Suppressive effect of regucalcin on cell differentiation and mineralization in osteoblastic MC3T3-E1 cells.
The role of regucalcin in the regulation of osteoblastic cell function was investigated. Osteoblastic MC3T3-E1 cells with subconfluent monolayers were cultured in a medium containing regucalcin (10(-10)-10(-8) M) without fetal bovine serum (FBS). The proliferation of osteoblastic cells was not significantly altered in the presence of regucalcin. The results of reverse transcription-polymerase chain reaction (RT-PCR) analysis with specific primers showed that the expression of Runx2 (Cbfa1) and insulin-like growth factor-I (IGF-I) mRNAs in osteoblastic cells was significantly suppressed in the presence of regucalcin (10(-10) or 10(-9) M). Transforming growth factor-beta1 mRNA levels were significantly enhanced in the 24 h-culture with regucalcin (10(-10) or 10(-9) M). Alpha1(I) collagen and glyceroaldehyde-3-phosphate dehydrogenase (G3PDH) mRNA levels were not significantly changed by culture with regucalcin (10(-10) or 10(-9) M). Alkaline phosphatase activity was significantly decreased in the lysate of cells cultured for 24 or 48 h with regucalcin (10(-10)-10(-8) M). Moreover, the expression of regucalcin in osteoblastic cells was demonstrated by RT-PCR and Western blot analysis. When regucalcin (10(-7) M) was added into the enzyme reaction mixture containing the lysate of osteoblastic cells cultured in the absence of regucalcin, alkaline phosphatase activity was significantly decreased. Also, Ca2+/calmodulin-dependent nitric oxide (NO) synthase activity in the cell lysate was significantly decreased by addition of regucalcin (10(-10)-10(-8) M) into the reaction mixture. The presence of anti-regucalcin monoclonal antibody (5 or 10 ng/ml) in the enzyme reaction mixture caused a significant increase in NO synthase activity in the cell lysate in the presence or absence of Ca2+/calmodulin, suggesting a role of endogenous regucalcin. When osteoblastic cells with subconfluency were cultured in the presence of regucalcin (10(-10) or 10(-9) M) for 3, 9, or 18 days, the results with Alizarin red staining showed that the mineralization was markedly suppressed by culture with regucalcin for 3, 9, or 18 days. This study demonstrates that regucalcin regulates the function of osteoblastic cells, and that the protein suppresses cell differentiation and mineralization.
Yamaguchi
Masayoshi
M
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan. yamaguch@u-shizuoka-ken.ac.jp
Kobayashi
Mayumi
M
Uchiyama
Satoshi
S
D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002113:Calcification, Physiologic / Q000187:drug effects* / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D002265:Carboxylic Ester Hydrolases; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases
D002135:Calcium-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C513324:Rgn protein, mouse; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D000469:Alkaline Phosphatase