Taupenot L, Mahata S K, Wu H, O'Connor D T
Department of Medicine and Center for Molecular Genetics, University of California at San Diego, San Diego, California 92161-9111, USA.
J Clin Invest. 1998 Feb 15;101(4):863-76. doi: 10.1172/JCI1129.
Pituitary adenylyl cyclase-activating polypeptide (PACAP) is a potent endogenous secretagogue for chromaffin cells. Chromogranin A is the major soluble core component in secretory vesicles. Since chromogranin A is secreted along with catecholamines, we asked whether PACAP regulates expression of the chromogranin A gene in PC12 rat chromaffin cells, so as to resynthesize the just-secreted protein, and whether such biosynthetic regulation is coupled mechanistically to catecholamine secretion. PACAP activated the endogenous chromogranin A gene by four- to fivefold. Proportional results (seven- to eightfold activation) were obtained with a transfected 1,200-bp mouse chromogranin A promoter/luciferase reporter construct. A series of chromogranin A promoter 5' deletion mutant/luciferase reporter constructs narrowed down the PACAP response element to a proximal region containing the cAMP response element (CRE box), at (-71 bp)5'-TGACGTAA-3'(-64 bp). Site-directed point mutations of the CRE site suppressed PACAP-induced trans-activation of the promoter. Thus, the proximal CRE box is entirely necessary for the chromogranin A promoter response to PACAP. Transfer of the CRE box to a neutral, heterologous promoter also conferred activation by PACAP, suggesting that the CRE domain is also sufficient to mediate the transcriptional response to PACAP. Expression of a dominant-negative mutant (KCREB) of the CRE-binding factor CREB markedly diminished trans-activation of the chromogranin A promoter by PACAP. Cotransfection of expression plasmids encoding the protein kinase A inhibitor, or an inactive protein kinase A (PKA) catalytic beta subunit, inhibited both forskolin and PACAP activation of chromogranin A transcription, revealing that PACAP-induced trans-activation is highly dependent on PKA. By contrast, inhibition of protein kinase C (by chronic exposure to phorbol ester) had no effect on transcriptional activation by PACAP. The potent PACAP/vasoactive intestinal peptide (VIP) type I receptor antagonist PACAP6-38 impaired both chromogranin A transcription or catecholamine secretion triggered by PACAP38, while the PACAP/VIP type II receptor antagonist (p-Chloro-D-Phe6, Leu17)-VIP had little or no ability to antagonize the PACAP38 effect. The agonist VIP was approximately 100- to 1,000-fold less potent than PACAP in stimulating either secretion or transcription. Thus, PACAP-evoked chromogranin A transcription and catecholamine secretion are likely mediated by the PACAP/VIP type I receptor isoform. Although the calcium channel antagonists Zn2+ (100 microM), nifedipine (10 microM), or ruthenium red (10 microM), or the cytosolic calcium chelator BAPTA-AM (50 microM) each strongly impaired PACAP-induced secretion, transcriptional activation of chromogranin A remained unaltered. Therefore, we propose that PACAP signals to chromogranin A transcription through the CRE in cis, and through PKA and CREB in trans. By contrast, a pathway involving cytosolic calcium entry through L-type voltage-dependent channels is required for PACAP to evoke catecholamine secretion.
垂体腺苷酸环化酶激活多肽（PACAP）是一种对嗜铬细胞有强大作用的内源性促分泌素。嗜铬粒蛋白A是分泌囊泡中的主要可溶性核心成分。由于嗜铬粒蛋白A与儿茶酚胺一起分泌，我们研究了PACAP是否调节PC12大鼠嗜铬细胞中嗜铬粒蛋白A基因的表达，以便重新合成刚分泌的蛋白质，以及这种生物合成调节在机制上是否与儿茶酚胺分泌相关联。PACAP使内源性嗜铬粒蛋白A基因激活了4至5倍。用转染的1200 bp小鼠嗜铬粒蛋白A启动子/荧光素酶报告基因构建体获得了成比例的结果（激活7至8倍）。一系列嗜铬粒蛋白A启动子5'缺失突变体/荧光素酶报告基因构建体将PACAP反应元件缩小到一个近端区域，该区域包含位于（-71 bp）5'-TGACGTAA-3'（-64 bp）的环磷酸腺苷反应元件（CRE框）。CRE位点的定点突变抑制了PACAP诱导的启动子反式激活。因此，近端CRE框对于嗜铬粒蛋白A启动子对PACAP的反应是完全必需的。将CRE框转移到一个中性的异源启动子上也赋予了PACAP激活作用，这表明CRE结构域也足以介导对PACAP的转录反应。CRE结合因子CREB的显性负性突变体（KCREB）的表达显著降低了PACAP对嗜铬粒蛋白A启动子的反式激活。共转染编码蛋白激酶A抑制剂或无活性蛋白激酶A（PKA）催化β亚基的表达质粒，抑制了福斯可林和PACAP对嗜铬粒蛋白A转录的激活，表明PACAP诱导的反式激活高度依赖PKA。相比之下，抑制蛋白激酶C（通过长期暴露于佛波酯）对PACAP的转录激活没有影响。强效的PACAP/血管活性肠肽（VIP）I型受体拮抗剂PACAP6-38损害了PACAP38触发的嗜铬粒蛋白A转录或儿茶酚胺分泌，而PACAP/VIP II型受体拮抗剂（对氯-D-苯丙氨酸6，亮氨酸17）-VIP几乎没有或没有能力拮抗PACAP38的作用。激动剂VIP在刺激分泌或转录方面的效力比PACAP低约100至1000倍。因此，PACAP诱发的嗜铬粒蛋白A转录和儿茶酚胺分泌可能由PACAP/VIP I型受体亚型介导。尽管钙通道拮抗剂锌离子（100 microM）、硝苯地平（10 microM）或钌红（10 microM），或胞质钙螯合剂BAPTA-AM（50 microM）都强烈损害了PACAP诱导的分泌，但嗜铬粒蛋白A的转录激活仍未改变。因此，我们提出PACAP通过顺式作用的CRE向嗜铬粒蛋白A转录发出信号，并通过反式作用的PKA和CREB发出信号。相比之下，PACAP诱发儿茶酚胺分泌需要一条涉及通过L型电压依赖性通道进入胞质钙的途径。
