Hypoxia-inducible factor regulation of ANK expression in nucleus pulposus cells: possible implications in controlling dystrophic mineralization in the intervertebral disc.
OBJECTIVE
Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins.


METHODS
Quantitative reverse transcription-polymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity.


RESULTS
ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1α and HIF-2α resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1β-null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1α and HIF-2α in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxia-responsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state.


CONCLUSIONS
Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions.
Skubutyte
Renata
R
Jefferson Medical College, Philadelphia, Pennsylvania, USA.
Markova
Dessislava
D
Freeman
Theresa A
TA
Anderson
D Greg
DG
Dion
Arnold S
AS
Williams
Charlene J
CJ
Shapiro
Irving M
IM
Risbud
Makarand V
MV
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism*; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D015153:Blotting, Western; D002114:Calcinosis / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression / Q000187:drug effects; D020868:Gene Silencing; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007403:Intervertebral Disc / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051379:Mice; D018345:Mice, Knockout; D008875:Middle Aged; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D000099162:Endothelial PAS Domain-Containing Protein 1
D017487:Ankyrins; D051792:Basic Helix-Loop-Helix Proteins; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D000099162:Endothelial PAS Domain-Containing Protein 1; C540195:Ank1 protein, rat; C497444:Hif1a protein, rat