Role of Protein Kinases in The Induction of Inducible Isoform of Cyclooxygenase-2 (Cox-2) by Endotoxin-Activated Endothelial Cells

Abstract

Cyclooxygenase (COX) exists in at least two isoforms. COX-1 is present constitutively under physiological condition. COX-2 is induced in various cell types by mitogens and cytokines including endotoxin (lipopolysaccharides, LPS). Recently, we have shown that COX-2 can be induced by endotoxin in endothelial cells. The signal transduction mechanism of COX-2 induction is still unclear. Some cell membrane receptors have an intracellular protein kinase domain, activation of which results in the phosphorylation of proteins following ligand binding. In the present report, protein kinase inhibitors (erbstatin and genistein for tyrosine kinase inhibitors, staurosporine and calphostin C for protein kinase C inhibitors) were used as pharmacological tools to investigate the potential role of protein kinase in COX-2 induction in bovine aortic endothelial cells (BAEC) activated with endotoxin. The predominant COX metabolite, 6-oxo-prostaglandin (PG) F_1a was measured by radioimmunoassay under the following experimental conditions: (i) accumulation of COX metabolites of endogenous arachidonic acid was measured at 24 h after addition of LPS (1 μg/ml)~ (ii) determination of "COX activity" by measuring COX metabolites generated by LPS-activated BAEC after incubation with exogenous arachidonic acid (30 μM) for 15 min. Erbstatin (0.25 to 25 μM) or genistein (0.15 to 150 μM) caused a dose-dependent inhibition of the accumulation of COX metabolites in the supernatant of LPS-activated BAEC. Erbstatin or genistein also caused a dose-dependent inhibition of "COX activity" in BAEC. Western blot analysis with a specific antibody to COX-2 which determined the expression of COX-2 protein induced by LPS in cell extracts showed that erbstatin (25 μM) or genistein (150 μM) inhibited the expression of COX-2 protein in LPS-activated BAEC. In contrast to tyrosine kinase inhibitors, COX-2 induction in BAEC stimulated with LPS was not inhibited by protein kinase C (PKC) inhibitors, either staurosporine (0.0002-0.2 μM) or calphostin C (0.0015-1.5 μM). These results showed that tyrosine phospho1ylation, not protein kinase C, was pai1 of the signal transduction mechanism that mediated the induction of COX-2 elicited by LPS in BAEC.