HIF-1α Protein Research Articles

The oxygen sensor prolyl hydroxylase domain 2 regulates the in vivo suppressive capacity of regulatory T cells.

The oxygen sensor prolyl hydroxylase domain 2 (PHD2) plays an important role in cell hypoxia adaptation by regulating the stability of HIF proteins (HIF1α and HIF2α) in numerous cell types, including T lymphocytes. The role of oxygen sensor on immune cells, particularly on regulatory T cell (Treg) function, has not been fully elucidated. The purpose of our study was to evaluate the role of PHD2 in the regulation of Treg phenotype and function. We demonstrate herein that selective ablation of PHD2 expression in Treg (PHD2ΔTreg mice) leads to a spontaneous systemic inflammatory syndrome, as evidenced by weight loss, development of a rectal prolapse, splenomegaly, shortening of the colon, and elevated expression of IFN-γ in the mesenteric lymph nodes, intestine, and spleen. PHD2 deficiency in Tregs led to an increased number of activated CD4 conventional T cells expressing a Th1-like effector phenotype. Concomitantly, the expression of innate-type cytokines such as Il1b, Il12a, Il12b, and Tnfa was found to be elevated in peripheral (gut) tissues and spleen. PHD2ΔTreg mice also displayed an enhanced sensitivity to dextran sodium sulfate-induced colitis and toxoplasmosis, suggesting that PHD2-deficient Tregs did not efficiently control inflammatory response in vivo, particularly those characterized by IFN-γ production. Further analysis revealed that Treg dysregulation was largely prevented in PHD2-HIF2α (PHD2-HIF2αΔTreg mice), but not in PHD2-HIF1α (PHD2-HIF1αΔTreg mice) double KOs, suggesting an important and possibly selective role of the PHD2-HIF2α axis in the control of Treg function. Finally, the transcriptomic analysis of PHD2-deficient Tregs identified the STAT1 pathway as a target of the PHD2-HIF2α axis in regulatory T cell phenotype and in vivo function.

Repeated sprint exercise in hypoxia stimulates HIF-1-dependent gene expression in skeletal muscle.

Our aim was to determine the effect of repeated sprint exercise in hypoxia on HIF-1 and HIF-1-regulated genes involved in glycolysis, mitochondrial turnover and oxygen transport. We also determined whether genes upregulated by exercise in hypoxia were dependent on the activation of HIF-1 in an in vitro model of exercise in hypoxia. Eight endurance athletes performed bouts of repeated sprint exercise in control and hypoxic conditions. Skeletal muscle was sampled pre, post and 3h post-exercise. HIF-1α protein and HIF1A, PDK1, GLUT4, VEGFA, BNIP3, PINK1 and PGC1A mRNA were measured. C2C12 myotubes were exposed to hypoxia and muscle contraction following treatment with a HIF-1α inhibitor to determine whether hypoxia-sensitive gene expression was dependent on HIF-1α. Sprint exercise in hypoxia increased HIF-1α protein expression immediately post-exercise [fold change (FC) = 3.5 ± 2.0]. Gene expression of PDK1 (FC = 2.1 ± 1.2), BNIP3 (FC = 2.4 ± 1.4) and VEGFA (FC = 2.7 ± 1.7) increased 3h post-exercise in hypoxia but not control. PGC1A mRNA increased 3h post-exercise in control (FC = 5.16) and hypoxia (FC = 5.7 ± 4.1) but there was no difference between the trials. Results from the in vitro experiment showed that hypoxia plus contraction also increased PDK1, BNIP3, and VEGFA gene expression. These responses were inhibited when HIF-1 protein activity was suppressed. Repeated sprint exercise in hypoxia upregulates some genes involved in glycolytic metabolism, mitochondrial turnover, and oxygen transport. HIF-1α is necessary for the expression of these genes in skeletal muscle cells.

Production of functional recombinant prolyl hydroxylase-2 enzyme in insect cells for small molecule inhibitor screening studies

Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates the hypoxia inducible factor (HIF) which then targets it for degradation in oxygenated cells. Inhibition of the activity of the PHD2 enzyme under hypoxic environmental conditions acts to upregulate HIF. Thus, PHD2 inhibitors may serve as a promising treatment for HIF-dependent diseases. In this study, recombinant PHD2 protein was successfully expressed using a baculovirus-insect cell expression secretory system. PHD2 was purified and in combination with bacterially expressed functional von Hippel Lindau protein-elongin B-elongin C (VBC) protein complex was used to successfully develop a fluorescence-based PHD2 activity assay. Myricetin was identified as a novel potent PHD2 inhibitor by high-throughput screening of a natural compound library. Further studies showed that treatment of human neuroblastoma SH-SY5Y cells with Myricetin increased HIF-1α protein levels. These results indicate that the insect cell expression system is capable of producing highly active recombinant PHD2 protein from which a fluorescence-based activity assay can be developed for high-throughput screening applications.

Adaptation of the Oxygen Sensing System during Lung Development.

During gestation, the most drastic change in oxygen supply occurs with the onset of ventilation after birth. As the too early exposure of premature infants to high arterial oxygen pressure leads to characteristic diseases, we studied the adaptation of the oxygen sensing system and its targets, the hypoxia-inducible factor- (HIF-) regulated genes (HRGs) in the developing lung. We draw a detailed picture of the oxygen sensing system by integrating information from qPCR, immunoblotting, in situ hybridization, and single-cell RNA sequencing data in ex vivo and in vivo models. HIF1α protein was completely destabilized with the onset of pulmonary ventilation, but did not coincide with expression changes in bona fide HRGs. We observed a modified composition of the HIF-PHD system from intrauterine to neonatal phases: Phd3 was significantly decreased, while Hif2a showed a strong increase and the Hif3a isoform Ipas exclusively peaked at P0. Colocalization studies point to the Hif1a-Phd1 axis as the main regulator of the HIF-PHD system in mouse lung development, complemented by the Hif3a-Phd3 axis during gestation. Hif3a isoform expression showed a stepwise adaptation during the periods of saccular and alveolar differentiation. With a strong hypoxic stimulus, lung ex vivo organ cultures displayed a functioning HIF system at every developmental stage. Approaches with systemic hypoxia or roxadustat treatment revealed only a limited in vivo response of HRGs. Understanding the interplay of the oxygen sensing system components during the transition from saccular to alveolar phases of lung development might help to counteract prematurity-associated diseases like bronchopulmonary dysplasia.

Estrogen-mediated downregulation of HIF-1α signaling in B lymphocytes influences postmenopausal bone loss

In the bone marrow, B cells and bone-resorbing osteoclasts colocalize and form a specific microenvironment. How B cells functionally influence osteoclasts and bone architecture is poorly understood. Using genetically modified mice and high-throughput analyses, we demonstrate that prolonged HIF-1α signaling in B cells leads to enhanced RANKL production and osteoclast formation. In addition, deletion of HIF-1α in B cells prevents estrogen deficiency-induced bone loss in mice. Mechanistically, estrogen controls HIF-1α protein stabilization through HSP70-mediated degradation in bone marrow B cells. The stabilization of HIF-1α protein in HSP70-deficient bone marrow B cells promotes RANKL production and osteoclastogenesis. Induction of HSP70 expression by geranylgeranylacetone (GGA) administration alleviates ovariectomy-induced osteoporosis. Moreover, RANKL gene expression has a positive correlation with HIF1A expression in human B cells. In conclusion, HIF-1α signaling in B cells is crucial for the control of osteoclastogenesis, and the HSP70/HIF-1α axis may serve as a new therapeutic target for osteoporosis.

Aggravating Effects of Psychological Stress on Ligature-Induced Periodontitis via the Involvement of Local Oxidative Damage and NF-κB Activation.

Periodontitis is the leading cause of tooth loss in adults, and psychological factors play an important role in the development of periodontitis. To elucidate the adverse effects of psychological stress on the inflammatory process and redox status of periodontitis tissue, fifty male Sprague-Dawley rats were divided into the control, experimental periodontitis, psychological stress, experimental periodontitis plus psychological stress, and experimental periodontitis plus psychological stress plus fluoxetine groups. Chronic unpredictable mild stress (CUMS) was used to establish psychological stress, and silk ligature was used to induce experimental periodontitis. Four weeks later, stressed rats showed altered behaviour, serum hormone levels, and sucrose preference. More obvious alveolar bone loss and attachment loss and higher protein expressions of inflammatory cytokines were observed in the experimental periodontitis plus psychological stress group. The combination of CUMS and periodontitis had synergistic effects on increasing hypoxia-inducible factor-1α (HIF-1α) protein expression and reactive oxygen species (ROS) and malondialdehyde (MDA) contents and decreasing antioxidant enzyme activities compared with those in the stress or periodontitis groups. Moreover, psychological stress further increased p-IκBα and p-NF-κB p65 protein levels and decreased IκBα protein levels in periodontitis rats. Fluoxetine administration alleviated the adverse effects of psychological stress on the progression of periodontitis in rats. These results hint us that psychological stress could aggravate inflammation in periodontitis tissues, which may be partly due to local worsening of oxidative damage and further activation of the nuclear factor kappa-B (NF-κB) signalling pathway.

IASPP is essential for HIF-1α stabilization to promote angiogenesis and glycolysis via attenuating VHL-mediated protein degradation.

Hypoxia-inducible factor-1α (HIF-1α) plays central roles in the hypoxia response. It is highly expressed in multiple cancers, but not always correlated with hypoxia. Mutation of the von Hippel-Lindau (VHL) gene, which encodes an E3 ligase, contributes to the constructive activation of HIF-1α in specific tumor types, as exemplified by renal cell carcinoma; but how VHL wild-type tumors acquire this ability is not completely understood. Here, we found that the oncogene iASPP (inhibitor of apoptosis-simulating protein of p53) plays essential roles in such a context. Genetic inhibition of iASPP reduced tumor growth, accompanied by impaired angiogenesis, increased areas of tumor necrosis, and reduced glycolysis that was HIF-1α-dependent. These abilities of iASPP were validated by in vitro assays. Mechanistically, iASPP directly binds VHL at its β domain, a region also involved in HIF-1α binding, therefore blocking VHL's binding and the subsequent degradation of HIF-1α protein under normoxia. iASPP levels correlate with HIF-1α protein and vascular endothelial growth factor (VEGF) and the glucose transporter protein type 1(GLUT1), representative HIF-1α target genes, in human colon cancer tissues. Furthermore, inhibition of iASPP expression synergizes with low toxic dose of the HIF-1α inhibitor YC-1 to inhibit HIF-1α expression and tumor growth. Our findings suggest that iASPP contributes to HIF-1α activation in cancers, and that iASPP-mediated HIF-1α stabilization has potential as a therapeutic approach against cancer.

The PER1/HIF-1alpha negative feedback loop promotes ferroptosis and inhibits tumor progression in oral squamous cell carcinoma

Current studies have proven that the decreased expression of the core circadian clock gene Period 1 (PER1) is closely related to the occurrence and progression of multiple malignant tumors, including oral squamous cell carcinoma (OSCC). But the mechanism involved is largely unknown. In this study, we found that PER1 was negatively correlated with the expression of the key ferroptosis-regulated proteins glutathione peroxidase (GPX4) and hypoxia inducible factor-1alpha (HIF-1α) in OSCC tissues. The expression of the ferroptosis related proteins GPX4, solute carrier family 7 member 11 (SLC7A11) and transferrin receptor (TFRC) and the levels of glutathione (GSH), malondialdehyde (MDA), reactive oxygen species (ROS) and Fe2+ were detected in OSCC cells with overexpression or silencing of PER1. Mitochondrial morphology changes were observed. We found that PER1 promotes ferroptosis depending on HIF-1α in OSCC cells. In vivo tumorigenicity assays proved that PER1 overexpression inhibits HIF-1α, promotes ferroptosis and suppresses OSCC growth. Mechanistically, coimmunoprecipitation and cycloheximide tracking assays proved that PER1 binds to HIF-1α to promote HIF-1α protein degradation. ChIP and dual luciferase reporter assays proved that HIF-1α binds to the PER1 promoter leading to feedback inhibition of PER1 transcription. Our findings suggest that targeting the PER1/HIF-1α negative feedback loop may provide a new strategy for OSCC treatment.

Kaempferol increases intracellular ATP content in C2C12 myotubes under hypoxic conditions by suppressing the HIF-1α stabilization and/or by enhancing the mitochondrial complex IV activity

Kaempferol (KMP) has numerous important biological functions, and we recently showed that it remarkably increased intracellular adenosine triphosphate (ATP) content in C2C12 myotubes under hypoxic conditions. Since intracellular ATP is generated by aerobic energy metabolism or anaerobic glycolysis, hypoxia inducible factor-1α (HIF-1α) has been shown to be associated with metabolic remodeling and causes metabolic shift from aerobic energy metabolism to anaerobic glycolysis in response to hypoxic conditions. Here, we investigate the effects of KMP under hypoxic conditions on the stabilization of HIF-1α in C2C12 myotubes and its underlying molecular mechanisms. Constitutive HIF-1α protein expression was observed in C2C12 myotubes, and its expression under hypoxic conditions was remarkably suppressed by KMP by reducing its stability; thus, resulting in an increase in ATP content. Furthermore, KMP strikingly increased the ubiquitination of HIF-1α and promoted its degradation via the ubiquitin proteasome system. Inhibition of HIF-1α by KMP resulted in the abrogation of the expression of glycolytic enzymes such as lactate dehydrogenase A and pyruvate dehydrogenase kinase isozyme 1. In addition, the metabolome profiling showed that KMP promoted oxidative energy production, while the mitochondrial complex activity assay indicated that KMP increased the activity of mitochondrial complex IV. Finally, we showed that KMP inhibited HIF-1α expression and increased intracellular ATP content in the soleus muscle of rats. Taken together, these results suggest that KMP increases intracellular ATP content under hypoxic conditions by suppressing the HIF-1α stabilization and/or by enhancing the mitochondrial complex IV activity in muscle.

Transcriptional Regulation of Yin-Yang 1 Expression through the Hypoxia Inducible Factor-1 in Pediatric Acute Lymphoblastic Leukemia.

Yin-Yang transcription factor 1 (YY1) is involved in tumor progression, metastasis and has been shown to be elevated in different cancers, including leukemia. The regulatory mechanism underlying YY1 expression in leukemia is still not understood. Bioinformatics analysis reveal three Hypoxia-inducible factor 1-alpha (HIF-1α) putative binding sites in the YY1 promoter region. The regulation of YY1 by HIF-1α in leukemia was analyzed. Mutation of the putative YY1 binding sites in a reporter system containing the HIF-1α promoter region and CHIP analysis confirmed that these sites are important for YY1 regulation. Leukemia cell lines showed that both proteins HIF-1α and YY1 are co-expressed under hypoxia. In addition, the expression of mRNA of YY1 was increased after 3 h of hypoxia conditions and affect several target genes expression. In contrast, chemical inhibition of HIF-1α induces downregulation of YY1 and sensitizes cells to chemotherapeutic drugs. The clinical implications of HIF-1α in the regulation of YY1 were investigated by evaluation of expression of HIF-1α and YY1 in 108 peripheral blood samples and by RT-PCR in 46 bone marrow samples of patients with pediatric acute lymphoblastic leukemia (ALL). We found that the expression of HIF-1α positively correlates with YY1 expression in those patients. This is consistent with bioinformatic analyses of several databases. Our findings demonstrate for the first time that YY1 can be transcriptionally regulated by HIF-1α, and a correlation between HIF-1α expression and YY1 was found in ALL clinical samples. Hence, HIF-1α and YY1 may be possible therapeutic target and/or biomarkers of ALL.

HIF-1α protects osteoblasts from ROS-induced apoptosis

The regulatory mechanism of hypoxia-inducible factor-1α (HIF-1α) is complex. HIF-1α may inhibit or promote apoptosis in osteoblasts under different physiological conditions, and induce bone regeneration and repair injury in coordination with angiogenesis. The relationship between H2O2 and HIFs is complex, and this study aimed to explore the role of HIF-1α in H2O2-induced apoptosis. Dimethyloxallyl glycine (DMOG) and 2-Methoxyestradiol (2ME) were used to stabilize and inhibit HIFs, respectively. Cell viability was assessed with CCK8. Apoptosis and ROS levels were detected by flow cytometry, and HIF mRNA expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Western blot was performed to detect HIF-1α, HIF-2α, Bax, Bak, Bcl-2, Bcl-XL, caspase-9, and PCNA protein amounts. Our data suggest that both HIF-1α and HIF-2α play a protective role in oxidative stress. HIF-1α reduces H2O2-induced apoptosis by upregulating Bcl-2 and Bcl-XL, downregulating Bax, Bak, and caspase-9, stabilizing intracellular ROS levels, and promoting the repair of H2O2-induced DNA damage to reduce apoptosis.

Effects of different types of exercise training on angiogenic responses in the left ventricular muscle of aged rats.

We evaluated angiogenic responses in the left ventricular muscle and aerobic capacity according to exercise type (aerobic, resistance, combined) in aged rats. In total, 24 male Sprague-Dawley rats (100weeks old) were used. To investigate the effect of regular training, the rats were divided into non-exercise (NE), aerobic exercise (AE), resistance exercise (RE), and combined exercise (CE) groups (six rats per group). Regular training tailored to each exercise type was performed for 8weeks (five times a week, 1h per day). After 8weeks of training, aerobic capacity was evaluated by a treadmill running test. Left ventricular muscle tissue was collected and the protein levels of angiogenesis indicators (eNOS, HIF-1α, PGC-1α, VEGF, FLK-1, Ang-1, Ang-2) were analyzed by Western blotting. Capillaries were observed by immunohistochemical staining for CD31. Body weight, heart weight, and heart/body weight ratio showed no difference among the groups. The AE and CE groups showed higher treadmill running capacity than the NE and RE groups. The eNOS, VEGF, HIF-1α, PGC-1α, and Ang-2 protein levels were significantly higher in the AE than NE group. The PGC-1α and FLK-1 protein levels were significantly higher in the RE than NE group. In addition, in the CE group, the eNOS, FLK-1, and PGC-1α protein levels were significantly higher than in the NE group. Expression of CD31 in cardiac tissue was higher in the AE and CE groups than in the other groups. Taken together, the results suggest that regular exercise training, irrespective of exercise type, might improve cardiovascular function by inducing angiogenic responses in the aged myocardium; however, AE may be the most effective.

The Role of Hypoxia in Endometrial Cancer.

Endometrial cancer represents the most frequent neoplasia from the corpus uteri and comprises the 14th leading cause of death in women worldwide. Risk factors that contribute to the disease include early menarche, late menopause, nulliparity, and menopausal hormone use, as well as hypertension and obesity comorbidities. The clinical effectiveness of chemotherapy is variable, suggesting that novel molecular targeted therapies against specific cellular processes associated with the maintenance of cancer cell survival and therapy resistance ameliorate the rates of success in endometrial cancer treatment. In the course of tumor growth, cancer cells must adapt to decreased oxygen availability in the microenvironment by upregulation of hypoxia-inducible factors, which orchestrate the activation of a transcriptional program leading to cell survival. During this adaptative process, the hypoxic cancer cells may acquire invasive and metastatic properties as well as increased cell proliferation and resistance to chemotherapy, enhanced angiogenesis, vasculogenic mimicry, and maintenance of cancer cell stemness, which contribute to more aggressive cancer phenotypes. Several studies have shown that hypoxia-inducible factor 1 alpha (HIF-1α) protein is aberrantly overexpressed in many solid tumors of the breast, prostate, ovarian, bladder, colon, brain, and pancreas. Thus, it has been considered an important therapeutic target. Here, we reviewed the current knowledge of the relevant roles of cellular hypoxia mechanisms and HIF-1α functions in diverse processes associated with endometrial cancer progression. In addition, we also summarize the role of microRNAs in the posttranscriptional regulation of protein-encoding genes involved in the hypoxia response in endometrial cancer. Finally, we pointed out the need for urgent targeted therapies to impair the cellular processes activated by hypoxia in the tumor microenvironment.

Melatonin affects hypoxia-inducible factor 1α and ameliorates delayed brain injury following subarachnoid hemorrhage via H19/miR-675/HIF1A/TLR4

ABSTRACT This study aimed to investigate the molecular mechanism of how melatonin (MT) interferes with hypoxia-inducible factor 1α (HIF1A) and toll-like receptor 4 (TLR4) expression, which is implicated in the management of delayed brain injury (DBI) after subarachnoid hemorrhage (SAH). Luciferase assay, real-time PCR, Western-blot analysis and immunohistochemistry (IHC) assays were utilized to explore the interaction among H19, miR-675, HIF1A and TLR4, and to evaluate the effect of MT on the expression of above transcripts in different groups. MT enhanced H19 expression by promoting the transcription efficiency of H19 promoter, and HIF1A was identified as a target of miR-675. HIF1A enhanced TLR4 expression via promoting the transcription efficiency of TLR4 promoter. Furthermore, administration of MT up-regulated miR-675 but suppressed the expressions of HIF1A and TLR4. Treatment with MT alleviated neurobehavioral deficits and apoptosis induced by SAH. According to the result of IHC, HIF1A and TLR4 protein levels in the SAH group were much higher than those in the SAH+MT group. Therefore, the administration of MT increased the levels of H19 and miR-675 which have been inhibited by SAH. In a similar way, treatment with MT decreased the levels of HIF1A and TLR4 which have been enhanced by SAH. MT could down-regulate the expression of HIF1A and TLR4 via the H19/miR-675/HIF1A/TLR4 signaling pathway, while TLR4 is crucial to the release of pro-inflammatory cytokines. Therefore, the treatment with MT could ameliorate post-SAH DBI.Running title: Melatonin ameliorates post-SAH DBI via H19/miR-675/HIF1A/TLR4 signaling pathways

Mesenteric Adipose Tissue Contributes to Intestinal Fibrosis in Crohn's Disease Through the ATX-LPA Axis.

Intestinal fibrostenosis is an important cause of surgical intervention in patients with Crohn's disease [CD]. Hypertrophic mesenteric adipose tissue [MAT] is associated with the disease process of CD. The purpose of this study was to investigate the contribution of MAT to intestinal fibrosis. MAT from surgical specimens of fibrostenotic CD patients and controls was collected for measurement of the levels of autotaxin [ATX] and lysophosphatidic acid [LPA]. ATX was inhibited in vivo in DNBS [dinitrobenzene sulfonic acid]-induced colitis mice, which were evaluated for colonic inflammation and fibrosis. 3T3-L1 cells and primary colonic fibroblasts were used in vitro to investigate the interaction between MAT and intestinal fibrosis, as well as the molecular mechanism underlying this interaction. MAT adjacent to the fibrostenotic intestine in CD patients showed an activated ATX-LPA axis. An in vivo study indicated that inhibition of ATX was associated with the improvement of morphology and function of diseased MAT, which was combined with ameliorated intestinal inflammation and fibrosis in DNBS-instilled mice. In vitro studies showed that hypoxia stimulated adipocyte ATX expression and that LPA stabilized adipocyte HIF-1α protein, forming an ATX-LPA-HIF-1α amplification loop and aggravating adipocyte dysfunction. LPA secreted by adipocytes bound to LPA1 on the surface of fibroblasts, promoted their proliferation and differentiation, and increased the expression of fibrosis-related factors. The ATX-LPA axis regulated intestinal fibrosis by influencing the proliferation and differentiation of intestinal fibroblasts. Inhibiting this axis may be a therapeutic target for intestinal fibrosis in CD.

USP5 promotes breast cancer cell proliferation and metastasis by stabilizing HIF2α.

Hypoxia-inducible factor 2α (HIF2α) plays a pivotal role in breast tumor growth and metastasis. However, the regulatory mechanisms of HIF2α protein stability remain poorly understood. The precise role of the deubiquitinase (DUB) ubiquitin-specific peptidase 5 (USP5) in breast cancer and the underlying mechanism remains largely unknown. Here, we identified USP5as a novel DUBfor HIF2α. Physically, USP5 interacts with HIF2α and protects HIF2α from ubiquitin-proteasome degradation, thereby promoting the transcription of HIF2α target genes, such as SLC2A1, PLOD2, P4HA1, and VEGFA. USP5 ablation impairs breast cancer cells proliferation, colony formation, migration, and invasion. Moreover, USP5 is highly expressed in breast cancer, and the protein levels of USP5 are positively correlated with HIF2α protein levels in human breast cancer tissues. Importantly, high levels of USP5 leads to poor clinical outcome in patients with breast cancer. Collectively, USP5 stabilizes HIF2α through its DUBactivity and provides a potential therapeutic target for breast cancer.

Oxygen sensors mediated HIF-1α accumulation and translocation: A pivotal mechanism of fine particles-exacerbated myocardial hypoxia injury

Epidemiological studies have demonstrated a strong association of ambient fine particulate matter (PM2.5) exposure with the increasing mortality by ischemic heart disease (IHD), but the involved mechanisms remain poorly understood. Herein, we found that the chronic exposure of real ambient PM2.5 led to the upregulation of hypoxia-inducible factor-1 alpha (HIF-1α) protein in the myocardium of mice, accompanied by obvious myocardial injury and hypertrophy. Further data from the hypoxia-ischemia cellular model indicated that PM2.5-induced HIF-1α accumulation was responsible for the promotion of myocardial hypoxia injury. Moreover, the declined ATP level due to the HIF-1α-mediated energy metabolism remodeling from β-oxidation to glycolysis had a critical role in the PM2.5-increased myocardial hypoxia injury. The in-depth analysis delineated that PM2.5 exposure decreased the binding of prolyl hydroxylase domain 2 (PHD2) and HIF-1α and subsequent ubiquitin protease levels, thereby leading to the accumulation of HIF-1α. Meanwhile, factor-inhibiting HIF1 (FIH1) expression was down-regulated by PM2.5, resulting in the enhanced translocation of HIF-1α to the nucleus. Overall, our study provides valuable insight into the regulatory role of oxygen sensor-mediated HIF-1α stabilization and translocation in PM-exacerbated myocardial hypoxia injury, we suggest this adds significantly to understanding the mechanisms of haze particles-caused burden of cardiovascular disease.

TRPM7 silencing modulates glucose metabolic reprogramming to inhibit the growth of ovarian cancer by enhancing AMPK activation to promote HIF-1\u03b1 degradation

BackgroundTumor cell metabolic reprogramming is crucial for the malignant behavior of cancer cells by promoting their proliferation. However, little is known on how transient receptor potential 7 (TRPM7) modulates metabolic reprogramming in ovarian cancer.MethodsThe effects of TRPM7 silencing on transcriptome profile, glucose uptake, lactic acid production, extracellular acidification rate (ECAR), oxygen consumption rate (OCR), intracellular ROS and ATP levels, and NAD+/NADH ratios in ovarian cancer cells were examined. The impacts of TRPM7 silencing on the levels of glycolysis-related HK2, PDK1 and oxidative phosphorylation (OXPHOS)-related IDH3B and UQCRC1, HIF-1α expression and AMPK phosphorylation were determined in ovarian cancer. The effect of AMPK activity on HIF-1α ubiquitination degradation was investigated in ovarian cancer cells.ResultsCompared with the control, TRPM7 silencing suppressed the proliferation of ovarian cancer cells by shifting preferable glycolysis to OXPHOS. In parallel, TRPM7 silencing decreased the glucose uptake of tumor-bearing mice and TRPM7 levels were negatively correlated with IDH3B and UQCRC1, but positively with HK2 and PDK1 expression in ovarian cancer tissues. Mechanistically, TRPM7 silencing significantly increased AMPK phosphorylation and decreased HIF-1α protein levels in ovarian cancer, particularly in HIF-1α silencing cells. The shifting from glycolysis to OXPHOS by TRPM7 silencing was abrogated by HIF-1α over-expression and impaired by inhibiting AMPK activity in ovarian cancer cells. Moreover, enhanced AMPK activation inhibited glycolysis, which was abrogated by HIF-1α over-expression in ovarian cancer cells. Moreover, the enhanced AMPK activation promoted HIF-1α ubiquitination degradation.ConclusionsTRPM7 silencing enhanced AMPK activation to shift glycolysis to oxidative phosphorylation by promoting HIF-1α ubiquitination degradation in ovarian cancer. Hence, TRPM7 may be a therapeutic target for intervention of ovarian cancer.

Aged Mouse Hippocampus Exhibits Signs of Chronic Hypoxia and an Impaired HIF-Controlled Response to Acute Hypoxic Exposures.

Altered hypoxia-inducible factor-alpha (HIF-α) activity may have significant consequences in the hippocampus, which mediates declarative memory, has limited vascularization, and is vulnerable to hypoxic insults. Previous studies have reported that neurovascular coupling is reduced in aged brains and that diseases which cause hypoxia increase with age, which may render the hippocampus susceptible to acute hypoxia. Most studies have investigated the actions of HIF-α in aging cortical structures, but few have focused on the role of HIF-α within aged hippocampus. This study tests the hypothesis that aging is associated with impaired hippocampal HIF-α activity. Dorsal hippocampal sections from mice aged 3, 9, 18, and 24 months were probed for the presence of HIF-α isoforms or their associated gene products using immunohistochemistry and fluorescent in situ hybridization (fISH). A subset of each age was exposed to acute hypoxia (8% oxygen) for 3 h to investigate changes in the responsiveness of HIF-α to hypoxia. Basal mean intensity of fluorescently labeled HIF-1α protein increases with age in the hippocampus, whereas HIF-2α intensity only increases in the 24-month group. Acute hypoxic elevation of HIF-1α is lost with aging and is reversed in the 24-month group. fISH reveals that glycolytic genes induced by HIF-1α (lactose dehydrogenase-a, phosphoglycerate kinase 1, and pyruvate dehydrogenase kinase 1) are lower in aged hippocampus than in 3-month hippocampus, and mRNA for monocarboxylate transporter 1, a lactose transporter, increases. These results indicate that lactate, used in neurotransmission, may be limited in aged hippocampus, concurrent with impaired HIF-α response to hypoxic events. Therefore, impaired HIF-α may contribute to age-associated cognitive decline during hypoxic events.

Enhanced Ca2+ Entry Sustains the Activation of Akt in Glucose Deprived SH-SY5Y Cells.

The two crucial cellular insults that take place during cerebral ischemia are the loss of oxygen and loss of glucose, which can both activate a cascade of events leading to neuronal death. In addition, the toxic overactivation of neuronal excitatory receptors, leading to Ca2+ overload, may contribute to ischemic neuronal injury. Brain ischemia can be simulated in vitro by oxygen/glucose deprivation, which can be reversible by the re-establishment of physiological conditions. Accordingly, we examined the effects of glucose deprivation on the PI3K/Akt survival signaling pathway and its crosstalk with HIF-1α and Ca2+ homeostasis in SH-SY5Y human neuroblastoma cells. It was found that glucose withdrawal decreased HIF-1α protein levels even in the presence of the ischemia-mimicking CoCl2. On the contrary, and despite neuronal death, we identified a strong activation of the master pro-survival kinase Akt, a finding that was also confirmed by the increased phosphorylation of GSK3, a direct target of p-Akt. Remarkably, the elevated Ca2+ influx recorded was found to promptly trigger the activation of Akt, while a re-addition of glucose resulted in rapid restoration of both Ca2+ entry and p-Akt levels, highlighting the plasticity of neurons to respond to ischemic challenges and the important role of glucose homeostasis for multiple neurological disorders.