COORDINATE REGULATION OF STRESS-ACTIVATED AND MITOGEN-ACTIVATED PROTEIN-KINASES IN THE APOPTOTIC ACTIONS OF CERAMIDE AND SPHINGOSINE

We characterized participation of the stress-activated protein kinase (SAPK) cascade in the lethal actions of the cytotoxic lipid messengers ceramide and sphingosine in U937 human monoblastic leukemia cells. Ac ute exposure of U937 cells to either lipid resulted in loss of prolife rative capacity, degradation of genomic DNA, and manifestation of apop totic cytoarchitecture. Ceramide robustly stimulated p46-JNK1/p54-JNK2 activity and increased expression of c-jun mRNA and c-Jun protein; in contrast, sphingosine moderately stimulated p46-JNK1/p54-JNK2 and fai led to modify c-jun/c-Jun expression. Dominant-negative blockade of no rmal c-Jun activity by transfection with the TAM-67 c-Jun NH2-terminal deletion mutant abolished the lethal actions of ceramide but was with out effect on those of sphingosine, indicating that ceramide-related a poptosis is directly dependent on activation of c-Jun, whereas sphingo sine-induced cell death proceeds via an unrelated downstream mechanism . Characterization of the mitogen-activated protein kinase (MAPK) casc ade in these responses revealed a further functional disparity between the two lipids: basal p42-ERK1/p44-ERK2 activity was gradually reduce d by ceramide but immediately and completely suppressed by sphingosine . Moreover, blockade of the MAPK cascade by the aminomethoxyflavone ME K1 inhibitor PD-98059 unexpectedly activated p46-JNK1/p54-JNK2 and ind uced apoptosis in a manner qualitatively resembling that of sphingosin e. Both lipids sharply increased p38-RK activity; selective pharmacolo gical inhibition of p38-RK by the pyridinyl imidazole SE-203580 failed to mitigate the cytotoxicity associated with either ceramide or sphin gosine, suggesting that p38-RK is not essential for lipid-induced apop tosis. These findings demonstrate that reciprocal alterations in the S ARK and MAPK cascades are associated with the apoptotic influence of e ither lipid inasmuch as (i) ceramide-mediated lethality is primarily a ssociated with strong stimulation of SARK and weak inhibition of MAPK, whereas (ii) sphingosine-mediated lethality is primarily associated w ith weak stimulation of SAPK and strong inhibition of MAPK. We therefo re propose that leukemic cell survival depends on the maintenance of a n imbalance of the outputs from the MAPK and SAPK systems such that th e dominant basal influence of the MAPK cascade allows sustained prolif eration, whereas acute redirection of this balance toward the SAPK cas cade initiates apoptotic cell death.