Hypoxic regulation of β-1,3-glucuronyltransferase 1 expression in nucleus pulposus cells of the rat intervertebral disc: role of hypoxia-inducible factor proteins

Arthritis Rheum. 2011 Jul;63(7):1950-60. doi: 10.1002/art.30342.

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.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Basic Helix-Loop-Helix Transcription Factors / genetics
  • Basic Helix-Loop-Helix Transcription Factors / metabolism*
  • Blotting, Western
  • Fluorescent Antibody Technique
  • Glucuronosyltransferase / genetics
  • Glucuronosyltransferase / metabolism*
  • Hypoxia / genetics
  • Hypoxia / metabolism*
  • Hypoxia-Inducible Factor 1, alpha Subunit / genetics
  • Hypoxia-Inducible Factor 1, alpha Subunit / metabolism*
  • Intervertebral Disc / metabolism*
  • Nitric Oxide Synthase Type II / genetics
  • Nitric Oxide Synthase Type II / metabolism
  • Promoter Regions, Genetic
  • Rats
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • Hif1a protein, rat
  • Hypoxia-Inducible Factor 1, alpha Subunit
  • endothelial PAS domain-containing protein 1
  • Nitric Oxide Synthase Type II
  • galactosylgalactoylxylosylprotein 3-beta-glucuronosyltransferase
  • Glucuronosyltransferase