陳兆煜， 袁 喬， 談 智

(中山大學(xué) 1中山醫(yī)學(xué)院臨床醫(yī)學(xué)系， 2光華口腔醫(yī)學(xué)院口腔醫(yī)學(xué)系, 3中山醫(yī)學(xué)院生理教研室，廣東 廣州 510080)

Caveolae-caveolin-1-PTRF/cavin-1系統(tǒng)與血管平滑肌細(xì)胞遷移：一種可能的機(jī)制*

陳兆煜1， 袁 喬2， 談 智3△

(中山大學(xué)1中山醫(yī)學(xué)院臨床醫(yī)學(xué)系，2光華口腔醫(yī)學(xué)院口腔醫(yī)學(xué)系,3中山醫(yī)學(xué)院生理教研室，廣東 廣州 510080)

動(dòng)脈粥樣硬化性疾病(如冠狀動(dòng)脈粥樣硬化和腦動(dòng)脈粥樣硬化)仍是全球范圍內(nèi)引起死亡的主要原因[1]。在動(dòng)脈粥樣硬化的演變過程中，血管壁首先表現(xiàn)為動(dòng)脈內(nèi)膜的增厚，內(nèi)膜增厚可以由于年齡和應(yīng)對(duì)逐漸升高的血壓導(dǎo)致內(nèi)膜損傷而產(chǎn)生[2]。在內(nèi)膜損傷后，多種細(xì)胞包括內(nèi)皮細(xì)胞、血小板和炎癥細(xì)胞釋放多種介質(zhì)(如生長因子和細(xì)胞因子)能夠激活血管平滑肌細(xì)胞(vascular smooth muscle cells, VSMCs)和單核-巨噬細(xì)胞，引起VSMCs從血管肌層遷移到內(nèi)膜層并且在內(nèi)膜層增殖和產(chǎn)生大量基質(zhì)和吸引單核巨噬細(xì)胞趨化、遷移至內(nèi)膜層[3-4]。內(nèi)膜增厚與內(nèi)膜層中VSMCs數(shù)目的增多和VSMCs相關(guān)的細(xì)胞外基質(zhì)的增加有關(guān)[2]。遷移到內(nèi)膜的VSMCs和巨噬細(xì)胞吞噬經(jīng)破損內(nèi)膜進(jìn)入的脂質(zhì)，分別成為VSMCs來源的泡沫細(xì)胞和巨噬細(xì)胞來源的泡沫細(xì)胞，從而使脂質(zhì)積聚于血管[4]。VSMCs脂質(zhì)聚積會(huì)導(dǎo)致其表面纖維蛋白和I型膠原下降[5]，這是引起動(dòng)脈粥樣硬化斑塊破裂，從而導(dǎo)致猝死的主要原因[6]。平滑肌細(xì)胞從血管肌層遷移到內(nèi)膜層是動(dòng)脈粥樣硬化演變過程中的主要因素[7]，因此認(rèn)識(shí)這一遷移過程的機(jī)制顯得尤為重要。

1 微囊(caveolae)與VSMCs遷移

細(xì)胞遷移過程中涉及多種細(xì)胞結(jié)構(gòu)及細(xì)胞結(jié)構(gòu)蛋白[8]，微囊也稱作小凹或陷窩是一類50～100 nm大小的細(xì)胞膜上呈長頸瓶樣(flask-shaped)內(nèi)陷入胞漿的細(xì)胞膜亞單位結(jié)構(gòu)[9-10]，存在于多種細(xì)胞的細(xì)胞膜上，但大量分布于內(nèi)皮細(xì)胞、心肌細(xì)胞、平滑肌細(xì)胞、上皮細(xì)胞、I型肺泡細(xì)胞等細(xì)胞的細(xì)胞膜。微囊結(jié)構(gòu)中富含糖基磷脂酰肌醇(glycosyl phosphatidylinositol, GPI)錨定蛋白、膽固醇和鞘糖脂，這種特殊的脂質(zhì)結(jié)構(gòu)在洗滌劑中難溶解,因而可以根據(jù)這個(gè)特性來提純微囊結(jié)構(gòu)[11]。研究發(fā)現(xiàn)微囊中存在大量與細(xì)胞遷移以及增殖相關(guān)的酶和受體如Src樣激酶、表皮生長因子受體(epidermal growth factor receptor，EGFR)和血小板源性生長因子受體(platelet-derived growth factor receptor，PDGFR),調(diào)控細(xì)胞內(nèi)鈣離子穩(wěn)態(tài)的分子如胞膜鈣泵和三磷酸肌醇(inositol 1,4,5-triphosphate, IP3)受體，以及G蛋白相關(guān)的信號(hào)分子如G蛋白偶聯(lián)受體和腺苷酸環(huán)化酶[10-11]。

2 小凹蛋白1(caveolin-1,Cav-1)與VSMCs遷移

在脊椎動(dòng)物中，微囊有3種跨膜結(jié)構(gòu)蛋白,分別是Cav-1、Cav-2和Cav-3。Caveolins主要存在于細(xì)胞膜上，也有小部分存在于高爾基復(fù)合體、內(nèi)質(zhì)網(wǎng)、囊泡和胞漿中。這3種caveolins含量最高的是Cav-1(又稱VIP21),主要分布于終末分化細(xì)胞如脂肪細(xì)胞、內(nèi)皮細(xì)胞、平滑肌細(xì)胞和I型肺泡細(xì)胞。Cav-2主要與Cav-1共表達(dá)，需要Cav-1提供靶位點(diǎn)才能結(jié)合在胞膜上。Cav-3的氨基酸序列與Cav-1較為相似，有組織特異性，主要表達(dá)在平滑肌細(xì)胞、骨骼肌細(xì)胞和心肌細(xì)胞[12]。

Cav-1是微囊形呈的重要的結(jié)構(gòu)，其表達(dá)與微囊的形成呈直接的平行關(guān)系，而當(dāng)微囊在細(xì)胞轉(zhuǎn)化過程中消失時(shí)，Cav-1的mRNA以及蛋白的表達(dá)停止或者減少。在敲除Cav-1的細(xì)胞株上，通過轉(zhuǎn)染富含Cav-1的質(zhì)粒使得上述細(xì)胞株的細(xì)胞膜上的微囊重新形成，并且微囊在細(xì)胞膜上所占的面積比例大大提高，但何種因素調(diào)控Cav-1聚集形成微囊仍未明確[11]。

已有研究提出Cav-1通過調(diào)控信號(hào)分子的極化在細(xì)胞的遷移中起重要的作用[9,13-14]。Cav-1和層黏連蛋白(lamin)與細(xì)胞骨架成分β-actin或γ-actin相連，同時(shí)也通過細(xì)絲蛋白(filamin)與整合素相聯(lián)系[15]。Caveolae和Cav-1在內(nèi)皮細(xì)胞遷移過程中顯示極化分布現(xiàn)象[16-17]。然而關(guān)于Cav-1在細(xì)胞的遷移中的調(diào)控作用存在著爭議，有研究表明Cav-1表達(dá)量的增加能夠抑制細(xì)胞遷移[18]。Cav-1缺陷細(xì)胞正常的極化現(xiàn)象和定向遷移能力消失，RhoA的表達(dá)下降而Rac和Cdc42的表達(dá)上升抑制細(xì)胞遷移所需要的結(jié)構(gòu)如黏著斑(focal adhesions, FAs)、絲狀偽足等成熟，從而抑制細(xì)胞的遷移[14]。RhoA、Rac1和Cdc42是Rho GTP激酶家族的3個(gè)重要成員，能夠調(diào)控細(xì)胞骨架的重構(gòu)FAs的形成和重新分布,肌動(dòng)蛋白的聚集,絲狀偽足、板狀偽足、細(xì)胞膜皺褶和調(diào)節(jié)應(yīng)力纖維的形成以及細(xì)胞的收縮性，從而實(shí)現(xiàn)對(duì)細(xì)胞遷移的調(diào)控[8,19]，阻斷Rho GTP激酶的表達(dá)，則VSMCs遷移的表型改變消失[20]，提示Cav-1通過調(diào)節(jié)Rho GTP酶來調(diào)控細(xì)胞的遷移。EGFR和PDGFR與Cav-1共分布于微囊中，Cav-1分別通過與EGFR和PDGFR結(jié)合，抑制EGFR和PDGFR的自我磷酸化，從而抑制EGFR和PDGFR的功能[21]，而EGFR信號(hào)通路和PDGFR信號(hào)通路與細(xì)胞的遷移和增殖密切相關(guān)，也提示了Cav-1對(duì)細(xì)胞的遷移起負(fù)性調(diào)控作用。與此相反，有研究表明Cav-1是負(fù)調(diào)控因子，恢復(fù)MTLn3細(xì)胞的Cav-1的表達(dá)，可以減少M(fèi)TLn3細(xì)胞板狀偽足的形成和趨化性遷移[22]，敲除Cav-1基因能增加內(nèi)皮細(xì)胞的定向遷移[23]。因此Cav-1在調(diào)控細(xì)胞遷移的作用機(jī)制仍不明確。

3 聚合酶I和轉(zhuǎn)錄釋放因子(polymerase I and transcript release factor, PTRF)與細(xì)胞遷移

PTRF又稱cavin-1,在過去被認(rèn)為是RNA核酸聚合酶I(RNA polymerase I，Pol I)轉(zhuǎn)錄終止作用的關(guān)鍵分子，甲狀腺轉(zhuǎn)錄因子I(thyroid transcription factor I, TTF-I)和PTRF/cavin-1是Pol I轉(zhuǎn)錄終止2個(gè)關(guān)鍵步驟的重要分子。TTF-I通過結(jié)合于rDNA下游的一個(gè)叫作“Sal box”的終止子使Pol I復(fù)合體的延伸作用中止，接著PTRF/cavin-1與Pol I 復(fù)合體中前體rRNA的3’末端結(jié)合，同時(shí)也與Pol I和TTF-I相互作用，從而使Pol I從模板中釋放出來[24-25]。隨后的研究發(fā)現(xiàn)，PTRF/cavin-1在微囊中大量存在，并且與Cav-1共分布于微囊中(位于微囊的胞漿側(cè))和細(xì)胞骨架蛋白有著密切的聯(lián)系[26-27]。過度表達(dá)PTRF/cavin-1或者下調(diào)PTRF/cavin-1的表達(dá)能引起Cav-1表達(dá)量相應(yīng)的改變，同時(shí)發(fā)現(xiàn)過度表達(dá)PTRF/cavin-1，細(xì)胞能形成明顯的細(xì)胞骨架樣的結(jié)構(gòu)，當(dāng)使微管或者肌動(dòng)蛋白絲解聚能減少PTRF/cavin-1結(jié)合于細(xì)胞膜的脂質(zhì)層，而對(duì)Cav-1沒有顯著的影響[27]。敲除PTRF/cavin-1能減少胞膜中微囊的密度，而Cav-1仍存在于細(xì)胞膜中，但不能聚集形成微囊，重新導(dǎo)入PTRF/cavin-1，發(fā)現(xiàn)微囊結(jié)構(gòu)的形成[28]。在PTRF/cavin-1基因敲除的小鼠組織細(xì)胞中，微囊結(jié)構(gòu)消失[29]。這些結(jié)果提示PTRF/cavin-1通過與細(xì)胞骨架分子相互作用在微囊的形成中起重要的作用。

在前列腺癌PC3細(xì)胞中，PTRF/cavin-1的穩(wěn)定表達(dá)可以抑制PC3細(xì)胞的遷移以及減少基質(zhì)金屬蛋白酶9(matrix metalloproteinase 9，MMP-9)的生成[30]，而在另一種不表達(dá)PTRF/cavin-1的前列腺癌細(xì)胞株LnCAP中富含MMP-9，MMP-9能夠降解細(xì)胞外基質(zhì)成分中的IV、V、VII、X和XIV型膠原以及纖連蛋白、層黏連蛋白、巢蛋白、蛋白聚糖連接蛋白、多功能蛋白聚糖[30-31]。 PTRF/cavin-1抑制PC3細(xì)胞的遷移和減少細(xì)胞外基質(zhì)的降解也與Cav-1的表達(dá)增加有關(guān)，因此抑制PC3細(xì)胞的遷移和減少細(xì)胞外基質(zhì)的降解與微囊和Cav-1也有關(guān)[30]。 在細(xì)絲環(huán)損傷小鼠動(dòng)脈后，與對(duì)照組相比，敲除MMP-9基因的小鼠VSMCs遷移能力下降，內(nèi)膜增厚不明顯[32]，這提示PTRF/cavin-1在VSMCs的遷移也存在與PC3細(xì)胞遷移的類似機(jī)制，但未見相關(guān)的報(bào)道。

4 結(jié)語

綜上所述，VSMCs遷移在動(dòng)脈粥樣硬化的發(fā)生、發(fā)展和演變的過程中起著重要的作用，而微囊在VSMCs的遷移中起著重要的作用，其結(jié)構(gòu)蛋白Cav-1在介導(dǎo)VSMCs遷移中的作用存在著爭議，目前無法明確這種爭議的原因。新近研究表明，細(xì)胞質(zhì)膜微囊結(jié)構(gòu)蛋白PTRF/cavin-1在細(xì)胞的遷移中起重要的作用，提示PTRF/cavin-1可能在Cav-1和caveolae的上游介導(dǎo)細(xì)胞的遷移。PTRF/cavin-1很有可能在介導(dǎo)VSMCs的遷移中起著重要作用，因此研究PTRF/cavin-1在介導(dǎo)VSMCs遷移的作用有可能可以闡明動(dòng)脈粥樣硬化發(fā)病的機(jī)制。

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Caveolae-caveolin-1-PTRF/cavin-1systemandVSMCmigration:apossiblemechanism

CHEN Zhao-yu1, YUAN Qiao2, TAN Zhi3

(1DepartmentofClinicalMedicine,ZhongshanSchoolofMedicine,2DepartmentofStomatologicalMedicine,GuanghuaSchoolofStomatology,3DepartmentofPhysiology,ZhongshanSchoolofMedicine,SunYat-senUniversity,Guangzhou510080,China.E-mail:tanzhi@mail.sysu.edu.cn)

The migration of vascular smooth muscle cells (VSMCs) from the media to the intima of artery plays an important role in the genesis and development of atherosclerosis. Caveolaes, the 50～100 nm membrane microdomains and flask-shaped organelles, contain several ingredients involved in the cell migration. Caveolin-1 (Cav-1), a 22-kD integral membrane protein and an important structural component of caveolae, mediates the migration of VSMCs. Polymerase I and transcript release factor (PTRF/cavin-1) co-localize with Cav-1 in caveolaes and directly interact with cytoskeleton network. PTRF/cavin-1 decreases the migration of prostate cancer PC3 cells, but without similar reports in VSMCs. Therefore, Caveolae-caveolin-1-PTRF/cavin-1 system might act as another mechanism of migration of VSMCs in the development of atherosclerosis.

動(dòng)脈粥樣硬化; 血管平滑肌細(xì)胞; 細(xì)胞遷移; 微囊; 小凹蛋白1; 聚合酶I和轉(zhuǎn)錄釋放因子

Atherosclerosis; Vascular smooth muscle cells; Cell migration; Caveolae; Caveolin-1; Polymerase I and transcript release factor

R363

A

10.3969/j.issn.1000- 4718.2013.05.034

1000- 4718(2013)05- 0957- 04

2012- 12- 11

2013- 03- 27

國家自然科學(xué)基金資助項(xiàng)目(No. 81270377);中山大學(xué)實(shí)驗(yàn)室開放基金資助項(xiàng)目(No.KF201245)

△通訊作者 Tel: 020-87332091； E-mail: tanzhi@mail.sysu.edu.cn