Regucalcin stimulates osteoclast‐like cell formation in mouse marrow cultures

The effect of regucalcin, a regulatory protein in intracellular signaling, on osteoclastic cell formation in mouse bone marrow culture is investigated. The bone marrow cells were cultured for 7 days in an α‐minimal essential medium containing either vehicle or regucalcin (10 −10 –10 −8 M). Osteoclast‐like cell formation was estimated by staining for tartrate‐resistant acid phosphatase (TRACP), a marker enzyme of osteoclasts. The presence of regucalcin (10 −10 –10 −8 M) caused a remarkable increase in osteoclast‐like multinucleated cells (MNCs). The effect of regucalcin in stimulating osteoclast‐like cell formation was significantly inhibited in the presence of calcitonin (CT; 10 −9 M), 17β‐estradiol (10 −9 M), β‐cryptoxanthin (CX; 10 −6 M), or zinc sulfate (10 −4 M), which is an anti‐bone resorbing factor. The effect of regucalcin on osteoclast‐like cell formation was not significantly blocked in the presence of cycloheximide, an inhibitor of protein synthesis, or 5,6‐dichloro‐1‐β‐ D ‐ribofuranosylbenzimidazole (DRB), an inhibitor of transcriptional activity. The effect of parathyroid hormone (10 −7 M), 1,25‐dihydroxyvitamin D 3 (10 −7 M), prostaglandin E 2 (10 −5 M), or tumor necrosis factor‐α (10 ng/ml) in increasing osteoclast‐like cell formation was significantly enhanced in the presence of regucalcin (10 −8 M). Moreover, when rat femoral‐diaphyseal or ‐metaphyseal tissues were cultured for 48 h in the presence of regucalcin (10 −10 –10 −8 M), the diaphyseal or metaphyseal calcium content was significantly decreased in the presence of regucalcin (10 −10 –10 −8 M) in vitro. The consumption of glucose and the production of lactic acid in culture medium by the diaphyseal or metaphyseal tissues was significantly raised in the presence of regucalcin (10 −10 –10 −8 M). This study demonstrates that regucalcin directly stimulates osteoclast‐like cell formation in mouse marrow culture in vitro, and that the protein stimulates bone resorption in rat femoral tissues in vitro. © 2004 Wiley‐Liss, Inc.