Budziszewska Bogusława, Jaworska-Feil Lucylla, Tetich Magdalena, Basta-Kaim Agnieszka, Kubera Marta, Leśkiewicz Monika, Lasoń Władysław
Department of Endocrinology, Institute of Pharmacology, Polish Academy of Sciences, Kraków, Smetna St, Poland.
Neuropsychopharmacology. 2004 Apr;29(4):785-94. doi: 10.1038/sj.npp.1300379.
Major depression is frequently associated with hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. Clinically effective therapy with antidepressant drugs normalizes the disturbed activity of HPA axis, in part, by decreasing corticotropin-releasing hormone (CRH) synthesis, but the mechanism of this action is poorly recognized. In order to find out whether antidepressants directly affect CRH gene promoter activity, we studied their effect on undifferentiated and differentiated Neuro-2A cells, and for comparison the effect of the selected antidepressants on AtT-20 cells was also determined. The cells were stably transfected with a human CRH promoter fragment (-663 to +124 bp) linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The regulation of CRH gene promoter activity is similar in Neuro-2A cells, both intact and differentiated, and in AtT-20 cell line, and cAMP/PKA-dependent pathway plays an important role in the stimulation of CRH gene. It was found that imipramine, amitryptyline, desipramine, fluoxetine, and mianserin, present in the culture medium for 5 days, in a concentration-dependent manner inhibited basal hCRH gene promoter activity in undifferentiated Neuro-2A cells, while other drugs under study (citalopram, tianeptine, moclobemide, venlafaxine, reboxetine, mirtazapine, and milnacipram) were inactive. In the differentiated cells, all examined antidepressants, except moclobemide (no effect) and tianeptine (increase), inhibited hCRH gene transcription. Moreover, in differentiated cells, the drugs acted stronger and were effective at lower concentrations. Forskolin-induced CAT activity was attenuated by imipramine and fluoxetine and to a lesser degree by amitriptyline and desipramine in differentiated cells, whereas other drugs were inactive. Moreover, imipramine and fluoxetine, but not tianeptine, showed moderate inhibitory effect on CRH gene promoter activity also in AtT-20 cell line, commonly used in CRH gene regulation studies. These results indicate that neuron-like differentiated Neuro-2A cells are a better model than pituitary and intact neuroblastoma to investigate the mechanism of psychotropic drug action. Inhibition of CRH gene promoter activity by antidepressant drugs may be a molecular mechanism by which these drugs inhibit the activity of HPA axis.
重度抑郁症常与下丘脑 - 垂体 - 肾上腺（HPA）轴功能亢进有关。抗抑郁药物的临床有效治疗可部分通过降低促肾上腺皮质激素释放激素（CRH）的合成使紊乱的HPA轴活动恢复正常，但其作用机制尚不清楚。为了探究抗抑郁药是否直接影响CRH基因启动子活性，我们研究了它们对未分化和分化的Neuro - 2A细胞的作用，并且为了进行比较，还测定了所选抗抑郁药对AtT - 20细胞的作用。这些细胞用与氯霉素乙酰转移酶（CAT）报告基因相连的人CRH启动子片段（-663至+124 bp）进行稳定转染。在完整的和分化的Neuro - 2A细胞以及AtT - 20细胞系中，CRH基因启动子活性的调节相似，并且cAMP/PKA依赖性途径在CRH基因的刺激中起重要作用。结果发现，培养基中存在5天的丙咪嗪、阿米替林、去甲丙咪嗪、氟西汀和米安色林以浓度依赖性方式抑制未分化Neuro - 2A细胞中基础hCRH基因启动子活性，而其他所研究的药物（西酞普兰、噻奈普汀、吗氯贝胺、文拉法辛、瑞波西汀、米氮平和米那普明）无活性。在分化细胞中，除了吗氯贝胺（无作用）和噻奈普汀（增加）外，所有检测的抗抑郁药均抑制hCRH基因转录。此外，在分化细胞中，这些药物作用更强且在较低浓度下有效。在分化细胞中，福斯高林诱导的CAT活性被丙咪嗪和氟西汀减弱，被阿米替林和去甲丙咪嗪在较小程度上减弱，而其他药物无活性。此外，丙咪嗪和氟西汀，但不是噻奈普汀，在常用于CRH基因调节研究的AtT - 20细胞系中也对CRH基因启动子活性表现出适度的抑制作用。这些结果表明，与垂体和完整的神经母细胞瘤相比，神经元样分化的Neuro - 2A细胞是研究精神药物作用机制的更好模型。抗抑郁药对CRH基因启动子活性的抑制可能是这些药物抑制HPA轴活性的分子机制。
