Abstract
ClC‐3 chloride channel has been speculated to contribute to the acidification of synaptic vesicles and endosomes. However, the biological function of ClC‐3 in osteogenesis remains to be determined. In this study, we first analyzed ClC‐3 expression in MC3T3‐E1 cells and primary mouse osteoblasts and then performed the osteoinductive procedure to determine the effects on gene expression. Subsequently, we transiently transfected ClC‐3 cDNA or ClC‐3‐siRNA into MC3T3‐E1 cells to determine the changed phenotype and gene expression. Lastly, we assessed the underlying mechanism responsible for ClC‐3‐induced osteodifferentiation. We found that ClC‐3 mRNA was expressed in primary mouse osteoblasts and MC3T3‐E1 cells and induced by using an osteoinductive procedure. We also found that overexpression of ClC‐3 contributed to osteodifferentiation, such as increase in the expression of osteogenic markers [alkaline phosphatase (Alp), osteocalcin (Oc), bone sialoprotein (Bsp), osterix (Osx), and runt‐related transcription factor 2 (Runx2)], morphological changes, and mineralized nodules in MC3T3‐E1 cells. ClC‐3 gene silencing suppressed gene expression of these osteogenic markers. Moreover, overexpressed ClC‐3 protein co‐localized with TGF‐β1 in intracellular organelles, inhibited TGF‐β1 protein expression and induced endosomal acidification. Nevertheless, knockdown of Runx2 expression antagonized the effects of ClC‐3 in osteodifferentiation and expression of osteogenic markers. The data from the current study suggest that the function of ClC‐3 in osteodifferentiation may be through the Runx2 pathway. J. Cell. Biochem. 111: 49–58, 2010. © 2010 Wiley‐Liss, Inc.