Idres Nadia, Marill Julie, Flexor Maria A, Chabot Guy G
INSERM UMR-496, Institut Universitaire d'Hématologie, Hôpital Saint-Louis, Paris 10, France.
J Biol Chem. 2002 Aug 30;277(35):31491-8. doi: 10.1074/jbc.M205016200. Epub 2002 Jun 17.
We have shown that four metabolites of all-trans-retinoic acid (ATRA) (4-oxo-, 4-OH-, 18-OH-, and 5,6-epoxy-RA) can induce maturation of NB4 promyelocytic leukemia cells (Idres, N., Benoit, G., Flexor, M. A., Lanotte, M., and Chabot, G. G. (2001) Cancer Res. 61, 700-705). To better understand the mechanism of action of ATRA metabolites and isomers, we assessed their binding to retinoic acid receptors (RARs) and activation of RAR-mediated transcription via a retinoic acid response element (RARE). Competition binding experiments with tritiated ATRA showed that all metabolites could bind to RARs with variable affinity. For transactivation studies, COS-7 cells were cotransfected with RAR alpha, beta, or gamma expression vectors and the reporter plasmid RARE-tk-Luc, and the retinoid concentrations for half-maximal luciferase activity (EC(50)) were determined. All retinoids tested could activate the three RAR isotypes. The lowest EC(50) value for RAR alpha was with 9-cis-RA (13 nM), followed by 4-oxo-RA (33 nM), 5,6-epoxy-RA (77 nM), 13-cis-RA (124 nM), 18-OH-RA (162 nM), ATRA (169 nM), and 4-OH-RA (791 nM). For RAR beta, the EC(50) values increased as follows: 4-oxo-RA (8 nM), ATRA (9 nM), 18-OH-RA (14 nM), 5,6-epoxy-RA (35 nM), 13-cis-RA (47 nM), 4-OH-RA (64 nM), and 9-cis-RA (173 nM). For RAR gamma the EC(50) values were: ATRA (2 nM), 5,6-epoxy-RA (4 nM), 18-OH-RA (14 nM), 13-cis-RA (36 nM), 9-cis-RA (58 nM), 4-oxo-RA (89 nM), and 4-OH-RA (94 nM). By comparing the -fold induction of luciferase activity, all retinoids tested were equipotent at transactivating RARE-tk-Luc whatever the RAR considered. However, the best induction of the transcription was obtained for RAR alpha, which was 5-fold higher than for RAR beta and 10-fold higher than for RAR gamma. In conclusion, these data show that ATRA metabolites can bind to and activate the three RARs with variable relative affinity but with similar efficacy. These results suggest that ATRA metabolites may activate several signaling pathways and probably play an important role in cellular physiology and cancer therapy.
我们已经证明，全反式维甲酸（ATRA）的四种代谢产物（4-氧代-、4-羟基-、18-羟基-和5,6-环氧-RA）可诱导NB4早幼粒细胞白血病细胞成熟（伊德雷斯，N.，贝努瓦，G.，弗莱克索尔，M. A.，拉诺特，M.，以及沙博，G. G.（2001年）《癌症研究》61卷，700 - 705页）。为了更好地理解ATRA代谢产物和异构体的作用机制，我们评估了它们与维甲酸受体（RARs）的结合以及通过维甲酸反应元件（RARE）对RAR介导的转录的激活。用氚标记的ATRA进行的竞争结合实验表明，所有代谢产物都能以不同的亲和力与RARs结合。对于反式激活研究，将COS - 7细胞与RARα、β或γ表达载体以及报告质粒RARE - tk - Luc共转染，并测定半数最大荧光素酶活性（EC(50)）时的类维生素A浓度。所有测试的类维生素A都能激活三种RAR亚型。RARα的最低EC(50)值是9 - 顺式 - RA（13 nM），其次是4 - 氧代 - RA（33 nM）、5,6 - 环氧 - RA（77 nM）、13 - 顺式 - RA（124 nM）、18 - 羟基 - RA（162 nM）、ATRA（169 nM）和4 - 羟基 - RA（791 nM）。对于RARβ，EC(50)值按以下顺序增加：4 - 氧代 - RA（8 nM）、ATRA（9 nM）、18 - 羟基 - RA（14 nM）、5,6 - 环氧 - RA（35 nM）、13 - 顺式 - RA（47 nM）、4 - 羟基 - RA（64 nM）和9 - 顺式 - RA（173 nM）。对于RARγ，EC(50)值分别为：ATRA（2 nM）、5,6 - 环氧 - RA（4 nM）、18 - 羟基 - RA（14 nM）、13 - 顺式 - RA（36 nM）、9 - 顺式 - RA（58 nM）、4 - 氧代 - RA（89 nM）和4 - 羟基 - RA（94 nM）。通过比较荧光素酶活性的诱导倍数，无论考虑哪种RAR，所有测试的类维生素A在反式激活RARE - tk - Luc方面都是等效的。然而，RARα的转录诱导效果最佳，比RARβ高5倍，比RARγ高10倍。总之，这些数据表明，ATRA代谢产物能够以不同的相对亲和力结合并激活三种RARs，但具有相似的效力。这些结果表明，ATRA代谢产物可能激活多种信号通路，并且可能在细胞生理学和癌症治疗中发挥重要作用。
