Acute postnatal ablation of <em>Hif-2</em>α results in anemia

Michaela Gruber

Cheng Hu

Randall Johnson

Eric Brown

Brian Keith

M Simon

^{Abramson Family Cancer Research Institute and}

^{Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104;}

^{University of Vienna, Dr. Bohr Gasse, 1030 Vienna, Austria; and}

^{Department of Biology, University of California at San Diego, La Jolla, CA 92093}

^{To whom correspondence should be addressed at: Howard Hughes Medical Institute, Abramson Family Cancer Institute, University of Pennsylvania School of Medicine, BRB II/III, Room 456, 421 Curie Boulevard, Philadelphia, PA 19104., E-mail:}ude.nnepu.dem.liam@2etselec

Edited by Philip W. Majerus, Washington University Medical School, St. Louis, MO, and approved December 18, 2006

Author contributions: M.G., C.-J.H., B.K., and M.C.S. designed research; M.G., C.-J.H., and B.K. performed research; M.G., R.S.J., E.J.B., B.K., and M.C.S. contributed new reagents/analytic tools; M.G., C.-J.H., and M.C.S. analyzed data; and M.G. wrote the paper.

Edited by Philip W. Majerus, Washington University Medical School, St. Louis, MO, and approved December 18, 2006

Received 2006 Sep 22

Abstract

Adaptive transcriptional responses to oxygen deprivation (hypoxia) are mediated by the hypoxia-inducible factors (HIFs), heterodimeric transcription factors composed of two basic helix–loop–helix–PAS family proteins. The transcriptional activity of HIF is determined by the hypoxic stabilization of the HIF-α proteins. HIF-1α and HIF-2α exhibit high sequence homology but have different mRNA expression patterns; HIF-1α is expressed ubiquitously whereas HIF-2α expression is more restricted to certain tissues, e.g., the endothelium, lung, brain, and neural crest derivatives. Germ-line deletion of either HIF subunit is embryonic lethal with unique features suggesting important roles for both HIF-α isoforms. Global deletion of Hif-2α results in distinct phenotypes depending on the mouse strain used for the mutation, clearly demonstrating an important role for HIF-2α in mouse development. The function of HIF-2α in adult life, however, remains incompletely understood. In this study, we describe the generation of a conditional murine Hif-2α allele and the effect of its acute postnatal ablation. Under very stringent conditions, we ablate Hif-2α after birth and compare the effect of acute global deletion of Hif-2α and Hif-1α. Our results demonstrate that HIF-2α plays a critical role in adult erythropoiesis, with acute deletion leading to anemia. Furthermore, although HIF-1α was first purified and cloned based on its affinity for the human erythropoietin (EPO) 3′ enhancer hypoxia response element (HRE) and regulates Epo expression during mouse embryogenesis, HIF-2α is the critical α isoform regulating Epo under physiologic and stress conditions in adults.

Keywords: Epo, hypoxia-inducible factor, hypoxia, red blood cell

Abstract

Hypoxia-inducible factors (HIFs), members of the basic helix–loop–helix (bHLH)-PAS family of transcription factors, are master regulators of oxygen (O₂) homeostasis and stimulate genes important for angiogenesis, erythropoiesis and glucose metabolism (1 –3). HIFs are heterodimeric factors consisting of α (HIF-1α, -2α, and -3α) and β subunits [HIF-1β or ARNT (arylhydrocarbon-receptor nuclear translocator)]. The activity of HIF is regulated via the labile α subunit, whereas HIF-β is expressed constitutively. At normal O₂ levels (≥5% or 36 mmHg) HIF-α is hydroxylated by prolyl hydroxylases (PHDs), allowing recognition by the tumor suppressor protein von Hippel-Lindau (pVHL), and targeting for proteosomal degradation (4 –6). Under conditions of low O₂ (< 5%), HIF-α is no longer degraded, and translocates into the nucleus where it forms a heterodimeric transcription factor with ARNT. HIF activates the transcription of >150 target genes by binding to HREs in their promoter/enhancer regions (7). The closely related HIF-1α and HIF-2α proteins activate common target genes (e.g., VEGF) but also regulate unique genes, such as glycolytic genes [glucose transporter 1 (Glut-1) and phosphoglycerate kinase (PGK)] or TGF-α and the stem cell factor Oct-4, respectively (8 –10, 35). The regulation of different target genes might be due to distinct α subunit expression patterns, as HIF-1α mRNA seems to be expressed ubiquitously whereas HIF-2α mRNA expression is more restricted to endothelial cells, the mesenchyme of the lung, and neural crest derivatives during development (11, 12). Immunohistochemical analysis of HIF-2α in adult rat organs also demonstrated hypoxic stabiliziation of HIF-2α in distinct cell populations in the brain, heart, lung, kidney, liver, pancreas, and intestine (13). HIF activity is essential during embryogenesis as deletion of Arnt, Hif-1α, and Hif-2α in murine models leads to embryonic lethality (14 –22). The phenotype of Hif-2α^{embryos ranges from embryonic lethality between embryonic day (E) 9.5 and E13.5 because of vascular disorganization in the yolk sac and embryo (}19), mid-gestational lethality because of bradycardia (17) and perinatal death as a result of defects in lung maturation (18). The variable phenotypes observed are likely due to modifier loci in different mouse strains. These results clearly demonstrate an important role for HIF-2α during embryogenesis and shortly after birth. The role of HIF-2α in the adult, however, has not yet been fully described. Scortegagna et al. (23) obtained a small number of Hif-2α^{mice that survived until adulthood by generating a unique background in the F}₁ generation of C57/Black6 and 129 interbred heterozygotes. These mice suffer from multiple organ pathologies, impaired homeostasis of reactive oxygen species (ROS), metabolic abnormalities, mitochondrial distress syndrome, and pancytopenia. However, as a result of strain dependent influences on Hif-2α mutant phenotypes, it is possible that some of the findings described in this study are due to additional loci other than Hif-2α or the impairment of mitochondrial metabolism.

To investigate the adult role of HIF-2α in a controlled fashion, we generated a conditional allele of Hif-2α. By crossing this allele to a ubiquitously expressed, inducible Cre transgene (Ubc-CreER^{) we ablated}Hif-2α postnatally, allowing us to investigate the physiologic role of HIF-2α in the adult while excluding possible secondary effects because of the lack of HIF-2α during embryogenesis. This study describes the role of HIF-2α in ensuring proper adult erythropoiesis and the development of anemia when HIF-2α is depleted. Acute deletion of Hif-2α resulted in reduced red blood cell numbers, hemoglobin and hematocrit values. Furthermore, the potential of bone marrow progenitors to form erythroid colonies in vitro was reduced in Hif-2α mutant animals. We also deleted Hif-1α in adults using the inducible Cre system. Surprisingly, we determined that HIF-2α, and not HIF-1α, is the critical HIF complex regulating erythropoietin expression in vivo under both physiologic and stress conditions.

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Acknowledgments

We thank Anja Runge, Mercy Gohil, Aneesa Sataur, and Yi Pan for technical assistance and members of the M.C.S. laboratory for thoughtful discussions and reading of the manuscript. M.G. is supported by a Ph.D. fellowship from the Boehringer Ingelheim Fonds. This research was supported by National Institutes of Health Grant 66310 and by the Abramson Family Cancer Research Institute. M.C.S. is an Investigator of the Howard Hughes Medical Institute.

Acknowledgments

Abbreviations

HIF	hypoxia-inducible factor
bHLH	basic helix–loop–helix
ARNT	arylhydrocarbon-receptor nuclear translocator
En	embryonic day n
ROS	reactive oxygen species
BFU-E	burst-forming unit-erythroid
CFU-E	colony-forming unit-erythroid
EPO	erythropoietin
PH	phenylhydrazine.

Abbreviations

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0608382104/DC1.

Footnotes

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