Open AccessHypoxic regulation of β‐1,3‐glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: Role of hypoxia‐inducible factor proteins
Author(s) -
Gogate Shilpa S.,
Nasser Rena,
Shapiro Irving M.,
Risbud Makarand V.
Publication year - 2011
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.30342
Subject(s) - hypoxia inducible factors , nucleus , chemistry , microbiology and biotechnology , transcription factor , hypoxia (environmental) , repressor , transfection , downregulation and upregulation , biology , gene , biochemistry , organic chemistry , oxygen
Objective To determine whether hypoxia and hypoxia‐inducible factor (HIF) proteins regulate expression of β‐1,3‐glucuronyltransferase 1 (GlcAT‐1), a key enzyme in glycosaminoglycan synthesis in nucleus pulposus cells. Methods Real‐time reverse transcriptase–polymerase chain reaction and Western blotting were used to measure GlcAT‐1 expression. Transfections were performed to determine the effect of HIF‐1α and HIF‐2α on GlcAT‐1 promoter activity. Results Under hypoxic conditions there was an increase in GlcAT‐1 expression; a significant increase in promoter activity was seen both in nucleus pulposus cells and in N1511 chondrocytes. We investigated whether HIF controlled GlcAT‐1 expression. Suppression of HIF‐1α and HIF‐2α induced GlcAT‐1 promoter activity and expression only in nucleus pulposus cells. Transfection with CA‐HIF‐1α as well as with CA‐HIF‐2α suppressed GlcAT‐1 promoter activity only in nucleus pulposus cells, suggesting a cell type–specific regulation. Site‐directed mutagenesis and deletion constructs were used to further confirm the suppressive effect of HIFs on GlcAT‐1 promoter function in nucleus pulposus cells. Although it was evident that interaction of HIF with hypoxia‐responsive elements resulted in suppression of basal promoter activity, it was not necessary for transcriptional suppression. This result suggested both a direct and an indirect mode of regulation, possibly through recruitment of a HIF‐dependent repressor. Finally, we showed that hypoxic expression of GlcAT‐1 was also partially dependent on MAPK signaling. Conclusion These studies demonstrate that hypoxia regulates GlcAT‐1 expression through a signaling network comprising both activator and suppressor molecules, and that this regulation is unique to nucleus pulposus cells.