title: Negative regulation of osteoblastogenesis through downregulation of runt-related transcription factor-2 in osteoblastic MC3T3-E1 cells with stable overexpression of the cystine/glutamate antiporter xCT subunit.
abstract: We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with intracellular GSH depletion mediated by the bidirectional cystine/Glu antiporter in osteoblastic MC3T3-E1 cells cultured in the absence of differentiation inducers. To further evaluate the possible role of the antiporter in osteoblastogenesis, in this study, we have established stable transfectants of the xCT subunit of the antiporter in MC3T3-E1 cells. Stable overexpression led to a significant facilitation of cellular proliferation determined by different indices with increased GSH levels and decreased ROS generation in addition to promoted [(14)C]cystine incorporation, while Glu failed to significantly inhibit cellular proliferation in stable xCT transfectants. In stable transfectants cultured under differentiation conditions, drastic decreases were invariably seen in Ca(2+) accumulation, alkaline phosphatase activity and several osteoblastic marker gene expressions, in addition to downregulation of mRNA and corresponding protein for runt-related transcription factor-2 (Runx2). Runx2 promoter activity was significantly promoted by the introduction of Runx2 expression vector in a manner sensitive to the prevention by the co-introduction of xCT expression vector in MC3T3-E1 cells. In both MC3T3-E1 cells and murine calvarial osteoblasts cultured with differentiation inducers, transient transfection with xCT siRNA significantly increased Runx2 protein expression along with decreases in xCT mRNA expression and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction. These results suggest that the cystine/Glu antiporter plays a pivotal role in cellular differentiation through a mechanism related to the regulation of transactivation of Runx2 essential for osteoblastogenesis toward maturation in osteoblastic cells.
authors.affiliation: Laboratory of Molecular Pharmacology, Division of Pharmaceutical Sciences, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa, Ishikawa, Japan.
authors.forename: Kyosuke
authors.initials: K
authors.lastname: Uno
authors.forename: Takeshi
authors.initials: T
authors.lastname: Takarada
authors.forename: Mika
authors.initials: M
authors.lastname: Takarada-Iemata
authors.forename: Yukari
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Fujita
authors.forename: Eiichi
authors.initials: E
authors.lastname: Hinoi
authors.forename: Yukio
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D027182:Amino Acid Transport System y+ / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D010012:Osteogenesis; D011401:Promoter Regions, Genetic / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D027182:Amino Acid Transport System y+; D050659:Core Binding Factor Alpha 1 Subunit; D017382:Reactive Oxygen Species; C493716:Runx2 protein, mouse; C492255:Slc7a11 protein, mouse; D013778:Tetrazolium Salts; D013844:Thiazoles; D000469:Alkaline Phosphatase; C022616:thiazolyl blue; D005978:Glutathione; D002118:Calcium