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

Rebecca J Appelhoff,Ya-Min Tian,Raju R Raval,Helen Turley,Adrian L Harris,Christopher W Pugh,Peter J Ratcliffe,Jonathan M Gleadle

doi:10.1074/jbc.m406026200

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 alpha-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-1alpha and HIF-2alpha 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-alpha subunit, and a degree of selectively between HIF-1alpha and HIF-2alpha isoforms, indicating that differential PHD inhibition has the potential to selectively alter the characteristics of HIF activation.

Highlights

Hypoxia-inducible factor (HIF) is a transcriptional regulator that plays a key role in many aspects of oxygen homeostasis
The response of cells to hypoxia is characterized by specific alterations in the expression of a large number of genes, many of which are regulated directly or indirectly by hypoxia-inducible factor (HIF),1 a transcriptional regulator that plays a key role in many aspects of oxygen homeostasis [1,2,3]
Whereas levels of PHD1 mRNA were unchanged or significantly decreased by hypoxia (ZR75–1, OVCAR-3, and JAR), levels of PHD2 and PHD3 were increased by hypoxia, induction being striking for PHD3 mRNA

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—The human cell lines studied were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 IU/ml penicillin, and 50 ␮g/ml streptomycin except for BT-474, OVCAR-3, 833K, and SuSa, which were cultured in RPMI 1640 and A549 in Ham’s F-12, both with identical supplements. Test bleeds were examined by immunoblot analysis of COS7 cell extract transfected with pcDNA3 PHD1. Hybridoma supernatants were screened by enzyme-linked immunosorbent assays against the fusion proteins and fusion tags alone and immunolabeling of COS7 cells, which were transfected with corresponding PHD plasmids. Positive clones were confirmed by immunoblot analysis of COS7 cell extracts of PHD transfectants and are designated mAb P1-112, mAb P2-76a, and mAb P3-188e. The cells were co-transfected using Fugene transfection reagent (Roche Applied Science) with plasmids expressing a Gal4responsive luciferase reporter (pUAS.tk.Luc, 0.4 ␮g), ␤-galactosidase (pCMV␤Gal, 0.12 ␮g), Gal4/HIF/VP16 fusions [35], and pcDNA3 PHD1, PHD2, or PHD3 [12]. Gal4/HIF/VP16 plasmid doses were in the range 1– 4 ng, based on preliminary titration experiments that aimed to generate approximately similar luciferase activities in cells not cotransfected with PHD expression plasmids (see Fig. 8, A and B, control). The dose of transfected plasmid encoding the PHD enzymes was ad-

HIF Regulation by Prolyl Hydroxylases

RESULTS

DISCUSSION