The Experts below are selected from a list of 525 Experts worldwide ranked by ideXlab platform
Ryuzo Sasaki - One of the best experts on this subject based on the ideXlab platform.
-
An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines
Cytotechnology, 2011Co-Authors: Ryuzo Sasaki, Seiji MasudaAbstract:Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high dose of EPO also inhibited the proliferation of various erythropoietin-dependent cell lines, suggesting that excess amount of EPO could not trigger the conformational change of the receptor. In the presence of an extra high dose of erythropoietin as well as in the absence of erythropoietin, the cells caused the DNA fragmentation, a typical symptom of apoptosis. The impairment of cell growth and the DNA fragmentation at the extremely high concentration of EPO was rescued by the addition of erythropoietin antibody or soluble form of erythropoietin receptor by titrating the excess erythropoietin. These results suggest that two erythropoietin binding sites on erythropoietin receptor dimer should be occupied by a single erythropoietin molecule for the proper conformational change of the receptor and the signal transduction of erythropoietin, instead, when two erythropoietin binding sites on the receptor are shared by two erythropoietin molecules, it fails to evoke the conformational change of erythropoietin receptor adequate for signal transduction.
-
Erythropoietin and erythropoietin-receptor producing cells demonstrated by in situ hybridization in mouse visceral yolk sacs.
Anatomical Science International, 2002Co-Authors: Yoshiko Yasuda, Seiji Masuda, Masaya Nagao, Yoshihiko Fujita, Masaki Okano, Ryuzo SasakiAbstract:We have previously demonstrated that mRNAs for erythropoietin and the erythropoietin receptor temporarily express on the visceral yolk sacs on days 9–11 of gestation in mice. In order to investigate the sites of expression, we performed in situ hybridization on visceral yolk sacs. Visceral yolk sacs from 10-day-old mice embryos were frozen in liquid nitrogen, and processed for cryosections. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for erythropoietin or erythropoietin receptor. Erythropoietin mRNA was detectable in 57.6% of the endodermal epithelial cells, while erythropoietin-receptor mRNA was discerned in 90.8% of the endodermal cells and mesodermal cells, including hemocyteblasts. Moreover, erythropoietin protein was detectable in 52.8% of the endodermal epithelial cells, and on the surface of hemocyteblasts and mesothelial cells. Erythropoietin-receptor protein was discernible in 87.2% of the endodermal cells and in the corresponding mesodermal cells to those where erythropoietin protein was expressed by immunohistochemical examinations. The results indicate that erythropoietin-synthesizing cells are located in half of the endodermal epithelial cells, while the majority of cells in the visceral yolk sac are erythropoietin-receptor-producing cells, indicating that almost all cell population in the visceral yolk sac is erythropoietin-responding cells via both autocrine and paracrine routes.
-
Erythropoietic, neurotrophic, and angiogenic functions of erythropoietin and regulation of erythropoietin production.
International Journal of Hematology, 1999Co-Authors: Seiji Masuda, Masaya Nagao, Ryuzo SasakiAbstract:: Stimulation of erythrocyte formation has been generally thought to be the sole physiological function of erythropoietin. Erythropoietin and its receptors, however, are also expressed in the central nervous system. In addition, erythropoietin is produced in the uterus in an estrogen-dependent manner. Novel functions of erythropoietin in these local sites are presented.
-
Neuroprotective Function of Erythropoietin in the Central Nervous System
Animal Cell Technology: Basic & Applied Aspects, 1998Co-Authors: Seiji Masuda, Masaya Nagao, Emi Morishita, Mariko Chikuma, Ryuzo SasakiAbstract:It has been thought that erythropoietin exclusively acts on erythroid precursor cells in vivo. However, we describe that erythropoietin is produced by astrocytes and its receptor is expressed in neurons. Erythropoietin production by astrocytes is dependent on oxygen tension and activated by insulin and insulin-like growth factors. Furthermore, erythropoietin protected the cultured hippocampal and cerebral cortical neurons from glutamate neurotoxicity. These results indicate that, in the central nervous system, erythropoietin acts on neurons in a paracrine fashion, and erythropoietin and its receptor system is independent of that for erythropoiesis.
-
erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons and erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract 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.
Seiji Masuda - One of the best experts on this subject based on the ideXlab platform.
-
An extra high dose of erythropoietin fails to support the proliferation of erythropoietin dependent cell lines
Cytotechnology, 2011Co-Authors: Ryuzo Sasaki, Seiji MasudaAbstract:Erythropoietin is responsible for the red blood cell formation by stimulating the proliferation and the differentiation of erythroid precursor cells. Erythropoietin triggers the conformational change in its receptor thereby induces the phosphorylation of JAK2. In this study, we show that an extra high dose of erythropoietin, however, fails to activate the erythropoietin receptor, to stimulate the phosphorylation of JAK2 and to support the cell proliferation of Ep-FDC-P2 cell. Moreover, high dose of EPO also inhibited the proliferation of various erythropoietin-dependent cell lines, suggesting that excess amount of EPO could not trigger the conformational change of the receptor. In the presence of an extra high dose of erythropoietin as well as in the absence of erythropoietin, the cells caused the DNA fragmentation, a typical symptom of apoptosis. The impairment of cell growth and the DNA fragmentation at the extremely high concentration of EPO was rescued by the addition of erythropoietin antibody or soluble form of erythropoietin receptor by titrating the excess erythropoietin. These results suggest that two erythropoietin binding sites on erythropoietin receptor dimer should be occupied by a single erythropoietin molecule for the proper conformational change of the receptor and the signal transduction of erythropoietin, instead, when two erythropoietin binding sites on the receptor are shared by two erythropoietin molecules, it fails to evoke the conformational change of erythropoietin receptor adequate for signal transduction.
-
Erythropoietin and erythropoietin-receptor producing cells demonstrated by in situ hybridization in mouse visceral yolk sacs.
Anatomical Science International, 2002Co-Authors: Yoshiko Yasuda, Seiji Masuda, Masaya Nagao, Yoshihiko Fujita, Masaki Okano, Ryuzo SasakiAbstract:We have previously demonstrated that mRNAs for erythropoietin and the erythropoietin receptor temporarily express on the visceral yolk sacs on days 9–11 of gestation in mice. In order to investigate the sites of expression, we performed in situ hybridization on visceral yolk sacs. Visceral yolk sacs from 10-day-old mice embryos were frozen in liquid nitrogen, and processed for cryosections. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for erythropoietin or erythropoietin receptor. Erythropoietin mRNA was detectable in 57.6% of the endodermal epithelial cells, while erythropoietin-receptor mRNA was discerned in 90.8% of the endodermal cells and mesodermal cells, including hemocyteblasts. Moreover, erythropoietin protein was detectable in 52.8% of the endodermal epithelial cells, and on the surface of hemocyteblasts and mesothelial cells. Erythropoietin-receptor protein was discernible in 87.2% of the endodermal cells and in the corresponding mesodermal cells to those where erythropoietin protein was expressed by immunohistochemical examinations. The results indicate that erythropoietin-synthesizing cells are located in half of the endodermal epithelial cells, while the majority of cells in the visceral yolk sac are erythropoietin-receptor-producing cells, indicating that almost all cell population in the visceral yolk sac is erythropoietin-responding cells via both autocrine and paracrine routes.
-
Erythropoietic, neurotrophic, and angiogenic functions of erythropoietin and regulation of erythropoietin production.
International Journal of Hematology, 1999Co-Authors: Seiji Masuda, Masaya Nagao, Ryuzo SasakiAbstract:: Stimulation of erythrocyte formation has been generally thought to be the sole physiological function of erythropoietin. Erythropoietin and its receptors, however, are also expressed in the central nervous system. In addition, erythropoietin is produced in the uterus in an estrogen-dependent manner. Novel functions of erythropoietin in these local sites are presented.
-
Neuroprotective Function of Erythropoietin in the Central Nervous System
Animal Cell Technology: Basic & Applied Aspects, 1998Co-Authors: Seiji Masuda, Masaya Nagao, Emi Morishita, Mariko Chikuma, Ryuzo SasakiAbstract:It has been thought that erythropoietin exclusively acts on erythroid precursor cells in vivo. However, we describe that erythropoietin is produced by astrocytes and its receptor is expressed in neurons. Erythropoietin production by astrocytes is dependent on oxygen tension and activated by insulin and insulin-like growth factors. Furthermore, erythropoietin protected the cultured hippocampal and cerebral cortical neurons from glutamate neurotoxicity. These results indicate that, in the central nervous system, erythropoietin acts on neurons in a paracrine fashion, and erythropoietin and its receptor system is independent of that for erythropoiesis.
-
erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons and erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract 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.
Masaya Nagao - One of the best experts on this subject based on the ideXlab platform.
-
Erythropoietin and erythropoietin-receptor producing cells demonstrated by in situ hybridization in mouse visceral yolk sacs.
Anatomical Science International, 2002Co-Authors: Yoshiko Yasuda, Seiji Masuda, Masaya Nagao, Yoshihiko Fujita, Masaki Okano, Ryuzo SasakiAbstract:We have previously demonstrated that mRNAs for erythropoietin and the erythropoietin receptor temporarily express on the visceral yolk sacs on days 9–11 of gestation in mice. In order to investigate the sites of expression, we performed in situ hybridization on visceral yolk sacs. Visceral yolk sacs from 10-day-old mice embryos were frozen in liquid nitrogen, and processed for cryosections. Sections were hybridized with a 35S-labeled RNA probe complementary to mRNA coding for erythropoietin or erythropoietin receptor. Erythropoietin mRNA was detectable in 57.6% of the endodermal epithelial cells, while erythropoietin-receptor mRNA was discerned in 90.8% of the endodermal cells and mesodermal cells, including hemocyteblasts. Moreover, erythropoietin protein was detectable in 52.8% of the endodermal epithelial cells, and on the surface of hemocyteblasts and mesothelial cells. Erythropoietin-receptor protein was discernible in 87.2% of the endodermal cells and in the corresponding mesodermal cells to those where erythropoietin protein was expressed by immunohistochemical examinations. The results indicate that erythropoietin-synthesizing cells are located in half of the endodermal epithelial cells, while the majority of cells in the visceral yolk sac are erythropoietin-receptor-producing cells, indicating that almost all cell population in the visceral yolk sac is erythropoietin-responding cells via both autocrine and paracrine routes.
-
Erythropoietic, neurotrophic, and angiogenic functions of erythropoietin and regulation of erythropoietin production.
International Journal of Hematology, 1999Co-Authors: Seiji Masuda, Masaya Nagao, Ryuzo SasakiAbstract:: Stimulation of erythrocyte formation has been generally thought to be the sole physiological function of erythropoietin. Erythropoietin and its receptors, however, are also expressed in the central nervous system. In addition, erythropoietin is produced in the uterus in an estrogen-dependent manner. Novel functions of erythropoietin in these local sites are presented.
-
Neuroprotective Function of Erythropoietin in the Central Nervous System
Animal Cell Technology: Basic & Applied Aspects, 1998Co-Authors: Seiji Masuda, Masaya Nagao, Emi Morishita, Mariko Chikuma, Ryuzo SasakiAbstract:It has been thought that erythropoietin exclusively acts on erythroid precursor cells in vivo. However, we describe that erythropoietin is produced by astrocytes and its receptor is expressed in neurons. Erythropoietin production by astrocytes is dependent on oxygen tension and activated by insulin and insulin-like growth factors. Furthermore, erythropoietin protected the cultured hippocampal and cerebral cortical neurons from glutamate neurotoxicity. These results indicate that, in the central nervous system, erythropoietin acts on neurons in a paracrine fashion, and erythropoietin and its receptor system is independent of that for erythropoiesis.
-
erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons and erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract 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.
-
erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons and erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract 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.
Patrick Mayeux - One of the best experts on this subject based on the ideXlab platform.
-
proteasomes regulate erythropoietin receptor and signal transducer and activator of transcription 5 stat5 activation possible involvement of the ubiquitinated cis protein
Journal of Biological Chemistry, 1998Co-Authors: Frederique Verdier, Sylvie Gisselbrecht, Stany Chrétien, Catherine Lacombe, Odile Muller, Paule Varlet, Akihiko Yoshimura, Patrick MayeuxAbstract:Abstract Cis is an Src homology 2 domain-containing protein, which binds to the erythropoietin receptor and decreases erythropoietin-stimulated cell proliferation. We show that Cis associates with the second tyrosine residue of the intracellular domain of the erythropoietin receptor (Tyr401). Two forms of Cis with molecular masses of 32 and 37 kDa were detected, and we demonstrate that the 37-kDa protein resulted from post-translational modifications of the 32-kDa form. Anti-ubiquitin antibodies recognized the 37-kDa form of Cis and the proteasome inhibitorsN-acetyl-leucyl-leucyl-norleucinal and lactacystin inhibited its degradation, showing that the 37-kDa form of Cis is a ubiquitinated protein, which seems to be rapidly degraded by the proteasome. In erythropoietin-stimulated UT-7 cells, the activation of the erythropoietin receptor and signal transducer and activator of transcription 5 (STAT5) was transient and returned to basal levels after 30–60 min of erythropoietin stimulation. In contrast, these proteins remained strongly phosphorylated, and STAT5 remained activated for at least 120 min in the presence of proteasome inhibitors. These experiments demonstrate that the proteasomes are involved in the down-regulation of the erythropoietin receptor activation signals. Because the proteasome inhibitors induced the accumulation of both the ubiquitinated form of Cis and the Cis-erythropoietin receptor complexes, our results suggest that the ubiquitinated form of Cis could be involved in the proteasome-mediated inactivation of the erythropoietin receptor.
-
Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.
The EMBO Journal, 1996Co-Authors: Stephanie Gobert, Isabelle Dusanter-fourt, Sylvie Gisselbrecht, Stany Chrétien, Fabrice Gouilleux, Christophe Pallard, Catherine Lacombe, Bernd Groner, O. Muller, Patrick MayeuxAbstract:Abstract FDCP-1 cells are hematopoietic progenitor cells which require interleukin-3 for survival and proliferation. FDCP-1 cells stably transfected with the murine erythropoietin receptor cDNA survive and proliferate in the presence of erythropoietin. Erythropoietin induces the activation of the short forms (80 kDa) of STAT5 in the cells. Erythropoietin-induced activation of STAT5 was strongly reduced in cells expressing mutated variants of the erythropoietin receptors in which tyrosine residues in their intracellular domain have been eliminated. We determined that the erythropoietin receptor tyrosine residues 343 and 401 are independently necessary for STAT5 activation. The amino acid sequences surrounding these two tyrosine residues are very similar. Peptides comprising either phosphorylated Tyr343 or phosphorylated Tyr401, but not their unphosphorylated counterparts, inhibited the STAT5 activation. We propose that these two tyrosine residues of the erythropoietin receptor constitute docking sites for the STAT5 SH2 domain. The growth stimulus mediated by erythropoietin was decreased in cells expressing erythropoietin receptors lacking both Tyr343 and Tyr401. This suggests that STAT5 activation could be involved in the growth control of FDCP-1 cells.
-
Structure of the erythropoietin receptor.
Experimental Biology and Medicine, 1994Co-Authors: Patrick Mayeux, Sylvie Pallu, Stephanie Gobert, Catherine Lacombe, Sylvie GisselbrechtAbstract:Despite extensive studies, the structure of the erythropoietin receptor remains little understood. cDNAs encoding the human and murine erythropoietin receptors have been cloned and the structure of these proteins is discussed. Although the proteins encoded by these cDNAs play key roles in erythropoietin binding and in erythropoietin signal transduction, increasing evidence strongly suggests that the erythropoietin receptor is a multimeric complex. The murine erythropoietin receptor has been solubilized under mild conditions and the molecular mass of the native receptor has been shown to be significantly higher than the molecular mass of the cloned chain. Cross-linking experiments have revealed the presence of three proteins covalently bound to erythropoietin by the cross-linking reagents; however, only one of them seems to derive from the cloned chain. Moreover, functional evidence also suggests the presence of other erythropoietin receptor subunits.
-
Erythropoietin induces the association of phosphatidylinositol 3′-kinase with a tyrosine-phosphorylated protein complex containing the erythropoietin receptor
FEBS Journal, 1993Co-Authors: Patrick Mayeux, Philippe Mauduit, Michèle Sabbah, Isabelle Dusanter-fourt, Brian J. Druker, Catherine Lacombe, William Vainchenker, O. Muller, S Fischer, Sylvie GisselbrechtAbstract:Stimulation of sensitive cells with erythropoietin results in rapid induction of protein tyrosine phosphorylation. Other than tyrosine phosphorylation of one chain of the erythropoietin receptor, the identities of the remaining tyrosine-phosphorylated proteins are undefined. In this report, we demonstrate that the stimulation of the erythropoietin-sensitive human UT7 cells by erythropoietin rapidly resulted in the appearance of phosphatidylinositol 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Erythropoietin action was rapid, detectable after as early as 1 min stimulation, transient, returning to control level after 30 min stimulation and was observed using the erythropoietin concentrations able to stimulate the cell proliferation. Anti-(phosphatidylinositol 3-kinase) antibodies specifically immunoprecipitated 125I-erythropoietin bound to its receptor, strongly suggesting that phosphatidylinositol 3-kinase associated with a protein complex containing the activated erythropoietin receptor. To confirm this result, phosphatidylinositol 3-kinase was immunoprecipitated from erythropoietin-stimulated cells using mild conditions followed by Western analysis using anti-phosphotyrosine antibodies. Five tyrosine phosphorylated proteins were revealed: the cloned chain of the erythropoietin receptor, the regulatory subunit of phosphatidylinositol 3-kinase and three unidentified proteins of 111, 97 and 64 kDa. None of these tyrosine phosphorylated proteins was detected in anti-(phosphatidylinositol 3-kinase) immunoprecipitates from unstimulated cells. Thus, our results show that phosphatidylinositol 3-kinase associates with a tyrosine-phosphorylated protein complex containing the activated erythropoietin receptor.
Emi Morishita - One of the best experts on this subject based on the ideXlab platform.
-
Neuroprotective Function of Erythropoietin in the Central Nervous System
Animal Cell Technology: Basic & Applied Aspects, 1998Co-Authors: Seiji Masuda, Masaya Nagao, Emi Morishita, Mariko Chikuma, Ryuzo SasakiAbstract:It has been thought that erythropoietin exclusively acts on erythroid precursor cells in vivo. However, we describe that erythropoietin is produced by astrocytes and its receptor is expressed in neurons. Erythropoietin production by astrocytes is dependent on oxygen tension and activated by insulin and insulin-like growth factors. Furthermore, erythropoietin protected the cultured hippocampal and cerebral cortical neurons from glutamate neurotoxicity. These results indicate that, in the central nervous system, erythropoietin acts on neurons in a paracrine fashion, and erythropoietin and its receptor system is independent of that for erythropoiesis.
-
erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons and erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract 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.
-
erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons and erythropoietin prevents in vitro glutamate induced neuronal death
Neuroscience, 1996Co-Authors: Emi Morishita, Masaya Nagao, Seiji Masuda, Yoshiko Yasuda, Ryuzo SasakiAbstract:Abstract 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.