Novel Role of MAPK14 in Regulation of VSMC Contractile Phenotype

描述(申请人提供)：血管平滑肌细胞(VSMC)表现出表型可塑性，导致人类血管疾病。血清反应因子(SRF)和Myocardin辅活化子家族(如MRTFA)已成为VSMC收缩表型的关键诱因。虽然在SRF-MRTFA依赖的基因表达方面已经有了很多工作，但对于汇聚到SRF-MRTFA从而对VSMC基因表达产生负面影响的信号通路，我们的理解存在一个关键的差距。我们和其他人认为p38MAPK信号是转化生长因子1诱导VSMC分化的关键信号转导通路；然而，到目前为止，所有的研究都依赖于p38MAPK的化学抑制剂(如SB203580)的单独使用。由于这些抑制剂可能具有与p38MAPK无关的未知作用，我们对Sb化合物靶向的VSMC中p38MAPK的主要亚型p38MAPK(MAPK14)进行了小干扰RNA击倒研究。我们没有像先前使用SB化合物预测的那样抑制VSMC基因的表达，而是发现MAPK14的敲除刺激了VSMC收缩基因的表达。利用几种明确的血管损伤模型，我们发现MAPK14总的和磷酸化的MAPK14在表型改变的VSMC所在的血管壁的新生内膜中丰富，这表明MAPK14的表达与合成的VSMC表型有关。新的令人兴奋的数据显示，VSMC特异的Mapk14基因敲除小鼠表现出诱导的VSMC收缩基因表达，并完全抵抗损伤诱导的新生内膜形成。在机制上，我们发现MAPK14调节MRTFA核质穿梭，这是依赖SRF的VSMC收缩基因表达的关键决定因素。我们最新的发现表明，MAPK14的缺失上调了一种新的平滑肌和内皮细胞富含的长非编码RNA(SENCR)，这对VSMC的收缩表型产生了积极的影响。基于这些发现，我们提出了MAPK14拮抗VSMC收缩表型，并通过MRTFA核转位失调和抑制SENCR促进血管疾病的全球假说。提出了三个相互关联的具体目标，以利用新的小鼠模型和创新的概念来解决这一假说。在目标1中，我们将阐明MAPK14在导致血管疾病的VSMC表型可塑性中的作用。在目标2中，我们将阐明MAPK14和MRTFA在调节VSMC分化中的整合作用。在第三个目标中，我们将阐明MAPK14依赖的SENCR抑制在VSMC中的调节和作用。总之，这些研究将阐明MAPK14在负向调控VSMC分化中的重要作用，从而为VSMC表型的分子调控提供新的视角。这些研究还挑战了p38MAPK作为前VSMC分化信号通路的范式，并将这一途径与两个关键下游靶点(MRTFA和SENCR)的拮抗联系起来。通过这些研究获得的信息对设计抗击血管疾病的新策略具有耐人寻味的治疗潜力。

DESCRIPTION (provided by applicant): Vascular smooth muscle cells (VSMC) exhibit phenotypic plasticity that contributes to human vascular disease. Serum response factor (SRF) and the Myocardin family of coactivators (e.g., MRTFA) have emerged as key inducers of the VSMC contractile phenotype. Although much work exists on SRF-MRTFA-dependent gene expression, a critical gap exists with respect to our understanding of signaling pathways that converge upon SRF-MRTFA to negatively affect VSMC gene expression. We and others have proposed p38MAPK signaling as a critical signal transducer of TGF¿1-induced VSMC differentiation; however, all studies to date have relied upon the exclusive use of chemical inhibitors of p38MAPK (e.g., SB203580). Since such inhibitors likely have unknown effects un-related to p38MAPK, we performed small interference RNA knockdown studies of p38MAPK¿ (MAPK14), the major isoform of p38MAPK in VSMC targeted by SB compounds. Rather than inhibiting VSMC gene expression, as predicted by prior studies using SB compounds, we found knockdown of MAPK14 stimulates VSMC contractile gene expression. Using several well-defined vascular injury models, we found total and phosphorylated MAPK14 are enriched in the neointima of the vessel wall where phenotypically altered VSMC reside suggesting MAPK14 expression is somehow linked to the synthetic VSMC phenotype. New, exciting data show VSMC-specific Mapk14 knockout mice exhibit induced VSMC contractile gene expression and are completely resistant to injury-induced neointimal formation. Mechanistically, we have found that MAPK14 regulates MRTFA nucleo-cytoplasmic shuttling, a critical determinant of SRF-dependent VSMC contractile gene expression. Our most recent finding reveals depletion of MAPK14 up-regulates a novel Smooth muscle and Endothelial cell enriched long Non-Coding RNA (SENCR), that exerts positive effects on the VSMC contractile phenotype. Based on these findings, we have formulated the global hypothesis that MAPK14 antagonizes VSMC contractile phenotype and promotes vascular disease through dysregu- lation of MRTFA nuclear translocation and inhibition of SENCR. Three inter-related specific aims are pro- posed to address this hypothesis using novel mouse models and innovative concepts. In Aim 1, we will elucidate the role of MAPK14 in VSMC phenotypic plasticity leading to vascular disease. In Aim 2, we will elucidate the integrative role of MAPK14 and MRTFA in regulating VSMC differentiation. In Aim 3, we will elucidate the regulation and function of MAPK14-dependent inhibition of SENCR in VSMC. Collectively, these studies will illuminate an important and heretofore unrecognized role for MAPK14 in negatively regulating VSMC differentiation, thus providing fresh insight into the molecular control of VSMC phenotype. These studies also challenge the paradigm of p38MAPK as a pro-VSMC differentiation signaling pathway and will link this pathway to the antagonism of two key downstream targets (MRTFA and SENCR). Information gained through these studies has intriguing therapeutic potential for designing novel strategies to combat vascular diseases.