Weill Cornell Graduate School of Medical Sciences of Cornell University, New York, New York.
Department of Pharmacology, Weill Cornell Medical College, New York, New York.
Alcohol Clin Exp Res. 2019 Sep;43(9):1859-1871. doi: 10.1111/acer.14146. Epub 2019 Aug 5.
Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which EtOH affects stem cell differentiation are unclear. Our goal here is to examine the effects of EtOH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation.
We designed ESC lines in which aldehyde dehydrogenase (ALDH2, NCBI#11669) and acyl-CoA synthetase short-chain family member 2 (ACSS2, NCBI#60525) were knocked out by CRISPR-Cas9 technology. We selected these genes because of their key roles in EtOH oxidation in order to dissect the effects of EtOH metabolism on differentiation.
By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH2 rather than ALDH1A2. We found increases in mRNAs of differentiation-associated genes (Hoxa1, Cyp26a1, and RARβ2) upon EtOH treatment of WT and Acss2 ESCs, but not Aldh2 ESCs. The absence of ALDH2 reduced mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). Treatment of WT ESCs with AcH or 4-hydroxynonenal (4-HNE), another substrate of ALDH2, increased differentiation-associated transcripts compared to levels in untreated cells. mRNAs of genes involved in retinoic acid (RA) synthesis (Stra6 and Rdh10) were also increased by EtOH, AcH, and 4-HNE treatment. Retinoic acid receptor-γ (RARγ) is required for both EtOH- and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARγ signaling in AcH-induced ESC differentiation.
ACSS2 knockouts showed no changes in differentiation phenotype, while pluripotency-related transcripts were decreased in ALDH2 knockout ESCs. We demonstrate that AcH increases differentiation-associated mRNAs in ESCs via RARγ.
乙醇（EtOH）是一种致畸物，可导致严重的出生缺陷，但乙醇影响干细胞分化的机制尚不清楚。我们的目标是研究乙醇及其代谢产物乙醛（AcH）和乙酸盐对胚胎干细胞（ESC）分化的影响。
我们使用 CRISPR-Cas9 技术设计了醛脱氢酶（ALDH2，NCBI#11669）和酰基辅酶 A 合成酶短链家族成员 2（ACSS2，NCBI#60525）敲除的 ESC 系。我们选择这些基因是因为它们在乙醇氧化中起关键作用，以便剖析乙醇代谢对分化的影响。
通过使用动力学测定法，我们证实 AcH 主要由 ALDH2 而不是 ALDH1A2 氧化。我们发现 WT 和 Acss2 ESC 在用 EtOH 处理后，分化相关基因（Hoxa1、Cyp26a1 和 RARβ2）的 mRNAs 增加，但 Aldh2 ESC 则不然。ALDH2 的缺失降低了一些多能性因子（Nanog、Sox2 和 Klf4）的 mRNAs。与未处理的细胞相比，用 AcH 或另一种 ALDH2 底物 4-羟基壬烯醛（4-HNE）处理 WT ESC 会增加分化相关转录物。乙醇、AcH 和 4-HNE 处理还增加了参与视黄酸（RA）合成的基因（Stra6 和 Rdh10）的 mRNAs。视黄酸受体-γ（RARγ）是 EtOH 和 AcH 介导的 Hoxa1 和 Stra6 增加所必需的，这表明 RA：RARγ 信号在 AcH 诱导的 ESC 分化中起关键作用。
ACSS2 敲除不会改变分化表型，而 ALDH2 敲除 ESC 中的多能性相关转录物减少。我们证明 AcH 通过 RARγ 增加 ESC 中的分化相关 mRNAs。
