整合素α5β1-Src信号轴调节肝实质细胞药物代谢功能成熟的机制研究

The drug metabolic functional maturation of hepatocyte is dependent on the regulation of extracellular matrix. However, the regulatory mechanism of extracellular matrix in this process is not very clear. Result in our previous studies shown that the expression of PXR and the activities of CYP450 in human adipose stem cell-derived hepatocyte-like cells (hASC-HLCs) were regulated by the integrin α5β1-src signal axis. Reducing the expression of α5 integrin decreased the ten-eleven translocation (Tet) family of dioxygenases 1 and Tet3 mRNA levels, which responsible for the demethylation of gene promoters. Studies have suggested that the DNA methylation of the PXR promoter was more densely detected in the PXR low expression cells. We wondered whether the expression of PXR regulated by the α5β1-Src signal axis is dependent on the DNA demethylation of the PXR promoter, which promoted the expression of nuclear receptor PXR and resulted in the metabolic functional maturation of hepatocyte. In this study, the regulation of the α5β1-Src signal axis on the expression of tet protein and the transcription factor c-Jun, the key transcription factor in Src signal pathway, the regulation of tet protein and c-Jun on the DNA demethylation of the PXR promoter, the expression of PXR and the CYP450 activities will be explored in the hASC-HLCs and human hepatocyte in vitro and the hepatic specific integrin α5 knock out mice in vivo using real-time RT-PCR, MeDIP-qPCR, ChIP-qPCR,western blot and P450-Glo assay.

肝实质细胞的药物代谢功能成熟依赖ECM的调节作用，但机制并不清楚。我们研究发现，整合素α5β1-Src信号轴介导ECM信号，促进人脂肪干细胞分化的肝实质样细胞中核受体PXR的表达及CYP450酶活性。敲减整合素α5可降低参与基因启动子DNA去甲基化的蛋白Tet1和Tet3的mRNA水平。因PXR的低表达与其启动子DNA高甲基化密切相关，由此推测，整合素α5β1-Src信号轴通过促进PXR启动子DNA的去甲基化，上调PXR的表达，促进肝实质细胞药物代谢功能的成熟。本研究拟利用定量PCR、MeDIP-qPCR、ChIP-qPCR、蛋白印迹和P450-Glo分析等方法，研究人肝实质细胞及肝实质细胞特异性敲除整合素α5小鼠中，整合素α5β1-Src信号轴对Tet及Src下游转录因子c-Jun表达的调节作用，明确Tet及c-Jun与PXR启动子DNA去甲基化、PXR的表达和CYP450酶活性的关系。

肝实质细胞的结构与功能成熟依赖于细胞外基质的调节作用，多种原因引起肝组织中细胞外基质的过度累积，形成的肝纤维化及硬化，损伤肝脏的结构与功能。但细胞外基质是通过何种机制调节肝实质细胞结构与功能，目前尚不清楚。本项目研究发现，特异性敲减小鼠肝实质细胞及人肝实质细胞的细胞外基质受体整合素α5亚基，可显著降低肝实质细胞紧密连接信号通路及关键蛋白claudin1的表达，破坏肝实质细胞的极性特征；但与肝脏代谢功能密切的基因变化并不明显。分析机制显示，整合素α5调节肝实质细胞claudin1的表达依赖于Src信号；下调整合素α5-Src信号通路引起肝实质细胞中TET1蛋白水平的显著降低，继而引起DNA5hmc水平的降低，DNA5mc水平则明显升高；hMeDIP-seq分析表明，特异性敲减小鼠肝实质细胞整合素α5，引起cldn1基因间2个位点（chr16：26279061-26279820和chr16：26325841-26326040）羟甲基化水平显著减低，但并不影响cldn1基因启动子区域的羟甲基化水平。进一步分析胆汁淤积性肝病患者肝脏组织证实，胆汁淤积性肝病患者肝脏纤维化区域整合素α5、claudin 1及DNA羟甲基化水平的表达呈明显正相关。本项目研究表明，整合素α5-Src-TET1信号轴通过调节肝实质细胞cldn1基因羟甲基化水平及claudin 1的表达，介导肝实质细胞紧密连接及肝脏结构完整性的维持，调控肝脏正常功能的实现。本项目研究表明，整合素α5-Src-TET1信号轴有希望成为治疗肝纤维化及硬化等慢性肝脏疾病的新靶点。