Cytokine signaling in activation of the endothelium

该项目的中心目标是定义信号调节因子和转录因子在 血管内皮细胞(EC)的细胞因子激活。我们的重点是肿瘤坏死因子-a受体-11(P75)和 同源框蛋白HOXA9。我们最近已经证明，这两个分子都是细胞因子诱导EC- 白细胞黏附分子E-选择素，以及其他激活基因。我们发现，肿瘤坏死因子刺激的白细胞 在p75缺失的小鼠中，与EC的牢固黏附显著减少，但在p55(另一种肿瘤坏死因子受体)缺失的小鼠中不受影响 老鼠。两种受体均参与了肿瘤坏死因子诱导的白细胞滚动和迁移。此外，p75和p55都是 E-选择素和VCAM-1的诱导需要p75，而ICAM-1的诱导需要p75。我们也 研究表明，在apoE基因缺失的小鼠中，p75基因缺失减少了动脉粥样硬化病变的发展。基于这些 结果，我们假设EC中p75的激活触发了细胞内信号通路，而不是由p55诱导的 是肿瘤坏死因子诱导的EC促炎基因表达所必需的。我们最近还表明， 转录因子HOXA9是肿瘤坏死因子诱导血管内皮细胞E-选择素的必需因子，因为它与一种新的 在启动子中发现了类似Abd-B的位点。导致HOXA9与E-选择素前体结合的肿瘤坏死因子信号 MOTER是由P75而不是P55触发的。我们推测HOXA9在血管内皮细胞的激活中起着新的作用。 对炎性刺激反应迟钝。我们建议通过追求三个目标来检验我们的假设。第一个是确定肿瘤坏死因子- 用肿瘤坏死因子处理分离的小鼠内皮细胞中诱导的、p75特异的信号通路和p75诱导的新基因 来自p55-/-、p75-/-和p55-p75-双零小鼠。我们还将确定肿瘤坏死因子诱导的p75和p75的结合伙伴 确定p75中负责诱导EC基因表达的结构域。第二个目的是阐明 HOXA9介导的E-选择素表达的分子机制及寻找HOXA9在EC中的新靶基因。 我们将继续研究HOXA9的翻译后修饰及其可能与其他专性转录因子的结合 用于E-选择素的诱导。基因和启动子阵列研究将用于确定HOXA9的新靶基因。在 第三个目的我们将研究HOXA9结合伙伴蛋白精氨酸甲基转移酶5(PRMT5)的作用。 在激活的EC中使用生化、基于siRNA和药理学的方法。我们还计划识别蛋白质 被PRMT5甲基化，可能有资格成为EC激活的新介体。 相关性(请参阅说明)： EC排列着体内的所有血管，在炎症中发挥着关键作用--炎症是许多疾病的基础过程， 包括动脉粥样硬化。我们的研究将揭示炎症调控中的新分子参与者。这些球员 将代表抗炎疗法发展的新靶点，可用于治疗这种 冠心病和类风湿性关节炎等疾病。

The central objective of this Project is to define signaling mediators and transcription factors of critical importance in cytokine activation of vascular endothelial cells (EC). Our specific focus is on TNF-a receptor-ll (p75) and the homeobox protein HOXA9. We have recently shown that both molecules are required for cytokine induction of the EC- leukocyte adhesion molecule E-selectin, as well as other activation genes. We showed that TNF-stimulated leukocyte firm adhesion to EC was dramatically reduced in p75-null mice, but not affected in p55 (the other TNF receptor)-null mice. Both receptors participated in TNF-induced leukocyte rolling and transmigration. Furthermore, both p75 and p55 were required for TNF induction of E-selectin and VCAM-1; whereas, p75 was sufficient for ICAM-1 induction. We also demonstrated that p75-deficiency in apoE-null mice reduced atherosclerotic lesion development. Based on these findings, we hypothesize that p75 activation in EC triggers intra-cellular signaling pathways, not induced by p55, that are essential for TNF-induced pro-inflammatory gene expression in EC. We have also recently shown that the transcription factor HOXA9 is required for TNF induction of E-selectin in EC due to its transient binding to a newly identified Abd-B-like site in the promoter. The TNF signal that leads to association of HOXA9 with the E-selectin pro- moter is triggered by p75 and not p55. We hypothesize that HOXA9 plays a novel role in the activation of EC in res- ponse to inflammatory stimuli. We propose to test our hypotheses by pursuing three aims. The first is to identify TNF- induced, p75-specific signaling pathways and novel p75-induced genes in EC using TNF-treated mouse EC isolated from p55-/-, p75-/- and p55-p75-double null mice. We will also identify TNF-induced binding partners of p75 and determine structural domains in p75 responsible for induced gene expression in EC. The second aim is to elucidate the molecular mechanism of HOXA9-mediated E-selectin expression and to identify novel target genes for HOXA9 in EC. We will pursue post-translational modifications of HOXA9 and its possible binding to other obligate transcription factors for E-selectin induction. Gene and promoter array studies will be used to identify novel target genes of HOXA9. In the third aim we will characterize the role of the HOXA9-binding partner protein arginine methyl transferase 5 (PRMT5) in activated EC using biochemical, siRNA-based and pharmacological approaches. We also plan to identify proteins methylated by PRMT5 that may qualify for novel mediators of EC activation. RELEVANCE (See instructions): EC, which line all blood vessels in the body, play a key role in inflammation - a process that underlies many diseases, including atherosclerosis. Our studies will reveal new molecular players in the regulation of inflammation. These players will represent novel targets for the development of anti-inflammatory therapeutics that may be used to treat such diseases as coronary artery disease and rheumatoid arthritis.