EPO,EPOR及其信号传递研究进展

EPO(Erythropoietin,促红细胞生成素)是促进哺乳动物红系祖细胞增殖和分化的主要细胞团子。EPO与红系祖细胞表面的EPOR(EPO Receptor)结合后,通过信号传递使细胞核内特定基因转录发生改变。与其它造血生长因子不同,EPO主要作用于相对成熟的红系细胞。近年来,随着对人和动物的EPOR分子克隆以及EPOR突变体分析,使我们对EPO与EPOR的相互作用及其信号传递路径有了比较深入的了解。     

花斑裸鲤Epo和Epor基因克隆及其低氧诱导的mRNA表达

促红细胞生成素(EPO)是一种糖蛋白激素,在动物低氧适应中发挥着重要作用。本研究采用RT-PCR方法克隆了花斑裸鲤(Gymncypris eckloni)Epo和Epor基因的编码序列,并开展了分子进化和低氧诱导表达研究。结果表明,花斑裸鲤Epo基因编码序列长度为552 bp,编码183个氨基酸;Epor基因编码序列长度为1 590 bp,编码529个氨基酸。氨基酸序列同源性分析显示,花斑裸鲤促红细胞生成素(EPO)和促红细胞生成素受体(EPOR)与鲤科其他鱼类均有较高的同源性,表明在进化过程中具有较强保守性,同时花斑裸鲤Epo和Epor基因均未经历正向选择。在重度低氧[溶解氧为(0.3±0.1)mg/L]和中度低氧[溶解氧为(3.0±0.1)mg/L]胁迫下,花斑裸鲤部分关键组织如红肌、脑组织中Epo m RNA表达量急剧升高,表明促红细胞生成素(EPO)可能在花斑裸鲤运动、神经营养和神经保护方面发挥了重要作用,是对低氧环境适应的一种调节机制。     

促红细胞生成素和受体在前列腺癌组织中共同过表达的研究

目的:探讨促红细胞生成素(EPO)和受体(EPOR)在前列腺癌(PCa)组织中的表达,并进一步阐述EPO和EPOR在前列腺癌发生发展中所起的作用。方法:应用免疫组化SP法检测30例前列腺癌根治术组织标本中癌与增生(BPH)组织的EPO和EPOR表达及30例正常前列腺组织(NP)中的EPO和EPOR表达。前列腺癌分级采用Gleason评分。半定量EPO和EPOR评分分析免疫组化结果。同时根据细胞染色强度区分为过表达和正常表达。统计学分析采用配对样本比较Wilcoxon的秩和检验及线形回归分析。结果:大部分前列腺癌均可见EPO和EPOR同时过表达,但是前列腺增生组织只有EPO过表达;正常前列腺组织没有EPO和EPOR过表达。前列腺癌和良性前列腺增生的EPO免疫组化评分中位数为2.38和0.93(P<0.01);前列腺癌和良性前列腺增生的EPOR免疫组化评分中位数为2.50和0.68(P<0.01)。前列腺增生和前列腺癌的EPO和EPOR表达密切相关,但是前列腺癌的Gleason评分和EPO以及EPOR评分没有相关性(P值均>0.05)。结论:EPO和EPOR同时过表达促进前列腺癌发生发展,但是相对于EPO的过表达,EPOR过表达是前列腺癌发生更为重要的早期事件;前列腺癌组织中EPO和EPOR表达差异,提示除了缺氧外,可能还有其它机制参与EPOR的过表达。     

EPO/EPOR 在非小性细胞肺癌发展中的作用研究

目的：研究促红细胞生成素（erythropoietin, EPO）及其受体（EPOR）在非小性细胞肺癌中的生物学作用。方法：收集27 例非小 性细胞肺癌（NSCLC）,免疫组织化学方法检测肺癌组织中EPO 和EPOR的表达;观察人源重组EPO（rhEPO）对HCC15 和 HCC1819 细胞活力和细胞周期的影响;分析缺氧对NSCLC细胞EPO 及EPOR 表达的影响。结果：27例非小细胞肺癌的组织标 本中13 例表达EPO,表达率为48 %,25 例表达EPOR,表达率为92 %。rhEPO明显增加了高表达EPOR 的HCC1819 细胞克隆 数,而对低表达EPOR 的HCC15 细胞的克隆形成没有影响。rhEPO增强了HCC1819 的细胞活力,但以siRNA干涉HCC1819 EPOR后,EPO对HCC1819 细胞活力增强作用消失。rhEPO 明显增加了HCC1819 细胞的细胞周期。缺氧促进了HCC1819 细胞的 EPO 的表达,增强了细胞活力。结论：EPO 和EPOR在非小性细胞肺癌中表达增高,EPO 通过EPOR 促进了NSCLC 细胞的增殖, 缺氧诱导了NSCLC 细胞EPO的表达。     

促红细胞生成素在低氧预适应中的神经保护作用

促红细胞生成素(EPO)是体内一种重要的糖蛋白激素,主要由胎肝和成人的肾脏产生。EPO的表达除受到转录因子的调控之外,还受到表观遗传学的调控。研究发现,EPO及其受体(EPOR)在中枢神经系统中广泛表达,提示其对中枢系统具有神经保护作用。低氧预适应是机体抗缺氧或缺血的一种内源性保护机制,它可以促进EPO表达,减轻低氧/缺血引起的神经元损伤。EPO主要通过激活一系列信号转导通路及多种可能的机制发挥神经保护作用。     

造血调控因子及其受体转录调控机制的研究进展

综述了5种集落刺激因子(CSF)、红细胞生成素(EPO)、5种白介素、干扰素、肿瘤坏死因子、白血病抑制因子、干细胞抑制因子、2种 CSF 受体 (R)、EPOR 和两种白介素 R 等转录调控分子机制的研究进展.     

人红细胞生成素受体的研究进展

人红细胞生成素受体(hEPOR)是位于相对成熟阶段人体红系祖细胞表面的跨膜蛋白,它能专一性结合人红细胞生成素(hEPO),将促进细胞生长、增殖和分化的信号传导到膜内,该过程涉及了hEPOR自身及部分相关蛋白的磷酸化.hEPOR具有一些与其功能相关的保守结构,它的氨基酸序列与鼠EPOR(mEPOP)高度同源.EPOR因为膜外R129C突变或结合特定蛋白质而具有组成型活性.EPOR还与红白血病等多种血液病密切相关.     

重组促红细胞生成素(rHu-EPO)及其应用

19世纪,法国生理学家 Paul Verte 发现低氧可以刺激血红细胞生长。1950年美国 Reissman 证实了体内存在一种能刺激血红细胞的物质,称为红细胞生成素(EPO)。1985年,美国的 Fritsch 等成功地从成百上千的基因中发现了能产生 EPO 的一种,运用遗传工程,得以从动物细胞中产生对人类 EPO 负责的基因,从而使大量生产 rHuEPO 成为可能。EPO 是由肾脏分泌的一种重要激素,它具有促进骨髓中红细胞系列的增殖、分化、成熟和释放作用,以及维持血中细胞数和血红蛋白量的稳定状态慢性肾功能衰竭(CRF)的贫血与许多因素有关,这些因素包括:促红细胞生成素(EPO)生成减少、血中存在红细胞生成抑制因子(Inhibitors of Erythropoicsis,IE)、红细胞寿命缩     

重组人促红细胞生成素研究成果通过专家鉴定

重组人促红细胞生成素(EPO)是调节和维持红细胞生理循环的重要激素,对慢性肾性贫血有特效,并有可能在癌症、血液系统恶变、AIDS病等疾病所致的贫血治疗中开拓广泛的临床应用领域。目前国内使用的EPO制剂基本上全部从美国Amgen公司进口,为此每年要支付巨额外汇。我国促红细胞生成素的研究起步较晚,第二军医大学分子遗传学教研室在上海市科委的支持下,从1991年开始进行重组人促红细胞生成素高表达CHO细胞株     

中国北方汉族人群EPO基因SNP/T3541G多态性研究

目的:研究促红细胞生成素(EPO)基因T3541G单核苷酸多态性(SNP/C1772T)在中国北方汉族人群中的分布特征.方法:通过PCR-RFLP实验方法,解析206名中国北方汉族人群EPO基因SNP/T3541G.结果:中国北方汉族人辟EPO基因SNP/T3541G多态位点的基因型符合Hardy-Weinberg遗传...     

Expression cloning of the murine erythropoietin receptor

Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.     

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor.

Two cDNA clones encoding a receptor for human granulocyte-macrophage colony-stimulating factor (hGM-CSF-R) were isolated by expression screening of a library made from human placental mRNA. Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM-CSF using a sensitive microscopic autoradiographic approach. The cloned GM-CSF-R precursor is a 400 amino acid polypeptide (Mr 45,000) with a single transmembrane domain, a glycosylated extracellular domain and a short (54 amino acids) intracytoplasmic tail. It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family, but does show some significant sequence homologies with receptors for several other haemopoietic growth factors, including those for interleukin-6, erythropoietin and interleukin-2 (beta-chain) and also to the prolactin receptor. When transfected into COS cells the cloned cDNA directed the expression of a GM-CSF-R showing a single class of affinity (KD = 2(-8) nM) and specificity for human GM-CSF but not interleukin-3. Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM-CSF binding, and cross-linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells.     

Mechanism of leukemogenesis induced by mink cell focus-forming murine leukemia viruses.

The Friend or Moloney mink cell focus-forming (MCF) virus encodes a recombinant-type envelope glycoprotein, gp70, that is closely related to the membrane glycoprotein, gp55, of Friend spleen focus-forming virus (SFFV). We have shown previously that gp55 has the ability to activate cell growth by binding to the cellular receptor for erythropoietin. Here we show that gp70 encoded by either the Friend or Moloney MCF virus also binds to the erythropoietin receptor and that coexpression of the receptor and gp70 in an interleukin-3 (IL-3)-dependent cell line can activate IL-3-independent growth. Furthermore, when the cDNA for the human IL-2 receptor beta chain, which is related by sequence to the erythropoietin receptor, was introduced into this cell line, it became growth factor independent after infection either with SFFV or with one of the two MCF viruses but not with an ecotropic virus. Based on these observations, we propose a mechanism for the early stage of leukemogenesis induced by the MCF-type murine leukemia viruses.     

Identification of tyrosine residues within the intracellular domain of the erythropoietin receptor crucial for STAT5 activation.

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.     

Interleukin-2 (IL-2) induces erythroid differentiation and tyrosine phosphorylation in ELM-I-1 cells transfected with a human IL-2 receptor beta chain cDNA.

The molecular mechanism of erythroid differentiation has been still ill-defined. In this study, we introduced a human interleukin-2 receptor (IL-2R) beta chain cDNA into ELM-I-1 cells which differentiated into hemoglobin-positive cells in the presence of erythropoietin (Epo), and established the transformant which expressed IL-2R beta chain. In this transformant, we revealed that IL-2 induced erythroid differentiation and the same pattern of tyrosine phosphorylation as Epo. These data suggest that tyrosine phosphorylation is involved in signal transduction pathway of erythroid differentiation. It is also implicated that the Epo and IL-2 receptor system share a common signal transduction pathway.     

Hamster estrogen receptor cDNA: cloning and mRNA expression

Estrogen-induced hamster kidney tumor model serves as a useful model to study the biochemical and molecular mechanisms of hormonal carcinogenesis. In this model, we have demonstrated an increased expression of estrogen receptor mRNA and protein in estrogen-treated kidneys and in estrogen-induced tumors. The sequence information for hamster estrogen receptor gene is not known and has been investigated in this study. A hamster uterus cDNA library was constructed and the 5'-region of the hamster estrogen receptor cDNA cloned from this library using polymerase chain reaction (PCR) methodology. Additionally, hamster kidney polyadenylated RNA was reverse transcribed and PCR amplified using primers that were designed based on maximum homology between mouse, rat and human estrogen receptor cDNAs. These PCR amplified fragments were cloned into plasmid vectors and clones with the expected size of the insert subjected to Southern blot analysis using human estrogen receptor cDNA as a probe. The positive clones on Southern blot analysis and the PCR amplified products from these clones were subjected to DNA sequence analysis. Using this strategy, a full length, 1978 bp hamster estrogen receptor cDNA has been cloned which shows 87% homology with human, 90% with rat and 91% with mouse estrogen receptor cDNA. The deduced amino acid shares 88% homology with human, and 93% with rat and mouse estrogen receptors. Hamster estrogen receptor domain C (DNA binding domain) shows a 100% homology with a similar domain from mouse, rat, human, pig, sheep, horse and chicken estrogen receptor (Genebank reference ID: AF 181077).     

Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3'-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200,000 U/mg), but was of lower Mr (23,000) than human erythropoietin produced in COS cells (30,000) or purified from urine (30,000 to 38,000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosaccharides from this Mr 23,000 hormone using N-Glycanase yielded an apo-erythropoietin (Mr 18,000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Sterol Composition in the Pollens of Citrus unshiu Marcov. and Citrus sinensis Osbeck

Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and an EPO-fixed gel for sEPOR. The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities. Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.     

Characterization of a human homologue of the murine peripheral lymph node homing receptor

Lymphocyte trafficking is a fundamental aspect of the immune system that allows B and T lymphocytes with diverse antigen recognition specificities to be exposed to various antigenic stimuli in spatially distinct regions of an organism. A lymphocyte adhesion molecule that is involved with this trafficking phenomenon has been termed the homing receptor. Previous work (Lasky, L., T. Yednock, M. Singer, D. Dowbenko, C. Fennie, H. Rodriguez, T. Nguyen, S. Stachel, and S. Rosen. 1989. Cell. 56:1045-1055) has characterized a cDNA clone encoding a murine homing receptor that is involved in trafficking of lymphocytes to peripheral lymph nodes. This molecule was found to contain a number of protein motifs, the most intriguing of which was a carbohydrate binding domain, or lectin, that is apparently involved in the adhesive interaction between murine lymphocytes and peripheral lymph node endothelium. In this study, we have used the murine cDNA clone to isolate a human homologue of this peripheral lymph node-specific adhesion molecule. The human receptor was found to be highly homologous to the murine receptor in overall sequence, but showed no sequence similarity to another surface protein that may be involved with human lymphocyte homing, the Hermes glycoprotein. The extracellular region of the human receptor contained an NH2 terminally located carbohydrate binding domain followed by an EGF-like domain and a domain containing two repeats of a complement binding motif. Transient cell transfection assays using the human receptor cDNA showed that it encoded a surface glycoprotein that cross reacted with a polyclonal antibody directed against the murine peripheral lymph node homing receptor. Interestingly, the human receptor showed a high degree of sequence homology to another human cell adhesion glycoprotein, the endothelial cell adhesion molecule ELAM.     

Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli.

We developed an assay to directly measure the ligand binding properties of the cloned human erythropoietin receptor (EpoR). The cDNA encoding the extracellular domain of the human EpoR was amplified by polymerase chain reaction and ligated into the prokaryotic expression vector pGEX3X. Synthesis in Escherichia coli was induced and a soluble glutathione S-transferase fusion protein, EREx, was purified by erythropoietin affinity chromatography. Purified EREx was bound to GSH agarose beads and used in a solid phase ligand binding assay. Specific binding of 125I-erythropoietin to EREx beads was demonstrated. A single affinity class (Kd = 1.5 nM) of the binding site was evident on Scatchard analysis. The Kd of this site is quantitatively equivalent to that of the "low" affinity cellular binding site. Kinetic analysis of ligand binding to EREx revealed both the on and off rates to be rapid, with t1/2 of 60 and 40 s, respectively. EREx ligand binding exhibits no obvious metal ion dependence or cross-competition by other hemopoietins. Antibodies to EREx block the binding of erythropoietin to the cellular EpoR. We conclude that the 66-kDa EpoR protein is capable of specific ligand binding and that no covalent modifications or associated molecules are required for this interaction. We speculate that the "high" affinity cellular binding site (Kd less than 0.2 nM) results from the interaction of the EpoR with another molecule, either additional EpoR or associated subunits, that decreases the ligand off rate.