KLF4与TMEM16J相互作用在调控VSMC增殖和分化中的作用研究

In the previous study, we found that the interaction of KLF4 with Sp1 and PPAR-γ plays a key role in inhibiting VSMC proliferation and inducing VSMC differentiation. In addition, we also found that TGF-β1 significantly induced the interaction of KLF4 with a cytoplasmic protein TMEM16J. Based on the fact that cytoplasmic and nuclear events are usually interconnected, we thought that the interaction of KLF4 with TMEM16J in the cytoplasm plays an important role in VSMC proliferation and differentiation. In this study, we sought to explore the role and mechanism of KLF4 and TMEM16J interaction in regulating VSMC proliferation and differentiation. We will focus on studying whether KLF4 inhibits VSMC proliferation and promotes VSMC differentiation by interacting with TMEM16J to block the contact of proproliferation signal proteins or enhance the contact of prodifferentiation signal proteins. In addition, we will further study whether KLF4 interaction with TMEM16J in the cytoplasm blocks KLF4 nuclear translocation, inhibiting the expression of proliferation-related genes or activating the expression of differentiation-related genes regulated by KLF4 and its cofactors. Further understanding of the role and mechanism of KLF4 in regulating VSMC proliferation and differentiation in the cytoplasm should help recognize KLF4 functions thoroughly in VSMC, and thus providing a new pathway to prevent proliferative vascular diseases.

我们在研究KLF4与Sp1和PPAR-γ相互作用抑制血管平滑肌细胞（VSMC）增殖和诱导VSMC分化的分子机制过程中，发现TGF-β除调节上述三者的组装外，还显著诱导KLF4与胞浆蛋白TMEM16J相互作用。基于同一细胞事件的完成需要胞浆和胞核协调联动、共同参与，我们认为，KLF4在胞浆中与TMEM16J相互作用也对VSMC增殖与分化产生影响。为此，本项目研究KLF4在胞浆中与TMEM16J相互作用是否能阻隔增殖信号转导分子间的相互接触或促进分化信号转导分子间的相互接触，探讨KLF4是否通过与TMEM16J相互作用，使KLF4核转位受限，导致受KLF4及其辅助因子激活的增殖相关基因表达受阻，受KLF4及其辅助因子阻抑的分化相关基因表达上调，来发挥抑制VSMC增殖及诱导VSMC分化的作用，明确KLF4 在VSMC不同区域中协同调节VSMC增殖与分化的分子机制，为防治血管增殖性疾病提供新思路。

跨膜蛋白TMEM16A是血管平滑肌细胞（vascular smooth muscle cells, VSMCs）钙激活氯通道（calcium-activated chloride channels, CaCCs）的分子基础。近年研究表明，TMEM16A能抑制基底动脉平滑肌细胞增殖。然而，TMEM16A是否在以VSMC增殖为特征的血管重塑性疾病中发挥作用以及如何发挥作用尚不清楚。本研究利用荧光素酶报告基因分析、Western blotting、实时定量PCR分析等研究方法，取得下述研究成果：第一、在人主动脉平滑肌细胞（human aortic smooth muscle cells, HASMCs）中，转录因子myocardin通过与血清应答因子SRF形成复合物结合于TMEM16A基因启动子区促进TMEM16A基因表达；TMEM16A反过来促进myocardin以及VSMCs分化标志基因表达，因此，myocardin与TMEM16A形成正反馈环路，促进VSMC分化、抑制VSMC增殖。第二、血管紧张素II（Ang II）通过上调KLF5表达抑制TMEM16A表达；体内实验证实，KLF5介导了Ang II诱导的血管重塑。第三、Ang II通过抑制myocardin与SRF结合、促进myocardin与KLF5结合抑制TMEM16A转录。本研究结果表明，Ang II通过促进KLF5表达及其与myocardin的相互作用抑制TMEM16A表达，打破TMEM16A与myocardin形成的正反馈环路，抑制VSMC分化、促进VSMC增殖和血管重塑。通过这些研究，首次阐明了钙激活氯离子通道蛋白TMEM16A在血管重塑中的作用及作用机制，并进一步阐明了Ang II促进血管重塑的机制，为防治以VSMC增殖为特征的血管重塑性疾病如高血压、动脉粥样硬化等提供干预靶点。