G蛋白偶联雌激素受体1介导的环境雌激素效应研究进展

刘帅，胡磊，杨明,*

1.上海大学环境与化学工程学院，上海200444

2.上海大学生命科学学院，上海200444

G蛋白偶联雌激素受体1介导的环境雌激素效应研究进展

刘帅1，胡磊2，杨明1,*

1.上海大学环境与化学工程学院，上海200444

2.上海大学生命科学学院，上海200444

环境雌激素进入生物体后,可通过多种方式介导发挥类似内源雌激素的作用,干扰生物体的正常功能,进而对生物体产生毒害作用。其中,基因组方式介导的雌激素效应主要通过与细胞核内的雌激素受体(如ERα和ERβ)结合；而非基因组方式介导的雌激素效应则主要通过与膜雌激素受体结合从而发挥作用。近年来对G蛋白偶联雌激素受体1(GPER1)的研究表明,该受体是区别于雌激素核受体的膜雌激素受体,可单独介导雌激素诱发的非基因组方式雌激素效应,然而目前对其介导的雌激素效应机制研究并不完善。综上,本文结合近年来对GPER1的研究进展,从该受体的发现、特性以及其介导的雌激素效应和相关通路进行了综述。

环境雌激素；膜雌激素受体；G蛋白偶联雌激素受体

环境内分泌干扰物(environmental endocrine disruptors,EEDs)是环境污染物的重要组成部分,可对生物体的内分泌系统产生复合干扰损害作用,其中最重要的一类物质环境雌激素(environmental estrogens)可诱导生物体产生雌激素效应,从而影响生物体的生殖系统[1]、神经系统[2]、免疫系统[3]等的正常功能。

雌激素介导细胞的效应机制主要分为2类:第一类是以传统雌激素核受体ERα和ERβ介导为主的基因组效应,细胞核内的雌激素受体不仅可直接结合目的DNA片段,还可以与其他核内蛋白如雌激素的激活剂或抑制剂相互作用,从而增强或抑制基因的激活[4]；第二类是通过膜雌激素受体介导的快速非基因组效应,与传统ER受体不同,膜性受体同细胞表面的内源或外源雌激素产生结合或拮抗等作用,可在极短时间内引发信号转导,如钾离子通道关闭、钙离子浓度升高以及相关通路的激活,从而影响有机体的正常功能,发挥其生物学效应[5-6]。近年来关注并研究最多的膜雌激素受体是G蛋白偶联雌激素受体1(G protein-coupled estrogen receptor 1, GPER1),文章对GPER1的研究进展进行了综述。

1 GPER1的发现及组织分布(The discovery and tissue distribution of GPER1)

G蛋白偶联雌激素受体1,最早称之为G蛋白偶联受体30(GPR30),是20世纪90年代从人体细胞中发现的一种膜雌激素受体,Carmeci等[7]从人体乳腺癌细胞系(MCF7)的cDNA文库中筛选出一个全长2 604 bp的cDNA序列,包括长度为1 128 bp的开放阅读框(open read frame,ORF),可编码成375个氨基酸的蛋白,分析发现,该蛋白与早前报道的G蛋白偶联受体(GPCRs)同源性很高,并命名为GPR30。该受体位于染色体7p22区域,具有7个跨膜疏水区域,但与大多数G蛋白偶联受体不同, GPER1蛋白序列在第三跨膜区后存在一个保守的Asp-Arg-Tyr三联体(DRY)序列,分析认为可能与其介导的信号转导有关[8]。其他研究团队同时期也发现并分离扩增得到该受体基因,其序列信息基本相似[9-11]。

研究者相继在哺乳类动物如人(Homo sapiens)[9-11]和小家鼠(Mus musculus)[12-13]等,鸟类如鸡(Gallus gallus)[14],两栖类如蟾蜍(Xenopus)[15]和黑斑蛙(Rana nigromaculataH.)[16]等,硬骨鱼类如细须石首鱼(Micropogonias undulatus)[17]、斑马鱼(Danio rerio)[18]、斜带石斑鱼(Epinephelus coioides)[19]、金头鲷(Sparus aurataL.)[20]等物种中均发现GPER1的存在。此外,GPER1在不同物种中的组织分布情况不同,虽然其在人体中的各组织中均有分布,包括心脏、脑、胎盘、肺、肝脏、肌肉、前列腺等,但在肺、肝脏和前列腺中表达较多,脑和性腺(卵巢、睾丸)中表达较少[21],然而在鱼类中[18],GPER1的分布情况相反,在脑中表达最高,性腺次之,推测其原因可能是该受体在不同物种中的作用不同。

2 GPER1蛋白序列比对及进化树分析(The multiple alignment of protein sequences and phylogenetic tree analysis of GPER1)

图1为基因库中常见物种如人(human,Homo sapiens)、牛(cattle,Bos taurus)、小鼠(mouse,Mus musculus)、马(horse,Equus caballus)、狗(dog,Canis lupus familiaris)、鸡(chicken,Gallus gallus)、蟾蜍(toad, Gallus gallus)、石首鱼(croaker,Micropogonias undulatus)、青鳉(medaka,Oryzias latipes)和斑马鱼(zebrafish,Danio rerio)的GPER1蛋白序列的序列比对(A)及进化树构建结果(B)。比对结果表明,GPER1在不同物种之间的跨膜区域同源性均很高,且在第三跨膜区后存在DRY三联体保守序列；此外,进化树分析发现,GPER1基因在脊椎动物中有明显的分化,包括哺乳动物、鸟类、两栖动物及硬骨鱼类等。

3 GPER1的特性(The characteristics of GPER1)

早期研究过程中发现,GPER1与传统ER核受体的相关配体如17β-雌二醇(E2)、tamoxifen及ICI 182,780具有选择性结合能力[22-26],但未找到GPER1的特异性配体,直到2006年Bologa等[27]通过虚拟和分子生物筛选融合法,从包括10 000个GPCRs结合配体的分子文库中筛选出一个GPER1的特异性配体G1,验证后发现,G1和雌激素一样,可有效激活SkBr3细胞(不表达ERα、ERβ,但表达GPER1)和MCF7细胞(3种受体均有表达)内磷脂酰肌醇激酶PI3K的表达,从而诱导磷脂酰肌醇三磷酸PIP3的产生。G1可特异性结合GPER1也可通过后续的研究得到证实[28-29]。

图1 不同常见物种间的GPER1蛋白序列比对(A)及其进化树分析(B)注:各相关物种蛋白序列的基因库登录号分别为人,NP_001091671.1；牛,DAA15165.1；鼠,NP_084047.2；马,XP_001488797；狗,XP_005621261.1；鸡,NP_001155877.1；蟾蜍,NP_001107725.1；石首鱼,B0F9W3.1；青鳉,XP_004071380.1；斑马鱼,NP_001122195.1。图A中横线所在位置代表7个跨膜区域(seven transmembrane domains,TM),方框位置代表DRY三联体序列。Fig.1 Multiple alignment of protein sequences(A)and phylogenetic tree analysis(B)of GPER1 between different common speciesNote:The GenBank accession number of several protein sequences are as follows:human GPER1(NP_001091671.1),cattle GPR30 (DAA15165.1),mouse GPER1(NP_084047.2),horse GPER1(XP_001488797.1),dog GPER1(XP_005621261.1),chicken GPER1 (NP_001 515877.1),toad GPER1(NP_001107725.1),croaker GPR30(B0F9W3.1),medaka GPER1(XP_004071380.1),zebrafish GPER1(NP_001122195.1).The seven-transmembrane domains and DRY triplets were indicated by horizontal lines and open box in Fig.1A.

He等[30]将不表达ER的人体子宫内膜癌KLE细胞系暴露于不同浓度的G1中,结果发现,G1刺激后可有效促进该细胞的增殖,并通过有丝分裂原活化蛋白激酶MAPK通路诱导基质金属蛋白酶的产生和白介素-6(IL-6)的分泌,表明GPER1可介导非基因组效应在子宫内膜癌研究中发挥重要作用；Sirianni等[31]发现在小鼠精原细胞系GC1中,G1、E2可通过GPER1和ERα的交互作用快速激活EGFR/ ERK/fos通路,进而诱导小鼠精原细胞的增殖,但当GPER1基因沉默后,增殖效应消失。

此外,在2009年,Dennis等[32]从分子库中筛选出一种化合物G15,并确认其是GPER1的拮抗剂。与G1的化学结构相比,G15化合物少一个乙酰基,由于结构的相似性而得到关注,通过配体结合实验表明,同G1类似,G15表现出较高的GPER1亲和性,而与ERα、ERβ亲和性很低,此外,G1、G15分别暴露和共暴露刺激小鼠子宫上皮细胞后,其增殖程度不同,G1组细胞显著增殖,G15组几乎无增殖,而共同刺激组较G1组增殖效果显著降低,表明G15可作为GPER1的拮抗剂应用于其在生物体内作用机制的进一步研究中。2011年,Dennis等[33]在G15的结构基础上增加一个异丙基,得到一种新的化合物G36,研究后发现也具有抑制GPER1表达的作用。

4 环境雌激素物质对生物体的效应机制研究(Mechanisms of actions of environmental estrogens on organisms)

雌激素的基因组效应主要从3个方面进行:(1)进入细胞核内的雌激素可激活核受体ERα或ERβ,与衔接蛋白和信号转导分子作用,进而影响下游通路；(2)激活目的基因启动子区域的ER二聚体从而调节细胞的基因表达变化；(3)促进相关转录因子的磷酸化作用[34]。而进一步的研究发现,有的雌激素引起的下游效应非常快速,表明此效应可能不需要经过细胞核内的雌激素受体,而在胞膜或质膜发挥作用,与膜上的受体结合,激活相关通路等,从而诱导下游信号转导,该效应被称为非基因组效应。

4.1 基因组效应

环境雌激素的基因组效应主要指由雌激素核受体(ER)作为雌激素类物质的配体与进入生物体内的雌激素(天然或环境雌激素)结合,而介导的胞内转录因子激活所引起的细胞内反应[5]。17α-乙炔基雌二醇(EE2)是一种合成激素,作为天然激素E2的衍生物而得到使用并大量进入环境中,对生物体可能造成危害。在早期活体研究中发现,在EE2长期暴露下,可降低性成熟时期虹鳟鱼的生育能力[35],破坏斑马鱼胚胎的性腺成熟[36]或降低成年斑马鱼的生殖腺指数[37],影响其正常生殖功能；离体实验也表明,较高浓度EE2暴露(100 nmol·L-1)可显著降低雄性青鳉生殖细胞的增殖[38]。最初观点认为,环境雌激素效应主要由ER介导。Cabas等[39]发现,EE2刺激可显著上调金头鲷器官和细胞内ERα的mRNA表达,从而调节其免疫功能和性激素水平。同样地, Yang等[40]以鲤鱼巨噬细胞作为研究对象发现,双酚A也可通过诱导ERα的表达对鱼体产生免疫毒性。此外,其他对双酚A的研究中也得到类似研究结果[41-42]。综上表明,ER作为转录因子,在雌激素诱导下所引起基因表达的变化足以影响细胞反应,进而影响生物体。

4.2 非基因组效应

在环境雌激素暴露下,传统核受体可介导基因组效应,从而参与下游的信号转导,类似地,膜雌激素受体GPER1也具有调节雌激素相关基因表达的功能,即非基因组效应机制。Rettew等[43]发现,E2和G1刺激小鼠巨噬细胞后,在10～60 min内TLR4的表达量显著性降低,而在敲除GPER1后,TLR4的表达量趋于正常。在不表达ERα和ERβ的人体乳腺癌 SkBr3细胞系中,Vivacqua等[44]发现,E2、tamoxifen和G1分别刺激1 h后,显著上调细胞中早期生长反应因子(early growth response protein 1, EGR-1)的表达,从而诱导结缔组织生长因子(connective tissue growth factor,CTGF)和细胞周期素D1的基因和蛋白表达,促进细胞增殖,而在RNA干扰沉默GPER1表达后,在同样处理条件下相关基因的表达不再上调。最新研究发现,tamoxifen和 ICI 182,780(ER拮抗剂)可在30 min内激活GPER1促进小鼠性兴奋[45]。上述结果表明,与 ER相比, GPER1的表达可快速介导雌激素的下游信号转导,符合非基因组效应的特点。

其他研究也表明,在不同浓度的环境雌激素类物质暴露下,可通过激活GPER1调节不同种类细胞中相关基因的表达量。Catalano等[46]的研究发现,对乳腺癌细胞长期tamoxifen暴露攻毒后,可上调芳香化酶的mRNA表达,促进细胞增殖,而GPER1敲除及与拮抗剂共暴露后,上调现象消失；Pupo等[47]研究双酚A对SkBr3细胞和癌症相关纤维细胞CAFs(均不表达 ER)的影响中发现,可显著上调GPER1目的基因c-fos、早期生长反应蛋白1和CTGF在细胞中的表达,在GPER1基因沉默后,相关基因不再上调；Teng等[48]的研究发现,GPER1过表达可消除因E2刺激而诱导的c-fos、c-jun及细胞周期蛋白D1的上调表达,而RNA干扰导致GPER1失活后,反而上调这些基因的表达。进一步地,环境雌激素可通过调节细胞内相关基因的表达,从而改变细胞的生长情况。Bouskine等[49]和Liu等[50]的研究分别证实了GPER1在双酚A促进人体精原瘤细胞JKT-1增殖和E2促进不表达ERα的卵巢癌细胞增殖过程中起关键作用。而Chimento等[51-52]则发现环境雌激素选择性激活GPER1,可引起小鼠精母细胞和睾丸间质肿瘤细胞的凋亡。由此可见,雌激素膜受体如GPER1介导的环境雌激素非基因组效应可快速但持久地激活细胞内不同的雌激素信号通路,从而影响细胞的增殖情况及其功能。

5 GPER1相关信号转导通路 (GPER1-related signal transduction pathways)

生物体中的GPER1受到雌激素刺激后,可快速介导非基因组效应,引起下游相关效应变化,如参与部分基因的转录调控,调节基因表达量,引起细胞增殖或凋亡等,而GPER1激活后,主要通过介导相关信号转导通路来引起上述效应。通过对多年的研究总结,其相关通路主要有以下4种:

5.1 EGFR-MAPKs通路

早期研究结果表明,雌激素在激活丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)的过程中需要GPER1的参与[53]。Zhang等[54]发现,雌激素可通过表皮生长因子受体(epidermal growth factor receptor,EGFR)信号来诱导细胞外信号调节激酶-1/-2(extracellular signal-regulated kinase-1/-2, Erk-1/-2)即MAPKs的激活,进而上调基质金属硫蛋白(matrix metalloproteinases,MMPs)的表达,促进细胞的迁移和侵袭能力,进一步研究表明,在抑制GPER1和EGFR表达后,A549肺癌细胞的迁移和侵袭作用受到抑制。相似地,在G1功能研究过程中发现,雌激素类化合物可通过GPER1反式激活EGFR,从而增强MAPK的磷酸化作用,在维持斑马鱼卵母细胞的减数分裂阻滞过程中起重要作用[55]。其他研究也证实GPER1参与MAPKs的激活过程[56-58]。

5.2 cAMP-PKA通路

作为细胞的“第二信使”,环磷酸腺苷(cyclic adenosine monophosphate,cAMP)是分子信号被刺激后,激活腺苷酸环化酶而催化ATP环化形成的。在精原瘤细胞JKT-1增殖实验中发现,双酚A与细胞接触15 min后,可快速激活蛋白激酶A(protein kinase A,PKA)信号通路,随即诱导cAMP反应元件结合蛋白(cAMP response-element-binding protein, CREB)和细胞周期调节蛋白的快速磷酸化作用,进而促进JKT-1细胞的增殖[59]。进一步的研究发现,该快速雌激素效应是由GPER1介导的非基因组效应。此外,Yu等[60-61]的研究发现,G1刺激可快速诱导cAMP的产生及增强PKA活性,进而通过GPER/ cAMP/PKA信号通路促进冠状动脉的舒张。此通路有时也与 MAPKs通路同时交互作用,通过GPER/PKA/ERK/CREB信号通路调节细胞的非正常效应[62]。

5.3 PI3K-Akt通路

E2和G1刺激胰腺β细胞后,除可激活EGFR和Erk,还可通过促进Akt即蛋白激酶B(protein kinase B,PKB)的磷酸化、提高磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)的活性,增强胰岛素的分泌能力[63]。Ruiz-Palmero等[64]研究GPER在小鼠海马神经元发育过程中的作用时发现, GPER1可介导E2、G1等雌激素类化合物促进Akt第473位的丝氨酸(ser473)磷酸化,而G15及PI3K抑制剂与雌激素共暴露刺激后,Akt激活效应消失,雌激素效应包括Ngn3基因的表达、神经突触形成等消失,表明GPER1可介导PI3K/Akt信号通路影响雌激素的生物效应。后续研究也证实了GPER1介导PI3K信号的激活[65]。

5.4 Ca2＋通道

钙离子通道(calcium channel)是跨越细胞膜上的控制钙离子流动的结构,该通道可调节细胞促炎效应[66],而胞内钙离子浓度升高或下降可诱导神经细胞功能障碍或死亡[67]。研究发现,在小鼠心肌细胞中核受体ERα和ERβ基因的缺失与雌激素引起的抑制钙离子内流现象无关[68],然而G1刺激却可显著提高下丘脑神经元[69]和COS7细胞[27]中的胞内Ca2+浓度。此外,不同细胞暴露于E2后,都可通过激活GPER1而出现胞内钙动员现象[70]。表明G1或雌激素可通过激活GPER1而引起细胞内Ca2+浓度上升,从而异常调节细胞或机体的生理反应。

6 结语(Conclusion)

G蛋白偶联雌激素受体1作为一类雌激素受体,对其研究可追溯至20世纪90年代,而随着时间的推移,GPER1的作用机制及功能研究也在不断深入和完善,它不仅可与ER核受体交互作用,也可单独介导雌激素诱发的信号转导,引发雌激素非基因组效应,进而参与相关基因的转录调控,影响生物体的正常功能。然而,目前对于GPER1介导的环境雌激素效应机制的研究主要集中在该受体高表达的各类癌化细胞中,而对其在正常生物体内特别是接触环境雌激素较多的水生生物中的作用机理研究还需进一步深入。

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Research Progress of the Effects of Environmental Estrogens Mediated by G Protein-Coupled Estrogen Receptor 1

Liu Shuai1,Hu Lei2,Yang Ming1,*
1.School of Environmental and Chemical Engineering,Shanghai University,Shanghai 200444,China
2.School of Life Sciences,Shanghai University,Shanghai 200444,China

17 April 2015 accepted 28 July 2015

Exogenous environmental estrogens can mimic endogenous estrogens in several ways.After entering the bodies,they may interfere with the normal function of organisms and produce some toxic effects on organisms.The genomic effects of estrogens are mainly mediated by estrogen receptors(ERs)in cell nucleus,such as ERα and ERβ;while the non-genomic effects of estrogens are mediated by a mechanism independent of the classical genomic pathway of estrogen action,which generally involves membrane-associated estrogen receptors.Recently,researchers have discovered that G protein-coupled estrogen receptor 1(GPER1),a membrane estrogen receptor different from the nuclear ERs,alone can mediate the effects on cells or organisms induced by estrogens.However,regarding the mechanism of GPER1 mediated estrogenic effects it is still illusive.In this review,the research progress of GPER1 was discussed in details including the discovery,and properties,as well as its mediated estrogenic effectsand the related pathways.

environmental estrogen;membrane-associated estrogen receptors;G protein-coupled estrogen receptor

2015-04-17 录用日期:2015-07-28

1673-5897(2016)1-052-9

X171.5

A

10.7524/AJE.1673-5897.20150417004

刘帅,胡磊,杨明.G蛋白偶联雌激素受体1介导的环境雌激素效应研究进展[J].生态毒理学报,2016,11(1):52-60

Liu S,Hu L,Yang M.Research progress of the effects of environmental estrogens mediated by G protein-coupled estrogen receptor 1[J].Asian Journal of Ecotoxicology,2016,11(1):52-60(in Chinese)

国家自然科学基金(No.31470554)；上海市教委创新项目(No.14YZ001)

刘帅(1993-),男,硕士研究生,研究方向为环境内分泌干扰物的毒性机制,E-mail:lslovetracyx@shu.edu.cn

),E-mail:mingyang@shu.edu.cn

简介:杨明(1979-)，女，博士，副研究员，主要研究方向水生生态毒理学。