Different Effects of Knockouts in ALDH2 and ACSS2 on Embryonic Stem Cell Differentiation

Abstract

Background: 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.Methods: 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.Results: 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 RARb2) upon EtOH treatment of WT and Acss2 À/À ESCs, but not Aldh2 À/À 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-c (RARc) is required for both EtOH-and AcH-mediated increases in Hoxa1 and Stra6, demonstrating the critical role of RA:RARc signaling in AcH-induced ESC differentiation.Conclusions: 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 RARc.

Silencing transcription factor FOXM1 represses proliferation, migration, and invasion while inducing apoptosis of liver cancer stem cells by regulating the expression of ALDH2

“…This procedure was repeated in six wells. Three time points (24,48, and 72 hr) were set for the subsequent experiments. Each well was added with 20 μL cell counting kit-8 (CCK-8) solution (5 mg/mL), which was incubated at 37 C for 2 hr, and then the culture was terminated.…”

“…Evidence has shown that inhibition of ALDH can significantly reduce the stemness characteristics of stem cells. 24 To investigate whether FOXM1 affects the stemness characteristics of LCSCs by regulating ALDH2, we tested the expression of ALDH2 protein after silencing FOXM1 by western blot analysis.…”

“…22,23 It is reported that the absence of acetaldehyde dehydrogenase-2 (ALDH2) is able to decrease mRNAs of some pluripotency factors (Nanog, Sox2, and Klf4). 24 Evidence has shown that inhibition of ALDH can significantly reduce the stemness characteristics of stem cells. 24 ALDH2 is a mitochondrial enzyme, which is activated only in the mitochondrial matrix, and it also plays a part in removing other reactive aldehydes that are derived from lipid peroxidatio and oxidative stress.…”

“…24 Evidence has shown that inhibition of ALDH can significantly reduce the stemness characteristics of stem cells. 24 ALDH2 is a mitochondrial enzyme, which is activated only in the mitochondrial matrix, and it also plays a part in removing other reactive aldehydes that are derived from lipid peroxidatio and oxidative stress. 25 Meanwhile, according to the International Agency for Research on Cancer, ALDH2 is the lowest Michaelis constant for acetaldehyde, and it is classified as a Group 1 carcinogen.…”

Modification of stem cell states by alcohol and acetaldehyde

“…Our lab has shown that AcH is preferentially metabolized by ALDH2, and not ALDH1A2, using ESC knockout cell lines for each gene and measuring the formation of NADH from NAD + in lysates [69]. ALDH2-null ESCs display lower transcript levels of several pluripotency markers (Nanog, Sox2, Klf4) than WT ESCs [69]. The responses of ALDH2-KO ESCs to EtOH are complex.…”