Dataset Information

Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-β signaling.

ABSTRACT: TGF-β receptors phosphorylate SMAD2 and SMAD3 transcription factors, which then form heterotrimeric complexes with SMAD4 and cooperate with context-specific transcription factors to activate target genes. Here we provide biochemical and structural evidence showing that binding of SMAD2 to DNA depends on the conformation of the E3 insert, a structural element unique to SMAD2 and previously thought to render SMAD2 unable to bind DNA. Based on this finding, we further delineate TGF-β signal transduction by defining distinct roles for SMAD2 and SMAD3 with the forkhead pioneer factor FOXH1 as a partner in the regulation of differentiation genes in mouse mesendoderm precursors. FOXH1 is prebound to target sites in these loci and recruits SMAD3 independently of TGF-β signals, whereas SMAD2 remains predominantly cytoplasmic in the basal state and set to bind SMAD4 and join SMAD3:FOXH1 at target promoters in response to Nodal TGF-β signals. The results support a model in which signal-independent binding of SMAD3 and FOXH1 prime mesendoderm differentiation gene promoters for activation, and signal-driven SMAD2:SMAD4 binds to promoters that are preloaded with SMAD3:FOXH1 to activate transcription.

SUBMITTER: Aragon E

PROVIDER: S-EPMC6824466 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-β signaling.

Aragón Eric E Wang Qiong Q Zou Yilong Y Morgani Sophie M SM Ruiz Lidia L Kaczmarska Zuzanna Z Su Jie J Torner Carles C Tian Lin L Hu Jing J Shu Weiping W Agrawal Saloni S Gomes Tiago T Márquez José A JA Hadjantonakis Anna-Katerina AK Macias Maria J MJ Massagué Joan J

Genes & development 20191003 21-22

TGF-β receptors phosphorylate SMAD2 and SMAD3 transcription factors, which then form heterotrimeric complexes with SMAD4 and cooperate with context-specific transcription factors to activate target genes. Here we provide biochemical and structural evidence showing that binding of SMAD2 to DNA depends on the conformation of the E3 insert, a structural element unique to SMAD2 and previously thought to render SMAD2 unable to bind DNA. Based on this finding, we further delineate TGF-β signal transdu ...[more]

PMID: 31582430

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Project description:TGF-βs regulate fibroblast responses, by activating Smad2 or Smad3 signaling, or via Smad-independent pathways. We have previously demonstrated that myofibroblast-specific Smad3 is critically implicated in repair of the infarcted heart. However, the role of fibroblast Smad2 in myocardial infarction remains unknown. This study investigates the role of myofibroblast-specific Smad2 signaling in myocardial infarction, and explores the mechanisms responsible for the distinct effects of Smad2 and Smad3. In a mouse model of non-reperfused myocardial infarction, Smad2 activation in infarct myofibroblasts peaked 7 days after coronary occlusion. In vitro, TGF-β1, -β2 and -β3, but not angiotensin 2 and bone morphogenetic proteins-2, -4 and -7, activated fibroblast Smad2. Myofibroblast-specific Smad2 and Smad3 knockout mice (FS2KO, FS3KO) and corresponding control littermates underwent non-reperfused infarction. In contrast to the increase in rupture rates and adverse remodeling in FS3KO mice, FS2KO animals had mortality comparable to Smad2 fl/fl controls, and exhibited a modest but transient improvement in dysfunction after 7 days of coronary occlusion. At the 28 day timepoint, FS2KO and Smad2 fl/fl mice had comparable adverse remodeling. Although both FS3KO and FS2KO animals had increased myofibroblast density in the infarct, only FS3KO mice exhibited impaired scar organization, associated with perturbed alignment of infarct myofibroblasts. In vitro, Smad3 but not Smad2 knockdown downmodulated fibroblast α2 and α5 integrin expression. Moreover, Smad3 knockdown reduced expression of the GTPase RhoA, whereas Smad2 knockdown markedly increased fibroblast RhoA levels. Smad3-dependent integrin expression may be important for fibroblast activation, whereas RhoA may transduce planar cell polarity pathway signals, essential for fibroblast alignment. Myofibroblast-specific Smad3, but not Smad2 is required for formation of aligned myofibroblast arrays in the infarct. The distinct in vivo effects of myofibroblast Smad2 and Smad3 may involve Smad3-dependent integrin synthesis, and contrasting effects of Smad2 and Smad3 on RhoA expression.

Project description:BackgroundProgression of pancreatic ductal adenocarcinoma (PDAC) is largely the result of genetic and/or epigenetic alterations in the transforming growth factor-beta (TGF-β)/Smad signalling pathway, eventually resulting in loss of TGF-β-mediated growth arrest and an increase in cellular migration, invasion, and metastasis. These cellular responses to TGF-β are mediated solely or partially through the canonical Smad signalling pathway which commences with activation of receptor-regulated Smads (R-Smads) Smad2 and Smad3 by the TGF-β type I receptor. However, little is known on the relative contribution of each R-Smad, the possible existence of functional antagonism, or the crosstalk with other signalling pathways in the control of TGF-β1-induced growth inhibition and cell migration. Using genetic and pharmacologic approaches we have inhibited in PDAC cells endogenous Smad2 and Smad3, as well as a potential regulator, the small GTPase Rac1, and have analysed the consequences for TGF-β1-mediated growth inhibition and cell migration (chemokinesis).ResultsSiRNA-mediated silencing of Smad3 in the TGF-β responsive PDAC cell line PANC-1 reduced TGF-β1-induced growth inhibition but increased the migratory response, while silencing of Smad2 enhanced growth inhibition but decreased chemokinesis. Interestingly, siRNA-mediated silencing of the small GTPase Rac1, or ectopic expression of a dominant-negative Rac1 mutant largely mimicked the effect of Smad2 silencing on both TGF-β1-induced growth inhibition, via upregulation of the cdk inhibitor p21WAF1, and cell migration. Inhibition of Rac1 activation reduced both TGF-β1-induction of a Smad2-specific transcriptional reporter and Smad2 C-terminal phosphorylation in PDAC cells while Smad3-specific transcriptional activity and Smad3 C-terminal phosphorylation appeared increased. Disruption of autocrine TGF-β signalling in PANC-1 cells rendered cells less susceptible to the growth-suppressive effect of Rac1 inhibition, suggesting that the decrease in "basal" proliferation upon Rac1 inhibition was caused by potentiation of autocrine TGF-β growth inhibition.ConclusionsIn malignant cells with a functional TGF-β signalling pathway Rac1 antagonizes the TGF-β1 growth inhibitory response and enhances cell migration by antagonistically regulating Smad2 and Smad3 activation. This study reveals that Rac1 is prooncogenic in that it can alter TGF-β signalling at the R-Smad level from a tumour-suppressive towards a tumour-promoting outcome. Hence, Rac1 might represent a viable target for therapeutic intervention to inhibit PDAC progression.

Dataset Information

Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-β signaling.

Publications

Structural basis for distinct roles of SMAD2 and SMAD3 in FOXH1 pioneer-directed TGF-β signaling.

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