阳性表达E-钙粘蛋白对整合蛋白α5β1及其下游信号分子的影响

【作者】 吴衡；

【导师】 查锡良；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2003， 博士

【摘要】 细胞粘附是细胞行使多种生物学功能的基础。E-钙粘蛋白介导细胞间的粘附，整合蛋白介导细胞和基底膜（ECM）间的粘附。胚胎发育或正常生理活动中的细胞的迁移，乃至肿瘤细胞的侵袭和转移都涉及细胞-ECM、细胞-细胞的粘附，这一系列过程必然伴随着E-钙粘蛋白和整合蛋白信号通路的相互串话(cross-talk)。本论文主要研究了E-钙粘蛋白表达缺失的人乳腺癌细胞转染E-钙粘蛋白后，对整合蛋白表达及其下游信号通路的影响。本论文将野生型E-钙粘蛋白cDNA真核细胞表达质粒转染至E-钙粘蛋白表达阴性的人乳腺癌细胞MDA-MB-435和MDA-MB-231，建立阳性表达细胞株—E-cad-435和E-cad-231，同时转染空质粒载体pcDNA3作为阴性对照（Mock-435和Mock-231）。E-cad-435和E-cad-231与对照细胞相比生长变慢，更多细胞停滞在G0/G1期，细胞之间更容易发生粘附，以及更容易发生凋亡。阳性表达E-钙粘蛋白后细胞对ECM的主要成分FN和LN粘附能力下降，尤其是FN。整合蛋白α5β1是FN的主要受体，结果发现细胞膜上的整合蛋白α5、β1亚基的蛋白和mRNA水平都下降了。在阳性表达细胞株E-cad-435和E-cad-231中，β-连环蛋白蛋白量下降；其分布主要集中在细胞膜内侧和细胞浆中，而细胞核中基本消失。Mock-435和Mock-231中的β-连环蛋白主要集中在细胞核。由于β-连环蛋白是Wnt信号通路的重要参与者，在细胞核中能与转录因子TCF/LEF家族成员结合，共同起始转录和调控很多基因。那么，E-钙粘蛋白是否能通过β-连环蛋白来影响整合蛋白的表达？为此，选用GSK-3β的抑制剂-LiCl，以抑制β-连环蛋白的降解来观察整合蛋白的变化。结果显示LiCl处理细胞36小时后β-连环蛋白明显增加，同时整合蛋白α5、β1亚基的蛋白量也随之增加。这说明E-钙粘蛋白能通过β-连环蛋白来影响整合蛋白α5β1表达量。同时发现阳性表达E-钙粘蛋白还能抑制FAK、PKB和ILK以及细胞周期蛋白D1的表达，并促进抑癌基因PTEN的表达。LiCl处理细胞36小时后FAK和PKB的表达量也随着增加，同样说明E-钙粘蛋白影响整合蛋白下游的信号分子FAK和PKB的蛋白量主要由β-连环蛋白介导。在转染丧失与β-连环蛋白结合能力的突变E-钙粘蛋白cDNAΔ71的细胞株的研究中，发现突变E-钙粘蛋白不能结合β-连环蛋白，引起β-连环蛋白的蛋白量下降，并其分布主要集中在细胞浆；进一步发现，突变E-钙粘蛋白cDNAΔ71同样能抑制整合蛋白α5、β1、亚基的蛋白量，尤其在MDA-MB-435细胞株中更<WP=5>加明显，α5亚基蛋白基本消失，此结果提示了在E-钙粘蛋白调节整合蛋白α5β1表达过程中，发挥作用的是游离的β-连环蛋白。有文献报道PKB能磷酸化GSK-3并抑制其活性，进而抑制β-连环蛋白的降解。用PI3K的抑制剂—Wortmannin处理细胞，发现Wortmannin能抑制PKB的473位Ser磷酸化。随着Wortmannin抑制浓度的增加，PKB Ser473磷酸化程度呈现下降趋势，并且β-连环蛋白的蛋白量也随之下降。说明在MDA-MB-231和MDA-MB-435细胞中降解β-连环蛋白的系统完整，只是受到了PKB高表达的抑制，同时也说明β-连环蛋白和PKB可相互影响。此外还发现，阳性表达E-钙粘蛋白能抑制乳腺癌细胞分泌雌激素能力，已有文献报道雌激素能通过非受体途径GPCR30，激活PKB，来影响整合蛋白的表达，为E-钙粘蛋白影响整合蛋白信号途径的解释又增加了一种选择。更多还原

【Abstract】 E-cadherin mediates adhesion between cells, while integrins mediates adhesion between cells and ECM. Tumor cells detach from the primary tumor tissue, metasising to distal sites and adhesing to distal organs, dissolving the ECM and forming new vascules. This serial precesses should be accompanied the cross-talks between E-cadherin and integrins. The cross-talks between E-cadherin and integrins should also take active part in normal cells and the embryo development process. In this experiment, wild-typed E-cadherin cDNA construct which can be expressed in eukaryotic cells, was transfected into two E-cadherin-negative breast carcinoma cell lines, MDA-MB-231 and MDA-MB-435. Positive transfectants were selected by G418 and named as E-cad-231 and E-cad-435, correspondingly. At the same time, pcDNA3, the empty vector, was also transfected and selected by G418 and named as Mock-231 and Mock-435 as negative control. E-cad-435 and E-cad-231 adhesion ability to fibronectin and laminin decreased compared with the control cells, ewpecially fibronectin. Integrin α5β1 is the typical receptor of fibronectin. Flow cytometry, Western blot and RT-PCR showed that bothα5 and β1 integrins protein level and mRNA decreased compared with the control cells. In negative cells, β-catenin accumulated in the nucleus, while β-catenin nearly dispeared from the necleus and mainly accumulated near the membrane and cytosol. Since β-catenin is one of important participants in Wnt pathway, binding with mombers of TCF/LEF family and initiating or inhibiting transcription of many genes. In order to cerfiticate whether E-cadherin Could influence integrins through β-catenin, LiCl treatement was used to inhibit β-catenin protein degradation. After LiCl treatment, β-catenin and integrin α5 and β1 protein level increased. It suggested that E-cadherin could influence integrinα5β1 expression through β-catenin. Furthermore, positive expression of E-cadherin could inhibit integrin downstream signal molecules, such as FAK,PKB and ILK and a cell cycle regulator, cyclinD1. LiCl treatment could increase FAK and PKB protein level, which suggested that E-cadherin could inhibit FAK and PKB.Δ71, mutant E-cadherin cDNA which lacks the core sequence binding with β-catenin, was also transfected into the two cell lines mention above. G418 was used to select the positive transfectants. Δ71, the mutant E-cadherin, could bind with β-catenin, but could still decrease β-catenin protein level and relocate β-catenin from mainly nucleus to<WP=7>cytosol. The results showed that E-cadherin could decrease β-catenin protein level and relocate β-catenin without the help of direct binding.Further research showed thatΔ71 could inhibit bothα5 and β1 integrin protein levels, especially in MDA-MB-435, in which positive expression ofΔ71 could inhibit mostα5 integrin expression. This result was consistent with other results and furtherly confirmed that E-cadherin could inhibit bothα5 and β1 integrin, but was not fulfilled by simply binding with β-catenin and influencing its location.It has been reported that PKB can phosphorylate GSK-3 and inhibit β-catenin protein degradation. Wortmannin is an inhibitor of PI3K. With the increase of Wortmannin concentration, Phosphorylation of PKB 473-threnine and β-catenin protein level decreased. It seemed to mean that β-catenin protein degradation system was intact and β-catenin accumulated in the nucleus was due to inhibition of high protein level of PKB in both MDA-MB-231 and MDA-MB-435. β-catenin and PKB protected each other or promote the other’s expression in a loop. In addition, positive expression of E-cadherin could inhibit the estrogen secretion ability in the human carcinoma cells. It has been reported that estrogen could active PKB through non-receptor pathway, such as GPCR30 and influence integrins expression. This provided another explanation for how E-cadherin influences integrin pathway. Above all, the positive expression of E-cadherin in human breast carcinoma cells not only binded with ?更多还原