GANGLIOSIDE GENE MODULATION EFFECT ON CELL MOTILITY

描述：（改编自申请人摘要）-人角质形成细胞 纤维连接蛋白的运动性在愈合的上皮再生中是至关重要的 伤口和皮肤恶性肿瘤的扩散。 的分子事件 人们对这种迁移影响知之甚少。 补充GT 1 b，a 角化细胞的多唾液酸神经节苷脂，抑制 图1示出了在nM浓度下在纤连蛋白基质上的角质形成细胞的细胞粘附。 帕勒博士和 他的同事提出神经节苷脂调节α 5 β 1/纤连蛋白 相互作用，可能是通过与α 5 β 1的直接相互作用。 的 这一假设得到以下研究结果的支持：a）GT 1b抑制 角质形成细胞迁移仅发生在纤连蛋白基质上; B）RGDS肽 与GT 1b竞争以抑制细胞与纤连蛋白的粘附; c）预孵育 GT 1b与纤连蛋白基质的结合不会改变角质形成细胞与 纤连蛋白; d）GT 1b仅在生长时诱导角质形成细胞凋亡 e）响应于纤连蛋白的FAK磷酸化， 当角质形成细胞用GT 1b处理时降低; f）GT 1b不能抑制 SCC 13和HaCaT细胞的迁移和增殖， 降低α 5 β 1的表达;和g）TGF-β 1增加HaCaT 细胞表达α 5 β 1，并诱导细胞对 GT 1b对纤维连接蛋白粘附的抑制作用。 帕勒博士和 同事们将探讨的生理意义和机制（S）， 神经节苷脂对表皮细胞的作用通过这些特定的目的：1. 调制 稳定转染表达神经节苷脂生物合成途径 将糖基转移酶基因导入角化细胞衍生系。 为了 改变神经节苷脂的内源性产生，包括增加 表达GT 1b、GalNAc转移酶和唾液酸转移酶基因，由一种 孕酮拮抗剂诱导系统，将被单独转染 SCC 12细胞，一种角化细胞衍生的GT 1b反应性细胞系。 2. 检查神经节苷脂表达变化对迁移的影响， 粘附、诱导凋亡和培养细胞中的整合素信号传导。 研究人员将记录形态学，增殖潜力， 细胞凋亡和伤口愈合能力。 他们还将培育转基因小鼠，以研究改变基因的影响。 神经节苷脂含量对增殖、分化和创伤愈合的影响 体内正常角质形成细胞。 3. 评估神经节苷脂的作用机制 α 5 β 1/纤连蛋白相互作用的抑制： 神经节苷脂/α 5 β 1相互作用将在两个研究中进行研究。 使用重组α 5和β 1蛋白的无细胞系统， 从转染的SCC 12细胞免疫沉淀α 5 β 1。 博士 Paller将提出以下问题：a）神经节苷脂，包括GT 1b， 直接与α 5 β 1结合？B）神经节苷脂是否干扰α 5 beta 1汇编？c）神经节苷脂是否下调α 5的表达 β 1？和d）神经节苷脂含量的改变是否影响膜 流动性，从而整合素受体的方向？ 了解神经节苷脂对纤维连接蛋白整合素的影响 相互作用可能导致正常人中糖基转移酶活性调节 或肿瘤性角质形成细胞作为治疗 慢性伤口、皮肤肿瘤和过度增生的表皮 牛皮癣等疾病。

DESCRIPTION: (Adapted from the applicant's abstract) - Human keratinocyte motility on fibronectin is critical in the reepithelialization of healing wounds and in the spread of cutaneous malignancy. The molecular events that influence this migration are poorly understood. Supplemental GT1b, a polysialyated ganglioside of keratinocytes, inhibits migration of keratinocytes on a fibronectin matrix at nM concentrations. Dr. Paller and colleagues propose that gangliosides modulate the alpha 5 beta 1/fibronectin interaction, probably by direct interaction with alpha 5 beta 1. The hypothesis is supported by the findings that: a)GT1b inhibition of keratinocyte migration occurs only on a fibronectin matrix; b)RGDS peptide competes with GT1b to inhibit cell adhesion to fibronectin; c)Preincubation of GT1b with the fibronectin matrix does not alter keratinocyte binding to the fibronectin; d)GT1b induces apoptosis of keratinocytes only when grown on a fibronectin matrix; e)FAK phosphorylation in response to fibronectin is decreased when keratinocytes are treated with GT1b; f)GT1b fails to inhibit the migration and proliferation of SCC13 and HaCaT cells, which have decreased expression of alpha 5 beta 1; and g)TGF-beta 1 increases HaCaT cell expression of alpha 5 beta 1, and induces cells to respond to the inhibitory effects of GT1b on adhesion to fibronectin. Dr. Paller and colleagues will explore the physiologic significance and mechanism(s) of the ganglioside action on epidermal cells by these specific aims: 1. Modulate the expression of ganglioside biosynthetic pathways by stable transfection of glycosyltransferase genes into a keratinocyte-derived line. In order to shift the endogenous production of gangliosides, including increasing expression GT1b, GalNAc transferase and sialyltransferase genes, driven by a progesterone antagonist-inducible system, will be transfected individually into SCC12 cells, a keratinocyte-derived GT1b-responsive cell line. 2. Examine the effects of changes in ganglioside expression on migration, adhesion, induction of apoptosis, and integrin signalling in cultured cells. The investigators will note changes in morphology, proliferative potential, apoptosis, and wound healing capability in an ex vivo transplantation model. They will also develop transgenic mice to study the effects of altered ganglioside content on proliferation, differentiation, and wound healing of normal keratinocytes in vivo. 3. Assess the mechanism of ganglioside inhibition of the alpha 5 beta 1/fibronectin interaction: The ganglioside/alpha 5 beta 1 interaction will be investigated in both a cell-free system using recombinant alpha 5 and beta 1 proteins, and with alpha 5 beta 1 immunoprecipitated from the transfected SCC12 cells. Dr. Paller will ask the following questions: a)Do gangliosides, including GT1b, bind directly to alpha 5 beta 1?; b)Do gangliosides interfere with alpha 5 beta 1 assembly?; c)Do gangliosides downregulate the expression of alpha 5 beta 1?; and d)Do alterations in ganglioside content affect membrane fluidity and thus the orientation of integrin receptors? Understanding the effect of gangliosides on the fibronectin-integrin interaction may lead to modulation of glycosyltransferase activity in normal or neoplastic keratinocytes as a novel therapeutic modality for treatment of chronic wounds, cutaneous neoplasia, and hyperproliferative epidermal disorders such as psoriasis.