Hansen T V, Rehfeld J F, Nielsen F C
Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
Mol Endocrinol. 1999 Mar;13(3):466-75. doi: 10.1210/mend.13.3.0257.
Cholecystokinin (CCK) is a potent neuropeptide expressed in the small intestine and in the central nervous system. We have examined the effect of basic fibroblast factor (bFGF) and forskolin on CCK gene transcription and depicted the signaling pathways that lead to promoter activation. bFGF and forskolin stimulated promoter activity via a cAMP response element (CRE)/12-O-tetradecanoylphorbol-13-acetate response element (TRE) located 80 bp upstream from the transcription initiation site. In nuclear extracts from unstimulated as well as stimulated cells, only CRE-binding protein (CREB) and activating transcription factor-1 (ATF-1) bound to the CRE/TRE, and activation was associated with phosphorylation of CREB serine-133 and ATF-1 serine-63. In murine F9 cells, CREB stimulated promoter activity 10-fold in the presence of protein kinase A (PKA), and in SK-N-MC cells activation was inhibited 60-70% by a dominant negative CREB mutant. In contrast, ATF-1 had no effect in F9 cells and exhibited a dominant negative effect in SK-N-MC cells. bFGF stimulation led to phosphorylation of the p38 mitogen-activated protein kinase (MAPK), and the extracellular signal-regulated kinase (ERK) MAPK and promoter activation, phosphorylation of CREB, and GAL4-CREB-dependent transcription were selectively prevented by a dominant negative Ras-mutant, the p38 MAPK-specific inhibitor SB203580, and the MAP/ERK kinase 1 (MEK1) inhibitor PD098059. Forskolin stimulation proceeded via the PKA pathway, and to a minor extent via the p38 and ERK MAPK pathways. We conclude that bFGF and forskolin stimulate the CCK gene promoter via the CRE/TRE(-80) in the proximal promoter region. Signaling proceeds through the p38 MAPK, the ERK MAPK, and the PKA-signaling pathways, which leads to cumulative phosphorylation and activation of CREB. We propose that bFGF in combination with neurotransmitters/neuropeptides coupling to the PKA-signaling pathway play an important role in the control of CCK gene expression.
胆囊收缩素（CCK）是一种在小肠和中枢神经系统中表达的强效神经肽。我们研究了碱性成纤维细胞生长因子（bFGF）和福斯高林对CCK基因转录的影响，并描绘了导致启动子激活的信号通路。bFGF和福斯高林通过位于转录起始位点上游80 bp处的环磷酸腺苷反应元件（CRE）/十四酰佛波醇乙酯反应元件（TRE）刺激启动子活性。在未刺激和刺激细胞的核提取物中，只有CRE结合蛋白（CREB）和激活转录因子-1（ATF-1）与CRE/TRE结合，且激活与CREB丝氨酸-133和ATF-1丝氨酸-63的磷酸化相关。在小鼠F9细胞中，在蛋白激酶A（PKA）存在的情况下，CREB刺激启动子活性增加10倍，而在SK-N-MC细胞中，显性负性CREB突变体抑制激活60-70%。相比之下，ATF-1在F9细胞中无作用，而在SK-N-MC细胞中表现出显性负性作用。bFGF刺激导致p38丝裂原活化蛋白激酶（MAPK）磷酸化，显性负性Ras突变体、p38 MAPK特异性抑制剂SB203580和丝裂原活化蛋白激酶/细胞外信号调节激酶1（MEK1）抑制剂PD098059可选择性地阻止细胞外信号调节激酶（ERK）MAPK、启动子激活、CREB磷酸化以及GAL4-CREB依赖性转录。福斯高林刺激通过PKA途径进行，在较小程度上通过p38和ERK MAPK途径进行。我们得出结论，bFGF和福斯高林通过近端启动子区域中的CRE/TRE（-80）刺激CCK基因启动子。信号通过p38 MAPK、ERK MAPK和PKA信号通路传递，导致CREB的累积磷酸化和激活。我们提出，bFGF与偶联至PKA信号通路的神经递质/神经肽结合，在CCK基因表达的控制中起重要作用。
