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Research Article

Protein kinase D mediates synergistic expression of COX-2 induced by TNF- and bradykinin in human colonic myofibroblasts

¹David Geffen School of Medicine at UCLA ²UCLA

Submitted 29 April 2009 ; revised 17 September 2009 ; accepted in final form 28 September 2009

Myofibroblasts have recently been identified as major mediators of tumor necrosis factor- (TNF-)-associated colitis but the precise mechanism(s) involved remains incompletely understood. In particular, the possibility that TNF- signaling cross talks with other pro-inflammatory mediators, including bradykinin (BK), has not been examined in these cells. Here, we show that treatment of 18Co cells, a model of human colonic myofibroblasts, with BK and TNF- induce striking synergistic COX-2 protein expression that was paralleled by increases in the levels of transcripts encoding COX-2 and m-PGES-1 and by the production of PGE₂. COX-2 expression in 18Co cells treated with BK and TNF- was prevented by the B₂ BK receptor antagonist HOE140, the preferential protein kinase C (PKC) inhibitors Ro31-8220 and GF109203X, and Go6976, an inhibitor of conventional PKCs and protein kinase D (PKD). In a parallel fashion, TNF-, while having no detectable effect on the activation of PKD when added alone, augmented PKD activation induced by BK, as measured by PKD phosphorylation at its activation loop (Ser⁷⁴⁴) and auto-phosphorylation site (Ser⁹¹⁶). BK-induced PKD activation was also inhibited by HOE140, Ro31-8220, and Go6976. Transfection of 18Co cells with siRNA targeting PKD completely inhibited the synergistic increase in COX-2 protein in response to BK and TNF- demonstrating, for the first time, a critical role of PKD in the pathways leading to synergistic expression of COX-2. Our results imply that cross talk between TNF- and BK amplifies a PKD phosphorylation cascade that mediates synergistic COX-2 expression in colonic myofibroblasts. It is plausible that PKD increases COX-2 expression in colonic myofibroblasts to promote an inflammatory microenvironment that supports tumor growth.

PKC; 18Co Cells; PGE2; PKD phosphorylation