Cited2 modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc.
OBJECTIVE
To determine whether nucleus pulposus cells of the intervertebral disc express hypoxia-inducible factor 2alpha (HIF-2alpha), and to assess the role of HIF-1 and HIF-2 in controlling cited2 and vascular endothelial growth factor (VEGF) expression.


METHODS
Rat cells were cultured under normoxic (21% O2) or hypoxic (2% O2) conditions, and expression and promoter activity of HIF-2 target genes were evaluated. Gain- or loss-of-function experiments were performed to investigate the contribution of HIF isoforms to cited2 activity as well as the role of cited2 in regulating VEGF expression.


RESULTS
We found that HIF-2alpha protein was expressed in vivo and that protein and messenger RNA expression were similar under both normoxic and hypoxic conditions. However, there was a significant increase in HIF-2alpha transactivation under hypoxic conditions. With respect to functional activity, unlike the case in most other tissues, HIF-2 failed to increase the transcriptional activities of superoxide dismutase 2 and frataxin, 2 common target genes involved in radical dismutation. However, under hypoxic conditions, HIF-2 preferentially regulated the expression and promoter activity of cited2, a p300 binding protein. When HIF-2alpha or HIF-1alpha was suppressed, cited2 promoter activity was inhibited. Finally, we showed that forced expression or suppression of cited2 resulted in corresponding changes in expression of VEGF, a common target gene for HIF-1 and HIF-2 in the nucleus pulposus cells.


CONCLUSIONS
Results of this study indicate that in nucleus pulposus cells, HIF-2 and HIF-1 modulate their own transcriptional activity through cited2. We suggest that the 2 arms of the regulatory circuit serve to maintain survival activities and inhibit angiogenesis in the healthy disc.
Agrawal
Amit
A
Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Gajghate
Sachin
S
Smith
Harvey
H
Anderson
D Greg
DG
Albert
Todd J
TJ
Shapiro
Irving M
IM
Risbud
Makarand V
MV
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D015870:Gene Expression / Q000502:physiology; D000860:Hypoxia / Q000378:metabolism / Q000503:physiopathology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007403:Intervertebral Disc / Q000166:cytology / Q000502:physiology*; D033862:Iron-Binding Proteins / Q000235:genetics; D008297:Male; D010100:Oxygen / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology; D013482:Superoxide Dismutase / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*; D000096923:Frataxin; D000099162:Endothelial PAS Domain-Containing Protein 1; D000099156:Superoxide Dismutase 2
D051792:Basic Helix-Loop-Helix Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D033862:Iron-Binding Proteins; D010100:Oxygen; D012333:RNA, Messenger; D013482:Superoxide Dismutase; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D000096923:Frataxin; D000099162:Endothelial PAS Domain-Containing Protein 1; D000099156:Superoxide Dismutase 2; C517260:CITED2 protein, rat; C497444:Hif1a protein, rat; C467498:vascular endothelial growth factor A, rat