MECHANISM OF PHORBOL ESTER-INDUCED LIPID METABOLISM

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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.