Remes Lenicov Federico, Lemonde Sylvie, Czesak Margaret, Mosher Tera M, Albert Paul R
Ottawa Health Research Institute (Neuroscience), University of Ottawa, Ottawa, Ontario, Canada.
J Neurochem. 2007 Dec;103(5):2047-57. doi: 10.1111/j.1471-4159.2007.04903.x. Epub 2007 Sep 14.
Alterations in brain serotonin levels are implicated in major depression and are regulated by tryptophan hydroxylase-2 (TPH2). To study its regulation, we measured TPH2 RNA by quantitative RT-PCR in differentiated serotonergic rat raphe RN46A and GH4C1 pituitary cells, which express TPH2. Upon calcium mobilization using KCl (40 mmol/L), TPH2 RNA was rapidly (1 h) and strongly (> 10-fold) induced in differentiated RN46A cells, but not in GH4C1 cells. This effect was blocked by actinomycin D, implicating transcriptional activation. Similarly, calcium ionophore ionomycin induced TPH2 RNA by threefold in RN46A cells. To address the promoter sites involved, the transcription start site was identified and a series of TATA-containing TPH2 promoter-luciferase constructs were analyzed. In differentiated RN46A cells, the TPH2 promoter was induced 2.5-fold by ionomycin, similar to its action on TPH2 RNA. By contrast, ionomycin had no effect on TPH2 promoter activity in GH4C1 cells or TPH2-negative L6 myoblasts. Ionomycin sensitivity was localized to within 88 bp of the start site, containing putative CCATT-enhancer binding protein element, activator protein-1 and -2 (AP-1, AP-2) elements. These results are the first to identify calcium-mediated regulation of the proximal TPH2 promoter as critical for cell-specific TPH2 expression.
大脑血清素水平的改变与重度抑郁症有关，且受色氨酸羟化酶-2（TPH2）调节。为研究其调节机制，我们通过定量逆转录聚合酶链反应（RT-PCR）在表达TPH2的分化的血清素能大鼠中缝核RN46A细胞和GH4C1垂体细胞中测量了TPH2 RNA。使用氯化钾（40 mmol/L）动员钙后，TPH2 RNA在分化的RN46A细胞中迅速（1小时）且强烈（>10倍）诱导，但在GH4C1细胞中未诱导。这种效应被放线菌素D阻断，提示转录激活。同样，钙离子载体离子霉素在RN46A细胞中使TPH2 RNA诱导了三倍。为确定所涉及的启动子位点，确定了转录起始位点并分析了一系列含TATA的TPH2启动子-荧光素酶构建体。在分化的RN46A细胞中，离子霉素使TPH2启动子诱导了2.5倍，与其对TPH2 RNA的作用相似。相比之下，离子霉素对GH4C1细胞或TPH2阴性的L6成肌细胞中的TPH2启动子活性没有影响。离子霉素敏感性定位于起始位点的88 bp内，包含假定的CCAAT增强子结合蛋白元件、激活蛋白-1和-2（AP-1、AP-2）元件。这些结果首次确定近端TPH2启动子的钙介导调节对细胞特异性TPH2表达至关重要。
