連接蛋白43調(diào)控成骨細(xì)胞分子機(jī)制

劉剛　薄占東

530000　南寧，　　廣西醫(yī)科大學(xué)第一附屬醫(yī)院骨關(guān)節(jié)外科

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連接蛋白43調(diào)控成骨細(xì)胞分子機(jī)制

劉剛薄占東

530000南寧，廣西醫(yī)科大學(xué)第一附屬醫(yī)院骨關(guān)節(jié)外科

摘要連接蛋白43(CX43)是骨細(xì)胞中表達(dá)最多的縫隙連接蛋白，由CX43構(gòu)成的縫隙連接或半通道在骨細(xì)胞間通訊中發(fā)揮至關(guān)重要的作用。大量研究表明，CX43可影響骨細(xì)胞、成骨細(xì)胞的功能，最終影響骨骼的發(fā)育和塑形。然而，關(guān)于CX43調(diào)控骨的分子機(jī)制研究較少。該文就CX43調(diào)控成骨細(xì)胞分子機(jī)制作一綜述。

關(guān)鍵詞連接蛋白43；骨細(xì)胞；成骨細(xì)胞；信號(hào)轉(zhuǎn)導(dǎo)

骨骼的正常發(fā)育和維持依賴于骨原細(xì)胞、成骨細(xì)胞、骨細(xì)胞和破骨細(xì)胞間的緊密協(xié)同作用。在構(gòu)建和維持骨骼系統(tǒng)時(shí)，為了實(shí)現(xiàn)骨骼系統(tǒng)結(jié)構(gòu)的完整性、機(jī)械能力和骨內(nèi)礦物質(zhì)平衡，4種細(xì)胞間的通訊不可或缺。研究發(fā)現(xiàn)，成骨細(xì)胞、破骨細(xì)胞和骨細(xì)胞之間存在一種直接信號(hào)通道，即縫隙連接?？p隙連接是一種跨膜通道，由細(xì)胞膜中連接蛋白單體排列成的六聚體(半通道)與相鄰細(xì)胞膜中的相似六聚體配對(duì)而成，進(jìn)而在兩細(xì)胞間形成親水性通道，相鄰細(xì)胞間的離子、小分子代謝物和第二信使等物質(zhì)可經(jīng)其進(jìn)行自由擴(kuò)散，從而實(shí)現(xiàn)相鄰細(xì)胞間的信號(hào)轉(zhuǎn)導(dǎo)[1]。Jordan等[2]研究發(fā)現(xiàn)，連接蛋白43(CX43)主要在細(xì)胞核周圍表達(dá)，在成骨細(xì)胞和骨細(xì)胞的非交界質(zhì)膜處也有表達(dá)，這種亞細(xì)胞定位提示骨中的CX43可能不僅作為縫隙連接通道起作用。Gonzalez-Nieto等[3]研究證實(shí)，在細(xì)胞液與胞外環(huán)境間的物質(zhì)交換過程中，縫隙連接還可作為半通道起作用。近期研究[4]發(fā)現(xiàn)，CX43蛋白C末端可與蛋白激酶結(jié)合，主動(dòng)參與信號(hào)轉(zhuǎn)導(dǎo)過程而發(fā)揮作用。

1CX43與骨骼疾病

研究[5]表明，人類GJA1基因突變可導(dǎo)致眼齒指發(fā)育不良(ODDD，一種以管狀長骨增寬、顱面骨畸形及中節(jié)指骨發(fā)育不良或并指為特征的骨骼疾病)。研究[6-7]證實(shí)，在小鼠ODDD模型中GJA1基因突變可引起骨骼幾何結(jié)構(gòu)、骨微結(jié)構(gòu)改變和骨量缺失。全部或部分敲除小鼠成骨細(xì)胞系GJA1基因后，小鼠表現(xiàn)出骨細(xì)胞分化延遲、骨質(zhì)疏松及與ODDD患者相同的骨骼結(jié)構(gòu)(如長骨周長增大伴骨髓腔擴(kuò)大和皮質(zhì)骨變薄)[8]，這種骨髓腔擴(kuò)大和皮質(zhì)變薄的表現(xiàn)與老齡及骨病患者骨表型極為相似[9]。此外，GJA1基因突變還可引起與ODDD骨表型不同的顱骨干骺端發(fā)育不良[10]。

2CX43與成骨細(xì)胞分化及骨形成

CX43可通過調(diào)控成骨細(xì)胞分化、骨細(xì)胞活動(dòng)來調(diào)節(jié)骨形成和重塑，也可調(diào)控某些因子如核因子-κ B受體活化因子配體(RANKL)、骨保護(hù)素(OPG)的表達(dá)來調(diào)節(jié)骨重吸收。Lecanda等[11]建立CX43缺陷小鼠模型發(fā)現(xiàn)，模型組中軸骨和四肢骨骨化明顯延遲，顱面骨畸形，從這些小鼠長骨和顱骨分離出的成骨細(xì)胞某些成骨基因表達(dá)減少，骨化延遲。此外，由CX43缺陷小鼠分離的成骨細(xì)胞或過表達(dá)CX45的成骨細(xì)胞系中，成骨標(biāo)志性物質(zhì)(如骨鈣素、Ⅰ型膠原α1、骨橋蛋白和Runx1)減少[12-13]。Loiselle等[14]建立小鼠CX43缺陷伴有股骨骨折模型，發(fā)現(xiàn)CX43可促進(jìn)骨折愈合。

CX43調(diào)控骨細(xì)胞功能和骨質(zhì)量的機(jī)制非常復(fù)雜，在不同條件下會(huì)產(chǎn)生不同的作用。CX43不僅可以傳導(dǎo)骨合成代謝信號(hào)，還可傳導(dǎo)骨分解代謝信號(hào)，這取決于年齡、負(fù)荷狀態(tài)，甚至其位置(骨膜或骨內(nèi)膜表面)[15]。CX43缺陷會(huì)降低力學(xué)負(fù)荷刺激引起的合成代謝效應(yīng)，還會(huì)鈍化去負(fù)荷甚至高齡引起的骨丟失[16-17]。 在CX43 fl/fl、Dermol-Cre小鼠軟骨母細(xì)胞中，缺乏CX43可致使全身骨骼骨礦物質(zhì)密度降低和皮質(zhì)骨變薄[9]；早期成骨細(xì)胞CX43缺陷只表現(xiàn)出骨體積、骨量和骨細(xì)胞數(shù)目輕微減少[12]。去除成熟骨細(xì)胞和成骨細(xì)胞中的CX43所導(dǎo)致的骨礦物質(zhì)密度變化幾乎不能用雙能X線骨密度測(cè)定法區(qū)別，而利用顯微CT僅檢測(cè)到股骨皮質(zhì)密度輕度降低[18-19]。

3骨組織中CX43信號(hào)轉(zhuǎn)導(dǎo)分子機(jī)制

包埋于骨基質(zhì)中的骨細(xì)胞、成骨細(xì)胞和破骨細(xì)胞經(jīng)CX43縫隙連接通道來協(xié)調(diào)相互間的功能，使骨骼對(duì)刺激產(chǎn)生精確的成骨和破骨反應(yīng)[20]。目前關(guān)于CX43調(diào)節(jié)成骨細(xì)胞分化的具體分子機(jī)制尚未明確。Niger等[21-22]研究證實(shí)，有絲分裂原激活蛋白激酶(MAPK)/細(xì)胞外調(diào)節(jié)蛋白激酶(ERK)及蛋白激酶C(PKC)信號(hào)轉(zhuǎn)導(dǎo)通路位于CX43下游，CX43高表達(dá)可激活MAPK/ERK及PKC，從而促進(jìn)成骨細(xì)胞分化相關(guān)基因的轉(zhuǎn)錄；反之，則抑制MAPK/ERK及PKC信號(hào)轉(zhuǎn)導(dǎo)通路，成骨相關(guān)基因轉(zhuǎn)錄減少。也有研究[23]認(rèn)為，CX43信號(hào)轉(zhuǎn)導(dǎo)與Wnt經(jīng)典信號(hào)轉(zhuǎn)導(dǎo)通路相關(guān)，其可促進(jìn)β-連環(huán)蛋白(β-catenin)累積、激活成骨分化和增加骨礦化。Wnt經(jīng)典信號(hào)轉(zhuǎn)導(dǎo)通路的激活又可促進(jìn)CX43表達(dá)[24]。

3.1CX43與骨合成代謝信號(hào)轉(zhuǎn)導(dǎo)通路

CX43調(diào)控成骨細(xì)胞分化和基因表達(dá)與轉(zhuǎn)錄因子Runx2和Osterix(Sp7)有關(guān)，而這兩個(gè)轉(zhuǎn)錄因子是骨形成的主要調(diào)控因子。在MC3T3-E1成骨細(xì)胞中，CX43過表達(dá)可增強(qiáng)Runx2報(bào)告基因的轉(zhuǎn)錄活性；相反，利用siRNA干擾CX43表達(dá)，Runx2依賴性轉(zhuǎn)錄則受到抑制[25]。CX43主要通過調(diào)節(jié)其下游PKC家族δ亞型(PKCδ)和ERK來調(diào)節(jié)Runx2的轉(zhuǎn)錄活性，在PKCδ轉(zhuǎn)移至細(xì)胞核內(nèi)之前，其與CX43的C末端結(jié)合，調(diào)節(jié)Runx2的轉(zhuǎn)錄[26-27]。當(dāng)用藥物抑制細(xì)胞間縫隙連接通訊或低密度培養(yǎng)細(xì)胞時(shí)，CX43對(duì)Runx2的作用減弱甚至消失；高密度培養(yǎng)時(shí)CX43才對(duì)Runx2發(fā)揮作用[22]。因此，在這一調(diào)節(jié)過程中，CX43是作為經(jīng)典縫隙連接通道而非其他功能如半通道發(fā)揮作用的。此外，成骨細(xì)胞中的Sp7也是CX43下游信號(hào)轉(zhuǎn)導(dǎo)通路的靶蛋白。在cKOTW2小鼠模型中，3月齡小鼠股骨中的Sp7 mRNA較對(duì)照組降低40%[9]。這一現(xiàn)象是繼發(fā)于Runx2轉(zhuǎn)錄活性降低還是CX43直接調(diào)節(jié)Sp7所致，至今仍不明了。

GJA1基因功能獲得性突變或功能缺失性突變可影響斑馬魚鰭片的長短，分別引起長鰭和短鰭表型[28-29]。進(jìn)一步研究表明，CX43可調(diào)節(jié)影響鰭片生長的關(guān)鍵分泌因子腦信號(hào)蛋白3d(Sema 3d)的表達(dá)，Sema 3d再經(jīng)叢蛋白A3(Plexin A3)和神經(jīng)纖毛蛋白-2a(NRP-2a)轉(zhuǎn)導(dǎo)信號(hào)，依次調(diào)控關(guān)節(jié)形成和骨細(xì)胞增殖，最終影響鰭長和骨形態(tài)發(fā)生[30]。這表明在鰭片生長過程中，Sema 3d是CX43下游的關(guān)鍵靶蛋白。

3.2CX43調(diào)控骨細(xì)胞凋亡和存活

在多種骨代謝性疾病中，骨細(xì)胞凋亡增多[31]。骨細(xì)胞凋亡增多可損害骨細(xì)胞對(duì)力學(xué)刺激的反應(yīng)和細(xì)胞間信號(hào)轉(zhuǎn)導(dǎo)功能，導(dǎo)致骨吸收增加和骨質(zhì)量降低[32]，以及破骨細(xì)胞形成增多，進(jìn)而引起皮質(zhì)骨重吸收增多。

在cKOhOC老鼠模型中，應(yīng)用原位末端標(biāo)記法檢測(cè)股骨干皮質(zhì)骨中骨細(xì)胞的凋亡情況，結(jié)果顯示模型組凋亡骨細(xì)胞陽性率是對(duì)照組的2倍，空骨陷窩數(shù)是對(duì)照組的6倍[8]。應(yīng)用shRNA敲除MLOY4骨細(xì)胞樣細(xì)胞的GJA1基因后，細(xì)胞生存能力降低，同時(shí)破骨細(xì)胞分化因子如RANKL增多，OPG表達(dá)下降[8,33]，破骨細(xì)胞形成增多。

研究[19,33]表明，甲狀旁腺激素(PTH)和雙膦酸鹽化合物發(fā)揮抗凋亡作用時(shí)都需要CX43的參與；CX43的C末端可與β-抑制蛋白結(jié)合，有效地將β-抑制蛋白與甲狀旁腺激素受體(PTHR)1隔開，使得PTHR1可以持續(xù)發(fā)揮作用，同時(shí)細(xì)胞內(nèi)環(huán)磷酸腺苷(cAMP)積累，從而增強(qiáng)骨細(xì)胞存活能力。雙膦酸鹽化合物是骨重吸收的強(qiáng)烈抑制劑，其主要藥理作用是抑制破骨細(xì)胞的功能和存活[34]。研究發(fā)現(xiàn)，雙膦酸鹽化合物可與細(xì)胞表面的磷酸酶結(jié)合[35-36]，磷酸酶再作用于CX43，引起CX43半通道開放，接觸激活Src/ERK信號(hào)級(jí)聯(lián)，抑制促凋亡蛋白Bad，激活CAAT/增強(qiáng)子結(jié)合蛋白β(C/EBPβ)的抗凋亡作用，從而抑制細(xì)胞凋亡，促進(jìn)成骨細(xì)胞和骨細(xì)胞存活[19,37]。

3.3CX43與機(jī)械力傳導(dǎo)

機(jī)械應(yīng)力是調(diào)節(jié)骨合成和分解代謝的重要因素。利用剪切力刺激體外活骨可產(chǎn)生 CX43依賴性鈣波[38-39]，提示在骨細(xì)胞通過縫隙連接傳導(dǎo)機(jī)械力信號(hào)時(shí)，鈣離子或鈣信號(hào)效應(yīng)器(如三磷酸肌醇)可能作為第二信使發(fā)揮作用。在MLO-Y4骨樣細(xì)胞中，流體剪切應(yīng)力可使α5β1-整合素磷酸化、構(gòu)象改變，然后與CX43的C末端物理性接觸，使CX43半通道開放，前列腺素E2(PGE2)由半通道釋放，PGE2信號(hào)通過其同源的EP2/4受體引起磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(AKT)通路活化和cAMP累積[40],促進(jìn)骨細(xì)胞存活。而cAMP/PKA和PI3K/AKT通路都匯聚至β-catenin[41]，從而活化Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路，促進(jìn)β-catenin累積，激活成骨分化，增加骨的礦化。

綜上所述，CX43在調(diào)節(jié)骨細(xì)胞功能、信號(hào)轉(zhuǎn)導(dǎo)、基因表達(dá)、骨細(xì)胞存活和凋亡以及機(jī)械傳導(dǎo)方面均發(fā)揮著至關(guān)重要的作用。CX43不僅可作為縫隙連接蛋白發(fā)揮作用(CX43高表達(dá)，可激活MAPK/ERK及PKCδ，促進(jìn)成骨細(xì)胞分化相關(guān)基因如Runx2、Sp7的轉(zhuǎn)錄)，還可作為半通道發(fā)揮作用(CX43半通道與其他蛋白如磷酸酶、整合素等相互作用，使其開放，釋放PGE2，從而活化Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路，促進(jìn)β-catenin的累積，激活成骨分化，增加骨的礦化)。相反，CX43的低表達(dá)可使成骨細(xì)胞分化延遲，進(jìn)而影響骨骼發(fā)育與塑性，最終導(dǎo)致骨骼畸形。然而，CX43發(fā)揮功能的途徑多而復(fù)雜，目前有關(guān)CX43調(diào)節(jié)成骨細(xì)胞分化的具體分子機(jī)制尚不十分清楚，通過CX43傳遞信息的第二信使與細(xì)胞功能之間的相互關(guān)系也需進(jìn)一步明確。明確CX43調(diào)節(jié)骨內(nèi)穩(wěn)態(tài)及對(duì)機(jī)械刺激、激素信號(hào)反應(yīng)的具體分子機(jī)制，可為某些骨骼疾病提供新的分子標(biāo)志物，為其治療提供新靶點(diǎn)。

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(收稿：2015-06-03; 修回：2015-10-28)

(本文編輯：盧千語)

DOI：10.3969/j.issn.1673-7083.2016.01.010

通信作者：薄占東E-mail： zdb71718@163.com

基金項(xiàng)目：國家自然科學(xué)基金(81460348)、廣西教育廳一般項(xiàng)目(2013YB314)