IN VIVO FUNCTIONS OF ADHESION AND SIGNALING MOLECULES

粘附和信号分子的体内功能

• 批准号: 6866593
• 负责人: RODGER PAUL MCEVER
• 金额: $ 42.42万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6866593
• 关键词: biological signal transduction; blood coagulation; cell adhesion; cytokine; fusion gene; gel mobility shift assay; gene targeting; genetically modified animals; glycoproteins; inflammation; laboratory mouse; ligands; protein purification; receptor binding; selectins; thrombosis; tissue /cell culture; vascular endothelium

Selectins initiate adhesion to the vessel wall during inflammation. P- selectin on activated platelets and endothelial cells binds to P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes. In vitro studies suggest that engagement on PSGL-1 transmits signals through the cytoplasmic domain. However, the adhesive and designing functions of PSGL-1 in vivo are poorly understood. In mice, TNF-alpha, IL-1beta, LPS, and oncostatin M increase synthesis of P-selectin in endothelial cells. In humans, only oncostatin M increase synthesis of P-selectin. The biological significance of this specifies difference is not known. Oncostatin M and other IL-6 family members bind to receptors that include gp130. In vitro studies suggest that signaling via gp130 induces synthesis of adhesion receptors and chemokines in endothelial cells. However, many cells express gp130, making it difficult to determine the function of gp130 signaling in endothelial cells in vivo. We will use gene targeting to study the expression and function of P-selectin, gp130 and PSGL- in vivo. First, we will make mice with a chimeric P-selectin gene in which we replace part of the 5' flanking region of the murine gene with the corresponding region from the human gene. We will determine whether the chimeric P-selectin gene responds to cytokines as in humans, and if so, will examine how P-selectin expressed in this manner contributes to models of inflammation and atherosclerosis. Second, we will use Cre-loxP methods to delete gp130 specifically in endothelial cells of mice. We will determine if oncostatin M fails to augment expression of P- and E-selectin and chemokines inflammation and atherosclerosis. Third, we will generate PSGL-1-null mice and mice expressing PSGL-1 without the cytoplasmic domain. We will use the mice to determine whether PSGL-12 couples adhesion to cytoplasmic- dependent signaling in models of inflammation and arterial injury. These studies will clarify the relationships between adhesion and signaling in the vasculature during inflammation.

在炎症期间，Selectins启动对血管壁的粘附。活化血小板和内皮细胞上的p-选择素与白细胞上的P-选择蛋白糖蛋白配体1（PSGL-1）结合。体外研究表明，对PSGL-1的参与通过细胞质结构域传输信号。然而，对体内PSGL-1的粘合剂和设计功能知之甚少。在小鼠中，TNF-ALPHA，IL-1BETA，LPS和ONOCOSTATIN M增加了内皮细胞中P-选择素的合成。在人类中，只有Oncostatin M增加了P-选择素的合成。该指定差异的生物学意义尚不清楚。 OnCostatin M和其他IL-6家族成员与包括GP130在内的受体结合。体外研究表明，通过GP130信号传导诱导内皮细胞中粘附受体和趋化因子的合成。但是，许多细胞表达GP130，因此很难确定体内内皮细胞中GP130信号的功能。我们将使用基因靶向研究P-选择素，GP130和PSGL-体内的表达和功能。首先，我们将用嵌合p-选择蛋白基因使小鼠取代鼠基因的5'侧翼区域的一部分，并用来自人类基因的相应区域。我们将确定嵌合p-选择素基因是否像人类一样对细胞因子反应，如果是的，则将检查P-选择素以这种方式表达的如何有助于炎症和动脉粥样硬化模型。其次，我们将使用CRE-LoxP方法在小鼠的内皮细胞中专门删除GP130。我们将确定Oncostatin M是否无法增强P-和E-选择素的表达以及趋化因子炎症和动脉粥样硬化。第三，我们将生成表达无细胞质结构域的PSGL-1小鼠的PSGL-1-NULL小鼠和小鼠。我们将使用小鼠来确定PSGL-12夫妇是否在炎症和动脉损伤模型中是否粘附于细胞质依赖性信号。这些研究将阐明炎症期间脉管系统中的粘附与信号传导之间的关系。