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. 2011 Nov 1;2(6):269–275. doi: 10.4161/trns.2.6.18619

Deletion of the oxygen-dependent degradation domain results in impaired transcriptional activity of hypoxia-inducible factors

Patricia Klinger 1,2, Ruth E Schietke 3, Christina Warnecke 4, Bernd Swoboda 2, Michael Wiesener 3,4, Friedrich F Hennig 1, Kolja Gelse 1,2,✉
PMCID: PMC3265788  PMID: 22223045

Abstract

Hypoxia-inducible factors (HIF1α/HIF2α) are key transcription factors that promote angiogenesis. The overexpression of degradation-resistant HIF mutants is considered a promising pro-angiogenic therapeutic tool. We compared the transcriptional activity of HIF1α/HIF2α mutants that obtained their resistance to oxygen-dependent degradation either by deletion of their entire oxygen-dependent degradation (ODD) domain or by replacement of prolyl residues that are crucial for oxygen-dependent degradation. Although all HIF mutants translocated into the nucleus, HIF1α and HIF2α mutants inclosing the point mutations were significantly more effective in trans-activating the target gene VEGF and in inducing tube formation of endothelial cells than mutants lacking the complete ODD domain.

Key words: hypoxia-inducible factor, oxygen-dependent degradation domain, mutation, transcription factor, angiogenesis, VEGF

Introduction

Hypoxia-inducible factors (HIFs) are α/β heterodimeric transcription factors that are centrally involved in angiogenesis, a crucial process not only for visceral organs but also for the development of the skeletal system.1–6 Many pathological situations are confronted with insufficient vascularization, which calls for pro-angiogenic therapeutic concepts. The overexpression of HIFs is considered a therapeutic tool to effectively stimulate angiogenesis. However, the α-subunits of HIF1 and HIF2 are rapidly degraded in the presence of molecular oxygen by hydroxylation of two conserved prolyl residues by members of the egg-laying-defective nine (EGLN)/prolyl-hydroxylase (PHD) family. Prolyl hydroxylation generates a binding site for the Von Hippel-Lindau protein (pVHL), followed by capture through an E3-ubiquitinligase complex and subsequent rapid proteasomal degradation.7 Consequently, overexpression or application of wild-type HIF1α or HIF2α would be inefficient. Therefore, degradation-resistant HIF1α/HIF2α mutants, lacking the so-called oxygen-dependent degradation (ODD) domain (HIF-1αΔODD/HIF2αΔODD) have been constructed.8,9 These mutants are resistant to oxygen-dependent hydroxylation of two prolyl residues located within the ODD domain of the HIF1α/HIF2α molecule and, thus, are resistant to pVHL-mediated ubiquitination and proteasomal degradation.10 Although positive biological effects for such HIFαΔODD mutants with deletion of the complete ODD domain have been reported in references 11–14, it remains doubtful if these constructs maintain their full transcriptional activity, since a functionally important N-terminal transactivation domain (NTAD) was identified within the ODD domain.9,15

Therefore, the aim of this study was to directly compare the transcriptional activity of HIF1α/HIF2α mutants lacking the complete ODD domain (HIF1αΔODD and HIF2αΔODD) to HIF1α/HIF2α mutants in which merely point mutations eliminate the critical prolyl residues within the ODD domain.16

In addition, all HIFα mutants contained an inactivated asparaginyl hydroxylation site within the C-terminal transactivation domain (CTAD), which prevents oxygen-dependent hydroxylation by the asparaginyl hydroxylase FIH-1 (factor-inhibiting HIF-1).21 This modification increases the transcriptional activity of HIFα by ascertaining binding of the co-activators p300 and CBP.17

For evaluation of the transcriptional activity of the different HIFα mutants, we focused on the expression of VEGF and on tube formation of endothelial cells.

Results

Characterization and detection of HIF1α and HIF2α mutants.

All HIFα mutants used in this study are resistant to oxygen-dependent degradation, which is achieved by elimination of two prolyl residues within the oxygen-dependent degradation (ODD) domain. In addition, all HIFα mutants contained an inactivated asparaginyl hydroxylation site within the CTAD, which prevents loss of transcriptional activity of the HIFα, mediated by the oxygen-dependent asparaginyl hydroxylase FIH-1. Thus, the HIFα triple-mutants (HIF1αTM and HIF2αTM) are characterized by two point mutations within the oxygen-dependent degradation (ODD) domain (P402A and P577G for HIF1αTM; P405A and P530G for HIF2αTM) and one inactivated asparaginyl hydroxylation site (N814A for HIF1αTM; N851A for HIF2αTM) within the CTAD, respectively. The HIFαΔODD mutants received their resistance to oxygen-dependent degradation by deletion of the complete ODD domain (Fig. 1A).

Figure 1.

Figure 1

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Characterization and detection of HIF mutants. Schemes of the degradation-resistant HIF1α/HIF2α-mutants (A) (bHLH, basic helix-loop-helix domain; PAS, Per-ARNT-Sim domain; NTAD, N-terminal transactivation domain; CTAD, C-terminal transactivation domain; NLS, nuclear localization signal). Detection of protein levels of HIF1α (B), HIF2α (D) and α-tubulin (loading control) by immunoblot analysis, and detection of mRNA levels of HIF1α (C) and HIF2α (E) by quantitative RT-PCR, following transient transfection with the respective HIF1α/HIF2α mutant expression vectors. Localization of the HIF1α mutants (F) or HIF2α mutants (G) by immunofluorescence within the nucleus of transfected of C28a2 cells under normoxia (21% O2) and hypoxia (1% O2). Quantitative RT-PCR results are the mean and SD from three independent experiments. Blots and stainings are representative results obtained from three independent experiments. *p < 0.05; ***p < 0.001.

The persistence of the HIFα mutants under normoxia was detected by immunoblot analysis following transfection of C28a2 cells. In nuclear extracts of transfected cells, HIF1αTM and HIF2αTM were detectable at 110–120 kDa, depending on their phosphorylation status. The deletion of the ODD-domain of HIF1αΔODD and HIF2αΔODD resulted in smaller proteins detected at 70–80 kDa. No endogenous HIF1α or HIF2α could be detected under normoxia (control) (Fig. 1B and D).

The detection of mRNA levels of HIF1α (Fig. 1C) and HIF2α (Fig. 1E) by quantitative RT-PCR revealed significant transgene expression following transfection with the different HIF1α/HIF2α mutant expression vectors, but also confirmed that there are no significant differences in the transgene expression between HIF1αTM/HIF2αTM mutants and HIF1αΔODD/HIF2αΔODD mutants, respectively.

Nuclear translocation of HIF mutants.

Nuclear translocation of the HIF1α/HIF2α-mutants was detected under normoxia and hypoxia by immunofluorescence following transfection of C28a2 cells (Fig. 1F and G). In control cells transfected with pcDNA3, endogenous HIF-1α and HIF-2α could be detected within the nucleus only under hypoxia but not under normoxia. Transfection with the different HIF1α/HIF2α mutant expression vectors resulted in strong signals within the nucleus both under hypoxia and normoxia. No difference in the subcellular localization of the signals could be detected between the different HIFα mutants.

Transcriptional activity of HIF1α/HIF2α mutants.

A luciferase reporter gene assay driven by a promoter containing six copies of hypoxia-responsive elements (HRE) from the human phosphoglycerate kinase promoter (6xHRE/tk/luc) served to compare the transcriptional activity of the different HIFα mutants. Overexpression of HIF1αTM or HIF2αTM yielded a 27.3-fold or 12.8-fold increased luciferase activity compared with transfection with the pcDNA3 plasmid (control). Overexpression of HIF1αΔODD or HIF2αΔODD resulted only in a 2.4-fold or 1.9-fold increase, respectively (Fig. 2A). Thus, the transcriptional activity of HIF1αTM and HIF2αTM was significantly higher than that of HIF1αΔODD or HIF2αΔODD, respectively. The stabilization of endogenous HIF1α by DMOG induced a 8.9-fold increased luciferase expression.

Figure 2.

Figure 2

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Reporter gene activity. Transcriptional activity of the HIF mutants applying a luciferase assay with a luciferase reporter plasmid containing six copies of mouse phosphoglycerate kinase HRE (6xHRE/tk/luc) (A) or a human VEGF-promoter luciferase reporter plasmid (B) in HKC-8 cells. As controls, the cells were transfected with pcDNA3 (control) or stimulated with 1 mM DMOG. Results are the mean and SD from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Similar results were observed using a luciferase reporter construct containing the full-length human VEGF promoter region.16 A significantly higher luciferase activity was observed following overexpression of HIF1αTM (5.9-fold increase) or HIF2αTM (10.9-fold increase) compared with overexpression of HIF1αΔODD (2.1-fold increase) or HIF2αΔOOD (3.7-fold increase) (Fig. 2B). Stabilization of endogenous HIF by DMOG resulted in a 1.7-fold induction compared with cells transfected with pcDNA3.

The results of the luciferase assays were verified by quantitative real-time PCR for expression of VEGF mRNA in HEK293 and C28a2 cells. In both cell lines, DMOG significantly increased the expression of VEGF (Fig. 3A and B). Overexpression of HIF1αTM or HIF2αTM resulted in significant higher expression of VEGF compared with that achieved by overexpression of HIF1αΔODD or HIF2αΔODD, respectively.

Figure 3.

Figure 3

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Induction of VEGF gene expression. mRNA of VEGF was quantified by qRT-PC R 48 h after transfection of the different HIFα mutants into HE K293 (A) or C28a2 cells (B). As a control, the cells were stimulated with 1 mM DMOG. Data are the mean and SD obtained from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Biological effects of HIF2a mutants.

The biological activity of the HIF2α mutants was investigated by measuring synthesis of VEGF protein and by evaluating their proangiogenic effects. Transient transfection of HIF2αTM significantly increased the amount of VEGF protein within the supernatant of C28a2 cells (12.8 ± 4.1 ng/ml) compared with cells transfected with HIF2αΔODD (3.8 ± 1.1 ng/ml) or compared with the control plasmid pcDNA3 (0.7 ± 0.2 ng/ml) (Fig. 4A). The VEGF levels following by administration of DMOG amounted to 11.9 ng/ml.

Figure 4.

Figure 4

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Biological effects of HIF2α mutants. VEGF protein secreted by C28a2 cells transfected by HIF2αTM, HIF2aΔODD or the pcDNA3 control plasmid was quantified by ELISA. As a control, the cells were stimulated by 1 mM DMOG (A). The pro-angiogenic effect of HIF2αTM and HIF2αΔODD was evaluated by the tube formation assay on a basement membrane-like matrix with human dermal microvascular endothelial cells (HDMECs) that were stimulated with the supernatants of c28a2 cells which were transiently transfected with either the respective hIF mutant or an empty pcDNa3 control-vector. Supernatant from non-transfected cells served as a negative control. As controls, 1 mM DMOG or 100 ng/ml recombinant VEGF were applied (B). Quantification of tube formation: the angiogenic index (AI = total length of connected tubes/surface of analysis) of the different culture systems was related to the AI of HDMEC cultured with supernatant of non-transfected cells (C). Data of VEGF ELISA and angiogenic index are the mean and SD from three independent experiments. The images of the tube formation assay are representative results obtained from three independent experiments *p < 0.05; **p < 0.01; ***p < 0.001.

The cultivation of HDMECs on a basement membrane-like matrix allows them to form typically tube-like structures, which reflects their angiogenic capacity. The formation of tube-like structures was moderately increased by applying 1 mM DMOG and strongly increased by application of 100 ng/ml VEGF with relative angiogenic indices (AI) of 1.4 ± 0.1 and 2.3 ± 0.1, respectively (Fig. 4B and C). A significant stimulatory effect on tube-formation could also be detected by applying conditioned medium from cells that were transfected with HIF2αTM with an AI of 2.3 ± 0.2. Application of conditioned medium from cells infected with HIF2αΔODD was significantly less effective on tube-formation with an AI of 1.4 ± 0.2 compared with HIF2αTM.

Discussion

This work directly compared different forms of degradation-resistant HIF1α and HIF2α mutants as therapeutic tools to induce VEGF expression and promoting angiogenesis.

Significant differences could be observed between the different HIF1α/HIF2α mutants that were characterized either by lacking the complete ODD domain (HIF1αΔODD, HIF2αΔODD) or by replacement of merely two prolyl residues within the ODD domain (HIF1αTM, HIF2αTM). Thus, for both HIF1α and for HIF2α mutants, the transcriptional activities of the respective TM mutant were significantly increased compared with those of the ΔODD mutant. The inferior transcriptional activity of ΔODD mutants was most impressive in HRE-driven reporter gene assays but also significant for VEGF expression and tube-formation of endothelial cells.

Thus, the deletion of the ODD domain resulted in a dramatically reduced transcriptional activity of the HIFα mutants. All HIFα mutants could strongly be detected within the nucleus; thus, inferior function of ΔODD mutants is neither a consequence of impaired nuclear translocation, nor is it due to deviant transgene expression. The reduced transcriptional activity can rather be ascribed to the loss of the N-terminal transactivation domain (NTAD), which is lost by deletion of the ODD domain. The NTAD was shown to functionally interact with other coactivators and transcription factors including CBP/p300, SRC-1 or Ref-1.18,19 Yan et al. could demonstrate that the selective deletion of the NTAD significantly impaired the transcriptional activity of HIF2α constructs in reporter gene assays.9 This implicates that the ODD domain, and in particular the NTAD, is important for full transcriptional activity of HIF1α and HIF2α.

It is true that some previous studies reported beneficial effects on angiogenesis by viral or non-viral overexpression of HIFα-ΔODD mutants.12–14,20 These effects may have been achieved by extensive overexpression to compensate for the low transcriptional activity. However, the data of this study suggest that the use of the NTAD-preserving HIF2αTM mutant would be more efficient since the replacement of the proline residues by point mutations maintained their transcriptional activity. The effect of HIF2αTM on tube formation of endothelial cells was comparable to the application of recombinant VEGF, which encourages the use of HIF2αTM for future pro-angiogenic therapeutic concepts.

Materials and Methods

Expression vectors.

Expression vectors for mouse HIF1αTM and HIF2αTM were obtained from C. Warnecke.16 HIF1αΔODD and HIF2αΔODD expression vectors lacking the complete ODD region were constructed on the basis of the plasmid HIF2αTM, which was digested by SfiI/XhoI to excise the ODD region. The resulting HIF2αΔODD cDNA was amplified by high-fidelity PCR with the oligonucleotides 5′-GGC CCA GTT GGC CGC CAT GGA CAC GGA GC-3′ and 5′-CTC TAG ATG CAT GCT CGA GC-3′ to insert the restriction sites SfiI/XhoI.

HIF1αΔODD cDNA was amplified with Phusion High-Fidelity PCR (NEB, UK) with 5′-GCA TGG TAC CAC AAC GCG GGC ACC GAT T-3′ and 5′-CAG CAG AGT GAG AGC ATC AG-3′ for the N-terminal part, and with 5′-CAG CAG ACC CAG TTA CAG AA-3′ and 5′-CTG GCT CGA GGG AAT GAG ATT AGG AAA CGC TC-3′ for the C-terminal part and cloned after ligation of the N- and C-terminal fragments by digestion with Kpn/XhoI into the backbone of HIF1αTM.16 The final constructs were controlled by DNA sequencing.

Transfection and stimulation procedures.

C28a2 cells22 were cultivated in DMEM-F12 and HEK293 cells in DMEM, supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were transfected with pcDNA3 (empty control vector) or the different HIF1α or HIF2α mutant expression vectors using Lipofectamine 2000 (Invitrogen, Darmstadt, Germany) in serum-free OptiMEM-1 medium (GIBCO, Darmstadt, Germany). After 24 h, the serum-free medium was replaced by full medium. As a control, the cells were either stimulated with 1 mM dimethyloxaloylglycine (DMOG) (Cayman Chemical, Ann Arbor, MI) or were cultivated under hypoxia. For hypoxic conditions, the cells were kept in a sealed incubator (Binder, Tuttlingen, Germany), flushed with a gas mixture containing 1% O2 and 5% CO2.

Immunoblot analysis.

Nuclear extracts from C28a2 cells transfected with the different HIFα mutant expression vectors or with pcDNA3 were prepared as described by Grimmer et al.23 After boiling, 50 µg nuclear extracts were separated by sodium dodecyl sulfate (SDS) PAGE on a 10% gel and transferred on nitrocellulose transfer membranes. The efficiency of protein transfer was controlled by Ponceau S staining. After blocking, the nitrocellulose membranes were incubated with rabbit polyclonal anti-human/mouse HIF1α antibodies (Cayman Chemical, Ann Arbor, MI) (diluted 1:200), or with rabbit polyclonal anti-human/mouse HIF2α antibodies (diluted 1:1,000) (Abcam, Cambridge, UK), or with rat anti-α-tubulin (diluted 1:1,000) (AbD Serotec, Kidlington, UK) overnight at 4°C. Horseradish peroxidase-conjugated goat anti-rabbit antibodies or goat anti-rat antibodies (Jackson Immuno Research, Baltimore, MD) (diluted 1:5,000) were used as secondary antibodies. The immunoreactive proteins were visualized using a chemiluminescence kit (Roche, Mannheim, Germany) followed by exposure to a chemiluminescent detection film.

Nuclear localization of HIF1α/HIF2α by immunofluorescence.

C28a2 cells were grown on Thermanox™; coverslips (Nunc, Langenselbold, Germany) and transfected with the HIFα mutant expression vectors, or the control vector pcDNA3. After 24 h, the cells were either incubated under normoxic (21% O2) or hypoxic conditions (1% O2) for further 24 h. The cells were then fixed in 3% PFA for 5 min, permeabilized with 0.1% TritonX100 for 4 min and treated with 0.2% Saponin (Sigma-Aldrich, Munich, Germany) for 30 min. After blocking with 5% BSA, the coverslips were incubated for 45 min with rabbit anti-human HIF2α antibodies (ab199; Abcam, Cambridge, UK) or with rabbit anti-human HIF1α antibodies (Cayman Chemical). The cells were washed with PBS and incubated with Cy2-labeled anti-rabbit antibodies (Dianova, Hamburg, Germany) for 45 min and counterstained with 4′,6-diamidino2-phenylindole (DAPI). Coverslips were finally fixed on glass slides and visualized by fluorescence microscopy.

Luciferase-reporter gene assays.

For luciferase reporter assays immortalized human renal proximal tubular kidney cells (HKC-8 cells) 24 were transfected with either the 6x(PGK)HRE-luciferase reporter plasmid or the VEGF-luciferase reporter plasmid (both vectors in pGL3; a gift from P. Ratcliffe)16 and additionally with the pCMV-β-galactosidase expression vector (Stratagene, La Jolla, CA) using jetPei™ transfection reagent (Polyplus, New York, NY). One day after transfection, the cells were supertransfected with equimolar amounts of HIF1αTM, HIF2αTM, HIF1αΔODD or HIF2αΔODD expression vectors. As controls, cells were transfected by an equimolar amount of pcDNA3, or were stimulated by 1 mM DMOG. After 16 h, luciferase activities were determined using the Luciferase Assay Reagent (Promega, Madison, WI) and normalized to the β-galactosidase expression. Data from three independent experiments are shown.

Gene expression analysis.

The mRNA levels of HIF1α, HIF2α and VEGF in C28a2 or HEK293 cells following transfection with the different HIFα mutant expression vectors were compared with cells transfected with the pcDNA3. Total RNA was isolated 48 h after transfection using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The expression was quantified by real-time RT-PCR using the ABI Prism 7900 sequence detection system (Applied Biosystems, Foster City, CA) and Verso One-Step QRT-PCR Rox Kit (Abgene, Hamburg, Germany). The amount of mRNA was quantified using samples of three independent experiments by the standard curve method using the primer sets: VEGF (forward 5′-TGA CTT TGT CAC AGC CCA AGA TA-3′; reverse 5′-AAT CCA AAT GCG GCA TCT TC-3′; probe 5′-TGA TGC TGC TTA CAT GTC TCG ATC CCA-3′); HIF2α (forward 5′-AGA GTG CTG AAT CCC TGG-3′; reverse 5′-GTC CTT TGC AGA CCT CAT C-3′; probe 5′-GGT CAT CGC AGT TGG AAC CTC CGA GC-3′). The primer and probe set for human HIF1α was purchased from Applied Biosystems (Foster City, CA). The relative amount of target mRNA was normalized to the expression of β2-microglobulin (β2m).23

VEGF-ELISA.

The protein concentration of soluble VEGF isoforms within the supernatants of C28a2 cells 48 h following transfection with HIF2αΔODD and HIF2αTM was determined by the DuoSet ELISA development kit for human VEGF (R&D Systems, Minneapolis, MN). As a control, supernatants from non-transfected cells, or from cells stimulated by 1 mM DMOG were analyzed. Triplicates of three different experiments were used.

Tube formation assay.

Human dermal microvascular endothelial cells (HDMECs; PromoCell, Heidelberg, Germany) were cultured in 12-well plates coated with a basement membrane-like matrix (2 × 104 cells/well) (Geltrex; Invitrogen, Karlsruhe, Germany). The culture medium (DMEM-F12, 10% FCS) was mixed at a ratio of 1:3 with the supernatant of non-transfected C28a2 cells or with the supernatant of cells transfected with HIF2αTM, HIF2αΔODD, or pcDNA3. As controls, the supernatant of C28a2 cells stimulated for 24 h with 1 mM DMOG was added, or recombinant VEGF (100 ng/ml) was directly applied to the culture medium. The endothelial cells were incubated for a total of 24 h to allow the formation of tube-like structures and the extent of tube formation in ten random fields per well was captured by a digital camera. Tube formation was quantified as described previously in reference 25. The angiogenic index (AI) was determined for each field: (AI = total length of connected tubes/surface of analysis). Representative samples of three independent experiments are shown.

Statistical analysis.

All data are presented as the mean ± standard deviation. Statistical analysis was performed by analysis of variance followed by Tukey-Kramer post-hoc test. p values less than 0.05 were considered significant.

Acknowledgments

This work was supported by the Interdisciplinary Center of Clinical Research (IZKF) at the University Hospital Erlangen (grant A36) and the German Research Foundation (DFG) (grant GE 1975/2-1).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

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