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MCB Accepts, published online ahead of print on 13 November 2006
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Mol. Cell. Biol. doi:10.1128/MCB.01223-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Multiple factors affecting cellular redox status and energy metabolism modulate HIF prolyl hydroxylase activity in vivo and in vitro

Yi Pan, Kyle D. Mansfield, Cara C. Bertozzi, Viktoriya Rudenko, Denise A. Chan, Amato J. Giaccia, and M. Celeste Simon*

Howard Hughes Medical Institute, University of Pennsylvania, Philadelphia, PA 19104; Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104; Center for Clinical Science Research, Department of Radiation Oncology, Stanford University, Stanford, CA 94305

* To whom correspondence should be addressed. Email: Celeste2{at}mail.med.upenn.edu.


  Abstract

Prolyl hydroxylation of HIF-{alpha} proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O2) replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O2 deprivation (hypoxia). Using a fusion protein containing the human HIF-1{alpha} O2-dependent degradation domain (ODD), we monitored PHD activity both in vivo and in cell free systems. This novel assay allows the simultaneous detection of both hydroxylated and non-hydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1{alpha} during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that levels of iron (Fe), ascorbate and various TCA cycle intermediates affect PHD activity. Intracellular levels of these factors also modulate PHD function and HIF-1{alpha} accumulation in vivo. Furthermore, cells treated with mitochondrial inhibitors, such as rotenone and myxothiazol, provided direct evidence that PHDs remain active in hypoxic cells lacking functional mitochondria. Our results suggest that multiple mitochondrial products, including TCA cycle intermediates and reactive oxygen species, can coordinate PHD activity, HIF stabilization, and cellular responses to O2 depletion.




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