Ganglioside Gene Modulation Effect on Cell Motility

描述（由申请人提供）： 神经节苷脂，细胞膜的鞘糖脂，已显示在体外影响正常角质形成细胞和角质形成细胞衍生的SCC 12细胞的几个生物过程和信号通路。这些细胞的功能，增殖和粘附，扩散，并在纤连蛋白基质上迁移，激活表皮生长因子受体和α 5 β 1整合素，并对伤口愈合至关重要。细胞膜中含有小窝蛋白-1的信号结构域是受体和信号分子偶联的位点。我们已经表明，神经节苷脂与小窝蛋白-1，α 5 β 1和EGFR共免疫沉淀，并表明神经节苷脂影响小窝蛋白-1介导的信号传导。我们最近已经证明，神经节苷脂GM 3转移小窝蛋白-1从洗涤剂不溶性域洗涤剂可溶性域与EGFR的接近。鉴于已知小窝蛋白-1对EGFR的抑制作用，这一发现表明神经节苷脂可能作为穿梭分子促进小窝蛋白-1与受体的相互作用。在这个竞争的应用程序中，我们将继续支持探索神经节苷脂，小窝蛋白-1，和α 5 β 1整合素之间的关系，以及这些相互作用在细胞运动中的作用。我们将调查：a）通过免疫共沉淀和免疫显微镜检查，神经节苷脂与小窝蛋白-1、受体和信号分子的共定位; B）神经节苷脂功能对完整小窝结构域和/或小窝蛋白-1的依赖性。我们将通过β-环糊精破坏小窝结构域，并通过引入反义寡聚体和突变小窝蛋白-1特异性干扰小窝蛋白-1的功能; c）小窝蛋白-1磷酸化对细胞运动性的影响;和d）神经节苷脂和小窝蛋白-1直接结合的可能性。将使用表皮特异性神经节苷脂耗竭的转基因小鼠模型评估神经节苷脂调节对伤口愈合的体内作用。这些研究将提供有关神经节苷脂在小窝蛋白-1介导的信号传导中的作用的信息，并可能为异常愈合反应的患者提供新的治疗方法。

DESCRIPTION (provided by applicant): Gangliosides, glycosphingolipids of the cell membrane, have been shown in vitro to impact several biologic processes and signaling pathways of normal keratinocytes and the keratinocyte-derived SCC12 cells. These cell functions, proliferation and adhesion, spreading, and migration on a fibronectin matrix, activation of the epidermal growth factor receptor and alpha5beta1integrin and are critical for wound healing. Caveolin-l-containing signaling domains in membranes are sites of coupling of receptors and signaling molecules. We have shown that gangliosides co-immunoprecipitate with caveolin-1, alpha5beta1 and the EGFR, and suggest that gangliosides affect caveolin-1 mediated signaling. We have recently demonstrated that ganglioside GM3 shifts caveolin-1 from detergent-insoluble domains into detergent-soluble domains in proximity with the EGFR. Given the known inhibitory effect of caveolin-1 on the EGFR, this finding suggests that ganglioside may serve as a shuttle molecule to facilitate caveolin-1 interaction with receptors. In this competing application for continuing support we will explore the relationships among gangliosides, caveolin-1, and alpha5beta1 integrin, and the role of these interactions in cell motility. We will investigate: a) the colocalization of gangliosides with caveolin-1, receptors, and signaling molecules by co-immunoprecipitation and immunomicroscopy; b) the dependence of ganglioside function on the intact caveolar domain and/or caveolin-l. We will disrupt caveolar domains with by beta-cyclodextrin and will interfere specifically with caveolin-1 function by introducing antisense oligomers and mutant caveolin-1; c) the effect of caveolin-1 phosphorylation on cell motility; and d) the possibility of direct binding of gangliosides and caveolin-l. The in vivo effect of ganglioside modulation on wound healing will be assessed using a transgenic mouse model of epidermis-specific ganglioside depletion. These studies will provide information about the role of gangliosides in caveolin-1 mediated signaling and may lead to novel therapies for patients with abnormal healing responses.