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 increased proenkephalin (proENK) mRNA; the significant increase began at 6 h after 30 microM C2 ceramide treatment (about 13-fold) and at 12 h after treatment (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 blocking of protein synthesis by cycloheximide (CHX) evokes a further increase of C2 ceramide-induced proENK mRNA and phospho-CREB level, while C2 ceramide-induced increases of AP-1 protein levels were reduced by CHX. The C2 ceramide-induced proENK mRNA expression was not changed significantly by the pretreatment with H89 (a PKA inhibitor), KN62 (a calcium/calmodulin-dependent protein kinase II inhibitor), and PD98059 (an ERK pathway inhibitor). However, calphostin C (a PKC inhibitor) and or SB203580 (a p38 inhibitor) partially but significantly reduced C2 ceramide-induced proENK mRNA expression as well as phospho-CREB level. These results suggest that, in the rat astrocyte-enriched culture, C2 ceramide increases proENK mRNA expression via phosphorylation of CREB rather than the increases of AP-1 protein levels. Additionally, the activations of PKC and p38, but not PKA, calcium/calmodulin-dependent protein kinase II, and ERK, by C2 ceramide play important regulatory roles in C2 ceramide-induced proENK mRNA expression via activating the CREB.