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.
Background Inflammatory bowel disease (IBD) is characterized by episodic intestinal injury and repair. Myofibroblasts are gastrointestinal (GI) tract stromal cells that regulate the reparative process, and are known targets of inflammatory mediators including bradykinin (BK). However, the mechanisms through which inflammation regulates myofibroblast-induced wound healing remain incompletely understood. Here, we demonstrate, for the first time, that BK stimulates myofibroblast migration through protein kinase D (PKD)-mediated activation of the COX-2 and Hsp27 pathways. Materials and Methods CCD-18Co is a human colonic myofibroblast cell line used from passages 8–14. An in vitro scratch assay assessed the effect of BK (100nM) on myofibroblast migration over 24h in the presence or absence of several inhibitors (CID755673 (10 µM) and NS398 (10 µM)). Hsp27 siRNA evaluated the effect of Hsp27 on colonic myofibroblast migration. Antibodies to pPKD, pHsp27, and COX-2 evaluated expression levels by Western blot. Results BK stimulated myofibroblast migration over 24h. BK also led to rapid and sustained phosphorylation of PKD at Ser-916, rapid phosphorylation of Hsp27 at Ser-82, and increased COX-2 expression over 4h. BK-mediated COX-2 expression and Hsp27 phosphorylation were both inhibited by the PKD inhibitor CID755673. Similarly, BK-induced myofibroblast migration was significantly inhibited by CID755673 (p<0.05), by the direct COX-2 inhibitor NS398 (p<0.05), and by Hsp27 siRNA (p<0.05). Conclusions BK stimulates myofibroblast migration through PKD-mediated activation of COX-2 and Hsp27. PKD, COX-2 and Hsp27 all appear to regulate myofibroblast cell migration, a stromal population that may play an important role in mucosal healing in the setting of inflammation.
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