Glutamate Inhibits Chondral Mineralization through Apoptotic Cell Death Mediated by Retrograde Operation of the Cystine/Glutamate Antiporter

Wang, Liyang; Hinoi, Eiichi; Takemori, Akihiro; Nakamichi, Noritaka; Yoneda, Yukio

doi:10.1074/jbc.m600939200

Cited by 43 publications

(43 citation statements)

References 33 publications

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“…This allowed us to investigate the effects of FK506 in an experimental system where chondrocytes retain their normal architecture of round and columnar prehypertrophic and hypertrophic zones. The absence of mesenchymal cells, other than chondrocytes, from any of the cell layers, except those in bone collar or perichondrium of cultured metatarsals, is already confirmed by in situ hybridization in our previous study (36).…”

Section: Discussionsupporting

confidence: 49%

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes

et al. 2009

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Abstract. Tacrolimus (FK506) has been used as a therapeutic drug beneficial for the treatment of rheumatoid arthritis in humans. In this study, we investigated the effects of FK506 on cellular differentiation in cultured chondrogenic cells. Culture with FK506 led to a significant and concentration-dependent increase in Alcian blue staining for matrix proteoglycan at 0.1 to 1,000 ng/ml, but not in alkaline phosphatase (ALP) activity, in ATDC5 cells, a mouse prechondrogenic cell line, cultured for 7 to 28 days, while the non-steroidal anti-inflammatory drug indomethacin significantly decreased Alcian blue staining in a concentration-dependent manner, without altering ALP activity. FK506 significantly increased the expression of mRNA for both type II and type X collagen, but not for osteopontin, in ATDC5 cells. Similar promotion was seen in chondrogenic differentiation in both mouse metatarsals and chondrocytes cultured with FK506. However, FK506 failed to significantly affect transcriptional activity of the reporter construct for either sry-type HMG box 9 (Sox9) or runt-related transcription factor-2 (Runx2), which are both transcription factors responsible for chondrocytic maturation as a master regulator. These results suggest that FK506 may predominantly promote cellular differentiation into proliferating chondrocytes through a mechanism not relevant to the transactivation by either Sox9 or Runx2 in chondrogenic cells.

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Section: Discussionsupporting

confidence: 49%

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes

et al. 2009

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“…To our knowledge, this is the first direct demonstration of a negative correlation between expression profiles of Runx2 and xCT subunit during osteoblastogenesis in ovariectomized mouse femurs in vivo and in pre-osteoblastic MC3T3-E1 cells in vitro. Although several previous studies including ours have already demonstrated the functional expression of the Xc − in several cells such as chondrocytes (20), dendritic cells (27), and meningeal cells (28), no direct evidence is available for a pivotal role of the Xc − in the cellular differentiation of osteoblas- Intracellular glutathione levels are regulated by the bidirectional Xc − system expressed at the cellular surface as a crucial determinant of the incorporation of extracellular cystine across plasma membranes to fuel the substrate cysteine in exchange for intracellular Glu in a variety of eukaryotic cells (12,15,16,29). Under the condition of high extracellular Glu levels, however, extracellular Glu is supposed to be taken up in exchange for intracellular cystine through the promoted retrograde operation of the Xc − , followed by decreased intracellular levels of GSH toward cell death mediated by GSH depletion (15,16).…”

Section: Discussionmentioning

confidence: 99%

“…Under the condition of high extracellular Glu levels, however, extracellular Glu is supposed to be taken up in exchange for intracellular cystine through the promoted retrograde operation of the Xc − , followed by decreased intracellular levels of GSH toward cell death mediated by GSH depletion (15,16). Although marked apoptosis is induced by extracellular Glu at a high concentration, but not by agonists for any Glu-receptor subtypes, in association with GSH depletion in cultured rat costal chondrocytes (20), exposure to Glu at a high concentration leads to marked inhibition of cellular proliferation through a mechanism related to GSH depletion without inducing apoptotic cell death in osteoblastic MC3T3-E1 cells (17). It is thus conceivable that extracellular Glu at a high concentration could induce apoptosis through a mechanism associated with the depletion of intracellular GSH as a consequence of the promoted retrograde operation of the Xc − in chondrocytes, but not in osteoblasts.…”

Section: Discussionmentioning

confidence: 99%

“…The reverse transcriptase reaction was run at 37°C for 50 min with bone and cell extracts, and an aliquot of synthesized cDNA was directly used for PCR performed in buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl 2 , 200 mM each of dNTP, 20 pmol of each primer, and 2 U of Taq DNA polymerase as reported elsewhere (20). Reactions were initiated by the incubation at 94°C for 5 min, and PCR (denaturation at 94°C for 1 min, annealing at 64°C for 1 min, and extension at 72°C for 1 min, respectively) was performed for 28 cycles with a final extension at 72°C for 10 min.…”

Section: Reverse Transcription Polymerase Chain Reaction (Rt-pcr) Anamentioning

confidence: 99%

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A Negative Correlation Between Expression Profiles of Runt-Related Transcription Factor-2 and Cystine/Glutamate Antiporter xCT Subunit in Ovariectomized Mouse Bone

et al. 2011

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Abstract.We have previously demonstrated that glutamate (Glu) suppresses cellular proliferation toward self-renewal through a mechanism associated with the depletion of intracellular GSH after promoting the retrograde operation of the bidirectional cystine/Glu antiporter in undifferentiated osteoblastic MC3T3-E1 cells. In this study, we investigated the expression profile of the xCT subunit of the antiporter as well as the master regulator of osteoblastogenesis runt-related transcription factor-2 (Runx2) in ovariectomized mouse bone. In spinal columns isolated 28 days after ovariectomy, a marked reduction was seen with the intensity of Von Kossa staining used as an index of ossification. In femurs of these ovariectomized mice, a significant decrease was seen in mRNA and protein levels of Runx2 along with increased expression of both mRNA and the corresponding protein for the xCT subunit. To evaluate the possible role of the antiporter in osteoblastogenesis, stable transfectants were established with the xCT subunit toward the culture with osteoblastic differentiation inducers in MC3T3-E1 cells. In stable xCT transfectants cultured under differentiation conditions, marked decreases were seen in nodule formation, Ca 2+ accumulation, and osteoblastic marker gene expression, in addition to downregulation of both mRNA and the corresponding protein for Runx2. Runx2 promoter activity was markedly stimulated in MC3T3-E1 cells transfected with a responsive promoter plasmid after the culture under differentiation conditions, while transient and stable transfection with xCT expression vector invariably prevented the stimulation through an activator protein-1 site. These results suggest that Runx2 expression would be negatively regulated by the cystine/glutamate antiporter expressed by osteoblastic cells at the level of gene transactivation.

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“…Determination of ALP activity and Alcian blue staining were done as described previously (21). In brief, chondrocytes were solubilized with 0.1% Triton X-100 followed by determination of the ALP activity in lysates using p-nitrophenol phosphate as a substrate.…”

Section: Determination Of Alp Activity and Alcian Blue Staining-mentioning

confidence: 99%

Positive Regulation by γ-Aminobutyric Acid B Receptor Subunit-1 of Chondrogenesis through Acceleration of Nuclear Translocation of Activating Transcription Factor-4

Takahata¹,

Hinoi²,

Takarada

et al. 2012

Journal of Biological Chemistry

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Background: GABA B R is functionally expressed in a variety of peripheral organs outside the central nervous system. Results: Mice defective in GABA B R subunit-1 (GABA B R1) show delayed calcification. Conclusion: GABA B R1 molecule alone positively regulates chondrogenesis through accelerating nuclear translocation of activating transcription factor-4. Significance: GABA B R1 is a target candidate for the drug therapy of bone diseases relevant to endochondral ossification impairments.

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Glutamate Inhibits Chondral Mineralization through Apoptotic Cell Death Mediated by Retrograde Operation of the Cystine/Glutamate Antiporter

Cited by 43 publications

References 33 publications

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes

Predominant Promotion by Tacrolimus of Chondrogenic Differentiation to Proliferating Chondrocytes

A Negative Correlation Between Expression Profiles of Runt-Related Transcription Factor-2 and Cystine/Glutamate Antiporter xCT Subunit in Ovariectomized Mouse Bone

Positive Regulation by γ-Aminobutyric Acid B Receptor Subunit-1 of Chondrogenesis through Acceleration of Nuclear Translocation of Activating Transcription Factor-4

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