Differential Function of the Prolyl Hydroxylases PHD1, PHD2, and PHD3 in the Regulation of Hypoxia-inducible Factor

doi:10.1074/jbc.M406026200

Differential Function of the Prolyl Hydroxylases PHD1, PHD2, and PHD3 in the Regulation of Hypoxia-inducible Factor*

^‡Henry Wellcome Bldg. of Genomic Medicine, Roosevelt Drive, Headington, Oxford, OX3 7BN, the ^**Tumour Pathology Group, Cancer Research UK (CRUK), Nuffield Department of Clinical Laboratory Sciences, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DU, and the ^‡‡Molecular Oncology Group, CRUK, Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, United Kingdom

§§ To whom correspondence should be addressed. Tel.: 44-1-865-287-530; Fax: 44-1-865-287-535; E-mail: jgleadle{at}well.ox.ac.uk.

Abstract

Hypoxia-inducible factor (HIF) is a transcriptional regulator that plays a key role in many aspects of oxygen homeostasis. The heterodimeric HIF complex is regulated by proteolysis of its α-subunits, following oxygen-dependent hydroxylation of specific prolyl residues. Although three HIF prolyl hydroxylases, PHD1, PHD2, and PHD3, have been identified that have the potential to catalyze this reaction, the contribution of each isoform to the physiological regulation of HIF remains uncertain. Here we show using suppression by small interference RNA that each of the three PHD isoforms contributes in a non-redundant manner to the regulation of both HIF-1α and HIF-2α subunits and that the contribution of each PHD under particular culture conditions is strongly dependent on the abundance of the enzyme. Thus in different cell types, isoform-specific patterns of PHD induction by hypoxia and estrogen alter both the relative abundance of the PHDs and their relative contribution to the regulation of HIF. In addition, the PHDs manifest specificity for different prolyl hydroxylation sites within each HIF-α subunit, and a degree of selectively between HIF-1α and HIF-2α isoforms, indicating that differential PHD inhibition has the potential to selectively alter the characteristics of HIF activation.

Footnotes

↵1 The abbreviations used are: HIF, hypoxia-inducible factor; PHD, prolyl hydroxylase domain; siRNA, small interfering RNA; HREs, hypoxia response elements; PBS, phosphate-buffered saline; ODD, oxygen-dependent degradation domain; NODD, N-terminal oxygen-dependent degradation domain; CODD, C-terminal oxygen-dependent degradation domain; VP16, transactivation domain from the herpes simplex virus protein 16 (amino acids 410–490); Gal, the N-terminal 147 amino acids of the yeast transcription factor Gal4; IVTT, in vitro transcription and translation; CA-IX, carbonic anhydrase IX; mAb, monoclonal antibody.
↵* This work was supported in part by the Wellcome Trust, the Medical Research Council, UK, and Cancer Research UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§ Both authors contributed equally to the work and are joint first authors.
↵¶ A recipient of a Wellcome Trust Prize Studentship.
↵∥ Supported by a Rhodes Scholarship.
- Received June 1, 2004.
- Revision received July 2, 2004.
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