The mineral dust-induced gene, mdig, regulates angiogenesis and lymphangiogenesis in lung adenocarcinoma by modulating the expression of VEGF-A/C/D via EGFR and HIF-1α signaling.

Mineral dust­induced gene (mdig) is a novel lung cancer­related oncogene. The aim of this study was to explore the effects of mdig on angiogenesis and lymphangiogenesis by vascular endothelial growth factor (VEGF) in lung adenocarcinoma. mdig­overexpressing A549, H1299 and 293T cells, mdig­silenced A549, human umbilical vein endothelial cells (HUVECs) and human lymphatic endothelial cells (HLECs) were cultured under normoxic and hypoxic conditions. Protein expression levels of mdig, epidermal growth factor receptor (EGFR), phospho(p)­EGFR Tyr1068, hypoxia­inducible factor­1α (HIF­1α), VEGF­A/C/D and VEGF­R1/R2/R3 were assessed using western blotting. mRNA expression levels of mdig, EGFR and HIF­1α were measured using RT­qPCR. Tube formation and xenograft tumor experiments were performed to examine the mechanism of mdig in angiogenesis and lymphangiogenesis. Protein expression levels of EGFR, HIF­1α and VEGF­A/C/D were significantly upregulated in cells cultured under hypoxic conditions compared with those cultured under normoxic conditions, whereas the levels of mdig were decreased. Protein expression levels of EGFR, p­EGFR and VEGF­A/R1/R2 were significantly increased in the mdig­overexpressing cells, whereas the levels of HIF­1α and VEGF­C/D/R3 were decreased compared with those in control cells, all of which were reversed in mdig­silenced cells. Tumor volumes and density of angiogenesis in the mdig­overexpressing group were significantly increased compared with those in the control group, whereas the density of lymphangiogenesis was decreased. No tumors formed in the mdig­silenced group after 3 weeks of assessment in vivo. Protein expression levels of EGFR, p­EGFR, VEGF­A and angiogenesis density were significantly reduced in the mdig­overexpressing cells treated with an EGFR inhibitor, whereas the levels of HIF­1α, VEGF­C/D and the lymphangiogenesis density were significantly increased in mdig­overexpressing cells treated with a HIF­1α agonist. All changes in protein expression were reversed in EGFR agonist and HIF­1α inhibitor treated mdig­silenced cells. In conclusion, mdig is an oxygen­sensitive protein that promotes tumor growth and angiogenesis by activating the EGFR/p­EGFR/VEGF­A/VEGF­R1/R2 pathway and inhibits lymphangiogenesis by blocking the HIF­1α/VEGF­C/D/VEGF­R3 pathway.

HIF-1α Stimulators Function Equally to Leading Hair Loss Agents in Enhancing Dermal Papilla Growth.

INTRODUCTION: Androgenic alopecia (AGA) occurs due to progressive miniaturization of the dermal papilla (DP). During this process the hair follicle loses nutrition over time and eventually dies, causing the hair to fall out. Recent evidence suggests that hypoxia-inducible factor-1a (HIF-1α) modulation may counteract hair loss. This study aims to evaluate the proliferation of dermal papilla cells (DPCs) under the influence of a selection of commercially available topical hair loss drugs, compared to HIF-1α-stimulating agents. MATERIALS AND METHODS: Using the hanging drop method, DPCs self-organized into spheroid shape, mirroring the three-dimensional (3D) structure of the DP in vivo. DP analogs were treated with established substances against AGA (minoxidil and caffeine) compared to HIF-1α-stimulating agents (deferoxamine [DFO] and deferiprone [DFP]), at 10 mM doses. DP analogs were simultaneously stained with 5-bromo-2'-deoxyuridine (BrdU) to evaluate impact of drug compounds on DP daughter cell production. Concurrently, fluorescent microscopy visualization of migration of daughter cells after 48 h in culture was performed. RESULTS: DPC proliferation within the spheroid structure was significantly enhanced by caffeine, minoxidil, and the HIF-1α-stimulating agent DFP when compared to control. Highest proliferation was seen in the DFP-treated DP analogs. Migration of peripheral DP daughter cells was highest in control and DFO groups. CONCLUSION: Here we demonstrate a significantly enhanced proliferative activity for both established substances against AGA (minoxidil and caffeine) and the HIF-1α-stimulating agent DFP in a 3D DPC spheroid culture model with equal results for DFP and minoxidil. These favorable characteristics make such compounds potential water-soluble alternatives to minoxidil.

A Hypoxia-Inducible Factor Stabilizer Improves Hematopoiesis and Iron Metabolism Early after Administration to Treat Anemia in Hemodialysis Patients.

Roxadustat (Rox), a hypoxia-inducible factor (HIF) stabilizer, is now available for the treatment of anemia in hemodialysis (HD) patients. To investigate hematopoietic effect and iron metabolism, this study involved 30 HD patients who were initially treated with darbepoetin (DA), a conventional erythropoietin-stimulating agent, and then switched to Rox. We measured erythrocyte, reticulocyte indices, and iron-related factors at every HD during the first two weeks after the treatment switch (Days 0-14) and again on Days 21 and 28. We measured erythropoietin (EPO) concentration every week and examined their changes from Day-0 values. The same variables were measured in 15 HD patients who continued DA at every HD for one week. Iron-related factors were also measured on Days 14 and 28. In the Rox group, hepcidin significantly decreased from Day 2. The reticulocyte hemoglobin content (CHr) significantly increased on Day 4, but decreased with a significant increase in reticulocyte count from Day 7. Log10(serum ferritin) significantly decreased after Day 11. Log10(EPO concentration) was lower at all time points. Compared with the DA group, the Rox group showed significant differences in all variables except CHr. These results suggest that Rox improves hematopoiesis and iron metabolism early after administration independent of EPO concentration.

Reactivation of Epstein-Barr Virus by HIF-1α Requires p53.

We previously reported that the cellular transcription factor hypoxia-inducible factor 1α (HIF-1α) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1α-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN-4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1α and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1α failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1α to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1α requires phosphorylated, wild-type p53 as a coactivator, with HIF-1α binding recruiting p53 to Zp.IMPORTANCE EBV, a human herpesvirus, is latently present in most nasopharyngeal carcinomas, Burkitt lymphomas, and some gastric cancers. To develop a lytic-induction therapy for treating patients with EBV-associated cancers, we need a way to efficiently reactivate EBV into lytic replication. EBV's BZLF1 gene product, Zta, usually controls this reactivation switch. We previously showed that HIF-1α binds the BZLF1 gene promoter, inducing Zta synthesis, and HIF-1α-stabilizing drugs can induce EBV reactivation. In this study, we determined which EBV-positive cell lines are reactivated by classes of HIF-1α-stabilizing drugs. We found, unexpectedly, that HIF-1α-stabilizing drugs only induce reactivation when they also induce accumulation of phosphorylated, wild-type p53. Fortunately, p53 phosphorylation can also be provided by drugs such as nutlin-3, leading to synergistic reactivation of EBV. These findings indicate that some HIF-1α-stabilizing drugs may be helpful as part of a lytic-induction therapy for treating patients with EBV-positive malignancies that contain wild-type p53.

Extracellular vesicles derived from fat-laden hepatocytes undergoing chemical hypoxia promote a pro-fibrotic phenotype in hepatic stellate cells.

BACKGROUND: The transition from steatosis to non-alcoholic steatohepatitis (NASH) is a key issue in non-alcoholic fatty liver disease (NAFLD). Observations in patients with obstructive sleep apnea syndrome (OSAS) suggest that hypoxia contributes to progression to NASH and liver fibrosis, and the release of extracellular vesicles (EVs) by injured hepatocytes has been implicated in NAFLD progression. AIM: To evaluate the effects of hypoxia on hepatic pro-fibrotic response and EV release in experimental NAFLD and to assess cellular crosstalk between hepatocytes and human hepatic stellate cells (LX-2). METHODS: HepG2 cells were treated with fatty acids and subjected to chemically induced hypoxia using the hypoxia-inducible factor 1 alpha (HIF-1α) stabilizer cobalt chloride (CoCl2). Lipid droplets, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic-associated genes were assessed. EVs were isolated by ultracentrifugation. LX-2 cells were treated with EVs from hepatocytes. The CDAA-fed mouse model was used to assess the effects of intermittent hypoxia (IH) in experimental NASH. RESULTS: Chemical hypoxia increased steatosis, oxidative stress, apoptosis and pro-inflammatory and pro-fibrotic gene expressions in fat-laden HepG2 cells. Chemical hypoxia also increased the release of EVs from HepG2 cells. Treatment of LX2 cells with EVs from fat-laden HepG2 cells undergoing chemical hypoxia increased expression pro-fibrotic markers. CDAA-fed animals exposed to IH exhibited increased portal inflammation and fibrosis that correlated with an increase in circulating EVs. CONCLUSION: Chemical hypoxia promotes hepatocellular damage and pro-inflammatory and pro-fibrotic signaling in steatotic hepatocytes both in vitro and in vivo. EVs from fat-laden hepatocytes undergoing chemical hypoxia evoke pro-fibrotic responses in LX-2 cells.

Involvement of HIF-1α-regulated miR-21, acting via the Akt/NF-κB pathway, in malignant transformation of HBE cells induced by cigarette smoke extract.

Although the relationship between cigarette smoke and lung cancer has been widely studied, the molecular mechanism for cigarette smoke-induced lung cancer remains largely unclear. The present study investigated the roles of hypoxia-inducible factor (HIF)-1α and miR-21 in the malignant transformation of human bronchial epithelial (HBE) cells induced by cigarette smoke extract (CSE). In case of acute and chronic treatment of HBE cells, CSE increased the levels of HIF-1α, p-Akt, p-NF-κB, and miR-21 and decreased PTEN levels. The increased miR-21 levels induced by CSE were prevented by down-regulation of HIF-1α. Further, elevated miR-21 suppressed PTEN levels, which decreased the levels of p-Akt and p-NF-κB. However, those changes were attenuated in cells co-transfected with HIF-1α siRNA and an miR-21 mimic. Silencing of HIF-1α or NF-κB decreased colony formation and the invasion and migration capacities of CSE-transformed HBE cells; however, up-regulation of miR-21 reversed these effects. These results indicate that the oncogenic capacity of HIF-1α in regulation of miR-21-inhibited PTEN in a manner dependent on the Akt/NF-κB pathway, a process that is involved in the CSE-induced malignant transformation of HBE cells. Thus, the present research has established a new mechanism for cigarette smoke-induced lung carcinogenesis.

Down-regulation of CASK in glucotoxicity-induced insulin dysfunction in pancreatic ß cells.

High-glucose level exerts deleterious effects on pancreatic ß cells, but the mechanisms remain unclear. Calcium/calmodulin-dependent serine protein kinase (CASK) plays a vital role in neural development and release of neurotransmitters, and probably plays a role in the anchoring of insulin on pancreatic ß cell membrane. Hypoxia-inducible factor 1α (HIF1α) is involved in ß-cell dysfunction. The aim of this study was to provide some basic evidence that CASK could be involved in glucotoxicity-induced insulin secretion dysfunction mediated by HIF1α in INS-1E cells. CASK overexpression plasmid, HIF1α agonist (CoCl2), and HIF1α selective inhibitor (KC7F2) were used. The results showed that chronic stimulation with high glucose could induce insulin secretion dysfunction in INS-1E ß cells. Overexpression of CASK partially reversed the effects of high glucose on insulin secretion. CoCl2 reduced the expression of CASK, but KC7F2 reversed the glucotoxicity-induced CASK level reduction. These results suggested that glucotoxicity-induced insulin secretion defects in INS-1E cells could be mediated by HIF1α via the down-regulation of CASK.

Tumor stressors induce two mechanisms of intracellular P-glycoprotein-mediated resistance that are overcome by lysosomal-targeted thiosemicarbazones.

Multidrug resistance (MDR) is a major obstacle in cancer treatment due to the ability of tumor cells to efflux chemotherapeutics via drug transporters (e.g. P-glycoprotein (Pgp; ABCB1)). Although the mechanism of Pgp-mediated drug efflux is known at the plasma membrane, the functional role of intracellular Pgp is unclear. Moreover, there has been intense focus on the tumor micro-environment as a target for cancer treatment. This investigation aimed to dissect the effects of tumor micro-environmental stress on subcellular Pgp expression, localization, and its role in MDR. These studies demonstrated that tumor micro-environment stressors (i.e. nutrient starvation, low glucose levels, reactive oxygen species, and hypoxia) induce Pgp-mediated drug resistance. This occurred by two mechanisms, where stressors induced 1) rapid Pgp internalization and redistribution via intracellular trafficking (within 1 h) and 2) hypoxia-inducible factor-1α expression after longer incubations (4-24 h), which up-regulated Pgp and was accompanied by lysosomal biogenesis. These two mechanisms increased lysosomal Pgp and facilitated lysosomal accumulation of the Pgp substrate, doxorubicin, resulting in resistance. This was consistent with lysosomal Pgp being capable of transporting substrates into lysosomes. Hence, tumor micro-environmental stressors result in: 1) Pgp redistribution to lysosomes; 2) increased Pgp expression; 3) lysosomal biogenesis; and 4) potentiation of Pgp substrate transport into lysosomes. In contrast to doxorubicin, when stress stimuli increased lysosomal accumulation of the cytotoxic Pgp substrate, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), this resulted in the agent overcoming resistance. Overall, this investigation describes a novel approach to overcoming resistance in the stressful tumor micro-environment.

Adrenomedullin serves a role in the humoral pathway of delayed remote ischemic preconditioning via a hypoxia-inducible factor-1α-associated mechanism.

Remote ischemic preconditioning (RIPC) is a minimally invasive method that provides protection by reducing injury to the heart, kidneys, brain and other tissues or organs. RIPC may improve the outcome in patients undergoing surgery. Although the role of RIPC has been studied, the results remain controversial. It is difficult to confirm whether RIPC has a kidney protective effect and the understanding of the preconditioning signal pathway involved remains unclear. In the present study, the effect of RIPC in urology was evaluated. The protection against renal damage was assessed by investigating the potential mediator, hypoxia­inducible factor­1α (HIF­1α), and the functional adrenomedullin (ADM) pathway. Male Sprague­Dawley (SD) rats were used in the present study. The animal model of kidney damage induced by ischemia reperfusion (IR) was used to investigate the protective effect of the acute and delayed phase RIPC. Furthermore, the protective effects of RIPC mediated by a HIF­1α­ADM pathway were assessed. The indexes of renal function and oxidative damage indicators were measured by Cr, BUN, mALB, ß2­MG, MPO, MDA and SOD assays, and the expression of HIF­1α and ADM were detected by western blot analysis, immunohistochemistry and ELISA assays. Tubular score, determined using hematoxylin and eosin staining, was used to evaluate renal tissue damage. Applying RIPC prevented IR­induced renal dysfunction and oxidative damage by decreasing Cr, BUN, mALB, ß2­MG, MPO, MDA levels and increasing SOD activity. Findings showed that delayed RIPC had an improved effect compared with acute treatment. Delayed RIPC also upregulated the expression of HIF­1α and ADM, indicating that the protective effect of the delayed RIPC may be associated with a HIF­1α­ADM­mediated mechanism. The effect of the delayed RIPC to reduce IR­induced renal damage and increase ADM expression was enhanced by HIF­1α agonists DMOG and BAY 85­3934, whereas the effect was whittled by HIF­1α antagonists YC­1 and 2­MeOE2. Furthermore, receiving ADM also offered protection to the kidney in comparison with the IR+Vehicle group. These findings suggest that RIPC prevents IR­mediated renal damage by HIF­1α via an ADM humoral pathway. In the present study, RIPC provided an effective renal protection. ADM could also offer protection regulated by HIF­1α in renal tissue. However, the mechanism of ADM as a protective factor in RIPC requires further research.

HIF-1alpha and infectious diseases: a new frontier for the development of new therapies.

The aim of this review is to show the significant role of HIF-1alpha in inflammatory and infectious diseases. Hypoxia is a physiological characteristic of a wide range of diseases from cancer to infection. Cellular hypoxia is sensed by oxygen-sensitive hydrolase enzymes, which control the protein stability of hypoxia-inducible factor alpha 1 (HIF-1alpha) transcription factors. When stabilized, HIF-1alpha binds with its cofactors to HIF-responsive elements (HREs) in the promoters of target genes to organize a broad ranging transcriptional program in response to the hypoxic environment. HIF-1alpha also plays a regulatory function in response to a diversity of molecular signals of infection and inflammation even under normoxic conditions. HIF-1alpha is stimulated by pro-inflammatory cytokines, growth factors and a wide range of infections. Its induction is a general element of the host response to infection. In this review, we also discuss recent advances in knowledge on HIF-1alpha and inflammatory responses, as well as its direct influence in infectious diseases caused by bacteria, virus, protozoan parasites and fungi.

Discovery and Preclinical Characterization of GSK1278863 (Daprodustat), a Small Molecule Hypoxia Inducible Factor-Prolyl Hydroxylase Inhibitor for Anemia.

Decreased erythropoietin (EPO) production, shortened erythrocyte survival, and other factors reducing the response to EPO contribute to anemia in patients who have a variety of underlying pathologies such as chronic kidney disease. Treatment with recombinant human EPO (rHuEPO) at supraphysiologic concentrations has proven to be efficacious. However, it does not ameliorate the condition in all patients, and it presents its own risks, including cardiovascular complications. The transcription factors hypoxia-inducible factor (HIF) 1α and HIF2α control the physiologic response to hypoxia and invoke a program of increased erythropoiesis. Levels of HIFα are modulated by oxygen tension via the action of a family of HIF-prolyl hydroxylases (PHDs), which tag HIFα for proteasomal degradation. Inhibition of these PHDs simulates conditions of mild hypoxia, leading to a potentially more physiologic erythropoietic response and presenting a potential alternative to high doses of rHuEPO. Here we describe the discovery and characterization of GSK1278863 [2-(1,3-dicyclohexyl-6-hydroxy-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamido) acetic acid], a pyrimidinetrione-glycinamide low nanomolar inhibitor of PHDs 1-3 that stabilizes HIFα in cell lines, resulting in the production of increased levels of EPO. In normal mice, a single dose of GSK1278863 induced significant increases in circulating plasma EPO but only minimal increases in plasma vascular endothelial growth factor (VEGF-A) concentrations. GSK1278863 significantly increased reticulocytes and red cell mass parameters in preclinical species after once-daily oral administration and has demonstrated an acceptable nonclinical toxicity profile, supporting continued clinical development. GSK1278863 is currently in phase 3 clinical trials for treatment of anemia in patients with chronic kidney disease.

Administration of follicle-stimulating hormone induces autophagy via upregulation of HIF-1α in mouse granulosa cells.

Recent studies reported the important role of autophagy in follicular development. However, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of follicle-stimulating hormone (FSH) on mouse granulosa cells (MGCs). Results indicated that autophagy was induced by FSH, which is known to be the dominant hormone regulating follicular development and granulosa cell (GC) proliferation. The activation of mammalian target of rapamycin (mTOR), a master regulator of autophagy, was inhibited during the process of MGC autophagy. Moreover, MHY1485 (an agonist of mTOR) significantly suppressed autophagy signaling by activating mTOR. The expression of hypoxia-inducible factor 1-alpha (HIF-1α) was increased after FSH treatment. Blocking hypoxia-inducible factor 1-alpha attenuated autophagy signaling. In vitro, CoCl2-induced hypoxia enhanced cell autophagy and affected the expression of beclin1 and BCL2/adenovirus E1B interacting protein 3 (Bnip3) in the presence of FSH. Knockdown of beclin1 and Bnip3 suppressed autophagy signaling in MGCs. Furthermore, our in vivo study demonstrated that the FSH-induced increase in weight was significantly reduced after effectively inhibiting autophagy with chloroquine, which was correlated with incomplete mitophagy process through the PINK1-Parkin pathway, delayed cell cycle, and reduced cell proliferation rate. In addition, chloroquine treatment decreased inhibin alpha subunit, but enhanced the expression of 3 beta-hydroxysteroid dehydrogenase. Blocking autophagy resulted in a significantly lower percentage of antral and preovulatory follicles after FSH stimulation. In conclusion, our results indicate that FSH induces autophagy signaling in MGCs via HIF-1α. In addition, our results provide evidence that autophagy induced by FSH is related to follicle development and atresia.

Metabolic reprogramming by HIF-1 activation enhances survivability of human adipose-derived stem cells in ischaemic microenvironments.

OBJECTIVES: Poor cell survival severely limits the beneficial effect of adipose-derived stem cell (ADSC)-based therapy for disease treatment and tissue regeneration, which might be caused by the attenuated level of hypoxia-inducible factor-1 (HIF-1) in these cells after having been cultured in 21% ambient oxygen in vitro for weeks. In this study, we explored the role of pre-incubation in dimethyloxalylglycine (DMOG, HIF-1 activator) in the survivability of human ADSCs in a simulated ischaemic microenvironment in vitro and in vivo. The underlying mechanism and angiogenesis were also studied. MATERIALS AND METHODS: Survivability of ADSCs was determined in a simulated ischaemic model in vitro and a nude mouse model in vivo. Cell metabolism and angiogenesis were investigated by tube formation assay, flow cytometry, fluorescence staining and real-time polymerase chain reaction (RT-PCR) after DMOG treatment. RESULTS: The results of the experimental groups showed significant enhancement of ADSC survivability in a simulated ischaemic microenvironment in vitro and transplanted model in vivo. Study of the underlying mechanisms suggested that the improved cell survival was regulated by HIF-1-induced metabolic reprogramming including decreased reactive oxygen species, increased intracellular pH, enhanced glucose uptake and increased glycogen synthesis. Tube formation assay revealed higher angiogenic ability in the DMOG-treated group than that in control group. CONCLUSIONS: The promotion of HIF-1 level in ADSCs induced by DMOG preconditioning suggests a potential strategy for improving the outcome of cell therapy due to increased survival and angiogenic ability.

Cobalt(II) Chloride Modifies the Phenotype of Macrophage Activation.

Cobalt (Co) is vital for cells in trace amounts, but excessive exposure to Co is possible due to surgical devices such as artificial metal-on-metal joints. Cobalt(II) chloride (CoCl2 ) has also been shown to imitate hypoxic conditions in cells by stabilizing the transcription factor hypoxia-inducible factor-1α (HIF-1α). The purpose of this study was to investigate the possible immunomodulatory action of CoCl2 by investigating its effects on the expression of inflammatory genes in macrophages. The following factors were assessed: inducible nitric oxidase synthase (iNOS), nicotinamide adenine dinucleotide phosphate-oxidase 2 (NOX2), interleukin-6 (IL-6), arginase-1 and HIF-1α. In the absence of exogenous cytokines, CoCl2 enhanced alternative (M2) macrophage activation as demonstrated by increased arginase-1 expression, but had no direct effect on inflammatory factors associated with classical (M1) activation. Interestingly, in lipopolysaccharide (LPS)-stimulated macrophages, CoCl2 modified the M1-type activation profile by increasing iNOS expression and nitric oxide production and decreasing NOX2 and IL-6. Also, CoCl2 increased HIF-1α levels in unstimulated and LPS-stimulated cells as expected. In conclusion, we showed that CoCl2 enhanced alternative (M2) activation in resting macrophages. In addition, CoCl2 was found to remodel the classical M1 phenotype of macrophage activation by changing the balance of iNOS, NOX2 and IL-6.

Coffee induces vascular endothelial growth factor (VEGF) expression in human neuroblastama SH-SY5Y cells.

Recent evidence indicates that hypoxia-inducible vascular endothelial growth factor (VEGF) has neurotrophic and neuroprotective effects on neuronal and glial cells. On the other hand, recent epidemiological studies showed that daily coffee consumption has been associated with a lower risk of several neuronal disorders. Therefore, we investigated the effect of coffee on VEGF expression in human neuroblastoma SH-SY5Y cells. We found that even low concentration of coffee (<2%) strongly induced VEGF expression via an activation of HIF-1α. The activation of HIF-1α by coffee was attributed to the coffee-dependent inhibition of prolyl hydroxylation of HIF1α, which is essential for proteolytic degradation of HIF-1α. However, no inhibition was observed at the catalytic activity in vitro. Coffee component(s) responsible for the activation of HIF-1α was not major constituents such as caffeine, caffeic acid, chlorogenic acid, and trigonelline, but was found to emerge during roasting process. The active component(s) was extractable with ethyl acetate. Our results suggest that daily consumption of coffee may induce VEGF expression in neuronal cells. This might be related to protective effect of coffee on neural disorders such as Alzheimer's disease and Parkinson's disease.

Proteomic profiling reveals that collismycin A is an iron chelator.

Collismycin A (CMA), a microbial product, has anti-proliferative activity against cancer cells, but the mechanism of its action remains unknown. Here, we report the identification of the molecular target of CMA by ChemProteoBase, a proteome-based approach for drug target identification. ChemProteoBase profiling showed that CMA is closely clustered with di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone, an iron chelator. CMA bound to both Fe(II) and Fe(III) ions and formed a 2:1 chelator-iron complex with a redox-inactive center. CMA-induced cell growth inhibition was completely canceled by Fe(II) and Fe(III) ions, but not by other metal ions such as Zn(II) or Cu(II). Proteomic and transcriptomic analyses showed that CMA affects the glycolytic pathway due to the accumulation of HIF-1α. These results suggest that CMA acts as a specific iron chelator, leading to the inhibition of cancer cell growth.

Entinostat up-regulates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-1α transcription factors.

Bacterial resistance against classical antibiotics is a growing problem and the development of new antibiotics is limited. Thus, novel alternatives to antibiotics are warranted. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can be induced by several compounds, including vitamin D and phenyl-butyrate (PBA). Utilizing a luciferase based assay, we recently discovered that the histone deacetylase inhibitor Entinostat is a potent inducer of the CAMP gene encoding the human cathelicidin LL-37. Here we investigate a mechanism for the induction and also find that Entinostat up-regulates human ß-defensin 1. Analysis of the CAMP promoter sequence revealed binding sites for the transcription factors STAT3 and HIF-1α. By using short hairpin RNA and selective inhibitors, we found that both transcription factors are involved in Entinostat-induced expression of LL-37. However, only HIF-1α was found to be recruited to the CAMP promoter, suggesting that Entinostat activates STAT3, which promotes transcription of CAMP by increasing the expression of HIF-1α. Finally, we provide in vivo relevance to our findings by showing that Entinostat-elicited LL-37 expression was impaired in macrophages from a patient with a STAT3-mutation. Combined, our findings support a role for STAT3 and HIF-1α in the regulation of LL-37 expression.

Cigarette smoke extract (CSE) induces transient receptor potential ankyrin 1(TRPA1) expression via activation of HIF1αin A549 cells.

We previously found that transient receptor potential ankyrin 1 (TRPA1) in guinea pig tracheal epithelial cells was elevated after 14 days of cigarette smoke (CS) exposure. However, the mechanism underlying CS-induced TRPA1 expression remains unknown. Here, we explored whether cigarette smoke extract (CSE)-induced TRPA1 expression is related with modulation of HIF1α in A549 cells. Our results showed that CSE increased TRPA1 expression in A549 cells, decreased Iκ B, PHD2, and HDAC2, and increased ROS release and nuclear translocation of NF-κ B and HIF1α. Moreover, HIF1α siRNA and/or MG132 (a proteasome inhibitor) pretreatment significantly inhibited CSE-induced TRPA1 expression and HIF1α nuclear translocation in A549 cells. However, HIF1α siRNA pretreatment did not affect CSE-induced NF-κ B nuclear translocation, suggesting that CSE-induced TRPA1 expression in A549 cells is directly mediated by HIF1α, but not by NF-κ B. Similar to CSE treatment, treatment of A549 cells with LPS caused significant increases in nuclear translocation of NF-κ B and HIF1α mRNA expression, but did not alter TRPA1 mRNA expression. However, pretreatment with PHD2 siRNA did result in increased TRPA1 mRNA expression in LPS-treated A549 cells; an effect that was inhibited by SN50 (a NF-κ B inhibitor). It suggests a role for NF-κ B to indirectly regulate TRPA1 mRNA expression via modulating HIF1α mRNA transcription. In addition, treatment cells with HDAC2 siRNA plus 2%CSE resulted in increased HIF1α nuclear translocation and TRPA1 expression, which was significantly inhibited by MG132 and HIF1α siRNA. These results suggest that HDAC2 indirectly modulates TRPA1 expression by promoting the DNA-binding activity of HIF1α. These findings show that CSE increases TRPA1 expression in airway epithelial cells by directly activating HIF1α, and that this increase in TRPA1 expression is indirectly regulated via NF-κ B, PHD2 and HDAC2 modulation of HIF1α activity.

Hypoxia enhances tenocyte differentiation of adipose-derived mesenchymal stem cells by inducing hypoxia-inducible factor-1α in a co-culture system.

OBJECTIVES: Tissue engineering is a promising approach for repair of tendon injuries. Adipose-derived mesenchymal stem cells (ADMSCs) have gained increasing research interest for their potential in improving healing and regeneration of injured tendons. The present study aimed to investigate effects of O2 tension and potential signalling pathways on AMDSC differentiation into tenocytes, in an indirect co-culture system. MATERIALS AND METHODS: Human ADMSCs were co-cultured under normoxia (20% O2 ) and also under hypoxia (2% O2 ). Tenocyte differentiation of AMDSCs and expression of hypoxia-inducible factor-1 (HIF-1α) were analysed by reverse transcription-PCR, Western blotting and immunohistochemistry. Furthermore, HIF-1α inhibitor and inducer (FG-4592) effects on differentiation of AMDSCs were studied using qPCR, immunofluorescence and Western blotting. RESULTS: Indirect co-culture with tenocytes increased differentiation of ADMSCs into tenocytes; furthermore, hypoxia further enhanced tenocyte differentiation of AMDSCs, accompanied by increased expression of HIF-1α. HIF-1α inhibitor attenuated effects of hypoxia on differentiation of ADMSCs; in contrast, FG-4592 increased differentiation of ADMSCs under both hypoxia and normoxia. CONCLUSIONS: Taken together, we found that growing ADMSCs under hypoxia, or activating expression of HIF-1α to be important in differentiation of ADMSCs, which provides a foundation for application of ADMSCs in vivo for tendon regeneration.

Studies on novel HIF activators, A-503451sII: biological activities of A-503451A.

In the course of our screening, we discovered a novel compound, A-503451A, as a potent hypoxia-inducible factor (HIF) activator. In human hepatocarcinoma HepG2 cells, A-503451A induced HIF-mediated luciferase reporter gene expression and stabilized HIF-1α protein. A-503451A increased the mRNA expression levels and the protein secretion of HIF-dependent genes, vascular endothelial growth factor and erythropoietin. Addition of excess ferric chloride to the culture medium suppressed the HIF-induction activity of A-503451A. A-503451A did not have iron-chelating activity in vitro, but decreased the intracellular labile iron pool concentration. These data indicate that A-503451A is a unique HIF activator.