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

Yoo J, Chung C, Slice L, Sinnett-Smith J, Rozengurt E. Protein kinase D mediates synergistic expression of COX-2 induced by TNFand bradykinin in human colonic myofibroblasts. Am J Physiol Cell Physiol 297: C1576–C1587, 2009. First published September 30, 2009; doi:10.1152/ajpcell.00184.2009.—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 TNFsignaling cross talks with other proinflammatory 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 TNFinduced striking synergistic COX-2 protein expression that was paralleled by increases in the levels of transcripts encoding COX-2 and microsomal prostaglandin E synthase 1 (mPGES-1) and by the production of PGE2. COX-2 expression in 18Co cells treated with BK and TNFwas prevented by the B2 BK receptor antagonist HOE-140, the preferential protein kinase C (PKC) inhibitors Ro31-8220 and GF-109203X, and Gö6976, 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 (Ser) and autophosphorylation site (Ser). BK-induced PKD activation was also inhibited by HOE-140, Ro31-8220, and Gö-6976. Transfection of 18Co cells with small interfering RNA 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 TNFand 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.