Inhibitor of nuclear factor-kappaB alpha derepresses hypoxia-inducible factor-1 during moderate hypoxia by sequestering factor inhibiting hypoxia-inducible factor from hypoxia-inducible factor 1&agr;

Shin, Dong Hoon,Li, Shan Hua,Yang, Seung-Won,Lee, Byung Lan,Lee, Myung Kyu,Park, Jong-Wan Blackwell Publishing Ltd 2009 The FEBS journal Vol.276 No.13

<P>Hypoxia and inflammation often develop concurrently in numerous diseases, and both hypoxia-inducible factor (HIF)-1&agr; and nuclear factor-kappaB (NF-&kgr;B) are key transcription factors of stress response genes. An NF-&kgr;B inhibitor, inhibitor of NF-&kgr;B&agr; (I&kgr;B&agr;), was found to interact with factor inhibiting HIF (FIH) and to be hydroxylated by FIH. However, FIH did not functionally regulate I&kgr;B&agr;, and the consequence of the FIH–I&kgr;B&agr; interaction thus remains uncertain. In the present study, we tested the possibility that I&kgr;B&agr; regulates FIH. FIH–I&kgr;B&agr; binding was confirmed by yeast two-hybrid and coimmunoprecipitation analyses. Functionally, I&kgr;B&agr; expression further enhanced the transcriptional activity of HIF-1&agr; under hypoxic conditions. Furthermore, I&kgr;B&agr; knockdown repressed HIF-1&agr; activity. Mechanistically, I&kgr;B&agr; derepressed HIF-1&agr; activity by inhibiting the FIH-mediated Asn803 hydroxylation of HIF-1&agr;. It was also found that I&kgr;B&agr; activated HIF-1&agr; by sequestering FIH from HIF-1&agr;. However, the effect of I&kgr;B&agr; on HIF-1&agr; activity was only observed in atmospheres containing 1% or more of oxygen. After tumor necrosis factor-&agr; treatment, I&kgr;B&agr; downregulation, Asn803 hydroxylation and HIF-1&agr; inactivation all occurred up to 8 h, but subsided later. On the basis of these results, we propose that I&kgr;B&agr; plays a positive regulatory role during HIF-1-mediated gene expression. Therefore, I&kgr;B&agr;, owing to its interactions with NF-&kgr;B and HIF-1&agr;, may play a pivotal role in the crosstalk between the molecular events that underlie inflammatory and hypoxic responses.</P>

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.

Endothelial Deletion of Phospholipase D2 Reduces Hypoxic Response and Pathological Angiogenesis

Ghim, Jaewang,Moon, Jin-Sook,Lee, Chang Sup,Lee, Junyeop,Song, Parkyong,Lee, Areum,Jang, Jin-Hyeok,Kim, Dayea,Yoon, Jong Hyuk,Koh, Young Jun,Chelakkot, Chaithanya,Kang, Byung Jun,Kim, Jung-Min,Kim, Ky American Heart Association, Inc. 2014 Arteriosclerosis, thrombosis, and vascular biology Vol.34 No.8

<P><B>Objective—</B></P><P>Aberrant regulation of the proliferation, survival, and migration of endothelial cells (ECs) is closely related to the abnormal angiogenesis that occurs in hypoxia-induced pathological situations, such as cancer and vascular retinopathy. Hypoxic conditions and the subsequent upregulation of hypoxia-inducible factor-1α and target genes are important for the angiogenic functions of ECs. Phospholipase D2 (PLD2) is a crucial signaling mediator that stimulates the production of the second messenger phosphatidic acid. PLD2 is involved in various cellular functions; however, its specific roles in ECs under hypoxia and in vivo angiogenesis remain unclear. In the present study, we investigated the potential roles of PLD2 in ECs under hypoxia and in hypoxia-induced pathological angiogenesis in vivo.</P><P><B>Approach and Results—</B></P><P><I>Pld2</I> knockout ECs exhibited decreased hypoxia-induced cellular responses in survival, migration, and thus vessel sprouting. Analysis of hypoxia-induced gene expression revealed that PLD2 deficiency disrupted the upregulation of hypoxia-inducible factor-1α target genes, including <I>VEGF</I>, <I>PFKFB3</I>, <I>HMOX-1</I>, and <I>NTRK2</I>. Consistent with this, PLD2 contributed to hypoxia-induced hypoxia-inducible factor-1α expression at the translational level. The roles of PLD2 in hypoxia-induced in vivo pathological angiogenesis were assessed using oxygen-induced retinopathy and tumor implantation models in endothelial-specific <I>Pld2</I> knockout mice. <I>Pld2</I> endothelial-specific knockout retinae showed decreased neovascular tuft formation, despite a larger avascular region. Tumor growth and tumor blood vessel formation were also reduced in <I>Pld2</I> endothelial-specific knockout mice.</P><P><B>Conclusions—</B></P><P>Our findings demonstrate a novel role for endothelial PLD2 in the survival and migration of ECs under hypoxia via the expression of hypoxia-inducible factor-1α and in pathological retinal angiogenesis and tumor angiogenesis in vivo.</P>

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 Induced Multidrug Resistance of Laryngeal Cancer Cells via Hypoxia-inducible Factor-1α

Li, Da-Wei,Dong, Pin,Wang, Fei,Chen, Xin-Wei,Xu, Cheng-Zhi,Zhou, Liang Asian Pacific Journal of Cancer Prevention 2013 Asian Pacific journal of cancer prevention Vol.14 No.8

Objectives: To investigate whether hypoxia has an effect on regulation of multidrug resistance (MDR) to chemotherapeutic drugs in laryngeal carcinoma cells and explore the role of hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$). Methods: Laryngeal cancer cells were cultured under normoxic and hypoxic conditions. The sensitivity of the cells to multiple drugs and levels of apoptosis induced by paclitaxel were determined by MTT assay and annexin-V/propidium iodide staining analysis, respectively. HIF-$1{\alpha}$ expression was blocked by RNA interference. The expression of HIF-$1{\alpha}$ gene was detected by real-time quantitative RT-PCR and Western blotting. The value of fluorescence intensity of intracellular adriamycin accumulation and retention in cells was evaluated by flow cytometry. Results: The sensitivity to multiple chemotherapy agents and induction of apoptosis by paclitaxel could be reduced by hypoxia (P<0.05). A the same time, the adriamycin releasing index of cells was increased (P<0.05). However, resistance acquisition subject to hypoxia in vitro was suppressed by down-regulating HIF-$1{\alpha}$ expression. Conclusion: HIF-$1{\alpha}$ could be considered as a key regulator for mediating hypoxia-induced MDR in laryngeal cancer cells via inhibition of drug-induced apoptosis and decrease in intracellular drug accumulation.

자궁내막증의 자궁내막에서 저산소증이 혈관내피성장인자 및 섬유모세포성장인자 발현에 미치는 영향

정경아,전선희,윤정미 이화여자대학교 의과학연구소 2008 EMJ (Ewha medical journal) Vol.31 No.1

Objectives : This study was performed to investigate the functional roles of hypoxia and HIF-1 α, leading to expression of VEGF and FGF in the pathogenesis of endometriosis. Methods : From September 2005 to february 2006, endometrial stromal cells were obtained from the patients with or without endometrosis at the Department of Obstetrics and Gynecology of Ewha Womans University Dongdaemun Hospital. These cells were cultured and treated with 100uM desferrioxamine (DFO) for 0hr and 2hr. After the extraction of total RNA, RT-PCR was performed and the expression level of HIF-1 α, VEGF and FGF mRNA were measured by β-actin as 1. Statistical analysis was performed by Wilcoxon signed rank test and Mann-Whitney U test (SPSS 12.0 version). A p value of 0.05 was considered as the limit for statistical significance. Results : Chemical hypoxia condition with DFO in normal endometrium results m the up-regulation of HIF-1 α, but it was significantly decreased in the eutopic endometrium of the patients with endometriosis. The expression of VEGF in normal control group was not changed, but it was increased in endometriosis group under chemical hypoxia. Hypoxia with DFO induced the overexpression of FGF in endometriosis group, compared that it was slightly decreased in normal endometrium. Conclusion : Hypoxia and subsequent production of HIF-1 α might regulate angiogenesis by the expression of VEGF and FGF, that is related to the pathogenesis of endometriosis. However, further studies are warranted to confirm. 목적 본 연구는 저산소증과 저산소증 유도인자(hypoxia inducible factor, HIF) -1 α의 역할에 따른 혈관내피성장인자(vascular endothelial growth factor, VEGF)와 섬유모세포성장인자(fibroblast growth factor, FGF) 발현이 자궁내막증의 발생기전에서 미치는 영향에 관하여 연구하고자 하였다. 방법 2005년 9월부터 2006년 2월까지 이화의대부속 동대문 병원 산부인과에서 자궁내막증으로 복강경 수술을 받은 환자의 자궁내막 조직과 자궁내막증이 없는 환자의 자궁내막 조직을 채취하였다. 자궁내막 세포를 분리 및 배양하고 desferrioxamine (DFO) 처리하였다. DFO는 100uM. 배양시간은 0시간과 2시간으로 정하여 실험하였다. 전체RNA 추출 후에, RT-PCR을 이용하여, HIF-1 α, VEGF, FGF mRNA 발현량을 측정하였으며, β-actin product 값을 1로 하였을 때의 상대적인 값으로 산출하였다. Wilcoxon signed rank test, Mann-Whitney U test(SPSS 12.0 version)를 이용하여 통계처리를 하였으며, p값은 0.05미만일 때 통계학적으로 유의하다고 하였다. 결과 DFO를 처리하여 화학적 저산소증 환경을 만들어주었을 때 HIF-1 α의 경우 대조군은 증가된 양상으로 나타났으나 자궁내막증군은 현저하게 감소되었다. 또한 대조군의 VEGF는 화학적 저산소증에 영향을 받지 않았으나 자궁내막증군에서는 증가됨을 알 수 있었다. 이에 비해 FGF는 대조군에서 화학적 저산소증에 의해 발현량이 약간 감소되었으나 자궁내막증군에서는 증가되었다. 결론 자궁내막증의 발생기전에서 저산소증으로 인한 HIF-1 α의 역할과 VEGP, FGF 발현으로 유도되는 혈관생성이 연관성이 있을 것으로 추정되며, 앞으로 이에 대한 많은 연구가 필요할 것이다.

Hypoxia Mediates Runt-Related Transcription Factor 2 Expression via Induction of Vascular Endothelial Growth Factor in Periodontal Ligament Stem Cells

Manjing Deng,Qian Xu,Zhihua Liu1,Ling Guo,Rui Liu,Rulei Li,Xiang Chu,Jiajia Yang,Jia Luo,Faming Chen 한국분자세포생물학회 2019 Molecules and cells Vol.42 No.11

Periodontitis is characterized by the loss of periodontal tissues, especially alveolar bone. Common therapies cannot satisfactorily recover lost alveolar bone. Periodontal ligament stem cells (PDLSCs) possess the capacity of self-renewal and multilineage differentiation and are likely to recover lost alveolar bone. In addition, periodontitis is accompanied by hypoxia, and hypoxia-inducible factor-1α (HIF-1α) is a master transcription factor in the response to hypoxia. Thus, we aimed to ascertain how hypoxia affects runt-related transcription factor 2 (RUNX2), a key osteogenic marker, in the osteogenesis of PDLSCs. In this study, we found that hypoxia enhanced the protein expression of HIF-1α, vascular endothelial growth factor (VEGF), and RUNX2 ex vivo and in situ. VEGF is a target gene of HIF-1α, and the increased expression of VEGF and RUNX2 proteins was enhanced by cobalt chloride (CoCl2, 100 μmol/L), an agonist of HIF-1α, and suppressed by 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl indazole (YC-1, 10 μmol/L), an antagonist of HIF-1α. In addition, VEGF could regulate the expression of RUNX2, as RUNX2 expression was enhanced by human VEGF (hVEGF165) and suppressed by VEGF siRNA. In addition, knocking down VEGF could decrease the expression of osteogenesis-related genes, i.e., RUNX2, alkaline phosphatase (ALP), and type I collagen (COL1), and hypoxia could enhance the expression of ALP, COL1, and osteocalcin (OCN) in the early stage of osteogenesis of PDLSCs. Taken together, our results showed that hypoxia could mediate the expression of RUNX2 in PDLSCs via HIF-1α-induced VEGF and play a positive role in the early stage of osteogenesis of PDLSCs

Hypoxia Induces Epithelial-Mesenchymal Transition in Follicular Thyroid Cancer: Involvement of Regulation of Twist by Hypoxia Inducible Factor-1α

양연주,장재원,나휘정,서지영,반명진,변형권,김원식,김재욱,최은창,권형주,고윤우 연세대학교의과대학 2015 Yonsei medical journal Vol.56 No.6

Purpose: Although follicular thyroid cancer (FTC) has a relatively fair prognosis, distant metastasis sometimes results in poorprognosis and survival. There is little understanding of the mechanisms contributing to the aggressiveness potential of thyroidcancer. We showed that hypoxia inducible factor-1α (HIF-1α) induced aggressiveness in FTC cells and identified the underlyingmechanism of the HIF-1α-induced invasive characteristics. Materials and Methods: Cells were cultured under controlled hypoxic environments (1% O2) or normoxic conditions. The effectof hypoxia on HIF-1α, and epithelial-to-mesenchymal transition (EMT) related markers were evaluated by quantitative real-timePCR, Western blot analysis and immunocytochemistry. Invasion and wound healing assay were conducted to identify functionalcharacter of EMT. The involvement of HIF-1α and Twist in EMT were studied using gene overexpression or silencing. After orthotopicnude mouse model was established using the cells transfected with lentiviral shHIF-1α, tissue analysis was done. Results: Hypoxia induces HIF-1α expression and EMT, including typical morphologic changes, cadherin shift, and increased vimentinexpression. We showed that overexpression of HIF-1α via transfection resulted in the aforementioned changes withouthypoxia, and repression of HIF-1α with RNA interference suppressed hypoxia-induced HIF-1α and EMT. Furthermore, we alsoobserved that Twist expression was regulated by HIF-1α. These were confirmed in the orthotopic FTC model. Conclusion: Hypoxia induced HIF-1α, which in turn induced EMT, resulting in the increased capacity for invasion and migrationof cells via regulation of the Twist signal pathway in FTC cells. These findings provide insight into a possible therapeutic strategyto prevent invasive and metastatic FTC.

Two Transactivation Domains of Hypoxia-inducible Factor-1a Regulated by the MEK-1/p42/p44 MAPK Pathway

Eunjung Lee,Sujin Yim,이승기,Hyunsung Park 한국분자세포생물학회 2002 Molecules and cells Vol.14 No.1

At a low-oxygen tension, cells increase the expression of several genes (such as erythropoietin, the vascular endothelial growth factor, and glycolytic enzymes) in order to adapt to hypoxic stress. A common transacti-vator, named the hypoxia-inducible factor 1 (HIF-1) activates these genes. HIF-1 is a heterodimeric trans-activator that is composed of a and b subunits. HIF-1 activity is primarily determined by the hypoxia-induced stabilization of the a subunit, whereas the HIF-1b subunit is expressed constitutively. Our previ-ous observation implied that the MEK-1/p42/p44 MAPK pathway is involved in the hypoxia-induced transactivation ability, but not in the stabilization and DNA binding of HIF-1a. In this paper, we dissected the transactivation domain of HIF-1a in more detail, and tested the correlation between specific domains of HIF-1a and specific signaling pathways. We designed several fusion proteins that contain deletion mutants of HIF-1a that is linked to the DNA binding domain of the yeast protein Gal4. By using the Gal4-driven re-porter system, we tested the transactivation activities of the Gal4/HIF-1a fusion proteins in Hep3B cells. Our findings suggest that tyrosine kinases, the MEK-1/p42/p44 MAPK pathway, but not the PI-3 kinase/ Akt pathway, are involved in the hypoxia-induced transactivation of HIF-1a. We have shown that the functional transactivation activities are located at both 522-649 and 650-822 amino acids of HIF-1a. Treat-ment of PD98059, a MEK-1 inhibitor, blocked the hy-poxia-induced transactivation abilities of both the 522 -649 and 650-822 amino acids of the C- terminal half of HIF-1a. This implies that the MEK-1/ p42/p44 MAPK signaling pathway cannot distinguish between the two hypoxia-induced transactivation domains.

Hypoxia Inducible Factor-1α Directly Induces the Expression of Receptor Activator of Nuclear Factor-κB Ligand in MLO-Y4 Osteocytes

Kyunghwa Baek,Hyun-Jung Park,Jeong-Hwa Baek 대한구강생물학회 2015 International Journal of Oral Biology Vol.40 No.1

Osteocytes may function as mechanotransducers byregulating local osteoclastogenesis. Reduced availability ofoxygen, i.e. hypoxia, could occur during disuse, bonedevelopment, and fracture. Receptor activator of nuclearfactor-κB ligand (RANKL) is an osteoblast/stromal cellderived essential factor for osteoclastogenesis. The hypoxiainduced osteoclastogenesis via increased RANKL expressionin osteoblasts was demonstrated. Hypoxic regulation of geneexpression generally involves activation of thehypoxia-inducible factor (HIF) transcription pathway. In thepresent study, we investigated whether hypoxia regulatesRANKL expression in murine osteocytes and HIF-1αmediates hypoxia-induced RANKL expression bytransactivating RANKL promoter, to elucidate the role ofosteocyte in osteoclastogenesis in the context of hypoxiccondition. The expression levels of RANKL mRNA andprotein, as well as hypoxia inducible factor-1α (HIF-1α)protein, were significantly increased in hypoxic condition inMLO-Y4s. Constitutively active HIF-1α alone significantlyincreased the levels of RANKL expression in MLO-Y4sunder normoxic conditions, whereas dominant negativeHIF-1α blocked hypoxia-induced RANKL expression. Tofurther explore to find if HIF-1α directly regulates RANKLtranscription, a luciferase reporter assay was conducted. Hypoxia significantly increased RANKL promoter activity,whereas mutations of putative HIF-1α binding elements inRANKL promoter prevented this hypoxia-induced RANKLpromoter activity in MLO-Y4s. These results suggest thatHIF-1α mediates hypoxia-induced up-regulation of RANKLexpression, and that in osteocytes of mechanically unloadedbone, hypoxia enhances osteoclastogenesis, at least in part, viaan increased RANKL expression in osteocytes.