Ganglioside Gene Modulation Effect on Cell Motility

描述(由申请人提供)： 神经节苷脂是细胞膜上的神经节苷脂，在体外可以影响正常角质形成细胞和角质形成细胞来源的SCC12细胞的多种生物学过程和信号通路。这些细胞的功能、增殖和黏附、在纤维连接蛋白基质上的扩散和迁移、表皮生长因子受体和α5β1整合素的激活，对伤口愈合至关重要。膜上含有小窝L的信号域是受体和信号分子的偶联部位。我们发现神经节苷脂与小窝蛋白-1、α5β1和EGFR共沉淀，提示神经节苷脂影响小窝蛋白-1介导的信号转导。我们最近已经证明神经节苷脂GM3将小窝蛋白-1从不溶于洗涤剂的区域转移到靠近EGFR的可溶于洗涤剂的区域。鉴于小窝蛋白-1对EGFR的已知抑制作用，这一发现表明神经节苷脂可能作为一种穿梭分子促进小窝蛋白-1与受体的相互作用。在这个相互竞争的持续支持应用中，我们将探索神经节苷脂、小窝蛋白-1和α5beta1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。我们将通过免疫共沉淀和免疫显微镜研究：a)神经节苷脂与小窝蛋白-1、受体和信号分子共定位；b)神经节苷脂功能依赖于完整的小窝结构域和/或小窝蛋白-L。我们将通过引入反义寡聚体和突变的小窝蛋白-1来特异性干扰小窝蛋白-1的功能；c)小窝蛋白-1磷酸化对细胞运动的影响；以及d)神经节苷脂和小窝蛋白-L直接结合的可能性。神经节苷脂调节对伤口愈合的体内影响将使用表皮特异性神经节苷脂耗竭的转基因小鼠模型进行评估。这些研究将提供有关神经节苷脂在小窝蛋白-1介导的信号转导中的作用的信息，并可能为有异常愈合反应的患者带来新的治疗方法。

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.