Bradykinin stimulates protein kinase D–mediated colonic myofibroblast migration via cyclooxygenase-2 and heat shock protein 27

Abstract

BackgroundInflammatory bowel disease is characterized by episodic intestinal injury and repair. Myofibroblasts are gastrointestinal 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 cyclooxygenase-2 (COX-2) and heat shock protein 27 (Hsp27) pathways. Materials and methodsCCD-18Co is a human colonic myofibroblast cell line used from passages 8 to 14. An in vitro scratch assay assessed the effect of BK (100 nM) on myofibroblast migration over 24 h in the presence or absence of several inhibitors (CID755673 [10 μM] and NS398 [10 μM]). Hsp27 small interfering RNA evaluated the effect of Hsp27 on colonic myofibroblast migration. Antibodies to pPKD, pHsp27, and COX-2 evaluated expression levels by Western blot. ResultsBK stimulated myofibroblast migration over 24 h. 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 4 h. 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 small interfering RNA (P < 0.05). ConclusionsBK 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.