In vivo analysis of signaling dynamics in the Notch interaction network

我们的项目重点是血管炎症。本项目的重点是单核细胞趋化蛋白-1(MCP-1)，这是一种促炎性CC趋化因子，在向动脉壁募集单核/巨噬细胞方面发挥关键作用，介导动脉粥样硬化的早期进展和动脉壁对损伤的反应。我们已经证明，MCP-1在SMC中的积累在很大程度上是通过改变mRNA的稳定性来调节的。生长激动剂，如PDGF和血管紧张素II(Ang II)，增加MCP-1 MRNA半衰期(t1/2)由45分钟缩短至3小时，而糖皮质激素则使t1/2缩短至15分钟。PDGF和Ang II稳定SMC中大量的mRNAs，而糖皮质激素地塞米松(Dex)选择性地破坏MCP-1mRNA的稳定。本项目的主要目标是确定参与调节SMC中MCP-1mRNA稳定性的分子和机制。在本奖项的当前任期内，我们已经取得了实质性的进展，并证明了地塞米松介导的MCP-1mRNA的失稳 涉及依赖糖皮质激素受体(GR)的新机制。与其更典型的转录因子作用不同，GR似乎是专门结合MCP-1mRNA的降解性复合体的一部分。利用RNA亲和方法，我们已经确定Y-box结合蛋白-1(YB-1)是该复合体的关键成分，Y-box结合蛋白-1是一种具有内切核酸酶特性的多功能DNA和RNA结合蛋白。我们认为，我们已经发现了一种新的抗炎途径，它是由糖皮质激素/GR介导的，涉及MCP-1mRNA的降解。这一更新将集中于充分阐明这条途径。我们提出了4个目标：1)确定YB-1和GR介导地塞米松诱导MCP-1mRNA降解的机制。2)确定参与调节MCP-1mRNA稳定性的GR结构域。3)确定在SMC中受YB-1和GR依赖、地塞米松介导的mRNA失稳调节的其他炎症介质。4)证实在地塞米松对体内MCP-1mRNA和蛋白的影响中，mRNA稳定性的变化起着重要的作用。这些研究可能允许开发出 模拟糖皮质激素的强大抗炎作用，而不会引起其无数的副作用。

Our program project focuses on vascular inflammation. This project focuses on Monocyte chemoattractant protein-1 (MCP-1), a pro-inflammatory CC chemokine that plays a pivotal role in recruiting monocytes/macrophages to the arterial wall, mediating both early progression of atherosclerosis and response of the arterial wall to injury. We have demonstrated that accumulation of MCP-1 in SMC is mediated in large part by changes in mRNA stability. Growth agonists, such as PDGF and angiotensin II (Ang II), increase MCP-1 mRNA half-life (t1/2) from ¿45 min to >3 hr, whereas glucocorticoids decrease the t1/2 to <15 min. Whereas PDGF and Ang II stabilize numerous mRNAs in SMC, the glucocorticoid Dexamethasone (Dex) selectively destabilizes MCP-1 mRNA. The primary goal of this project is to identify the molecules and mechanisms involved in mediating MCP-1 mRNA stability in SMC. We have made substantial progress during the current term of this award, and have demonstrated that Dex-mediated destabilization of MCP-1 mRNA involves a novel mechanism dependent upon the glucocorticoid receptor (GR). Unlike its more typical role as a transcription factor, the GR appears to be part of a degradative complex that specifically binds MCP-1 mRNA. Using an RNA affinity approach, we have identified Y-box binding protein-1 (YB-1), a multifunctional DNA and RNA binding protein with endoribonuclease properties, as a key component of this complex. We believe that we have identified a novel anti-inflammatory pathway mediated by glucocorticoids/GR that involves degradation of MCP-1 mRNA. This renewal will focus on fully elucidating this pathway. We propose 4 aims: 1) Determine the mechanism by which YB-1 and the GR mediate degradation of MCP-1 mRNA in response to Dex. 2) Identify the domains of the GR involved in regulating MCP-1 mRNA stability. 3) Identify other mediators of inflammation that are regulated in SMC by YB-1- and GR-dependent, Dex-mediated mRNA destabilization. 4) Establish that changes in mRNA stability plays an important role in mediating the effect of Dex on MCP-1 mRNA and protein in vivo. These studies may allow development of agents that mimic the potent anti-inflammatory effects of glucocorticoids without inducing their myriad side effects.