SPHINGOLIPIDS AS SIGNALS FOR TUMOR NECROSIS FACTOR-A

这项拨款提案的总体目标是定义一种信号转导 肿瘤坏死因子-α途径。这种细胞因子是一种 针对外来抗原的主要宿主防御系统，可服务于多种 生理调节功能。尽管有一些报道称 建议其中一个已知的信号系统扮演一个角色，没有连贯的 第二个信使途径的画面已经浮出水面，这可能解释了 肿瘤坏死因子-α的全部多效性效应。这项建议是基于 根据最近的观察，鞘磷脂降解为神经酰胺是一种 人早幼粒细胞白血病(HL-60)中肿瘤坏死因子-α作用的早期事件 神经酰胺可替代单核细胞中的肿瘤坏死因子-α 差异化。在过去的几年里，这个实验室描述了 一种新的涉及鞘磷脂及其衍生物的代谢途径。这 通过质膜鞘磷脂的水解来启动这一途径 神经酰胺通过鞘磷脂酶的作用。随后，神经酰胺可 脱酰化为鞘氨醇，一种蛋白激酶C的抑制剂，或 磷酸化成神经酰胺1-磷酸，一种新发现的化合物 实验室。因为这种新陈代谢之间的明显相似性 途径和肌醇磷脂途径，推测神经酰胺 可以起到第二信使作用。事实上，一种细胞通透性神经酰胺 类似物诱导的表皮生长因子选择性磷酸化 苏氨酸669的受体。基于这个观察，这个实验室有 最近鉴定了一种新的激酶，它介导了 神经酰胺，称为神经酰胺活化蛋白激酶，在衍生的膜中 来自HL-60和A431人表皮样癌细胞。高程 肿瘤坏死因子-α引起的神经酰胺水平也导致脑组织中的激酶活性增强。 从受刺激的细胞衍生的膜。这笔赠款的具体目的是 目的是：(1)证明这个鞘磷脂途径是紧密偶联的 对完整细胞和体外细胞中肿瘤坏死因子受体的激活：(2) 表明该途径足以介导肿瘤坏死因子-α诱导的蛋白 磷酸化；以及(3)将该途径推广到各种模型的肿瘤坏死因子- α作用，特别是确定它可能如何与肿瘤坏死因子诱导的 细胞毒性和细胞停滞。希望这些研究将提供 对肿瘤坏死因子-α作用机制的基本见解，并允许 对这一系统的最终药理学操作。

The general goal of this grant proposal is to define a signal transduction pathway for tumor necrosis factor (TNF)-alpha. This cytokine is a principal host defense against foreign antigen and may serve a variety of physiologic regulatory functions. Although a number of reports have suggested a role for one of the known signalling systems, no coherent picture has emerged for a second messenger pathway that may account for the entirety of the pleiotropic effects of TNF-alpha. This proposal is based on the recent observation that sphingomyelin degradation to ceramide is an early event in TNF-alpha action in human promyelocytic leukemia (HL-60) cells and that ceramide may substitute for TNF-alpha in monocytic differentiation. During the past few years, this laboratory has described a new metabolic pathway involving sphingomyelin and its derivatives. This pathway is initiated by hydrolysis of plasma membrane sphingomyelin to ceramide by the action of a sphingomyelinase. Subsequently, ceramide may be de-acylated to sphingosine, an inhibitor of protein kinase C, or phosphorylated to ceramide 1-phosphate, a compound newly discovered in this laboratory. Because of apparent similarities between this metabolic pathway and the phosphoinositide pathway, it was postulated that ceramide may serve second messenger function. Indeed, a cell-permeable ceramide analog induced selective phosphorylation of the epidermal growth factor receptor on threonine 669. Based on this observation, this laboratory has recently characterized a novel kinase that mediates this action of ceramide, termed ceramide-activated protein kinase, in membranes derived from HL-60 and A431 human epidermoid carcinoma cells. Elevation of ceramide levels by TNF-alpha also resulted in enhanced kinase activity in membranes derived from stimulated cells. The specific aims of this grant are to: (1) demonstrate that this sphingomyelin pathway is tightly coupled to activation of the TNF receptor both in intact cells and in vitro: (2) show that this pathway is sufficient to mediate TNF-alpha-induced protein phosphorylation; and (3) generalize this pathway to various models of TNF- alpha action and in particular determine how it might relate to TNF-induced cytotoxicity and cytostasis. Hopefully, these studies will provide fundamental insights into the mechanism of TNF-alpha action and allow for eventual pharmacologic manipulation of this system.