Successive occupancy by immediate early transcriptional factors of the tyrosine hydroxylase gene TRE and CRE sites in PACAP-stimulated PC12 pheochromocytoma cells.
We previously demonstrated that pituitary adenylate cyclase-activating polypeptide (PACAP) coordinately upregulates the expression of the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) genes by activating the cyclic AMP (cAMP) and protein kinase C (PKC) signaling pathways. In this study, we examined the effects of PACAP on the expression of fos and jun immediate early gene (IEG) families, expression of which can be up-regulated by both PKC and cAMP signaling pathways, in rat pheochromocytoma cell line PC12 cells. PACAP potently stimulated the expression of c-fos, fosB junB and junD, but not c-jun mRNAs, at doses of 0.1-10 nM, as revealed by Northern blot analysis. The effects of PACAP on the expression of these mRNAs in PC12 cells was rapid (30-60 min) and dose-dependent. PACAP administration induced maximum expression of c-fos, fosB and junB mRNA after 60 min, and of junD mRNA after 8 h. Gel mobility shift assays using synthetic DNA oligonucleotides corresponding to the TH 5'-flanking region and nuclear extracts from PC12 cells demonstrated that PACAP enhanced formation of the specific protein complexes which bind to the TPA-responsive element (TRE) and cAMP-responsive element (CRE), respectively. Gel shift and supershift analyses showed that the TRE-binding factors and CRE-binding factors comprised fosB, c-fos, junB, and junD, and CRE-binding protein (CREB) and junD, respectively. JunB was dominant in the TRE-binding complexes at 4 h after addition of PACAP, whereas both JunD and JunB were dominant at 12 h. These results suggest that agonist occupancy of PACAP receptors activates transcriptional factors (Fos/Jun families and CREB) that interact with the TRE and CRE sites of the TH 5'-flanking region, contributing to transcriptional activation of TH gene.
Yukimasa
N
N
Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Isobe
K
K
Nagai
H
H
Takuwa
Y
Y
Nakai
T
T
D000818:Animals; D015152:Blotting, Northern; D002273:Carcinogens / Q000494:pharmacology; D016384:Consensus Sequence; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017781:Genes, Immediate-Early / Q000502:physiology*; D008934:Mitogens / Q000494:pharmacology*; D009474:Neurons / Q000166:cytology / Q000201:enzymology; D009479:Neuropeptides / Q000494:pharmacology*; D016716:PC12 Cells; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D011493:Protein Kinase C / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020218:Response Elements / Q000187:drug effects / Q000235:genetics*; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics*
C495403:Adcyap1 protein, rat; D002273:Carcinogens; D017362:Cyclic AMP Response Element-Binding Protein; D008934:Mitogens; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D000242:Cyclic AMP; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate