We have shown that extracellular calcium [Ca+2](e) induces cyclooxygenase-2 (COX-2) expression and prostaglandin E-2 (PGE(2)) production via an ERK signaling pathway in osteoblasts. In this study, we examined the roles of protein kinase C (PKC) and A (PKA) signaling pathways in the [Ca+2], induction of COX-2 in primary calvarial osteoblasts from mice transgenic for -371 bp of the COX-2 promoter fused to a luciferase reporter. Neither PKC specific inhibitors nor downregulation of the PKC pathway by phorbol myristate acetate (PMA) affected the [Ca+2], stimulation of COX-2 mRNA or promoter activity. In contrast, PKA inhibitors, used at doses that inhibited forskolin-stimulated luciferase activity by 90%, reduced [Ca+2](e)-stimulated COX-2 mRNA expression and promoter activity by 80-90%. [Ca+2](e) also stimulated a 2- to 3-fold increase in cAMP production. Hence, the [Ca+2](e) induction of COX-2 mRNA expression and promoter activity was independent of the PKC pathway and dependent on the PKA signaling pathway. (C) 2004 Elsevier Inc. All rights reserved.