Multiple factors affecting cellular redox status and energy metabolism modulate hypoxia-inducible factor prolyl hydroxylase activity in vivo and in vitro

Abstract

Prolyl hydroxylation of hypoxible-inducible factor alpha (HIF-alpha) proteins is essential for their recognition by pVHL containing ubiquitin ligase complexes and subsequent degradation in oxygen (O(2))-replete cells. Therefore, HIF prolyl hydroxylase (PHD) enzymatic activity is critical for the regulation of cellular responses to O(2) deprivation (hypoxia). Using a fusion protein containing the human HIF-1alpha O(2)-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 nonhydroxylated PHD substrates in cells and during in vitro reactions. Importantly, the ODD fusion protein is regulated with kinetics identical to endogenous HIF-1alpha during cellular hypoxia and reoxygenation. Using in vitro assays, we demonstrated that the levels of iron (Fe), ascorbate, and various tricarboxylic acid (TCA) cycle intermediates affect PHD activity. The intracellular levels of these factors also modulate PHD function and HIF-1alpha 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 O(2) depletion.

FIG. 1.

A fusion protein containing the human HIF-1α ODD exhibits differential mobility during SDS-PAGE when hydroxylated. (A) Schematic illustration of the prolyl hydroxylation reaction. (B) Proposed hydroxylation of the GHO fusion protein by PHDs. GHO contains a single proline residue that can be hydroxylated (red bold). (C) Bacterially produced GST-wtPHD2 was used to hydroxylate wheat germ IVT-produced GHO protein in vitro. HA antibody was used to detect both the hydroxylated and the unhydroxylated GHO species. Samples from identical experiments were also run on duplicate gels, probed with anti-HA and anti-HIF hydroxyproline antibody, respectively. In vitro-translated GHO(P→A) protein with the critical proline residue mutated to alanine was assayed in in vitro hydroxylation reactions and detected by anti-HA antibody. (D) HEK 293T cell lysates were prepared in HEB buffer and used to hydroxylate wheat germ IVT GHO protein (upper panel). Reactions included the indicated amounts of FeCl₂ and ascorbate under normoxic conditions with 1 mM DFX or inside a hypoxia workstation at 1.5% O₂. Densitometry analysis was performed to quantify relative GHO hydroxylation levels under each condition, and the results were plotted in a histogram. In the lower panel, GHO protein was transiently expressed in HEK 293T cells. In lanes 2 to 4, Flag-tagged human PHD1, PHD2, and PHD3 were individually coexpressed with the GHO protein. In lanes 5 to 7, transfected cells were treated with 100 μM DFX, 100 μM CoCl₂, and 1.5% O₂, respectively, for 4 h prior to harvest.

FIG. 2.

Iron (Fe), ascorbate, and TCA cycle intermediates promote GHO prolyl hydroxylation in vitro. (A) Bacterially produced GST-wtPHD2 was used to hydroxylate GHO protein in HEB buffer with or without FeCl₂, ascorbate, and α-ketoglutarate at the indicated concentrations. (B) HEK 293T cell lysate was used to hydroxylate in vitro-translated GHO protein in the presence of different concentrations of additional FeCl₂, ascorbate, and α-ketoglutarate, respectively. (C) HEK 293T cell lysate supplemented with 100 μM ascorbate was used in the in vitro hydroxylation assay at the indicated conditions. (D) HEK 293T cell lysate supplemented with 100 μM ascorbate was used to hydroxylate GHO in vitro with the indicated concentrations of glucose metabolites added.

FIG. 3.

GHO protein stability mimics that of endogenous HIF-1α protein in HEK 293T cells. (A) GHO and GHO(P>A) protein were stably expressed in HEK 293T cells. Cells were treated with 100 μM ALLN, 100 μM DFX, and 1.5% O₂ for 4 h prior to harvest. (B) HEK 293T stable transformants expressing GHO protein were treated with 1.5% O₂ for 6 h or 24 h, respectively. Cells were removed from the hypoxia workstation and lysed at the indicated times after reoxygenation. Cells harvested inside the workstation were used as time zero. Untreated normoxic cells (N) and cells exposed to 100 μM DFX for 4 h (D) were used as controls. A nonspecific band (NS) serves as a loading control. (C) HEK 293T cells expressing GHO protein were exposed to 1.5% O₂ for 24 h. At 1 h prior to reoxygenation, 100 μM ALLN was added to the medium. Cells were harvested at the indicated times after reoxygenation. Normoxic cells treated with ALLN only (N) were used as a control. Four independent experiments were performed, and the results of GHO hydroxylation were quantified and are presented as a bar graph.

FIG. 4.

Cells lacking a functional mitochondrial respiratory chain retain PHD activity under low O₂. (A) Wild-type and cyt c-null embryonic cells were exposed to 1.5% O₂ or 100 μM DFX for 4 h in the presence or absence of 10 μM MG132 as indicated. Total HIF-1α protein and hydroxy HIF-1α protein levels were measured by Western blotting. Of note, samples assayed for HIF-1α accumulation demonstrated multiple alternatively phosphorylated species. Identical lysates were rerun on SDS-polyacrylamide gels to probe for hydroxylated HIF-1α so that HIF-1α appears as a single species at 100 kDa. (B) HEK 293T cells stably expressing the GHO protein were exposed to 1.5% O₂ or 100 μM DFX for 4 h in the presence of different concentrations of mitochondrial inhibitors rotenone or myxothiazol. (C) The inhibition of HEK 293T cell respiration by different doses of rotenone or myxothiazol was measured as cellular KCN-dependent O₂ consumption. All respiration rates were normalized to that of untreated cells.

FIG. 5.

Hypoxic 768-O cells treated with mitochondrial inhibitors resume GHO hydroxylation. (A) 786-O stable transformants expressing GHO protein were exposed to 21% O₂ or 1.5% O₂ for 4 h in the presence or absence of DFX, rotenone, or myxothiazol as indicated. GHO protein was detected by Western analysis with HA antibody; densitometry analysis was used to measure GHO protein hydroxylation levles. (B) GHO-expressing 786-O cells were cultured at 1.5% O₂ overnight; rotenone or myxothiazol were added in the hypoxic workstation, and cells were harvested at different times after drug treatment as indicated. Normoxic and DFX-treated cells were used as controls. Levels of GHO protein hydroxylation were measured by Western blot and densitometry analyses. The inhibition of 786-O cellular respiration by different doses of rotenone or myxothiazol was measured as cellular KCN-dependent O₂ consumption. All respiration rates were normalized to that of untreated cells. (C) The indicated concentrations of MitoQ were added to culture medium for 4 h. After an additional 4 h of hypoxic and normoxic incubation, cells were harvested for analysis. Three parallel sets of samples were used. One set was harvested for HIF-1α and actin Western analysis. Another was treated with 100 μM ALLN to block protein degradation and harvested for GHO protein Western analysis. The third set was harvested at the same time for respiration measurements.

FIG. 6.

Exogenous hydrogen peroxide (H₂O₂) inhibits GHO prolyl hydroxylation in vivo and in vitro. (A) Diagram of H₂O₂ production by glucose oxidase and subsequent metabolism by catalase. (B) HEK 293T cells stably expressing GHO were treated with increasing amounts of glucose oxidase in the presence or absence of catalase for 2 h as indicated. The experiment was carried out in the presence of 100 μM ALLN to prevent GHO protein degradation. Densitometry analysis estimated GHO hydroxylation. (C) 786-O cells stably expressing GHO protein were treated with various amounts of glucose oxidase with or without catalase as indicated. Western blots were probed for GHO protein, and the levels of hydroxylation were measured by densitometry. (D) RCC4 cells stably expressing GHO protein were treated with various amounts of glucose oxidase or tert-butyl H₂O₂ as indicated. DFX treatment was used as a control. Western blots were probed for GHO protein, and the levels of hydroxylation were measured by densitometry. (E) Bacterially produced GST-wtPHD2 was used to hydroxylate IVT-produced GHO protein in vitro. All reactions contained 1 mM α-ketoglutarate, 100 μM ascorbate, and 100 μM FeCl₂. Additional glucose oxidase, glucose, and/or catalase were added to different reactions as indicated. GHO prolyl hydroxylation was detected by Western blotting and quantitated by densitometry assay.

FIG. 7.

Exogenous H₂O₂ does not stimulate HIF-1α transcription but stabilizes HIF-1α protein via PHD inhibition. (A) Hep3B cells were treated with 100 mM DFX and different doses of H₂O₂ for 60 min or various concentrations of glucose oxidase for 8 h. HIF-1α protein was detected by Western blotting, and a nonspecific band provided a loading control. RNA was extracted for quantitative RT-PCR analysis to measure HIF-1α and PGK-1 transcript levels. All data are presented as levels relative to untreated cells. (B) Hep3B cells were grown on coverslips and treated as indicated. Immunofluorescence analysis was performed with antibody recognizing human HIF-1α. DAPI containing mounting medium was used to stain for nuclei. (C) Vhl-null ES cells were treated with 100 mM DFX or 1 mM H₂O₂ for 90 min in the presence or absence of 100 μM ascorbate. GHO protein was assessed by Western blot analysis.