Regulatory effect of exogenous regucalcin on cell function in osteoblastic MC3T3-E1 cells: Involvement of intracellular signaling factor

Bone loss is induced in regucalcin transgenic rats. The role of exogenous regucalcin in the regulation of osteoblastic cell function was investigated. Osteoblastic MC3T3-E1 cells with subconfluent monolayers were cultured for 24-72 h in medium containing regucalcin (10−10 or 10−9 M) without fetal bovine serum. The presence of regucalcin did not have a significant effect on cell number. Culture with regucalcin (10−9 M) for 24 h caused a significant decrease in protein and DNA contents in osteoblastic cells. The effect of regucalcin in decreasing cellular protein content was significantly inhibited in the presence of various kinase inhibitors including staurosporine (10−7 M), dibucaine (10−6 M), PD98059 (10−8 M), or wortmannin (10−8 M). Meanwhile, culture with regucalcin caused a significant decrease in cellular DNA content in the presence of various kinase inhibitors. The presence of regucalcin did not have a significant effect on protein and DNA contents in the cells cultured with cycloheximide (10−7 M), an inhibitor of protein synthesis, or 5,6-dichloro -1-β-D-ribofuranosylbenzimidazole (10−6 M), an inhibitor of transcription activity; which each inhibitor caused a significant decrease in those contents. The effect of regucalcin in decreasing cellular protein content was seen in the presence of insulin-like growth factor-I (IGF-I; 10−9 or 10−8 M). Such an effect was not observed in cellular DNA content. The results of reverse transcription-polymerase chain reaction analysis with specific primers showed that the expression of Runx 2 (Cbfa 1) and alkaline phosphatase mRNAs in osteoblastic cells was significantly suppressed in the presence of regucalcin (10−10 or 10−9 M). Glyceraldhyde-3-phosphate dehydrogenase mRNA level was not significantly changed with culture of regucalcin (10−10 or 10−9 M). This study supports the view that exogenous regucalcin regulates the function of osteoblastic cells, and that the effect of protein is mediated through signaling factors.