Stimulation of astrocyte-enriched culture with C2 ceramide increases proenkephalin mRNA: involvement of cAMP-response element binding protein and mitogen activated protein kinases

In rat astrocyte-enriched culture, C2 ceramide dose- and time-dependently i ncreased proenkephalin (proENK) mRNA; the significant increase began at 6 h after 30 muM C2 ceramide treatment (about 13-fold) and at 12 h after treat ment (about 21-fold). In addition, C2 ceramide also increased AP-1 proteins , such as Fra-1, c-Jun. JunB and JunD, and phosphorylation of CREB. The blo cking of protein synthesis by cycloheximide (CHX) evokes a further increase of C2 ceramide-induced proENK mRNA and phospho-CREB level, while C2 cerami de-induced increases of AP-1 protein levels were reduced by CHX. The C2 cer amide-induced proENK mRNA expression was not changed significantly by the p retreatment with H89 (a PKA inhibitor), KN62 (a calcium/calmodulin-dependen t protein kinase II inhibitor), and PD98059 tan ERK pathway inhibitor). How ever, calphostin C (a PKC inhibitor) and or SB203580 (a p38 inhibitor) part ially but significantly reduced C2 ceramide-induced proENK mRNA expression as well as phospho-CREB level. These results suggest that, in the rat astro cyte-enriched culture, C2 ceramide increases proENK mRNA expression via pho sphorylation of CREB rather than the increases of AP-I protein levels. Addi tionally, the activations of PKC and p38, but not PKA, calcium/calmodulin-d ependent protein kinase II, and ERK, by C2 ceramide play important regulato ry roles in C2 ceramide-induced proENK mRNA expression via activating the C REB. (C) 2001 Elsevier Science B.V. All rights reserved.