康海軍綜述，康剛勁審校

（西南醫(yī)科大學(xué)附屬醫(yī)院眼科，四川瀘州646000）

綜述

Wnt信號(hào)通路在晶狀體發(fā)育中的作用

康海軍綜述，康剛勁審校

（西南醫(yī)科大學(xué)附屬醫(yī)院眼科，四川瀘州646000）

Wnt信號(hào)通路；晶狀體上皮細(xì)胞；發(fā)育

在眼球的發(fā)育過(guò)程中，Wnt（wingless-type MMTV integration site family members）信號(hào)轉(zhuǎn)導(dǎo)通路起著重要作用，晶狀體上皮細(xì)胞的分化以及晶狀體的發(fā)育與Wnt信號(hào)轉(zhuǎn)導(dǎo)通路有著密切的關(guān)系[1]。在脊椎動(dòng)物晶狀體的早期發(fā)育階段，經(jīng)典Wnt信號(hào)轉(zhuǎn)導(dǎo)通路通過(guò)抑制視泡和表面外胚層的形成來(lái)調(diào)節(jié)晶狀體的發(fā)育[2-4]，隨后通過(guò)影響晶狀體上皮細(xì)胞和晶狀體纖維細(xì)胞分化調(diào)節(jié)晶狀體的發(fā)育[5-6]。非經(jīng)典的Wnt信號(hào)轉(zhuǎn)導(dǎo)通路主要在晶狀體上皮細(xì)胞延長(zhǎng)和晶狀體纖維細(xì)胞規(guī)律的排列中起重要作用[7-8]。本文結(jié)合目前該領(lǐng)域研究進(jìn)展就Wnt信號(hào)轉(zhuǎn)導(dǎo)通路在晶狀體的發(fā)育分化中的作用進(jìn)行詳細(xì)綜述。

1 Wnt信號(hào)通路

最初在果蠅胚胎發(fā)育的研究中發(fā)現(xiàn)了無(wú)翅基因（wingless，wg），wg基因突變將導(dǎo)致無(wú)翅。隨后在用小鼠乳頭瘤病毒（MMTV）誘導(dǎo)小鼠產(chǎn)生乳腺癌的過(guò)程中，發(fā)現(xiàn)MMTV常常固定整合于宿主染色體的特定位置，命名為整合基因（integration，int）。隨著研究的不斷深入，證明小鼠的int基因就是果蠅的wg基因，現(xiàn)已將相關(guān)的二類(lèi)基因統(tǒng)一命名為wnt基因家族。wnt基因編碼的Wnt蛋白，可啟動(dòng)細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)途徑，傳遞生長(zhǎng)刺激信號(hào)，參與不同的發(fā)育機(jī)制，如細(xì)胞分化、移行，以及決定細(xì)胞命運(yùn)的增殖等，因其啟動(dòng)蛋白為Wnt蛋白，故命名為Wnt信號(hào)轉(zhuǎn)導(dǎo)通路。目前對(duì)Wnt信號(hào)轉(zhuǎn)導(dǎo)通路研究較為清楚的有3條途徑：根據(jù)LRP5/6是否參與信號(hào)轉(zhuǎn)導(dǎo)分為經(jīng)典Wnt/β-catenin信號(hào)通路（canonical pathway）和非經(jīng)典途徑，Wnt/細(xì)胞極化通路（the planar cell polarity pathway，Wnt/PCP）以及Wnt/Ca2+通路。

2 Wnt信號(hào)轉(zhuǎn)導(dǎo)通路對(duì)晶狀體發(fā)育的調(diào)控

一個(gè)世紀(jì)以來(lái)，通過(guò)對(duì)晶狀體發(fā)育的研究，讓研究者能夠從基因、生化水平研究胚胎誘導(dǎo)、細(xì)胞調(diào)控、信號(hào)轉(zhuǎn)導(dǎo)等過(guò)程來(lái)研究眼發(fā)育進(jìn)程與發(fā)育機(jī)制。晶狀體的形成是一系列信號(hào)因子對(duì)預(yù)定晶狀體外胚層細(xì)胞連續(xù)誘導(dǎo)和作用的結(jié)果。Wnt信號(hào)轉(zhuǎn)導(dǎo)通路和FGF信號(hào)轉(zhuǎn)導(dǎo)通路、BMP/TGF-β信號(hào)轉(zhuǎn)導(dǎo)通路一樣在晶狀體和視網(wǎng)膜發(fā)育中起著關(guān)鍵作用[9]。

2.1 Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路

現(xiàn)已證實(shí)Wnt/β-catenin信號(hào)通路對(duì)胚胎發(fā)育和成體組織中細(xì)胞命運(yùn)的調(diào)控起著關(guān)鍵作用[10]，當(dāng)然包括晶狀體上皮和晶狀體纖維細(xì)胞的發(fā)育分化。當(dāng)細(xì)胞分泌的Wnt蛋白與人卷曲/低密度脂蛋白受體相關(guān)蛋白6（frizzled/low-density lipoprotein receptor-related protein 6，F(xiàn)ZD/LRP 6）受體結(jié)合后，可磷酸化散亂蛋白（dishevelled，Dsh），磷酸化的Dsh抑制β-catenin與Axin-APC-GSK-3β形成降解復(fù)合物，使βcatenin在細(xì)胞質(zhì)內(nèi)積累，并最終結(jié)合核蛋白Nup62、Nup153和RanBP2[11]，促進(jìn)其核轉(zhuǎn)移然后移至細(xì)胞核內(nèi)與TCF/LEF相結(jié)合并調(diào)控下游cyclinD1、c-myc等靶基因的轉(zhuǎn)錄和表達(dá)，從而調(diào)控細(xì)胞增殖和分化[12]。Carpenter AC等[13]通過(guò)小鼠的研究發(fā)現(xiàn)在表面外胚層的晶狀體基板表達(dá)12種Wnt配體,在晶狀體發(fā)育早期階段，Wnt/β-catenin信號(hào)通路抑制外胚層的發(fā)育調(diào)控晶狀體發(fā)育。抑制E9.5區(qū)的β-catenin會(huì)導(dǎo)致晶狀體的異位與晶狀體纖維細(xì)胞間的連接異常[2-3]。通過(guò)實(shí)驗(yàn)去除整個(gè)晶狀體中的β-catenin則會(huì)導(dǎo)致晶狀體上皮細(xì)胞內(nèi)鈣黏蛋白和Pax6表達(dá)的下調(diào)，同時(shí)使晶狀體上皮細(xì)胞的周期停止在G1-S期，但是已經(jīng)分化的細(xì)胞并不會(huì)受到影響[14]。同時(shí)Pax6也會(huì)直接調(diào)控Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路中相關(guān)成分的表達(dá)，比如Bcl9l,Ccnd1,Dkk3,Lrp6和Wnt7a[15-16]；Pax6與Wnt/β-catenin也可以共同調(diào)控α1-laminin基因的表達(dá)來(lái)控制晶狀體細(xì)胞骨架的形成[17]。在晶狀體隨后的發(fā)育階段，如果沒(méi)有β-catenin的調(diào)控將引起晶狀體纖維細(xì)胞的不規(guī)則延長(zhǎng)、β-晶狀體蛋白表達(dá)的下降以及細(xì)胞間不正常的連接[18]。Hayashi等[14]用RT-PCR技術(shù)在晶狀體發(fā)育的不同時(shí)間點(diǎn)收集虹膜組織，發(fā)現(xiàn)整個(gè)過(guò)程中Wnt 2b和Fz4表達(dá)上調(diào),特別是第8、12d時(shí)在虹膜組織上調(diào)更為顯著。用含有FGF2和Wnt信號(hào)通路阻斷劑（DKK1、SFRP1）的培養(yǎng)基研究晶狀體的發(fā)育時(shí)，發(fā)現(xiàn)晶狀體發(fā)育速度顯著下降；但是用含有FGF2和Wnt信號(hào)通路蛋白（WNT3A）培養(yǎng)基則會(huì)出現(xiàn)截然不同的效果，甚至可以從虹膜腹側(cè)部觀察到大量的晶狀體在虹膜背部重建。這項(xiàng)研究結(jié)果顯示由FGF2觸發(fā)的Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路，對(duì)虹膜背部的晶狀體發(fā)育起著關(guān)鍵作用。同時(shí)由FGF2觸發(fā)的Wnt/β-catenin信號(hào)轉(zhuǎn)導(dǎo)通路也對(duì)晶狀體蛋白表達(dá)起著促進(jìn)作用[19]。

2.2 Wnt/細(xì)胞極化通路

Wnt信號(hào)激活Rho或者Rac，這些GTP酶通過(guò)Rho-associated protein kinase（ROCK）調(diào)控細(xì)胞骨架變化或者通過(guò)Jun N-terminal kinase（JNK）調(diào)控基因的轉(zhuǎn)錄[20],稱(chēng)為Wnt/細(xì)胞極化通路（the planar cell polarity pathway，Wnt/PCP）。晶狀體纖維的延長(zhǎng)以及規(guī)則的排列需要Wnt/PCP的準(zhǔn)確調(diào)控[7,21]，錯(cuò)誤的晶狀體上皮細(xì)胞遷移運(yùn)動(dòng)可導(dǎo)致晶狀體形狀的異常和晶狀體縫的異常。研究發(fā)現(xiàn)在晶狀體中PCP相關(guān)蛋白不對(duì)稱(chēng)地分布在頂端晶狀體纖維表面[21]：Fz6和Vangl2主要分布在晶狀體前面，Pk1在晶狀體后表面細(xì)胞和一些基質(zhì)較少的細(xì)胞中占據(jù)優(yōu)勢(shì)，Dvl2和Dvl3則在每個(gè)細(xì)胞中都有分布。這種不均勻的分布提示W(wǎng)nt/PCP信號(hào)轉(zhuǎn)導(dǎo)通路在晶狀體纖維規(guī)則排列起著重要作用。其中FGF通過(guò)上調(diào)Wnt/PCP信號(hào)通路相關(guān)蛋白Fz3、Fz6、Dvl2、Dvl3來(lái)調(diào)控晶狀體纖維的分化；在添加Sfrp1或IWP-2的FGF培養(yǎng)基中體外培養(yǎng)晶狀體纖維可以發(fā)現(xiàn)晶狀體纖維的延長(zhǎng)被抑制、晶狀體絲蛋白和β-晶狀體蛋白的表達(dá)下降；同時(shí)Wnt/PCP信號(hào)通路相關(guān)蛋白表達(dá)也下降，Dvl2的激活被抑制[22]。用Van Gogh-like 2（Vangl2）和Celsr1基因突變（PCP核心基因）的小鼠會(huì)導(dǎo)致晶狀體不規(guī)則排列和晶狀體縫的形成[23]。在小鼠中過(guò)量表達(dá)的Sfrp2（Wnt/PCP調(diào)節(jié)蛋白）通過(guò)下調(diào)Wnt/PCP信號(hào)轉(zhuǎn)導(dǎo)通路中相關(guān)組成分子的表達(dá)/活化，打亂規(guī)則排列的晶狀體纖維[8,21]。Wnt/PCP信號(hào)通路激活Rho,Rac和Cdc42等GTP蛋白激酶后，這些激酶再調(diào)節(jié)細(xì)胞骨架變化來(lái)決定細(xì)胞的遷移運(yùn)動(dòng)，同時(shí)激活JNK以調(diào)控一系列轉(zhuǎn)錄因子的表達(dá)[24]。這些研究揭示W(wǎng)nt/PCP信號(hào)轉(zhuǎn)導(dǎo)通路對(duì)晶狀體發(fā)育的重要作用主要表現(xiàn)在對(duì)晶狀體纖維規(guī)則排列和遷移的調(diào)控。

2.3 Wnt/Ca2+信號(hào)轉(zhuǎn)導(dǎo)通路

鈣離子是細(xì)胞內(nèi)功能最為廣泛的第二信使之一，幾乎在所有的細(xì)胞生理過(guò)程中發(fā)揮作用。從細(xì)胞的分化到程序性凋亡、從興奮收縮耦聯(lián)到興奮釋放耦聯(lián)、從基因表達(dá)到突觸可塑性，鈣離子都在其中發(fā)揮著作用[25-26]，Wnt蛋白與受體結(jié)合后導(dǎo)致細(xì)胞內(nèi)Ca2+濃度增加，從而激活CaM依賴(lài)性蛋白激酶II（CAMKII）和蛋白激酶C（PKC）來(lái)調(diào)控細(xì)胞的遷移以及分化[20,27]；在胚胎發(fā)育時(shí)期，細(xì)胞的遷移需要細(xì)胞骨架蛋白和整合蛋白的共同調(diào)節(jié)，Wnt/Ca2+信號(hào)通路通過(guò)調(diào)控這些蛋白的表達(dá)從而控制細(xì)胞的遷移。Wnt/Ca2+的激活需要Wnt4a、Wnt5a的參與[28-29]。Wnt5a在神經(jīng)脊細(xì)胞高度表達(dá)，從而對(duì)晶狀體的早期發(fā)育起著調(diào)控作用。Wnt5a與Frizzled蛋白受體結(jié)合后，通過(guò)使磷脂酰肌醇4,5-二磷酸（PIP2）水解而影響細(xì)胞內(nèi)Ca2+水平。PIP2水解生成甘油二脂（DG）和肌醇1,4,5-三磷脂（IP3），其中DG可使PKC活化，IP3可作用于細(xì)胞內(nèi)質(zhì)網(wǎng)IP3受體，引起Ca2+從內(nèi)質(zhì)網(wǎng)中釋放。細(xì)胞內(nèi)升高的Ca2+反過(guò)來(lái)激活PKC蛋白激酶Ⅱ[30-31]。PKC通過(guò)與細(xì)胞骨架蛋白的相互作用來(lái)調(diào)節(jié)細(xì)胞的粘附和遷移[32]。

2.4 三條信號(hào)轉(zhuǎn)導(dǎo)通路間的相互作用與關(guān)系

Wnt與Frizzled配體結(jié)合后，激活Wnt信號(hào)通路，其中Wnt/β-catenin信號(hào)通路調(diào)控細(xì)胞增殖和分化[12]，不需要LRP5/6參與的PCP信號(hào)通路直接參與對(duì)晶狀體纖維發(fā)育過(guò)程中的規(guī)則排列和遷移的調(diào)控。Dkkl與LRP6特異性結(jié)合能阻斷Wnt/β-catenin信號(hào)通路,但不能阻斷PCP信號(hào)[33],表明其功能也不依賴(lài)于Wnt/β-catenin信號(hào)通路，Wnt/β-catenin信號(hào)通路與Wnt/PCP在控制晶狀體的發(fā)育調(diào)節(jié)中起著不同作用，相互獨(dú)立；非經(jīng)典Wnt/Ca2+信號(hào)通路能阻斷經(jīng)典Wnt信號(hào)通路,它可以促使細(xì)胞內(nèi)鈣的釋放并激活CamKII和PKC兩種激酶調(diào)節(jié)細(xì)胞的粘附和遷移[34]。只有三條信號(hào)通路在空間、時(shí)間上準(zhǔn)確調(diào)控才能保證形成良好的視覺(jué)。

3 展望

在晶狀體的發(fā)育過(guò)程中，細(xì)胞的增殖和分化受到精確的調(diào)控，這個(gè)過(guò)程的調(diào)節(jié)受到基質(zhì)成分及粘附蛋白的表達(dá)和信號(hào)轉(zhuǎn)導(dǎo)通路的影響。Wnt/β-catenin信號(hào)通路和非經(jīng)典Wnt/ Ca2+信號(hào)通路主要通過(guò)調(diào)控晶狀體發(fā)育相關(guān)基因的表達(dá)來(lái)調(diào)控晶狀體的早期發(fā)育，Wnt/PCP信號(hào)轉(zhuǎn)導(dǎo)通路則對(duì)晶狀體纖維細(xì)胞發(fā)育過(guò)程中的規(guī)則排列和遷移的調(diào)控起著重要作用。Wnt信號(hào)通路對(duì)晶狀體每一步發(fā)育過(guò)程的精確調(diào)控是形成良好視覺(jué)質(zhì)量的前提。盡管人們對(duì)此進(jìn)行了大量的研究,但此通路中各種蛋白相互作用的分子機(jī)制仍未完全闡明。因此,進(jìn)一步研究Wnt信號(hào)轉(zhuǎn)導(dǎo)通路之間的相互作用與關(guān)系，以及各基因及其表達(dá)蛋白的相互作用,對(duì)于從更深層次闡明晶狀體的發(fā)育有著重大意義，并有望成為研究晶狀體發(fā)育的新方向。

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（2016-04-05收稿）

R776.1

A

10.3969/j.issn.1000-2669.2016.06.023

康海軍（1988-），男，碩士生。E-mail:705094775@qq.com