S100A4蛋白的生物學(xué)功能研究進展

尚禹東，楊艷玲，趙海平，李春義

(中國農(nóng)業(yè)科學(xué)院特產(chǎn)研究所特種動物分子生物學(xué)省部共建國家重點實驗室，長春130112)

1　引言

S100家族是一類Ca2+結(jié)合蛋白，通過與其他目標蛋白的相互作用來調(diào)節(jié)細胞各種功能，其生物學(xué)活性的發(fā)揮需要借助Ca2+依賴性所引起的構(gòu)象改變，在此過程中，Ca2+對于S100的活化是必需的[1]。因此，S100蛋白被認為是一種Ca2+傳感器蛋白，通過Ca2+信號轉(zhuǎn)導(dǎo)途徑，在細胞通訊聯(lián)系、增殖、生長、分化、收縮、基因表達、分泌及凋亡中發(fā)揮重要作用[2]。S100A4是S100蛋白家族中的一員，人類的S100A4基因定位于染色體的1q21，而小鼠和大鼠的基因則分別位于3f3和2q34[3]。S100A4是由101個氨基酸組成的多肽，在哺乳動物中高度保守，分子量為115ku[1]。分子當(dāng)中的EF臂型結(jié)構(gòu)域具有結(jié)合Ca2+的能力，當(dāng)其蛋白部分與Ca2+結(jié)合后，S100A4蛋白構(gòu)象發(fā)生改變，暴露出其與靶蛋白結(jié)合的位點，進而通過相應(yīng)的靶蛋白發(fā)揮其生物學(xué)效應(yīng)[4]。與其他S100家族蛋白不同的是，S100A4與目標分子發(fā)生相互作用時，可表現(xiàn)為Ca2+依賴性和非依賴性2種不同的結(jié)合方式[1]。

2　S100A4的生物學(xué)活性

S100A4兼具細胞內(nèi)和細胞外活性，并具有調(diào)節(jié)某些信號通路蛋白的功能[5]，其介導(dǎo)的Ca2+信號通路在細胞的增殖、分化、粘附、遷移、形態(tài)發(fā)生以及凋亡等生理過程中起到了決定性的作用；S100A4通常與絲狀肌動蛋白共聚，并與非肌性肌球蛋白原和非肌性肌球蛋白相互作用[6，7]；S100A4還可與細胞骨架蛋白直接結(jié)合，或通過調(diào)節(jié)鈣黏蛋白/連環(huán)蛋白、CD44/細胞骨架復(fù)合物的細胞骨架連接，活化細胞外基質(zhì)水解酶，來改變細胞動力學(xué)模式和遷移能力，參與細胞運動；在哺乳動物發(fā)育期間，它廣泛存在于胚胎巨噬細胞和分化的間充質(zhì)組織當(dāng)中[8]；S100A4的高表達還發(fā)生在細胞類型從上皮向間充質(zhì)轉(zhuǎn)變的過程中[9，10]；S100A4在腦損傷、心肌損傷、類風(fēng)濕性關(guān)節(jié)炎和組織纖維化等病變組織中也存在過度表達情況[11]。

2.1　S100A4與腫瘤發(fā)生、發(fā)展的關(guān)系

S100A4蛋白的表達與細胞的異常增生及腫瘤的發(fā)生、發(fā)展有關(guān)，其在正常人體的肺、泌尿系統(tǒng)、乳腺、甲狀腺、胰腺及結(jié)腸等組織細胞中均不表達，而在乳腺癌、胃癌、結(jié)直腸癌、胰腺癌、甲狀腺癌、膀胱癌、前列腺癌、口腔上皮細胞癌、非小細胞肺癌、神經(jīng)膠質(zhì)瘤細胞以及多種基質(zhì)細胞(如成纖維細胞、淋巴細胞和巨噬細胞等)中高度表達，在惡性腫瘤中比良性腫瘤中表達量更高[12～15]。S100A4也可在體外培養(yǎng)的相關(guān)細胞系中正常表達，并可存在于腫瘤細胞間充質(zhì)液中[16]。腫瘤患者的組織學(xué)檢查顯示，癌變組織中的S100A4表達水平要遠高于鄰近的正常組織[17]。腫瘤間充質(zhì)中S100A4的表達同樣會使腫瘤細胞發(fā)生遷移和侵入，在S100A4基因敲除的小鼠體內(nèi)，轉(zhuǎn)移性癌細胞并沒有發(fā)生遷移，而將表達S100A4的腫瘤間充質(zhì)細胞注射入腫瘤組織內(nèi)，癌細胞的轉(zhuǎn)移能力有所恢復(fù)[16]。將人或大鼠的S100A4基因?qū)肓夹缘拇笫笕橄偌毎麅?nèi)，S100A4蛋白高水平的表達導(dǎo)致細胞遷移的出現(xiàn)[18，19]。將S100A4基因轉(zhuǎn)染至不能轉(zhuǎn)移的小鼠乳腺癌細胞系，其癌細胞即發(fā)生轉(zhuǎn)移，轉(zhuǎn)染至小鼠黑色素瘤細胞系B16和人乳腺癌細胞系MCF-7中后，其發(fā)生肺轉(zhuǎn)移的機率明顯增加[20]，而轉(zhuǎn)染S100A4反義基因，則可以明顯降低具有高轉(zhuǎn)移特性惡性腫瘤細胞系的轉(zhuǎn)移[21]。有研究表明，人乳腺癌中S100A4表達水平與尿激酶型纖溶酶原激活物(UPA)的表達呈顯著性正相關(guān)，而UPA是乳腺癌高侵襲性的標志，可見S100A4與乳腺癌的高侵襲性相關(guān)[22，23]。通過對骨肉瘤細胞系MG-63和U-2OS進行RNA干擾發(fā)現(xiàn)[24]，S100A4通過調(diào)節(jié)一些可降低細胞外基質(zhì)粘附性并使其降解的蛋白的表達來促進骨肉瘤細胞的增殖、侵襲和轉(zhuǎn)移。因此，S100A4可作為腫瘤細胞遷移的標志物[11，12]。

2.2　S100A4促進腫瘤、干細胞增殖的作用機制及影響因素

S100A4與特定的目標蛋白相結(jié)合，會對腫瘤細胞、多能干細胞、專能干細胞的增殖、遷移起著非常重要的促進作用[25～28]。據(jù)報道，S100A4能夠參與活化基質(zhì)金屬蛋白酶(MMPs)和血管內(nèi)皮生長因子(VEGF)來誘導(dǎo)血管的生成[29，30]。S100A4還可與腫瘤抑制蛋白p53相互作用，在腫瘤發(fā)生的早期階段加速野生型p53功能的喪失，以致細胞周期失去調(diào)控，使一些已發(fā)生編碼或翻譯錯誤的細胞得以繼續(xù)生存并繁殖，這樣就可能會導(dǎo)致細胞惡變甚至誘發(fā)腫瘤，并影響細胞壽命[31]，而敲除S100A4的基因會導(dǎo)致依賴p53的細胞周期停滯[32]。S100A4的表達會被某些胞外生長因子所激活，如表皮生長因子(EGF)、成纖維細胞生長因子(FGF)和轉(zhuǎn)化生長因子(TGF)等[33，34]。隨后，S100A4會增強MMPs的表達和活化，而水解蛋白酶(包括纖溶酶和MMPs)可釋放細胞外基質(zhì)中的TGF-β前體蛋白和堿性成纖維細胞生長因子(BFGF)并進行活化[35，36]，這會引起細胞中S100A4表達的上調(diào)，由此建立起了一個正反饋的調(diào)節(jié)機制。其中，在TGF-β1信號通路的作用下，S100A4的表達會發(fā)生明顯上調(diào)，某些細胞中，S100A4與TGF-β信號通路中重要的信號蛋白Smad3相結(jié)合，進而增強了細胞的增殖能力[37，38]，下調(diào)S100A4表達則會抑制上述細胞的行為[39～41]。干擾素-γ可抑制S100A4的基因轉(zhuǎn)錄。將小鼠乳腺細胞通過S100A4基因轉(zhuǎn)染后，會明顯降低鈣黏著蛋白的表達量，從而增強了細胞的遷移能力，這是由于鈣黏著蛋白不能正常介導(dǎo)細胞間的黏附力所致[42]。

2.3　S100A4與胚胎發(fā)育的關(guān)系

在胚胎發(fā)育期間，S100A4能夠通過調(diào)節(jié)肌球蛋白-IIA纖維的組裝來改變細胞極性，進而介導(dǎo)細胞遷移[43]。S100A4與Liprin 1相互作用可影響細胞的運動能力，與Ⅱ型膜聯(lián)蛋白[44]接觸會使細胞外基質(zhì)發(fā)生重構(gòu)。在小鼠個體發(fā)育的第12天左右，可檢測到表達S100A4的細胞，其中，在小鼠的胚胎發(fā)生階段，S100A4參與了骨、胎兒巨噬細胞的發(fā)育以及間充質(zhì)組織的分化[45]。

研究表明，在胚胎發(fā)育期，S100A4在多核的破骨細胞內(nèi)表達，除此以外，在骨形成過程中，S100A4同樣在成軟骨細胞和成骨細胞內(nèi)表達，這一表達進程與軟骨形成細胞向骨原細胞轉(zhuǎn)化的時間點是一致的，骨原細胞(Osteogenic cell)可進一步分化為成骨細胞[46]。S100A4可在軟骨向成骨細胞轉(zhuǎn)化時或骨原細胞侵入軟骨的早期發(fā)揮其功能[47]。另外，在毛囊發(fā)育的過程中，S100A4存在短暫的、空間限制性的表達[48]。在表皮的發(fā)育毛囊中，上皮細胞鈣黏蛋白的免疫染色要明顯強于基底膜附近的毛囊周圍細胞[49]。

S100A4在胚胎巨噬細胞中表達，這些巨噬細胞存在于胚胎間充質(zhì)中，其某些性質(zhì)與轉(zhuǎn)移癌細胞相似[50]，例如：侵入能力、較強的細胞運動活性、對細胞外基質(zhì)的降解作用。此外，S100A4也存在于血管周圍間充質(zhì)的星狀或圓形細胞中，這說明在小鼠胚胎發(fā)育期間，S100A4對于間充質(zhì)組織細胞的分化與形態(tài)發(fā)生起著非常重要的作用[51]。S100A4表達過程一直延續(xù)到出生，實際上，S100A4在成年小鼠和人類體內(nèi)的表達形式與胚胎中的類似[52]，表達S100A4的巨噬細胞可遷移至腸間充質(zhì)，隨后，進入腸上皮組織，說明這些細胞具有穿越基底膜的能力，對基底膜的降解是這一轉(zhuǎn)移過程的起始[53]。有研究表明，S100A4基因在淋巴組織，如脾、胸腺、骨髓、活化巨噬細胞以及淋巴細胞中均有所表達，胸腺組織的S100A4表達量要比其他淋巴組織中的高[54]。

在小鼠發(fā)育期間，表達S100A4的間充質(zhì)組織有鼻囊(Nasal capsule)周圍分化中的間充質(zhì)、發(fā)育中的指(趾)、膀胱上皮組織、發(fā)育中毛囊和牙囊的間充質(zhì)聚集區(qū)(Mesenchymal condensation)以及骨發(fā)育中侵入的間充質(zhì)組織。S100A4的表達與組織的形態(tài)發(fā)生是相關(guān)的，在毛囊的形態(tài)發(fā)育過程中，可被誘導(dǎo)的間充質(zhì)細胞能夠表達S100A4，具有促進細胞生長、細胞間解粘附和基底膜重構(gòu)的作用，其多數(shù)生理過程與腫瘤細胞中的相一致[8]。

2.4　S100A4在鹿茸發(fā)生與再生過程中的作用

鹿茸的發(fā)生和再生是依賴干細胞驅(qū)動的過程，其中，鹿茸發(fā)生干細胞(Antlerogenic Periosteum cells，AP細胞)決定著鹿茸發(fā)生過程，鹿茸再生干細胞(Pedicle Periosteum cells，PP細胞)特別是末端PP細胞是鹿茸再生的締造者。本研究組已有的研究結(jié)果表明，S100A4在AP細胞中大量表達，在AP細胞形成角柄的過程中，S100A4參與了AP細胞的遷移[55]。而我們的最新研究發(fā)現(xiàn)，在PP細胞中S100A4同樣存在表達情況。S100A4可上調(diào)TP53的表達，進而介導(dǎo)細胞凋亡，這對于鹿茸的再生是非常重要的[56]。

在鹿茸的快速生長過程中，伴隨著血管系統(tǒng)的完全再生，血管的再生是組織成功再生的基礎(chǔ)，而S100A4可明顯促進增殖細胞周圍血管的生成[57]，這就為深入研究S100A4在鹿茸發(fā)育中的作用提供了另外一個切入點。有幾種途徑可以導(dǎo)致鹿茸血管的生成，一種可能是由機械牽拉引起的血管伸長刺激全長或局部血管的血管內(nèi)皮細胞和支持細胞出現(xiàn)分裂繁殖[58]；另一種可能是在生長因子等的誘導(dǎo)下，新血管分枝由原來血管長出，或由于血管重建，一枝血管內(nèi)部融合形成兩枝血管[59，60]。目前在鹿茸中，已知與S100A4有關(guān)的血管形成因子有成纖維細胞生長因子(FGF)、血管內(nèi)皮生長因子(VEGF)等[61，62]。但是截至目前，S100A4在鹿茸發(fā)生與再生過程中的相關(guān)功能尚未見報道，與鹿茸發(fā)育的關(guān)系也不十分清楚。

3　展望

鹿茸是哺乳動物中唯一失去后還能完全再生的器官，最重要的原因是其中的鹿茸干細胞具備自我更新和多重分化潛能，可以向多種體細胞分化，最終形成具有特定結(jié)構(gòu)的器官——鹿茸，而不是像無分化或低分化的腫瘤細胞群那樣，發(fā)生不可控制的無限制的增殖。為了維持自身的干細胞狀態(tài)和完成鹿茸的發(fā)生/再生，鹿茸干細胞要受到體內(nèi)、外多種分泌因子的調(diào)控。已有的蛋白質(zhì)組學(xué)研究表明，S100A4通過與其他功能蛋白的結(jié)合來行使自身生物學(xué)功能，因此未來將重點關(guān)注在鹿茸干細胞中，能夠與S100A4發(fā)生結(jié)合的相關(guān)蛋白，并比較其在與S100A4結(jié)合前后結(jié)構(gòu)與功能所發(fā)生的變化。通過此研究，可得知在鹿茸中某些與S100A4相關(guān)活性因子的生物學(xué)功能及其作用機制。

綜上所述，基于干細胞的鹿茸再生，為我們提供了一個研究肢體再生、血管形成、組織快速增殖而不癌變的極好模型。通過對S100A4及相關(guān)蛋白的深入了解，可以此作為今后研究相關(guān)生物學(xué)功能以及信號通路的基礎(chǔ)，并為最終揭示鹿茸再生的奧秘進行理論和實踐的鋪墊。

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