Regulation and Function of AKAP12A in the Vessel Wall

描述(由申请人提供)：A-Kinase锚定蛋白(AKAP)作为分子支架，指导与细胞生长、迁移和分化相关的上下文相关信号事件。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的视黄醇诱导基因筛选中发现了AKAP12，并证明了AKAP12A是VSMC分化的标志，这是基于其通过血清反应因子(SRF)和强大的SRF辅助激活因子Myocardin(MYOCD)的mRNA表达调控以及在实验和临床血管疾病中的下调；另一种异构体(Akap12b)在体内VSMC中低表达，但在细胞培养中增加。整个AKAP12基因座的基因失活会导致VSMC迁移、增殖和IL6表达的增加。体外基因敲除研究表明，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协调VSMC中依赖CREB的基因转录，包括新的VSMC特异性CNN1的激活，该CNN1在体外对脂质堆积和炎性基因表达以及在转基因小鼠中过表达时对新生内膜疾病具有深刻的抵抗作用。此外，AKAP12A增强了MYOCD依赖的反式激活，这是VSMC分化的关键分子过程。基于这些来自申请人实验室和其他实验室的强大的初步和已发表的数据，我们试图测试依赖SRF的AKAP12A维持正常的VSMC动态平衡的新假设。我们提出了一系列相互关联的特定目标，将通过以下方式直接检验这一假说：(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(特异性目标1)；(2)通过与我们实验室培育或最近获得的SMC特异性CRE重组酶小鼠杂交的创新遗传小鼠模型(特异性目标2)，阐明AKAP12A在血管疾病中的作用；以及(3)通过对来自野生型或Akap12a基因敲除小鼠的最新一代RNA或染色质免疫沉淀CREB结合序列的测序来定义AKAP12A调控的VSMC中的“CREBome”。预计通过这些重点研究获得的信息将确立AKAP12A在对抗正常血管壁内稳态扰动方面的新的和重要的作用。这些信息反过来将引发开发药物或遗传干预措施的努力，这些干预措施要么可以阻止动脉粥样硬化或医源性血管闭塞等疾病中AKAP12A基因的急剧丢失，要么可以诱导特定的下游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表达和下游活性受到影响的其他疾病背景(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).