P116 PERIOSTIN PROMOTES ENDOPLASMIC RETICULUM STRESS-MEDIATED FIBROSIS IN CROHN’S DISEASE

Abstract

Fibrostenosis complicates Crohn’s disease in the 30-50% of patients with a Montreal B2 phenotype. Current medical therapies can delay but no prevent critical fibrostenosis and the need for surgery. Transforming growth factor-β1 (TGF-β1)-induced excess extracellular matrix (ECM) protein production in ileal subepithelial myofibroblasts (SEMF) are a major structural component of fibrosis. The role of endoplasmic reticulum (ER) stress in the pathogenesis of Crohn’s disease has been delineated in epithelial and lamina propria mononuclear cells and more recently we have delineated the role of ER stress in SEMF of fibrostenotic ileum. ER stress marker and chaperone GRP78 can bind to type I collagen in a stimulated extracellular environment, which suggests the potential link between ER stress and fibrosis formation. The secreted matricellular protein periostin, typically promotes fibrosis in a TGF-β1-dependent fashion and interacts with plasma membrane integrin family members, including αVβ3. We have previously shown αVβ3 integrin is crucial for LAP-TGF-β1 activation in ileal mesenchymal cells, SEMF and smooth muscle cells. Our RNAseq data identified TGFB1, ITGAV, ITGB3 COL1A1 and all as significantly upregulated (25-200 fold) in SEMF of strictured ileum. However, the role of periostin in SEMF during the ER stress-mediated component of intestinal fibrosis in Crohn’s disease remains unknown. To determine the role of a periostin during the development of intestinal fibrosis in fibrostenotic Crohn’s disease, SEMF were isolated from the patients with each Crohn’s disease phenotype and place them into primary culture or 3D culture. Periostin expression was determined by qRT-PCR and immunoblot analysis. Binding of periostin to αvβ3 integrin was determined by proximity ligation hybridization assay. Knockdown of periostin in cells was accomplished with siRNA. Active and total TGF-β1 protein levels were measured by ELISA. Our data show that periostin expression and production are increased in SEMF from strictured ileum compared to normal ileum in the same patient. IL-6 promotes the interaction between secreted periostin and integrin αvβ3 on the SEMF cell membrane. This was associated with increased levels of active TGF-β1 and increased the resulting production of collagen 1α1. Induction of ER stress in SEMF from normal ileum with tunicamycin elicited increased expression of periostin and αVβ3. The ER stress-induced increase in αVβ3, however, was inhibited by siRNA-mediated knockdown of periostin. The increase in active TGF-β1 seen following ER stress was also decreased in the SEMF in which periostin was knocked down. Our data suggest ER stress-induced periostin links increased αVβ3 integrin and αVβ3-dependent activation of latent TGF-β1 and the resulting increase in collagen production (Figure 1).