Role of ceramide-activated protein phosphatase in ceramide-mediated signal transduction.

Abstract

Extracellular agonists such as tumor necrosis factor-a (=-a) activate the sphingomyelin cycle leading to the generation of ceramide.Ceramide has been suggested as an important mediator of the effects of TNF"a on growth inhibition, c-myc down-regulation, apoptosis, and the activation of the nuclear factor KB.Although there is no clearly defined intracellular target for ceramide activity, previous studies have demonstrated the existence of a ceramide-activated protein phosphatase (CAPP) in uitro.Since c-myc is an early downstream cellular target for TNF-a, we examined the role of ceramide and CAPP in c-myc down-regulation.In intact HL-60 cells ceramide induced down-regulation of c-myc RNA levels.C,-ceramide was active at 1-10 p~ and caused 4040% inhibition of c-myc RNA levels at 30-120 min of treatment.In nuclear run-on studies, C,-ceramide induced a block to transcription elongation of the c-myc transcript without affecting transcription through the first exon.Therefore, ceramide appeared to inhibit c-myc expression via a mechanism identical with that of TNF-a.HL-60 cells contained CAPP which was inhibited by okadaic acid (0.1-10 nm).CAPP in HL-60 cells was activated by Derythro-ceramide but not D-erythro-dihydroceramide. The specificity of activation of CAPP by ceramide in uitro was matched by a similar specificity of c-myc downregulation in cells.Moreover, okadaic acid inhibited the effects of ceramide and TNF-a on c-myc down-regulation.On the other hand, okadaic acid did not inhibit the ability of phorbol12-myristate 13-acetate to down-regulate c-myc, demonstrating the existence of at least two distinct pathways in the regulation of c-myc expression.These results demonstrate that CAPP is important for ceramide-induced down-regulation of c-myc in myeloid leukemia cells.The implications of these findings in further delineating a sphingomyelin signaling pathway with important anti-proliferative effects are discussed.Recently, a sphingomyelin cycle has emerged as a candidate signal transduction pathway with important biologic consequences.Tumor necrosis factor-a (TNF-a)' and other extracellular agents, such as 1,25-dihydroxyvitamin D,, y-interferon, and interleukin-1, activate a neutral sphingomyelinase resulting in hydrolysis of membrane sphingomyelin (1-4).Ceramide, the product of sphingomyelin cleavage, inhibits cell growth (1) *These studies were supported in part by National Institutes of Health Grant GM-43825.The costs of publication of this article were