Backround
Crohn’s disease (CD) is a chronic inflammatory enteropathy characterized by fibrotic strictures. Myofibroblasts (MFB) are stromal cells of the GI tract found in increased numbers in patients with CD and represent the key effector cells involved in pathologic fibrosis. MFB are a known target of TNF-α, a 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 migration through the COX-2 and Hsp27 pathways.
Materials and Methods
The human colonic MFB cell line 18Co was grown to confluence on 35×10mm cell culture dishes and used from passages 8–14. An in vitro scratch assay assessed the effect of TNF-α (10 ng/ml) on MFB migration over 24 hours in the presence or absence of several inhibitors (NS398, SB203580, Hsp27 siRNA).
Results
TNF-α significantly increased MFB migration over 24 h. TNF-α also led to the increased expression of COX-2 and stimulated rapid phosphorylation of Hsp27 at Ser-82. TNF-α-induced-COX-2 expression, Hsp27 phosphorylation, and MFB migration were all significantly inhibited by the P38 MAPK inhibitor SB203580 (p<0.05). TNF-α-induced MFB migration was also significantly inhibited by NS398 (p<0.05), a direct inhibitor of COX-2, and by siRNA targeting Hsp27 (p<0.05).
Conclusions
TNF-α stimulates colonic myofibroblast migration through P38 MAPK-mediated activation of COX-2 and Hsp27. Further elucidating these inflammatory signaling pathways may lead to novel therapeutic targets for the treatment of Crohn’s disease related fibrosis and strictures.