Yukimasa N, Isobe K, Nagai H, Takuwa Y, Nakai T
Department of Clinical Pathology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Neuropeptides. 1999 Dec;33(6):475-82. doi: 10.1054/npep.1999.0765.
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.
我们先前证明，垂体腺苷酸环化酶激活多肽（PACAP）通过激活环磷酸腺苷（cAMP）和蛋白激酶C（PKC）信号通路，协同上调酪氨酸羟化酶（TH）和多巴胺β羟化酶（DBH）基因的表达。在本研究中，我们检测了PACAP对大鼠嗜铬细胞瘤细胞系PC12细胞中fos和jun即刻早期基因（IEG）家族表达的影响，PKC和cAMP信号通路均可上调其表达。Northern印迹分析显示，PACAP在0.1 - 10 nM剂量下能有效刺激c-fos、fosB、junB和junD的表达，但不刺激c-jun mRNA的表达。PACAP对PC12细胞中这些mRNA表达的影响迅速（30 - 60分钟）且呈剂量依赖性。给予PACAP后，60分钟时c-fos、fosB和junB mRNA表达达到最大值，8小时时junD mRNA表达达到最大值。使用与TH 5'-侧翼区域对应的合成DNA寡核苷酸和PC12细胞核提取物进行凝胶迁移率变动分析表明，PACAP增强了分别与佛波酯反应元件（TRE）和cAMP反应元件（CRE）结合的特异性蛋白复合物的形成。凝胶迁移和超迁移分析表明，TRE结合因子和CRE结合因子分别由fosB、c-fos、junB和junD，以及CRE结合蛋白（CREB）和junD组成。添加PACAP后4小时，JunB在TRE结合复合物中占主导地位，而12小时时JunD和JunB均占主导地位。这些结果表明，PACAP受体的激动剂占据激活了与TH 5'-侧翼区域的TRE和CRE位点相互作用的转录因子（Fos/Jun家族和CREB），有助于TH基因的转录激活。
