title: Hypoxic regulation of β-1,3-glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: role of hypoxia-inducible factor proteins.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania, USA.
authors.forename: Shilpa S
authors.initials: SS
authors.lastname: Gogate
authors.forename: Rena
authors.initials: R
authors.lastname: Nasser
authors.forename: Irving M
authors.initials: IM
authors.lastname: Shapiro
authors.forename: Makarand V
authors.initials: MV
authors.lastname: Risbud
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D007403:Intervertebral Disc / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099162:Endothelial PAS Domain-Containing Protein 1
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D014453:Glucuronosyltransferase; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D052247:Nitric Oxide Synthase Type II; D000099162:Endothelial PAS Domain-Containing Protein 1; C497444:Hif1a protein, rat; C111364:galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase