Characterization of the cocaine- and amphetamine-regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP-dependent signaling pathway in GH3 cells
Characterization of the cocaine- and amphetamine-regulated transcript (CART) peptide gene promoter and its activation by a cyclic AMP-dependent signaling pathway in GH3 cells
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DOI：
10.1046/j.0022-3042.2002.00775.x
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发表时间：
2002-03-01
影响因子：
4.7
通讯作者：
Kuhar, MJ
中科院分区：
文献类型：
--
作者：
Dominguez, G;Lakatos, A;Kuhar, MJ
可卡因和苯丙胺调节的转录物（CART）肽是调节的神经肽，在各种生理过程中起作用。卡特mRNA也受到高度调节，因为其水平因精神刺激药物和瘦素而变化。为了了解调节卡车mRNA水平涉及的机制，研究了小鼠手推车5'Fanking调节区域。 3.4 kb的小鼠卡车5'频浮动区的序列揭示了一个近端启动子，其中包含一个转录因子结合位点，包括重叠的STAT/CRE/AP1位点。此外，小鼠和人之间的5'最多320 bp的手推车启动子具有83％的核苷酸身份。生成了三个荧光素酶表达含有不同数量的CART 5'上游序列的构建体并测试了启动子活性。用含有641和3451 bp的上游序列的构造对GH3细胞的瞬时转染表现出强启动子活性，分别产生29倍和51倍刺激，而含有102 bp上游序列的构建体显示了5.4倍的活性增加。含有复合统计/CRE/AP1位点的构建体对GH3细胞中的福斯科林诱导循环诱导响应。 Forskolin的治疗也导致6小时后的CART mRNA水平增加了4.5倍，H89（一种蛋白激酶A的抑制剂）添加了50％的水平。这些研究表明，CART近端启动子位于最高的641 bp之内，而在GH3细胞中，CART基因通过环状AMP依赖性途径进行调节。
Cocaine- and amphetamine-regulated transcript (CART) peptides are regulated neuropeptides that play a role in a variety of physiological processes. CART mRNA is also highly regulated as its levels change in response to psychostimulant drugs and leptin. To understand the mechanisms involved in regulating CART mRNA levels, the mouse CART 5'-flanking regulatory region was studied. The sequence of 3.4 kb of the mouse CART 5'-flanking region revealed a proximal promoter that contains a cluster of transcription factor binding sites, including an overlapping STAT/CRE/AP1 site. In addition, the 5'-most 320 bp of the CART promoter shares 83% nucleotide identity between mouse and human. Three luciferase expressing constructs containing varying amounts of CART 5' upstream sequence were generated and tested for promoter activity. Transient transfection of GH3 cells with constructs containing 641 and 3451 bp of upstream sequence displayed strong promoter activity, producing 29-fold and 51-fold stimulation, respectively, while, a construct containing 102 bp of upstream sequence displayed a 5.4-fold increase in activity. A construct containing the composite STAT/CRE/AP1 site was responsive to cyclic AMP induction by forskolin in GH3 cells. Forskolin treatment also resulted in a 4.5-fold increase in CART mRNA levels after 6 h and the addition of H89, an inhibitor of protein kinase A, reduced the levels by 50%. These studies indicate that the CART proximal promoter lies within the 5'-most 641 bp and that in GH3 cells the CART gene is regulated via a cyclic AMP-dependent pathway.