Abstract
Hypoxia-inducible factor-1 (HIF-1) regulates the transcription of genes whose products play critical roles in energy metabolism, erythropoiesis, angiogenesis, and cell survival. Limited information is available concerning its function in mammalian hematopoiesis. Previous studies have demonstrated that homozygosity for a targeted null mutation in the Hif1alpha gene, which encodes the hypoxia-responsive alpha subunit of HIF-1, causes cardiac, vascular, and neural malformations resulting in lethality by embryonic day 10.5 (E10.5). This study revealed reduced myeloid multilineage and committed erythroid progenitors in HIF-1alpha-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1alpha-deficient yolk sacs at E9.5. Dysregulation of erythropoietin (Epo) signaling was evident from a significant decrease in mRNA levels of Epo receptor (EpoR) in Hif1alpha-/- yolk sac as well as Epo and EpoR mRNA in Hif1alpha-/- embryos. The erythropoietic defects in HIF-1alpha-deficient erythroid colonies could not be corrected by cytokines, such as vascular endothelial growth factor and Epo, but were ameliorated by Fe-SIH, a compound delivering iron into cells independently of iron transport proteins. Consistent with profound defects in iron homeostasis, Hif1alpha-/- yolk sac and/or embryos demonstrated aberrant mRNA levels of hepcidin, Fpn1, Irp1, and frascati. We conclude that dysregulated expression of genes encoding Epo, EpoR, and iron regulatory proteins contributes to defective erythropoiesis in Hif1alpha-/- yolk sacs. These results identify a novel role for HIF-1 in the regulation of iron homeostasis and reveal unexpected regulatory differences in Epo/EpoR signaling in yolk sac and embryonic erythropoiesis.
Highlights
Hypoxia-inducible factor-1 (HIF-1)3 regulates a variety of adaptive physiological responses to hypoxia, including glucose transport, glycolysis, angiogenesis, erythropoiesis, and iron metabolism [1,2,3,4]
HIF-1 activates the transcription of numerous genes, including vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut1), Epo, transferrin (Tf), and transferrin receptor (TfR)
We demonstrate that HIF-1␣-deficient embryos have reduced numbers of erythroid progenitors and impaired terminal erythroid differentiation in the yolk sac, which differ from the defects described in HIF-1-deficient embryonic stem cells
Summary
Mice and Genotyping—Hif1␣ϩ/Ϫ mice were previously generated by gene targeting and have been maintained on a mixed background (C57BL/6 ϫ 129 genetic background) by brothersister mating as described [5]. Condition 4 is FBS-free methylcellulose medium (M3236, MethoCult) supplemented with 10% fetal platelet derived serum (Animal Technologies, Tyler, TX), 5% protein-free hybridoma medium (PFHM-II, Invitrogen), 3 units/ml Epo, 10 ng/ml murine IL3, 10 ng/ml murine IL6, 50 ng/ml murine SCF, 3 ng/ml murine granulocyte-macrophage colony-stimulating factor (PeproTech, Rocky Hill, NJ), as described previously [16], and 100 l of a 1 mM solution of salicylaldehyde isonicotinoyl hydrazone saturated with iron (Fe-SIH) [17], an iron chelate that delivers iron for heme synthesis without involving the TfR/ DMT1 pathway [17]. One l of cDNA was used in a 20-l reaction mixture using the TaqMan Universal PCR master mix, No AmpErase UNG reagent kit (Applied Biosystem), 900 nM of each primer, and 100 nM of the TaqMan probe using FAM and VIC fluorescence dyes (Applied Biosystems). Mm00489837_m1 F: GAC ACT GGC TGT GGA CAT CTA C R: CAG CAG GCC CAA AGT AAC ATC P: 6-FAM-CAG CAC AAC ACC CCC TTT-MGBNFQ (3Ј–5Ј) Mm00801417_m1
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