Rac1 Stimulation in Adenosine-Induced Barrier Protection

项目总结(见说明)： 血管内皮细胞(EC)在血管内部空间和底层组织之间形成半透屏障。在急性肺损伤中，EC屏障减弱，导致通透性增加。在调节EC屏障功能的机制中，Rho/vRad平衡似乎是重要的，其中RhoA激活是屏障破坏性的，Rad激活是屏障保护的。本项目将阐明Rad屏障保护作用的潜在机制。我们发现，腺苷诱导Rad的激活迅速增加，这与在体外和体内显着降低脂多糖诱导的EC通透性有关。腺苷增强屏障功能的能力似乎依赖于MLCP，并与cAMP水平的升高相关。然而，cAMP与Rad激活偶联的机制环节增加，MLCP的刺激和屏障保护作用尚不清楚。 因此，在这个项目中，我们提出了一种新的机制，通过Epac1-Rap1-和蛋白激酶A(PKA)-信号介导的Rad和MLCP的协同激活和相互作用，来研究腺苷诱导的EC屏障增强/保护。我们还将确定Rad和MYPT1之间的相互作用是否参与调节MLCP的激活，以及Epac1-Rap1和PKA在这一过程中的作用。最后，我们将检验MLCP激活通过新的细胞骨架蛋白：ERM蛋白(Ezrin、Radix和Moesin)和caldesmon的去磷酸化而导致EC屏障增强/保护的假设。因此，本项目的总体目标是阐明 Rad参与腺苷对EC屏障增强/保护的分子机制。具体的具体目标是： 明确腺苷诱导的Rad和MLCP激活之间的联系，并确定它们在体外和体内EC屏障保护中的作用。2.明确MLCP细胞骨架靶点在Rad诱导的EC屏障增强/保护中的作用。

PROJECT SUMMARY (See instructions): Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury the EC barrier is weakened leading to increased permeability. Among the mechanisms regulating EC barrier function, Rho/VRad balance appears to be of major importance with RhoA activation being barrier-destructive and Rad activation is barrier-protective. This Project will elucidate the mechanisms underlying the barrier protective effects of Rad. We show that adenosine induces rapid increases in Rad activation and this correlates with a significant attenuation of LPS-induced EC permeability in vitro and in vivo. The ability of adenosine to enhance barrier function appears to be dependent on MLCP and correlates with increased levels of cAMP. However, the mechanistic links coupling increases in cAMP with the activation of Rad, MLCP stimulation and barrier protection are unknown. Thus, in this project we propose to investigate a novel mechanism of adenosine-induced EC barrier enhancement/protection via the coordinated activation, and interaction, of Rad and MLCP that is mediated by Epac1-Rap1- and protein kinase A (PKA)-signaling. We will also determine if the interactions between Rad and MYPT1 are involved in regulating MLCP activation and if the role of Epac1-Rap1 and PKA in this process. Finally, we will test the hypothesis that MLCP activation leads to EC barrier enhancement/protection via the dephosphorylation of the novel cytoskeletal proteins: the ERM proteins (ezrin, radixin, and moesin), and caldesmon. Thus, the overall objective of this project is to elucidate the molecular mechanisms through which Rad is involved in EC barrier enhancement/protection in response to adenosine. The detailed specific aims are: SA #1. To define the links between adenosine-induced Rad and MLCP activation and determine their role in EC barrier protection in vitro and in vivo SA #2. To define the involvement of MLCP cytoskeletal targets in Rad-induced EC barrier enhancement/protection in vitro and in vivo.