Naoshi Obara

Frequent pathway mutations of splicing machinery in myelodysplasia.

Myelodysplastic syndromes and related disorders (myelodysplasia) are a heterogeneous group of myeloid neoplasms showing deregulated blood cell production with evidence of myeloid dysplasia and a predisposition to acute myeloid leukaemia, whose pathogenesis is only incompletely understood. Here we report whole-exome sequencing of 29 myelodysplasia specimens, which unexpectedly revealed novel pathway mutations involving multiple components of the RNA splicing machinery, including U2AF35, ZRSR2, SRSF2 and SF3B1. In a large series analysis, these splicing pathway mutations were frequent (∼45 to ∼85%) in, and highly specific to, myeloid neoplasms showing features of myelodysplasia. Conspicuously, most of the mutations, which occurred in a mutually exclusive manner, affected genes involved in the 3′-splice site recognition during pre-mRNA processing, inducing abnormal RNA splicing and compromised haematopoiesis. Our results provide the first evidence indicating that genetic alterations of the major splicing components could be involved in human pathogenesis, also implicating a novel therapeutic possibility for myelodysplasia.

Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms

Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.

Repression via the GATA box is essential for tissue-specific erythropoietin gene expression

In response to anemia, erythropoietin (Epo) gene transcription is markedly induced in the kidney and liver. To elucidate how Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of a 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of the kidney and hepatocytes surrounding the central vein. Surprisingly, renal Epo-producing cells had a neuronlike morphology and expressed neuronal marker genes. Furthermore, the regulatory mechanisms of Epo gene expression were explored using transgenes containing mutations in the GATA motif of the promoter region. A single nucleotide mutation in this motif resulted in constitutive ectopic expression of transgenic GFP in renal distal tubules, collecting ducts, and certain populations of epithelial cells in other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in distal tubular cells, both factors are likely to repress constitutively ectopic Epo gene expression in these cells. Thus, GATA-based repression is essential for the inducible and cell type-specific expression of the Epo gene.

Interleukin-6 and Tumor Necrosis Factor-α in Patients with Obstructive Sleep Apnea-Hypopnea Syndrome

Background: In previous studies, significantly elevated levels of vascular endothelial growth factor (VEGF) have been reported in patients with severe obstructive sleep apnea-hypopnea syndrome (OSAHS). On the other hand, plasma tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) have been significantly higher in mild sleep apneics than in normal controls. However, this study included a small number of patients and milder cases of OSAHS. Objectives andMethods: To assess the involvement of IL-6 and TNF-α in VEGF increases in patients with severe OSAHS, serum levels of IL-6 and TNF-α were determined in patients with severe OSAHS (n = 110) and compared to those of controls (n = 45) using an enzyme-linked immunosorbent assay. Results: No significant increase in IL-6 or TNF-α was detected in the present study cohort. However, the body mass index was significantly correlated with the severity of the apnea-hypopnea index. Conclusions: These data suggest that the elevation in VEGF is not directly related to IL-6 or TNF-α levels. However, the question of whether VEGF is the cause or the result of OSAHS remains to be determined. Further studies are needed to clarify the role of IL-6 and TNF-α in the pathogenesis of OSAHS, in which obesity should be entered as an independent factor.

A GATA-specific inhibitor (K-7174) rescues anemia induced by IL-1β, TNF-α, or L-NMMA

Interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), or NG‐monomethyl‐l‐arginine (l‐NMMA) are increased in patients with chronic disease‐related anemia. They increase the binding activity of GATA and inhibit erythropoietin (Epo) promoter activity. In this study, we examined the ability of K‐7174 (a GATA‐specific inhibitor) to improve Epo production when inhibited by treatment with IL‐1β, TNF‐α, or l‐NMMA. Epo protein production and promoter activity were induced in Hep3B cells with 1% O2. However, 15 U/ml IL‐1β, 220 U/ml TNF‐α, or 10−3 M l‐NMMA inhibited Epo protein production and promoter activity, respectively. Addition of 10 µM K‐7174 rescued these inhibitions of Epo protein production and promoter activity induced by IL‐1β, TNF‐α, or l‐NMMA, respectively. Electrophoretic mobility shift assays revealed that addition of K‐7174 decreased GATA binding activity, which was increased with the addition of IL‐1β, TNF‐α, or l‐NMMA. Furthermore, intraperitoneal injection of mice with IL‐1β or TNF‐α decreased the hemoglobin concentrations and reticulocyte counts. However, the addition of K‐7174 reversed these effects. These results raise the possibility of using K‐7174 as therapy to treat anemia.

Specific Contribution of the Erythropoietin Gene 3′ Enhancer to Hepatic Erythropoiesis after Late Embryonic Stages

ABSTRACT Erythropoietin (Epo) is secreted from the liver and kidney, where Epo production is strictly regulated at the transcriptional level in a hypoxia- and/or anemia-inducible manner. Here, we examined the in vivo function of the enhancer located 3′ to the Epo gene (EpoE-3′). Reporter transgenic-mouse analyses revealed that the EpoE-3′ enhancer is necessary and sufficient for the liver-specific and hypoxia-responsive expression of the gene after embryonic day 14.5 (E14.5). However, the enhancer is dispensable for Epo gene expression in the kidney and early-stage embryonic liver. Genetic removal of EpoE-3′ from the endogenous Epo gene resulted in mice with severe anemia at late embryonic and neonatal stages due to defects in hepatic erythropoiesis, but early hepatic and splenic erythropoiesis was not affected. The mutant mice recover from the anemia in the juvenile period when major Epo production switches from the liver to the kidney. These results demonstrate that EpoE-3′ is necessary for late hepatic erythropoiesis by specifically supporting paracrine production of Epo in the liver. In contrast, Epo production in the kidney utilizes distinct regulatory machinery and supports erythropoiesis in the bone marrow and spleen in adult animals.

Suppression of erythropoietin gene expression by cadmium depends on inhibition of HIF-1, not stimulation of GATA-2

Long-term exposure of rats to cadmium (Cd) resulted in a marked suppression of erythropoietin (Epo) mRNA expression in the kidneys and the development of severe anemia. A recent report revealed that Cd inhibited hypoxia-inducible factor 1 (HIF-1) binding activity and Epo mRNA expression and protein production. However, Epo gene expression is also regulated by transcription factor GATA-2, which binds to the GATA binding site of the Epo promoter. To elucidate the mechanism of suppression of Epo by Cd, the effect of Cd on GATA-2 function was studied. Epo promoter/enhancer luciferase constructs, one with the wild-type promoter and another with a promoter with a mutant GATA site, were transfected into Hep3B cells. No significant difference in Epo promoter activity in these two types of cells was observed in the presence of Cd. The binding activity of GATA-2 was not affected by Cd. This study showed that Cd inhibited HIF-1 binding activity and Epo promoter activity, and then suppressed Epo protein production. Inhibition of Epo gene expression by Cd depends on suppression of HIF-1 binding activity, not on alteration of GATA function.

Reduced TET2 function leads to T-cell lymphoma with follicular helper T-cell-like features in mice

TET2 (Ten Eleven Translocation 2) is a dioxygenase that converts methylcytosine (mC) to hydroxymethylcytosine (hmC). TET2 loss-of-function mutations are highly frequent in subtypes of T-cell lymphoma that harbor follicular helper T (Tfh)-cell-like features, such as angioimmunoblastic T-cell lymphoma (30–83%) or peripheral T-cell lymphoma, not otherwise specified (10–49%), as well as myeloid malignancies. Here, we show that middle-aged Tet2 knockdown (Tet2gt/gt) mice exhibit Tfh-like cell overproduction in the spleen compared with control mice. The Tet2 knockdown mice eventually develop T-cell lymphoma with Tfh-like features after a long latency (median 67 weeks). Transcriptome analysis revealed that these lymphoma cells had Tfh-like gene expression patterns when compared with splenic CD4-positive cells of wild-type mice. The lymphoma cells showed lower hmC densities around the transcription start site (TSS) and higher mC densities at the regions of the TSS, gene body and CpG islands. These epigenetic changes, seen in Tet2 insufficiency-triggered lymphoma, possibly contributed to predated outgrowth of Tfh-like cells and subsequent lymphomagenesis. The mouse model described here suggests that TET2 mutations play a major role in the development of T-cell lymphoma with Tfh-like features in humans.

Antitumor effects of 2‐oxoglutarate through inhibition of angiogenesis in a murine tumor model

Hypoxia‐inducible factor 1 (HIF‐1) plays essential roles in tumor angiogenesis and growth by regulating the transcription of several key genes in response to hypoxic stress and growth factors. HIF‐1 is a heterodimeric transcriptional activator consisting of inducible α and constitutive β subunits. In oxygenated cells, proteins containing the prolyl hydroxylase domain (PHD) directly sense intracellular oxygen concentrations. PHDs tag HIF‐1α subunits for polyubiquitination and proteasomal degradation by prolyl hydroxylation using 2‐oxoglutarate (2‐OX) and dioxygen. Our recent studies showed that 2‐OX reduces HIF‐1α, erythropoietin, and vascular endothelial growth factor (VEGF) expression in the hepatoma cell line Hep3B when under hypoxic conditions in vitro. Here, we report that similar results were obtained in Lewis lung cancer (LLC) cells in in vitro studies. Furthermore, 2‐OX showed potent antitumor effects in a mouse dorsal air sac assay and a murine tumor xenograft model. In the dorsal air sac assay, 2‐OX reduced the numbers of newly formed vessels induced by LLC cells. In a murine tumor xenograft model, intraperitoneal injection of 2‐OX significantly inhibited tumor growth and angiogenesis in tumor tissues. Moreover, 5‐fluorouracil combined with 2‐OX significantly inhibited tumor growth in this model, which was accompanied by reduction of Vegf gene expression and inhibited angiogenesis in tumor tissues. These results suggest that 2‐OX is a promising anti‐angiogenic therapeutic agent. (Cancer Sci 2009; 100: 1639–1647)

2-oxoglutarate downregulates expression of vascular endothelial growth factor and erythropoietin through decreasing hypoxia-inducible factor-1α and inhibits angiogenesis

In oxygenated cells, hypoxia‐inducible factor‐1 (HIF‐1) α subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel–Lindau tumor suppressor E3 ligase complex using 2‐oxoglutarate as a substrate. We examined the effect of 2‐oxoglutarate on the production of erythropoietin and vascular endothelial growth factor (VEGF). The expression of erythropoietin and VEGF protein were dose‐dependently downregulated in Hep3B cells by the addition of 2‐oxoglutarate. The promoter activity of VEGF‐luciferase was dose‐dependently downregulated by the addition of 2‐oxoglutarate. Gel mobility shift assays revealed that the addition of 2‐oxoglutarate dose‐dependently inhibited HIF‐1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2‐oxoglutarate dose‐dependently inhibited the HIF‐1α protein level in Hep3B cells in hypoxic conditions. However, MG132 (the proteasome inhibitor) rescued the inhibition of HIF‐1α protein expression by 2‐oxoglutarate. Furthermore, under hypoxic conditions, 2‐oxoglutarate dose‐dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2‐oxoglutarate treatment may be useful for the inhibition of angiogenesis. J. Cell. Physiol. 209: 333–340, 2006.