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

项目摘要/摘要 高血压是心血管疾病最可预防的危险因素，每3人中就有1人受到影响 美国人。心血管疾病是全球每年导致死亡的主要原因，平均每年 每年直接医疗费用近10亿美元的经济负担和经济生产力的损失。健康 血管功能是由一氧化氮(NO)信号通路维持的，该通路在允许 用于适当扩张血管以应对较高的血压。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着关键作用， 环鸟苷-3‘，5’-单磷酸(CGMP)，诱导SMC下游松弛。尽管扮演着不可或缺的角色 SGC在血管张力调节中的作用，SMC中sGC基因表达的调控机制仍然存在。 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 结合在sGca和b启动子上。我们的初步数据表明，FoxO的药理抑制作用 携带AS1842856基因的大鼠主动脉系膜细胞中的转录因子导致sGCα和sGCβ表达下降90-95%。 SGCα和sGCβ蛋白表达下降70-80%，cGMP产量下降90%。 不是捐献者，不是戒毒者。同样，用FoxO抑制剂治疗小鼠离体主动脉环可导致48%的死亡率。 免疫荧光检测sGCβ蛋白表达缺失，并显著钝化NO依赖 血管松弛。因此，我们试图确定三个FoxO转录因子中的哪一个在SMC中表达 (Foxo1、FOXO3a和FOXO4)通过发展腺病毒负责调节SMC中sGC的表达 FOXO shRNA构建瞬时下调每个转录因子的表达。我们的初步数据显示 在FoxO1或FOXO3a shRNA处理后，sGC的表达增加。相反，对大鼠的治疗 经FOXO4shRNA处理的主动脉系膜细胞sGCα、sGCβ和sGCβ蛋白表达均下降50%。这些 初步数据导致了以下目标的形成：1)阐明FOXO4是否对sGC负责 SMC中的转录调控以及这一途径是如何调节的，以及2)决定了 调控FOXO4表达对NO介导的血管反应性体外血管松弛的影响。在目标1中，我们将尝试 1.1)确定FOXO4 shRNA敲除后对下游sGC信号的影响，1.2)产生sGC 启动子-荧光素酶构建并利用染色质免疫沉淀(CHIP)实验来确定 FOXO4能够结合并启动SMC内sGC的转录，以及1.3)确定是否为瞬时的 FoxO蛋白的敲除/挽救足以调节sGC mRNA和蛋白的表达。为了评估目标2， 我们将2.1)用FOXO4 shRNA处理小鼠离体主动脉环，以确定FOXO4表达缺失的影响 在体外，2.2)我们将用AS1842856(FoxO抑制剂)处理分离的小鼠主动脉，以诱导NO-1的丢失 依赖血管反应性，继而诱导人FOXO4过表达，并阐明FOXO4 过表达可以在体外挽救下游的sGC信号。我的指导团队和 匹兹堡大学可用的资源将使我能够定义FOXO4是否是主要的 SGC在维管系统中的转录调节因子。完成这些目标将对过剩产生广泛的影响 并有助于确定基因研究和治疗的潜在新靶点 高血压的症状。

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.