MECHANISM OF PHORBOL ESTER-INDUCED LIPID METABOLISM

通过蛋白激酶C途径的紊乱信号转导已经被发现， 与肿瘤的发展有关。 的目的 这一提议是为了确定蛋白质的潜在抑制途径， 激酶C 游离鞘氨醇碱已被证明可以拮抗 体外和完整细胞中蛋白激酶C的活化。 然而，在这方面， 目前还没有证据表明，一个途径， 鞘脂代谢为鞘氨醇碱， 在生理条件下的激酶C。 最近的研究表明， 实验室证明，1，2-二酰基甘油，内源性 蛋白激酶C的激活剂，刺激降解的蛋白激酶C， GH3垂体细胞鞘磷脂。 该事件发生 通过鞘磷脂酶（EC www.example.com）。 佛波酯，肿瘤- 促进蛋白激酶C的激活剂，未能刺激这一点， 活动 此外，鞘磷脂酶的作用足以抑制 蛋白激酶C 这些研究表明，1，2-二酰基甘油可能 激活未利用的蛋白激酶C的负效应通路 被佛波醇酯污染了 这项建议的目的是探讨 这个概念的细节。 这些研究将使用三种不同的细胞系：GH3大鼠 垂体细胞，3T3小鼠成纤维细胞和HL-60，人白血病细胞。 1，2-二酰基甘油和佛波酯对合成的影响 以及含胆碱磷脂的降解， 磷脂酰胆碱和鞘磷脂。 的作用 这些过程中的cAMP和蛋白激酶C系统将被 测定 将通过鞘磷脂酶降解鞘磷脂。 与蛋白激酶C激活/失活相关。 受体介 鞘磷脂酶活化和游离类鞘氨醇碱的产生 将评估利用磷酸肌醇途径的配体 用于信号传导。 将进行研究，以确定是否 在1，2- 二酰基甘油和佛波醇酯可归因于鞘磷脂酶 行动上 希望这些研究能揭示一种生理学上相关的 蛋白激酶C的抑制途径，并解释一些 在1，2-二酰基甘油的细胞活化之间观察到的差异 和佛波醇酯。

Disordered signal transduction via the protein kinase C pathway has been implicated in the development of the neoplastic process. The purpose of this proposal is to define a potential inhibitory pathway for protein kinase C. The free sphingoid bases have been shown to antagonize activation of protein kinase C in vitro and in intact cells. However, evidence has not been presented to suggest that a pathway involving sphingolipid metabolism to sphingoid bases serves to inactivate protein kinase C under physiologic conditions. Recent studies from this laboratory demonstrated that 1,2-diacylglycerols, the endogenous activators of protein kinase C, stimulated degradation of the sphingolipid sphingomyelin in GH3 pituitary cells. This event occurred via a sphingomyelinase (EC 3.1.4.12). Phorbol esters, the tumor- promoting activators of protein kinase C, failed to stimulate this event. Further, sphingomyelinase action was sufficient to inactivate protein kinase C. These studies suggest that 1,2-diacylglycerols may activate a negative effector pathway for protein kinase C not utilized by the phorbol esters. The intention of this proposal is to explore this concept in detail. Three distinct cell lines will be utilized for these studies; GH3 rat pituitary cells, 3T3 mouse fibroblasts and HL-60, human leukemia cells. The effect of 1,2-diacylglycerols and phorbol esters on the synthesis and degradation of the choline-containing phospholipids, phosphatidylcholine and sphingomyelin, will be evaluated. The role of the cAMP and protein kinase C systems in these processes will be determined. Sphingomyelin degradation via a spingomyelinase will be related to protein kinase C activation/inactivation. Receptor-mediated sphingomyelinase activation and the generation of free sphingoid bases will be assessed for ligands that utilize the phosphoinositide pathway for signal transduction. Studies will be performed to determine if differences in the pattern of biologic responses after 1,2- diacylglycerols and phorbol esters can be ascribed to sphingomyelinase action. Hopefully, these studies will reveal a physiologically relevant inhibitory pathway for protein kinase C and explain some of the differences observed between cellular activation by 1,2-diacylglycerols and phorbol esters.