PKC与Rab蛋白协同调控整合素αvβ6内吞胞吐循环的分子机制研究

Integrin αvβ6 is a special subtype which has close relationship with malignant behavior of colon carcinomas. In our previous studies, we have found that there was a direct binding between integrin β6 subunit and ERK2. This binding was able to facilitate the phosphorylation of ERK2 and maintain its active state. We tested the internalization and recycling rates of integrin αvβ6 in colon cancer cells, and the starting procedure of its internalization required the phosphorylation of ERK2. PKC activation was able to accelerate the internalization and recycling of integrin αvβ6, while the 11 amino acid at the C terminal of integrin β6 subunit was able to affect the malignant behavior of colon cancer cells. In this study, the regulatory mechanism of integrin αvβ6 trafficking was focused. Fusion proteins and dominant negative mutants were designed, and the technique of laser scanning confocal microscopy and GST pull down assay were applied. We would proved that PKCδ was able to bind with special sequence of β6 subunit, activate ERK2 and promote the internalization of integrin αvβ6. Rab family was involved in regulating intracellular transport. Multiple factors co-regulated integrin αvβ6 trafficking and promoted the migration, invasion and metastasis of colon cancer cells. Above all, we hope that these studies were able to provide experimental foundation and theoretical basis for the future targeting therapy.

整合素αvβ6是一类与结肠癌恶性行为关系密切的特殊整合素亚型，我们前期研究发现整合素β6与ERK2存在直接连接，该连接可促进ERK2磷酸化，并维持其激活状态；在结肠癌细胞中整合素αvβ6以内吞胞吐循环形式存在，其内吞启动过程受与之直接连接的ERK2磷酸化程度影响；PKC活化能加快整合素αvβ6内吞和胞吐过程；整合素β6胞内段C末端11个氨基酸序列可在一定程度上影响结肠癌细胞的恶性行为。本项目拟通过构建融合蛋白、显性负效应突变体，采用激光共聚焦检测、GST pull down实验等方法，证实PKCδ可以与整合素αvβ6胞内段特定氨基酸序列结合，激活ERK2的同时，促进整合素αvβ6内吞，Rab蛋白参与调控整合素αvβ6胞内转运，多因素协同调控整合素αvβ6内吞胞吐循环过程，促进结肠癌细胞迁移、侵袭和转移。该项研究可为进一步以整合素αvβ6内吞胞吐循环为靶点进行临床治疗提供实验基础和理论依据。

整合素αvβ6是一种与结肠癌恶性行为密切相关的特殊整合素亚型，它的表达以及在细胞内外的存在方式影响着肿瘤的发生发展并受多种因素调控。我们前期研究发现整合素αvβ6在结肠癌细胞内部是以内吞胞吐循环的方式快速运转，介导了结肠癌细胞的迁移，这种内吞胞吐循环模式受细胞内PKC活性的影响。在此基础上，我们进一步通过质谱分析、免疫共沉淀等方法发现整合素αvβ6可以与PKCβ、vimentin等直接结合，后者参与了整合素αvβ6胞内转运的调控。我们成功构建并鉴定了肠道上皮特异性过表达整合素αvβ6的转基因小鼠，通过肠道上皮自发成瘤模型、炎症性肠病相关肿瘤发生模型等发现整合素αvβ6的表达更容易促进肿瘤发生，并且在DSS诱导的急性炎症反应中，整合素αvβ6的表达明显加重了肠道炎症反应，表现在体重严重下降、便血较早出现、出血量增多、肠道长度缩短、肠道微环境中巨噬细胞增多等方面。除了可以在胞内转运外，整合素αvβ6还可以以exosome的形式被释放到细胞外并被其他细胞摄取，影响局部微环境，我们在结肠癌细胞中首次发现了这个现象，并通过电镜和激光共聚焦显微镜进行了观察验证。综上所述，本项目利用质谱分析、免疫共沉淀、转基因小鼠等实验手段，结合整合素胞内转运以及exosome释放等当今研究热点，揭示了整合素αvβ6在结肠癌发生发展中的重要作用和分子机制，为临床以整合素αvβ6为基础的分子诊断和靶向治疗提供了实验基础和理论依据。