Kinuko Ohneda

Abnormal Heart Development and Lung Remodeling in Mice Lacking the Hypoxia-Inducible Factor-Related Basic Helix-Loop-Helix PAS Protein NEPAS

ABSTRACT Hypoxia-inducible factors (HIFs) are crucial for oxygen homeostasis during both embryonic development and postnatal life. Here we show that a novel HIF family basic helix-loop-helix (bHLH) PAS (Per-Arnt-Sim) protein, which is expressed predominantly during embryonic and neonatal stages and thereby designated NEPAS (neonatal and embryonic PAS), acts as a negative regulator of HIF-mediated gene expression. NEPAS mRNA is derived from the HIF-3α gene by alternative splicing, replacing the first exon of HIF-3α with that of inhibitory PAS. NEPAS can dimerize with Arnt and exhibits only low levels of transcriptional activity, similar to that of HIF-3α. NEPAS suppressed reporter gene expression driven by HIF-1α and HIF-2α. By generating mice with a targeted disruption of the NEPAS/HIF-3α locus, we found that homozygous mutant mice (NEPAS/HIF-3α−/−) were viable but displayed enlargement of the right ventricle and impaired lung remodeling. The expression of endothelin 1 and platelet-derived growth factor β was increased in the lung endothelial cells of NEPAS/HIF-3α-null mice. These results demonstrate a novel regulatory mechanism in which the activities of HIF-1α and HIF-2α are negatively regulated by NEPAS in endothelial cells, which is pertinent to lung and heart development during the embryonic and neonatal stages.

Hypoxia-inducible Transcription Factor-2α in Endothelial Cells Regulates Tumor Neovascularization through Activation of Ephrin A1

The hypoxia-inducible transcription factors (HIF)-1α and -2α mediate responses to hypoxia, such as tumor neovascularization. To determine the function of HIF-2α in vascular endothelial cells (ECs), we examined vascular formation in HIF-2α knockdown (kd/kd) mice transplanted with tumors. We observed that both the tumor size and the number of large vessels growing within transplanted melanomas were significantly reduced in kd/kd recipients compared with wild-type (WT) mice. In contrast, we observed a similar extent of vascular formation within fibrosarcomas transplanted from either kd/kd or WT mice into WT recipients. Thus, HIF-2α expression in host animal ECs, but not in the tumor cells, is crucial for tumor neovascularization. HIF-2α may function through ephrin A1 as the expression of ephrin A1 and related genes was markedly reduced in kd/kd ECs, and HIF-2α specifically bound a hypoxia-response element sequence in the ephrin A1 promoter. Treatment of WT ECs with an ephrin A1 inhibitor (ephrin A1-Fc) also impaired neovascularization. We conclude that in ECs, HIF-2α plays an essential role in vascular remodeling during tumor vascularization through activation of at least ephrin A1.

Leukemogenesis Caused by Incapacitated GATA-1 Function

ABSTRACT GATA-1 is essential for the development of erythroid and megakaryocytic lineages. We found that GATA-1 gene knockdown female (GATA-1.05/X) mice frequently develop a hematopoietic disorder resembling myelodysplastic syndrome that is characterized by the accumulation of progenitors expressing low levels of GATA-1. In this study, we demonstrate that GATA-1.05/X mice suffer from two distinct types of acute leukemia, an early-onset c-Kit-positive nonlymphoid leukemia and a late-onset B-lymphocytic leukemia. Since GATA-1 is an X chromosome gene, two types of hematopoietic cells reside within heterozygous GATA-1 knockdown mice, bearing either an active wild-type GATA-1 allele or an active mutant GATA-1.05 allele. In the hematopoietic progenitors with the latter allele, low-level GATA-1 expression is sufficient to support survival and proliferation but not differentiation, leading to the accumulation of progenitors that are easily targeted by oncogenic stimuli. Since such leukemia has not been observed in GATA-1-null/X mutant mice, we conclude that the residual GATA-1 activity in the knockdown mice contributes to the development of the malignancy. This de novo model recapitulates the acute crisis found in preleukemic conditions in humans.

Ablation of Gatal in adult mice results in aplastic crisis, revealing its essential role in steady-state and stress erythropoiesis

The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre-mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre-mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.

Hypoxia Responsive Mesenchymal Stem Cells Derived from Human Umbilical Cord Blood Are Effective for Bone Repair

Mesenchymal stem cells (MSCs) are highly useful in a variety of cell therapies owing to their multipotential differentiation capability. MSCs derived from umbilical cord blood are generally isolated by their plastic adherence without using specific cell surface markers and examined for their osteogenic, adipogenic, and chondrogenic differentiation properties retrospectively. Here, we report 2 subpopulations of MSCs, separated based on aldehyde dehydrogenase (ALDH) activity. MSCs with a high ALDH activity (Alde-High) proliferated more than those with a low ALDH activity (Alde-Low). Alde-High MSCs had a greater ability to differentiate than Alde-Low MSCs in in vitro culture. Transplantation of Alde-High MSCs into fractured mouse femurs enabled early repair of tissues and rapid bone substitution. Alde-High MSCs were also more responsive to hypoxia than Alde-Low MSCs, with the upregulation of Flt-1, CXCR4, and Angiopoietin-2. Thus, MSCs with a high ALDH activity might serve as an effective therapeutic tool for healing fractures within a short period of time.

Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation.

ABSTRACT GATA1 is a key regulator of erythroid cell differentiation. To examine how Gata1 gene expression is regulated in a stage-specific manner, transgenic mouse lines expressing green fluorescent protein (GFP) reporter from the Gata1 locus in a bacterial artificial chromosome (G1BAC-GFP) were prepared. We found that the GFP reporter expression faithfully recapitulated Gata1 gene expression. Using GFP fluorescence in combination with hematopoietic surface markers, we established a purification protocol for two erythroid progenitor fractions, referred to as burst-forming units-erythroid cell-related erythroid progenitor (BREP) and CFU-erythroid cell-related erythroid progenitor (CREP) fractions. We examined the functions of the Gata1 gene hematopoietic enhancer (G1HE) and the highly conserved GATA box in the enhancer core. Both deletion of the G1HE and substitution mutation of the GATA box caused almost complete loss of GFP expression in the BREP fraction, but the CREP stage expression was suppressed only partially, indicating the critical contribution of the GATA box to the BREP stage expression of Gata1. Consistently, targeted deletion of G1HE from the chromosomal Gata1 locus provoked suppressed expression of the Gata1 gene in the BREP fraction, which led to aberrant accumulation of BREP stage hematopoietic progenitor cells. These results demonstrate the physiological significance of the dynamic regulation of Gata1 gene expression in a differentiation stage-specific manner.

Identification of human placenta-derived mesenchymal stem cells involved in re-endothelialization†

Human placenta is an attractive source of mesenchymal stem cells (MSC) for regenerative medicine. The cell surface markers expressed on MSC have been proposed as useful tools for the isolation of MSC from other cell populations. However, the correlation between the expression of MSC markers and the ability to support tissue regeneration in vivo has not been well examined. Here, we established several MSC lines from human placenta and examined the expression of their cell surface markers and their ability to differentiate toward mesenchymal cell lineages. We found that the expression of CD349/frizzled‐9, a receptor for Wnt ligands, was positive in placenta‐derived MSC. So, we isolated CD349‐negative and ‐positive fractions from an MSC line and examined how successfully cell engraftment repaired fractured bone and recovered blood flow in ischemic regions using mouse models. CD349‐negative and ‐positive cells displayed a similar expression pattern of cell surface markers and facilitated the repair of fractured bone in transplantation experiments in mice. Interestingly, CD349‐negative, but not CD349‐positive cells, showed significant effects on recovering blood flow following vascular occlusion. We found that induction of PDGFβ and bFGF mRNAs by hypoxia was greater in CD349‐negative cells than in CD349‐positive cells while the expression of VEGF was not significantly different in CD349‐negative and CD349‐positive cells. These findings suggest the possibility that CD349 could be utilized as a specialized marker for MSC isolation for re‐endothelialization. J. Cell. Physiol. 226: 224–235, 2010.

GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation

Transcription factor GATA2 is highly expressed in hematopoietic stem cells and progenitors, whereas its expression declines after erythroid commitment of progenitors. In contrast, the start of GATA1 expression coincides with the erythroid commitment and increases along with the erythroid differentiation. We refer this dynamic transition of GATA factor expression to as the ‘GATA factor switching’. Here, we examined contribution of the GATA factor switching to the erythroid differentiation. In Gata1‐knockdown embryos that concomitantly express Gata2‐GFP reporter, high‐level expression of GFP reporter was detected in accumulated immature hematopoietic cells with impaired differentiation, demonstrating that GATA1 represses Gata2 gene expression in hematopoietic progenitors in vivo. We have conducted chromatin immunoprecipitation (ChIP) on microarray analyses of GATA2 and GATA1, and results indicate that the GATA1‐binding sites widely overlap with the sites pre‐occupied by GATA2 before the GATA1 expression. Importantly, erythroid genes harboring GATA boxes bound by both GATA1 and GATA2 tend to be expressed in immature erythroid cells, whereas those harboring GATA boxes to which GATA1 binds highly but GATA2 binds only weakly are important for the mature erythroid cell function. Our results thus support the contention that preceding binding of GATA2 helps the following binding of GATA1 and thereby secures smooth expression of the transient‐phase genes.

The microenvironment for erythropoiesis is regulated by HIF-2α through VCAM-1 in endothelial cells

Erythropoiesis is a dynamic process regulated by oxygen in vertebrates. Recent evidence has indicated that erythropoietin (Epo) expression is regulated by hypoxia-inducible transcription factors (HIFs), HIF-2alpha in particular. In this study, we report that knockdown mutation of HIF-2alpha in mice (kd/kd) results in normocytic anemia, despite Epo induction in response to hypoxia not being severely affected. Transplantation analyses clearly demonstrated that the hematopoietic microenvironment, but not the hematopoietic cells, was altered in kd/kd. Furthermore, cell-type specific recovery of HIF-2alpha expression in endothelial cells (ECs) abrogated the anemic condition of the kd/kd mice, indicating that HIF-2alpha in EC plays an essential role in supporting erythropoiesis. In the absence of HIF-2alpha, the expression of vascular adhesion molecule-1 (VCAM-1) was reduced significantly and restoration of VCAM-1 expression in kd/kd ECs enhanced the development of erythroid progenitors. Finally, a chromatin immunoprecipitation assay and a reporter assay indicated that VCAM-1 gene transcription is directly regulated by HIF-2alpha. These data suggest that the hematopoietic microenvironment required for erythropoiesis is dynamically regulated by oxygen through the functions of HIF-2alpha in ECs.

A Chemokine Receptor, CXCR4, Which Is Regulated by Hypoxia-Inducible Factor 2α, Is Crucial for Functional Endothelial Progenitor Cells Migration to Ischemic Tissue and Wound Repair

Endothelial progenitor cells (EPCs) have the ability to form new blood vessels and protect ischemic tissues from damage. We previously reported that EPCs with low activity of aldehyde dehydrogenase (Alde-Low EPCs) possess the greater ability to treat ischemic tissues compared with Alde-High EPCs. The expression level of the hypoxia-inducible factors (HIFs), HIF-1α and HIF-2α, was found to be greater in Alde-Low EPCs than in Alde-High EPCs. However, the precise role of the HIF factors in the regulation of EPC activity remains obscure. In this study, we demonstrate a critical role of HIF-2α and its target gene CXCR4 for controlling the migratory activity of EPC to ischemic tissue. We found that coculture of Alde-High EPCs with microvesicles derived from Alde-Low EPCs improved their ability to repair an ischemic skin flap, and the expression of CXCR4 and its ligand SDF1 was significantly increased following the coculture. In Alde-Low EPCs, the expression of CXCR4 was suppressed by short hairpin RNA (shRNA)-mediated HIF-2α, but not HIF-1α downregulation. Chromatin immunoprecipitation assays showed that HIF-2α, but not HIF-1α, binds to the promoter region of CXCR4 gene. The CXCR4 shRNA treatment in Alde-Low EPCs almost completely abrogated their migratory activity to ischemic tissues, whereas the reduction of vascular endothelial growth factor (VEGF) showed much less effect. The CXCR4 overexpression in Alde-High EPCs resulted in a partial, but significant improvement in their repairing ability in an ischemic skin flap. Collectively, these findings indicate that the CXCR4/SDF-1 axis, which is specifically regulated by HIF-2α, plays a crucial role in the regulation of EPC migration to ischemic tissues.