Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin in osteoblasts

Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotege rin (OPG) produced by osteoblasts/stromal cells are involved as positive an d negative regulators in osteoclast formation. Three independent signals ha ve been proposed to induce RANKL expression in osteoblasts/stromal cells: v itamin D receptor-, cAMP-, and gp130-mediated signals. We previously report ed that intracellular calcium-elevating compounds such as ionomycin, cyclop iazonic acid, and thapsigargin induced osteoclast formation in cocultures o f mouse bone marrow cells and primary osteoblasts. Increases in calcium con centration in culture medium also induced osteoclast formation in coculture s. Treatment of primary osteoblasts with these compounds or with high calci um medium stimulated the expression of both RANKL and OPG messenger RNAs (m RNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acet oxymethyl)ester, an intracellular calcium chelator, suppressed both ionamyc in-induced osteoclast formation in cocultures and expression of RANKL and O PG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in thes e cocultures and the expression of RANKL and OPG mRNAs in primary osteoblas ts. Protein kinase C inhibitors such as calphostin and staurosporin suppres sed ionomycin- and PMA-induced osteoclast formation in cocultures and expre ssion of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated R ANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast format ion in response to ionomycin in cocultures with these stromal cell lines. O PG strongly inhibited osteoclast formation induced by calcium-elevating com pounds and PMA in cocultures, suggesting that RANKL expression in osteoblas ts is a rate-limiting step for osteoclast induction. Forskolin, an activato r of cAMP signals, also stimulated osteoclast formation in cocultures. Fors kolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RAN KL mRNA expression, which, in turn, stimulates osteoclast formation.