G蛋白偶聯雌激素受體1介導的環境雌激素效應研究進展

劉帥，胡磊，楊明,*

1.上海大學環境與化學工程學院，上海200444

2.上海大學生命科學學院，上海200444

G蛋白偶聯雌激素受體1介導的環境雌激素效應研究進展

劉帥1，胡磊2，楊明1,*

1.上海大學環境與化學工程學院，上海200444

2.上海大學生命科學學院，上海200444

環境雌激素進入生物體后,可通過多種方式介導發揮類似內源雌激素的作用,干擾生物體的正常功能,進而對生物體產生毒害作用。其中,基因組方式介導的雌激素效應主要通過與細胞核內的雌激素受體(如ERα和ERβ)結合；而非基因組方式介導的雌激素效應則主要通過與膜雌激素受體結合從而發揮作用。近年來對G蛋白偶聯雌激素受體1(GPER1)的研究表明,該受體是區別于雌激素核受體的膜雌激素受體,可單獨介導雌激素誘發的非基因組方式雌激素效應,然而目前對其介導的雌激素效應機制研究并不完善。綜上,本文結合近年來對GPER1的研究進展,從該受體的發現、特性以及其介導的雌激素效應和相關通路進行了綜述。

環境雌激素；膜雌激素受體；G蛋白偶聯雌激素受體

環境內分泌干擾物(environmental endocrine disruptors,EEDs)是環境污染物的重要組成部分,可對生物體的內分泌系統產生復合干擾損害作用,其中最重要的一類物質環境雌激素(environmental estrogens)可誘導生物體產生雌激素效應,從而影響生物體的生殖系統[1]、神經系統[2]、免疫系統[3]等的正常功能。

雌激素介導細胞的效應機制主要分為2類:第一類是以傳統雌激素核受體ERα和ERβ介導為主的基因組效應,細胞核內的雌激素受體不僅可直接結合目的DNA片段,還可以與其他核內蛋白如雌激素的激活劑或抑制劑相互作用,從而增強或抑制基因的激活[4]；第二類是通過膜雌激素受體介導的快速非基因組效應,與傳統ER受體不同,膜性受體同細胞表面的內源或外源雌激素產生結合或拮抗等作用,可在極短時間內引發信號轉導,如鉀離子通道關閉、鈣離子濃度升高以及相關通路的激活,從而影響有機體的正常功能,發揮其生物學效應[5-6]。近年來關注并研究最多的膜雌激素受體是G蛋白偶聯雌激素受體1(G protein-coupled estrogen receptor 1, GPER1),文章對GPER1的研究進展進行了綜述。

1 GPER1的發現及組織分布(The discovery and tissue distribution of GPER1)

G蛋白偶聯雌激素受體1,最早稱之為G蛋白偶聯受體30(GPR30),是20世紀90年代從人體細胞中發現的一種膜雌激素受體,Carmeci等[7]從人體乳腺癌細胞系(MCF7)的cDNA文庫中篩選出一個全長2 604 bp的cDNA序列,包括長度為1 128 bp的開放閱讀框(open read frame,ORF),可編碼成375個氨基酸的蛋白,分析發現,該蛋白與早前報道的G蛋白偶聯受體(GPCRs)同源性很高,并命名為GPR30。該受體位于染色體7p22區域,具有7個跨膜疏水區域,但與大多數G蛋白偶聯受體不同, GPER1蛋白序列在第三跨膜區后存在一個保守的Asp-Arg-Tyr三聯體(DRY)序列,分析認為可能與其介導的信號轉導有關[8]。其他研究團隊同時期也發現并分離擴增得到該受體基因,其序列信息基本相似[9-11]。

研究者相繼在哺乳類動物如人(Homo sapiens)[9-11]和小家鼠(Mus musculus)[12-13]等,鳥類如雞(Gallus gallus)[14],兩棲類如蟾蜍(Xenopus)[15]和黑斑蛙(Rana nigromaculataH.)[16]等,硬骨魚類如細須石首魚(Micropogonias undulatus)[17]、斑馬魚(Danio rerio)[18]、斜帶石斑魚(Epinephelus coioides)[19]、金頭鯛(Sparus aurataL.)[20]等物種中均發現GPER1的存在。此外,GPER1在不同物種中的組織分布情況不同,雖然其在人體中的各組織中均有分布,包括心臟、腦、胎盤、肺、肝臟、肌肉、前列腺等,但在肺、肝臟和前列腺中表達較多,腦和性腺(卵巢、睪丸)中表達較少[21],然而在魚類中[18],GPER1的分布情況相反,在腦中表達最高,性腺次之,推測其原因可能是該受體在不同物種中的作用不同。

2 GPER1蛋白序列比對及進化樹分析(The multiple alignment of protein sequences and phylogenetic tree analysis of GPER1)

圖1為基因庫中常見物種如人(human,Homo sapiens)、牛(cattle,Bos taurus)、小鼠(mouse,Mus musculus)、馬(horse,Equus caballus)、狗(dog,Canis lupus familiaris)、雞(chicken,Gallus gallus)、蟾蜍(toad, Gallus gallus)、石首魚(croaker,Micropogonias undulatus)、青鳉(medaka,Oryzias latipes)和斑馬魚(zebrafish,Danio rerio)的GPER1蛋白序列的序列比對(A)及進化樹構建結果(B)。比對結果表明,GPER1在不同物種之間的跨膜區域同源性均很高,且在第三跨膜區后存在DRY三聯體保守序列；此外,進化樹分析發現,GPER1基因在脊椎動物中有明顯的分化,包括哺乳動物、鳥類、兩棲動物及硬骨魚類等。

3 GPER1的特性(The characteristics of GPER1)

早期研究過程中發現,GPER1與傳統ER核受體的相關配體如17β-雌二醇(E2)、tamoxifen及ICI 182,780具有選擇性結合能力[22-26],但未找到GPER1的特異性配體,直到2006年Bologa等[27]通過虛擬和分子生物篩選融合法,從包括10 000個GPCRs結合配體的分子文庫中篩選出一個GPER1的特異性配體G1,驗證后發現,G1和雌激素一樣,可有效激活SkBr3細胞(不表達ERα、ERβ,但表達GPER1)和MCF7細胞(3種受體均有表達)內磷脂酰肌醇激酶PI3K的表達,從而誘導磷脂酰肌醇三磷酸PIP3的產生。G1可特異性結合GPER1也可通過后續的研究得到證實[28-29]。

圖1 不同常見物種間的GPER1蛋白序列比對(A)及其進化樹分析(B)注:各相關物種蛋白序列的基因庫登錄號分別為人,NP_001091671.1；牛,DAA15165.1；鼠,NP_084047.2；馬,XP_001488797；狗,XP_005621261.1；雞,NP_001155877.1；蟾蜍,NP_001107725.1；石首魚,B0F9W3.1；青鳉,XP_004071380.1；斑馬魚,NP_001122195.1。圖A中橫線所在位置代表7個跨膜區域(seven transmembrane domains,TM),方框位置代表DRY三聯體序列。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]將不表達ER的人體子宮內膜癌KLE細胞系暴露于不同濃度的G1中,結果發現,G1刺激后可有效促進該細胞的增殖,并通過有絲分裂原活化蛋白激酶MAPK通路誘導基質金屬蛋白酶的產生和白介素-6(IL-6)的分泌,表明GPER1可介導非基因組效應在子宮內膜癌研究中發揮重要作用；Sirianni等[31]發現在小鼠精原細胞系GC1中,G1、E2可通過GPER1和ERα的交互作用快速激活EGFR/ ERK/fos通路,進而誘導小鼠精原細胞的增殖,但當GPER1基因沉默后,增殖效應消失。

此外,在2009年,Dennis等[32]從分子庫中篩選出一種化合物G15,并確認其是GPER1的拮抗劑。與G1的化學結構相比,G15化合物少一個乙酰基,由于結構的相似性而得到關注,通過配體結合實驗表明,同G1類似,G15表現出較高的GPER1親和性,而與ERα、ERβ親和性很低,此外,G1、G15分別暴露和共暴露刺激小鼠子宮上皮細胞后,其增殖程度不同,G1組細胞顯著增殖,G15組幾乎無增殖,而共同刺激組較G1組增殖效果顯著降低,表明G15可作為GPER1的拮抗劑應用于其在生物體內作用機制的進一步研究中。2011年,Dennis等[33]在G15的結構基礎上增加一個異丙基,得到一種新的化合物G36,研究后發現也具有抑制GPER1表達的作用。

4 環境雌激素物質對生物體的效應機制研究(Mechanisms of actions of environmental estrogens on organisms)

雌激素的基因組效應主要從3個方面進行:(1)進入細胞核內的雌激素可激活核受體ERα或ERβ,與銜接蛋白和信號轉導分子作用,進而影響下游通路；(2)激活目的基因啟動子區域的ER二聚體從而調節細胞的基因表達變化；(3)促進相關轉錄因子的磷酸化作用[34]。而進一步的研究發現,有的雌激素引起的下游效應非常快速,表明此效應可能不需要經過細胞核內的雌激素受體,而在胞膜或質膜發揮作用,與膜上的受體結合,激活相關通路等,從而誘導下游信號轉導,該效應被稱為非基因組效應。

4.1 基因組效應

環境雌激素的基因組效應主要指由雌激素核受體(ER)作為雌激素類物質的配體與進入生物體內的雌激素(天然或環境雌激素)結合,而介導的胞內轉錄因子激活所引起的細胞內反應[5]。17α-乙炔基雌二醇(EE2)是一種合成激素,作為天然激素E2的衍生物而得到使用并大量進入環境中,對生物體可能造成危害。在早期活體研究中發現,在EE2長期暴露下,可降低性成熟時期虹鱒魚的生育能力[35],破壞斑馬魚胚胎的性腺成熟[36]或降低成年斑馬魚的生殖腺指數[37],影響其正常生殖功能；離體實驗也表明,較高濃度EE2暴露(100 nmol·L-1)可顯著降低雄性青鳉生殖細胞的增殖[38]。最初觀點認為,環境雌激素效應主要由ER介導。Cabas等[39]發現,EE2刺激可顯著上調金頭鯛器官和細胞內ERα的mRNA表達,從而調節其免疫功能和性激素水平。同樣地, Yang等[40]以鯉魚巨噬細胞作為研究對象發現,雙酚A也可通過誘導ERα的表達對魚體產生免疫毒性。此外,其他對雙酚A的研究中也得到類似研究結果[41-42]。綜上表明,ER作為轉錄因子,在雌激素誘導下所引起基因表達的變化足以影響細胞反應,進而影響生物體。

4.2 非基因組效應

在環境雌激素暴露下,傳統核受體可介導基因組效應,從而參與下游的信號轉導,類似地,膜雌激素受體GPER1也具有調節雌激素相關基因表達的功能,即非基因組效應機制。Rettew等[43]發現,E2和G1刺激小鼠巨噬細胞后,在10～60 min內TLR4的表達量顯著性降低,而在敲除GPER1后,TLR4的表達量趨于正常。在不表達ERα和ERβ的人體乳腺癌 SkBr3細胞系中,Vivacqua等[44]發現,E2、tamoxifen和G1分別刺激1 h后,顯著上調細胞中早期生長反應因子(early growth response protein 1, EGR-1)的表達,從而誘導結締組織生長因子(connective tissue growth factor,CTGF)和細胞周期素D1的基因和蛋白表達,促進細胞增殖,而在RNA干擾沉默GPER1表達后,在同樣處理條件下相關基因的表達不再上調。最新研究發現,tamoxifen和 ICI 182,780(ER拮抗劑)可在30 min內激活GPER1促進小鼠性興奮[45]。上述結果表明,與 ER相比, GPER1的表達可快速介導雌激素的下游信號轉導,符合非基因組效應的特點。

其他研究也表明,在不同濃度的環境雌激素類物質暴露下,可通過激活GPER1調節不同種類細胞中相關基因的表達量。Catalano等[46]的研究發現,對乳腺癌細胞長期tamoxifen暴露攻毒后,可上調芳香化酶的mRNA表達,促進細胞增殖,而GPER1敲除及與拮抗劑共暴露后,上調現象消失；Pupo等[47]研究雙酚A對SkBr3細胞和癌癥相關纖維細胞CAFs(均不表達 ER)的影響中發現,可顯著上調GPER1目的基因c-fos、早期生長反應蛋白1和CTGF在細胞中的表達,在GPER1基因沉默后,相關基因不再上調；Teng等[48]的研究發現,GPER1過表達可消除因E2刺激而誘導的c-fos、c-jun及細胞周期蛋白D1的上調表達,而RNA干擾導致GPER1失活后,反而上調這些基因的表達。進一步地,環境雌激素可通過調節細胞內相關基因的表達,從而改變細胞的生長情況。Bouskine等[49]和Liu等[50]的研究分別證實了GPER1在雙酚A促進人體精原瘤細胞JKT-1增殖和E2促進不表達ERα的卵巢癌細胞增殖過程中起關鍵作用。而Chimento等[51-52]則發現環境雌激素選擇性激活GPER1,可引起小鼠精母細胞和睪丸間質腫瘤細胞的凋亡。由此可見,雌激素膜受體如GPER1介導的環境雌激素非基因組效應可快速但持久地激活細胞內不同的雌激素信號通路,從而影響細胞的增殖情況及其功能。

5 GPER1相關信號轉導通路 (GPER1-related signal transduction pathways)

生物體中的GPER1受到雌激素刺激后,可快速介導非基因組效應,引起下游相關效應變化,如參與部分基因的轉錄調控,調節基因表達量,引起細胞增殖或凋亡等,而GPER1激活后,主要通過介導相關信號轉導通路來引起上述效應。通過對多年的研究總結,其相關通路主要有以下4種:

5.1 EGFR-MAPKs通路

早期研究結果表明,雌激素在激活絲裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)的過程中需要GPER1的參與[53]。Zhang等[54]發現,雌激素可通過表皮生長因子受體(epidermal growth factor receptor,EGFR)信號來誘導細胞外信號調節激酶-1/-2(extracellular signal-regulated kinase-1/-2, Erk-1/-2)即MAPKs的激活,進而上調基質金屬硫蛋白(matrix metalloproteinases,MMPs)的表達,促進細胞的遷移和侵襲能力,進一步研究表明,在抑制GPER1和EGFR表達后,A549肺癌細胞的遷移和侵襲作用受到抑制。相似地,在G1功能研究過程中發現,雌激素類化合物可通過GPER1反式激活EGFR,從而增強MAPK的磷酸化作用,在維持斑馬魚卵母細胞的減數分裂阻滯過程中起重要作用[55]。其他研究也證實GPER1參與MAPKs的激活過程[56-58]。

5.2 cAMP-PKA通路

作為細胞的“第二信使”,環磷酸腺苷(cyclic adenosine monophosphate,cAMP)是分子信號被刺激后,激活腺苷酸環化酶而催化ATP環化形成的。在精原瘤細胞JKT-1增殖實驗中發現,雙酚A與細胞接觸15 min后,可快速激活蛋白激酶A(protein kinase A,PKA)信號通路,隨即誘導cAMP反應元件結合蛋白(cAMP response-element-binding protein, CREB)和細胞周期調節蛋白的快速磷酸化作用,進而促進JKT-1細胞的增殖[59]。進一步的研究發現,該快速雌激素效應是由GPER1介導的非基因組效應。此外,Yu等[60-61]的研究發現,G1刺激可快速誘導cAMP的產生及增強PKA活性,進而通過GPER/ cAMP/PKA信號通路促進冠狀動脈的舒張。此通路有時也與 MAPKs通路同時交互作用,通過GPER/PKA/ERK/CREB信號通路調節細胞的非正常效應[62]。

5.3 PI3K-Akt通路

E2和G1刺激胰腺β細胞后,除可激活EGFR和Erk,還可通過促進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,增強胰島素的分泌能力[63]。Ruiz-Palmero等[64]研究GPER在小鼠海馬神經元發育過程中的作用時發現, GPER1可介導E2、G1等雌激素類化合物促進Akt第473位的絲氨酸(ser473)磷酸化,而G15及PI3K抑制劑與雌激素共暴露刺激后,Akt激活效應消失,雌激素效應包括Ngn3基因的表達、神經突觸形成等消失,表明GPER1可介導PI3K/Akt信號通路影響雌激素的生物效應。后續研究也證實了GPER1介導PI3K信號的激活[65]。

5.4 Ca2＋通道

鈣離子通道(calcium channel)是跨越細胞膜上的控制鈣離子流動的結構,該通道可調節細胞促炎效應[66],而胞內鈣離子濃度升高或下降可誘導神經細胞功能障礙或死亡[67]。研究發現,在小鼠心肌細胞中核受體ERα和ERβ基因的缺失與雌激素引起的抑制鈣離子內流現象無關[68],然而G1刺激卻可顯著提高下丘腦神經元[69]和COS7細胞[27]中的胞內Ca2+濃度。此外,不同細胞暴露于E2后,都可通過激活GPER1而出現胞內鈣動員現象[70]。表明G1或雌激素可通過激活GPER1而引起細胞內Ca2+濃度上升,從而異常調節細胞或機體的生理反應。

6 結語(Conclusion)

G蛋白偶聯雌激素受體1作為一類雌激素受體,對其研究可追溯至20世紀90年代,而隨著時間的推移,GPER1的作用機制及功能研究也在不斷深入和完善,它不僅可與ER核受體交互作用,也可單獨介導雌激素誘發的信號轉導,引發雌激素非基因組效應,進而參與相關基因的轉錄調控,影響生物體的正常功能。然而,目前對于GPER1介導的環境雌激素效應機制的研究主要集中在該受體高表達的各類癌化細胞中,而對其在正常生物體內特別是接觸環境雌激素較多的水生生物中的作用機理研究還需進一步深入。

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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 錄用日期:2015-07-28

1673-5897(2016)1-052-9

X171.5

A

10.7524/AJE.1673-5897.20150417004

劉帥,胡磊,楊明.G蛋白偶聯雌激素受體1介導的環境雌激素效應研究進展[J].生態毒理學報,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)

國家自然科學基金(No.31470554)；上海市教委創新項目(No.14YZ001)

劉帥(1993-),男,碩士研究生,研究方向為環境內分泌干擾物的毒性機制,E-mail:lslovetracyx@shu.edu.cn

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

簡介:楊明(1979-)，女，博士，副研究員，主要研究方向水生生態毒理學。