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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

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Authors
이현직
Advisor
한호재
Major
수의과대학 수의학과
Issue Date
2018-02
Publisher
서울대학교 대학원
Keywords
Stem cellsHypoxiaLipogenesisO-GlcNAcylationMitophagyFASNGPAT1BNIP3
Description
학위논문 (박사)-- 서울대학교 대학원 : 수의과대학 수의학과, 2018. 2. 한호재.
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.
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
Language
English
URI
https://hdl.handle.net/10371/140932
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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Theses (Ph.D. / Sc.D._수의학과)
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