Article

The DNA Binding Activities of Smad2 and Smad3 Are Regulated by Coactivator-mediated Acetylation

Ludwig Institute for Cancer Research, Uppsala University, Biomedical Center, Box 595, Husargatan 3, S-751 24 Uppsala, Sweden.
Journal of Biological Chemistry (Impact Factor: 4.57). 01/2007; 281(52):39870-80. DOI: 10.1074/jbc.M607868200
Source: PubMed

ABSTRACT

Phosphorylation-dependent activation of the transcription factors Smad2 and Smad3 plays an important role in TGFbeta-dependent signal transduction. Following phosphorylation of Smad2 and Smad3, these molecules are translocated to the nucleus where they interact with coactivators and/or corepressors, including p300, CBP, and P/CAF, and regulate the expression of TGFbeta target genes. In the current study, we demonstrate that both Smad2 and Smad3 are acetylated by the coactivators p300 and CBP in a TGFbeta-dependent manner. Smad2 is also acetylated by P/CAF. The acetylation of Smad2 was significantly higher than that of Smad3. Lys(19) in the MH1 domain was identified as the major acetylated residue in both the long and short isoform of Smad2. Mutation of Lys(19) also reduced the p300-mediated acetylation of Smad3. By generating acetyl-Lys(19)-specific antibodies, we demonstrate that endogenous Smad2 is acetylated on this residue in response to TGFbeta signaling. Acetylation of the short isoform of Smad2 improves its DNA binding activity in vitro and enhances its association with target promoters in vivo, thereby augmenting its transcriptional activity. Acetylation of Lys(19) also enhanced the DNA binding activity of Smad3. Our data indicate that acetylation of Lys(19) induces a conformational change in the MH1 domain of the short isoform of Smad2, thereby making its DNA binding domain accessible for interactions with DNA. Thus, coactivator-mediated acetylation of receptor-activated Smad molecules could represent a novel way to regulate TGFbeta signaling.

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Available from: Eva Gronroos, Jan 21, 2016
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    • "Numerous protein-coding genes are up-regulated by TGF-β through Smad signaling while few genes are down-regulated and the underlined molecular mechanism is poorly understood. Smads can recruit transcription co-activators such as CBP, p300, and P/CAF, which contain intrinsic histone acetyltransferase, to promote trans-activation[34]. On the other hand, a Smad repression model suggests that Smads can recruit transcription co-repressors such as p107[35], Ski, or NCoR/mSin3/HDAC[23,36]to repress the downstream genes. "
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    ABSTRACT: The microRNA-30 family plays important roles in maintaining kidney homeostasis. Patients with focal segmental glomerulosclerosis (FSGS) have reduced miR-30 levels in glomerulus. TGF-β represses miR-30s in kidney podocytes, which leads to cytoskeleton damage and podocyte apoptosis. In this study, we investigated the mechanism by which TGF-β represses miR-30d in vitro. The human miR-30d promoter contains multiple copies of Smad binding element-like sequences. A fragment of 150 base pairs close to the transcription start site was negatively regulated by TGF-β to a similar extent as the 1.8 kb promoter, which was blocked by histone-deacetylase inhibition. TGF-β specifically enhanced HDAC3 expression. Knockdown of HDAC3 by shRNA or a selective inhibitor RGFP966 significantly relieved the repression of miR-30d mRNA and the promoter transcription. TGF-β promoted HDAC3 association with Smad2/3 and NCoR and caused their accumulation at the putative Smad binding site on the miR-30d promoter, which was prohibited by TSA or RGFP966. Furthermore, TSA or RGFP966 treatment reversed TGF-β-induced up-regulation of miR-30d targets Notch1 and p53 and alleviated the podocyte cytoskeleton damage and apoptosis. Taken together, these findings pinpoint that TGF-β represses miR-30d through a Smad2/3-HDAC3-NCoR repression complex and provide novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies. Key message MiR-30d promoter is negatively regulated by TGF-β. TGF-β down-regulates miR-30 through Smad signaling pathway. HDAC3 and NCoR are recruited by Smad2/3 to mediate miR-30d repression by TGF-β. HDAC3 acts as a critical player in TGF-β-induced miR-30d repression and podocyte injuries.
    Full-text · Article · Oct 2015 · Journal of Molecular Medicine
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    • "Canonical TGF-β1 signaling involves the receptor activated Smad proteins (Smad2 and Smad3), which, upon phosphorylation, associate with Smad4, translocate to the nucleus and act as transcription factors [41,51,52]. However recent data demonstrates that a further level of transcriptional regulation is necessary to mediate TGF-β downstream signaling, involving Smad acetylation [28,29,42]. Indeed, it has come to be appreciated that the post translational modifications of proteins by acetylation and de-acetylation is ubiquitous, comparable to other well described post translational modifications as a key regulator of protein and therefore cell function [19,43]. "
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    ABSTRACT: Background Despite advances in the treatment of heart failure, mortality remains high, particularly in individuals with diabetes. Activated transforming growth factor beta (TGF-β) contributes to the pathogenesis of the fibrotic interstitium observed in diabetic cardiomyopathy. We hypothesized that high glucose enhances the activity of the transcriptional co-activator p300, leading to the activation of TGF-β via acetylation of Smad2; and that by inhibiting p300, TGF-β activity will be reduced and heart failure prevented in a clinically relevant animal model of diabetic cardiomyopathy. Methods p300 activity was assessed in H9c2 cardiomyoblasts under normal glucose (5.6 mmol/L—NG) and high glucose (25 mmol/L—HG) conditions. 3H-proline incorporation in cardiac fibroblasts was also assessed as a marker of collagen synthesis. The role of p300 activity in modifying TGF-β activity was investigated with a known p300 inhibitor, curcumin or p300 siRNA in vitro, and the functional effects of p300 inhibition were assessed using curcumin in a hemodynamically validated model of diabetic cardiomyopathy – the diabetic TG m(Ren-2)27 rat. Results In vitro, H9c2 cells exposed to HG demonstrated increased p300 activity, Smad2 acetylation and increased TGF-β activity as assessed by Smad7 induction (all p < 0.05 c/w NG). Furthermore, HG induced 3H-proline incorporation as a marker of collagen synthesis (p < 0.05 c/w NG). p300 inhibition, using either siRNA or curcumin reduced p300 activity, Smad acetylation and TGF-β activity (all p < 0.05 c/w vehicle or scrambled siRNA). Furthermore, curcumin therapy reduced 3H-proline incorporation in HG and TGF-β stimulated fibroblasts (p < 0.05 c/w NG). To determine the functional significance of p300 inhibition, diabetic Ren-2 rats were randomized to receive curcumin or vehicle for 6 weeks. Curcumin treatment reduced cardiac hypertrophy, improved diastolic function and reduced extracellular matrix production, without affecting glycemic control, along with a reduction in TGF-β activity as assessed by Smad7 activation (all p < 0.05 c/w vehicle treated diabetic animals). Conclusions These findings suggest that high glucose increases the activity of the transcriptional co-regulator p300, which increases TGF-β activity via Smad2 acetylation. Modulation of p300 may be a novel strategy to treat diabetes induced heart failure.
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    ABSTRACT: Transforming growth factor-β1, the key ligand of Smad-dependent signaling pathway, is critical for epithelial-mesenchymal transition during embryo-morphogenesis, fibrotic diseases, and tumor metastasis. In this study, we found that activation of p300/CBP, a histone acetyltransferase, by TGF-β1 mediates Epithelial-mesenchymal transition (EMT) via acetylating Smad2 and Smad3 in TGF-β1 signaling pathway. We demonstrated that treatment with EGCG inhibited p300/CBP activity in human lung cancer cells. Also, we observed that EGCG potently inhibited TGF-β1-induced EMT and reversed the up-regulation of various genes during EMT. Our findings suggest that EGCG inhibits the induction of p300/CBP activity by TGF-β1. Therefore, EGCG inhibits TGF-β1-mediated EMT by suppressing the acetylation of Smad2 and Smad3 in human lung cancer cells.
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