ALBERT

Description: Erythropoietin (Epo) gene expression is controlled by hypoxia-inducible factor-1 (HIF-1); it is positively regulated through the HIF-1 binding site in the Epo enhancer and negatively regulated by GATA, which binds to the GATA site in the Epo promoter. Drugs that inhibit GATA or activate HIF-1 might increase the production of Epo and restore hemoglobin concentration. Epo gene doping and the illicit use of HIF-PHD inhibitor (FG-2216) and GATA inhibitor (K-11706) or HIF-1 activators may be used as new doping practices to increase the number of red blood cells. The broad scope of this research is to develop a system for detecting illegal hypoxia-inducible gene manipulation. We performed hematologic analyses, a treadmill exercise test, microarray and real-time RT-PCR (reverse transcriptase-polymerase chain reaction) to examine the effects of K-11706 and FG-2216 on mice, and compared these effects with those induced by recombinant human Epo (rhEpo) as a positive control. Oral administration of K-11706 for 5 and 8 days, FG-2216 for 5 days, and 14-day intra-peritoneal injection of rhEpo on alternate days significantly increased Epo and hemoglobin concentrations, hematocrit, and endurance performance, respectively, compared with the control. Transgenic lines of mice were generated to evaluate erythropoiesis in living mice. Firefly luciferase reporter line generated with the human β-globin locus control region (Hbb-LCR) is referred as Hbb-LCR-Luc; this mouse model provides a sensitive, noninvasive, and real-time method to monitor erythropoiesis in vivo in response to the administration of K-11706 and FG-2216. The oral administration of K-11706 and FG-2216 for 5 days significantly accumulated Hbb-LCR-Luc activity in the spleen. DNA chip technology was used to investigate the molecular mechanisms responsible for hypoxia-inducible gene manipulation. Total RNA was prepared from the bone marrow cells of mice treated with K-11706 for 28 days, FG-2216 for 5 days, and rhEpo for 14 days, and in untreated mice. The genes that appeared to exhibit a difference in the expression level (increase more or decrease less than five times for K-11706 and rhEpo, two times for FG-2216) were 251 genes for K-11706, 343 genes for FG-2216, and 142 genes for rhEpo. Twenty-eight genes showed a significant difference between the 3 groups, and 4 genes showed it with K-11706 and FG-2216. Among them, real-time RT-PCR revealed that FG-2216 significantly decreased the expression levels of FOS, OSM, IL8rb, Ms4a8a, and Arl5c genes to 0.58, 0.54, 0.56, 0.46, and 0.53-fold compared with the control, respectively. A panel of several candidate genes to detect hypoxia-inducible gene manipulation by real-time RT-PCR will provide a new detection system.

Description: Abstract 3614 Poster Board III-550 To maintain tissue oxygen homeostasis, erythropoietin (Epo) regulates the erythropoiesis. During embryonic development Epo is produced in the liver, but after birth and throughout adulthood Epo is produced and secreted from the kidney and support definitive erythropoiesis in the bone marrow. Anemia or hypoxic stress provokes transcription of the Epo gene and the mechanisms how the Epo gene expression is activated in a tissue-specific and hypoxia-inducible manner are one of the research topics of the molecular biology or gene regulation study. Since Epo-null mice die in utero of definitive erythropoiesis failure in the fetal liver, physiological significance of the Epo production for the adult erythropoiesis remains to be clarified. We have found that transgenic reporter gene expression driven by a BAC clone containing 180-kb mouse Epo gene locus recapitulates the Epo gene expression in the liver and kidney. To examine whether the Epo gene transcriptional activity within the 180-kb BAC is sufficient to recapitulate physiological Epo production, we attempted to rescue Epo-null mice from embryonic lethality with a BAC transgene harboring a floxed allele of the Epo gene. We found that transgene-derived Epo recovered the erythropoiesis in the fetal liver of Epo-null mice and Epo-null mice with the BAC transgene were born showing a Mendelian ratio. The rescued mice showed no hematological abnormality and were fertile. In the rescued mice bleeding anemia transiently increased plasma Epo level and after recovery from anemia plasma Epo level declined similarly to wild-type mice. In an RT-PCR analysis Epo production in the liver and kidney of the rescued mice was practically equivalent to that in the wild-type counterpart, while other tissues produced only marginal amount of Epo. These results thus demonstrate that the BAC transgene contains the Epo gene regulatory domain region sufficient to fully recapitulate the endogenous Epo gene expression and to compensate for the Epo-deficiency. We also found that the Epo gene deletion from the BAC transgene by general deletor-Cre transgene provoked the Epo-null phenotype, demonstrating that conditional inactivation of the Epo gene is attained in the BAC-rescued mice. The latter system will be a precious tool to investigate adult stage-specific roles of Epo. Disclosures: Yamamoto: Chugai Pharmaceutical, Co., Ltd. : Research Funding.

Description: In response to anemia erythropoietin ( Epo) gene transcription is markedly induced in the kidney and liver. This regulation has been ascribed for the promoter GATA motif and 3′ enhancer. To elucidate how the Epo gene expression is regulated in vivo, we established transgenic mouse lines expressing green fluorescent protein (GFP) under the control of 180-kb mouse Epo gene locus. GFP expression was induced by anemia or hypoxia specifically in peritubular interstitial cells of renal cortex and hepatocytes surrounding the central vein. Surprisingly, the renal Epo-producing cells were identified with neuron-like morphology and expression of neuronal markers. We also examined the regulatory mechanism of Epo gene using the transgenes in which mutations had been introduced in the GATA promoter motif, since we have reported the motif as a negative regulatory element for the inducible Epo gene expression. A single nucleotide mutation in the motif resulted in constitutive ectopic expression of GFP in the renal distal tubules, collecting ducts and certain epithelial cells of other tissues. Since both GATA-2 and GATA-3 bind to the GATA box in the distal tubular cells, these factors are likely to constitutively repress 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.

Description: 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 (pVHL) E3 ligase complex using 2-oxoglutarate as a substrate. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. The interaction between human pVHL and a specific domain of the HIF-1α subunit is regulated through hydroxylation of proline residues 402 and 564 by HIF-1α proryl-hydroxylase (PHD). N-oxalyl glycine acts as a competitive inhibitor of HIF-PHDs and this inhibition is in competition with 2-oxoglutarate. We examined the effect of 2-oxoglutarate on the production of vascular endothelial growth factor (VEGF) and erythropoietin (Epo). The expression of VEGF and Epo protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The enhancer activity of the HIF-1 binding site of Epo and the promoter activity of VEGF-luciferase were also 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 HIF-1α protein in Hep3B, Hela and SW480 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 tumor angiogenesis through decreasing HIF-1α protein, HIF-1 binding activity, the promoter activity of VEGF and enhancer activity of Epo, and the production of VEGF and Epo. More studies to determine if 2-oxoglutarate inhibits tumor angiogenesis in vivo mouse assay are in progress.

Description: Erythropoietin (Epo) is produced from the kidney and liver under anemic and/or hypoxic conditions. This inducible and tissue-specific production of Epo is regulated at the Epo gene transcription level. To elucidate the mechanisms how hypoxia inducible gene expression is regulated, a number of studies have been carried out and the function of the hypoxia-responsible enhancer sequence located in the 3′ flanking region of the Epo gene (3′HRE) were identified using the Epo-producing human hepatoma cell lines, Hep3B and HepG2. In order to clarify in vivo function of 3′HRE, in this study we deleted 500-bp mouse genomic region (referred to as BS region) containing 3′HRE by the gene targeting strategy. The homozygous BS-null mutant neonates suffered from severe anemia (hematocrit 17±2%) compared with wild-type neonate (hematocrit 28±2%). This anemic phenotype was observed from embryonic day 15 (E15). To our surprise, however, erythropoiesis in the adult BS-null mice was within the normal range. We then examined the expression level of Epo gene in the kidney and liver of BS-null mouse line in the embryonic stage, and found that the Epo gene expression was completely silenced in the BS-null embryonic liver after E15 to adulthood. The BS deletion did not affect the Epo gene expression in the liver up to midgestation stage (E10 to E13) and in the kidney throughout life. These results thus demonstrate that the BS region including 3′HRE is the enhancer for Epo gene that is functional after the late embryonic stage and in the hepatocytes. BS-null mice also showed that the hepatic erythropoiesis in the embryonic and neonatal stages requires the paracrine Epo secretion from the hepatocytes, and the Epo production in the kidney is insufficient to compensate the loss of hepatic Epo production.

Description: (Introduction) Aplastic anemia (AA) is characterized by a fatty bone marrow and the reduced number of hematopoietic stem cell (HSC)s, resulting in peripheral pancytopenia. Several clinical studies have reported that the expression of transcription factor GATA−2 is significantly decreased in CD34 positive cells in AA, suggesting that downregulation of GATA−2 in HSCs may cause the reduction of number of HSCs in AA. Of note, GATA−2 has been shown to be essential for the maintenance of immaturity of preadipocytes as well as HSCs. If GATA−2 functions in the differentiation of stromal preadipocytes in bone marrow, GATA−2 may be involved in the formation of a fatty bone marrow in AA. In order to explore the role of GATA−2 for development of AA, phenotypic changes of HSCs and stromal preadipocytes by aberrant expression of GATA−2 were examined in this study. (Method) First, to analyze phenotypic changes of HSCs by decreased expression of GATA-2, lineage negative, sca-1 positive, c-kit positive (LSK) cells were sorted both from wild-type and GATA-2 haplodeficient mice (kindly provided Dr SH Orkin), and the gene expression profile was compared by cDNA array. The difference of expression level on cDNA array was confirmed by RQ-PCR. Next, in order to examine the role of GATA-2 in the differentiation of stromal preadipocytes, the GATA-2-GFP expression vector was transfected into TBR343, a mouse stromal preadipocyte cell line, and GFP positive cells were sorted. The differentiation capacity of these GATA-2 overexpressing TBR343 cells was compared to that of control cells. In addition, GATA-2 expression was suppressed by using siRNA in TBR343, and the phenotypic change was also examined. (Results) By comparing the gene expression profile, the expression levels of cyclin D1, D3, p21, E2F, c-Myc were found to be decreased in GATA-2 haplodeficient LSKs, suggesting GATA-2 down-regulation in HSCs may inhibit the cell cycle progression. When GATA-2 was overexpressed in TBR343, the oil drop formation and adipocyte-specific gene expression was significantly suppressed. Conversely, When GATA-2 was suppressed in TBR343 by siRNA, the oil drop formation and adipocyte-specific gene expression was significantly accelerated, suggesting that decrease of GATA-2 expression accelerates the differentiation of stromal preadipocytes. (Conclusion) These results suggest that suppression of GATA-2 in the bone marrow leads to an inactive cell cycle of HSCs and the accumulation of mature adipocytes, which may contribute to the expression of the representative features of AA, a fatty bone marrow and the reduced number of HSCs.

Description: 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.