BACKGROUND: Our recent work showed that in patients with Montreal B2 Crohn's disease TGF-[beta]1 and TGF-[beta]1-dependent collagen I are increased specifically in the intestinal smooth muscle of strictures compared to normal resection margin in the same patient. TGF-[beta]1 and collagen I was not similarly increased in B1 or B3 phenotype Crohn's. In muscle cells of normal intestine or normal resection margin in B2 patients STAT3(Y705) phosphorylation was high and STAT3(S727) phosphorylation was low. The opposite pattern was seen in muscle cells from strictures: phosphorylation of STAT3(Y705) decreased and STAT3(S727) increased. Since a consensus STAT3 binding element is present in the promoter of the TGFB gene, we hypothesized that IL-6-dependent STAT3 activity regulates increased TGF-[beta]1 and Collagen I gene expression and could be blocked by the selective STAT3(S727) inhibitor, Stattic.
METHODS: Smooth muscle cells isolated from muscularis propria of patients undergoing ileal resection for Montreal B1, B2 and B3 Crohn's disease were used to prepare RNA, cell lysates or primary cell culture. IL-6 was measured by ELISA. STAT3 phosphorylation was measured by immunoblot analysis and immunofluorescence. STAT3-TGFB DNA binding activity was measured by ChIP; transcriptional activity of TGFB gene was measured using a luciferase reporter assay. TGF-[beta]1 and Collagen I[alpha]I RNA was measured by qRT-PCR. STAT3(Ser727) phosphorylation was selectively inhibited using 10[micro]M Static. The participation of STAT3 in regulation of TGF-[beta]1 and Collagen I[alpha]I expression was investigated in cells transfected with either a dominant negative STAT3(S727A), a constitutively active STAT3(S727E) or a dominant negative STAT3(Y705F) gene.
RESULTS: IL-6 production by muscle cells from strictured intestine is significantly increased compared to normal resection margin in the same patient and significantly higher than that in affected regions of B1 or B3 patients or from non-Crohn's disease patients (Fig. 1). Treatment of muscle cells with 10[micro]g/mL IL-6 increased STAT3(S727) phosphorylation by 250% while basal STAT3(Y705) phosphorylation was unaffected. In cells treated with 10 [micro]M Static, IL-6-induced STAT3(727) phosphorylation was inhibited 80% while STAT3(Y705) was unaffected. STAT3-TGFB DNA binding activity increased 5.5-fold in muscle cells in strictures compared to the normal resection margin and reflected increased STAT3(S727) and not STAT3(Y205) binding to TGFB. The same pattern of increased STAT3 and STAT3(S727) but not STAT3(Y705) binding activity to the SBE in the TGFB was seen in cells treated with 10 [micro]g/mL IL-6. Treatment of cells with IL-6 increased TGFB transcriptional activity by 2.5-fold which was inhibited 60% by the STAT3(S727) inhibitor, Static. Transfect of cell with dnSTAT3(S727A) abolished and transfection of constitutively active STAT3(S727E) enhanced IL-6-induced STAT3(S727) phosphorylation TGFB transcriptional activity (Fig. 2) and TGF-[beta]1 and collagen 1[alpha]I gene expression (Fig. 3). These findings indicate the STAT3(S727) phosphorylation mediates IL-6-induced upregulation of TGF-[beta]1 and Collagen I[alpha]I in muscle cells of strictured intestine.
CONCLUSIONS: The pathophysiology of IL-6-dependent STAT3(727) regulated TGF-[beta]1 and collagen expression is unique in Crohn's patients with a B2 phenotype compared to B1 and B3 disease. Increased STAT3(S727) activation, TGFB gene transcription and expression of TGF-[beta]1 and Collagen I[alpha]I that result from smooth muscle cell IL-6 production can be inhibited using Static.
(C) Crohn's & Colitis Foundation of America, Inc.