BK-induced COX-2 expression via PKC-δ-dependent activation of p42/p44 MAPK and NF-κB in astrocytes

Bradykinin (BK) is an inflammatory mediator, elevated levels in the region of several brain injury and inflammatory diseases. It has been shown to induce cyclooxygenase-2 (COX-2) expression implicating in inflammatory responses in various cell types. However, the signaling mechanisms underlying BK-induced COX-2 expression in astrocytes remain unclear. First, RT-PCR and Western blotting analysis showed that BK induced the expression of COX-2 mRNA and protein, which was inhibited by B₂ BK receptor antagonist Hoe140, suggesting the involvement of B₂ BK receptors. BK-induced COX-2 expression and translocation of PKC-δ from cytosol to membrane fraction were inhibited by rottlerin, suggesting that PKC-δ might be involved in these responses. This hypothesis was further supported by the transfection with a dominant negative plasmid of PKC-δ significantly blocked BK-induced COX-2 expression. BK-stimulated p42/p44 MAPK phosphorylation, COX-2 mRNA expression, and prostaglandin E₂ (PGE₂) release were attenuated by PD98059, indicating the involvement of MEK/p42/p44 MAPK in this pathway. Accordingly, BK-stimulated phosphorylation of p42/p44 MAPK was attenuated by rottlerin, indicating that PKC-δ might be an upstream component of p42/p44 MAPK. Moreover, BK-induced COX-2 expression might be mediated through the translocation of NF-κB into nucleus which was blocked by helenalin, rottlerin and PD98059, implying the involvement of NF-κB. These results suggest that in RBA-1 cells, BK-induced COX-2 expression and PGE₂ release was sequentially mediated through PKC-δ-dependent activation of p42/p44 MAPK and NF-κB. Understanding the regulation of COX-2 expression and PGE₂ release induced by BK in astrocytes might provide a new therapeutic strategy of brain injury and inflammatory diseases.