緊密連接蛋白Claudin-2研究進(jìn)展

馮燕海 綜述，王鳳君 審校

(陸軍軍醫(yī)大學(xué)西南醫(yī)院全軍燒傷研究所/創(chuàng)傷、燒傷與復(fù)合傷國(guó)家重點(diǎn)實(shí)驗(yàn)室，重慶 400038)

緊密連接蛋白Claudin-2是構(gòu)成細(xì)胞間緊密連接的重要蛋白分子，在維持細(xì)胞極性和緊密連接的屏障功能方面具有重要作用。Claudin-2在細(xì)胞旁路孔通道形成、陽(yáng)離子通透、離子大小選擇性及水分轉(zhuǎn)運(yùn)方面均有重要作用，并參與多種疾病如腫瘤、炎癥、細(xì)菌或病毒感染等的發(fā)生與發(fā)展。本文對(duì)Claudin-2的相關(guān)研究進(jìn)展進(jìn)行綜述，以便對(duì)Claudin-2有一較全面的了解。

1 Claudin-2概述

緊密連接是細(xì)胞間連接的一種常見形式，廣泛存在于上皮、內(nèi)皮和間皮細(xì)胞，在維持上皮細(xì)胞極性及屏障功能中具有重要作用。在眾多的緊密連接相關(guān)蛋白中，最重要的跨膜蛋白是Claudin蛋白家族。人類Claudin蛋白家族至少有24個(gè)成員，均包含4個(gè)跨膜區(qū)域和2條細(xì)胞外鏈，2條細(xì)胞外鏈中均包含有帶不同電荷的氨基酸殘基和2個(gè)細(xì)胞內(nèi)尾巴[1]。第1條細(xì)胞外鏈由大約50個(gè)氨基酸組成，是細(xì)胞旁路孔道形成的主要結(jié)構(gòu)基礎(chǔ)，對(duì)跨細(xì)胞電阻和細(xì)胞旁路的電荷選擇性均具有重要作用[2]；第2條細(xì)胞外鏈有大約25個(gè)氨基酸，主要具有支持功能，有助于緊密連接束的形成，使細(xì)胞旁路的裂縫更加緊密[3]。不同的Claudin家族成員在細(xì)胞旁路通透性的調(diào)控中有著不同的功能，如Claudin-2、-7、-10、-15、-16被稱作是孔道形成蛋白，而Claudin-1、-4、-5、-8、-11、-14、-19則在腸黏膜屏障中表現(xiàn)出封閉作用，可降低細(xì)胞旁路通透性[4]。因此，Claudin-2在孔道形成等方面發(fā)揮重要作用，進(jìn)而調(diào)節(jié)腸屏障功能。

2 Claudin-2的生物學(xué)作用

2.1Claudin-2與細(xì)胞旁路孔通道陽(yáng)離子選擇性的關(guān)系 Claudin家族蛋白細(xì)胞外鏈的帶電殘基決定了孔道的電荷選擇性[5]，并且2條細(xì)胞外鏈間的相互作用是離子通透性和跨上皮電阻(TER)形成的分子基礎(chǔ)[4]；在不同細(xì)胞中，孔道密度隨著Claudin-2的高表達(dá)而增加[6]。因此，Claudin-2是孔道形成的必要蛋白。在犬腎傳代細(xì)胞(madin darby canine kidney，MDCK)Ⅱ細(xì)胞中，Claudin-2缺失可導(dǎo)致其對(duì)Na+通透性降低[7]，且在無內(nèi)源性Claudin-2表達(dá)的MDCK Ⅰ細(xì)胞敲入外源性Claudin-2可使其對(duì)Na+和K+通透性增加，但不改變其對(duì)Cl-和大分子物質(zhì)如甘露醇及4×103葡聚糖的通透性[8]。有研究表明，Claudin-2蛋白細(xì)胞外鏈第65位天冬氨酸所帶負(fù)電荷及第67位的絡(luò)氨酸殘基可通過陽(yáng)離子-π相互作用(Cation-π interaction)或腸腔空間效應(yīng)(Luminal steric effect)賦予其陽(yáng)離子通透性的特點(diǎn)[9-10]。因此，Claudin-2以其特殊的氨基酸組成為基礎(chǔ)，成為了細(xì)胞旁路孔道對(duì)Na+等陽(yáng)離子通透的主要決定因素。除Na+外，Claudin-2形成的孔道對(duì)其他單價(jià)陽(yáng)離子如K+、Rb+、Li+和Cs+等也存在通透性，其通透性大小依次為K+>Rb+>Na+>Li+>Cs+[9]。

2.2Claudin-2與水轉(zhuǎn)運(yùn) 用環(huán)孢霉素A處理人腎小管上皮細(xì)胞后，Claudin-2 mRNA表達(dá)及細(xì)胞旁路水轉(zhuǎn)運(yùn)均明顯降低[11]；敲除Claudin-2基因后，小鼠表現(xiàn)出喝水行為增加、近端腎小管液體重吸收量降低、尿量增加及尿液滲透壓降低[12]。這些研究提示，Claudin-2確實(shí)可增加上皮細(xì)胞對(duì)水的通透性，該效應(yīng)可能與Claudin-2對(duì)Na+通透所引起的細(xì)胞內(nèi)外滲透壓梯度有關(guān)。

2.3Claudin-2與TER 有學(xué)者發(fā)現(xiàn)，小鼠CMT93-Ⅰ與CMT93-Ⅱ均可表達(dá)Claudin-4、-6、-7、-12，但CMT93-Ⅱ細(xì)胞株的TER僅為CMT93-Ⅰ細(xì)胞株的1/7[13]，究其原因可能是Claudin-2蛋白只表達(dá)于CMT93-Ⅱ細(xì)胞，而不表達(dá)于CMT93-Ⅰ細(xì)胞。同樣，高TER的MDCKⅠ細(xì)胞與低TER的MDCKⅡ細(xì)胞間的區(qū)別在于MDCKⅡ細(xì)胞有Claudin-2表達(dá)，而MDCKⅠ細(xì)胞不表達(dá)Claudin-2[8]。因此，Claudin-2被認(rèn)為可降低細(xì)胞TER。此外，多種因子可通過影響Claudin-2的表達(dá)來調(diào)控細(xì)胞TER，如饑餓誘導(dǎo)的Caco-2腸上皮細(xì)胞自噬可通過降解Claudin-2而引起TER明顯增加，從而保護(hù)腸上皮屏障功能[14]。但是，Claudin-2降低細(xì)胞TER的分子機(jī)制仍有待闡明。

3 Claudin-2與疾病

3.1Claduin-2與腫瘤 目前的研究發(fā)現(xiàn)，在胃癌、結(jié)直腸癌和乳腺癌等均有Claudins蛋白的異常表達(dá)，且Claudin-2在不同癌細(xì)胞的表達(dá)量也不同，在肝細(xì)胞癌、結(jié)直腸腺癌的肝轉(zhuǎn)移及胰腺癌，Claudin-2表達(dá)降低[15]；而在纖維板樣肝細(xì)胞癌、結(jié)直腸癌、胃癌、乳腺癌肝轉(zhuǎn)移等，Claudin-2表達(dá)則明顯增加[16]。(1)Claudin-2與消化道腫瘤：有研究表明，在胃癌中可檢測(cè)到Claudin-2的高表達(dá)[17]，而幽門螺桿菌的CagA可通過Cdx2，在轉(zhuǎn)錄和翻譯水平均增加Claudin-2表達(dá)，進(jìn)而破壞緊密連接和誘導(dǎo)胃上皮細(xì)胞的去分化，導(dǎo)致幽門螺桿菌相關(guān)胃癌的發(fā)生[18]。與正常組織相比，Claudin-2 mRNA水平在結(jié)直腸癌組織中明顯上調(diào)并與腫瘤進(jìn)展有關(guān)，這可能是結(jié)直腸癌微環(huán)境通過激活表皮生長(zhǎng)因子受體(EGFR)或者偶對(duì)蛋白(Symplekin)通過與ZO-1相關(guān)核酸結(jié)合蛋白的相互作用，增加Claudin-2的蛋白表達(dá)，從而促進(jìn)腫瘤細(xì)胞生長(zhǎng)[19-20]。因此，Claudin-2在消化道腫瘤的發(fā)生、發(fā)展中具有重要作用，可能是新的治療靶點(diǎn)。(2) Claudin-2與乳腺癌：與正常組織相比，原發(fā)性乳腺癌的Claudin-2表達(dá)降低，但在乳腺癌肝轉(zhuǎn)移中，Claudin-2表達(dá)卻明顯增加[21-23]；肝轉(zhuǎn)移時(shí)Claudin-2表達(dá)增加，一方面通過增強(qiáng)α2β1-和 α5β1-整合素復(fù)合物表達(dá)，利于乳腺癌細(xì)胞與細(xì)胞外基質(zhì)如纖連蛋白或Ⅳ型膠原黏附，進(jìn)而促進(jìn)乳腺癌肝轉(zhuǎn)移[24]；另一方面，通過其第一條細(xì)胞外鏈形成Claudin-2-Claudin-2同源復(fù)合物，介導(dǎo)乳腺癌細(xì)胞與肝細(xì)胞間黏附進(jìn)而促進(jìn)乳腺癌細(xì)胞的肝轉(zhuǎn)移[25]。由此可見，Claudin-2是乳腺癌肝轉(zhuǎn)移的重要調(diào)節(jié)子，在乳腺癌肝轉(zhuǎn)移的發(fā)生中具有重要作用。因此，有學(xué)者認(rèn)為可將Claudin-2作為乳腺癌肝轉(zhuǎn)移的生物標(biāo)記物[23]。(3)Claudin-2與肺癌：據(jù)報(bào)道，Claudin-2在不同類型肺癌的表達(dá)不盡相同，如在鱗癌、腺癌和良性腫瘤有Claudin-2表達(dá)[26]，但有學(xué)者在小細(xì)胞肺癌中卻未檢測(cè)到Claudin-2表達(dá)。對(duì)A549細(xì)胞的研究發(fā)現(xiàn)，Claudin-2表達(dá)呈時(shí)間依賴性增加，其可能機(jī)制是基質(zhì)金屬蛋白酶分泌EGF后，激活EGFR/MEK/ERK/c-Fos信號(hào)通路，引起c-Fos入核與Claudin-2 基因啟動(dòng)子的AP-1位點(diǎn)結(jié)合，增強(qiáng)其轉(zhuǎn)錄活性[27]。

3.2Claduin-2與腸道炎癥 有研究發(fā)現(xiàn)，在腸道炎癥發(fā)生時(shí)Claudin-2表達(dá)明顯增加[28]。炎癥性腸病(IBD)時(shí)Claudin-2表達(dá)增加可能與炎癥局部組織細(xì)胞因子增加有關(guān)，如潰瘍性結(jié)腸炎時(shí)，腸黏膜Th2淋巴細(xì)胞釋放白細(xì)胞介素(IL)-13增加，由IL-13誘導(dǎo)Claudin-2表達(dá)增加[29]。IBD時(shí)Claudin-2表達(dá)增加可能是機(jī)體自身的一種保護(hù)性反應(yīng)，研究表明，在腫瘤壞死因子α(TNF-α)或硫酸葡聚糖鈉鹽誘發(fā)的腸道炎癥中，Claudin-2(-/-)小鼠的炎癥程度均較Claudin-2(+/+)小鼠明顯嚴(yán)重，其機(jī)制可能與Claudin-2抑制肌球蛋白輕鏈激酶依賴性的信號(hào)通路活化有關(guān)，也可能與Claudin-2通過PI-3K/Bcl-2通路阻止腸細(xì)胞死亡有關(guān)[30-31]。

3.3Claudin-2與感染 發(fā)生細(xì)菌或病毒感染時(shí)，受侵組織Claudin-2表達(dá)也會(huì)發(fā)生相應(yīng)變化，但不同細(xì)菌或病毒對(duì)Claudin-2表達(dá)的影響截然不同。有學(xué)者在對(duì)HT29C19A進(jìn)行的研究發(fā)現(xiàn)，幽門螺桿菌由其編碼的CagA通過Cdx2連接于Claudin-2基因的3′側(cè)翼區(qū)，在轉(zhuǎn)錄和翻譯水平均增加Claudin-2表達(dá)[18]。相反，鼠疫桿菌卻抑制HT29/B6腸上皮細(xì)胞Claudin-2表達(dá)[32]。病毒如人類免疫缺陷病毒1型(HIV-1)則可能是通過增加炎癥因子如TNF-α等的產(chǎn)生，來破壞包括Claudin-2在內(nèi)的緊密連接蛋白，導(dǎo)致黏膜上皮緊密連接結(jié)構(gòu)破壞，從而有利于病毒自身或其他細(xì)菌的入侵[33]。

4 結(jié) 語(yǔ)

緊密連接蛋白Claudin-2是緊密連接的重要組成成分，在機(jī)體發(fā)育、屏障的離子選擇性、水轉(zhuǎn)運(yùn)及跨細(xì)胞電阻的調(diào)控等方面都具有重要作用，并與多種疾病如腫瘤、炎癥、細(xì)菌或病毒感染等密切相關(guān)。然而，Claudin-2與這些疾病的發(fā)生、發(fā)展及轉(zhuǎn)歸的復(fù)雜關(guān)系，以及這些疾病狀態(tài)下Claudin-2表達(dá)的調(diào)控機(jī)制仍需更深入的研究。

[1]GUNZEL D,FROMM M.Claudins and other tight junction proteins[J].Compr Physiol,2012,2(3):1819-1852.

[2]WEN H J,WATRY D D,MARCONDES M C,et al.Selective decrease in paracellular conductance of tight junctions:role of the first extracellular domain of claudin-5[J].Mol Cell Biol,2004,24(19):8408-8417.

[3]PIONTEK J,WINKLER L,WOLBURG H,et al.Formation of tight junction:determinants of homophilic interaction between classic claudins[J].FASEB J,2008,22(1):146-158.

[4]KRAUSE G,WINKLER L,MUELLER S L,et al.Structure and function of claudins[J].Biochim Biophys Acta Biomembr,2008,1778(3):631-645.

[5]VAN ITALLIE C M,HOLMES J,BRIDGES A,et al.Claudin-2-dependent changes in noncharged solute flux are mediated by the extracellular domains and require attachment to the PDZ-scaffold[J].Ann N Y Acad Sci,2009,1165(1):82-87.

[6]VAN ITALLIE C M,HOLMES J,BRIDGES A,et al.The density of small tight junction pores varies among cell types and is increased by expression of claudin-2[J].J Cell Sci,2008,121(Pt 3):298-305.

[7]HOU J,GOMES A S,PAUL D L,et al.Study of claudin function by RNA interference[J].J Biol Chem,2006,281(47):36117-36123.

[8]FURUSE M,FURUSE K,SASAKI H,et al.Conversion of zonulae occludentes from tight to leaky Strand type by introducing claudin-2 into Madin-Darby canine kidney I cells[J].J Cell Biol,2001,153(2):263-272.

[9]YU A S,CHENG M H,ANGELOW S,et al.Molecular basis for cation selectivity in claudin-2-based paracellular pores:identification of an electrostatic interaction site[J].J Gen Physiol,2009,133(1):111-127.

[10]LI J,ZHUO M,PEI L,et al.Conserved aromatic residue confers cation selectivity in claudin-2 and claudin-10b[J].J Biol Chem,2013,288(31):22790-22797.

[11]WILMES A,ASCHAUER L,LIMONCIEL A,et al.Evidence for a role of claudin 2 as a proximal tubular stress responsive paracellular water Channel[J].Toxicol Appl Pharmacol,2014,279(2):163-172.

[12]MUTO S,HATA M,TANIGUCHI J,et al.Claudin-2-deficient mice are defective in the leaky and cation-selective paracellular permeability properties of renal proximal tubules[J].Proc Natl Acad Sci U S A,2010,107(17):8011-8016.

[13]INAI T,SENGOKU A,HIROSE E,et al.Comparative characterization of mouse rectum CMT93-Ⅰ and-Ⅱ cells by expression of claudin isoforms and tight junction morphology and function[J].Histochem Cell Biol,2008,129(2):223-232.

[14]NIGHOT P K,HU C A,MA T Y.Autophagy enhances intestinal epithelial tight junction barrier function by targeting claudin-2 protein degradation[J].J Biol Chem,2015,290(11):7234-7246.

[15]HOLCZBAUER A,GYOENGYOESI B,LOTZ G,et al.Distinct claudin expression profiles of hepatocellular carcinoma and metastatic colorectal and pancreatic carcinomas[J].J Histochem Cytochem,2013,61(4):294-305.

[16]PATONAI A,ERDELYI-BELLE B,KOROMPAY A,et al.Claudins and tricellulin in fibrolamellar hepatocellular carcinoma[J].Virchows Arch,2011,458(6):679-688.

[17]JUNG H,JUN K H,JUNG J H,et al.The expression of claudin-1,claudin-2,claudin-3,and claudin-4 in gastric cancer tissue[J].J Surg Res,2011,167(2):e185-191.

[18]SONG X,CHEN H X,WANG X Y,et al.H.pylori-encoded CagA disrupts tight junctions and induces invasiveness of AGS gastric carcinoma cells via Cdx2-dependent targeting of Claudin-2[J].Cell Immunol,2013,286(1/2):22-30.

[19]DHAWAN P,AHMAD R,CHATURVEDI R,et al.Claudin-2 expression increases tumorigenicity of colon cancer cells:role of epidermal growth factor receptor activation[J].Oncogene,2011,30(29):3234-3247.

[20]BUCHERT M,PAPIN M,BONNANS C,et al.Symplekin promotes tumorigenicity by up-regulating claudin-2 expression[J].Proc Natl Acad Sci U S A,2010,107(6):2628-2633.

[21]KIM T H,HUH J H,LEE S,et al.Down-regulation of claudin-2 in breast carcinomas is associated with advanced disease[J].Histopathology,2008,53(1):48-55.

[22]FLORES A R,REMA A,CARVALHO F,et al.Reduced expression of claudin-2 is associated with high histological grade and metastasis of feline mammary carcinomas[J].J Comp Pathol,2014,150(2/3):169-174.

[23]KIMBUNG S,KOVCS A,BENDAHL P O,et al.Claudin-2 is an independent negative prognostic factor in breast cancer and specifically predicts early liver recurrences[J].Mol Oncol,2014,8(1):119-128.

[26]MOLDVAY J,JCKEL M,PSKA C,et al.Distinct claudin expression profile in histologic subtypes of lung cancer[J].Lung Cancer,2007,57(2):159-167.

[27]IKARI A,SATO T,WATANABE R,et al.Increase in claudin-2 expression by an EGFR/MEK/ERK/c-Fos pathway in lung adenocarcinoma A549 cells[J].Biochim Biophys Acta Mol Cell Res,2012,1823(6):1110-1118.

[28]LUETTIG J,ROSENTHAL R,BARMEYER C,et al.Claudin-2 as a mediator of leaky gut barrier during intestinal inflammation[J].Tissue Barriers,2015,3(1/2):e977176.

[29]WATSON A J,DUCKWORTH C A,GUAN Y,et al.Mechanisms of epithelial cell shedding in the Mammalian intestine and maintenance of barrier function[J].Ann N Y Acad Sci,2009,1165(1):135-142.

[30]NISHIDA M,YOSHIDA M,NISHIUMI S,et al.Claudin-2 regulates colorectal inflammation via myosin light chain kinase-dependent signaling[J].Dig Dis Sci,2013,58(6):1546-1559.

[31]AHMAD R,CHATURVEDI R,OLIVARES-VILLAGMEZ D,et al.Targeted colonic claudin-2 expression renders resistance to epithelial injury,induces immune suppression,and protects from colitis[J].Mucosal Immunol,2014,7(6):1340-1353.

[32]HERING N A,RICHTER J F,KRUG S M,et al.Yersinia enterocolitica induces epithelial barrier dysfunction through regional tight junction changes in colonic HT-29/B6 cell monolayers[J].Lab Invest,2011,91(2):310-324.

[33]NAZLI A,CHAN O,DOBSON-BELAIRE W N,et al.Exposure to HIV-1 directly impairs mucosal epithelial barrier integrity allowing microbial translocation[J].PLoS Pathog,2010,6(4):e1000852.