Cdc42bpg signaling in arteriosclerosis and vascular fibrosis

心血管疾病是美国人的主要死因。努力控制血压、胆固醇、 糖尿病通过减轻动脉粥样硬化和血栓栓塞症产生了显著但不完全的好处。 动脉硬化性管道血管硬化是一种重叠但不同的疾病，也会增加中风的风险， 心力衰竭和截肢风险。目前还没有治疗动脉硬化或病理性血管的疗法。 钙化和纤维化。我们发现血管平滑肌(VSM)甲状旁腺激素受体 (PTH1R)信号限制纤维化，下调由混合CT(A/T)6GG MEF/SRF驱动的转录 Col3a1和Cola1基因中的同源物。PTH1R抑制麦卡菌素相关转录因子-a(MKL1) 这些元素的激活。我们确定了肌营养不良症蛋白激酶家族成员-包括 ROCK2和CDC42结合蛋白(CDC42bps)-在支持VSM Col3a1基因表达中发挥关键作用 下游的PTH1R介导的抑制。Cdc42bpg尤其重要-击倒几乎 完全逆转VSM PTH1R缺乏症的Col3a1诱导-并选择性上调 血管紧张素II(AngII)静脉注射。此外，cdc42bpg的表达显著刺激 依赖于MKL1的Col3a1启动子的激活--首个已知的对CDC42bpg的上下文信息分析 生物活性。根据Pharos的说法，CDC42bpg上只有8种出版物，对其功能的分析是基本的 根本不存在。我们为这一年的R03提出了以下任务：任务1：推进和完善我们的新 基于转录的cdc42bpg活性分析，用于系统的结构-功能分析。这个 在HEK293T细胞中应用CDC42bpg转录调控分析来鉴定CDC42bpg结构域 通过MKL1支持Col3a1基因表达所必需的。在A7r5中独立确认了关键观察结果 主动脉VSM。任务2：建立受PTH1R调控的CDC42bpg相互作用组。Cdc42不太可能 与纤维化相关的CDC42bpg是唯一的，甚至是主要的调节因子。Cdc42bpg互动组将有所帮助 确定相关监管机构，提供有助于细化结构活动的见解。串联液 色质联用法将鉴定在与蛋白质发生交联后共沉淀的蛋白质 表位标记的CDC42bpg&在HEK和A7r5细胞中表达的关键亚域。重点将放在那些 受PTH1R信号调控的CDC42bpg相互作用调节Col3a1基因的表达。任务3： 研究CDC42bpg缺乏对血管紧张素转换酶诱导的心血管纤维化的影响。CDC42bpg-/- KOMP2-BCM已生成基因敲除小鼠(C57BL/6N背景)，正在导入到 托勒实验室。B6N小鼠对血管紧张素转换酶诱导的纤维化非常敏感。因此，动脉、心肌和肾脏 B6N.CDC42bpg-/-小鼠的纤维化将与对照组B6N动物进行比较，这些动物接受血管紧张素转换酶抑制剂的挑战 第一次活体评估。Cdc42bpg-空等位基因将被交叉到LDLR-/-背景上，从而启用 Cdc42bpg在饮食诱导的动脉粥样硬化、糖尿病和代谢综合征中的未来研究。

Cardiovascular disease is leading cause of death in the U.S. Assiduous control of blood pressure, cholesterol, and diabetes has yielded significant but incomplete benefit by mitigating atherosclerosis & thromboembolism. Arteriosclerotic conduit vessel stiffening, an overlapping yet distinct disease, also increases risk for stroke, heart failure and amputation risk. No therapies currently treat arteriosclerosis, or the pathological vascular calcification and fibrosis. We've identified that vascular smooth muscle (VSM) parathyroid hormone receptor (PTH1R) signaling limits fibrosis, down regulating transcription driven by hybrid CT(A/T)6GG MEF/SRF cognates in Col3a1 and Cola1 genes. PTH1R restrains mycardin-related transcription factor – a (Mkl1) activation of these elements. We identified that muscular dystrophy protein kinase family members – including ROCK2 & Cdc42 binding proteins (Cdc42bps) – play critical roles in supporting VSM Col3a1 gene expression downstream of PTH1R – mediated inhibition. Cdc42bpg is particularly important - with knockdown almost completely reversing Col3a1 induction occurring with VSM PTH1R deficiency – and is selectively upregulated in aorta by profibrotic angiotensin II (AngII) infusion. Moreover, Cdc42bpg expression significantly stimulates Mkl1-dependent activation of the Col3a1 promoter – the first known, context-informed assay of Cdc42bpg bioactivity. Per Pharos only 8 publications exist on Cdc42bpg, and assays of its functions are rudimentary to non-existent. We propose the following Tasks for this 1-year R03: Task1: Advance and refine our new transcription-based assay of Cdc42bpg activity for systematic domain structure-function analyses. The Cdc42bpg transcriptional regulation assay is deployed in HEK293T cells to identify Cdc42bpg domains necessary to support Col3a1 gene expression via Mkl1. Key observations are independently confirmed in A7r5 aortic VSM. Task2: Establish the Cdc42bpg interactome as regulated by the PTH1R. Cdc42 is unlikely to be the only, or even primary, regulator of Cdc42bpg as relevant to fibrosis. The Cdc42bpg interactome will help identify relevant regulators, providing insights useful for detailed structure-activity refinement. Tandem liquid chromatography – mass spectrometry will identify proteins that co-precipitate following cross-linking with epitope-tagged Cdc42bpg & key subdomains expressed in HEK and A7r5 cells. Focus will be upon those Cdc42bpg interactions that are regulated by PTH1R signals & modulate Col3a1 gene expression. Task3: Study the impact of Cdc42bpg deficiency in AngII - induced cardiovascular fibrosis. The Cdc42bpg-/- knockout mouse (C57BL/6N background) has been generated by KOMP2-BCM, & is being imported to the Towler lab. B6N mice are very susceptible to AngII-induced fibrosis. Thus, arterial, myocardial, and renal fibrosis in the B6N.Cdc42bpg-/- mice will be compared to control B6N animals challenged with AngII infusion in the first in vivo assessment. Cdc42bpg-null alleles will be crossed onto the LDLR-/- background, enabling future studies of Cdc42bpg in diet-induced atherosclerosis, diabetes, & metabolic syndrome.