S. Saini1, T. Liu1, L. Chen1, J. Yoo1 1Tufts Medical Center,Colon And Rectal Surgery,Boston, MA, USA
Introduction: Crohn’s disease (CD) is a chronic inflammatory enteropathy characterized by the presence of fibrotic strictures. Myofibroblasts (MFB) are stromal cells of the GI tract that are found in increased numbers in the lamina propria of patients with CD and represent the key effector cells involved in pathologic fibrosis during chronic inflammation. MFB are a known target of TNF-α, a key pro-inflammatory cytokine strongly implicated in the pathophysiology of CD. However, the precise mechanisms through which TNF-α contributes to fibrosis remain incompletely understood. Here, we demonstrate for the first time that TNF-α increases MFB-induced collagen production and MFB migration through the COX-2 and Hsp27 pathways.
Methods: The human colonic MFB cell line 18Co was grown to confluence on 35x10mm cell culture dishes and was used from passages 8-14. An in vitro wound-healing assay was used to assess the effect of TNF-α (12 ng/ml) on MFB migration over a period of 24 hours in the presence or absence of several inhibitors (NS398 (10 μM) and SB203580(10 μM)). Hsp27 siRNA was used to block the expression of Hsp27 in the 18Co cells, and to evaluate the influence of Hsp27 on colonic MFB migration. Antibodies to collagen (COL1A2), pHsp27, and COX-2 were used to evaluate their expression levels using western blotting.
Results:Exposure of 18Co cells to TNF-α increased collagen type I expression in a time-dependent fashion and increased MFB migration over 24 hrs. Incubation of 18Co cells with TNF-α for 4 hours also led to the increased expression of COX-2 and stimulated rapid phosphorylation of Hsp27 at Ser82. TNF-α-mediated expression of COX-2 and phosphorylation of Hsp27 were both inhibited by the P38 MAPK inhibitor SB203580. TNF- α-induced MFB migration was significantly inhibited by SB203580 (p<0.05), as well as NS398 (p<0.05), a direct inhibitor of COX-2. Hsp27 siRNA was effective in blocking the expression of Hsp27, and also inhibited MFB migration.
Conclusion:TNF-α increases collagen production and stimulates cell migration in human colonic myofibroblasts through P38 MAPK-mediated activation of COX-2 and Hsp27. Both COX-2 and Hsp27 appear to regulate myofibroblast cell migration, which may play an important role in mucosal healing in the setting of inflammation. Further elucidating these inflammatory signaling pathways may lead to novel therapeutic targets for the treatment of Crohn’s related fibrosis and strictures.
