Cystatin C Induces Mineralized Tissue Formation in Primary Mouse Osteoblasts

Objectives: Cystatin C (CC) is an endogenous cysteine proteinase inhibitor in cerebrospinal fluid, urine and milk. Recent studies reveal that a bovine whey basic protein induces bone formation, and also reported that CC is major component in the milk basic protein. However, the effect of CC on mineralized tissue formation. Therefore, we investigated the direct effect of CC on mineralized tissue formation in primary osteoblast culture, and analyzed mineralized nodule formation, as well as the expression of osteoblast specific genes. Methods: Osteoblasts were isolated from calvaria of neonatal C57BL/6J mice, and cultured in aMEM containing 10% FBS, 50 µg/ml ascorbic acid, and 5 mM a-glycerophosphate in the presence or absence of CC or chemical cysteine proteinase inhibitor E-64 by different concentrations (10-7`10-12 M). Staining for alkaline phosphatase (ALP), von-Kossa and Alizarin Red, measurement of both the ALP activity of cells and the total area and calcium content of nodules, and RT-PCR for the expression of osteoblast specific genes were examined. Results: CC- or E-64-treatment has no effect for both mRNA expression and enzyme activity of ALP compared with control group. Appearance of von-Kossa-positive or Alizarin Red-positive nodules in CC-treated group was earlier than that in control and E-64-treated groups. Total area and calcium accumulation of nodules were markedly increased by CC addition, but not by E-64. Moreover, the mRNA level of osterix, runx-2, and osteocalcin was decreased in CC treatment, but not in E-64 addition and control. Conclusion: Our findings indicated that CC inhibits differentiation into osteoblasts, and showed that CC stimulates osteoblasts to deposit mineralized tissue. It is also suggested that these effects of CC to osteoblasts is via an unknown mechanism instead of proteinase inhibitory effect. Furthermore, it is proposed that CC is a strong inducer of bone formation in bone injury and bone defect.