CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1α expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition

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Abstract

Vascular endothelial growth factor (VEGF) is essential for establishing vascularization and regulating chondrocyte development and survival. We have demonstrated that VEGF regulates the expression of CCN2/connective tissue growth factor (CCN2/CTGF) an essential mediator of cartilage development and angiogenesis, suggesting that CCN2 functions in down-stream of VEGF, and that VEGF function is mediated in part by CCN2. On the other hand, the phenotype of Ccn2 mutant growth plates, which exhibit decreased expression of VEGF in the hypertrophic zone, indicates that Vegf expression is dependent on Ccn2 expression as well. Therefore, we investigated the molecular mechanisms underlying the induction of VEGF by CCN2 using a human chondrocytic cell line, HCS-2/8. Hypoxic stimulation (5% O2) of HCS-2/8 cells increased VEGF mRNA levels by ∼ 8 fold within 6 h as compared with the cells cultured under normoxia. In addition, VEGF expression was further up-regulated under hypoxia in HCS-2/8 cells transfected with a Ccn2 expression plasmid. Hypoxia-inducible factor (HIF)-1α mRNA and protein levels were increased by stimulation with recombinant CCN2 (rCCN2). Furthermore, the activity of a VEGF promoter that contained a HIF-1 binding site was increased in HCS-2/8, when the cells were stimulated by rCCN2. These results suggest that CCN2 regulates the expression of VEGF at a transcriptional level by promoting HIF-1α activity. In fact, HIF-1α was detected in the nuclei of proliferative and pre-hypertrophic chondrocytes of wild-type mice, whereas it was not detected in Ccn2 mutant chondrocytes in vivo. This activation cascade from CCN2 to VEGF may therefore play a critical role in chondrocyte development and survival.

Original languageEnglish
Pages (from-to)24-31
Number of pages8
JournalBone
Volume44
Issue number1
DOIs
Publication statusPublished - Jan 2009

Fingerprint

Connective Tissue Growth Factor
Vascular Endothelial Growth Factor A
Cell Line
Hypoxia-Inducible Factor 1
Chondrocytes
Messenger RNA
Growth Plate
Survival
Cartilage
Cultured Cells

Keywords

  • CCN family 2/connective tissue growth factor (CCN2/CTGF)
  • Chondrocyte differentiation
  • Hypoxia-inducible factor (HIF)-1α
  • Signal conductor
  • Vascular endothelial growth factor (VEGF)

ASJC Scopus subject areas

  • Physiology
  • Endocrinology, Diabetes and Metabolism
  • Histology

Cite this

@article{8a4514958ce442c68ebae5a582a24f67,
title = "CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1α expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition",
abstract = "Vascular endothelial growth factor (VEGF) is essential for establishing vascularization and regulating chondrocyte development and survival. We have demonstrated that VEGF regulates the expression of CCN2/connective tissue growth factor (CCN2/CTGF) an essential mediator of cartilage development and angiogenesis, suggesting that CCN2 functions in down-stream of VEGF, and that VEGF function is mediated in part by CCN2. On the other hand, the phenotype of Ccn2 mutant growth plates, which exhibit decreased expression of VEGF in the hypertrophic zone, indicates that Vegf expression is dependent on Ccn2 expression as well. Therefore, we investigated the molecular mechanisms underlying the induction of VEGF by CCN2 using a human chondrocytic cell line, HCS-2/8. Hypoxic stimulation (5{\%} O2) of HCS-2/8 cells increased VEGF mRNA levels by ∼ 8 fold within 6 h as compared with the cells cultured under normoxia. In addition, VEGF expression was further up-regulated under hypoxia in HCS-2/8 cells transfected with a Ccn2 expression plasmid. Hypoxia-inducible factor (HIF)-1α mRNA and protein levels were increased by stimulation with recombinant CCN2 (rCCN2). Furthermore, the activity of a VEGF promoter that contained a HIF-1 binding site was increased in HCS-2/8, when the cells were stimulated by rCCN2. These results suggest that CCN2 regulates the expression of VEGF at a transcriptional level by promoting HIF-1α activity. In fact, HIF-1α was detected in the nuclei of proliferative and pre-hypertrophic chondrocytes of wild-type mice, whereas it was not detected in Ccn2 mutant chondrocytes in vivo. This activation cascade from CCN2 to VEGF may therefore play a critical role in chondrocyte development and survival.",
keywords = "CCN family 2/connective tissue growth factor (CCN2/CTGF), Chondrocyte differentiation, Hypoxia-inducible factor (HIF)-1α, Signal conductor, Vascular endothelial growth factor (VEGF)",
author = "Takashi Nishida and Seiji Kondo and Azusa Maeda and Satoshi Kubota and Lyons, {Karen M.} and Masaharu Takigawa",
year = "2009",
month = "1",
doi = "10.1016/j.bone.2008.08.125",
language = "English",
volume = "44",
pages = "24--31",
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T1 - CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1α expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition

AU - Nishida, Takashi

AU - Kondo, Seiji

AU - Maeda, Azusa

AU - Kubota, Satoshi

AU - Lyons, Karen M.

AU - Takigawa, Masaharu

PY - 2009/1

Y1 - 2009/1

N2 - Vascular endothelial growth factor (VEGF) is essential for establishing vascularization and regulating chondrocyte development and survival. We have demonstrated that VEGF regulates the expression of CCN2/connective tissue growth factor (CCN2/CTGF) an essential mediator of cartilage development and angiogenesis, suggesting that CCN2 functions in down-stream of VEGF, and that VEGF function is mediated in part by CCN2. On the other hand, the phenotype of Ccn2 mutant growth plates, which exhibit decreased expression of VEGF in the hypertrophic zone, indicates that Vegf expression is dependent on Ccn2 expression as well. Therefore, we investigated the molecular mechanisms underlying the induction of VEGF by CCN2 using a human chondrocytic cell line, HCS-2/8. Hypoxic stimulation (5% O2) of HCS-2/8 cells increased VEGF mRNA levels by ∼ 8 fold within 6 h as compared with the cells cultured under normoxia. In addition, VEGF expression was further up-regulated under hypoxia in HCS-2/8 cells transfected with a Ccn2 expression plasmid. Hypoxia-inducible factor (HIF)-1α mRNA and protein levels were increased by stimulation with recombinant CCN2 (rCCN2). Furthermore, the activity of a VEGF promoter that contained a HIF-1 binding site was increased in HCS-2/8, when the cells were stimulated by rCCN2. These results suggest that CCN2 regulates the expression of VEGF at a transcriptional level by promoting HIF-1α activity. In fact, HIF-1α was detected in the nuclei of proliferative and pre-hypertrophic chondrocytes of wild-type mice, whereas it was not detected in Ccn2 mutant chondrocytes in vivo. This activation cascade from CCN2 to VEGF may therefore play a critical role in chondrocyte development and survival.

AB - Vascular endothelial growth factor (VEGF) is essential for establishing vascularization and regulating chondrocyte development and survival. We have demonstrated that VEGF regulates the expression of CCN2/connective tissue growth factor (CCN2/CTGF) an essential mediator of cartilage development and angiogenesis, suggesting that CCN2 functions in down-stream of VEGF, and that VEGF function is mediated in part by CCN2. On the other hand, the phenotype of Ccn2 mutant growth plates, which exhibit decreased expression of VEGF in the hypertrophic zone, indicates that Vegf expression is dependent on Ccn2 expression as well. Therefore, we investigated the molecular mechanisms underlying the induction of VEGF by CCN2 using a human chondrocytic cell line, HCS-2/8. Hypoxic stimulation (5% O2) of HCS-2/8 cells increased VEGF mRNA levels by ∼ 8 fold within 6 h as compared with the cells cultured under normoxia. In addition, VEGF expression was further up-regulated under hypoxia in HCS-2/8 cells transfected with a Ccn2 expression plasmid. Hypoxia-inducible factor (HIF)-1α mRNA and protein levels were increased by stimulation with recombinant CCN2 (rCCN2). Furthermore, the activity of a VEGF promoter that contained a HIF-1 binding site was increased in HCS-2/8, when the cells were stimulated by rCCN2. These results suggest that CCN2 regulates the expression of VEGF at a transcriptional level by promoting HIF-1α activity. In fact, HIF-1α was detected in the nuclei of proliferative and pre-hypertrophic chondrocytes of wild-type mice, whereas it was not detected in Ccn2 mutant chondrocytes in vivo. This activation cascade from CCN2 to VEGF may therefore play a critical role in chondrocyte development and survival.

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KW - Vascular endothelial growth factor (VEGF)

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