Retinoic acid, dibutyryl-cAMP, and differentiation affect the expression of retinoic acid receptors in F9 cells.

Expression of the retinoic acid receptors alpha and beta (RAR-alpha and RAR-beta) was examined in F9 cells, an embryonal carcinoma cell model established for the study of retinoid metabolism and function. Addition of retinoic acid to F9 cell medium caused a dose-dependent increase in RAR-beta mRNA within 3 hr that reached 5- to 30-fold greater than the constitutively expressed mRNA by 24 hr. The elevation in mRNA resulted from increased transcription, as demonstrated by nuclear run-on transcription, did not require protein synthesis, and required the constant presence of retinoic acid. N6,O2'-Dibutyryl-cAMP attenuated the retinoic acid-induced increase in RAR-beta mRNA by a post-transcriptional mechanism. In contrast, RAR-alpha mRNA in F9 stem cells was affected less (1.2- to 1.4-fold increase) by retinoic acid and decreased 3-fold transiently when fresh serum was added to the medium. Differentiation of F9 cells resulted in increased steady-state levels of RAR-beta mRNA in primitive (4-fold), parietal (3-fold), and visceral (8-fold) endoderm but decreased steady-state levels of RAR-alpha mRNA in primitive (2-fold), parietal (3-fold), and visceral (1.4-fold) endoderm. These data demonstrate that RAR-beta is a primary target gene for retinoic acid in a characterized model of retinoid function, indicate that constitutive expression of both RAR-beta and RAR-alpha is dependent upon the differentiation state, and suggest hormonal modulation of RAR-beta by cAMP and modulation of RAR-alpha by serum factors. These results distinguish the effects of serum, cAMP, and retinoic acid on the expression of RAR from the effects mediated by differentiation. IMAGES:

本研究以F9细胞——一种用于类视黄醇（retinoid）代谢与功能研究的胚胎癌细胞模型（embryonal carcinoma cell model）——为对象，检测了视黄酸受体α（RAR-α）与视黄酸受体β（RAR-β）的表达情况。向F9细胞培养基中添加视黄酸（retinoic acid）后，3小时内即可引发RAR-β mRNA的剂量依赖性升高，至24小时时其表达量较组成型表达的mRNA提升5至30倍。该mRNA水平的升高源于转录速率提升，核连缀转录实验（nuclear run-on transcription）证实了这一点，且该过程无需蛋白质合成，同时需要视黄酸的持续存在。N6,O2'-二丁酰-cAMP通过转录后机制（post-transcriptional mechanism）减弱了视黄酸诱导的RAR-β mRNA水平升高。与之相反，F9干细胞中的RAR-α mRNA受视黄酸的影响较弱（仅升高1.2至1.4倍），且当向培养基中添加新鲜血清时，其表达会短暂下调3倍。F9细胞的分化可使原始内胚层（primitive endoderm）、壁内胚层（parietal endoderm）与脏内胚层（visceral endoderm）中的RAR-β mRNA稳态水平（steady-state levels）分别提升4倍、3倍与8倍，但同时会使上述三种内胚层中的RAR-α mRNA稳态水平分别下调2倍、3倍与1.4倍。上述数据表明，RAR-β是视黄酸在类视黄醇功能特征化模型中的初级靶基因（primary target gene）；同时提示RAR-β与RAR-α的组成型表达均依赖于细胞分化状态，且RAR-β的表达受cAMP的激素调控（hormonal modulation），而RAR-α的表达则受血清因子（serum factors）调控。本研究结果区分了血清、cAMP与视黄酸对RAR表达的影响，以及由细胞分化所介导的相关效应。IMAGES: