title: Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation.
abstract: Oxygen signaling is critical for stem cell regulation, and oxidative stress-induced stem cell apoptosis decreases the efficiency of stem cell therapy. Hypoxia activates O-linked β-N-acetyl glucosaminylation (O-GlcNAcylation) of stem cells, which contributes to regulation of cellular metabolism, as well as cell fate. Our study investigated the role of O-GlcNAcylation via glucosamine in the protection of hypoxia-induced apoptosis of mouse embryonic stem cells (mESCs). Hypoxia increased mESCs apoptosis in a time-dependent manner. Moreover, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by O-GlcNAc transferase inhibitors. In addition, hypoxia regulated several lipid metabolic enzymes, whereas glucosamine increased expression of glycerol-3-phosphate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). In addition, glucosamine-increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by gpat1 siRNA transfection reduced glucosamine-mediated anti-apoptosis in mESCs and reduced mammalian target of rapamycin (mTOR) phosphorylation. Indeed, LPA prevented mESCs from undergoing hypoxia-induced apoptosis and increased phosphorylation of mTOR and its substrates (S6K1 and 4EBP1). Moreover, mTOR inactivation by rapamycin (mTOR inhibitor) increased pro-apoptotic proteins expressions and mESC apoptosis. Furthermore, transplantation of non-targeting siRNA and glucosamine-treated mESCs increased cell survival and inhibited flap necrosis in mouse skin flap model. Conversely, silencing of GPAT1 expression reversed those glucosamine effects. In conclusion, enhancing O-GlcNAcylation of Sp1 by glucosamine stimulates GPAT1 expression, which leads to inhibition of hypoxia-induced mESC apoptosis via mTOR activation.
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute for Veterinary Science and BK21 Creative Veterinary Research Center, Seoul National University, Seoul, Korea.
authors.forename: H J
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Korea.
authors.forename: J M
authors.initials: JM
authors.lastname: Ryu
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute for Veterinary Science and BK21 Creative Veterinary Research Center, Seoul National University, Seoul, Korea.
authors.forename: Y H
authors.initials: YH
authors.lastname: Jung
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute for Veterinary Science and BK21 Creative Veterinary Research Center, Seoul National University, Seoul, Korea.
authors.forename: K H
authors.initials: KH
authors.lastname: Lee
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute for Veterinary Science and BK21 Creative Veterinary Research Center, Seoul National University, Seoul, Korea.
authors.forename: D I
authors.initials: DI
authors.lastname: Kim
authors.affiliation: Department of Veterinary Physiology, College of Veterinary Medicine, Research Institute for Veterinary Science and BK21 Creative Veterinary Research Center, Seoul National University, Seoul, Korea.
authors.forename: H J
authors.initials: HJ
authors.lastname: Han
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015687:Cell Hypoxia; D002467:Cell Nucleus / Q000378:metabolism; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005944:Glucosamine / Q000494:pharmacology; D005992:Glycerol-3-Phosphate O-Acyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000637:transplantation; D010766:Phosphorylation / Q000187:drug effects; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D012871:Skin Diseases / Q000473:pathology / Q000628:therapy; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D004791:Enzyme Inhibitors; D034741:RNA, Small Interfering; D016329:Sp1 Transcription Factor; D005992:Glycerol-3-Phosphate O-Acyltransferase; D058570:TOR Serine-Threonine Kinases; D005944:Glucosamine; D020123:Sirolimus