GANGLIOSIDE GENE MODULATION EFFECT ON CELL MOTILITY

描述：(改编自申请者的摘要)-人类角质形成细胞 纤维连接蛋白的运动性在愈合的再上皮化过程中起关键作用 伤口和皮肤恶性肿瘤的扩散。这些分子事件 人们对这种迁移的影响知之甚少。补充GT1b，a 角质形成细胞聚唾液酸神经节苷脂抑制细胞迁移 NM浓度下纤维连接蛋白基质上的角质形成细胞。帕勒博士和 同事们提出神经节苷脂调节α5β1/纤维连接蛋白 相互作用，可能是通过与阿尔法5β1直接相互作用。 支持这一假说的发现是：a)GT1b抑制 角质形成细胞仅在纤维连接蛋白基质上发生迁移；b)RGDS肽 与GT1b竞争抑制细胞与纤维连接蛋白的黏附；c)预培养 GT1b与纤维连接蛋白基质的结合不会改变角质形成细胞与 纤维连接蛋白GT1b只有在生长时才能诱导角质形成细胞的凋亡 在纤维连接蛋白基质上；e)纤维连接蛋白对FAK的磷酸化反应 角质形成细胞用GT1b处理后减少；f)GT1b未能抑制 SCC13和HaCaT细胞的迁移和增殖 α5β1表达降低；和g)转化生长因子β1增加HaCaT 细胞表达α5β1，并诱导细胞对 GT1b对纤维连接蛋白黏附的抑制作用帕勒博士和 同事们将探索血管紧张素转换酶的生理意义和机制(S) 神经节苷脂通过以下特定目的作用于表皮细胞：1.调节 稳定转染法表达神经节苷脂生物合成途径 将糖基转移酶基因转化为角质形成细胞来源的细胞系。为了 转移神经节苷脂的内源性生产，包括增加 GT1b、GalNAc转移酶和唾液酸转移酶基因的表达 孕酮拮抗剂诱导系统，将单独转染 转化SCC12细胞，角质形成细胞来源的GT1b反应细胞系。2. 研究神经节苷脂表达变化对迁移的影响， 培养细胞的黏附、诱导凋亡和整合素信号传递。 研究人员将注意到形态、增殖潜力、 体外移植模型中的细胞凋亡和伤口愈合能力。 他们还将开发转基因小鼠来研究改变的效果 神经节苷脂含量对细胞增殖、分化及创面愈合的影响 体内正常角质形成细胞。3.评价神经节苷脂的作用机制 抑制α-5-β1/纤维连接蛋白相互作用： 神经节苷脂/α5β1相互作用将在两个 使用重组α5和β1蛋白的无细胞系统，以及 转染组SCC12细胞中有α-5-β-1免疫沉淀。Dr。 帕勒将提出以下问题：a)神经节苷脂，包括GT1b， 直接与α5β1结合？；b)神经节苷脂是否干扰α5 β1组装；c)神经节苷脂是否下调α5的表达 和d)神经节苷脂含量的改变是否影响细胞膜 流动性和整合素受体的定位？ 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糖基转移酶活性的调节 或肿瘤性角质形成细胞作为一种新的治疗方式治疗 慢性创面、皮肤肿瘤和过度增殖的表皮 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.