S100A4: A Negative Regulator of Mineralization and Osteoblast Differentiation

S100A4 is an intracellular calcium-binding protein expressed by osteoblastic cells. We have previously found that inhibition of S100A4 synthesis in osteoblastic cells increased the production of mineralized nodules in vitro and up-regulated the expression of osteoblastic phenotypic markers. Thus, we hypothesized that S100A4 negatively regulates the mineralization process by altering osteoblastic cell differentiation. Objective: To investigate the effects of S100A4 overexpression on osteoblast differentiation and in vitro mineralized nodule formation. Methods: MC3T3-E1 osteoblastic cells were transfected with the pcDNA3HA3/S100A4 construct or with an empty pcDNA3HA3 expression vector (control, Ev clones). Stably transfected cell clones were isolated and the expression of S100A4 was examined by Western blotting. Cell clones overexpressing S100A4, MC3T3-E1 cells, and Ev clones were cultured in mineralization medium, and the mineralized nodules formed were stained with Alizarin Red S. Alkaline phosphatase (ALP) activity was histochemically examined and the mRNA expression of bone sialoprotein (BSP) and osteocalcin (OC) were examined by RT-PCR. Results: Significant amounts of mineralized nodules were formed in both MC3T3-E1 cells and Ev clones after 20 days, however, cell clones synthesizing high levels of S100A4 failed to form mineralized nodules even after extended culture periods (30 days). ALP activity was markedly inhibited and the expression of BSP and OC mRNAs was significantly downregulated in these clones. Ev clones were similar to MC3T3-E1 cells in these aspects. Conclusions: These results indicate that S100A4 may act as a negative regulator of osteoblast differentiation and, therefore, as an inhibitor of matrix mineralization. Supported by NIH-NIDCR grant DE014290.