Endoplasmic Reticulum Stress in Subepithelial Myofibroblasts Increases the TGF-β1 Activity That Regulates Fibrosis in Crohn’s Disease

Abstract

Background Endoplasmic reticulum (ER) stress is an essential response of epithelial and immune cells to inflammation in Crohn’s disease. The presence and mechanisms that might regulate the ER stress response in subepithelial myofibroblasts (SEMFs) and its role in the development of fibrosis in patients with Crohn’s disease have not been examined. Methods Subepithelial myofibroblasts were isolated from the affected ileum and normal ileum of patients with each Montreal phenotype of Crohn’s disease and from normal ileum in non-Crohn’s subjects. Binding of GRP78 to latent TGF-β1 and its subcellular trafficking was examined using proximity ligation-hybridization assay (PLA). The effects of XBP1 and ATF6 on TGF-β1 expression were measured using DNA-ChIP and luciferase reporter assay. Endoplasmic reticulum stress components, TGF-β1, and collagen levels were analyzed in SEMF transfected with siRNA-mediated knockdown of DNMT1 and GRP78 or with DNMT1 inhibitor 5-Azacytidine or with overexpression of miR-199a-5p. Results In SEMF of strictured ileum from patients with B2 Crohn’s disease, expression of ER stress sensors increased significantly. Tunicamycin elicited time-dependent increase in GRP78 protein levels, direct interaction with latent TGF-β1, and activated TGF-β1 signaling. The TGFB1 DNA-binding activity of ATF-6α and XBP1 were significantly increased and elicited increased TGFB1 transcription in SEMF-isolated from affected ileum. The levels of ER stress components, TGF-β1, and collagen expression in SEMF were significantly decreased following knockdown of DNMT1 or GRP78 by 5-Azacytidine treatment or overexpression of miR-199a-5p. Conclusions Endoplasmic reticulum stress is present in SEMF of patients susceptible to fibrostenotic Crohn’s disease and can contribute to development of fibrosis. Targeting ER stress may represent a novel therapeutic target to prevent fibrosis in patients with fibrostenotic Crohn’s disease.

Mechanism of fibrosis and stricture formation in Crohn's disease

“…It has recently been shown that endoplasmic reticulum (ER) stress, which occurs following the accumulation of misfolded proteins, induces TGF-β1 gene expression. 73 4.11.3 | Pro-inflammatory cytokines favour matrix remodelling and decrease wound healing Stricture myofibroblasts exposed experimentally to the proinflammatory cytokines IL17A and TNF release MMP-3 and MMP-12, in addition to increasing TIMP-1 and collagen production. 42 Moreover, IL-17E increased MMP-3 and MMP-12 without concomitant stimulation of TIMP and collagen.…”

Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

“…Moreover, our recent study showed that the UPR and its downstream signaling pathways can be manipulated through epigenetic regulations. We showed that expression of ER stress sensors increased significantly in subepithelial myofibroblasts of strictured intestine from patients with fibrostenotic Crohn's disease [9]. Increase in ER stress response featured with overexpression of GRP78, XBP1s, and ATF6α can be also reproduced in the normal intestinal subepithelial myofibroblasts when treated with tunicamycin, which is an ER stress agonist [9].…”

“…These stresses commonly include changes in calcium homeostasis, viral or bacterial infection, inflammation, nutrition or energy deficiency, hypoxia, lipid overload, altered redox status, as well as oncogene activation in cancer [7,10]. During UPR, transcription factors such as ATF6, XBP-1 are activated and translocated to the nucleus to initiate transcription of genes involved in inflammation, cell proliferation and fibrosis [9,11]. In addition, ER stress plays a role in cellular differentiation, antigen presentation, and stem cell renewal capacity [8].…”

“…In gastrointestinal system, it has been studied in epithelial cell differentiation and function, as well as Crohn's disease during the process of intestinal epithelial cell damage [5][6][7]. The role of ER stress in mesenchymal cells during the development of intestinal fibrosis has been recently reported and is discussed in details in subsequent sections [9].…”

“…Therefore, it is understandable that the UPR with its downstream signaling pathways is required in the maintenance of intestinal homeostasis. Moreover, dysfunction of ER stress response contributes to the pathogenesis of inflammatory bowel diseases (IBD) and the complications such as intestinal fibrosis [9].…”