Endogenous IGFBP-3 regulates excess collagen expression in intestinal smooth muscle cells of Crohnʼs disease strictures

Abstract

Background Stricture formation occurs in ≈30% of patients with Crohn’s disease (CD) and is a significant cause of morbidity. Strictures are characterized by intestinal smooth muscle cell hyperplasia, smooth muscle cell hypertrophy, and fibrosis due to excess net extracellular matrix production, including collagen. Transforming growth factor-β1 (TGF-β1) has profibrotic effects in many tissues due to its ability to regulate collagen expression and extracellular matrix dynamics. We previously showed that both insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) and TGF-β1 are expressed by normal human intestinal smooth muscle cells, bind to, and activate TGF-βRII/I receptors in these cells. Methods Smooth muscle cells isolated from the muscularis propria of patients were used to prepare RNA, protein lysates, or placed into primary culture. IGFBP-3, TGF-β1, and collagen IαI expression was measured with quantitative reverse-transcription polymerase chain reaction (RT-PCR) and protein levels by enzyme-linked immunosorbent assay (ELISA) or immunoblot. Results Expression and production of IGFBP-3, TGF-β1, and collagen IαI were significantly increased specifically in smooth muscle cells isolated from regions of strictured intestine in CD compared to nonstrictured histologically normal resection margin. IGFBP-3 and TGF-β1 regulated collagen IαI expression and production via a TGF-βRII/I-dependent and Smad2/3-dependent mechanism. Upregulated (excess) collagen IαI expression and production in smooth muscle cells of strictures and basal collagen IαI in smooth muscle cells of normal margin were inhibited by immunoneutralization of IGFBP-3 or TGF-β1. Conclusions The findings indicate that upregulated endogenous IGFBP-3 and TGF-β1 expression regulates excess collagen IαI production and contributes to fibrosis and stricture formation in CD.

Mechanisms That Mediate the Development of Fibrosis in Patients With Crohnʼs Disease

“…The expression of all three are increased in muscle cells of strictured intestine 34, 59 . Production of fibronectin and vitronectin, activating ligands of αVβ3 integrin (the cognate vitronectin receptor), is increased in stricturing Crohn’s disease.…”

“…Expression and production of TGF-β1, is increased specifically in smooth muscle cells of ileal strictures compared to histologically normal proximal resection margin. 34, 59 This is in contrast to patients expressing a B1 or B3 phenotype where TGF-β1 expression is unchanged from non-Crohn’s subjects in either affected regions or normal resection margin. TGF-β1, in addition, stimulates expression of other fibrogenic factors including: fibronectin, CTGF and IGF-I.…”

“…TGF-βRI/II receptors expressed by mesenchymal cells are linked to canonical Smad2/3 signaling. 54 Binding of active TGF-β1 to TGF-βRI/II receptors in human intestinal muscle activates canonical Smad signaling, r-Smad2/3 phosphorylation and increased collagen I production which accounts for ~70% of collagen present in the intestine 33, 55 . With the exception of Smad3 deletion, deletion of TGF-β protein, its receptors or other Smad proteins are generally lethal mutations or a feature of neoplastic cells.…”

“…The central role of IGF-I in the development of fibrosis in Crohn’s disease was shown by the increased expression of IGF-I, IGFBP-3 and IGFBP-5 in smooth muscle cells of strictured intestine 34, 59, 70–72 . In the muscularis propria of human intestine, increased autocrine IGF-I production results increased smooth muscle cell proliferation, inhibition of apoptosis as well a increased ECM production including collagen I, fibronectin and vitronectin.…”

Intestinal smooth muscle phenotype determines enteric neuronal survival via GDNF expression

“…While normally reversible, continued proliferation is well recognized to lead to progressively poorer reversion to the contractile state, and is associated with a number of disease pathologies. Intestinal smooth muscle hyperplasia occurs in animal models of intestinal inflammation (Stanzel et al, 2010;Nair et al, 2011) and is associated with the development of intestinal strictures in human IBD (Flynn et al, 2010(Flynn et al, , 2011. Therefore, we used a standardized protocol to obtain high-passage (P10) CSMC cell and examined the effect of protracted proliferation on the expression of GDNF.…”