Normoxic stabilization of HIF-1alpha drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk

Abstract

The nucleus pulposus is an aggrecan-rich, avascular tissue that permits the intervertebral disk to resist compressive loads. In the disk, nucleus pulposus cells express hypoxia-inducible factor (HIF)-1alpha, a transcription factor that responds to oxygen tension and regulates glycolysis. The goal of the present study was to examine the importance of HIF-1alpha in rat nucleus pulposus cells and to probe the function of this transcription factor in terms of regulating aggrecan gene expression. We found that HIF-1alpha protein levels and mRNA stability were similar at 20 and 2% O(2); there was a small, but significant increase in HIF-1alpha transactivation domain activity in hypoxia. With respect to HIF-1alpha target genes GAPDH, GLUT-1, and GLUT-3, mRNA and protein levels were independent of the oxygen tension. Other than a modest increase in 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase reporter activity, the oxemic state did not change GAPDH, GLUT-1, and GLUT-3 promoter activities. Treatment of cells with 2-deoxyglucose (2-DG), a glycolytic inhibitor, resulted in a significant suppression in ATP synthesis in normoxia, whereas treatment with mitochondrial inhibitors did not affect ATP production and cell viability. However, measurement of the rate of fatty acid oxidation indicated that these cells contained functioning mitochondria. Finally, we showed that when HIF-1alpha was suppressed, irrespective of the oxemic state, there was a partial loss of aggrecan expression and promoter activity. Moreover, when cells were treated with 2-DG, there was inhibition in aggrecan promoter activity. Results of this study indicate that oxygen-independent stabilization of HIF-1alpha in nucleus pulposus cells is a metabolic adaptation that drives glycolysis and aggrecan expression.