To investigate signal transduction pathways leading to apoptosis during the early phase of neurogenesis, we employed PCC7-Mz1 cells, which cease to proliferate and begin to differentiate into a stable pattern of neurons, astroglial cells, and fibroblasts upon incubation with retinoic acid (RA). As part of lineage determination, a sizable fraction of RA-treated cultures die by apoptosis. Applying natural long-chain C16-ceramides as well as membrane-permeable C2/C6-ceramide analogs caused apoptosis, whereas the biologically nonactive C2-dihydroceramide did not. Treating PCC7-Mz1 stem cells with a neutral sphingomyelinase or with the ceramidase inhibitor N-oleoylethanolamine elevated the endogenous ceramide levels and concomitantly induced apoptosis. Addition of RA caused an increase in ceramide levels within 3–5 h, which reached a maximum (up to 3.5-fold of control) between days 1 and 3 of differentiation. Differentiated PCC7-Mz1 cells did not respond with ceramide formation and apoptosis to RA treatment. The acidic sphingomyelinase contributed only weakly and the neutral Mg2+-dependent and Mg2+-independent sphingomyelinases not at all to the RA-mediated production of ceramides. However, ceramide increase was sensitive to the ceramide synthase inhibitor fumonisin B1, suggesting a crucial role for the de novo synthesis pathway. Enzymatic assays revealed that ceramide synthase activity remained unaltered, whereas serine palmitoyltransferase (SPT), a key enzyme in ceramide synthesis, was activated ∼2.5-fold by RA treatment. Activation of SPT seemed to be mediated via a post-translational mechanism because levels of the mRNAs coding for the two SPT subunits were unaffected. Expression of marker proteins shows that ceramide regulates apoptosis, rather than differentiation, during early neural differentiation.
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Published, JBC Papers in Press, June 20, 2000, DOI 10.1074/jbc.M000714200
This work was supported in part by Deutsche Forschungsgemeinschaft Grants He 2557.1-2, Ma 599.1-1, and SFB 415 (to S. S.) and by the Naturwissenschaftlich-Medizinische Forschungszentrum of the University of Mainz (to T. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.