KLF4同步调节VSMC标志基因和细胞周期调节基因表达的分子机制

Our recent studies demonstrate that KLF4 plays dual roles in the regulation of proliferation and differentiation through its acetylation/deacetylation modifications in vascular smooth muscle cells (VSMC). Based on KLF4's pleiotropic properties and its roles in the regulation of VSMC marker genes and cell cycle regulatory genes, we propose a novel idea that KLF4 couples VSMC marker gene expressions with expressions of cell cycle regulatory genes. In present study, we shall test this hypothesis using various methods for molecular and cell biology. We shall identify KLF4 cofactors for synchronously activating/repressing VSMC marker genes and cell cycle regulatory genes, elucidate the effects of pro-proliferation and pro-differentiation factors on the interactions of KLF4 with its cofactors as well as KLF4 modification states, explore the dynamic changes of protein molecules in the transcriptional regulatory complex for controlling VSMC marker gene and cell cycle-regulatory gene expressions, and examine the role of KLF4 in histone H3 and H4 acetylation in the promoter region of VSMC marker genes and cell cycle regulatory genes. Our study will uncover a novel mechanism whereby KLF4 couples VSMC marker gene expressions with expressions of cell cycle regulatory genes. The results will help to further understand the mechanism of VSMC phenotypic modulation, thereby revealing a new strategy for the treatment of vascular proliferative disorders.

在我们前期发现KLF4通过乙酰化/脱乙酰基修饰对血管平滑肌细胞（VSMC）发挥促增殖与促分化双重作用的基础上，基于KLF4的多能性及其在VSMC标志基因和细胞周期调节基因表达中的作用，提出KLF4使VSMC标志基因和细胞周期调节基因表达相耦联的新观点。本项目采用现代分子与细胞生物学实验技术，研究KLF4激活/阻抑VSMC标志基因和细胞周期正、负调节基因所依赖的辅助因子，阐明促增殖和促分化因素对KLF4与辅助因子相互作用及其对KLF4修饰状态的调节机制，揭示在VSMC表型转化过程中，对VSMC标志基因和细胞周期调节基因表达进行调控的转录调节复合物中蛋白质分子的动态变化，明确KLF4在VSMC标志基因和细胞周期调节基因启动子周围组蛋白乙酰化中的地位和作用，提出VSMC标志基因和细胞周期调节基因表达调节的耦联机制，进一步深化对VSMC表型调制机制的认识，为防治血管重塑性疾病提供新思路。

KLF4既促进VSMC标志基因表达，又作为细胞周期负调节因子激活细胞周期负调节基因（例如p21）。细胞分化/增殖与细胞周期阻滞/进展相耦联，然而，目前尚不清楚KLF4如何协同激活VSMC标志基因和细胞周期负调节基因。我们发现，促分化因子TGF-β1/ATRA诱导细胞周期负调节基因p21表达依赖于Smad和p38信号对KLF4的磷酸化，以及KLF4和p300相互作用及其被p300的乙酰化。同时，KLF4通过与p300相互作用将其募集到p21启动子上，p300催化组蛋白H3乙酰化，使染色质去稳定，从而实现p21激活与染色质重塑的耦联。促分化因素诱导VSMC标志基因表达依赖于JNK和p38信号诱导的KLF4磷酸化及其与p300结合，KLF4被p300乙酰化后定位到VSMC标志基因（SM22α）启动子上，上调SM22α表达。促增殖因素阻抑VSMC标志基因表达需要ERK和PI3K/Akt信号对KLF4脱磷酸及KLF4与HDAC2结合，KLF4被HDAC2脱乙酰基后从SM22α启动子上脱落，SM22α表达受阻。重要的是，我们还发现，表观遗传学调节因子miR-200c、miR-29a和miR-155通过调节KLF4和KLF5的表达与修饰参与对VSMC 标志基因和细胞周期调节基因表达的调控。miR-200c诱导KLF4苏素化并与苏素化型KLF4之间形成正反馈环，介导PDGF的促增殖效应；miR-155通过靶向抑制MST2激活Raf-1-MEK-ERK1/2信号，上调细胞周期正调节基因表达；miR-29a下调cyclin D1的负调节因子YY1、上调cyclin D1的正调节因子KLF5，从正、负两个方面协同促进cyclin D1表达。该研究成果明确了VSMC标志基因和细胞周期调节基因表达调节的耦联机制，进一步深化了对VSMC表型调制机制的认识，为防治血管重塑性疾病提供了新靶点。