NF-κB Inhibitor Suppresses Hypoxia-induced Apoptosis of Mouse Pancreatic β-cell Line MIN6

Hyun Sook Koh,Jae Young Kim 대한의생명과학회 2014 Biomedical Science Letters Vol.20 No.1

Hypoxia is one of the main reasons for islet apoptosis after transplantation as well as during isolation. In this study, we attempted to determine the potential usefulness of NF-κB inhibitor for suppression of hypoxia-induced β-cell apoptosis as well as the relationship between IP-10 induction and β-cell apoptosis in hypoxia. To accomplish this, we cultured the mouse pancreatic β-cell line MIN6 in hypoxia (1% O₂). Among several examined chemokines, only IP-10 mRNA expression was induced under hypoxia, and this induced IP-10 expression was due to NF-κB activity. Since a previous study suggested that IP-10 mediates β-cell apoptosis, we measured hypoxia-induced IP-10 protein and examined the effect of anti-IP-10 neutralizing Ab on hypoxia-induced β-cell apoptosis. However, IP-10 protein was not detected, and anti-IP-10 neutralizing Ab did not rescue hypoxia-induced MIN6 apoptosis, indicating that there is no relationship between hypoxia-induced IP-10 mRNA expression and hypoxia-induced β-cell apoptosis. Since it was still not clear if NF-κB functions as an apoptotic or anti-apoptotic mediator in hypoxia-induced β-cell apoptosis, we examined possible involvement of NF-κB in hypoxia-induced β-cell apoptosis. Treatment with 1 μM NF-κB inhibitor suppressed hypoxiainduced apoptosis by more than 50%, while 10 μM AP-1 or 4 μM NF-AT inhibitor did not, indicating involvement of NF-κB in hypoxia-induced β-cell apoptosis. Overall, these results suggest that IP-10 is not involved in hypoxia-induced β-cell apoptosis, and that NF-κB inhibitor can be useful for ameliorating hypoxia-induced β-cell apoptosis.

Effects of hypoxia on the concentration of circulating miR-210 in serum and the expression of HIF-1α and HSP90α in tissues of olive flounder (Paralichthys olivaceus)

( Najib Abdellaoui ),( Jun Soung Kwak ),( Ki Hong Kim ) 한국어병학회 2020 한국어병학회지 Vol.33 No.1

Hypoxia is a serious problem in the marine ecosystem causing a decline in aquatic resources. MicroRNAs (miRNAs) regulate the expression of genes through binding to the corresponding sequences of their target mRNAs. Especially, miRNAs in the cytoplasm can be secreted into body fluids, which called circulating miRNAs, and the availability of circulating miRNAs as biomarkers for hypoxia has been demonstrated in mammals. However, there has been no report on the hypoxiamediated changes in the circulating miRNAs in fish. miR-210 is known as the representative hypoxia- responsive circulating miRNA in mammals. To know whether fish miR-210 also respond to hypoxia, we analyzed the change of circulating miR-210 quantity in the serum of olive flounder (Paralichthys olivaceus) in response to hypoxia. The expression of hypoxia related genes, hypoxia inducible factor 1α (HIF-1α) and the heat shock protein 90α (HSP90α) was also analyzed. Similar to the reports from mammals, miR-210-5p and miR-210-3p were significantly increased in the serum of olive flounder in response to hypoxia, suggesting that circulating miR-210 levels in the serum can be used as a noninvasive prognostic biomarker for fish suffered hypoxia. The target genes of miR-210 were related to various biological processes, which explains the major regulatory role of miR-210 in response to hypoxia. The expression of HIF-1α and HSP90α in the tissues was also up-regulated by hypoxia. Considering the critical role of HIF-1α in miR-210 expression and HSP90 in miRNAs function, the present up-regulation of HIF-1α and HSP90α might be related to the increase of circulatory miR-210, and the interaction mechanism among HIF-1α, HSP90α, and hypoxia-responsive microRNAs in fish should be further studied.

Hypoxia-Induced EDNO Release is Further Augmented by Previous Hypoxia and Reoxygenation in Rabbit Aortic Endothelium

Jae Jin Han,Suk Hyo Suh,Kyung Phil Suh,Ki Whan Kim 대한생리학회-대한약리학회 1998 The Korean Journal of Physiology & Pharmacology Vol.2 No.2

<P> The present study was designed: (1) to determine whether or not hypoxia stimulates the release of endothelium-derived relaxing factors (EDRFs) from endothelial cells, and (2) to examine whether or not the hypoxia-induced EDRFs release is further augmented by previous hypoxia-reoxygenation, using bioassay system. In the bioassay experiment, rabbit aorta with endothelium was used as EDRFs donor vessel and rabbit carotid artery without endothelium as a bioassay test ring. The test ring was contracted by prostaglandin F<SUB>2α</SUB> (3×10<SUP>-6</SUP> M/L), which was added to the solution perfusing through the aortic segment. Hypoxia was evoked by switching the solution aerated with 95% O<SUB>2</SUB>/5% CO<SUB>2</SUB> mixed gas to one aerated with 95% N<SUB>2</SUB>/5% CO<SUB>2</SUB> mixed gas. When the contraction induced by prostaglandin F<SUB>2α </SUB>reached a steady state, the solution was exchanged for hypoxic one. And then, hypoxia and reoxygenation were interchanged at intervals of 2 minutes (intermittent hypoxia). The endothelial cells were also exposed to single 10-minute hypoxia (continuous hypoxia). When the bioassay ring was superfused with the perfusate through intact aorta, hypoxia relaxed the precontracted bioassay test ring markedly. Whereas, when bioassay ring was superfused with the perfusate through denuded aorta or polyethylene tubing, hypoxia relaxed the precontracted ring slightly. The relaxation was not inhibited by indomethacin but by nitro-L-arginine or methylene blue. The hypoxia-induced relaxation was further augmented by previous hypoxia-reoxygenation and the magnitude of the relaxation by intermittent hypoxia was significantly greater than that of the relaxation by continuous hypoxia. The results suggest that hypoxia stimulates EDNO release from endothelial cells and that the hypoxia-induced EDNO release is further augmented by previous hypoxia-reoxygenation.

Hypoxia Inducible Factor-1α Directly Induces then Expression of Receptor Activator of Nuclear Factor-κB Ligand in Periodontal Ligament Fibroblasts

Hyun-Jung Park,백정화,Kyung Hwa Baek,Hye-Lim Lee,권아랑,Hyo Rin Hwang,Abdul S. Qadir,우경미,류현모 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.6

During orthodontic tooth movement, local hypoxia and enhanced osteoclastogenesis are observed in the compression side of periodontal tissues. The receptor activator of nuclear factor-κB ligand (RANKL) is an osteoblast/stromal cell-derived factor that is essential for osteoclastogenesis. In this study, we examined the effect of hypoxia on RANKL expression in human periodontal ligament fibroblasts (PDLFs) to investigate the relationship between local hypoxia and enhanced osteoclastogenesis in the compression side of periodontal tissues. Hypoxia significantly enhanced the levels of RANKL mRNA and protein as well as hypoxia inducible factor-1α (HIF-1α) protein in PDLFs. Constitutively active HIF-1α alone significantly increased the levels of RANKL expression in PDLFs under normoxic conditions, whereas dominant negative HIF-1αblocked hypoxia-induced RANKL expression. To investigate further whether HIF-1α directly regulates RANKL transcription, a luciferase reporter assay was performed using the reporter vector containing the RANKL promoter sequence. Exposure to hypoxia or overexpression of constitutively active HIF-1α significantly increased RANKL promoter activity, whereas dominant negative HIF-1αblocked hypoxia-induced RANKL promoter activity. Furthermore,mutations of putative HIF-1α binding elements in RANKL promoter prevented hypoxia-induced RANKL promoter activity. The results of chromatin immunoprecipitation showed that hypoxia or constitutively active HIF-1α increased the DNA binding of HIF-1α to RANKL promoter. These results suggest that HIF-1α mediates hypoxia-induced up-regulation of RANKL expression and that in compression side periodontal ligament, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in PDLFs.

Hypoxia-dependent accumulation of hypoxia-inducible factor-1 alpha induces transient cell cycle arrest in porcine trophectoderm cells

Jeong, Wooyoung,Jung, Seoungo,Bazer, Fuller W.,Kim, Jinyoung Elsevier 2018 Theriogenology Vol.115 No.-

<P><B>Abstract</B></P> <P>In the uterine environment, the pre-implantation embryo adapts to low oxygen concentrations through intracellular responses including modification of gene expression, progression via the cell cycle and metabolism. In this study, we determined mechanisms underlying the adaptation of pig embryos to oxygen deficiency in the maternal-conceptus microenvironment in <I>in vitro</I> experiments using our established porcine trophectoderm (pTr) cells in culture. The transition from G<SUB>1</SUB> to S phase in pTr cells was reduced in response to 2% oxygen during a short period (<24 h), and the hypoxia-induced G<SUB>1</SUB> arrest was reversible during prolonged hypoxia exposure. Acute hypoxia up-regulated expression of transcription factors p21 and p27 and down-regulated cell cycle regulators associated with the G<SUB>1</SUB>/S phase transition including cyclin D1, cyclin E1 and E2F1 <I>mRNAs</I> and proteins. Furthermore, hypoxia exposure for 24 h markedly increased the abundance of HIF-1α protein. Even under acute hypoxia, by HIF-1α silencing reduced the hypoxia-induced transient G<SUB>1</SUB> arrest and expression of p21 and p27 genes was restored. Contrary to acute hypoxia, the accumulation of HIF-1α protein decreased as the length of the hypoxic period increased. Overall results of the present study suggest that increases in HIF-1α are responsible for initial response to hypoxia that results in a transient cell cycle arrest in pTr cells and cell cycle progression is restored by increasing degradation of HIF-1α during prolonged hypoxia. These findings advance understating of cellular adaptation of developing pre-implantation porcine conceptuses to hypoxic stress.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Hypoxia induces transient cell cycle arrest at G1 phase in pTr cells. </LI> <LI> Hypoxia-induced cell cycle arrest is alleviated during prolonged hypoxia exposure. </LI> <LI> Hypoxia-induced HIF-1α regulates expression of p21, p27, E2F1, cyclin D1 and cyclin E1. </LI> <LI> HIF-1α stability diminishes from acute to chronic hypoxic stress. </LI> <LI> G1 cell cycle arrest is induced even under chronic hypoxia by PHDs silencing. </LI> </UL> </P>

고온환경에서 일정한 동맥이산화탄소분압 저산소호흡이 안정시 인체의 환기 및 심혈관계반응에 미치는 영향

김경태(Kyong-Tae Kim) 한국생활환경학회 2010 한국생활환경학회지 Vol.17 No.6

The purpose of this study was to examine the influence of hot environment on ventilatory and cardiovascular response to isocapnic hypoxia at rest. Six volunteers (mean (±Sd): 30 ± 8 yrs, 176 ± 1 ㎝, 74 ± 11 ㎏) underwent two separate testing sessions which were normothermic (21℃, 40% RH) and hyperthermic (50℃, 40% RH) hypoxia. Each participant breathed 12% O₂ in N₂ mixture while the end tidal partial pressure of carbon dioxide (P<SUB>ET</SUB>CO₂) was kept approximately 1 ㎜Hg above normothermic resting levels. Ventilation (V<SUB>E</SUB>) was higher in hyperthermic condition than normothermic condition during pre-hypoxia and isocapnic hypoxia (p < .05) and there were significant increases in VE at hyperthermic group for pre-hypoxia and isocapnic hypoxia (p < .05) except the resting. For brething frequency (f<SUB>R</SUB>), there were significant increases in hyperthermic condition during pre-hypoxia and isocapnic hypoxia (p < .05) and also were significant increases in f<SUB>R</SUB> at hyperthermic group than normothermic group for pre-hypoxia and isocapnic hypoxia (p < .05). Tidal Volume (V<SUB>T</SUB>) was higher in hyperthermic condition than normothermic condition during pre-hypoxia and isocapnic hypoxia (p < .05) and there were significant increases in V<SUB>T</SUB> at hyperthermic group for pre-hypoxia (p < .05). There were significant increases in heart rate (HR) at hyperthermic condition during pre-hypoxia and isocapnic hypoxia (p < .05) but not at normothermic and also were significant increases in HR at hyperthermic group than nor-mothermic group for pre-hypoxia and isocapnic hypoxia (p < .05). For oxygen saturation (SaO₂), there were significant decreases at isocapnic hypoxia relative to the resting in hyperthermic and normothermic condition (p < .05) and there was no difference between hyperthermic and normothermic group during rest, pre-hypoxia, and isocapnic hypoxia (p > .05). In conclusion, there were effects for the change of cardiovascular responses and the increase of ventilatory responses to duplicate stimuli both hyperthermia and hypoxia.

Novel Pathway for Hypoxia-Induced Proliferation and Migration in Human Mesenchymal Stem Cells: Involvement of HIF-1α, FASN, and mTORC1.

Lee, Hyun Jik,Ryu, Jung Min,Jung, Young Hyun,Oh, Sang Yub,Lee, Sei-Jung,Han, Ho Jae AlphaMed Press 2015 Stem Cells Vol.33 No.7

<P>The control of stem cells by oxygen signaling is an important way to improve various stem cell physiological functions and metabolic nutrient alteration. Lipid metabolism alteration via hypoxia is thought to be a key factor in controlling stem cell fate and function. However, the interaction between hypoxia and the metabolic and functional changes to stem cells is incompletely described. This study aimed to identify hypoxia-inducible lipid metabolic enzymes that can regulate umbilical cord blood (UCB)-derived human mesenchymal stem cell (hMSC) proliferation and migration and to demonstrate the signaling pathway that controls functional change in UCB-hMSCs. Our results indicate that hypoxia treatment stimulates UCB-hMSC proliferation, and expression of two lipogenic enzymes: fatty acid synthase (FASN) and stearoyl-CoA desaturase-1 (SCD1). FASN but not SCD1 is a key enzyme for regulation of UCB-hMSC proliferation and migration. Hypoxia-induced FASN expression was controlled by the hypoxia-inducible factor-1 alpha (HIF-1)/SCAP/SREBP1 pathway. Mammalian target of rapamycin (mTOR) was phosphorylated by hypoxia, whereas inhibition of FASN by cerulenin suppressed hypoxia-induced mTOR phosphorylation as well as UCB-hMSC proliferation and migration. RAPTOR small interfering RNA transfection significantly inhibited hypoxia-induced proliferation and migration. Hypoxia-induced mTOR also regulated CDK2, CDK4, cyclin D1, cyclin E, and F-actin expression as well as that of c-myc, p-cofilin, profilin, and Rho GTPase. Taken together, the results suggest that mTORC1 mainly regulates UCB-hMSC proliferation and migration under hypoxia conditions via control of cell cycle and F-actin organization modulating factors. In conclusion, the HIF-1/FASN/mTORC1 axis is a key pathway linking hypoxia-induced lipid metabolism with proliferation and migration in UCB-hMSCs. Stem Cells 2015;33:2182-2195</P>

Hypoxia Increases β-Cell Death by Activating Pancreatic Stellate Cells within the Islet

김종진,이에스더,류경렬,고승현,안유배,송기호 대한당뇨병학회 2020 Diabetes and Metabolism Journal Vol.44 No.6

Background Hypoxia can occur in pancreatic islets in type 2 diabetes mellitus. Pancreatic stellate cells (PSCs) are activated during hypoxia. Here we aimed to investigate whether PSCs within the islet are also activated in hypoxia, causing β-cell injury. Methods Islet and primary PSCs were isolated from Sprague Dawley rats, and cultured in normoxia (21% O2) or hypoxia (1% O2). The expression of α-smooth muscle actin (α-SMA), as measured by immunostaining and Western blotting, was used as a marker of PSC activation. Conditioned media (hypoxia-CM) were obtained from PSCs cultured in hypoxia. Results Islets and PSCs cultured in hypoxia exhibited higher expressions of α-SMA than did those cultured in normoxia. Hypoxia increased the production of reactive oxygen species. The addition of N-acetyl-L-cysteine, an antioxidant, attenuated the hypoxia-induced PSC activation in islets and PSCs. Islets cultured in hypoxia-CM showed a decrease in cell viability and an increase in apoptosis. Conclusion PSCs within the islet are activated in hypoxia through oxidative stress and promote islet cell death, suggesting that hypoxia-induced PSC activation may contribute to β-cell loss in type 2 diabetes mellitus.

저산소환경에 의한 송사리(Oryzias dancena)의 Stanniocalcin-2와 Hypoxia-Lnducible Factor-1α mRNA 발현의 변화

신지혜 ( Ji Hye Shin ),손영창 ( Young Chang Sohn ) 한국수산과학회 2013 한국수산과학회지 Vol.46 No.1

Some fish live in aquatic environments with low of temporally changing O2availability. Variation in dissolved oxy- gen(DO) levels requinres behavioral, physiological, and biochemical adaptations to ensure the uptake of suffocoent O2. Several spccies are relatively well adapted to tolerate low O2 partial pressures(hypoxia). The medaka(Oryzias dancena) is an important model organism for biomedical research that shows remarkable tolerance to hypoxia. We investigated the regulation and role of hypoxia-inducible factor-1(HIF-1α)as a general hypoxia-response gene and stanniocalein-2(STC2), which is one of the genes regulated by HIF-1α in mammals under hypoxia. The changes adult male medaka to the following three acute hypoxia regimes: 1, 24, and 72 h at DO=1.8±0.5PPM. The changes inSTC2 and HIF-1α mRNA were monitored using quantitative real-time reverse-transcription PCR. We fownd strong upregulation of HIF-1α mRNA in the livers of fish exposed to hypoxia. Hypoxia rapidly upregulated STC. 2 mRNA expression in muscle, but not in the brain, gills, liver, of intestine. Therefore, unlike in mammals, hypoxia minght regulale O. dancena STC-2 expression in an HIF-1α-independent manner.

토끼 대동맥 혈관내피세포에서 저산소증이 내피세포성 이완인자의 분비에 미치는 영향

최수승 대한흉부외과학회 2009 Journal of Chest Surgery (J Chest Surg) Vol.42 No.5

Background: To clarify the effect of hypoxia on vascular contractility, we tried to show whether hypoxia induced the release of endothelium-derived relaxing factor (EDRF) and the nature of the underlying mechanism for this release. Material and Method: Isometric contractions were observed in rabbit aorta, and the released EDRF from the rabbit aorta was bioassayed by using rabbit denuded carotid artery. The intracellular Ca2+ concentration ([Ca2+]i) in the cultured rabbit aortic endothelial cells was recorded by a microfluorimeter with using Fura-2/AM. Hypoxia was evoked to the blood vessels or endothelial cells by eliminating the O2 in the aerating gases in the external solution. Chemical hypoxia was evoked by applying deoxyglucose or CN−. Result: Hypoxia relaxed the precontracted rabbit thoracic aorta that had its endothelium, and the magnitude of the relaxation was gradually increased by repetitive bouts of hypoxia. In contrast, hypoxia-induced relaxation was not evoked in the aorta that was denuded of endothelium. In a bioassay experiment, hypoxia released endothelium-derived relaxing factor (EDRF) and the release was inhibited by L-NAME or the K+ channel blocker tetraethylammonium (TEA). In the cultured endothelial cells, hypoxia augmented the ATP-induced increase of the intracellular Ca2+ concentration ([Ca2+]i) and this increase was inhibited by TEA. Furthermore, chemical hypoxia also increased the Ca2+ influx. Conclusion: From these results, it can be concluded that hypoxia might induce the release of NO from rabbit aortic endothelial cells by increasing [Ca2+]i. 배경: 저산소증이 혈관평활근 수축성에 미치는 영향을 규명하기 위하여 저산소증이 혈관내피세포에서 내피세포성 이완인자의 분비에 미치는 영향과 그 기전을 규명하고자 하였다. 대상 및 방법: 토끼 대동맥에서 내피세포 의존성 이완과 관찰하고, 토끼 대동맥에서 내피세포성 이완인자 분비 정도를 내피세포를 제거한 경동맥의 수축에 미치는 영향으로 생물검증을 하였다. 마지막으로, 배양한 토끼 대동맥 혈관내피세포에서 세포내 Ca2+ 변화를 측정하였다. 저산소증은 세포외 용액에 공급되는 산소를 질소로 대체하여 제거한 후 이 용액을 혈관 혹은 세포에 공급하여 유발시거나, deoxyglucose 혹은 CN−를 투여하여 화학적인 저산소증을 유발시켰다. 결과: 노에피네프린으로 토끼 대동맥을 수축시킨 다음 저산소증에 노출시키면 대동맥이 이완을 하였으며 저산소증에 반복하여 노출시키면 저산소증에 의한 이완이 더 크게 증가하였다. 이러한 저산소증에 의한 이완은 혈관내피세포를 제거한 대동맥에서는 관찰되지 않았다. 토끼 대동맥에서 분비되는 내피세포성 이완인자 분비를 내피세포를 제거한 경동맥을 이용하여 생물검증한 결과 저산소증에 의하여 내피세포성 이완인자의 분비가 증가하였는데 반복된 노출에 의하여 더 크게 증가하였다. 그리고 저산소증에 의한 내피세포성 이완인자 분비는 NO 생성을 억제하는 경우와 K+ 통로 억제제인 tetraethyl ammonium (TEA)에 의하여 억제되었다. 배양한 혈관내피세포에서 ATP에 의하여 증가한 세포내 Ca2+은 저산소증에 의하여 유의하게 증가하였으며 TEA에 의하여 억제되었다. Deoxyglucose에 의하여 세포내 Ca2+이 증가하였으며 세포외 Ca2+을 제거하면 감소하였다. CN− 역시 혈관내피세포 Ca2+ 유입을 증가시켰다. 결론: 이러한 실험 결과로 미루어 토끼 대동맥에서 저산소증은 내피세포 의존성 이완을 유발하는데 이는 저산소증에 의한 세포내 Ca2+ 유입 증가에 의하여 NO 생성이 증가되어 일어난 것으로 추정할 수 있었다.