HIF prolyl-hydroxylase 2 is the key oxygen sensor setting low steady-state levels of HIF-1alpha in normoxia

Abstract

Hypoxia-inducible factor (HIF), a transcriptional complex conserved from Caenorhabditis elegans to vertebrates, plays a pivotal role in cellular adaptation to low oxygen availability. In normoxia, the HIF-alpha subunits are targeted for destruction by prolyl hydroxylation, a specific modification that provides recognition for the E3 ubiquitin ligase complex containing the von Hippel-Lindau tumour suppressor protein (pVHL). Three HIF prolyl-hydroxylases (PHD1, 2 and 3) were identified recently in mammals and shown to hydroxylate HIF-alpha subunits. Here we show that specific 'silencing' of PHD2 with short interfering RNAs is sufficient to stabilize and activate HIF-1alpha in normoxia in all the human cells investigated. 'Silencing' of PHD1 and PHD3 has no effect on the stability of HIF-1alpha either in normoxia or upon re-oxygenation of cells briefly exposed to hypoxia. We therefore conclude that, in vivo, PHDs have distinct assigned functions, PHD2 being the critical oxygen sensor setting the low steady-state levels of HIF-1alpha in normoxia. Interestingly, PHD2 is upregulated by hypoxia, providing an HIF-1-dependent auto-regulatory mechanism driven by the oxygen tension.

Fig. 1. Specific silencing of PHD isoforms using siRNAs. Northern blot showing total RNA isolated from HeLa cells after 48 h of transfection with the indicated siRNAs (20 nM). Blots were hybridized with ³²P- labelled probes specific for PHD1, PHD2, PHD3 or 36B4 (loading control). Inset shows localization of the PHD region targeted by the two independent sets of siRNAs (filled squares and double asterisks).

Fig. 2. Role of PHD isoforms in HIF-1α regulation. (A) Silencing of PHD2 upregulates HIF-1α expression in normoxia. HeLa cells were transfected with siRNAs (20 nM) and, 48 h later, were analysed by immunofluorescence microscopy using the anti-HIF-1α antibody. As a control for HIF-1α induction, cells were incubated in the presence of Co²⁺ (200 µM) for 4 h. (B) PHD2 silencing upregulates HIF-1α in a dose-dependent manner. HeLa cells were transfected with either human HIF-1α siRNA (200 nM) or increasing doses of PHD2 siRNA, and HIF-1α expression was analysed by western blotting after 48 h of transfection (left panel). Extracts of cells incubated in normoxia (N; 20% O₂) or hypoxia (H; 1–2% O₂) for 4 h were resolved as a control (right panel). Western blots were re-probed using the anti-p42MAPK antibody to check for total protein loading.

Fig. 2. Role of PHD isoforms in HIF-1α regulation. (A) Silencing of PHD2 upregulates HIF-1α expression in normoxia. HeLa cells were transfected with siRNAs (20 nM) and, 48 h later, were analysed by immunofluorescence microscopy using the anti-HIF-1α antibody. As a control for HIF-1α induction, cells were incubated in the presence of Co²⁺ (200 µM) for 4 h. (B) PHD2 silencing upregulates HIF-1α in a dose-dependent manner. HeLa cells were transfected with either human HIF-1α siRNA (200 nM) or increasing doses of PHD2 siRNA, and HIF-1α expression was analysed by western blotting after 48 h of transfection (left panel). Extracts of cells incubated in normoxia (N; 20% O₂) or hypoxia (H; 1–2% O₂) for 4 h were resolved as a control (right panel). Western blots were re-probed using the anti-p42MAPK antibody to check for total protein loading.

Fig. 3. PHD2 silencing upregulates HIF-1α in all of the human cell lines so far analysed. Immunofluorescence staining showing cells transfected with 20 nM of an irrelevant siRNA (Control or C) or the PHD2 siRNA (PHD2) using the anti-HIF-1α antibody. As a positive control, cells were incubated in the presence of Co²⁺ (200 µM) for 4 h. The following cells are shown: CAL51 (breast cancer cell line), RCC4/pVHL (RCC4 stably transfected with pCDNA3pVHL), WM9 (melanoma cell line) and FHN (primary fibroblasts).

Fig. 4. HIF-1α induced by PHD2 silencing is functionally active. The indicated siRNAs (20 nM) were transfected into HeLa cells with 50 ng of a reporter vector (pRE-Δtk-LUC) containing three copies of the HRE from the erythropoietin gene. In all cases, 100 ng of a plasmid for β-galactosidase were co-transfected to normalize for transfection efficiency. After 48 h of transfection, luciferase activity was measured. As a control for HIF-1 activation, cells were incubated in the presence of Co²⁺ (200 µM) or in hypoxia (1–2% O₂) for 16 h. Results are representative of three independent experiments performed in triplicate.

Fig. 5. PHD2 controls HIF-1α stability upon re-oxygenation of hypoxic cells. HeLa cells transfected with siRNAs (200 nM) were incubated in normoxia (N; 20% O₂) or hypoxia (H; 1–2% O₂) for 4 h, after which hypoxic cells were returned to 20% O₂ for 0–180 min. Total cellular extracts were immunoblotted with the anti-HIF-1α antibody.

Fig. 6. phd2 is a hypoxia-inducible gene. (A) Specificity of the anti-PHD2 antibody. Western blot of total cell extracts of HeLa cells after 48 h of transfection with the indicated siRNAs (20 nM) using an anti-PHD2 antibody. (B) PHD2 is upregulated by hypoxia. Western blot showing the hypoxic induction of PHD2. Immunoblotting was performed using total cellular extracts of HeLa cells incubated in normoxia (N; 20% O₂) or hypoxia (1–2% O₂) for different periods of time. (C) Hypoxic induction of PHD2 is HIF-1 dependent. Western blot of total cell extracts of HeLa cells transfected in either the absence or presence of a human HIF-1α siRNA. Immunoblotting was performed with antibodies against HIF-1α and PHD2.