Hypoxia-inducible factor–2 (HIF-2) regulates hepatic erythropoietin in vivo
Erinn B. Rankin, Mangatt P. Biju, Qingdu Liu, Travis L. Unger, Jennifer Rha, Randall S. Johnson, M. Celeste Simon, Brian Keith, Volker H. Haase
Erinn B. Rankin, Mangatt P. Biju, Qingdu Liu, Travis L. Unger, Jennifer Rha, Randall S. Johnson, M. Celeste Simon, Brian Keith, Volker H. Haase
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Research Article

Hypoxia-inducible factor–2 (HIF-2) regulates hepatic erythropoietin in vivo

Abstract

Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1α and/or Hif-2α (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel–Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1α. Here we show that conditional inactivation of Hif-2α in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2α but not Hif-1α and that the hypoxic induction of liver Epo in anemic adults was Hif-2α dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1α dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.

Authors

Erinn B. Rankin, Mangatt P. Biju, Qingdu Liu, Travis L. Unger, Jennifer Rha, Randall S. Johnson, M. Celeste Simon, Brian Keith, Volker H. Haase

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Figure 7

HIF-2α preferentially binds to the endogenous EPO 3′ HRE in hepatocytes.

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HIF-2α preferentially binds to the endogenous EPO 3′ HRE in hepatocytes....
(A) Western blot analysis for HIF-1 and HIF-2 in normoxic (21% O2; NX) and hypoxic (1% O2; HX) Hep3B cells. (B) EPO and PGK1 mRNA levels in normoxic and hypoxic Hep3B cells as determined by real-time PCR. (C) The hypoxic induction of EPO expression in Hep3B cells is HIF-2 dependent. Real-time PCR analysis of EPO expression in normoxic and hypoxic Hep3B cells treated with control, HIF-1α, HIF-2α, or control siRNA oligonucleotides. Error bars represent SD. (D) HIF-1α preferentially binds to the EPO HRE in vitro as determined by EMSA. s.s.HIF, HIF supershift. + and – indicate the presence and absence, respectively, of the antibody used in a supershift reaction. (E) ChIP analysis of the EPO and PGK1 HREs in normoxic and hypoxic Hep3B cells using antibodies directed against HIF-1α, HIF-2α, and CBP/p300. Coprecipitated DNA fragments were detected by PCR using primers spanning the EPO and PGK1 HREs. mAb-HIF-1α, mAb against HIF-1α; pAb-HIF-2α, polyclonal antibody against human HIF-2α; pAb-p300, polyclonal antibody against human p300.