Schuchardt Jan P, Wahlström David, Rüegg Joëlle, Giese Norbert, Stefan Madalina, Hopf Henning, Pongratz Ingemar, Håkansson Helen, Eichele Gregor, Pettersson Katarina, Nau Heinz
Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine, Hannover, Germany.
FEBS J. 2009 Jun;276(11):3043-59. doi: 10.1111/j.1742-4658.2009.07023.x. Epub 2009 Apr 22.
Retinoic acid receptor (RAR) and retinoid X receptor are ligand-induced transcription factors that belong to the nuclear receptor family. The receptors are activated by small hydrophobic compounds, such as all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Interestingly, these receptors are also targets for a number of exogenous compounds. In this study, we characterized the biological activity of the 9-cis-substituted retinoic acid metabolite, S-4-oxo-9-cis-13,14-dihydro-retinoic acid (S-4o9cDH-RA). The endogenous levels of this metabolite in wild-type mice and rats were found to be higher than those of all-trans-retinoic acid, especially in the liver. Using cell-based luciferase reporter systems, we showed that S-4o9cDH-RA activates the transcription of retinoic acid response element-containing genes in several cell types, both from a simple 2xDR5 element and from the promoter of the natural retinoid target gene RARbeta2. In addition, quantitative RT-PCR analysis demonstrated that S-4o9cDH-RA treatment significantly increases the endogenous mRNA levels of the RAR target gene RARbeta2. Utilizing a limited proteolytic digestion assay, we showed that S-4o9cDH-RA induces conformational changes to both RARalpha and RARbeta in the same manner as does all-trans-retinoic acid, suggesting that S-4o9cDH-RA is indeed an endogenous ligand for these receptors. These in vitro results were corroborated in an in vivo system, where S-4o9cDH-RA induced morphological changes similar to those of all-trans-retinoic acid in the developing chicken wing bud. When locally applied to the wing bud, S-4o9cDH-RA induced digit pattern duplications in a dose-dependent fashion. The results illustrate that S-4o9cDH-RA closely mimics all-trans-retinoic acid with regard to pattern respecification. Finally, using quantitative RT-PCR analysis, we showed that S-4o9cDH-RA induces the transcription of several retinoic acid-regulated genes in chick wing buds, including Hoxb8, RARbeta2, shh, Cyp26 and bmp2. Although S-4o9cDH-RA was less potent when compared with all-trans-retinoic acid, the findings clearly demonstrate that S-4o9cDH-RA has the capacity to bind and activate nuclear retinoid receptors and regulate gene transcription both in vitro and in vivo.
维甲酸受体（RAR）和维甲酸X受体是属于核受体家族的配体诱导型转录因子。这些受体分别被小的疏水性化合物激活，如全反式维甲酸和9-顺式维甲酸。有趣的是，这些受体也是许多外源性化合物的作用靶点。在本研究中，我们对9-顺式取代的维甲酸代谢物S-4-氧代-9-顺式-13,14-二氢维甲酸（S-4o9cDH-RA）的生物学活性进行了表征。发现野生型小鼠和大鼠体内这种代谢物的内源性水平高于全反式维甲酸，尤其是在肝脏中。使用基于细胞的荧光素酶报告系统，我们表明S-4o9cDH-RA在几种细胞类型中均能激活含维甲酸反应元件基因的转录，无论是来自简单的2xDR5元件还是天然维甲酸靶基因RARbeta2的启动子。此外，定量RT-PCR分析表明，S-4o9cDH-RA处理显著增加了RAR靶基因RARbeta2的内源性mRNA水平。利用有限蛋白酶消化试验，我们表明S-4o9cDH-RA与全反式维甲酸一样，以相同的方式诱导RARalpha和RARbeta的构象变化，这表明S-4o9cDH-RA确实是这些受体的内源性配体。这些体外结果在体内系统中得到了证实，在发育中的鸡翼芽中，S-4o9cDH-RA诱导的形态变化与全反式维甲酸相似。当局部应用于翼芽时，S-4o9cDH-RA以剂量依赖性方式诱导指型重复。结果表明，在模式重新指定方面，S-4o9cDH-RA与全反式维甲酸非常相似。最后，使用定量RT-PCR分析，我们表明S-4o9cDH-RA在鸡翼芽中诱导了几种维甲酸调节基因的转录，包括Hoxb8、RARbeta2、shh、Cyp26和bmp2。尽管与全反式维甲酸相比，S-4o9cDH-RA的效力较低，但这些发现清楚地表明，S-4o9cDH-RA具有在体外和体内结合并激活核维甲酸受体以及调节基因转录的能力。
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