S100A4蛋白的生物学功能研究进展

尚禹东，杨艳玲，赵海平，李春义

(中国农业科学院特产研究所特种动物分子生物学省部共建国家重点实验室，长春130112)

1　引言

S100家族是一类Ca2+结合蛋白，通过与其他目标蛋白的相互作用来调节细胞各种功能，其生物学活性的发挥需要借助Ca2+依赖性所引起的构象改变，在此过程中，Ca2+对于S100的活化是必需的[1]。因此，S100蛋白被认为是一种Ca2+传感器蛋白，通过Ca2+信号转导途径，在细胞通讯联系、增殖、生长、分化、收缩、基因表达、分泌及凋亡中发挥重要作用[2]。S100A4是S100蛋白家族中的一员，人类的S100A4基因定位于染色体的1q21，而小鼠和大鼠的基因则分别位于3f3和2q34[3]。S100A4是由101个氨基酸组成的多肽，在哺乳动物中高度保守，分子量为115ku[1]。分子当中的EF臂型结构域具有结合Ca2+的能力，当其蛋白部分与Ca2+结合后，S100A4蛋白构象发生改变，暴露出其与靶蛋白结合的位点，进而通过相应的靶蛋白发挥其生物学效应[4]。与其他S100家族蛋白不同的是，S100A4与目标分子发生相互作用时，可表现为Ca2+依赖性和非依赖性2种不同的结合方式[1]。

2　S100A4的生物学活性

S100A4兼具细胞内和细胞外活性，并具有调节某些信号通路蛋白的功能[5]，其介导的Ca2+信号通路在细胞的增殖、分化、粘附、迁移、形态发生以及凋亡等生理过程中起到了决定性的作用；S100A4通常与丝状肌动蛋白共聚，并与非肌性肌球蛋白原和非肌性肌球蛋白相互作用[6，7]；S100A4还可与细胞骨架蛋白直接结合，或通过调节钙黏蛋白/连环蛋白、CD44/细胞骨架复合物的细胞骨架连接，活化细胞外基质水解酶，来改变细胞动力学模式和迁移能力，参与细胞运动；在哺乳动物发育期间，它广泛存在于胚胎巨噬细胞和分化的间充质组织当中[8]；S100A4的高表达还发生在细胞类型从上皮向间充质转变的过程中[9，10]；S100A4在脑损伤、心肌损伤、类风湿性关节炎和组织纤维化等病变组织中也存在过度表达情况[11]。

2.1　S100A4与肿瘤发生、发展的关系

S100A4蛋白的表达与细胞的异常增生及肿瘤的发生、发展有关，其在正常人体的肺、泌尿系统、乳腺、甲状腺、胰腺及结肠等组织细胞中均不表达，而在乳腺癌、胃癌、结直肠癌、胰腺癌、甲状腺癌、膀胱癌、前列腺癌、口腔上皮细胞癌、非小细胞肺癌、神经胶质瘤细胞以及多种基质细胞(如成纤维细胞、淋巴细胞和巨噬细胞等)中高度表达，在恶性肿瘤中比良性肿瘤中表达量更高[12～15]。S100A4也可在体外培养的相关细胞系中正常表达，并可存在于肿瘤细胞间充质液中[16]。肿瘤患者的组织学检查显示，癌变组织中的S100A4表达水平要远高于邻近的正常组织[17]。肿瘤间充质中S100A4的表达同样会使肿瘤细胞发生迁移和侵入，在S100A4基因敲除的小鼠体内，转移性癌细胞并没有发生迁移，而将表达S100A4的肿瘤间充质细胞注射入肿瘤组织内，癌细胞的转移能力有所恢复[16]。将人或大鼠的S100A4基因导入良性的大鼠乳腺细胞内，S100A4蛋白高水平的表达导致细胞迁移的出现[18，19]。将S100A4基因转染至不能转移的小鼠乳腺癌细胞系，其癌细胞即发生转移，转染至小鼠黑色素瘤细胞系B16和人乳腺癌细胞系MCF-7中后，其发生肺转移的机率明显增加[20]，而转染S100A4反义基因，则可以明显降低具有高转移特性恶性肿瘤细胞系的转移[21]。有研究表明，人乳腺癌中S100A4表达水平与尿激酶型纤溶酶原激活物(UPA)的表达呈显著性正相关，而UPA是乳腺癌高侵袭性的标志，可见S100A4与乳腺癌的高侵袭性相关[22，23]。通过对骨肉瘤细胞系MG-63和U-2OS进行RNA干扰发现[24]，S100A4通过调节一些可降低细胞外基质粘附性并使其降解的蛋白的表达来促进骨肉瘤细胞的增殖、侵袭和转移。因此，S100A4可作为肿瘤细胞迁移的标志物[11，12]。

2.2　S100A4促进肿瘤、干细胞增殖的作用机制及影响因素

S100A4与特定的目标蛋白相结合，会对肿瘤细胞、多能干细胞、专能干细胞的增殖、迁移起着非常重要的促进作用[25～28]。据报道，S100A4能够参与活化基质金属蛋白酶(MMPs)和血管内皮生长因子(VEGF)来诱导血管的生成[29，30]。S100A4还可与肿瘤抑制蛋白p53相互作用，在肿瘤发生的早期阶段加速野生型p53功能的丧失，以致细胞周期失去调控，使一些已发生编码或翻译错误的细胞得以继续生存并繁殖，这样就可能会导致细胞恶变甚至诱发肿瘤，并影响细胞寿命[31]，而敲除S100A4的基因会导致依赖p53的细胞周期停滞[32]。S100A4的表达会被某些胞外生长因子所激活，如表皮生长因子(EGF)、成纤维细胞生长因子(FGF)和转化生长因子(TGF)等[33，34]。随后，S100A4会增强MMPs的表达和活化，而水解蛋白酶(包括纤溶酶和MMPs)可释放细胞外基质中的TGF-β前体蛋白和碱性成纤维细胞生长因子(BFGF)并进行活化[35，36]，这会引起细胞中S100A4表达的上调，由此建立起了一个正反馈的调节机制。其中，在TGF-β1信号通路的作用下，S100A4的表达会发生明显上调，某些细胞中，S100A4与TGF-β信号通路中重要的信号蛋白Smad3相结合，进而增强了细胞的增殖能力[37，38]，下调S100A4表达则会抑制上述细胞的行为[39～41]。干扰素-γ可抑制S100A4的基因转录。将小鼠乳腺细胞通过S100A4基因转染后，会明显降低钙黏着蛋白的表达量，从而增强了细胞的迁移能力，这是由于钙黏着蛋白不能正常介导细胞间的黏附力所致[42]。

2.3　S100A4与胚胎发育的关系

在胚胎发育期间，S100A4能够通过调节肌球蛋白-IIA纤维的组装来改变细胞极性，进而介导细胞迁移[43]。S100A4与Liprin 1相互作用可影响细胞的运动能力，与Ⅱ型膜联蛋白[44]接触会使细胞外基质发生重构。在小鼠个体发育的第12天左右，可检测到表达S100A4的细胞，其中，在小鼠的胚胎发生阶段，S100A4参与了骨、胎儿巨噬细胞的发育以及间充质组织的分化[45]。

研究表明，在胚胎发育期，S100A4在多核的破骨细胞内表达，除此以外，在骨形成过程中，S100A4同样在成软骨细胞和成骨细胞内表达，这一表达进程与软骨形成细胞向骨原细胞转化的时间点是一致的，骨原细胞(Osteogenic cell)可进一步分化为成骨细胞[46]。S100A4可在软骨向成骨细胞转化时或骨原细胞侵入软骨的早期发挥其功能[47]。另外，在毛囊发育的过程中，S100A4存在短暂的、空间限制性的表达[48]。在表皮的发育毛囊中，上皮细胞钙黏蛋白的免疫染色要明显强于基底膜附近的毛囊周围细胞[49]。

S100A4在胚胎巨噬细胞中表达，这些巨噬细胞存在于胚胎间充质中，其某些性质与转移癌细胞相似[50]，例如：侵入能力、较强的细胞运动活性、对细胞外基质的降解作用。此外，S100A4也存在于血管周围间充质的星状或圆形细胞中，这说明在小鼠胚胎发育期间，S100A4对于间充质组织细胞的分化与形态发生起着非常重要的作用[51]。S100A4表达过程一直延续到出生，实际上，S100A4在成年小鼠和人类体内的表达形式与胚胎中的类似[52]，表达S100A4的巨噬细胞可迁移至肠间充质，随后，进入肠上皮组织，说明这些细胞具有穿越基底膜的能力，对基底膜的降解是这一转移过程的起始[53]。有研究表明，S100A4基因在淋巴组织，如脾、胸腺、骨髓、活化巨噬细胞以及淋巴细胞中均有所表达，胸腺组织的S100A4表达量要比其他淋巴组织中的高[54]。

在小鼠发育期间，表达S100A4的间充质组织有鼻囊(Nasal capsule)周围分化中的间充质、发育中的指(趾)、膀胱上皮组织、发育中毛囊和牙囊的间充质聚集区(Mesenchymal condensation)以及骨发育中侵入的间充质组织。S100A4的表达与组织的形态发生是相关的，在毛囊的形态发育过程中，可被诱导的间充质细胞能够表达S100A4，具有促进细胞生长、细胞间解粘附和基底膜重构的作用，其多数生理过程与肿瘤细胞中的相一致[8]。

2.4　S100A4在鹿茸发生与再生过程中的作用

鹿茸的发生和再生是依赖干细胞驱动的过程，其中，鹿茸发生干细胞(Antlerogenic Periosteum cells，AP细胞)决定着鹿茸发生过程，鹿茸再生干细胞(Pedicle Periosteum cells，PP细胞)特别是末端PP细胞是鹿茸再生的缔造者。本研究组已有的研究结果表明，S100A4在AP细胞中大量表达，在AP细胞形成角柄的过程中，S100A4参与了AP细胞的迁移[55]。而我们的最新研究发现，在PP细胞中S100A4同样存在表达情况。S100A4可上调TP53的表达，进而介导细胞凋亡，这对于鹿茸的再生是非常重要的[56]。

在鹿茸的快速生长过程中，伴随着血管系统的完全再生，血管的再生是组织成功再生的基础，而S100A4可明显促进增殖细胞周围血管的生成[57]，这就为深入研究S100A4在鹿茸发育中的作用提供了另外一个切入点。有几种途径可以导致鹿茸血管的生成，一种可能是由机械牵拉引起的血管伸长刺激全长或局部血管的血管内皮细胞和支持细胞出现分裂繁殖[58]；另一种可能是在生长因子等的诱导下，新血管分枝由原来血管长出，或由于血管重建，一枝血管内部融合形成两枝血管[59，60]。目前在鹿茸中，已知与S100A4有关的血管形成因子有成纤维细胞生长因子(FGF)、血管内皮生长因子(VEGF)等[61，62]。但是截至目前，S100A4在鹿茸发生与再生过程中的相关功能尚未见报道，与鹿茸发育的关系也不十分清楚。

3　展望

鹿茸是哺乳动物中唯一失去后还能完全再生的器官，最重要的原因是其中的鹿茸干细胞具备自我更新和多重分化潜能，可以向多种体细胞分化，最终形成具有特定结构的器官——鹿茸，而不是像无分化或低分化的肿瘤细胞群那样，发生不可控制的无限制的增殖。为了维持自身的干细胞状态和完成鹿茸的发生/再生，鹿茸干细胞要受到体内、外多种分泌因子的调控。已有的蛋白质组学研究表明，S100A4通过与其他功能蛋白的结合来行使自身生物学功能，因此未来将重点关注在鹿茸干细胞中，能够与S100A4发生结合的相关蛋白，并比较其在与S100A4结合前后结构与功能所发生的变化。通过此研究，可得知在鹿茸中某些与S100A4相关活性因子的生物学功能及其作用机制。

综上所述，基于干细胞的鹿茸再生，为我们提供了一个研究肢体再生、血管形成、组织快速增殖而不癌变的极好模型。通过对S100A4及相关蛋白的深入了解，可以此作为今后研究相关生物学功能以及信号通路的基础，并为最终揭示鹿茸再生的奥秘进行理论和实践的铺垫。

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