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The effect of hypoxia-induced lipid metabolic enzymes expression on mTOR signaling-regulated behavior of stem cells : 저산소 유도 지질대사 효소 발현이 mTOR 신호에 의한 줄기세포 행동에 미치는 영향
DC Field | Value | Language |
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dc.contributor.advisor | 한호재 | - |
dc.contributor.author | 이현직 | - |
dc.date.accessioned | 2018-05-28T16:49:10Z | - |
dc.date.available | 2018-05-28T16:49:10Z | - |
dc.date.issued | 2018-02 | - |
dc.identifier.other | 000000150100 | - |
dc.identifier.uri | https://hdl.handle.net/10371/140932 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 수의과대학 수의학과, 2018. 2. 한호재. | - |
dc.description.abstract | 7:e2158]
In conclusion, present study presented that 1) the HIF- 1α/FASN/mTORC1 axis is a key pathway linking hypoxia-induced lipogenesis with UCB-hMSC behavior, 2) BNIP3 is a major factor regulating mitophagy and lipogenesis induced by hypoxia, and 3) O-GlcNAc signaling enhanced by glucosamine suppresses hypoxia-induced mESC apoptosis through GPAT1 upregulation. | - |
dc.description.abstract | Nutrient metabolic regulation by hypoxia is an essential
physiological process to maintain the behavior of stem cell. Especially, lipid metabolism in the stem cells plays key roles in the regulations of cellular energetics, stemness and behavior. Previous investigators suggested that mitophagy and O-GlcNAc signaling induced by hypoxia are closely associated with metabolic regulation. However, the effect of hypoxia-induced mitpohagy and O-GlcNAc signaling on lipid metabolism of stem cell and the mechanism how lipid metabolism controls behavior are not completely described yet. Threfore, present study aimed to 1) investigate the effect of hypoxia on lipid metabolic enzymes expression and the mechanism how lipid metabolite controls stem cell behavior, and 2) demonstrate the effect of hypoxia-regulated mitophagy and OGlcNAc signaling on the lipid metabolic enzymes expression in stem cells and its associated mechanism. Results were as followings: 1. I investigated the effect of hypoxia on lipid metabolic enzyme in umbilical cord blood-derived human mesenchymal stem cells (UCB-hMSCs). In the present study, hypoxia treatment induces UCB-hMSC proliferation, and expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). I further confirmed that FASN but not SCD1 is a key enzyme for regulation of UCB-hMSC proliferation and migration. - 3 - This finding indicates that FASN-produced palmitic acid stimulates proliferation and migration of UCB-hMSC under hypoxia. I demonstrated that hypoxia increased FASN expression via HIF- 1α/SREBP1 pathway. In addition, I observed that hypoxia stimulated mTOR phosphorylation at Ser2481 and Ser2448 residues, whereas inhibition of FASN by cerulenin blocked hypoxia-induced mTOR phosphorylation, proliferation and migration in UCB-hMSCs. RAPTOR siRNA transfection significantly inhibited hypoxiainduced proliferation and migration. Hypoxia-induced mTOR also regulated cell cycle and cytoskeletal regulatory proteins. Taken together, these results suggest that hypoxia-induced FASN controls proliferation and migration in UCB-hMSCs through mTORC1 activation. [Stem Cells. 2015 33(7):2182-2195] 2. To identify the major mitophagy regulator involving in hypoxia-induced lipid metabolic enzyme expression, I investigated the effect of hypoxia on mitophagy regulator expressions including PINK1, BNIP3, NIX and FUNDC1. And, my data presented that hypoxia reduced mitochondria marker expression in a timedependent manner and increased mRNA and protein expression levels of BNIP3 and NIX. In addition, BNIP3 silencing induced abberent regulation of mitochondrial ROS production, mitochondrial membrane potential and ER stress markers expression. I demonstrated that hypoxia-induced BNIP3 expression was regulated by CREB binding protein-mediated transcriptional actions - 4 - of HIF-1α and FOXO3. Silencing of BNIP3 expression by siRNA transfection inhibited hypoxia-induced SREBP1/FASN-dependent free fatty acid synthesis and mTOR activation. In addition, BNIP3- silenced UCB-hMSC lost hypoxia preconditioning-induced phosphorylation of cofilin-1 and migration. In mouse skin wound healing model, transplantation of BNIP3-silenced UCB-hMSC delayed wound healing, recovered by palmitic acid. Collectively, these data suggest that hypoxia-induced BNIP3 expression via HIF1α and FOXO3 activation is a major mitophagy regulator for inducing the FASN-dependent lipogenesis, which is critical for migration and survival of UCB-hMSCs. [Redox Biol. 2017 13:426- 443] 3. I examined the effect of glucosamine-induced OGlcNAcylation on lipid metabolic enzyme expression and survival of mESCs under hypoxia. My data showed that hypoxia treatment increased mESCs apoptosis in a time-dependent manner. And, hypoxia also slightly increased the O-GlcNAc level. Glucosamine treatment as an O-GlcNAc inducer further enhanced the O-GlcNAc level and prevented hypoxia-induced mESC apoptosis, which was suppressed by an O-GlcNAc transferase inhibitor ST045849. Hypoxia regulated several lipid metabolic enzymes while glucosamine increased expression of glycerol-3-phophate acyltransferase-1 (GPAT1), a lipid metabolic enzyme producing lysophosphatidic acid (LPA). I further investigated signaling - 5 - pathway how glucosamine controls GPAT1 expression. Glucosamine increased O-GlcNAcylation of Sp1, which subsequently leads to Sp1 nuclear translocation and GPAT1 expression. Silencing of GPAT1 by Gpat1 siRNA transfection reduced glucosaminemediated anti-apoptosis in mESCs with mTOR dephosphorylation. 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 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 protective effects of glucosamine. Based upon these findings, present study suggests that upregulation of O-GlcNAc level by glucosamine treatment enhances hypoxiainduced GPAT1 expression through Sp1 activation, which leads to mTOR-mediated protection of mESCs against hypoxic damage. [Cell Death Dis. 2016 24 | - |
dc.description.tableofcontents | BACKGROUND 1
I. Regulation of proliferation and migration in UCB-hMSCs by hypoxia-induced HIF-1α/FASN/mTORC1 pathway 23 1.1 INTRODUCTION 25 1.2 MATERIALS & METHODS 27 1.3 RESULTS 41 1.4 DISCUSSION 73 II. Regulatory role of BNIP3-mediated mitophagy under hypoxia in FASN-dependent free fatty acid production enhancing therapeutic potential of UCB-hMSCs 79 2.1 INTRODUCTION 80 2.2 MATERIALS & METHODS 84 2.3 RESULTS 100 2.4 DISCUSSION 150 III. Protective effect of GPAT1 induction by O-GlcNAcylation on hypoxia-induced mESCs apoptosis 161 3.1 INTRODUCTION 162 3.2 MATERIALS & METHODS 166 3.3 RESULTS 176 3.4 DISCUSSION 210 GENERAL CONCLUSION 216 REFERENCES 220 국문초록 266 | - |
dc.format | application/pdf | - |
dc.format.extent | 7053782 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Stem cells | - |
dc.subject | Hypoxia | - |
dc.subject | Lipogenesis | - |
dc.subject | O-GlcNAcylation | - |
dc.subject | Mitophagy | - |
dc.subject | FASN | - |
dc.subject | GPAT1 | - |
dc.subject | BNIP3 | - |
dc.subject.ddc | 636.089 | - |
dc.title | The effect of hypoxia-induced lipid metabolic enzymes expression on mTOR signaling-regulated behavior of stem cells | - |
dc.title.alternative | 저산소 유도 지질대사 효소 발현이 mTOR 신호에 의한 줄기세포 행동에 미치는 영향 | - |
dc.type | Thesis | - |
dc.description.degree | Doctor | - |
dc.contributor.affiliation | 수의과대학 수의학과 | - |
dc.date.awarded | 2018-02 | - |
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