Extracellular calcium is a potent inducer of cyclo-oxygenase-2 in murine osteoblasts through an ERK signaling pathway

[Ca2+](e) may be important in bone turnover. We found [Ca2+](e) induces COX-2 transcription and PGE(2) production in primary calvarial osteoblasts through an ERK signaling pathway. Inhibition of PGE(2) production inhibited the [Ca2+](e) stimulation of osteoblastic differentiation but not the increase in cell number. Hence, some effects of [Ca2+](e) on bone may be mediated by COX-2. Introduction: Local changes in extracellular calcium ([Ca2+](e)) may play an important role in bone turnover. We examined the possibility that prostaglandins produced by cyclo-oxygenase-2 (COX-2) could mediate some of the effects of [Ca2+](e) on osteoblasts. Methods: We examined the [Ca2+](e) induction of COX-2 expression and prostaglandin E-2 (PGE(2)) production in primary osteoblasts (POBs) obtained by sequential enzymatic digestion of mouse calvariae. We measured mRNA and protein levels by Northern and Western analyses and PGE(2) production in culture medium by radioimmunoassay (RIA). COX-2 promoter activity was measured as luciferase activity in calvarial osteoblasts derived from mice transgenic for 371 bp of the COX-2 promoter fused to a luciferase reporter gene. Results and Conclusions: COX-2 mRNA and protein expression were induced by 3-40 mM of [Ca2+](e)(.) [Ca2+](e) (5 mM) induced COX-2 mRNA within 30 minutes; levels peaked at 6-9 h and remained elevated at 24 h. Cumulative medium PGE(2) was increased at 3 h, with levels rising to 30 nM at 24 h. PGE(2) production in POBs from mice with only COX-1 gene expression was 1/40th of that in POBs from mice with both COX-1 and COX-2 gene expression. [Ca2+](e) increased alkaline phosphatase activity and osteocalcin mRNA, and this increase was blocked by inhibiting PGE(2) production. [Ca2+](e) stimulation of COX-2 promoter activity correlated with the induction of COX-2 mRNA expression. [Ca2+](e) induced rapid and transient phosphorylation of extracellular signal-regulated kinase (ERK) in POBs, which peaked at 5-10 minutes. Inhibition of ERK phosphorylation with the specific inhibitors, PD-98059 and U-0126, decreased the [Ca2+](e) induction of both COX-2 mRNA and luciferase activity by 70-80%. Although less effective than [Ca2+](e) strontium [Sr2+], also induced COX-2 mRNA and promoter activity in POBs through an ERK signaling pathway. We conclude that [Ca2+](e) is a potent transcriptional inducer of COX-2 expression and PGE(2) production in osteoblasts through an ERK signaling pathway.