Cell-type-specific regulation of degradation of hypoxia-inducible factor 1 alpha: role of subcellular compartmentalization

Abstract

The hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a transcription factor that mediates adaptive cellular responses to decreased oxygen availability (hypoxia). At normoxia, HIF-1 alpha is targeted by the von Hippel-Lindau tumor suppressor protein (pVHL) for degradation by the ubiquitin-proteasome pathway. In the present study we have observed distinct cell-type-specific differences in the ability of various tested pVHL-interacting subfragments to stabilize HIF-1 alpha and unmask its function at normoxia. These properties correlated with differences in subcellular compartmentalization and degradation of HIF-1 alpha. We observed that the absence or presence of nuclear localization or export signals differently affected the ability of a minimal HIF-1 alpha peptide spanning residues 559 to 573 of mouse HIF-1 alpha to stabilize endogenous HIFalpha and induce HIF-driven reporter gene activity in two different cell types (primary mouse endothelial and HepG2 hepatoma cells). Degradation of HIF-1 alpha occurred mainly in the cytoplasm of HepG2 cells, whereas it occurs with equal efficiency in nuclear and cytoplasmic compartments of primary endothelial cells. Consistent with these observations, green fluorescent protein-HIF-1 alpha is differently distributed during hypoxia and reoxygenation in hepatoma and endothelial cells. Consequently, we propose that differential compartmentalization of degradation of HIF-1 alpha and the subcellular distribution of HIF-1 alpha may account for cell-type-specific differences in stabilizing HIF-1 alpha protein levels under hypoxic conditions.

FIG. 1.

Activation of HIFα function at normoxia. (A) Schematic representation of mHIF-1α(546-574) and deletion mutants. Asterisks indicate mutated residues. (B and D) Relative reporter gene activity obtained following expression of mHIF-1α(546-574) mutants. pT81/HRE-luc and increasing amounts of pFLAG-GAL4-mHIF-1α fusion constructs indicated in panel A were cotransfected into MBECs. Cells were cultured under normoxic conditions or treated with hypoxia (H), 100 μM 2,2′-dipyridyl (DP), or 100 μM CoCl₂ as indicated. Data are presented as luciferase activity relative to cells transfected with pFLAG-CMV-2 (FLAG). Values represent means ± standard deviations of three independent experiments performed in duplicate. *, P < 0.05, compared to the luciferase activity in cells overexpressing pFLAG-GAL4 (GAL4) (two-way analysis of variance test); ^, P < 0.05, compared to the cells overexpressing pFLAG-CMV-2 cultured at normoxia (Student's t test). (C and E) Expression levels of mHIF-1α-derived peptides detected by immunoblotting using an anti-FLAG antibody. (F) Schematic representation of chemically synthesized peptides spanning mHIF-1α residues 559 to 573 fused to the HIV-1 TAT protein transduction domain (PTD). (G) Peptide competition assay. In vitro translated FLAG-tagged full-length mHIF-1α was immunoprecipitated (IP) with anti-FLAG antibody and incubated with in vitro translated [³⁵S]methionine-labeled pVHL in the presence of either vehicle (lane 2); 10 μΜ TAT peptide (lane 3); 0.5, 1.0, 2.0, 5.0, or 10.0 μM THIF15 peptide (lanes 4 to 8); or 0.05, 0.1, 0.2, 0.5, or 1.0 μM THIF15OH peptide (lanes 9 to 13). Peptides were preincubated with either lysis buffer (−WCE) or whole-cell extracts from HepG2 cells (+WCE) for 1 h at room temperature. Precipitated pVHL was analyzed by 12.5% SDS-PAGE, followed by autoradiography. (H) Mouse corneal angiogenesis assay. Micropellets of the peptides were implanted into mouse cornea pockets (arrows), and 5 days later corneal angiogenesis was monitored. Photographs represent ×20 magnification of the mouse eye. Quantification (graph at right) of corneal neovascularization, presented as maximal areas of neovascularization on day 5. Graph represents mean values ± standard deviation. ***, P < 0.001 in comparison to the neovascularization area induced with TAT peptide alone (Student's t test).

FIG. 2.

Subcellular localization of the mHIF-1α(559-573) peptide affects activation of HIFα function in a cell-type-specific manner. (A) Schematic representation of the GST-GFP-HIF-1α fusion peptides. (B) Subcellular localization of the mHIF-1α fusion peptides. Plasmids encoding wild-type or mutant FLAG-GST-GFP/mHIF-1α(559-573) were transiently transfected into MBECs and HepG2 cells cultured under normoxic conditions. The subcellular localization of fusion peptides was detected by confocal microscopy. Representative images are shown. Categorization and quantitative evaluation of the subcellular localization pattern of the GST-GFP fusion peptides are presented as percentages of cells belonging to the categories N, N>C, N=C, and N+-BCE (F) cells. Cells were cultured under normoxic conditions. Data are presented as luciferase activity relative to cells transfected with pFLAG-CMV-2 (FLAG) alone. Values represent means ± standard deviations of three independent experiments performed in duplicate.

FIG. 3.

Compartmentalization of proteasome activity in MBECs and HepG2 cells. (A) Treatment of cells with the proteasome inhibitor MG132 leads to stabilization of the ZsProSensor-1 protein. pZsProSensor-1 was transfected into MBECs or HepG2 cells cultured at normoxia. The cells were treated with dimethyl sulfoxide (−MG132) or 10 μM MG132 (+MG132) for 8 h before fixation. Cells were observed with lower magnification. Representative images are shown. (B) Differential proteasome-mediated degradation of ZsProSensor-1 protein occurs in MBECs and HepG2 subcellular compartments. Cells were transfected with pZsProSensor-1 and cultured at normoxia. After treatment with 10 μM MG132 for 8 h, the cells were fixed (0 h) or washed three times with culture medium to remove MG132 and then fixed at 4 h, 6 h, 8 h, or 10 h of incubation. Cells were analyzed by confocal microscopy, and representative images are presented. Quantitative evaluation and categorization was performed as described in the legend of Fig. 2B. (C) FRAP analysis of cells transfected with pZsProSensor-1. Fluorescence before bleaching was considered 100%. Data are shown as the ratio between the fluorescence observed within the nuclear and cytoplasmic compartments or between cytoplasmic and nuclear compartments following bleaching of the nucleus or cytoplasm, respectively. The results presented are the average of the analysis of MBECs or HepG2 cells bleached in the nuclear compartment (MBEC_BN or HepG2_BN) or in the cytoplasm (MBEC_BC or HepG2_BC) after 4 h following MG132 removal. (D) Overexpressed HIF-1α stabilizes endogenous Nrf2. MBECs and HepG2 cells were cultured at normoxia (−), exposed to hypoxia or treated with 10 μM MG132 for 8 h, or transfected with pFLAG or pFLAG-mHIF-1α. Endogenous Nrf2 was detected by immunoblotting. (E) Overexpressed HIF-1α increased the expression of ZsProSensor-1 protein. MBECs and HepG2 cells were transfected with expression vector encoding ZsProSensor-1 alone (−) or treated with 10 μM MG132 (MG132) for 8 h or cotransfected with pFLAG or pFLAG-mHIF-1α as indicated. Levels of ZsProSensor-1 protein were determined by FACS analysis. The percentage of fluorescent cells is indicated. (F) Stabilization of the ZsProSensor-1 protein by expression of HIF-1α. MBECs or HepG2 cells transfected with pZsProSensor-1 and pFLAG-mHIF-1α or pCMV2-FLAG was observed at lower magnification.

FIG. 4.

Compartmentalized proteasome-dependent degradation of HIF-1α. (A) Competition of HIF-1α and ZsProSensor-1 protein for proteasomal degradation at normoxia and during reoxygenation. Cells were cotransfected with pZsProSensor-1 and pFLAG-mHIF-1α and cultured at normoxia (Nor) or hypoxia (H) for 8 h or exposed to reoxygenation for 30 min (R30) before fixation. The intracellular localization of ZsProSensor-1 protein was detected and evaluated using confocal microscopy. Quantitative evaluation and categorization of the cells were performed as described in the legend of Fig. 2B. (B and C) Degradation rate of endogenous pools of HIF-1α in MBECs and HepG2 cells in the nuclear and cytoplasmic compartments. Cells were treated with either dimethyl sulfoxide (-MG) or 10 μM MG132 (+MG) and grown under hypoxic conditions for 8 h, followed by reoxygenation for 0.1, 2, 4, 6, 8, or 10 min before harvest. Nuclear (Nuc) and cytosolic (Cyt) extracts were prepared, and immunoblot analysis using anti-HIF-1α, antipaxillin, and anti-YY1 antibodies was performed. To facilitate detection in the cytosolic extracts, HIF-1α was immunoprecipitated using an anti-HIF-1α antibody before immunoblotting. (C) Quantification of endogenous HIF-1α protein levels was normalized by YY1 or paxillin expression levels (control) in the nucleus or cytoplasm, respectively. Relative HIF-1α protein levels at hypoxia were considered 100%.

FIG. 5.

Subcellular distribution of GFP-fused HIF-1α in MBECs and HepG2 cells. (A) Intracellular localization of GFP-HIF-1α. GFP-HIF-1α expression vector was transiently transfected into MBECs or HepG2 cells. Cells exposed to hypoxia were fixed immediately or after 10, 20, or 30 min of reoxygenation (R10, R20, or R30, respectively). The intracellular localization of GFP-HIF-1α was detected and categorized by confocal microscopy. Representative images are shown. (B) Intracellular localization of GFP-HIF-1α deletion mutants. MBECs or HepG2 cells were transfected with GFP expression vectors of HIF-1α deletion mutants (shown schematically) and cultured at normoxia (Nor) or hypoxia (H). Quantitative evaluation and categorization of the subcellular distribution were done as described in Materials and Methods. (C) Protein levels of endogenous HIF-1α in MBECs and HepG2 cells. Cells were cultured at normoxia (N) or hypoxia (H) or treated with 100 μM 2,2′-dipyridyl (DP) in the presence or absence of 10 μM MG132 for 8 h as indicated. Endogenous HIF-1α levels were analyzed by immunoblot analysis using anti-HIF-1α antibodies. (D) Protein levels of endogenous HIF-2α in MBECs. MBECs were cultured at normoxia (N) or hypoxia (H) or treated with 100 μM 2,2′-dipyridyl (DP) for 8 h as indicated. Endogenous HIF-2α levels in whole-cell extracts were analyzed by immunoblotting using anti-HIF-2α antibodies. Whole-cell extracts from HEK293 cells transfected with pFLAG-HIF-2α (F-HIF-2α) were used as a positive control.

FIG. 6.

Cell-type-specific regulation of subcellular compartmentalization and degradation of HIF-1α. In primary endothelial cells (MBECs) (A and B), HIF-1α is present in both nuclear and cytoplasmic compartments under conditions of normoxia, hypoxia, and reoxygenation, and proteasome-dependent degradation occurs at a similar rate in both compartments. In this context pVHL-interacting peptides are able to inhibit pVHL-mediated degradation of HIF-1α in both compartments. In contrast, in highly proliferating or transformed cell types (e.g., HepG2 cells) (C and D), proteasome activity dominates in the cytoplasmic compartment. HIF-1α translocates from the cytoplasm to the nucleus in hypoxia. Upon reoxygenation, the protein needs to be transported back to the cytoplasm to be efficiently degraded by the proteasome. In this type of cells, pVHL-interacting peptides can only inhibit pVHL-mediated degradation of HIF-1α when targeted to the cytoplasmic compartment.