title: Chloride channel ClC-3 promotion of osteogenic differentiation through Runx2.
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-beta1 in intracellular organelles, inhibited TGF-beta1 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.
authors.affiliation: Department of Orthodontics, School of Stomatology, Fourth Military Medical University, Xi'an 710032, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Mao
authors.forename: Bin
authors.initials: B
authors.lastname: Zhang
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Feifei
authors.initials: F
authors.lastname: Li
authors.forename: Shanmin
authors.initials: S
authors.lastname: Fu
authors.forename: Yang
authors.initials: Y
authors.lastname: Xue
authors.forename: Ting
authors.initials: T
authors.lastname: Yang
authors.forename: Xuan
authors.initials: X
authors.lastname: Wen
authors.forename: Yin
authors.initials: Y
authors.lastname: Ding
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Duan
mesh_terms: D016475:3T3 Cells / Q000166:cytology / Q000502:physiology; D000818:Animals; D002454:Cell Differentiation / Q000502:physiology*; D018118:Chloride Channels / Q000235:genetics / Q000378:metabolism*; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013234:Stem Cells / Q000166:cytology / Q000502:physiology
chemical_list: D018118:Chloride Channels; C115376:ClC-3 channel; D050659:Core Binding Factor Alpha 1 Subunit; D034741:RNA, Small Interfering