Delescluse C, Cavey M T, Martin B, Bernard B A, Reichert U, Maignan J, Darmon M, Shroot B
Centre International de Recherches Dermatologiques Galderma (CIRD Galderma), Sophia Antipolis, Valbonne, France.
Mol Pharmacol. 1991 Oct;40(4):556-62.
Biological effects of retinoic acid (RA) are mediated through its binding to three closely related nuclear receptors (RAR alpha, RAR beta, and RAR gamma) belonging to the steroid-thyroid nuclear receptor family. RARs are able to modulate the transcription of specific genes by binding to responsive elements located in the promoter-enhancer region of these genes. As demonstrated by in situ hybridization, the distribution of each RAR type in the developing embryo, as well as in the adult, is not uniform. In this context, synthetic retinoids that would behave as selective ligands would be invaluable for studying the respective roles of each RAR type in cultured cells, whole animals, and embryos. Moreover, from a pharmacological point of view, such selective compounds may possess a higher therapeutic index and a lower teratogenic risk, because they might affect specific tissues and spare some others. As an approach to this problem, we have set up two complementary assays, (i) an in vitro binding assay to determine the Kd values of retinoids for RAR alpha, RAR beta, and RAR gamma and (ii) a functional assay in cultured cells to evaluate the potential of retinoids to transactivate, through their binding to one type of RAR, a reporter gene. The binding assay uses nuclear extracts of COS-7 cells transfected with vectors expressing RAR alpha, RAR beta, or RAR gamma. The functional assay is a measure of chloramphenicol acetyltransferase (CAT) activity in HeLa cells co-transfected with the expression vectors used in the binding assay and the reporter gene TRE-tk-CAT. Selective agonists for RAR alpha (Am80 and Am580) and RAR beta-RAR gamma (CD495 and CD564) were identified. However, compounds with pure RAR beta or RAR gamma selectivity have not yet been identified.
视黄酸（RA）的生物学效应是通过其与类固醇 - 甲状腺核受体家族中三个密切相关的核受体（RARα、RARβ和RARγ）结合来介导的。RAR能够通过与位于这些基因启动子 - 增强子区域的反应元件结合来调节特定基因的转录。原位杂交表明，每种RAR类型在发育中的胚胎以及成体中的分布并不均匀。在这种情况下，作为选择性配体的合成类视黄醇对于研究每种RAR类型在培养细胞、整体动物和胚胎中的各自作用将是非常宝贵的。此外，从药理学角度来看，这种选择性化合物可能具有更高的治疗指数和更低的致畸风险，因为它们可能只影响特定组织而不影响其他组织。作为解决这个问题的一种方法，我们建立了两种互补的检测方法：（i）一种体外结合检测方法，用于确定类视黄醇与RARα、RARβ和RARγ的解离常数（Kd值）；（ii）一种在培养细胞中的功能检测方法，用于评估类视黄醇通过与一种类型的RAR结合来反式激活报告基因的潜力。结合检测使用转染了表达RARα、RARβ或RARγ载体的COS - 7细胞的核提取物。功能检测是通过与结合检测中使用的表达载体和报告基因TRE - tk - CAT共转染的HeLa细胞中氯霉素乙酰转移酶（CAT）活性来衡量的。我们鉴定出了RARα的选择性激动剂（Am80和Am580）以及RARβ - RARγ的选择性激动剂（CD495和CD564）。然而，尚未鉴定出具有纯RARβ或RARγ选择性的化合物。
