Cdc42bpg signaling in arteriosclerosis and vascular fibrosis

在美国，心血管疾病是导致死亡的主要原因。严格控制血压、胆固醇， 糖尿病通过减轻动脉粥样硬化和血栓栓塞而产生了显著但不完全的益处。 动脉导管血管硬化是一种重叠但不同的疾病，也会增加中风的风险， 心脏衰竭和截肢风险。目前没有治疗动脉硬化或病理性血管病变的疗法。 钙化和纤维化。我们已经确定血管平滑肌（VSM）甲状旁腺激素受体 （PTH 1 R）信号传导限制纤维化，下调杂交CT（A/T）6 GG MEF/SRF驱动的转录 Col 3a 1和Cola 1基因的同源物。PTH 1 R抑制Mycardin相关转录因子- a（Mkl 1） 激活这些元素。我们发现肌营养不良蛋白激酶家族成员-包括 ROCK 2和Cdc 42结合蛋白（Cdc 42 bps）-在支持VSM Col 3a 1基因表达中起关键作用 PTH 1 R介导的抑制的下游。cdc 42 bpg是特别重要的-与击倒几乎 完全逆转VSM PTH 1 R缺乏时发生的Col 3a 1诱导-并选择性上调 血管紧张素II（AngII）。此外，Cdc 42 bpg表达显著刺激了 Col 3a 1启动子的Mkl 1依赖性激活-Cdc 42 bpg的第一个已知的上下文信息测定 生物活性根据Pharos，关于Cdc 42 bpg只有8篇出版物，其功能的测定是初步的， 根本不存在我们为这一年的R 03提出以下任务：任务1：推进和完善我们的新 Cdc 42 bpg活性的基于转录的测定用于系统结构域结构-功能分析。的 在HEK 293 T细胞中部署Cdc 42 bpg转录调控测定以鉴定Cdc 42 bpg结构域 需要通过Mkl 1支持Col 3a 1基因表达。关键观察结果在A7 r5中得到独立证实 主动脉VSM。任务2：建立由PTH 1 R调控的Cdc 42 bpg相互作用组。cdc 42不太可能 是与纤维化相关的Cdc 42 bpg的唯一或甚至主要调节剂。Cdc 42 bpg相互作用组将有助于 确定相关的监管机构，提供有用的见解详细的结构-活性细化。串联液相 色谱-质谱法将鉴定在交联后共沉淀的蛋白质， 表位标记的Cdc 42 bpg &在HEK和A7 r5细胞中表达的关键亚结构域。重点将放在那些 Cdc 42 bpg相互作用由PTH 1 R信号调节并调节Col 3a 1基因表达。任务3： 研究Cdc 42 bpg缺陷在AngII诱导的心血管纤维化中的影响。Cdc42bpg-/- 敲除小鼠（C57 BL/6 N背景）已由KOMP 2-EXP生成，并正在导入到 托勒实验室。B6 N小鼠对AngII诱导的纤维化非常敏感。因此，动脉、心肌和肾 将B6N.Cdc42bpg-/-小鼠中的纤维化与用AngII输注激发的对照B6 N动物进行比较， 第一次体内评估。Cdc 42 bpg-无效等位基因将与LDLR-/-背景杂交， Cdc 42 bpg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.