Epigenetic Mechanisms Inhibit Smad7 and Contribute to Fibrosis in Crohn's Disease

Abstract

Background & Aims: TGF-beta1 is a central regulator of profibrotic gene expression, extracellular matrix deposition, and the development of intestinal fibrosis in patients with fibrostenotic Crohns disease (CD). Unlike the increased expression of Smad7 in mucosal and lamina propria monocytes, where it impairs Treg function, mesenchymal cells from patients with fibrostenotic CD do not exhibit elevated Smad7 expression and instead display sustained TGF-beta1 signaling. We therefore sought to elucidate the mechanisms underlying the unexpected silencing of Smad7 in fibrostenotic CD. Methods: Subepithelial myofibroblasts (SEMFs) and muscularis smooth muscle cells were isolated from paired normal and affected ileal tissues obtained from the same patients with each Montreal phenotype of CD, as well as from non-CD controls. Gene expression, RNA-binding affinity, transcriptional activity, promoter methylation, protein levels, and the mechanisms that regulate Smad7 expression were evaluated in ex vivo tissues and primary cultured cells. MicroRNA-21 (miR-21) localization was assessed by RNAscope. miR-21-mediated suppression of Smad7 was investigated using antagomiR-21. Smad7 silencing through promoter hypermethylation was confirmed using 5-Azacytidine treatment and DNA methyltransferase 1 (DNMT1) knockdown. Smad7 lysine acetylation and Smurf1/2-mediated ubiquitination were examined using selective inhibitors and proximity ligation assays. Results: In SEMFs from patients with fibrostenotic CD, aberrant silencing of Smad7 resulted from DNMT1-dependent promoter hypermethylation and pSmad3-driven, miR-21-mediated degradation of Smad7 mRNA. In addition, Smurf1/2 promoted ubiquitination and proteasomal degradation of acetylated Smad7 protein. Inhibition of miR-21 with antagomiR-21 restored Smad7 expression and reduced TGF-beta 1-dependent profibrotic gene expression. Likewise, demethylation with 5-Azacytidine or knockdown of DNMT1 induced Smad7 promoter hypomethylation and restored Smad7 expression. Furthermore, modulation of the balance between p300 acetyltransferase and histone deacetylase 1 (HDAC1) stabilized Smad7 protein by protecting it from degradation. Conclusions: A distinctive pattern of epigenetic silencing and post-translational regulation of Smad7 was identified in patients with fibrostenotic CD. These mechanisms collectively promote unrestrained TGF-beta1 signaling by suppressing Smad7 expression and stability in SEMFs, thereby contributing to the development of intestinal fibrosis.