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

Abstract

Agrawal A, Guttapalli A, Narayan S, Albert TJ, Shapiro IM, Risbud MV. Normoxic stabilization of HIF-1␣ drives glycolytic metabolism and regulates aggrecan gene expression in nucleus pulposus cells of the rat intervertebral disk. Am J Physiol Cell Physiol 293: C621-C631, 2007. First published April 18, 2007; doi:10.1152/ajpcell.00538.2006.-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)-1␣, a transcription factor that responds to oxygen tension and regulates glycolysis. The goal of the present study was to examine the importance of HIF-1␣ 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-1␣ protein levels and mRNA stability were similar at 20 and 2% O2; there was a small, but significant increase in HIF-1␣ transactivation domain activity in hypoxia. With respect to HIF-1␣ 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-1␣ 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-1␣ in nucleus pulposus cells is a metabolic adaptation that drives glycolysis and aggrecan expression. hypoxia; hypoxia-inducible factor-1␣ THE LOCAL OXYGEN TENSION promotes cellular differentiation and regulates tissue energy metabolism. When the oxygen tension is low, there is almost complete reliance on glycolysis to generate ATP and reducing equivalents (12,29,30). In specialized tissues such as the intervertebral disk, the mechanism by which cells conserve energy has received little attention. Nonetheless, it is known that in the intervertebral disk, the nucleus pulposus has no direct vascular supply (8,24,33), and ATP is probably generated by anaerobic glycolysis (1, 10). The dependence on glycolysis reflects the low oxygen tension in the disk, which has been reported to be low as low as 2%, and the observation that modulation in the oxygen supply can cause vertebral defects (15). Regulation of glycolysis is mediated by hypoxia-inducible factor (HIF)-1␣, a transcription factor that responds to the local oxygen tension, and has been show...

Cited2 modulates hypoxia‐inducible factor–dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc

“…In specialized tissues such as the intervertebral disc, the mechanism by which cells conserve energy has received little attention. Nonetheless, since the gelatinous center, the nucleus pulposus, has no vascular supply (4,5), it is probable that ATP is generated by anaerobic glycolysis (6,7). Regulation of glycolysis is mediated by hypoxia-inducible factor 1␣ (HIF-1␣), a key transcription factor that is expressed by the nucleus pulposus cells (8).…”

“…Regulation of glycolysis is mediated by hypoxia-inducible factor 1␣ (HIF-1␣), a key transcription factor that is expressed by the nucleus pulposus cells (8). Unlike other skeletal cells such as chondrocytes, in the nucleus pulposus there is normoxic stabilization of this otherwise oxygen-labile protein (6,9). Moreover, despite an absence of HIF-1␣ protein induction in hypoxia, an increase in its transcriptional activity was seen (6,9).…”

“…Unlike other skeletal cells such as chondrocytes, in the nucleus pulposus there is normoxic stabilization of this otherwise oxygen-labile protein (6,9). Moreover, despite an absence of HIF-1␣ protein induction in hypoxia, an increase in its transcriptional activity was seen (6,9). In a recent study of nucleus pulposus cells, Agrawal et al demonstrated that this transcription factor controlled both glycolysis and matrix synthesis (6).…”

“…Moreover, despite an absence of HIF-1␣ protein induction in hypoxia, an increase in its transcriptional activity was seen (6,9). In a recent study of nucleus pulposus cells, Agrawal et al demonstrated that this transcription factor controlled both glycolysis and matrix synthesis (6).…”

New evidence of the role of the hypoxia‐regulated pathway in nucleus pulposus cell survival: Comment on the article by Hiyama et al

“…"Despite all that has changed in the practice of rheumatology over the past 34 years, I am actually amazed at how much has stayed the same," wrote Dr. Neal S. Birnbaum, President of the ACR in 2007 (4). The future of rheumatology certainly will be brighter because of the legacy of these early rheumatologists, some of whom unfortunately are no longer with us.…”