Investigating the role of FoxO transcriptional regulation of sGC in smooth muscle

研究 FoxO 转录调控 sGC 在平滑肌中的作用

• 批准号: 10388277
• 负责人: Joseph Carl Galley
• 金额: $ 4.68万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10388277
• 关键词: Affect; American; Aorta; Arteries; Binding; Blood Pressure; Blood Vessels; Cardiovascular Diseases; Cause of Death; Cell physiology; Complex; Critical Pathways; Cyclic GMP; DNA; DNA Binding; DNA Polymerase II; DNA Sequence; Data; Defect; Development; Disease; Economic Burden; Economics; Elements; Endothelium; Etiology; FOXO1A gene; Gene Expression; Genetic; Genetic Research; Genetic Transcription; Goals; Guanosine; Guanosine Triphosphate; Health Care Costs; Human; Hypertension; Immunofluorescence Immunologic; Impairment; Individual; Lead; Link; Luciferases; Measures; Mediating; Mentorship; Messenger RNA; Metabolism; Mus; Myography; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Signaling Pathway; Pathway interactions; Periodicity; Persons; Pharmacology; Play; Production; Productivity; Promoter Regions; Proteins; RNA; Rattus; Regulation; Relaxation; Reporter; Resources; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Soluble Guanylate Cyclase; Testing; Therapeutic; Transcription Repressor; Transcriptional Regulation; Universities; Vascular Smooth Muscle; Vasodilation; base; cGMP production; cardiovascular risk factor; chromatin immunoprecipitation; design; experimental study; hypertension treatment; improved; in silico; inhibitor; knock-down; mRNA Expression; novel; overexpression; promoter; protein expression; response; small hairpin RNA; transcription factor

PROJECT SUMMARY/ABSTRACT Hypertension constitutes the most preventable risk factor for cardiovascular disease, affecting nearly 1 out of every 3 Americans. Cardiovascular diseases are the leading causes of death each year across the globe, creating an average economic burden of nearly $1 billion dollars/year in direct healthcare costs and loss of economic productivity. Healthy vascular function is maintained by the nitric oxide (NO) signaling pathway, which plays a necessary role in allowing for proper dilation of blood vessels in response to higher blood pressure. Within vascular smooth muscle cells (SMC), the NO receptor, soluble guanylyl cyclase (sGC) plays a pivotal role in producing the second messenger molecule, cyclic guanosine-3’,5’-monophosphate (cGMP), to induce downstream relaxation of SMC. Despite the integral role that sGC plays in the regulation of vascular tone, the mechanisms that regulate sGC gene expression in SMC remain unknown. We recently found evidence that the Forkhead box class O (FoxO) transcription factors are capable of binding on both the sGCa and b promoters. Our preliminary data indicate that pharmacological inhibition of FoxO transcription factors in rat aortic SMC with AS1842856 results in a 90-95% loss of sGCα and sGCβ mRNA expression, a 70-80% loss of sGCα and sGCβ protein expression, and a 90% loss of cGMP production after stimulation by the NO-donor, DEA-NONOate. Likewise, treatment of isolated murine aortic rings with the FoxO inhibitor resulted in a 48% loss of sGCβ protein expression as assessed by immunofluorescence and significantly blunted NO-dependent vasorelaxation. Therefore, we sought to identify which of the three FoxO transcription factors expressed in SMC (FoxO1, FoxO3a, and FoxO4) is responsible for the regulation of sGC expression in SMC by developing adenoviral FoxO shRNA constructs to transiently knock down expression of each transcription factor. Our preliminary data show that sGC expression increases following treatment by either FoxO1 or FoxO3a shRNA. Conversely, treatment of rat aortic SMC with FoxO4 shRNA resulted in a 50% loss of sGCα and sGCβ mRNA and sGCβ protein expression. These preliminary data have led to the formation of the following aims: 1) Elucidate whether FoxO4 is responsible for sGC transcriptional regulation in SMC and how this pathway is modulated, and 2) Determine the consequences of manipulating FoxO4 expression on NO-mediated vasoreactivity ex vivo vasorelaxation. In Aim 1, we will attempt to 1.1) determine the downstream sGC signaling effects following FoxO4 shRNA knockdown, 1.2) generate sGC promoter-luciferase constructs and utilize chromatin immunoprecipitation (ChIP) experiments to determine whether FoxO4 is capable of binding and initiating transcription of sGC within SMC, and 1.3) determine if transient knockdown/rescue of FoxO proteins is sufficient to modulate sGC mRNA and protein expression. To assess Aim 2, we will 2.1) treat isolated mouse aortic rings with FoxO4 shRNA to determine the effect of loss of FoxO4 expression ex vivo, and 2.2) we will treat isolated murine aortas with AS1842856 (FoxO inhibitor) to induce the loss of NO- dependent vasoreactivity and subsequently induce overexpression of human FoxO4 and elucidate whether FoxO4 overexpression can rescue downstream sGC signaling ex vivo. The combination of my mentorship team and the available resources available at the University of Pittsburgh will allow me to define whether FoxO4 is the predominant transcriptional regulator of sGC in the vasculature. Completion of these aims will provide broad impact on a plethora of cardiovascular diseases and help to identify a potentially novel target for genetic research and therapeutic treatment of hypertension.

项目总结/摘要 高血压是心血管疾病最可预防的危险因素，每3个人中就有1个受到影响。 美国人心血管疾病是地球仪每年的主要死因， 直接医疗保健费用和经济生产力损失造成的经济负担近10亿美元/年。健康 血管功能是由一氧化氮（NO）信号通路维持的，其在允许 以适当地扩张血管来应对高血压。在血管平滑肌细胞（SMC）内， NO受体可溶性鸟苷酸环化酶（sGC）在产生第二信使分子中起关键作用， 环鸟苷-3 '，5'-单磷酸（cGMP），以诱导SMC的下游松弛。尽管不可或缺的作用 sGC在调节血管紧张性中起作用，但调控SMC中sGC基因表达的机制仍然存在 未知我们最近发现的证据表明，叉头盒O类（FoxO）转录因子能够 结合sGCa和B启动子。我们的初步数据表明，FoxO的药理学抑制 使用AS 1842856的大鼠主动脉SMC中的转录因子导致sGCα和sGCβ mRNA表达丢失90-95%， 在刺激后，sGCα和sGCβ蛋白表达损失70-80%，cGMP产生损失90%。 NO-供体，DEA-NONOate。同样，用FoxO抑制剂处理分离的鼠主动脉环导致48%的 通过免疫荧光评估sGCβ蛋白表达的缺失， 血管舒张因此，我们试图鉴定三种FoxO转录因子中哪一种在SMC中表达， （FoxO 1，FoxO 3a和FoxO 4）负责通过发育腺病毒介导的SMC中sGC表达的调节 FoxO shRNA构建体瞬时敲低每种转录因子的表达。我们的初步数据显示 在FoxO 1或FoxO 3a shRNA处理后sGC表达增加。相反，大鼠的治疗 FoxO 4 shRNA转染主动脉SMC后，sGCα和sGCβ mRNA及蛋白表达均下降50%。这些 初步的数据导致了以下目标的形成：1）阐明FoxO 4是否负责sGC SMC中的转录调控以及该途径是如何调节的，以及2）确定 操纵FoxO 4表达对NO介导的离体血管舒张的血管反应性。在目标1中，我们将尝试 1.1)确定FoxO 4 shRNA敲低后的下游sGC信号传导效应，1.2）产生sGC 启动子-荧光素酶构建体，并利用染色质免疫沉淀（ChIP）实验来确定是否 FoxO 4能够在SMC内结合并启动sGC的转录，并且1.3）确定是否是瞬时的 FoxO蛋白的敲低/拯救足以调节sGC mRNA和蛋白表达。为了评估目标2， 我们将2.1）用FoxO 4 shRNA处理分离的小鼠主动脉环以确定FoxO 4表达缺失的影响 离体，和2.2）我们将用AS 1842856（FoxO抑制剂）处理分离的小鼠睾丸，以诱导NO- 依赖性血管反应性并随后诱导人FoxO 4过表达，并阐明FoxO 4是否 过表达可以离体拯救下游sGC信号传导。我的导师团队和 匹兹堡大学的可用资源将使我能够确定FoxO 4是否是主要的 在脉管系统中sGC的转录调节因子。这些目标的实现将对许多国家产生广泛影响。 心血管疾病，并帮助确定一个潜在的新目标，遗传研究和治疗治疗 高血压