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

Kyosuke Uno, Takeshi Takarada, Mika Takarada-Iemata, Yukari Kyumoto, Hiroyuki Fujita, Eiichi Hinoi, Yukio Yoneda

Research output: Contribution to journalArticle

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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 [ 14C]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.

Original languageEnglish
Pages (from-to)2953-2964
Number of pages12
JournalJournal of Cellular Physiology
Volume226
Issue number11
DOIs
Publication statusPublished - Nov 2011
Externally publishedYes

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Antiporters
Cystine
Glutamic Acid
Transcription Factors
Down-Regulation
Cell Proliferation
Core Binding Factor Alpha 1 Subunit
Messenger RNA
Osteoblasts
Bromides
Gene expression
Small Interfering RNA
Transcriptional Activation
Transfection
Alkaline Phosphatase
Cultured Cells
Gene Expression
Proteins

All Science Journal Classification (ASJC) codes

  • Physiology
  • Clinical Biochemistry
  • Cell Biology

Cite this

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. / Uno, Kyosuke; Takarada, Takeshi; Takarada-Iemata, Mika; Kyumoto, Yukari; Fujita, Hiroyuki; Hinoi, Eiichi; Yoneda, Yukio.

In: Journal of Cellular Physiology, Vol. 226, No. 11, 11.2011, p. 2953-2964.

Research output: Contribution to journalArticle

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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 [ 14C]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.",
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