Intracellular Calcium and Protein Kinase C Mediate Expression of Receptor Activator of Nuclear Factor- B Ligand and Osteoprotegerin in Osteoblasts

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Abstract
Receptor activator of nuclear factor-kB ligand (RANKL) and os- teoprotegerin (OPG) produced by osteoblasts/stromal cells are in- volved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracel- lular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in cal- cium concentration in culture medium also induced osteoclast for- mation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o- aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid)-tetra(acetoxy- methyl)ester, an intracellular calcium chelator, suppressed both iono- mycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myris- tate 13-acetate (PMA), an activator of protein kinase C, also stimu- lated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stim- ulated RANKL 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 formation in response to ionomycin in co- cultures with these stromal cell lines. OPG strongly inhibited oste- oclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin 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 RANKL mRNA expression, which, in turn, stimulates osteoclast formation. (Endocrinology 141: 4711- 4719, 2000)

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