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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.
Nonstandard abbreviations used: ARNT, aryl-hydrocarbon-receptor nuclear translocator; bHLH, basic helix-loop-helix; Bnip3, BCL2/adenovirus E1B-interacting protein 1, NIP3; ChIP, chromatin immunoprecipitation; DMOG, dimethyloxalylglycine; EPO, erythropoietin; HIF-1, hypoxia-inducible factor-1; HRE, hypoxia response element; PGK, phosphoglycerate kinase 1; PHD, prolyl-4-hydroxylase domain; PHZ, phenylhydrazine; Trf, transferrin; VHL, von Hippel-Lindau; Vhlh, VHL syndrome homolog.
Introduction
The glycoprotein erythropoietin (EPO) is essential for the regulation of red blood cell mass in response to changes in tissue oxygenation. EPO induces erythropoiesis through the stimulation of erythroid precursor cell viability, proliferation, and differentiation, thus enhancing the oxygen-carrying capacity of blood (review in ref. 1). Lack of Epo during murine development results in embryonic lethality at E13.5 as a result of cardiac failure and anemia (2, 3). Clinically, dysregulated EPO expression results in the development of anemia when serum EPO levels are inadequately low or polycythemia as a result of EPO overproduction.
EPO expression is tightly regulated by developmental, tissue-specific, and physiological cues (1). During development, the physiological source of EPO switches from the fetal liver to the kidney in adults, the timing of which is species dependent (4-7)....