Hypoxia-induced HIF-1α Protein Research Articles

Abstract 15056: Cardiac P62/sqstm1 Deficiency Exacerbates Hypoxic Stress vi a Impaired Hypoxia Inducible Factor 1α Signaling

Ischemic heart disease (IHD), a major cause of heart failure, is characterized by metabolic dysfunction and myocardial cell death. Cellular hypoxia activates hypoxia inducible factor 1α (HIF1α) to initiate metabolic, angiogenic, and growth-factor related responses. Under normoxia, HIF1α is degraded by prolyl hydroxylase (PHD) domain-containing proteins via the proteasome. Due to the short half-life of HIF1α (<5mins), PHD inhibitors are utilized to stabilize HIF1α and improve cardiac function in pre-clinical models of ischemia/infarction. However, evidence exists that PHD inhibitors exert off-target effects. Based on prior studies in cancer cells demonstrating that the autophagy adaptor protein p62 prevents HIF1α degradation, we hypothesized that p62 stabilizes cardiac HIF1α and provides cardioprotection from hypoxia. We generated mice with tamoxifen-inducible cardiomyocyte-specific p62 deletion (p62icKO mice; 8 week male and female) and exposed them to 7% O 2 for 6h. Hypoxia increased cardiac mRNA expression of Sqstm1 , Bnip3 , and Hmox1 in wild-type (WT) but not p62icKO mice. Underscoring functional relevance, ejection fraction and fractional shortening were less, oxidative stress was elevated, and apoptosis was greater, in p62icKO vs. WT mice exposed to hypoxia. Mechanisms were evaluated using cultured H9c2 cardiomyoblasts exposed to 21% (normoxia) or 1% (hypoxia) O 2 for 24h. Hypoxia increased both HIF1α and p62 protein expression. To determine the role of p62, H9c2 cells were transfected with p62 or scrambled (ctrl) siRNA for 48h, followed by 24 h hypoxia. Hypoxia-induced HIF1α protein accrual and expression of HIF1α downstream targets Egln1 , Vegfa , Bnip3 and Hmox1 mRNA was attenuated in H9c2 cells transfected with p62 vs. control siRNA. These data indicate p62 stabilizes cardiac HIF1α and regulates its transcriptional activation. Thus, p62 could be a novel therapeutic target for treatment of IHD.

Abstract P466: Sqstm1/p62 Regulates Hypoxia Inducible Factor 1 Alpha Stabilization And Protects The Heart From Hypoxic Injury

Ischemic heart disease (IHD) is characterized by cardiac tissue hypoxia, dysregulatedmetabolism, and cell death. Hypoxia inducible factor 1a (HIF1α) is a major component of thehypoxia pathway that regulates metabolic and angiogenic genes. Under normoxia, HIF1α getshydroxylated by the prolyl hydroxylases (PHDs), followed by its ubiquitination, and degradationby the Ubiquitin Proteasome System (UPS). Given the short half-life of HIF1α (>5mins), PHDinhibitors are employed to stabilize HIF1α and improve cardiac function in animal models ofischemia/infarction. Because PHD inhibitors exert off-target effects, alternative strategies tostabilize HIF1α are needed. In cancer cells, p62 stabilizes HIF1α by binding PHD3. Wehypothesized that p62 would stabilize HIF1α and provides cardioprotection from hypoxia. Wegenerated mice with tamoxifen-inducible cardiomyocyte-specific p62 deletion (cip62KO mice)and exposed them to 7% O2 for 6h. Compared to wild-type (WT) littermates, cip62KO micedisplayed increased oxidative stress and enhanced cell death. Hypoxia caused contractiledysfunction in cip62KO vs. WT mice. To gain mechanistic insights as to how lack of p62exacerbated hypoxic injury, cultured H9c2 cardiac myoblasts were exposed to 21% (normoxia)or 1% (hypoxia) O2 for 24h. Hypoxia increased HIF1α and p62 protein expression in H9c2 cells(p<0.05). To determine whether hypoxia-induced p62 accumulation is required for HIF1αstabilization, H9c2 cells were transfected with p62 or scrambled (ctrl) siRNA for 48h, andexposed to hypoxia. Compared to ctrl siRNA cells, hypoxia-induced HIF1α protein accumulationwas reduced (p<0.05) after p62 knockdown. Additionally, hypoxia increased expression ofHIF1α downstream targets: Egln1 , Vegfa , Bnip3 and Hmox1 mRNA, in ctrl siRNA cells, but theresponse was blunted (p<0.05) after p62 knockdown. These data indicate that p62 contributes tohypoxia-induced HIF1α stabilization and transcriptional activation. Defining how p62contributes to HIF1α stabilization and hypoxia tolerance is relevant clinically and could identifyp62 as a therapeutic target for treating IHD.

Polysulfide inhibits hypoxia-elicited hypoxia-inducible factor activation in a mitochondria-dependent manner

In cellular signaling, the diverse physiological actions of biological gases, including O2, CO, NO, and H2S, have attracted much interest. Hypoxia-inducible factors (HIFs), including HIF-1 and HIF-2, are transcription factors that respond to reduced intracellular O2 availability. Polysulfides are substances containing varying numbers of sulfur atoms (H2Sn) that are generated endogenously from H2S by 3-mercaptopyruvate sulfurtransferase in the presence of O2, and regulate ion channels, specific tumor suppressors, and protein kinases. However, the effect of polysulfides on HIF activation in hypoxic mammalian cells is largely unknown. Here, we have investigated the effect of polysulfide on cells in vitro. In established cell lines, polysulfide donors reversibly reduced cellular O2 consumption and inhibited hypoxia-induced HIF-1α protein accumulation and the expression of genes downstream of HIFs; however, these effects were not observed in anoxia. In Von Hippel-Lindau tumor suppressor (VHL)- and mitochondria-deficient cells, polysulfides did not affect HIF-1α protein synthesis but destabilized it in a VHL- and mitochondria-dependent manner. For the first time, we show that polysulfides modulate intracellular O2 homeostasis and regulate HIF activation and subsequent hypoxia-induced gene expression in a VHL- and mitochondria-dependent manner.

Comprehensive Analysis of mRNA and lncRNA Transcriptomes Reveals the Differentially Hypoxic Response of Preadipocytes During Adipogenesis.

Local hypoxia has recently been reported to occur in the white adipose tissue (WAT) microenvironment during obesity. Adipocytes have a unique life cycle that reflects the different stages of adipogenesis in the WAT niche. Long non-coding RNAs (lncRNAs) play an important role in the cellular response to hypoxia. However, the differentially hypoxic responses of preadipocytes during adipogenesis and the potential role of lncRNAs in this process remain to be elucidated. Here, we evaluated the differentially hypoxic responses of primary hamster preadipocytes during adipogenesis and analyzed mRNA and lncRNA expression in same Ribo-Zero RNA-seq libraries. Hypoxia induced HIF-1α protein during adipogenesis and caused divergent changes of cell phenotypes. A total of 10,318 mRNAs were identified to be expressed in twenty libraries (five timepoints), and 3,198 differentially expressed mRNAs (DE mRNAs) were detected at five timepoints (hypoxia vs. normoxia). Functional enrichment analysis revealed the shared and specific hypoxia response pathways in the different stages of adipogenesis. Hypoxia differentially modulated the expression profile of adipose-associated genes, including adipokines, lipogenesis, lipolysis, hyperplasia, hypertrophy, inflammatory, and extracellular matrix. We also identified 4,296 lncRNAs that were expressed substantially and detected 1,431 DE lncRNAs at five timepoints. Two, 3, 5, 13, and 50 DE mRNAs at D0, D1, D3, D7, and D11, respectively, were highly correlated and locus-nearby DE lncRNAs and mainly involved in the cell cycle, vesicle-mediated transport, and mitochondrion organization. We identified 28 one-to-one lncRNA-mRNA pairs that might be closely related to adipocyte functions, such as ENSCGRT00015041780-Hilpda, TU2105-Cdsn, and TU17588-Ltbp3. These lncRNAs may represent the crucial regulation axis in the cellular response to hypoxia during adipogenesis. This study dissected the effects of hypoxia in the cell during adipogenesis, uncovered novel regulators potentially associated with WAT function, and may provide a new viewpoint for interpretation and treatment of obesity.

Inhibition of hypoxia-inducible factor 1α accumulation by glyceryl trinitrate and cyclic guanosine monophosphate.

A key mechanism mediating cellular adaptive responses to hypoxia involves the activity of hypoxia-inducible factor 1 (HIF-1), a transcription factor composed of HIF-1α, and HIF-1β subunits. The classical mechanism of regulation of HIF-1 activity involves destabilisation of HIF-1α via oxygen-dependent hydroxylation of proline residues and subsequent proteasomal degradation. Studies from our laboratory revealed that nitric oxide (NO)-mediated activation of cyclic guanosine monophosphate (cGMP) signalling inhibits the acquisition of hypoxia-induced malignant phenotypes in tumour cells. The present study aimed to elucidate a mechanism of HIF-1 regulation involving NO/cGMP signalling. Using human DU145 prostate cancer cells, we assessed the effect of the NO mimetic glyceryl trinitrate (GTN) and the cGMP analogue 8-Bromo-cGMP on hypoxic accumulation of HIF-1α. Concentrations of GTN known to primarily activate the NO/cGMP pathway (100 nM–1 µM) inhibited hypoxia-induced HIF-1α protein accumulation in a time-dependent manner. Incubation with 8-Bromo-cGMP (1 nM–10 µM) also attenuated HIF-1α accumulation, while levels of HIF-1α mRNA remained unaltered by exposure to GTN or 8-Bromo-cGMP. Furthermore, treatment of cells with the calpain (Ca2+-activated proteinase) inhibitor calpastatin attenuated the effects of GTN and 8-Bromo-cGMP on HIF-1α accumulation. However, since calpain activity was not affected by incubation of DU145 cells with various concentrations of GTN or 8-Bromo-cGMP (10 nM or 1 µM) under hypoxic or well-oxygenated conditions, it is unlikely that NO/cGMP signalling inhibits HIF-1α accumulation via regulation of calpain activity. These findings provide evidence for a role of NO/cGMP signalling in the regulation of HIF-1α, and hence HIF-1-mediated hypoxic responses, via a mechanism dependent on calpain.

Hypoxia-inducible factor-1\u03b1 is the therapeutic target of the SGLT2 inhibitor for diabetic nephropathy

Previous studies have demonstrated intrarenal hypoxia in patients with diabetes. Hypoxia-inducible factor (HIF)-1 plays an important role in hypoxia-induced tubulointerstitial fibrosis. Recent clinical trials have confirmed the renoprotective action of SGLT2 inhibitors in diabetic nephropathy. We explored the effects of an SGLT2 inhibitor, luseogliflozin on HIF-1α expression in human renal proximal tubular epithelial cells (HRPTECs). Luseogliflozin significantly inhibited hypoxia-induced HIF-1α protein expression in HRPTECs. In addition, luseogliflozin inhibited hypoxia-induced the expression of the HIF-1α target genes PAI-1, VEGF, GLUT1, HK2 and PKM. Although luseogliflozin increased phosphorylated-AMP-activated protein kinase α (p-AMPKα) levels, the AMPK activator AICAR did not changed hypoxia-induced HIF-1α expression. Luseogliflozin suppressed the oxygen consumption rate in HRPTECs, and subsequently decreased hypoxia-sensitive dye, pimonidazole staining under hypoxia, suggesting that luseogliflozin promoted the degradation of HIF-1α protein by redistribution of intracellular oxygen. To confirm the inhibitory effect of luseogliflozin on hypoxia-induced HIF-1α protein in vivo, we treated male diabetic db/db mice with luseogliflozin for 8 to 16 weeks. Luseogliflozin attenuated cortical tubular HIF-1α expression, tubular injury and interstitial fibronectin in db/db mice. Together, luseogliflozin inhibits hypoxia-induced HIF-1α accumulation by suppressing mitochondrial oxygen consumption. The SGLT2 inhibitors may protect diabetic kidneys by therapeutically targeting HIF-1α protein.

Discovery of Novel Aryl Carboxamide Derivatives as Hypoxia-Inducible Factor 1α Signaling Inhibitors with Potent Activities of Anticancer Metastasis.

In order to discover novel hypoxia-inducible factor 1 (HIF-1) inhibitors for the cancer metastasis treatment, 68 new aryl carboxamide compounds were synthesized and evaluated for their inhibitory effect by dual luciferase-reporter assay. Based on five rounds of investigation on structure-activity relationships step by step, compound 30m was discovered as the most active inhibitor (IC50 = 0.32 μM) with no obvious cytotoxicity (CC50 > 50 μM). It effectively attenuated hypoxia-induced HIF-1α protein accumulation and reduced transcription of vascular epidermal growth factor in a dose-dependent manner, which was further demonstrated by its inhibitory potency on capillary-like tube formation, angiogenesis of zebrafish as well as cellular migration and invasion. Importantly, compound 30m exhibited antimetastatic potency in breast cancer lung metastasis in the mice model, indicating its promising therapeutic potential for prevention and treatment of tumor metastasis. These results definitely merit attention for further rational design of more efficient HIF-1 inhibitors in the future.

Luseogliflozin Inhibits HIF-1α Expression in Renal Proximal Tubular Epithelial Cells

Recent clinical trials have demonstrated the renoprotective effects of SGLT2 inhibitors in diabetic nephropathy (DN). However, the mechanisms of SGLT2 inhibitors on prevention of DN have not been fully elucidated. Hypoxia-induced tubulointerstitial fibrosis is considered to be a common pathway for various progressive kidney diseases including DN. Hypoxia inducible factor (HIF)-1α plays an important role in these pathological processes. In this study, we assessed the effects of luseogliflozin, an SGLT2 inhibitor, on HIF-1α expression in both cultured human renal proximal tubular epithelial cells (HRPTECs) and kidney of diabetic mice. Hypoxia (1%O2 for 24h) markedly increased HIF-1α protein in HRPTEC, whereas luseogliflozin inhibited expression of hypoxia-induced HIF-1α protein. In addition, luseogliflozin inhibited mRNA expression for HIF-1α-targeted genes, PAI-1, VEGF and GLUT-1 under hypoxia. The inhibitors of mitochondrial respiratory complex I, rotenone or complex III, antimycin A equally suppressed hypoxia-induced HIF-1α expression. We next treated db/db mice with 15 mg/kg/day luseogliflozin for 8 weeks. Luseogliflozin administration lowered plasma glucose levels and decreased glomerular mesangial matrix expansion as well as glomerular and interstitial fibronectin accumulation in db/db mice. Electron microscopic study revealed that luseogliflozin attenuated the thickening of glomerular basement membrane in db/db mice. Intriguingly, luseogliflozin decreased the length of mitochondria in the renal proximal tubules of the S1 segment. Furthermore, luseogliflozin attenuated diabetes-induced HIF-1α accumulation in the kidney of db/db mice. These results suggest that luseogliflozin inhibits hypoxia-induced HIF-1α accumulation at least partly by suppressing mitochondrial respiration. SGLT2 inhibitors may protect diabetic kidney from hypoxia-induced renal fibrosis by attenuating the expression of HIF-1α and profibrotic molecules. Disclosure R. Bessho: None. Y. Takiyama: None. T. Ota: None.

Hypoxic inactivation of glycogen synthase kinase-3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia-inducible factor-1 signaling.

Since the molecular mechanism of hypoxic adaptation in cancer cells is cell-type specific, we investigated whether glycogen synthase kinase-3β (GSK-3β) activation is involved in hypoxia-induced gastric tumor promotion. Stable gastric cancer cell lines (SNU-638, SNU-484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild-type GSK-3β (WT-GSK-3β) or kinase-dead mutant of GSK-3β (KD-GSK-3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK-3β activation in gastric cancer cells. Cell viability and the expressions of HIF-1α protein and VEGF mRNA in gastric cancer cells were higher in KD-GSK-3β transfectants than in WT-GSK-3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF-1α activation, and VEGF expression were higher in KD-GSK-3β tumors than in WT-GSK-3β tumors in vivo. In addition, the expression of hypoxia-induced HIF-1α protein was regulated by GSK-3β at the translational level. Our data suggest that GSK-3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF-1α/VEGF signaling pathway.

Resveratrol inhibits hypoxia-driven ROS-induced invasive and migratory ability of pancreatic cancer cells via suppression of the Hedgehog signaling pathway.

A hypoxic microenvironment is commonly found in the central region of solid tumors, including pancreatic cancer. Our previous study revealed that resveratrol plays an important role in suppressing the proliferation and EMT of pancreatic cancer cells. However, whether resveratrol could suppress hypoxia-induced cancer progression and the underlying mechanisms have not been fully elucidated. The aim of the present study was to evaluate whether resveratrol affects hypoxia-induced reactive oxygen species (ROS) production and the activation of the Hedgehog (Hh) signaling pathway as well as the invasion of pancreatic cancer. The human pancreatic cancer cell lines, BxPC-3 and Panc-1, were subjected to a hypoxic condition and three different concentrations of resveratrol. The intracellular ROS were determined using 2,7-dichlorodihydrofluorecein diacetate. Wound healing and Transwell invasion assays were used to detect the migratory and invasive potential of the cancer cells. Metastatic-related and Hh signaling-related factors were detected by qRT-PCR and western blot analysis. Immunofluorescence staining was used to test the nuclear translocation of GLI1. The results showed that the hypoxia-induced production of ROS was decreased by resveratrol in a concentration-dependent manner. Resveratrol significantly inhibited the hypoxia-stimulated invasion and migration of pancreatic cancer cells. Resveratrol inhibited hypoxia-induced HIF-1α protein expression. Resveratrol also suppressed hypoxia‑induced expression of metastatic-related factors, uPA and MMP2. In addition, resveratrol markedly inhibited hypoxia-mediated activation of the Hh signaling pathway. Furthermore, the antioxidant N-acetylcysteine (NAC) significantly suppressed the invasive and migratory ability of pancreatic cancer cells during hypoxia. Taken together, these data indicate that resveratrol plays an important role in suppressing hypoxia-driven ROS-induced pancreatic cancer progression by inhibiting the Hh signaling pathway, providing evidence that resveratrol may be a potential candidate for the chemoprevention of cancer.

Sulforaphane inhibits hypoxia-induced HIF-1α and VEGF expression and migration of human colon cancer cells.

The effects of sulforaphane (a natural product commonly found in broccoli) was investigated on hypoxia inducible factor-1α (HIF-1α) expression in HCT116 human colon cancer cells and AGS human gastric cancer cells. We found that hypoxia-induced HIF-1α protein expression in HCT116 and AGS cells, while treatment with sulforaphane markedly and concentration-dependently inhibited HIF-1α expression in both cell lines. Treatment with sulforaphane inhibited hypoxia-induced vascular endothelial growth factor (VEGF) expression in HCT116 cells. Treatment with sulforaphane modulated the effect of hypoxia on HIF-1α stability. However, degradation of HIF-1α by sulforaphane was not mediated through the 26S proteasome pathway. We also found that the inhibition of HIF-1α by sulforaphane was not mediated through AKT and extracellular signal-regulated kinase phosphorylation under hypoxic conditions. Finally, hypoxia-induced HCT116 cell migration was inhibited by sulforaphane. These data suggest that sulforaphane may inhibit human colon cancer progression and cancer cell angiogenesis by inhibiting HIF-1α and VEGF expression. Taken together, these results indicate that sulforaphane is a new and potent chemopreventive drug candidate for treating patients with human colon cancer.

Chlorogenic acid inhibits hypoxia-induced angiogenesis via down-regulation of the HIF-1α/AKT pathway

The hypoxia-inducible factor-1 (HIF-1) is known to play an important role in cellular responses to hypoxia, including the transcriptional activation of a number of genes involved in tumor angiogenesis. Chlorogenic acid (CGA), one of the most abundant polyphenols in the human diet, has been reported to inhibit cancer cell growth. The effect of CGA on tumor angiogenesis and its underlying mechanisms are, as yet, unknown. The effect of CGA on HIF-1α expression was assessed by Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR) assays in A549 lung cancer cells. The transcriptional activity of the HIF-1 complex was confirmed using a luciferase assay. To assess whether angiogenic factors are increased under hypoxic conditions in these cells, vascular endothelial growth factor (VEGF) expression levels were measured by RT-PCR and Western blotting. The direct effect of CGA on human vascular endothelial cells (HUVEC) under hypoxic conditions was analyzed using in vitro assays, including tube-formation, wound healing and Transwell invasion assays. To investigate the effect of CGA on angiogenesis in vivo, we performed a Matrigel plug assay in a mouse model. Finally, the effect of CGA on AKT and ERK activation (phosphorylation) as a putative mechanism underlying the effect of CGA on VEGF-mediated angiogenesis inhibition was assessed using Western blotting. We found that CGA significantly decreases the hypoxia-induced HIF-1α protein level in A549 cells, without changing its mRNA level. CGA was, however, found to suppress the transcriptional activity of HIF-1α under hypoxic conditions, leading to a decrease in the expression of its downstream target VEGF. We also found that CGA can block hypoxia-stimulated angiogenesis in vitro and VEGF-stimulated angiogenesis in vivo using HUVEC cells. In addition, we found that CGA can inhibit the HIF-1α/AKT signaling pathway, which plays an important role in VEGF activation and angiogenesis. Our data indicate that CGA plays a role in the suppression of angiogenesis via inhibition of the HIF-1α/AKT pathway. CGA may represent a novel therapeutic option for the treatment of (lung) cancer.

Effect of Ca2EDTA on zinc mediated inflammation and neuronal apoptosis in hippocampus of an in vivo mouse model of hypobaric hypoxia.

BackgroundCalcium overload has been implicated as a critical event in glutamate excitotoxicity associated neurodegeneration. Recently, zinc accumulation and its neurotoxic role similar to calcium has been proposed. Earlier, we reported that free chelatable zinc released during hypobaric hypoxia mediates neuronal damage and memory impairment. The molecular mechanism behind hypobaric hypoxia mediated neuronal damage is obscure. The role of free zinc in such neuropathological condition has not been elucidated. In the present study, we investigated the underlying role of free chelatable zinc in hypobaric hypoxia-induced neuronal inflammation and apoptosis resulting in hippocampal damage.MethodsAdult male Balb/c mice were exposed to hypobaric hypoxia and treated with saline or Ca2EDTA (1.25 mM/kg i.p) daily for four days. The effects of Ca2EDTA on apoptosis (caspases activity and DNA fragmentation), pro-inflammatory markers (iNOS, TNF-α and COX-2), NADPH oxidase activity, poly(ADP ribose) polymerase (PARP) activity and expressions of Bax, Bcl-2, HIF-1α, metallothionein-3, ZnT-1 and ZIP-6 were examined in the hippocampal region of brain.ResultsHypobaric hypoxia resulted in increased expression of metallothionein-3 and zinc transporters (ZnT-1 and ZIP-6). Hypobaric hypoxia elicited an oxidative stress and inflammatory response characterized by elevated NADPH oxidase activity and up-regulation of iNOS, COX-2 and TNF-α. Furthermore, hypobaric hypoxia induced HIF-1α protein expression, PARP activation and apoptosis in the hippocampus. Administration of Ca2EDTA significantly attenuated the hypobaric hypoxia induced oxidative stress, inflammation and apoptosis in the hippocampus.ConclusionWe propose that hypobaric hypoxia/reperfusion instigates free chelatable zinc imbalance in brain associated with neuroinflammation and neuronal apoptosis. Therefore, zinc chelating strategies which block zinc mediated neuronal damage linked with cerebral hypoxia and other neurodegenerative conditions can be designed in future.

Hypoxia-induced autophagy contributes to radioresistance via c-Jun-mediated Beclin1 expression in lung cancer cells.

Reduced radiosensitivity of lung cancer cells represents a pivotal obstacle in clinical oncology. The hypoxia-inducible factor (HIF)-1α plays a crucial role in radiosensitivity, but the detailed mechanisms remain elusive. A relationship has been suggested to exist between hypoxia and autophagy recently. In the current study, we studied the effect of hypoxia-induced autophagy on radioresistance in lung cancer cell lines. A549 and H1299 cells were cultured under normoxia or hypoxia, followed by irradiation at dosage ranging from 0 to 8 Gy. Clonogenic assay was performed to calculate surviving fraction. EGFP-LC3 plasmid was stably transfected into cells to monitor autophagic processes. Western blotting was used to evaluate the protein expression levels of HIF-1α, c-Jun, phosphorylated c-Jun, Beclin 1, LC3 and p62. The mRNA levels of Beclin 1 were detected by qRT-PCR. We found that under hypoxia, both A549 and H1299 cells were radio-resistant compared with normoxia. Hypoxia-induced elevated HIF-1α protein expression preferentially triggered autophagy, accompanied by LC3 induction, EGFP-LC3 puncta and p62 degradation. In the meantime, HIF-1α increased downstream c-Jun phosphorylation, which in turn upregulated Beclin 1 mRNA and protein expression. The upregulation of Beclin 1 expression, instead of HIF-1α, could be blocked by SP600125 (a specific inhibitor of c-Jun NH2-terminal kinase), followed by suppression of autophagy. Under hypoxia, combined treatment of irradiation and chloroquine (a potent autophagy inhibitor) significantly decreased the survival potential of lung cancer cells in vitro and in vivo. In conclusion, hypoxia-induced autophagy through evaluating Beclin1 expression may be considered as a target to reverse the radioresistance in cancer cells.

4-hydroxyphenylacetic acid attenuated inflammation and edema via suppressing HIF-1α in seawater aspiration-induced lung injury in rats.

4-Hydroxyphenylacetic acid (4-HPA) is an active component of Chinese herb Aster tataricus which had been widely used in China for the treatment of pulmonary diseases. The aim of this study is to investigate the effect of 4-HPA on seawater aspiration-induced lung injury. Pulmonary inflammation and edema were assessed by enzyme-linked immunosorbent assay (ELISA), bronchoalveolar lavage fluid (BALF) white cell count, Evans blue dye analysis, wet to dry weight ratios, and histology study. Hypoxia-inducible factor-1α (HIF-1α) siRNA and permeability assay were used to study the effect of 4-HPA on the production of inflammatory cytokines and monolayer permeability in vitro. The results showed that 4-HPA reduced seawater instillation-induced mortality in rats. In lung tissues, 4-HPA attenuated hypoxia, inflammation, vascular leak, and edema, and decreased HIF-1α protein level. In primary rat alveolar epithelial cells (AEC), 4-HPA decreased hypertonicity- and hypoxia-induced HIF-1α protein levels through inhibiting the activations of protein translational regulators and via promoting HIF-1α protein degradation. In addition, 4-HPA lowered inflammatory cytokines levels through suppressing hypertonicity- and hypoxia-induced HIF-1α in NR8383 macrophages. Moreover, 4-HPA decreased monolayer permeability through suppressing hypertonicity and hypoxia-induced HIF-1α, which was mediated by inhibiting vascular endothelial growth factor (VEGF) in rat lung microvascular endothelial cell line (RLMVEC). In conclusion, 4-HPA attenuated inflammation and edema through suppressing hypertonic and hypoxic induction of HIF-1α in seawater aspiration-induced lung injury in rats.

MAPK and PI3K pathways regulate hypoxia-induced atrial natriuretic peptide secretion by controlling HIF-1 alpha expression in beating rabbit atria

Mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways are pivotal and intensively studied signaling pathways in hypoxic conditions. However, the roles of MAPK and PI3K in the regulation of hypoxia-induced atrial natriuretic peptide (ANP) secretion are not well understood. The purpose of the present study was to investigate the mechanism by which the MAPK/ERK (extracellular signal-regulated kinase) and PI3K signaling pathways regulate the acute hypoxia-induced ANP secretion in isolated beating rabbit atria. An acute hypoxic perfused beating rabbit atrial model was used. The ANP levels in the atrial perfusates were measured by radioimmunoassay, and the hypoxia-inducible factor-1α (HIF-1α) mRNA and protein levels in the atrial tissue were determined by RT-PCR and Western blot. Acute hypoxia significantly increased ANP secretion and HIF-1α mRNA and protein levels. Hypoxia-induced ANP secretion was markedly attenuated by the HIF-1α inhibitors, rotenone (0.5μmol/L) and CAY10585 (10μmol/L), concomitantly with downregulation of the hypoxia-induced HIF-1α mRNA and protein levels. PD098059 (30μmol/L) and LY294002 (30μmol/L), inhibitors of MAPK and PI3K, markedly abolished the hypoxia-induced ANP secretion and atrial HIF-1α mRNA and protein levels. The hypoxia-suppressed atrial dynamics were significantly attenuated by PD098059 and LY294002. Acute hypoxia in isolated perfused beating rabbit atria, markedly increased ANP secretion through HIF-1α upregulation, which was regulated by the MAPK/ERK and PI3K pathways. ANP appears to be part of the protective program regulated by HIF-1α in the response to acute hypoxic conditions.

Hyperbaric oxygen therapy suppresses osteoclast formation and bone resorption

The cellular and molecular mechanism through which hyperbaric oxygen therapy (HBO) improves osteonecrosis (ON) is unclear. The present study therefore examined the effect of HBO, pressure and hyperoxia on RANKL-induced osteoclast formation in RAW 264.7 cells and human peripheral blood monocytes (PBMC). Daily exposure to HBO (2.4 ATA, 97% O2 , 90 min), hyperbaric pressure (2.4 ATA, 8.8% O2 , 90 min) or normobaric hyperoxia (1 ATA, 95% O2 , 90 min) significantly decreased RANKL-induced osteoclast formation and bone resorption in normoxic conditions. HBO had a more pronounced anti-osteoclastic effect than hyperoxia or pressure alone and also directly inhibited osteoclast formation and resorption in hypoxic conditions a hallmark of many osteolytic skeletal disorders. The suppressive action of HBO was at least in part mediated through a reduction in RANK, NFATc1, and Dc-STAMP expression and inhibition of hypoxia-induced HIF-1α mRNA and protein expression. This data provides mechanistic evidence supporting the use of HBO as an adjunctive therapy to prevent osteoclast formation and bone loss associated with low oxygen partial pressure.

Prolyl Hydroxylase Domain Protein 2 (PHD2) Binds a Pro-Xaa-Leu-Glu Motif, Linking It to the Heat Shock Protein 90 Pathway

Prolyl hydroxylase domain protein 2 (PHD2, also known as Egg Laying Defective Nine homolog 1) is a key oxygen-sensing protein in metazoans. In an oxygen-dependent manner, PHD2 site-specifically prolyl hydroxylates the master transcription factor of the hypoxic response, hypoxia-inducible factor-α (HIF-α), thereby targeting HIF-α for degradation. In this report we show that the heat shock protein 90 (HSP90) co-chaperones p23 and FKBP38 interact via a conserved Pro-Xaa-Leu-Glu motif (where Xaa = any amino acid) in these proteins with the N-terminal Myeloid Nervy and DEAF-1 (MYND)-type zinc finger of PHD2. Knockdown of p23 augments hypoxia-induced HIF-1α protein levels and HIF target genes. We propose that p23 recruits PHD2 to the HSP90 machinery to facilitate HIF-1α hydroxylation. These findings identify a link between two ancient pathways, the PHD:HIF and the HSP90 pathways, and suggest that this link was established concurrent with the emergence of the PHD:HIF pathway in evolution.

Melatonin antagonizes hypoxia‐mediated glioblastoma cell migration and invasion via inhibition of HIF‐1α

Hypoxia is a crucial factor in tumor aggressiveness and resistance to therapy, especially in glioblastoma. Our previous results have shown that melatonin exerts antimigratory and anti-invasive action in glioblastoma cells under normoxia. However, the effect of melatonin on migration and invasion of glioblastoma cells under hypoxic condition remains poorly understood. Here, we show that melatonin strongly reduced hypoxia-mediated invasion and migration of U251 and U87 glioblastoma cells. In addition, we found that melatonin significantly blocked HIF-1α protein expression and suppressed the expression of downstream target genes, matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF). Furthermore, melatonin destabilized hypoxia-induced HIF-1α protein via its antioxidant activity against ROS produced by glioblastoma cells in response to hypoxia. Along with this, HIF-1α silencing by small interfering RNA markedly inhibited glioblastoma cell migration and invasion, and this appeared to be associated with MMP-2 and VEGF under hypoxia. Taken together, our findings suggest that melatonin suppresses hypoxia-induced glioblastoma cell migration and invasion via inhibition of HIF-1α. Considering the fact that overexpression of the HIF-1α protein is often detected in glioblastoma multiforme, melatonin may prove to be a potent therapeutic agent for this tumor.

Activation of Peroxisome Proliferator-activated Receptor α (PPARα) Suppresses Hypoxia-inducible Factor-1α (HIF-1α) Signaling in Cancer Cells

Activation of peroxisome proliferator-activated receptor α (PPARα) has been demonstrated to inhibit tumor growth and angiogenesis, yet the mechanisms behind these actions remain to be characterized. In this study, we examined the effects of PPARα activation on the hypoxia-inducible factor-1α (HIF-1α) signaling pathway in human breast (MCF-7) and ovarian (A2780) cancer cells under hypoxia. Incubation of cancer cells under 1% oxygen for 16 h significantly induced HIF-1α expression and activity as assayed by Western blotting and reporter gene analysis. Treatment of the cells with PPARα agonists, but not a PPARγ agonist, prior to hypoxia diminished hypoxia-induced HIF-1α expression and activity, and addition of a PPARα antagonist attenuated the suppression of HIF-1α signaling. Activation of PPARα attenuated hypoxia-induced HA-tagged HIF-1α protein expression without affecting the HA-tagged HIF-1α mutant protein level, indicating that PPARα activation promotes HIF-1α degradation in these cells. This was further confirmed using proteasome inhibitors, which reversed PPARα-mediated suppression of HIF-1α expression under hypoxia. Using the co-immunoprecipitation technique, we found that activation of PPARα enhances the binding of HIF-1α to von Hippel-Lindau tumor suppressor (pVHL), a protein known to mediate HIF-1α degradation through the ubiquitin-proteasome pathway. Following PPARα-mediated suppression of HIF-1α signaling, VEGF secretion from the cancer cells was significantly reduced, and tube formation by endothelial cells was dramatically impaired. Taken together, these findings demonstrate for the first time that activation of PPARα suppresses hypoxia-induced HIF-1α signaling in cancer cells, providing novel insight into the anticancer properties of PPARα agonists.