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

Abstract

Background. Glomerular inflammation is characterized by a consecutive infiltration of immunoreactive cells. To mimic the early phase of glomerular injury, a coculture system of platelets and rat renal mesangial cells was established. 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, transient induction of Cox-2 mRNA, which peaked at two hours, whereas OPN and monocyte chemoattractant protein-1 (MCP- 1) were induced at later time points. The induction of Cox-2 mRNA was concentration dependent and highly reproducible when platelets of different donors were investigated. Partial Cox-2 induction was observed when supernatants of preactivated platelets were incubated with mesangial cells. The inhibition of the signaling pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) or interference with G1-protein signaling partially inhibited platelet-induced Cox-2 expression. Down- regulation of protein kinase C (PKC), which is a common signaling module in many pathways leading to Cox-2 induction, almost completely abrogated platelet-induced Cox-2 expression. The time pattern of Cox-2 and OPN expression suggested that Cox-2 might play a role in OPN induction. The up- regulation of OPN was dependent on de novo protein synthesis and was induced by high levels of exogenous prostaglandin E2 (PGE2; 10 μmol/L). Endogenous PGE2, however, proved not to be essential for OPN mRNA expression, because inhibition of Cox activity did not change OPN mRNA levels. Dexamethasone inhibited Cox-2 mRNA induction but increased OPN mRNA and protein expression. Conclusion. These data indicate that Cox-2 and OPN are independently up- regulated upon interaction of platelets and mesangial cells.