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Cited by 143Cancer, Hypoxia, and MetabolismErythropoietin and Anemia TreatmentAngiogenesis and VEGF in Cancer

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The transition from HIF-1 to HIF-2 during prolonged hypoxia results from reactivation of PHDs and HIF1A mRNA instability

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Jaśkiewicz, Maciej, Moszyńska, Adrianna, Króliczewski, Jarosław, Cabaj, Aleksandra, Bartoszewska, Sylwia, Charzyńska, Agata, Gebert, Magda, Dąbrowski, Michał, Collawn, James F., Bartoszewski, Rafal·2022·Cellular & Molecular Biology Letters
DOI 10.1186/s11658-022-00408-7PMC PMC9730601

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Abstract

The hypoxia-inducible factors (HIF) are transcription factors that activate the adaptive hypoxic response when oxygen levels are low. The HIF transcriptional program increases oxygen delivery by inducing angiogenesis and by promoting metabolic reprograming that favors glycolysis. The two major HIFs, HIF-1 and HIF-2, mediate this response during prolonged hypoxia in an overlapping and sequential fashion that is referred to as the HIF switch. Both HIF proteins consist of an unstable alpha chain and a stable beta chain. The instability of the alpha chains is mediated by prolyl hydroxylase (PHD) activity during normoxic conditions, which leads to ubiquitination and proteasomal degradation of the alpha chains. During normoxic conditions, very little HIF-1 or HIF-2 alpha–beta dimers are present because of PHD activity. During hypoxia, however, PHD activity is suppressed, and HIF dimers are stable. Here we demonstrate that HIF-1 expression is maximal after 4 h of hypoxia in primary endothelial cells and then is dramatically reduced by 8 h. In contrast, HIF-2 is maximal at 8 h and remains elevated up to 24 h. There are differences in the HIF-1 and HIF-2 transcriptional profiles, and therefore understanding how the transition between them occurs is important and not clearly understood. Here we demonstrate that the HIF-1 to HIF-2 transition during prolonged hypoxia is mediated by two mechanisms: (1) the HIF-1 driven increase in the glycolytic pathways that reactivates PHD activity and (2) the much less stable mRNA levels of HIF-1α ( HIF1A ) compared to HIF-2α ( EPAS1 ) mRNA. We also demonstrate that the alpha mRNA levels directly correlate to the relative alpha protein levels, and therefore to the more stable HIF-2 expression during prolonged hypoxia.

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Fig. 1 Hypoxia results in the accumulation of HIF-1α and HIF-2α in HUVECs. Cells were exposed to hypoxia for the time periods specified, and total RNA and protein lysates were collected. The changes in HIF-1α and HIF-2α protein levels were evaluated by western blot (A) normalized to β-actin and total protein levels and related to the normoxic control (B, C). Data represent the mean ± SD of at least four independent experiments. * P < 0.05 was considered significant. HIF1A and EPAS1 mRNA levels were quantified by qRT-PCR and normalized to RPLP0 mRNA levels, and expressed as a fold change over normoxic samples (D, E). Data represent the mean ± SD of at least four independent experiments (3 replicates each). * P < 0.05 was considered significant. The ratios between HIF1A and EPAS1 mRNAs in HUVECs cultured in normoxia and hypoxia, based on qRT-PCR quantification in the same samples, the HIF1A mRNA level was considered as one for both conditions (F). Data represent the mean ± SD of five independent experiments (3 replicates each). * P < 0.05 was considered significant
Fig. 1 Hypoxia results in the accumulation of HIF-1α and HIF-2α in HUVECs. Cells were exposed to hypoxia for the time periods specified, and total RNA and protein lysates were collected. The changes in HIF-1α and HIF-2α protein levels were evaluated by western blot (A) normalized to β-actin and total protein levels and related to the normoxic control (B, C). Data represent the mean ± SD of at least four independent experiments. * P < 0.05 was considered significant. HIF1A and EPAS1 mRNA levels were quantified by qRT-PCR and normalized to RPLP0 mRNA levels, and expressed as a fold change over normoxic samples (D, E). Data represent the mean ± SD of at least four independent experiments (3 replicates each). * P < 0.05 was considered significant. The ratios between HIF1A and EPAS1 mRNAs in HUVECs cultured in normoxia and hypoxia, based on qRT-PCR quantification in the same samples, the HIF1A mRNA level was considered as one for both conditions (F). Data represent the mean ± SD of five independent experiments (3 replicates each). * P < 0.05 was considered significantCC BY · PubMed Central
Fig. 2 Hypoxia effect on HIF-1α protein half-live. A HIF-1α half-live measurements were taken in HUVECs exposed to hypoxia for 4, 8, and 16 h (cells cultured in normoxia were the negative control). Cycloheximide was added to stop translation, after which protein lysates were collected, and HIF-1α levels at each time point were measured by western blot and normalized to total protein levels. Values for each time point were calculated from three individual samples generated in at least three independent experiments. The mathematical representation of HIF-1α protein levels for 4 h (B), 8 h (C) and 16 (D) hypoxia in HUVECs. The time points indicating relative HIF-1α levels were plotted as differences from HIF-1α levels at the initial time point (t = 0). The protein half-lives were calculated from the exponential decay using the trend line equation C/C0 = e–kdt (where C and C0 are protein amounts at time t and at the t0, respectively, and kd is the protein decay constant). The error bars represent SD. * P < 0.05 was considered significant
Fig. 2 Hypoxia effect on HIF-1α protein half-live. A HIF-1α half-live measurements were taken in HUVECs exposed to hypoxia for 4, 8, and 16 h (cells cultured in normoxia were the negative control). Cycloheximide was added to stop translation, after which protein lysates were collected, and HIF-1α levels at each time point were measured by western blot and normalized to total protein levels. Values for each time point were calculated from three individual samples generated in at least three independent experiments. The mathematical representation of HIF-1α protein levels for 4 h (B), 8 h (C) and 16 (D) hypoxia in HUVECs. The time points indicating relative HIF-1α levels were plotted as differences from HIF-1α levels at the initial time point (t = 0). The protein half-lives were calculated from the exponential decay using the trend line equation C/C0 = e–kdt (where C and C0 are protein amounts at time t and at the t0, respectively, and kd is the protein decay constant). The error bars represent SD. * P < 0.05 was considered significantCC BY · PubMed Central
Fig. 3 Hypoxia effect on HIF-2α protein half-live. A HIF-2α half-live measurements were taken in HUVECs exposed to hypoxia for 4, 8, and 16 h (cells cultured in normoxia were negative control). Cycloheximide was added to stop translation, after which protein lysates were collected, and HIF-2α levels at each time point were measured by western blot and normalized to total protein levels. Values for each time point were calculated from three individual samples generated in at least three independent experiments. The mathematical representation of HIF-2α protein levels for 4 h (B), 8 h (C) and 16 (D) hypoxia in HUVECs. The time points indicating relative HIF-1α levels were plotted as differences from HIF-2α levels at the initial time point (t = 0). The protein half-lives were calculated from the exponential decay using the trend line equation C/C0 = e–kdt (where C and C0 are protein amounts at time t and at the t0, respectively, and kd is the protein decay constant). The error bars represent SD. * P < 0.05 was considered significant
Fig. 3 Hypoxia effect on HIF-2α protein half-live. A HIF-2α half-live measurements were taken in HUVECs exposed to hypoxia for 4, 8, and 16 h (cells cultured in normoxia were negative control). Cycloheximide was added to stop translation, after which protein lysates were collected, and HIF-2α levels at each time point were measured by western blot and normalized to total protein levels. Values for each time point were calculated from three individual samples generated in at least three independent experiments. The mathematical representation of HIF-2α protein levels for 4 h (B), 8 h (C) and 16 (D) hypoxia in HUVECs. The time points indicating relative HIF-1α levels were plotted as differences from HIF-2α levels at the initial time point (t = 0). The protein half-lives were calculated from the exponential decay using the trend line equation C/C0 = e–kdt (where C and C0 are protein amounts at time t and at the t0, respectively, and kd is the protein decay constant). The error bars represent SD. * P < 0.05 was considered significantCC BY · PubMed Central
Fig. 4 Prolyl hydroxylase inactivation in HUVECs influences ODD domains stability during hypoxia. A Schematic presentation of firefly luciferase (FLuc) and renilla luciferase reporter cloning vector pEZX-FR02 (GeneCopoeia) containing the HIF1A human (NM_181054) gene region comprising ODD (amino acids 401–653, including the Prolines 402 and 564 (a PHD substrate) or human EPAS1 (NM_001430) gene region comprising ODD (amino acids 403–607, including the Prolines 404 and 531 (a PHD substrate). These gene regions were fused with firefly luciferase (without the ATG start codon) downstream of the HIF-α. B Hypoxia-related stabilization of the HIF-1α and HIF-2 α ODD reporter. HUVECs following the transfection with the HIF-α-ODD reporter vectors were exposed to hypoxia (1% O2) for indicated time points in the presence or absence of 2.5 mM DMOG, or exposed to 0.3% O2 (both DMOG addition and the exposure to 0.3% O2 were performed after 4 h form the experiments start) and luciferase activity measured with Dual-Luciferase Reporter Assay System. The locations of the proline hydroxyl addition that destabilizes the HIF alpha subunits is illustrated in the panel A box. The firefly luciferase data were normalized to Renilla signal and expressed as a fold change over the control vector (without HIF-α-ODD) in the same experimental conditions. R.L.U. – relative light units. Data represent the mean ± SD of five independent experiments (3 replicates each). The P < 0.05 was considered significant and depicted as follows: * (for pairwise comparison to normoxia), # (for pairwise comparison to hypoxia 8 h), ## (for pairwise comparison to hypoxia 24 h)
Fig. 4 Prolyl hydroxylase inactivation in HUVECs influences ODD domains stability during hypoxia. A Schematic presentation of firefly luciferase (FLuc) and renilla luciferase reporter cloning vector pEZX-FR02 (GeneCopoeia) containing the HIF1A human (NM_181054) gene region comprising ODD (amino acids 401–653, including the Prolines 402 and 564 (a PHD substrate) or human EPAS1 (NM_001430) gene region comprising ODD (amino acids 403–607, including the Prolines 404 and 531 (a PHD substrate). These gene regions were fused with firefly luciferase (without the ATG start codon) downstream of the HIF-α. B Hypoxia-related stabilization of the HIF-1α and HIF-2 α ODD reporter. HUVECs following the transfection with the HIF-α-ODD reporter vectors were exposed to hypoxia (1% O2) for indicated time points in the presence or absence of 2.5 mM DMOG, or exposed to 0.3% O2 (both DMOG addition and the exposure to 0.3% O2 were performed after 4 h form the experiments start) and luciferase activity measured with Dual-Luciferase Reporter Assay System. The locations of the proline hydroxyl addition that destabilizes the HIF alpha subunits is illustrated in the panel A box. The firefly luciferase data were normalized to Renilla signal and expressed as a fold change over the control vector (without HIF-α-ODD) in the same experimental conditions. R.L.U. – relative light units. Data represent the mean ± SD of five independent experiments (3 replicates each). The P < 0.05 was considered significant and depicted as follows: * (for pairwise comparison to normoxia), # (for pairwise comparison to hypoxia 8 h), ## (for pairwise comparison to hypoxia 24 h)CC BY · PubMed Central
Fig. 5 Inhibition of PHDs activity during hypoxia with DMOG results in accumulation of HIF-1α and HIF-2α in HUVECs. Cells were exposed to hypoxia in the presence or absence of 2.5 mM DMOG that was added (grey bars) after 4 h from the experiments start and for the time periods specified, the total RNA and protein lysates were collected. The changes in HIF-1α and HIF-2α protein levels were evaluated by western blot (A) normalized to total protein levels and related to the normoxic control (B, C). Data represent the mean ± SD of four independent experiments * P < 0.05 was considered significant. HIF1A and EPAS1 mRNA levels were quantified by qRT-PCR and normalized to RPLP0 mRNA levels and expressed as a fold change over normoxic samples (D, E). Data represent the mean ± SD of five independent experiments (3 replicates each). * P < 0.05 was considered significant
Fig. 5 Inhibition of PHDs activity during hypoxia with DMOG results in accumulation of HIF-1α and HIF-2α in HUVECs. Cells were exposed to hypoxia in the presence or absence of 2.5 mM DMOG that was added (grey bars) after 4 h from the experiments start and for the time periods specified, the total RNA and protein lysates were collected. The changes in HIF-1α and HIF-2α protein levels were evaluated by western blot (A) normalized to total protein levels and related to the normoxic control (B, C). Data represent the mean ± SD of four independent experiments * P < 0.05 was considered significant. HIF1A and EPAS1 mRNA levels were quantified by qRT-PCR and normalized to RPLP0 mRNA levels and expressed as a fold change over normoxic samples (D, E). Data represent the mean ± SD of five independent experiments (3 replicates each). * P < 0.05 was considered significantCC BY · PubMed Central
Fig. 6 Inhibition of PHD activity during hypoxia by reducing oxygen levels results in accumulation of HIF-1α and HIF-2α in HUVECs. Cells were exposed to 1% O2 for 4 h, and next moved to 0.3% O2 (grey bars) or remained in the same conditions for the time periods specified, and total RNA and protein lysates were collected. The changes in HIF-1α and HIF-2α protein levels were evaluated by western blot (A) normalized to total protein levels and related to the normoxic control (B, C). Data represent the mean ± SD of four independent experiments. * P < 0.05 was considered significant. HIF1A and EPAS1 mRNA levels were quantified by qRT-PCR and normalized to RPLP0 mRNA levels and expressed as a fold change over normoxic samples (D, E). Data represent the mean ± SD of five independent experiments (3 replicates each). * P < 0.05 was considered significant
Fig. 6 Inhibition of PHD activity during hypoxia by reducing oxygen levels results in accumulation of HIF-1α and HIF-2α in HUVECs. Cells were exposed to 1% O2 for 4 h, and next moved to 0.3% O2 (grey bars) or remained in the same conditions for the time periods specified, and total RNA and protein lysates were collected. The changes in HIF-1α and HIF-2α protein levels were evaluated by western blot (A) normalized to total protein levels and related to the normoxic control (B, C). Data represent the mean ± SD of four independent experiments. * P < 0.05 was considered significant. HIF1A and EPAS1 mRNA levels were quantified by qRT-PCR and normalized to RPLP0 mRNA levels and expressed as a fold change over normoxic samples (D, E). Data represent the mean ± SD of five independent experiments (3 replicates each). * P < 0.05 was considered significantCC BY · PubMed Central

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  1. Smirnova NA, Osipyants AI, Khristichenko AY, Hushpulian DM, Nikulin SV, Chubar TA, et al. HIF2 ODD-luciferase reporter: the most sensitive assay for HIF prolyl hydroxylase inhibitors. Russ Chem Bull. 2018;67(1):150–6. · DOI

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Metadata

Authors
Jaśkiewicz, M., Moszyńska, A., Króliczewski, J., Cabaj, A., Bartoszewska, S., Charzyńska, A., Gebert, M., Dąbrowski, M., Collawn, J. F., & Bartoszewski, R.
Affiliations
  • Gdańsk Medical University (PL)
  • Instytut Biologii Doświadczalnej im. Marcelego Nenckiego (PL)
  • University of Alabama at Birmingham (US)
ORCID
Venue
Cellular & Molecular Biology Letters
Publisher
Springer Science and Business Media LLC
Published
2022-12
Volume
27
Issue
1
Pages
109-109
ISSN
  • 1425-8153 (ISSN-L)
  • 1689-1392
DOI
10.1186/s11658-022-00408-7
PMC
PMC9730601
OpenAlex
W4311079783
Cited by
143
References
58
Citations by year
2026: 37 · 2025: 46 · 2024: 39 · 2023: 20 · 2022: 1
FWCI
11.17
Type
journal article
Language
en
Open access
Licence
Locations
  • Cellular & Molecular Biology Letters (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Narodowe Centrum Nauki (2015/18/E/NZ3/00687)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Health Sciences › Medicine › Hematology › Erythropoietin and Anemia Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Angiogenesis and VEGF in Cancer
Keywords
hypoxia-inducible factor, HIF-1, HIF-2, prolyl hydroxylase, EPAS1, HIF-1α, HIF-2α, glycolysis, endothelial cells, hypoxia response, transcriptional regulation, mRNA stability
MeSH terms
Endothelial PAS Domain-Containing Protein 1, Humans, Oxygen, RNA, Messenger, Cell Hypoxia, RNA Stability, Endothelial Cells, Basic Helix-Loop-Helix Proteins, Basic Helix-Loop-Helix Transcription Factors, Hypoxia-Inducible Factor 1, alpha Subunit

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