Novel Role of MAPK14 in Regulation of VSMC Contractile Phenotype

描述（由申请方提供）：血管平滑肌细胞（VSMC）表现出导致人类血管疾病的表型可塑性。血清反应因子（SRF）和辅活化剂的Myocardin家族（例如，MRTFA）已经成为VSMC收缩表型的关键诱导剂。虽然SRF-MRTFA依赖的基因表达存在很多工作，一个关键的差距存在于我们的理解信号通路，收敛于SRF-MRTFA的负面影响VSMC基因表达。我们和其他人已经提出p38 MAPK信号传导作为TGF β 1诱导的VSMC分化的关键信号转导子;然而，迄今为止的所有研究都依赖于p38 MAPK的化学抑制剂的专门使用（例如，SB203580）。由于这种抑制剂可能具有与p38 MAPK无关的未知作用，我们对SB化合物靶向的VSMC中p38 MAPK的主要亚型p38 MAPK？（MAPK 14）进行了小干扰RNA敲低研究。我们发现MAPK 14的敲低刺激VSMC收缩基因的表达，而不是抑制VSMC基因的表达，如先前使用SB化合物的研究所预测的。使用几种定义明确的血管损伤模型，我们发现总的和磷酸化的MAPK 14在血管壁的新生内膜中富集，在那里表型改变的VSMC驻留，表明MAPK 14表达在某种程度上与合成的VSMC表型相关。新的，令人兴奋的数据显示，VSMC特异性Mapk 14敲除小鼠表现出诱导VSMC收缩基因表达，并完全抵抗损伤诱导的新生内膜形成。从机制上讲，我们发现MAPK 14调节MRTFA核质穿梭，这是SRF依赖的VSMC收缩基因表达的关键决定因素。我们最近的发现揭示了MAPK 14的缺失上调了一种新的平滑肌和内皮细胞富集的长非编码RNA（SENCR），其对VSMC收缩表型产生积极影响。基于这些发现，我们提出了MAPK 14拮抗VSMC收缩表型并通过MRTFA核转位失调和抑制SENCR促进血管疾病的总体假设。提出了三个相互关联的具体目标，以解决这一假设，使用新的小鼠模型和创新的概念。在目的1中，我们将阐明MAPK 14在导致血管疾病的VSMC表型可塑性中的作用。在目的2中，我们将阐明MAPK 14和MRTFA在调节VSMC分化中的整合作用。目的3：阐明MAPK 14依赖性抑制VSMC中SENCR的调节和功能。总的来说，这些研究将阐明MAPK 14在负调控VSMC分化中的重要且迄今未被认识的作用，从而为VSMC表型的分子控制提供新的见解。这些研究还挑战了p38 MAPK作为促VSMC分化信号通路的范例，并将该通路与两个关键下游靶点（MRTFA和SENCR）的拮抗作用联系起来。通过这些研究获得的信息对于设计对抗血管疾病的新策略具有有趣的治疗潜力。

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.