Erythropoietin Lacking Hypoxia Responsive Element in 3′ Untranslated Region Stimulates Hepatic Erythropoiesis in Anemic Xenopus.

Recent studies on EPO-EPOR systems in non-mammalian vertebrate including frog and teleost fishes demonstrate extramedullary adult erythropoiesis instead of bone marrow. In adult Xenopus laevis (African clawed frog), an animal model of hepatic erythropoiesis should give an opportunity for understanding the microenvironment of vascular niche. Therefore, we investigate on erythrocyte development, gene expressions and gene regulations of EPO and EPOR molecules in adult Xenopus liver. In situ hybridization and immunostaining revealed that Xenopus EPOR (xlEPOR) expressing erythrocyte progenitors, which have low hemoglobin content, were localized among liver sinusoids. The maximum number of xlEPOR expressing cells was observed when peripheral RBC count reached a nadir after hemolytic anemia. Flowcytometric analysis of peripheral blood cells and dispersed liver cells using anti-xlEPOR antibodies also indicate that the xlEPOR⁺ cells in liver were increased as decreasing count of peripheral RBC. After a nadir of RBC count, xlEPOR⁺ immature nucleated erythrocytes were emerged in the circulation. The count of xlEPOR⁺ immature erythrocytes was gradually decreased as increasing count of xlEPOR⁻ mature erythrocytes. Since mature erythrocytes are still nucleated in Xenopus, we used xlEPOR molecules as erythroid differentiation marker. Meanwhile real time RT-PCR analysis of Xenopus EPO (xlEPO) mRNA revealed that xlEpo gene expression was significantly induced in anemic liver compared to normal liver. These data suggest that xlEPO-xlEPOR signaling between erythrocyte progenitors and liver cells progress the proliferation and differentiation of erythrocyte progenitors, and the mobilization of immature erythrocytes into the circulation. Our previous study showed that the anemic serum of phenylhydrazine administrated Xenopus contains erythroid colony forming activity; however, there is no information about the relationship between anemia and hypoxia enough to stimulate erythropoiesis in adult Xenopus. In mammalian species, Epo gene expression is upregulated by binding of hypoxia inducible factor-1a (HIF-1a) and ARNT complex to hypoxia response element (HRE) located in 3′ enhancer region of Epo gene. In frog and fishes, EPO mRNAs are expressed even in normoxia condition. In fish Epo genes, consensus HRE sequence (ACGTG) were not found in 3′UTR, as well as the reporter assay failed to show Epo upregulation respond to hypoxia. Since any consensus HRE sequence was not found in 3′ UTR of xlEpo gene, we examined whether HIF-1a mediates xlEpo gene regulation. By western blot analysis of HIF-1a, we assessed whether HIF-1a is stabilized in anemia; meanwhile binding capacity of HIF-1a to 5′, 3′ UTRs and intron regions of xlEpo gene was analyzed by gel shift mobility assay. The findings in non-mammalian animal model demonstrate the basis of erythropoietic gene regulations, as well as molecular mechanism underlying adult extramedullary erythropoiesis.