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Different Effects of Knockouts in ALDH 2 and ACSS 2 on Embryonic Stem Cell Differentiation
Author(s) -
Serio Ryan N.,
Lu Changyuan,
Gross Steven S.,
Gudas Lorraine J.
Publication year - 2019
Publication title -
alcoholism: clinical and experimental research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.267
H-Index - 153
eISSN - 1530-0277
pISSN - 0145-6008
DOI - 10.1111/acer.14146
Subject(s) - aldehyde dehydrogenase , retinoic acid , homeobox protein nanog , sox2 , klf4 , cellular differentiation , embryonic stem cell , chemistry , stem cell , acetaldehyde , tretinoin , biochemistry , biology , microbiology and biotechnology , induced pluripotent stem cell , gene , ethanol
Background Ethanol (EtOH) is a teratogen that causes severe birth defects, but the mechanisms by which Et OH affects stem cell differentiation are unclear. Our goal here is to examine the effects of Et OH and its metabolites, acetaldehyde (AcH) and acetate, on embryonic stem cell (ESC) differentiation. Methods We designed ESC lines in which aldehyde dehydrogenase ( ALDH 2, NCBI #11669) and acyl‐CoA synthetase short‐chain family member 2 ( ACSS 2, NCBI #60525) were knocked out by CRISPR ‐Cas9 technology. We selected these genes because of their key roles in Et OH oxidation in order to dissect the effects of Et OH metabolism on differentiation. Results By using kinetic assays, we confirmed that AcH is primarily oxidized by ALDH 2 rather than ALDH 1A2. We found increases in mRNA s of differentiation‐associated genes ( Hoxa1 , Cyp26a1 , and RAR β2 ) upon Et OH treatment of WT and Acss2 −/− ESC s, but not Aldh2 −/− ESC s. The absence of ALDH 2 reduced mRNA s of some pluripotency factors ( Nanog , Sox2 , and Klf4 ). Treatment of WT ESC s with AcH or 4‐hydroxynonenal (4‐ HNE ), another substrate of ALDH 2, increased differentiation‐associated transcripts compared to levels in untreated cells. mRNA s of genes involved in retinoic acid ( RA ) synthesis ( Stra6 and Rdh10 ) were also increased by Et OH , AcH, and 4‐ HNE treatment. Retinoic acid receptor‐ γ ( RAR γ ) is required for both Et OH ‐ and AcH‐mediated increases in Hoxa1 and Stra6 , demonstrating the critical role of RA : RAR γ signaling in AcH‐induced ESC differentiation. Conclusions ACSS 2 knockouts showed no changes in differentiation phenotype, while pluripotency‐related transcripts were decreased in ALDH 2 knockout ESC s. We demonstrate that AcH increases differentiation‐associated mRNA s in ESC s via RAR γ .