Attardi B, Winters S J
Department of Medicine, University of Pittsburgh, School of Medicine, PA 15213, USA.
Mol Cell Endocrinol. 1998 Feb;137(2):97-107. doi: 10.1016/s0303-7207(98)00006-9.
We showed previously that pituitary adenylate cyclase-activating polypeptide (PACAP) increases glycoprotein hormone alpha-subunit gene expression and secretion in alphaT3-1 cells. We have now used 5'-flanking deletion and clustered point mutations of the mouse alpha-subunit promoter fused to the luciferase (LUC) reporter gene in transient transfection assays to further characterize the cell signaling pathways and sequences involved in responsiveness to PACAP. PACAP stimulated LUC activity at a lower concentration than VIP, supporting the notion that PACAP acts through type 1 receptors. The effect of PACAP on LUC activity was observed by 2 h, peaked at 4-12 h, and persisted until at least 20 h. alphaT3-1 cells were transfected with mouse alpha-LUC constructs truncated at -507, -424, -288, -205, -146, and -133, and treated with PACAP, a cell-permeable cAMP analog (8Br-cAMP), phorbol myristate acetate (PMA), or control medium. Transcriptional activation by PACAP was highest with the -288 and -205 mouse alpha-LUC vectors (7-8-fold stimulation) and decreased significantly with truncation of the 5'-flanking region to -146 or -133. The pattern of alpha-subunit stimulation by cAMP closely paralleled that of PACAP. With PMA, stepwise decrements in LUC activity were observed between -507 and -424 and, especially, -424 and -288, and there was no further loss of activity with deletion to -205, -146, or -133. Clustered point mutations in the pituitary glycoprotein hormone basal element (-337 to -330) or the gonadotropin-releasing hormone response element (GnRH-RE)(-406 to -399) of the -507 to +46 mouse alpha-promoter significantly (P < 0.05) increased and decreased, respectively, PACAP's effect on transcriptional activity. These results indicate that there are several regions of the mouse alpha-subunit promoter that mediate responsiveness to PACAP. The co-localization of PACAP and cAMP responsiveness as well as the results of studies involving specific inhibitors of protein kinase A (H-89) or protein kinase C (PKC) (bisindolylmaleimide) suggests that the action of PACAP on alpha-subunit transcription is mediated primarily by the protein kinase A (PKA) pathway.
我们之前的研究表明，垂体腺苷酸环化酶激活多肽（PACAP）可增加αT3-1细胞中糖蛋白激素α亚基基因的表达及分泌。现在，我们在瞬时转染实验中使用了与荧光素酶（LUC）报告基因融合的小鼠α亚基启动子的5'侧翼缺失和簇状点突变，以进一步表征参与对PACAP反应的细胞信号通路和序列。与血管活性肠肽（VIP）相比，PACAP在较低浓度时就能刺激LUC活性，这支持了PACAP通过1型受体发挥作用的观点。PACAP对LUC活性的影响在2小时时即可观察到，4至12小时达到峰值，并至少持续至20小时。用截短至-507、-424、-288、-205、-146和-133的小鼠α-LUC构建体转染αT3-1细胞，并用PACAP、一种细胞可渗透的环磷酸腺苷类似物（8-溴环磷酸腺苷）、佛波酯肉豆蔻酸酯乙酸酯（PMA）或对照培养基处理。PACAP对转录激活的作用在-288和-205小鼠α-LUC载体中最高（刺激7至8倍），当5'侧翼区域截短至-146或-133时显著降低。环磷酸腺苷对α亚基的刺激模式与PACAP的模式密切平行。使用PMA时，在-507和-424之间，尤其是-424和-288之间观察到LUC活性逐步下降，而截短至-205、-146或-133时活性不再进一步降低。在-507至+46小鼠α启动子的垂体糖蛋白激素基础元件（-337至-330）或促性腺激素释放激素反应元件（GnRH-RE）（-406至-399）中的簇状点突变分别显著（P<0.05）增强和降低了PACAP对转录活性的影响。这些结果表明，小鼠α亚基启动子有几个区域介导对PACAP的反应。PACAP和环磷酸腺苷反应性的共定位以及涉及蛋白激酶A（H-89）或蛋白激酶C（PKC）（双吲哚基马来酰亚胺）特异性抑制剂的研究结果表明，PACAP对α亚基转录的作用主要由蛋白激酶A（PKA）途径介导。
