Regulation and Function of AKAP12A in the Vessel Wall

描述（由申请人提供）：a -激酶锚定蛋白（AKAPs）作为分子支架，指导与细胞生长、迁移和分化相关的环境依赖性信号事件。AKAP信号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VSMC的类维甲酸诱导基因筛选中发现了Akap12，并发现AKAP12A的特异性异构体AKAP12A通过血清反应因子（SRF）和SRF的有效协同激活因子心肌素（心肌素）的mRNA表达控制及其在实验和临床血管疾病中的下调，从而表明AKAP12A是VSMC分化的标志；另一种异构体（Akap12b）在VSMC体内含量较低，但在细胞培养中含量增加。整个Akap12基因座的基因失活导致VSMC迁移、增殖和IL6表达升高。体外敲除研究表明，AKAP12A在控制脂质摄取和炎症基因表达方面发挥着重要作用。重要的是，AKAP12A在VSMC中协调creb依赖的基因转录，包括VSMC特异性CNN1的新激活，这在体外对脂质积累和炎症基因表达以及在转基因小鼠中过度表达时对新内膜疾病具有深远的抵抗作用。此外，AKAP12A增强了心肌依赖的转激活，这是VSMC分化的一个关键分子过程。基于这些来自申请人实验室和其他实验室的强有力的初步和已发表的数据，我们试图验证srf依赖性AKAP12A维持正常VSMC稳态的新假设。我们提出了一系列相互关联的特异性目标，通过(1)利用转基因小鼠模型和microRNA分析阐明Akap12a的转录和转录后调控（specific Aim 1）来直接验证这一假设；(2)利用创新的遗传小鼠模型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，阐明AKAP12A在血管疾病中的作用（Specific Aim 2）；(3)通过对野生型或Akap12a敲除小鼠主动脉SMC衍生的RNA或染色质免疫沉淀CREB结合序列进行下一代测序，确定VSMC中Akap12a调控的“creome”。期望通过这些重点研究获得的信息将确立AKAP12A在对抗正常血管壁稳态扰动中的新的重要作用。反过来，这一信息将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在动脉粥样硬化或病源性血管闭塞等疾病中AKAP12A的急剧丧失，或诱导特定的下游AKAP12A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（CNN1）。我们还设想，这里获得的信息将直接应用于AKAP12A表达和下游活动受损的其他疾病环境（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).