Lee Po-Wei, Lee Yu-May
Institute of Biological Chemistry, Academia Sinica, 128 Section 2, Academy Road, Nankang, Taipei 115, Taiwan.
Mol Pharmacol. 2003 Dec;64(6):1410-8. doi: 10.1124/mol.64.6.1410.
The utility of morphine for the treatment of chronic pain is hindered by the development of tolerance. Fentanyl has been shown to be a potent analgesic with a lower propensity to produce tolerance and physical dependence in the clinical setting. Previous finding has shown that fentanyl induces mu opioid receptor gene expression in PC-12 cells (Brain Res 859:217-223, 2000). In this report, we aim to identify the molecular mechanism of mu-opioid receptor (MOR) gene regulation by fentanyl. We demonstrated that the 4.7-kilobase MOR promoter could be induced by fentanyl in PC-12 cells, and we defined a partial cAMP response element (CRE) located at -106/-111 in 5'-untranslated region of the MOR gene. In electrophoretic mobility shift assay, cAMP response element-binding protein (CREB) was found in the protein-DNA complex formed on the CRE box. CREB was phosphorylated after forskolin induction, and both CREB and CREB-binding protein (CBP) binding to the endogenous MOR promoter was increased by forskolin in chromatin immunoprecipitation assay. The functional role of CREB in the induction of MOR gene was further elucidated by an experiment in which a dominant-negative mutant CREB, CREB-S133A, abolished the forskolin-mediated MOR induction. Moreover, we found that this CRE box is conserved in mouse, rat, and human MOR gene, implying physiological relevance in different species. Collectively, this study demonstrated that fentanyl-triggered MOR gene induction was mediated by the sequential activation of CREB and the binding of CREB and CBP to MOR promoter, thus provides direct evidence for lower propensity of fentanyl to produce tolerance.
吗啡用于治疗慢性疼痛的效用因耐受性的产生而受到阻碍。芬太尼已被证明是一种强效镇痛药，在临床环境中产生耐受性和身体依赖性的倾向较低。先前的研究发现表明，芬太尼可诱导PC-12细胞中μ阿片受体基因表达（《脑研究》859:217 - 223，2000）。在本报告中，我们旨在确定芬太尼对μ阿片受体（MOR）基因调控的分子机制。我们证明了4.7千碱基的MOR启动子可被芬太尼在PC-12细胞中诱导，并且我们确定了位于MOR基因5'非翻译区 - 106 / - 111处的部分环磷酸腺苷反应元件（CRE）。在电泳迁移率变动分析中，在CRE框上形成的蛋白质 - DNA复合物中发现了环磷酸腺苷反应元件结合蛋白（CREB）。福司可林诱导后CREB被磷酸化，并且在染色质免疫沉淀分析中，福司可林增加了CREB和CREB结合蛋白（CBP）与内源性MOR启动子的结合。通过一项实验进一步阐明了CREB在MOR基因诱导中的功能作用，在该实验中，显性负性突变体CREB，即CREB - S133A，消除了福司可林介导的MOR诱导。此外，我们发现该CRE框在小鼠、大鼠和人类MOR基因中是保守的，这意味着在不同物种中具有生理相关性。总体而言，本研究表明芬太尼触发的MOR基因诱导是由CREB的顺序激活以及CREB和CBP与MOR启动子的结合介导的，从而为芬太尼产生耐受性的倾向较低提供了直接证据。
