3 has an important role in TGF-beta-mediated regulation of T cell activation and mucosal immunity, and that the loss of these functions is responsible for chronic infection and the lethality of Smad3-null mice.  (+info)

No mutations of the Smad2 gene in human sporadic gastric carcinomas. (5/1304)

BACKGROUND: The majority of cancer cells escape from TGF-beta-mediated growth control. However, the mechanism of resistance to the growth inhibitory effects by TGF-beta is not clear. TGF-beta signaling is initiated when the type I receptor phosphorylates the SMAD proteins, Smad2 and Smad3. Recently, mutations of Smad2 have been detected in human colon and lung cancers. Mutation of coding sequences of Smad2 in gastric carcinomas has not yet been elucidated adequately. METHODS: PCR-SSCP analysis of the entire coding region of Smad2 in 35 human sporadic gastric cancers and eight gastric cancer cell lines was performed using 11 sets of intron-based primers. RESULTS: No mutations of Smad2 were detected in any tumor or cell line. CONCLUSIONS: The results suggest that mutation of Smad2 does not play a key role in human stomach carcinogenesis.  (+info)

Targeted disruption of Smad3 reveals an essential role in transforming growth factor beta-mediated signal transduction. (6/1304)

The Smads are a family of nine related proteins which function as signaling intermediates for the transforming growth factor beta (TGF-beta) superfamily of ligands. To discern the in vivo functions of one of these Smads, Smad3, we generated mice harboring a targeted disruption of this gene. Smad3 null mice, although smaller than wild-type littermates, are viable, survive to adulthood, and exhibit an early phenotype of forelimb malformation. To study the cellular functions of Smad3, we generated Smad3 null mouse embryonic fibroblasts (MEFs) and dermal fibroblasts. We demonstrate that null MEFs have lost the ability to form Smad-containing DNA binding complexes and are unable to induce transcription from the TGF-beta-responsive promoter construct, p3TP-lux. Using the primary dermal fibroblasts, we also demonstrate that Smad3 is integral for induction of endogenous plasminogen activator inhibitor 1. We subsequently demonstrate that Smad3 null MEFs are partially resistant to TGF-beta's antiproliferative effect, thus firmly establishing a role for Smad3 in TGF-beta-mediated growth inhibition. We next examined cells in which Smad3 is most highly expressed, specifically cells of immune origin. Although no specific developmental defect was detected in the immune system of the Smad3 null mice, a functional defect was observed in the ability of TGF-beta to inhibit the proliferation of splenocytes activated by specific stimuli. In addition, primary splenocytes display defects in TGF-beta-mediated repression of cytokine production. These data, taken together, establish a role for Smad3 in mediating the antiproliferative effects of TGF-beta and implicate Smad3 as a potential effector for TGF-beta in modulating immune system function.  (+info)

ATF-2 is a common nuclear target of Smad and TAK1 pathways in transforming growth factor-beta signaling. (7/1304)

Upon transforming growth factor-beta (TGF-beta) binding to its cognate receptor, Smad3 and Smad4 form heterodimers and transduce the TGF-beta signal to the nucleus. In addition to the Smad pathway, another pathway involving a member of the mitogen-activated protein kinase kinase kinase family of kinases, TGF-beta-activated kinase-1 (TAK1), is required for TGF-beta signaling. However, it is unknown how these pathways function together to synergistically amplify TGF-beta signaling. Here we report that the transcription factor ATF-2 (also called CRE-BP1) is bound by a hetero-oligomer of Smad3 and Smad4 upon TGF-beta stimulation. ATF-2 is one member of the ATF/CREB family that binds to the cAMP response element, and its activity is enhanced after phosphorylation by stress-activated protein kinases such as c-Jun N-terminal kinase and p38. The binding between ATF-2 and Smad3/4 is mediated via the MH1 region of the Smad proteins and the basic leucine zipper region of ATF-2. TGF-beta signaling also induces the phosphorylation of ATF-2 via TAK1 and p38. Both of these actions are shown to be responsible for the synergistic stimulation of ATF-2 trans-activating capacity. These results indicate that ATF-2 plays a central role in TGF-beta signaling by acting as a common nuclear target of both Smad and TAK1 pathways.  (+info)

Cooperation between SMAD and NF-kappaB in growth factor regulated type VII collagen gene expression. (8/1304)

We have previously demonstrated that transforming growth factor-beta (TGF-beta) and pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, synergistically enhance the expression of type VII collagen gene (COL7A1) in human dermal fibroblasts in culture (Mauviel et al., 1994). Recently, we identified a SMAD-containing complex, rapidly induced by TGF-beta and binding the region [-496/-444] of the COL7A1 promoter, responsible for COL7A1 gene transactivation (Vindevoghel et al., 1998a). In this report, we demonstrate that TGF-beta and TNF-alpha response elements are distinct entities within the COL7A1 promoter. In particular, we demonstrate that the TNF-alpha effect is mediated by NF-kappaB1/RelA (p50/p65) and RelA/RelA (p65/p65) NF-kappaB complexes binding the TNF-alpha response element (TaRE) located in the region [-252/-230], with RelA acting as the transcriptional activator. Finally, we provide definitive evidence for the role of both TGF-beta and TNF-alpha response elements as enhancer sequences, functioning in the context of a heterologous promoter in an additive manner in response to TGF-beta and TNF-alpha. This study provides the first identification of a functional interaction between the two immediate-early transcription factors, SMAD and NF-kappaB, to activate the expression of an extracellular matrix-related gene, COL7A1.  (+info)