Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells .1. Effects of the common secretagogues
Regulation of the rat proopiomelanocortin gene expression in AtT-20 cells .1. Effects of the common secretagogues
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DOI：
10.1210/en.138.5.1923
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发表时间：
1997-05-01
期刊：
影响因子：
4.8
通讯作者：
Saito, H
中科院分区：
文献类型：
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作者：
Aoki, Y;Iwasaki, Y;Saito, H
虽然各种促分泌剂对促肾上腺皮质激素(ACTH)分泌的影响已经被很好地研究，但它们对POMC基因表达的影响还没有得到充分的表征。本研究利用含有0.7kb大鼠POMC 5‘启动子-荧光素酶融合基因的质粒，建立了一种新的小鼠促肾上腺皮质激素肿瘤细胞株模型。低血清(1%胎牛血清)培养细胞对外源性CRH的反应性明显高于高血清培养(10%)。在此培养条件下，我们不仅检测了CRH，而且还检测了其他促分泌剂，如儿茶酚胺、血管加压素和血管紧张素II对POMC基因转录活性的影响。CRH以剂量和时间依赖的方式刺激POMC启动子活性(增加3.5倍)、cAMP生成和ACTH分泌，孵育3-5h后效果最好。儿茶酚胺，特别是肾上腺素(10 NM及以上)，也刺激所有参数，尽管不如CRH有效，这种作用可被β-肾上腺素能激动剂模拟，但不能被α-肾上腺素能激动剂所模拟，这表明β-肾上腺素能受体参与其中。肾上腺素和CRH在所有参数中的联合作用均大于CRH单独作用，两种激素的作用均被蛋白激酶A抑制剂H89完全阻断，加压素和血管紧张素II对POMC表达的影响最小。我们的结果表明：1)儿茶酚胺和CRH在生理浓度下正向调节POMC基因；2)cAMP-PKA。系统是CRH和儿茶酚胺的共同细胞内信号通路；3)加压素和血管紧张素II对POMC启动子的活性也有微弱但显著的刺激作用。
Although the effects of the various secretagogues on corticotropin (ACTH) secretion have been well studied, their effects on the POMC gene expression have not been thoroughly characterized. In this study, we established a new model system using the AtT20 mouse corticotroph tumor cell line transfected stably with a plasmid containing 0.7 kb of the rat POMC 5' promoter-luciferase fusion gene. The responsiveness to exogenous CRH improved markedly when the cells were cultured with low serum medium (1% FBS) compared with serum rich medium (10%). Using this culture condition, we examined the effects of not only CRH but also other secretagogues such as catecholamines, vasopressin, and angiotensin II, upon the transcriptional activity of the POMC gene. CRH stimulated POMC promoter activity (3.5-fold increase) as well as cAMP generation and ACTH secretion in a dose- and time-dependent manner, with the maximal effect being observed 3-5 h after the start of incubation. Catecholamines, especially epinephrine (10 nM and above), also stimulated all parameters, although less potently than CRH, and the effect was mimicked by the beta-, but not alpha-adrenergic, agonist, suggesting the involvement of the beta-adrenergic receptor. The combined effects of epinephrine and CRH were greater in all parameters than those of CRH alone, and the effects of both hormones were completely blocked by H89, an inhibitor of protein kinase A. Vasopressin and angiotensin II showed minimal effects on POMC expression. Our results suggest that 1) catecholamines, as well as CRH, positively regulate the POMC gene at physiological concentrations; 2) the cAMP-PKA. system is the common intracellular signaling pathway for CRH and catecholamines; and 3) vasopressin and angiotensin II also have weak but significant stimulatory effects on POMC promoter activity.