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Am J Physiol Cell Physiol 293: C621-C631, 2007. First published April 18, 2007; doi:10.1152/ajpcell.00538.2006
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CELLULAR METABOLISM

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

Amit Agrawal,1 Asha Guttapalli,1 Srinivas Narayan,2 Todd J. Albert,1 Irving M. Shapiro,1 and Makarand V. Risbud1

1Department of Orthopaedic Surgery and Graduate Program in Tissue Engineering and Regenerative Medicine, Thomas Jefferson University, Philadelphia; and 2Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

Submitted 18 October 2006 ; accepted in final form 15 April 2007

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{alpha}, 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{alpha} 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{alpha} protein levels and mRNA stability were similar at 20 and 2% O2; there was a small, but significant increase in HIF-1{alpha} transactivation domain activity in hypoxia. With respect to HIF-1{alpha} 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{alpha} 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{alpha} in nucleus pulposus cells is a metabolic adaptation that drives glycolysis and aggrecan expression.

hypoxia; hypoxia-inducible factor-1{alpha}


Address for reprint requests and other correspondence: M. V. Risbud, Dept. of Orthopaedic Surgery, 1015 Walnut St., Suite 501 Curtis Bldg., Thomas Jefferson Univ., Philadelphia, PA 19107 (e-mail: makarand.risbud{at}jefferson.edu)






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