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>

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.

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>

Role of Poly (ADP-ribose) Polymerase Activation in Chemical Hypoxia-Induced Cell Injury in Renal Epithelial Cells

Jung Soon-Hee The Korean Society for Biomedical Laboratory Scien 2005 Journal of biomedical laboratory sciences Vol.11 No.4

The molecular mechanism of ischemia/reperfusion injury remains unclear. Reactive oxygen species (ROS) are implicated in cell death caused by ischemia/reperfusion in vivo or hypoxia in vitro. Poly (ADP-ribose) polymerase (PARP) activation has been reported to be involved in hydrogen peroxide-induced cell death in renal epithelial cells. This study was therefore undertaken to evaluate the role of P ARP activation in chemical hypoxia in opossum kidney (OK) cells. Chemical hypoxia was induced by incubating cells with antimycin A, an inhibitor of mitochondrial electron transport. Exposure of OK cells to chemical hypoxia resulted in a time-dependent cell death. In OK cells subjected to chemical hypoxia, the generation of ROS was increased, and this increase was prevented by the $H_2O_2$ scavenger catalase. Chemical hypoxia increased P ARP activity and chemical hypoxia-induced cell death was prevented by the inhibitor of PARP activation 3-aminobenzamide. Catalase prevented OK cell death induced by chemical hypoxia. $H_2O_2$ caused PARP activation and $H_2O_2-induced$ cell death was prevented by 3-aminobenzamide. Taken together, these results indicate that chemical hypoxia-induced cell injury is mediated by PARP activation through H202 generation in renal epithelial cells.

Effects of Hypoxia on Epithelial-to-Mesenchymal Transition in Oral Squamous Cell Carcinoma

김경아,김지영,유미현,박봉수,정진,박혜련 대한구강악안면병리학회 2011 대한구강악안면병리학회지 Vol.35 No.1

Malignant tumor cells outgrow new blood vessel formation and tend to be in hypoxic state. Hypoxic cancer cells adapt to hypoxic conditions by transforming its characteristics. On the other hand, one of the most important features of cancer cells is that carcinoma cells loses its inherent epithelial phenotype and acquires mesenchymal characteristics, called as epithelial-mesenchymal transition(EMT). It has been already well known that EMT contributes to tumor invasion and metastasis. The present study investigated whether hypoxia play a major role in induction of phenotypic changes of oral squamous cell carcinoma(OSCC). Furthermore, the mechanism of EMT in oral squamous cell carcinoma cells by hypoxia has been clarified. To mimic hypoxic condition, cobalt chloride and desferoxamine, well-known hypoxic mimetic agents, were used. This study shows that hypoxia suppresses the expression of E-cadherin(epithelial marker) and increases vimentin and N-cadherin(mesenchymal markers) in OSCC. In addition, α5 integrin protein, which is a receptor for fibronectin and an important molecule for tumor invasion, is prominently induced by hypoxia. Malignant tumor cells outgrow new blood vessel formation and tend to be in hypoxic state. Hypoxic cancer cells adapt to hypoxic conditions by transforming its characteristics. On the other hand, one of the most important features of cancer cells is that carcinoma cells loses its inherent epithelial phenotype and acquires mesenchymal characteristics, called as epithelial-mesenchymal transition(EMT). It has been already well known that EMT contributes to tumor invasion and metastasis. The present study investigated whether hypoxia play a major role in induction of phenotypic changes of oral squamous cell carcinoma(OSCC). Furthermore, the mechanism of EMT in oral squamous cell carcinoma cells by hypoxia has been clarified. To mimic hypoxic condition, cobalt chloride and desferoxamine, well-known hypoxic mimetic agents, were used. This study shows that hypoxia suppresses the expression of E-cadherin(epithelial marker) and increases vimentin and N-cadherin(mesenchymal markers) in OSCC. In addition, α5 integrin protein, which is a receptor for fibronectin and an important molecule for tumor invasion, is prominently induced by hypoxia.

Angiopoietin-1 Protects Endothelial Cells From Hypoxia-Induced Apoptosis via Inhibition of Phosphatase and Tensin Homologue Deleted From Chromosome Ten

이세원,윤석원,김태윤,서정원,고규영,권유욱,채인호,박영배,김효수 대한심장학회 2009 Korean Circulation Journal Vol.39 No.2

Background and Objectives: Angiopoietin-1 (Ang1) is a regulator of blood vessel growth and maturation, and prevents radiation-induced or serum deprivation-induced apoptosis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN), a well-known tumor suppressor, regulates cell cycle arrest and apoptosis. Hypoxia induces apoptosis by increasing the expression of PTEN. We hypothesized that Ang1 may regulate PTEN expression and, thus, reduce endothelial apoptosis under hypoxia in vitro and in vivo. Materials and Methods: In vitro, human umbilical vein endothelial cells (HUVECs) were treated with Ang1, and signaling pathways were investigated. In vivo, eight-week-old C57BL/6 mice were used for a hind limb ischemia model. Ang1 or normal saline was intramusculary injected. Blood flow was evaluated by a laser Doppler perfusion analyzer and tissue histology. Results: The expression of PTEN was markedly upregulated in HUVECs after hypoxic stimulation, whereas Ang1 suppressed PTEN expression. Tie2-Fc, a soluble form of Tie2 (sTie2) that blocks Ang1, reversed the Ang1 effect on PTEN reduction under hypoxia. Ang1 inhibited the nuclear translocation of nuclear transcription factor-kB (NFkB), a binding factor for the PTEN promoter and Foxo1. Hypoxia-induced p27 expression and apoptosis were also suppressed by Ang1. In the mouse hind limb ischemia model, we observed a high capillary density, numerous proliferating cells and diminished cell death in skeletal muscle tissue in the Ang1 injected group. Conclusion: Ang1 enhanced endothelial cell survival by reducing apoptosis via PTEN down-regulation in HUVECs under hypoxia. Local injection of Ang1 significantly reduced apoptotic cells in vivo, and prevented limb loss for ischemic hind limb mice. Thus, Ang1 may be an effective therapeutic for protection from ischemic-endothelial cell injury. Background and Objectives: Angiopoietin-1 (Ang1) is a regulator of blood vessel growth and maturation, and prevents radiation-induced or serum deprivation-induced apoptosis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN), a well-known tumor suppressor, regulates cell cycle arrest and apoptosis. Hypoxia induces apoptosis by increasing the expression of PTEN. We hypothesized that Ang1 may regulate PTEN expression and, thus, reduce endothelial apoptosis under hypoxia in vitro and in vivo. Materials and Methods: In vitro, human umbilical vein endothelial cells (HUVECs) were treated with Ang1, and signaling pathways were investigated. In vivo, eight-week-old C57BL/6 mice were used for a hind limb ischemia model. Ang1 or normal saline was intramusculary injected. Blood flow was evaluated by a laser Doppler perfusion analyzer and tissue histology. Results: The expression of PTEN was markedly upregulated in HUVECs after hypoxic stimulation, whereas Ang1 suppressed PTEN expression. Tie2-Fc, a soluble form of Tie2 (sTie2) that blocks Ang1, reversed the Ang1 effect on PTEN reduction under hypoxia. Ang1 inhibited the nuclear translocation of nuclear transcription factor-kB (NFkB), a binding factor for the PTEN promoter and Foxo1. Hypoxia-induced p27 expression and apoptosis were also suppressed by Ang1. In the mouse hind limb ischemia model, we observed a high capillary density, numerous proliferating cells and diminished cell death in skeletal muscle tissue in the Ang1 injected group. Conclusion: Ang1 enhanced endothelial cell survival by reducing apoptosis via PTEN down-regulation in HUVECs under hypoxia. Local injection of Ang1 significantly reduced apoptotic cells in vivo, and prevented limb loss for ischemic hind limb mice. Thus, Ang1 may be an effective therapeutic for protection from ischemic-endothelial cell injury.

Hypoxia Enhances Cell Properties of Human Mesenchymal Stem Cells

권세윤,전소영,하윤석,김대환,김정식,송필현,김현태,유은상,김범수,권태균 한국조직공학과 재생의학회 2017 조직공학과 재생의학 Vol.14 No.5

Atmospheric (in vitro) oxygen pressure is around 150 mm Hg (20% O2), whereas physiologic (in vivo) oxygen pressure ranges between 5 and 50 mm Hg (0.7–7% O2). The normoxic environment in cell culture does not refer to a physiological stem cell niche. The aim of this study is to investigate the effect of oxygen concentration on cell properties of human mesenchymal stem cells (MSCs). We analyzed cell proliferation rate, senescence, immunophenotype, stemness gene expression and differentiation potency with human urine stem cells (USCs), dental pulp stem cells (DPSCs), amniotic fluid stem cells (AFSCs), and bone marrow stromal cells (BMSCs). USCs, DPSCs, AFSCs and BMSCs were cultured under either 5% O2 hypoxic or 20% O2 normoxic conditions for 5 days. MSCs cultured under hypoxia showed significantly increased proliferation rate and high percentage of S-phase cells, compared to normoxic condition. In real-time PCR assay, the cells cultured under hypoxia expressed higher level of Oct4, C-Myc, Nanog, Nestin and HIF-1a. In immunophenotype analysis, MSCs cultured under hypoxia maintained higher level of the MSC surface markers, and lower hematopoietic markers. Senescence was inhibited under hypoxia. Hypoxia enhances osteogenic differentiation efficiency compared to normoxia. Hypoxia showed enhanced cell proliferation rate, retention of stem cell properties, inhibition of senescence, and increased differentiation ability compared to normoxia.

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.

Endothelial Cell Products as a Key Player in Hypoxia-Induced Nerve Cell Injury after Stroke

Cho, Chul-Min,Ha, Se-Un,Bae, Hae-Rahn,Huh, Jae-Taeck The Korean Neurosurgical Society 2006 Journal of Korean neurosurgical society Vol.40 No.2

Objective : Activated endothelial cells mediate the cascade of reactions in response to hypoxia for adaptation to the stress. It has been suggested that hypoxia, by itself, without reperfusion, can activate the endothelial cells and initiate complex responses. In this study, we investigated whether hypoxia-induced endothelial products alter the endothelial permeability and have a direct cytotoxic effect on nerve cells. Methods : Hypoxic condition of primary human umbilical vein endothelial cells[HUVEC] was induced by $CoCl_2$ treatment in culture medium. Cell growth was evaluated by 3,4,5-dimethyl thiazole-3,5-diphenyl tetrazolium bromide [MTT] assay Hypoxia-induced products [$IL-1{\beta},\;TGF-{\beta}1,\;IFN-{\gamma},\;TNF-{\alpha}$, IL-10, IL-6, IL-8, MCP-l and VEGF] were assessed by enzyme-linked immunosorbent assay. Endothelial permeability was evaluated by Western blotting. Results : Prolonged hypoxia caused endothelial cells to secrete IL -6, IL -8, MCP-1 and VEGF. However, the levels of IL -1, IL -10, $TNF-{\alpha},\;TGF-{\beta},\;IFN-{\gamma}$ and nitric oxide remained unchanged over 48 h hypoxia. Hypoxic exposure to endothelial cells induced the time-dependent down regulation of the expression of cadherin and catenin protein. The conditioned medium taken from hypoxic HUVECs had the cytotoxic effect selectively on neuroblastoma cells, but not on astroglioma cells. Conclusion : These results suggest the possibility that endothelial cell derived cytokines or other secreted products with the increased endothelial permeability might directly contribute to nerve cell injury followed by hypoxia.

Pharmacological Properties of CDBT in Hypoxia-induced Neuronal Cell Injury and Their Underlying Mechanisms

박상규,정은선,차지윤,조현경,유호룡,김윤식,설인찬 대한한방내과학회 2019 大韓韓方內科學會誌 Vol.40 No.3

Objectives: This study aimed to reveal the pharmacological properties of the newly prescribed herbal mixture, Chenmadansam- gamibokhap-tang (CDBT), against hypoxia-induced neuronal cell injury (especially mouse hippocampal neuronal cell line, HT-22 cells) and their corresponding mechanisms. Methods: A cell-based in vitro experiment, in which a hypoxia condition induced neuronal cell death, was performed. Various concentrations of the CDBT were pre-treated to the HT-22 cells for 4 h before 18 h in the hypoxia chamber. The glial cell BV-2 cells were stimulated with IFNγ and LSP to produce inflammatory cytokines and reactive oxygen species. When the neuronal HT-22 cells were treated with this culture solution, the drug efficacy against neuronal cell death was examined. Results: CDBT showed cytotoxicity in the normal condition of HT-22 cells at a dose of 125 μg/mL and showed a protective effect against hypoxia-induced neuronal cell death at a dose of 31.3 μg/mL. CDBT prevented hypoxia-induced neuronal cell death in a dose-dependent manner in the HT-22 cells by regulating HIF1α and cell death signaling. CDBT prevented neuronal cell death signals and DNA fragmentation due to the hypoxia condition. CDBT significantly reduced cellular oxidation, cell death signals, and caspase-3 activities due to microglial cell activations. Moreover, CDBT significantly ameliorated LPS-induced BV-2 cell activation and evoked cellular oxidation through the recovery of redox homeostasis. Conclusions: CDBT cam be considered as a vital therapeutic agent against neuronal cell deaths. Further studies are required to reveal the other functions of CDBT in vivo or in the clinical field.