From the Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York 10065 and.
Department of Pharmacology, Weill Cornell Medical College, New York, New York 10065.
J Biol Chem. 2019 Apr 5;294(14):5536-5548. doi: 10.1074/jbc.RA118.007153. Epub 2019 Feb 8.
Ethanol (EtOH) is a teratogen, but its teratogenic mechanisms are not fully understood. The alcohol form of vitamin A (retinol/ROL) can be oxidized to all--retinoic acid (RA), which plays a critical role in stem cell differentiation and development. Using an embryonic stem cell (ESC) model to analyze EtOH's effects on differentiation, we show here that EtOH and acetaldehyde, but not acetate, increase differentiation-associated mRNA levels, and that EtOH decreases pluripotency-related mRNAs. Using reporter assays, ChIP assays, and retinoic acid receptor-γ (RARγ) knockout ESC lines generated by CRISPR/Cas9 and homologous recombination, we demonstrate that EtOH signals via RARγ binding to RA response elements (RAREs) in differentiation-associated gene promoters or enhancers. We also report that EtOH-mediated increases in homeobox A1 () and cytochrome P450 family 26 subfamily A member 1 () transcripts, direct RA target genes, require the expression of the RA-synthesizing enzyme, aldehyde dehydrogenase 1 family member A2 (Aldh1a2), suggesting that EtOH-mediated induction of and requires ROL from the serum. As shown with CRISPR/Cas9 knockout lines, the retinol dehydrogenase gene and a functional RARE in the ROL transporter stimulated by retinoic acid 6 () gene are required for EtOH induction of and We conclude that EtOH stimulates stem cell differentiation by increasing the influx and metabolism of ROL for downstream RARγ-dependent transcription. In stem cells, EtOH may shift cell fate decisions to alter developmental outcomes by increasing endogenous ROL/RA signaling via increased Stra6 expression and ROL oxidation.
乙醇（EtOH）是一种致畸物，但它的致畸机制尚未完全阐明。维生素 A 的酒精形式（视黄醇/ROL）可以被氧化为全反式视黄酸（RA），后者在干细胞分化和发育中起着关键作用。我们使用胚胎干细胞（ESC）模型来分析 EtOH 对分化的影响，结果表明 EtOH 和乙醛，但不是乙酸盐，会增加与分化相关的 mRNA 水平，而 EtOH 会降低与多能性相关的 mRNA。通过报告基因检测、染色质免疫沉淀检测以及 CRISPR/Cas9 和同源重组产生的 RARγ 敲除 ESC 系，我们证明 EtOH 通过 RARγ 与分化相关基因启动子或增强子中的 RA 反应元件（RARE）结合来发出信号。我们还报告说，EtOH 介导的同源盒 A1（）和细胞色素 P450 家族 26 亚家族 A 成员 1（）转录物的增加，直接是 RA 靶基因，需要表达 RA 合成酶醛脱氢酶 1 家族成员 A2（Aldh1a2），这表明 EtOH 介导的和的诱导需要血清中的 ROL。如通过 CRISPR/Cas9 敲除系显示，视黄醇脱氢酶基因和受视黄酸 6（）基因刺激的 ROL 转运蛋白中的功能性 RARE 对于 EtOH 诱导和是必需的。我们得出结论，EtOH 通过增加 ROL 的流入和代谢来刺激干细胞分化，从而促进下游 RARγ 依赖性转录。在干细胞中，EtOH 可能通过增加 Stra6 表达和 ROL 氧化来增加内源性 ROL/RA 信号，从而改变细胞命运决定，改变发育结果。
