An early and transient period of protein synthesis is required for induction of neuropeptide Y-mRNA by phorbol ester and forskolin in aggregate cultures of fetal brain cells

Abstract

We have previously shown that both forskolin (F) and phorbol ester (P) induce neuropeptide Y (NPY) production by aggregate cultures formed from dissociated fetal rat brains. In this study, we addressed the question: Do F/P induce NPY-mRNA in aggregate cultures and if so, does induction require active protein kinases and on-going protein synthesis? On Northern blots, the NPY cDNA hybridized to a single species of about 0.8 kb. F and P each induced a time-dependent increase in NPY-mRNA relative abundance, and this was inhibited by staurosporin, an inhibitor of both protein kinase A and C. Cycloheximide (CHX) inhibited F/P induction of mRNA in a time-dependent manner. When aggregates were incubated with F + P for a total 12 h period, CHX added along with F + P (0 h) completely inhibited, CHX added 1.5 h after F + P partially inhibited, and CHX added 6 h after F + P did not inhibit the increase in NPY-mRNA. To rule out the possibility that this inhibitory profile reflects toxicity of CHX, blots were re-hybridized with a SRIF cRNA probe and, as expected for SRIF gene, a 12 h exposure to CHX did not inhibit F + P induction of SRIF-mRNA. Close inspection of the blots (derived from 1.5% agarose gels) suggested the presence in F/P-treated aggregates of 2 species NPY-mRNA [ ≈ 0.75 and ≈ 0.85 kb, most likely differing in the length of poly(A) tail (Jamal et al., 1991)]; size-fractionation on a higher resolution gel (3% agarose) resolved the F/P induced mRNA into 2 distinct bands, while mRNA from untreated cultures migrated as a single band - the 0.75 kb. Thus, F and P each induces NPY-mRNA abundance in a process requiring protein synthesis and the activity of protein kinases. The fact that the requirement for protein synthesis is restricted to the early ( < 6 h) period of induction of mRNA is consistent with the involvement of immediate-early response genes and with the classification of NPY gene as a slow responding gene. The increase in NPY-mRNA abundance and the preferential induction of a larger molecular size NPY-mRNA suggests regulation of NPY expression at a transcriptional and a post-transcriptional level.