Stat3 in Post-Ischemic Myocardial Inflammation

心肌再灌注导致通过激活氧化还原敏感的 核转录因子我们发现信号转导子和转录激活子-3 （Stat 3）在心肌缺血-再灌注后被激活，其方式依赖于小GT3受体Rac 1， 并与细胞间粘附分子-1（ICAM-1）基因中的GAS启动子元件结合，该基因 编码与白细胞相互作用的重要内皮细胞粘附分子。磷酸化后 在其丝氨酸727残基中，StatS与转录激活因子Sp1结合。我们假设这代表了 缺血再灌注后内皮细胞ICAM-1转录增强的新机制。在 抑制培养的内皮细胞中Stat 3/Sp1的活性显著降低了ICAM-1的转录， 缺氧复氧后1例。在这个建议中，我们将确定StatS在ICAM-1调节中的作用 以及心肌缺血再灌注后血管内皮中的其他促炎基因。在目标1中， 将研究StatS与Sp1相互作用的分子机制，包括 参与两种分子之间的结合以及StatS S727残基的磷酸化作用。我们 将确定Stat 3-Sp1相互作用的抑制是否影响EC中ICAM-1的基因调控，以及是否 Stat 3/Sp1改变缺血后微血管炎症（通过视频显微镜评估）或心肌 缺血-再灌注损伤，使用体内鼠模型。在目标2中，我们将研究负责的机制 缺氧-复氧后Rac 1依赖性StatS激活，包括两种分子如何结合在一起， 这种结合如何促进StatS的Y 705和S727残基的磷酸化，以及哪些激酶 涉案在目的3中，我们将确定缺氧-复氧EC中StatS的激活是否是由一个 负反馈环参与StatS转录调控T细胞淋巴瘤侵袭和转移2 （TIAM 2），一种鸟嘌呤交换因子，和RacGAPI，一种GTP酶激活蛋白，两者又 下调Rac 1的激活状态，从而下调StatS本身的激活状态。

Myocardial reperfusion results in the induction of pro-inflammatory genes through the activation of redoxsensitive nuclear transcription factors. We have found that signal transducer and activator of transcription-3 (Stat3) is activated after myocardial ischemia-reperfusion, in a manner dependent on the small GTPase Rac1, and binds to a GAS promoter element in the intercellular adhesion molecule-1 (ICAM-1) gene, a gene which codes for an important endothelial cell adhesion molecule which interacts with leukocytes. After phosphorylation of its serine 727 residue, StatS binds to the transcriptional activator Sp1. We hypothesize that this represents a novel mechanism for enhancing ICAM-1 transcription in endothelial cells (ECs) after ischemia-reperfusion. In support of this idea, inhibition of Stat3/Sp1 activity in cultured ECs significantly reduces the transcription of ICAM- 1 after hypoxia-reoxygenation. In this proposal, we will determine the role of StatS in the regulation of ICAM-1 and other pro-inflammatory genes in vascular endothelium after myocardial ischemia-reperfusion. In Aim 1 we will investigate the molecular mechanisms responsible for StatS interaction with Sp1, including the mechanisms involved in binding between the two molecules and the role of phosphorylation of the StatS S727 residue. We will determine whether inhibition of the Stat3-Sp1 interaction affects ICAM-1 gene regulation in ECs, and whether Stat3/Sp1 modifies post-ischemic microvascular inflammation (assessed by videomicroscopy) or myocardial ischemia-reperfusion injury, using in vivo murine models. In Aim 2 we will examine the mechanisms responsible for Rac1-dependent StatS activation after hypoxia-reoxygenation, including how the two molecules bind together, how this binding promotes the phosphorylation of the Y705 and S727 residues of StatS, and which kinases are involved. In Aim 3 we will determine whether activation of StatS in hypoxic-reoxygenated ECs is self-limited by a negative feedback loop involving StatS transcriptional regulation of T-cell lymphoma invasion and metastasis 2 (TIAM2), a guanine exchange factor, and RacGAPI, a GTPase-activating protein, both of which in turn downregulate the activation state of Rac1, and thereby of StatS itself.