Abstract
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.