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

Abstract

Myofibroblasts have recently been identified as major mediators of tumor necrosis factor-α (TNF-α)-associated colitis, but the precise mechanism(s) involved remains incompletely understood. In particular, the possibility that TNF-α signaling 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 TNF-α induced 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 TNF-α was prevented by the B₂BK receptor antagonist HOE-140, the preferential protein kinase C (PKC) inhibitors Ro31-8220 and GF-109203X, and Gö-6976, an inhibitor of conventional PKCs and protein kinase D (PKD). In a parallel fashion, TNF-α, 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 autophosphorylation site (Ser⁹¹⁶). 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-α, 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-α 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.