Hypoxic Regulation of β-1,3-Glucuronyltransferase 1 Expression in Nucleus Pulposus Cells of the Rat Intervertebral Disc Role of Hypoxia-Inducible Factor Proteins

Objective. To determine whether hypoxia and hypoxia-inducible factor (HIF) proteins regulate expression of beta-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-l alpha and HIF-2 alpha 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-l alpha and HIF-2 alpha induced GlcAT-1 promoter activity and expression only in nucleus pulposus cells. Transfection with CA-HIF-1 alpha as well as with CA-HIF-2 alpha 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.