Cox-2 and osteopontin in cocultured platelets and mesangial cells: Role ofglucocorticoids

Background. Glomerular inflammation is characterized by a consecutive infil tration of immunoreactive cells. To mimic the early phase of glomerular inj ury, a coculture system of platelets and rat renal mesangial cells was esta blished. As prototypes, the inflammation-related proteins cyclooxygenase-2 (Cox-2) and the chemotactic protein osteopontin (OPN) were investigated. Methods. The expression of OPN and Cox-2 mRNA and protein was determined by Northern and Western blot analyses. Results. Coincubation of platelets and mesangial cells led to a rapid; tran sient induction of Cox-2 mRNA, which peaked at two hours, whereas OPN and m onocyte chemoattractant protein-1 (MCP-1) were induced at later time points . The induction of Cox-2 mRNA was concentration dependent and highly reprod ucible when platelets of different donors were investigated. Partial Cox-2 induction was observed when supernatants of preactivated platelets were inc ubated with mesangial cells. The inhibition of the signaling pathways of pl atelet-derived growth factor (PDGF) and epidermal growth factor (EGF) or in terference with Gi-protein signaling partially inhibited platelet-induced C ox-2 expression. Down-regulation of protein kinase C (PKC), which is a comm on signaling module in many pathways leading to Cox-2 induction, almost com pletely abrogated platelet-induced Cox-2 expression. The time pattern of Co x-2 and OPN expression suggested that Cox-2 might play a role in OPN induct ion. The up-regulation of OPN was dependent on de novo protein synthesis an d was induced by high levels of exogenous prostaglandin E-2 (PGE(2); 10 mu mol/L). Endogenous PGE(2), however, proved not to be essential for OPN mRNA expression, because inhibition of Cox activity did not change OPN mRNA lev els. Dexamethasone inhibited Cox-2 mRNA induction but increased OPN mRNA an d protein expression. Conclusion. These data indicate that Cox-2 and OPN are independently up-reg ulated upon interaction of platelets and mesangial cells.