Donghoon Yoon

Hypoxia-inducible factor-1 deficiency results in dysregulated erythropoiesis signaling and iron homeostasis in mouse development.

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 Hif1α gene, which encodes the hypoxia-responsive α 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-1α-deficient embryos, as well as decreased hemoglobin content in erythroid colonies from HIF-1α-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 Hif1α-/- yolk sac as well as Epo and EpoR mRNA in Hif1α-/- embryos. The erythropoietic defects in HIF-1α-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, Hif1α-/- 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 Hif1α-/- 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.

miR-451 enhances erythroid differentiation in K562 cells

Erythropoiesis is a multistep process regulated at the molecular level by intrinsic and extrinsic factors including microRNAs (miRNAs). We previously identified aberrant expression of miR-451 and miR-150 in polycythemia vera (PV) erythroid differentiating cells. To address the functional relevance of these miRNAs in erythroid differentiation, we employed synthetic mimics and inhibitors of miR-451 and miR-150 in erythroid differentiating K562 cells. We observed that miR-451 up-regulation and miR-150 down-regulation are associated with progression of erythroid maturation in K562 cells. Further, enforced expression of miR-451 promoted erythroid differentiation. Inhibition of miR-150 reduced hemoglobinization of K562 cells. Microarray data suggested potential targets regulated by miR-451: UBE2H, ARPP-19; and by miR-150: MS4A3, AGA, PTPRR. Our results demonstrate that miR-451 is involved in the regulation of erythroid differentiation and functions as an enhancer of differentiation. These data support the concept that aberrant expression of miRNAs may contribute to abnormal erythropoiesis such as that of PV.

Hypoxia. 5. Hypoxia and hematopoiesis

Our understanding of organismal responses to hypoxia has stemmed from studies of erythropoietin regulation by hypoxia that led to the discovery of the master regulator of the hypoxic response, i.e., hypoxia-inducible factor (HIF). This is a transcription factor that is now known to induce the expression of a battery of genes in response to hypoxia. HIF-1 and HIF-2 regulate many genes that are involved in erythropoiesis and iron metabolism, which are essential for tissue oxygen delivery.

Stat3 promotes the development of erythroleukemia by inducing Pu.1 expression and inhibiting erythroid differentiation

Leukemogenesis requires two classes of mutations, one that promotes proliferation and one that blocks differentiation. The erythroleukemia induced by Friend virus is a multistage disease characterized by an early proliferative stage driven by the interaction of the viral glycoprotein, gp55, with Sf-Stk and the EpoR, and a late block to differentiation resulting from retroviral insertion in the Pu.1 locus. We demonstrate here that activation of Stat3 by Sf-Stk in the early stage of disease is essential for the progression of erythroleukemia in the presence of differentiation signals induced by the EpoR, but is dispensable in the late stages of the disease. Furthermore, we identify Pu.1 as a Stat3 target gene in the early stages of erythroleukemia development. Our results support a model whereby the activation of Stat3 in the early stage of disease plays a pivotal role in regulating differentiation through the upregulation of Pu.1, thus inhibiting differentiation and favoring the expansion of infected erythroblasts and enhancing the pool of progenitors available for the acquisition of additional mutations, including insertional activation of Pu.1, resulting in full leukemic transformation.

Decreased serum glucose and glycosylated hemoglobin levels in patients with Chuvash polycythemia: a role for HIF in glucose metabolism

In Chuvash polycythemia, a homozygous 598C>T mutation in the von Hippel–Lindau gene (VHL) leads to an R200W substitution in VHL protein, impaired degradation of α-subunits of hypoxia-inducible factor (HIF)-1 and HIF-2, and augmented hypoxic responses during normoxia. Chronic hypoxia of high altitude is associated with decreased serum glucose and insulin concentrations. Other investigators reported that HIF-1 promotes cellular glucose uptake by increased expression of GLUT1 and increased glycolysis by increased expression of enzymes such as PDK. On the other hand, inactivation of Vhl in murine liver leads to hypoglycemia associated with a HIF-2-related decrease in the expression of the gluconeogenic enzyme genes Pepck, G6pc, and Glut2. We therefore hypothesized that glucose concentrations are decreased in individuals with Chuvash polycythemia. We found that 88 Chuvash VHLR200W homozygotes had lower random glucose and glycosylated hemoglobin A1c levels than 52 Chuvash subjects with wild-type VHL alleles. Serum metabolomics revealed higher glycerol and citrate levels in the VHLR200W homozygotes. We expanded these observations in VHLR200W homozygote mice and found that they had lower fasting glucose values and lower glucose excursions than wild-type control mice but no change in fasting insulin concentrations. Hepatic expression of Glut2 and G6pc, but not Pdk2, was decreased, and skeletal muscle expression of Glut1, Pdk1, and Pdk4 was increased. These results suggest that both decreased hepatic gluconeogenesis and increased skeletal uptake and glycolysis contribute to the decreased glucose concentrations. Further study is needed to determine whether pharmacologically manipulating HIF expression might be beneficial for treatment of diabetic patients.

Anti-inflammatory effect of methyl dehydrojasmonate (J2) is mediated by the NF-κB pathway

Inflammation as a major defense mechanism against pathogens is modulated by diverse microbial products. A variety of plant and microbial products interacting with Toll-like receptors initiate a wide spectrum of responses from phagocytosis to cytokine production, which modulates inflammation. Jasmonates are fatty acid-derived cyclopentanones produced by plants and lower eukaryotes that play an important role in the defense against insects. In this study, we are set up to define the molecular targets of J2 action. While the lipopolysaccharide (LPS) stimulation of macrophage cell line RAW264.7 induced TNF-α, IL-6, iNOS, and COX-2 that were associated with an increase in miR-155 and miR-146a, the J2 suppressed the induction of these inflammatory cytokines and enzymes as well as miR-155 in a dose-dependent manner. To assess the associations of miR-155 with inflammatory markers, we overexpressed miR-155 and found attenuation of COX-2 suppression with J2 treatment. Furthermore, J2 inhibited NF-κB, p65, and IκB but had no or only minimal effects on the mitogen-activated protein kinase pathway. In conclusion, the present study demonstrates that J2 suppresses LPS stimulation of RAW264.7 cells by targeting NF-κB pathways.

The phenotype of polycythemia due to Croatian homozygous VHL (571C>G:H191D) mutation is different from that of Chuvash polycythemia (VHL 598C>T:R200W)

Mutations of VHL (a negative regulator of hypoxia-inducible factors) have position-dependent distinct cancer phenotypes. Only two known inherited homozygous VHL mutations exist and they cause polycythemia: Chuvash R200W and Croatian H191D. We report a second polycythemic Croatian H191D homozygote distantly related to the first propositus. Three generations of both families were genotyped for analysis of shared ancestry. Biochemical and molecular tests were performed to better define their phenotypes, with an emphasis on a comparison with Chuvash polycythemia. The VHL H191D mutation did not segregate in the family defined by the known common ancestors of the two subjects, suggesting a high prevalence in Croatians, but haplotype analysis indicated an undocumented common ancestor ∼six generations ago as the founder of this mutation. We show that erythropoietin levels in homozygous VHL H191D individuals are higher than in VHL R200W patients of similar ages, and their native erythroid progenitors, unlike Chuvash R200W, are not hypersensitive to erythropoietin. This observation contrasts with a report suggesting that polycythemia in VHL R200W and H191D homozygotes is due to the loss of JAK2 regulation from VHL R200W and H191D binding to SOCS1. In conclusion, our studies further define the hematologic phenotype of VHL H191D and provide additional evidence for phenotypic heterogeneity associated with the positional effects of VHL mutations.

EPO Receptor Gain-of-Function Causes Hereditary Polycythemia, Alters CD34+ Cell Differentiation and Increases Circulating Endothelial Precursors

Background Gain-of-function of erythropoietin receptor (EPOR) mutations represent the major cause of primary hereditary polycythemia. EPOR is also found in non-erythroid tissues, although its physiological role is still undefined. Methodology/Principal Findings We describe a family with polycythemia due to a heterozygous mutation of the EPOR gene that causes a G→T change at nucleotide 1251 of exon 8. The novel EPOR G1251T mutation results in the replacement of a glutamate residue by a stop codon at amino acid 393. Differently from polycythemia vera, EPOR G1251T CD34+ cells proliferate and differentiate towards the erythroid phenotype in the presence of minimal amounts of EPO. Moreover, the affected individuals show a 20-fold increase of circulating endothelial precursors. The analysis of erythroid precursor membranes demonstrates a heretofore undescribed accumulation of the truncated EPOR, probably due to the absence of residues involved in the EPO-dependent receptor internalization and degradation. Mutated receptor expression in EPOR-negative cells results in EPOR and Stat5 phosphorylation. Moreover, patient erythroid precursors present an increased activation of EPOR and its effectors, including Stat5 and Erk1/2 pathway. Conclusions/Significance Our data provide an unanticipated mechanism for autosomal dominant inherited polycythemia due to a heterozygous EPOR mutation and suggest a regulatory role of EPO/EPOR pathway in human circulating endothelial precursors homeostasis.

Extent of hematopoietic involvement by TET2 mutations in JAK2V617F polycythemia vera

TET2 mutations are found in polycythemia vera and it was initially reported that there is a greater TET2 mutational burden than JAK2V617F in polycythemia vera stem cells and that TET2 mutations precede JAK2V617F. We quantified the proportion of TET2, JAK2V617F mutations and X-chromosome allelic usage in polycythemia vera cells, BFU-Es and in vitro expanded erythroid progenitors and found clonal reticulocytes, granulocytes, platelets and CD34+ cells. We found that TET2 mutations may also follow rather than precede JAK2V617F as recently reported by others. Only a fraction of clonal early hematopoietic precursors and largely polyclonal T cells carry the TET2 mutation. We showed that in vitro the concomitant presence of JAK2V617F and TET2 mutations favors clonal polycythemia vera erythroid progenitors in contrast with non-TET2 mutated progenitors. We conclude that loss-of-function TET2 mutations are not the polycythemia vera initiating events and that the acquisition of TET2 somatic mutations may increase the aggressivity of the polycythemia vera clone.

Erythropoiesis in Polycythemia Vera is Hyper-Proliferative and has Accelerated Maturation

Polycythemia vera (PV) is an acquired myeloproliferative clonal disorder, characterized by augmented erythropoiesis. To better define PV pathogenesis, we performed an in vitro erythroid expansion from peripheral blood mononuclear cells of controls and PV patients and evaluated the cells for proliferation, apoptosis, erythroid differentiation, and morphology at the defined time points. PV erythroid progenitors exhibited increased proliferation at days 9-14 and accelerated maturation at days 7-14, with a larger S-phase population (40%) than controls (20%) at day 11; however, the proportion of apoptotic cells was comparable to controls. Previously, we have identified PV-specific dysregulation of several microRNAs (i.e. miR-150, 451, 222, 155, 378). We had analyzed expression profiles of selected target genes of these microRNAs based on in silico prediction and their known function pertinent to the observed PV-specific erythropoiesis differences. p27, cMYB and EPOR showed differential expression in PV erythroid progenitors at the specific stages of erythroid differentiation. In this study, we identified accelerated maturation and hyper-proliferation at early stages of PV erythropoiesis. We speculate that aberrant expression of p27, c-MYB, and EPOR may contribute to these abnormal features in PV erythropoiesis.