Synergistic induction of pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression by nerve growth factor and PACAP in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) gene expression was analyzed in PC12 cells. PC12 cells transfected with a PACAP promoter-lu ciferase reporter construct were utilized to investigate the effects of PAC AP, either alone or in combination with nerve growth factor (NGF), on PACAP transcriptional response. PACAP induced transcription from the PACAP promo ter through PACAP type I receptor(PAC, receptor). PACAP gene transcription was also induced by NGF, Simultaneous treatment with PACAP and NGF resulted in a synergistic transcriptional response that was more than three times t he predicted response, based on a simple additive effect of both agents. Th is synergism in transcriptional response paralleled the PACAP mRNA levels, as determined by RT-PCR and northern blotting. The level of PACAP mRNA peak ed 3 h after stimulation and gradually returned to basal levels by 48 h, PC 12 cells are known to express predominantly the hop isoform of the PAC, rec eptor, which positively couples to both adenylate cyclase and phospholipase C, To determine the role of the cyclic AMP and protein kinase C pathways i n PACAP gene expression, the effects of forskolin and phorbol 12-myristate 13-acetate (PMA) were then examined. PMA did not alter PACAP mRNA levels bu t enhanced forskolin-induced PACAP mRNA expression. Down-regulation of prot ein kinase C blocked the ability of PACAP to stimulate PACAP mRNA expressio n. The mitogen-activated protein kinase extracellular signal-regulated kina se (ERK) kinase 1/2 (MEK1/2) inhibitor PD98059 also blocked the PACAP mRNA expression induced by either PACAP or NGF but not that induced by a combina tion of PACAP and NGF. These results suggest that PACAP stimulates the PACA P gene expression in PC12 cells at least in part through activation of aden ylate cyclase and protein kinase C signaling pathways and that the ERK1/2 c ascade is involved in PACAP and NGF-induced PACAP gene expression, although redundant signaling pathways may also be involved. The present finding sho wing that PACAP in combination with NGF causes a synergistic increase in PA CAP gene expression in PC12 cells supports the idea that PACAP acts as an a utocrine regulatory factor.