TRANSCRIPTIONAL REGULATION OF SECRETOGRANIN-II AND CHROMOGRANIN-B BY CYCLIC-AMP IN A RAT PHEOCHROMOCYTOMA CELL-LINE

When PC-12 cells were treated with 10 mu M forskolin, the expression o f two members of the granin family, secretogranin II (SgII) and chromo granin B (CgB), were differentially regulated. SgII mRNA levels declin ed progressively after forskolin treatment to reach a level of 22 +/- 1% of control after 48 hr, whereas CgB mRNA levels increased more rapi dly, reaching a maximum of 3-fold above control after 24 hr. The depen dence of these changes on an increase in cellular cAMP revels, activat ion of cAMP-dependent protein kinase (PKA), protein synthesis, and cha nges in the rate of transcription was investigated. The effects of for skolin on SgII and CgB mRNAs were reproduced by 1 mM 8-bromo-cAMP but not by 10 mu M 1,9-dideoxyforskolin, an inactive analog of forskolin. The actions of forskolin on SgII and CgB mRNAs were blocked by treatme nt with 60 mu M H-89, a selective PKA inhibitor, and were blunted in P KA-deficient PC-12 cell clones. To examine whether forskolin action wa s dependent on ongoing protein synthesis, PC-12 cells were treated wit h 1 mu g/ml cycloheximide before the addition of forskolin. The reduct ion in SgII mRNA levels by forskolin was not evident in PC-12 cells tr eated with cycloheximide. Rather, in the presence of cycloheximide, fo rskolin stimulated SgII mRNA levels 3.6 +/- 0.7-fold above control. Th e induction of CgB mRNA by forskolin was not affected by cycloheximide treatment. The superinduction of SgII mRNA by cycloheximide and forsk olin was related to the extent of protein synthesis inhibition, was ob served in cells treated with forskolin and other protein synthesis inh ibitors, and was blunted in PKA-deficient PC-12 cells, suggesting that this effect was dependent on inhibition of protein synthesis and acti vation of PKA. To determine whether changes in SgII and CgB mRNA level s resulted from changes in the rate of transcription, nuclear run-on a ssays were performed in nuclei isolated from PC-12 cells that had been treated for 2 hr with cycloheximide, forskolin, or the two combined. Transcription of the SgII gene was not significantly affected by treat ment with either forskolin or cycloheximide alone but was increased 12 .9 +/- 1.0-fold above control in nuclei from cells treated with cycloh eximide and forskolin together. Forskolin caused a 3.8 +/- 0.8-fold in duction of CgB transcription. The half-lives of SgII and CgB mRNAs are long (33 +/- 8 and 19 +/- 6 hr, respectively), suggesting that the st imulatory effects of forskolin on CgB mRNA levels and on SgII mRNA lev els in the absence of protein synthesis are mediated predominantly by an increase in the transcription of these genes. These results also in dicate that enhanced transcription of the SgII gene is repressed, such that a stimulatory effect of cAMP is not revealed unless protein synt hesis is inhibited.