Ryuzo Sasaki

Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death

Recently, erythropoietin has been shown to be produced by astrocytes and its production is hypoxia-inducible. In the present study, we demonstrated, using a reverse transcription-polymerase chain reaction assay and immunostaining of the cells, that the erythropoietin receptor was expressed in cultured hippocampal and cerebral cortical neurons of day 19 rat embryo. Erythropoietin protected the cultured neurons from glutamate neurotoxicity. Neurons cultured for seven to 10 days were exposed to glutamate for 15 min and after culture for a further 24 h in the absence of glutamate the neuron survival was assayed. Significant protection was observed with erythropoietin from 3 pM (c. 100 pg/ml) in a dose-dependent manner. The protection was completely reversed by co-application of a soluble erythropoietin receptor, an extracellular domain capable of binding with erythropoietin. For exhibition of the neuroprotective effect, exposure of neurons to erythropoietin approximately 8 h prior to exposure to glutamate was required. Experiments with the inhibitors indicated that RNA and protein syntheses were necessary for the protection. However, exposure to erythropoietin for a short period (5 min or less) was sufficient to elicit the protective effect. The protective effect of erythropoietin was blocked by the simultaneous addition of EGTA. These findings and the previous finding that erythropoietin induces a rapid and transient increase in intracellular Ca2+ concentration in neuronal cells suggest that erythropoietin plays a neuroprotective role in brain injury caused by hypoxia or ischemia and that erythropoietin-induced Ca2+ influx from outside of the cells is a critical initial event yielding an enhanced resistance of the neurons to glutamate toxicity.

ESTROGEN-DEPENDENT PRODUCTION OF ERYTHROPOIETIN IN UTERUS AND ITS IMPLICATION IN UTERINE ANGIOGENESIS

Although erythropoietin (Epo) has been shown to possess in vitro angiogenic activity, its physiological significance has not been demonstrated. Normally angiogenesis does not occur actively in adults but an exception is the female reproductive organ. In the uterine endometrium, angiogenesis takes place actively for supporting the endometrial growth that occurs during transition from the diestrus to estrous stage. This transition is under control of 17β-estradiol (E2), an ovarian hormone, and can be mimicked by injection of E2 to ovariectomized (OVX) mouse. Thus, the uterus is a pertinent site to examine the Epo function in angiogenesis. We found that Epo protein and its mRNA were produced in an E2-dependent manner, when the uterus from OVX mouse was cultured in vitro. The de novoprotein synthesis was not needed for E2 induction of Epo mRNA. Administration of E2 to OVX mouse induced a rapid and transient increase in Epo mRNA in the uterus. Injection of Epo into the OVX mouse uterine cavity promoted blood vessel formation in the endometrium. Furthermore, injection of the soluble Epo receptor capable of binding with Epo into the uterine cavity of non-OVX mouse in diestrus stage inhibited the endometrial transition to proestrus stage, whereas heat-inactivated soluble Epo receptor allowed the transition to occur. These results, combined with our finding that the endothelial cells in uterine endometrium express Epo receptor, strongly suggest that Epo is an important factor for the E2-dependent cyclical angiogenesis in uterus.

Identification of Cell-Active Lysine Specific Demethylase 1-Selective Inhibitors

Lysine specific demethylase 1 (LSD1) plays a key role in the regulation of gene expression by removing the methyl groups from methylated Lys4 of histone H3 (H3K4). Here we report the identification of the first small-molecule LSD1-selective inhibitors. These inhibitors show in vivo H3K4-methylating activity and antiproliferative activity and should be useful as lead structures for anticancer drugs and as tools for studying the biological roles of LSD1.

Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells

Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer. Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells. However, it remained attached to the cell wall and was not released into the culture medium. Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells. Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells. However, it had no in vivo biological activities. A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.

Design, Synthesis, Enzyme-Inhibitory Activity, and Effect on Human Cancer Cells of a Novel Series of Jumonji Domain-Containing Protein 2 Histone Demethylase Inhibitors

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.

Erythropoietin: Multiple Physiological Functions and Regulation of Biosynthesis

Erythropoietin (Epo), which is produced by the kidney in the adult and by the liver in the fetus, increases red blood cells by supporting the survival of erythroid progenitor cells and stimulating their differentiation and proliferation via binding to Epo receptor (EpoR). The main signal in the control of Epo production is oxygen; hypoxia stimulates Epo production through activation of Epo gene transcription. Tremendous progress in our understanding of molecular mechanisms of Epo action on erythroid cells and regulation of the Epo production has been made by manipulation of cDNAs and genes of Epo and EpoR. Studies on hypoxic induction of Epo gene transcription led to the identification of hypoxia-inducible factor (HIF-1), a transcriptional factor, that functions as a global regulator of hypoxic gene expression. Paracrine Epo/EpoR systems that are independent of the endocrine erythropoietic system (kidney/bone marrow) have been found in the central nervous system and uterus. Novel functions of Epo at these local sites and tissue-specific regulation of Epo production including a newly found potent regulator (estrogen) have been proposed. The tissue-specific regulation rationalizes the specific functions of Epo produced by individual tissues.

Inhibition of erythropoietin signalling destroys xenografts of ovarian and uterine cancers in nude mice

We have recently shown that malignant tumours from the ovary and uterus expressed erythropoietin (Epo) and its receptor (EpoR), and that deprivation of Epo signal in tumour blocks induced death of malignant cells and capillary endothelial cells in vitro (Yasuda et al, submitted). These in vitro results prompted us to examine the effect of Epo-signal withdrawal on tumours in vivo. RT-PCR analysis demonstrated the expression of mRNAs for Epo and EpoR in the transplants of uterine and ovarian tumours in nude mice. Then we injected locally anti-Epo antibody or soluble form of EpoR into the transplants. At 12 h, 1, 7 or 14 days after the injection, all transplants were resected and examined macro- and microscopically. Tumour size was reduced in Epo signal-deprived transplants. Immunohistochemical examinations revealed destruction of Epo-responding malignant and capillary endothelial cells through apoptotic death. The degree of tumour regression correlated well with the dose and frequency of the injections. Control xenografts with saline injection or needle insertion showed well-developed tumour masses. This Epo response pathway will have profound implications for our understanding of the development and progression of malignant tumours and for the use of Epo-signal deprivation as an effective therapy.

Insulin-like growth factors and insulin stimulate erythropoietin production in primary cultured astrocytes

Erythropoietin (EPO) is established as a major regulator of erythropoiesis. However, we and others have shown that neurons express erythropoietin receptor (EPO-R), that astrocytes produce EPO and that EPO may act as a neurotrophic factor in the CNS. We also found that EPO production is activated by insulin and insulin-like growth factors (IGFs) in astrocytes in a dose-dependent manner and that IGF-I was the most potent activator. The concentrations required for half-maximal activation were 3 nM IGF-I, 10 nM IGF-II and 100 nM insulin. The oxygen concentration regulates EPO production; hypoxia stimulates EPO production in astrocytes. The stimulatory effect of IGFs and insulin on EPO production in astrocytes was not affected by the oxygen concentration of astrocyte culture. Insulin and IGFs did not increase the total protein synthesis of astrocytes but increased EPO mRNA levels, indicating that EPO production is stimulated at the mRNA level. It appeared that the growth factor-induced accumulation of EPO mRNA in astrocytes was caused by activation of the tyrosine kinase-signal transduction pathway, because tyrosine phosphorylation of receptors for IGF-I and insulin was activated when astrocytes were stimulated by these growth factors.

Isolation of human erythropoietin with monoclonal antibodies.

Human erythropoietin was isolated from urine of aplastic anemic patients in a high yield with a simple purification procedure using an immunoadsorbent column of monoclonal antibodies and a Sephadex G-100 column. About 6 mg of erythropoietin was isolated from 700 liters of urine and the specific activity was estimated to be 81,600 units/mg of protein with an in vivo 59Fe incorporation assay method, using starved rats. Activity measurement of the extracts from sliced gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the Western blotting technique revealed heterogeneity of the isolated erythropoietin, which is probably caused by variable amounts of carbohydrates attached to the polypeptide chain. Thirty amino acids in the NH2-terminal portion of the isolated hormone were sequenced.

Cross-linking of a synthetic partial-length (1-28) peptide of the Alzheimer β/A4 amyloid protein by transglutaminase

Cerebral deposits of β/A4 amyloid protein is a pathologic sign of Alzheimers disease. A synthetic partial‐length (1–28) peptide of this protein contains one glutamine and two lysine residues. Here we show that this peptide can be a substrate of transglutaminase, which catalyzes cross‐linking between glutamine and lysine residues in peptides, by demonstrating the formation of multimeric peptides due to the action of this enzyme. A modified (LyS28 to l‐norleucine) version of the synthetic peptide was also cross‐linked, but another modified version (Lys16 to l‐norleucine) was very poorly cross‐linked, indicating that Lys16 is involved exclusively in the cross‐linking of the partial‐length peptide catalyzed by transglutaminase.