Regulation and Function of AKAP12A in the Vessel Wall

描述（由申请人提供）：A-激酶介导蛋白（AKAP）作为分子支架，指导与细胞生长、迁移和分化相关的背景依赖性信号传导事件。AKAP信号传导的主要下游底物之一是CREB转录因子，其在血管平滑肌细胞（VSMC）中的功能尚不清楚，特别是关于下游靶基因。我们在筛选VSMC中类维生素A诱导的基因中鉴定了Akap 12，并显示了一种特异性同种型AKAP 12 A是VSMC分化的标志物，这是基于其mRNA表达通过血清反应因子（SRF）和有效的SRF辅激活剂Myocardin（MYOCD）控制及其在实验和临床血管疾病中的下调;另一种同种型（Akap 12 b）在体内VSMC中低，但在细胞培养时增加。整个Akap 12基因座的基因失活导致VSMC迁移、增殖和IL 6表达升高。体外敲低研究表明AKAP 12 A在控制脂质摄取和炎症基因表达中具有重要作用。重要的是，AKAP 12 A协调VSMC中CREB依赖性基因转录，包括VSMC特异性CNN 1的新激活，其在转基因小鼠中过表达时赋予对脂质蓄积和体外炎症基因表达以及新生内膜疾病的深刻抗性。此外，AKAP 12 A增强MYOCD依赖性反式激活，这是VSMC分化中的关键分子过程。基于来自申请人实验室和其他实验室的这些强有力的初步和公开数据，我们试图检验SRF依赖性AKAP 12 A维持正常VSMC稳态的新假设。我们提出了一系列相互关联的具体目标，这些目标将通过以下方式直接验证这一假设：（1）使用转基因小鼠模型和microRNA分析阐明Akap 12 a的转录和转录后调控（具体目标1）;（2）使用与我们实验室开发或最近获得的SMC特异性Cre重组酶小鼠杂交的创新遗传小鼠模型阐明AKAP 12 A在血管疾病中的作用（具体目标2）;和（3）通过对来自野生型或Akap 12 a敲除小鼠主动脉SMC的RNA或染色质免疫沉淀的CREB结合序列进行最新一代测序，确定VSMC中AKAP 12 A调节的“CREBome”。预期通过这些集中研究获得的信息将确立AKAP 12 A在拮抗正常血管壁稳态的扰动中的新的重要作用。这些信息将反过来激发开发药理学或遗传干预措施的努力，这些干预措施将阻止动脉粥样硬化或医源性诱导的血管闭塞等疾病中AKAP 12 A的急剧损失，或诱导特定的下游AKAP 12 A底物，如预防或逆转疾病进展所必需的关键CREB依赖性靶基因（CNN 1）。我们还设想，这里获得的信息将直接应用于AKAP 12 A表达和下游活性受损的其他疾病背景（例如，癌症）。

DESCRIPTION (provided by applicant): A-Kinase Anchoring Proteins (AKAPs) act as molecular scaffolds to direct context-dependent signaling events associated with cell growth, migration, and differentiation. One of the major downstream substrates for AKAP signaling is the CREB transcription factor, whose functionality in vascular smooth muscle cells (VSMC) is not well understood, particularly with respect to downstream target genes. We identified Akap12 in a screen for retinoid-induced genes in VSMC and have shown a specific isoform, AKAP12A, to be a marker for VSMC differentiation based on its mRNA expression control through serum response factor (SRF) and the potent SRF coactivator, Myocardin (MYOCD) and its down-regulation in experimental and clinical vascular diseases; another isoform (Akap12b) is low in VSMC in vivo, but increases upon cell culture. Genetic inactivation of the entire Akap12 locus results in elevated VSMC migration, proliferation, and IL6 expression. In vitro knockdown studies demonstrate an important role for AKAP12A in the control of lipid uptake and inflammatory gene expression. Importantly, AKAP12A coordinates CREB-dependent gene transcription in VSMC, including novel activation of VSMC-specific CNN1, which confers profound resistance to lipid accumulation and inflammatory gene expression in vitro and neointimal disease when overexpressed in transgenic mice. Further, AKAP12A augments MYOCD-dependent transactivation, a key molecular process in the differentiation of VSMC. Based on these strong preliminary and published data from the applicant's lab and those of other labs, we seek to test the novel hypothesis that SRF-dependent AKAP12A maintains normal VSMC homeostasis. We propose a series of inter-related specific aims that will directly test this hypothesis by (1) elucidating the transcriptional and post-transcriptional regulatory control of Akap12a using transgenic mouse models and microRNA analyses (Specific Aim 1); (2) elucidating the role of AKAP12A in vascular disease using innovative genetic mouse models crossed with SMC-specific Cre recombinase mice our lab has developed or recently acquired (Specific Aim 2); and (3) defining the AKAP12A-regulated "CREBome" in VSMC through state-of-the-art next generation sequencing of RNA or chromatin immunoprecipitated CREB binding sequences derived from wildtype or Akap12a knockout mouse aortic SMC. It is expected that the information obtained through these focused studies will establish a new and important role for AKAP12A in antagonizing perturbations to normal vessel wall homeostasis. This information will, in turn, spark efforts to develop pharmacological or genetic interventions that would either thwart the dramatic loss in AKAP12A in such diseases as atherosclerosis or iatrogenic-induced vascular occlusion or induce specific downstream AKAP12A substrates such as critical CREB-dependent target genes (CNN1) necessary to prevent or reverse disease progression. We also imagine that information obtained here will have direct applications in other disease contexts where AKAP12A expression and downstream activities are compromised (e.g., cancer).