P118 TARGETING ENDOPLASMIC RETICULUM STRESS FOR THE TREATMENT OF FIBROSIS IN CROHN’S DISEASE

Abstract

Progressive tissue remodeling characterized by extensive collagen production and excess extracellular matrix deposition leads to intestinal fibrosis and stricture formation in patients with fibrostenotic Crohn’s disease. Currently medical therapies can delay but not prevent the development of critical fibrostenosis necessitating surgery. Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) are associated with intestinal epithelial cells damage and apoptosis in Crohn’s disease. However, the role of ER stress and UPR signaling in subepithelial myofibroblasts (SEMF) during the development of intestinal fibrosis awaits further clarification. Previously, we reported that increase in ER stress and UPR signaling in SEMF isolated from strictured ileum contributes to activation of TGF-β1 and fibrosis. Increased interaction between GRP78 and latent TGF-β complex on the cell membrane leads to αVβ3 integrin-mediated activation of latent TGF-β. The ER stress response was augmented in SEMF by silencing of inhibitory miR-199a-5p by DNA methyltransferase-1 (DNMT1). In current study we investigated the effects of selective targeting of activated ER stress sensors on intestinal fibrosis in vitro. SEMF were prepared from cells isolated from affected ileum and from normal ileum in the same patient undergoing resection for Crohn’s disease. SEMF were transfected with siRNA to the ER stress proteins: Grp78, ATF6, XBP1 and IRE1α, and to DNMT1 to knockdown these proteins and compared to scramble sense siRNA treatment. Some cells were co-transfected with a Smad-binding element (SBE) luciferase reporter and TGFB1 transcriptional activity measured. The protein levels of GRP78, DNMT1 and collagen I were measured by immunoblot and normalized to β-actin. Active and total TGF-β1 was measured by ELISA. Our results showed that knockdown of DNMT1 decreased GRP78 protein production, diminished activation of TGF-β1, and blocked ER stress-induced TGF-β1-dependent collagen production. Knockdown of Grp78, Atf6α, Xbp1, and Ire1α in normal SEMF decreased ER stress-induced TGFB1 transcriptional activity by 30 ± 2%, 33 ± 1.5%, 35 ± 1.8%, and 23 ± 1.2%, respectively. In 3D culture of normal SEMF with Grp78 knocked down, tunicamycin failed to induce ER stress, increase activation of TGF-β1 or increase collagen production. Taken together, our results indicate that ER stress in part mediates development of intestinal fibrosis in Crohn’s disease by where DNMT1-induced silencing of miR-199a-5p allows a heighted ER Stress response that results in greater TGF-β1 activation and collagen production in the SEMF.