Roles Of Clc-3, ClC-4 And ClC-5 Chloride Channels On Osteoblast Differentiation

BackgroundBone continues remodeling throughout the life. Bone formatioan and remodeling are the basic foundation of orthodontic treatment. The mechanisms of bone resorption and formation are the most important issue of orthodontics researches. Osteoblast is the predominant cell to anticipate the bone formation and remodeling. There are so many factors which influence on the osteogenic differentiation.The family of voltage gated chloride channels (ClCs) has nine known members in mammals. The ClC chloride channels control the excitability, transepithelial transport, ionic homeostasis, endocytotic trafficking and acidification of vesicles. Basing on their homology similarity, ClCs can be grouped into three branches. The first branch encodes plasma membrane members including ClC-1 and ClC-2. The branch of ClC-3, ClC-4 and ClC-5 are localized in intracellular vesicular system. The third branch including ClC-6 and ClC-7, resides predominantly in intracellular membranes.The second branch has drawn more attention during recent years due to their contribution to some genetic diseases. Clcn3 gene knockout mice exhibit a complex phenotype including poor growth and kyphosis. The mutations of CLCN5 cause Dent's disease and Clcn5 gene knockout mice display the identical symptoms of Dent's disease, such as hyperphosphaturia, hypercalciuria and kidney stones. Clcn5 gene knockout mice also show the abnormal spine and backward growth of teeth. The relationship between ClC-3, ClC-4, and ClC-5 and bone development has been explored recently. ClC-3 contributes to osteoclastic bone resorption in vitro through organelle acidification. ClC-4 and ClC-5 show Cl--H+ antiport activity, like the bacterial homolog ClC-ec1. ClC-5 regulates tooth development through TGF-β1 signal pathway. In this context, we hypothesized that endosomal ClCs might be involved in osteogenic differentiation.ObjectiveWe conducted the following experiments to examine the expression of Clcn3, Clcn4 and Clcn5 in osteogenic cells and to find out the possible functions of ClC-3, ClC-4 and ClC-5 in osteogenic differentiation.MethodsHere we used mouse bone marrow stromal stem cells, MC3T3-E1 osteoprogenitor cell line and primarily cultured mouse osteoblasts and detected the expression of Clcn3, Clcn4 and Clcn5 in these cells with RT-PCR. We analyzed the relationships between three endosomal ClCs and the osteogenic phenotype using osteoinductive treatment, overexpressing of ClCs and RNAi of ClCs. We used Alizarin red S staining and intracellular pH staining to explore the biological changes of MC3T3-E1 cells after overexpression of endosomal ClCs. Then, we used RNAi, Western blot and immunofluorescence analysis to explore the molecular mechanisms of ClCs-related function in osteogenic differentiation. ResultsWe have detected the positive expression of Clcn3, Clcn4 and Clcn5 in these cells. We found the increased osteogenic markers were in parallel to the increased mRNA levels of ClC-3, ClC-4 and ClC-5 with osteoinductive treatment and overexpressed ClCs. Overexpressed ClCs also promoted the mineralization of MC3T3-E1 cells in vitro and enhanced the acidification of endosomes of cells. Whilst RNAi mediated gene silencing of ClC-3, ClC-4 and ClC-5 down regulated the expression of the four osteogenic markers. Moreover, overexpressed CLC-3 protein co-localized with TGF-β1 in intracellular organelles and downregulated the expression of TGF-β1. Nevertheless, knockdown of Runx2 expression antagonized the effects of ClC-3 in osteodifferentiation and expression of osteogenic markers.ConclusionsClC-3, ClC-4 and ClC-5, the subfamily of endosomal ClCs, are expressed in MC3T3-E1 osteoprogenitor cells,primary mouse osteoblasts and mouse bone marrow stem cells. ClC-3, ClC-4 and ClC-5 and some osteogenic marker genes have a positive relationship during osteodifferentiation of MC3T3-E1 cells. ClC-3, ClC-4 and ClC-5 promote the mineralization of MC3T3-E1 cells and take part in endosomal acidification. Furthermore, Runx2 siRNA blocked the function of ClC-3 in the upregulation of gene expression in osteodifferentiation. Further investigation will elucidate the underlying mechanisms and potential use of ClC-3, ClC-4 and ClC-5 as therapeutic targets for treatment of osteogenesis-related diseases.