Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-alpha-prolyl-4-hydroxylases - PubMed (original) (raw)

Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-alpha-prolyl-4-hydroxylases

Jan H Marxsen et al. Biochem J. 2004.

Abstract

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.

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Figures

Figure 1

Figure 1. PHD induction in HepG2 cells by hypoxia

(a) Microarray analysis. Cells were incubated in an atmosphere of 1% O2 for 4 h. cDNA probes were generated as described in the Materials and methods section. Normoxic (NOX) and hypoxic (HOX) probes were combined and hybridized to the array overnight at 65 °C. Three replicate spots on each slide were averaged. (b) PHD2 Western blot. Cells were treated as for microarrays. Whole cell-protein extracts were subjected to SDS/PAGE and immunoblot analysis.

Figure 2

Figure 2. PHD induction in renal proximal tubule cells by hypoxia

Real-time quantitative RT–PCR was performed for PHD1, PHD2, PHD3 of normoxic (NOX), short-term hypoxic (HOX 1 h) and long-term hypoxic (HOX 18 h) RPTEC cultures. Bars indicate means±S.D. of three samples from one experiment. Results are given normalized to 60 S aRP mRNA as a housekeeping gene. These results are representative of two independent experiments.

Figure 3

Figure 3. Normoxic and hypoxic PHD levels in VHL-deficient cells

Quantitative RT–PCR of different RCC4 sublines was performed. cDNA levels were corrected for 60 S aRP. Results are presented for (a) PHD2 and (c) PHD3 in wild-type (wt), mock-transfected (Va) and VHL-recompleted (VHL) RCC4 cells. Bars indicate means±S.D. of three independent samples from one experiment. (b) Samples were prepared from three independent cultures of both cell lines in normoxia and in hypoxia and analysed for PHD2 and SP1 protein expression. SP1 serves as a loading and transfer control.

Figure 4

Figure 4. Western-blot analysis of HIF-α in U2OS cells

(a) Western-blot analysis of HIF-1α protein levels in U2OS cells treated with siRNA oligonucleotides. Cells were incubated with scramble or HIF-1α or HIF-2α oligonucleotides for 4 h and then subjected to hypoxia for 18 h. (b) Western-blot analysis of HIF-2α after 4 h of hypoxia demonstrating hypoxic induction of HIF-2α in VHL-transfected 786–0 cells but not in U2OS cells.

Figure 5

Figure 5. PHD expression in normoxia and in hypoxia after RNAi

Real-time quantitative RT–PCR for (a) PHD2 and (b) PHD3 in U2OS cultures transfected with the indicated siRNA oligonucleotides. Bars indicate the normalized means±S.D. of three samples from one experiment.

Figure 6

Figure 6. In vitro hydroxylase activity assay

Equal amounts of nuclear and cytoplasmic protein extracts from normoxic (N), short-term hypoxic (H, 1 h) and long-term hypoxic (H, 18 h) U2OS cultures hydroxylated Gal-HIF-1α549–582 bound to agarose beads. After stopping hydroxylation by adding desferrioxamine, 35S-labelled IVTT-pVHL was added and incubated overnight. Supernatants were subjected to SDS/PAGE and 35S-pVHL was detected by autoradiography. Captured 35S-VHL indicates hydroxylase activity of the protein extracts modifying the HIF-ODD. IVTT-pVHL appears in two isoforms due to an internal in-frame start codon. ‘neg.’ and ‘pos.’ indicate hydroxylase activity of unprogrammed reticulocyte lysate and IVTT-PHD2 respectively.

Figure 7

Figure 7. Western-blot analysis of PHD2 and HIF-1α after hypoxic incubation and reoxygenation

U2OS cultures were lysed at the indicated time of hypoxia and reoxygenation after long-term (18 h) and short-term (1 h) hypoxia. SDS/PAGE was performed with a total of 40 μg protein loaded in each lane: (a) PHD2; (b) time course of HIF-1α degradation; (c) line plot showing levels of HIF-1α protein at the time points shown in (b). Data originate from four repetitions of the hypoxia/reoxygenation experiments. HIF-1α bands were analysed by densitometry; results are expressed as means±S.D. Lines were calculated by non-linear regression.

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