Accumulation Of Hypoxia-inducible Factor Research Articles

Aqp-1 Gene Knockout Attenuates Hypoxic Pulmonary Hypertension of Mice.

Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.

Expression and epigenetic regulatory mechanism of BNIP3 in clear cell renal cell carcinoma.

The majority of clear cell renal cell carcinomas (ccRCCs) are caused by an accumulation of hypoxia-inducible factor (HIF) and the overexpression of downstream genes in response to the von Hippel-Lindau (VHL) gene becoming inactivated. In the present study, our hypothesis was that BNIP3, a gene positioned downstream of HIF, would be expressed at a higher level in ccRCC; however, instead, lower levels of BNIP3 expression were identified in RCC tumor tissues compared with adjacent non-tumor tissues. These changes were associated with lower levels of VHL, and higher levels of HIF and vascular endothelial growth factor. BNIP3 was also undetectable in three investigated RCC cell lines (786-O, ACHN, A498) and GRC-1-1 cells. Methylation of the BNIP3 promoter was not detected, and neither did treatment with a methylation inhibitor cause cell proliferation. However, treatment with a histone deacetylation inhibitor, trichostatin A (TSA), inhibited cultured RCC cell proliferation, promoted apoptosis and restored BNIP3 expression. Furthermore, histone deacetylation of the BNIP3 promoter was identified in ACHN and 786-O cells, and the acetylation status was restored following TSA treatment. Taken together, the results of the present study suggest that histone deacetylation, but not methylation, is most likely to cause BNIP3 inactivation in RCC. The data also indicated that restoration of BNIP3 expression by a histone deacetylation inhibitor led to growth inhibition and apoptotic promotion in RCC.

Reactive Oxygen Species Formation in the Brain at Different Oxygen Levels: The Role of Hypoxia Inducible Factors.

Hypoxia inducible factor (HIF) is the master oxygen sensor within cells and is central to the regulation of cell responses to varying oxygen levels. HIF activation during hypoxia ensures optimum ATP production and cell integrity, and is associated both directly and indirectly with reactive oxygen species (ROS) formation. HIF activation can either reduce ROS formation by suppressing the function of mitochondrial tricarboxylic acid cycle (TCA cycle), or increase ROS formation via NADPH oxidase (NOX), a target gene of HIF pathway. ROS is an unavoidable consequence of aerobic metabolism. In normal conditions (i.e., physioxia), ROS is produced at minimal levels and acts as a signaling molecule subject to the dedicated balance between ROS production and scavenging. Changes in oxygen concentrations affect ROS formation. When ROS levels exceed defense mechanisms, ROS causes oxidative stress. Increased ROS levels can also be a contributing factor to HIF stabilization during hypoxia and reoxygenation. In this review, we systemically review HIF activation and ROS formation in the brain during hypoxia and hypoxia/reoxygenation. We will then explore the literature describing how changes in HIF levels might provide pharmacological targets for effective ischaemic stroke treatment. HIF accumulation in the brain via HIF prolyl hydroxylase (PHD) inhibition is proposed as an effective therapy for ischaemia stroke due to its antioxidation and anti-inflammatory properties in addition to HIF pro-survival signaling. PHD is a key regulator of HIF levels in cells. Pharmacological inhibition of PHD increases HIF levels in normoxia (i.e., at 20.9% O2 level). Preconditioning with HIF PHD inhibitors show a neuroprotective effect in both in vitro and in vivo ischaemia stroke models, but post-stroke treatment with PHD inhibitors remains debatable. HIF PHD inhibition during reperfusion can reduce ROS formation and activate a number of cellular survival pathways. Given agents targeting individual molecules in the ischaemic cascade (e.g., antioxidants) fail to be translated in the clinic setting, thus far, HIF pathway targeting and thereby impacting entire physiological networks is a promising drug target for reducing the adverse effects of ischaemic stroke.

Hypoxia, Hypoxia-Inducible Factor-1α, and Innate Antileishmanial Immune Responses

Low oxygen environments and accumulation of hypoxia-inducible factors (HIFs) are features of infected and inflamed tissues. Here, we summarize our current knowledge on oxygen levels found in Leishmania-infected tissues and discuss which mechanisms potentially contribute to local tissue oxygenation in leishmanial lesions. Moreover, we review the role of hypoxia and HIF-1 on innate antileishmanial immune responses.

Insulin and IGF-1 receptor autocrine loops are not required for Exendin-4 induced changes to pancreatic β-cell bioenergetic parameters and metabolism in BRIN-BD11 cells

Pharmacological long lasting Glucagon-like peptide-1 (GLP-1) analogues, such as Exendin-4, have become widely used diabetes therapies. Chronic GLP-1R stimulation has been linked to β-cell protection and these pro-survival actions of GLP-1 are dependent on the activation of the mammalian target of rapamycin (mTOR) leading to accumulation of Hypoxia inducible factor 1 alpha (HIF-1α). Recent studies from our lab indicate that prolonged GLP-1R stimulation promotes metabolic reprograming of β-cells towards a highly glycolytic phenotype and activation of the mTOR/HIF-1α pathway was required for this action. We hypothesised that GLP-1 induced metabolic changes depend on the activation of mTOR and HIF-1α, in a cascade that occurs after triggering of a potential Insulin-like growth factor 1 receptor (IGF-1R) or the Insulin receptor (IR) autocrine loops. Loss of function of these receptors, through the use of small interfering RNA, or neutralizing antibodies directed towards their products, was undertaken in conjunction with functional assays. Neither of these strategies mitigated the effect of GLP-1 on glucose uptake, protein expression or bioenergetic flux. Our data indicates that activation of IGF-1R and/or the IR autocrine loops resulting in β-cell protection and function, involve mechanisms independent to the enhanced metabolic effects resulting from sustained GLP-1R activation.

Donor Treatment With a Hypoxia-Inducible Factor-1 Agonist Prevents Donation After Cardiac Death Liver Graft Injury in a Rat Isolated Perfusion Model.

The protective role of hypoxia-inducible factor-1 (HIF-1) against liver ischemia-reperfusion injury has been well proved. However its role in liver donation and preservation from donation after cardiac death (DCD) is still unknown. The objective of this study was to test the hypothesis that pharmaceutical stabilization of HIF-1 in DCD donors would result in a better graft liver condition. Male SD rats (6 animals per group) were randomly given the synthetic prolyl hydroxylase domain inhibitor FG-4592 (Selleck, 6 mg/kg of body weight) or its vehicle (dimethylsulfoxide). Six hours later, cardiac arrest was induced by bilateral pneumothorax. Rat livers were retrieved 30 min after cardiac arrest, and subsequently cold stored in University of Wisconsin solution for 24 h. They were reperfused for 60 min with Krebs-Henseleit bicarbonate buffer in an isolated perfused liver model, after which the perfusate and liver tissues were investigated. Pretreatment with FG-4592 in DCD donors significantly improved graft function with increased bile production and synthesis of adenosine triphosphate, decreased perfusate liver enzyme release, histology injury scores and oxidative stress-induced cell injury and apoptosis after reperfusion with the isolated perfused liver model. The beneficial effects of FG-4592 is attributed in part to the accumulation of HIF-1 and ultimately increased PDK1 production. Pretreatment with FG-4592 in DCD donors resulted in activation of the HIF-1 pathway and subsequently protected liver grafts from warm ischemia and cold-stored injury. These data suggest that the pharmacological HIF-1 induction may provide a clinically applicable therapeutic intervention to prevent injury to DCD allografts.

The Role of Hypoxia-Inducible Factors in Cancer Resistance

Diminished oxygen availability (hypoxia) is a hallmark of the tumor microenvironment. A major regulator of cellular adaptation to hypoxia is the hypoxia-inducible factor (HIF) family of transcription factors, which play key roles in many crucial aspects of cancer biology including angiogenesis, stem cell maintenance, metabolic reprogramming, resistance to apoptosis, autocrine growth factor signaling, the EMT program, invasion and metastasis. Resistance to chemotherapy/radiotherapy is the primary cause for treatment failure in clinical oncology. Hypoxia and accumulation of hypoxia-inducible factors (HIFs) in solid tumors have been associated with resistance to treatment and poor prognosis. HIFs causes autophagy establishment to promote survival of cancer cells, and is also associated with the promotion and maintenance of cancer stem cells, a minority subpopulation within the tumor responsible for tumor recurrence and resistance to chemotherapy. In this review, we provide a concise comparative description of the structure, regulation, transcribed genes and roles played by each HIFα subunit in coordinating the transcriptional responses to hypoxia and on the roles they play in the promotion of resistance to anti-cancer therapy.

Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells

IntroductionExtracellular vesicles (EVs) are small, spherical particles enclosed by a phospholipid bilayer (∼30–1000 nm) released from multiple cell types, and have been shown to have pathophysiological roles in a plethora of disease states. The transcription factor hypoxia-inducible factor-1 (HIF-1) allows for adaptation of cellular physiology in hypoxia and may permit the enhanced release of EVs under such conditions. Nitric oxide (NO) plays a pivotal role in vascular homeostasis, and can modulate the cellular response to hypoxia by preventing HIF-1 accumulation. We aimed to selectively target HIF-1 via sodium nitrite (NaNO2) addition, and examine the effect on endothelial EV, size, concentration and function, and delineate the role of HIF-1 in EV biogenesis. MethodsEndothelial (HECV) cells were exposed to hypoxic conditions (1% O2, 24 h) and compared to endothelial cells exposed to normoxia (21% O2) with and without the presence of sodium nitrite (NaNO2) (30 μM). Allopurinol (100 μM), an inhibitor of xanthine oxidoreductase, was added both alone and in combination with NaNO2 to cells exposed to hypoxia. EV and cell preparations were quantified by nanoparticle tracking analysis and confirmed by electron microscopy. Western blotting and siRNA were used to confirm the role of HIF-1α and HIF-2α in EV biogenesis. Flow cytometry and time-resolved fluorescence were used to assess the surface and intravesicular protein content. ResultsEndothelial (HECV) cells exposed to hypoxia (1% O2) produced higher levels of EVs compared to cells exposed to normoxia. This increase was confirmed using the hypoxia-mimetic agent desferrioxamine. Treatment of cells with sodium nitrite (NaNO2) reduced the hypoxic enhancement of EV production. Treatment of cells with the xanthine oxidoreductase inhibitor allopurinol, in addition to NaNO2 attenuated the NaNO2-attributed suppression of hypoxia-mediated EV release. Transfection of cells with HIF-1α siRNA, but not HIF-2α siRNA, prior to hypoxic exposure prevented the enhancement of EV release. ConclusionThese data provide evidence that hypoxia enhances the release of EVs in endothelial cells, and that this is mediated by HIF-1α, but not HIF-2α. Furthermore, the reduction of NO2− to NO via xanthine oxidoreductase during hypoxia appears to inhibit HIF-1α-mediated EV production.

Dedifferentiation of Glioma Cells to Glioma Stem-like Cells By Therapeutic Stress-induced HIF Signaling in the Recurrent GBM Model.

Increasing evidence exposes a subpopulation of cancer cells, known as cancer stem cells (CSCs), to be critical for the progression of several human malignancies, including glioblastoma multiforme. CSCs are highly tumorigenic, capable of self-renewal, and resistant to conventional therapies, and thus considered to be one of the key contributors to disease recurrence. To elucidate the poorly understood evolutionary path of tumor recurrence and the role of CSCs in this process, we developed patient-derived xenograft glioblastoma recurrent models induced by anti-glioma chemotherapy, temozolomide. In this model, we observed a significant phenotypic shift towards an undifferentiated population. We confirmed these findings in vitro as sorted CD133-negative populations cultured in differentiation-forcing media were found to acquire CD133 expression following chemotherapy treatment. To investigate this phenotypic switch at the single-cell level, glioma stem cell (GSC)-specific promoter-based reporter systems were engineered to track changes in the GSC population in real time. We observed the active phenotypic and functional switch of single non-stem glioma cells to a stem-like state and that temozolomide therapy significantly increased the rate of single-cell conversions. Importantly, we showed the therapy-induced hypoxia-inducible factors (HIF) 1α and HIF2α play key roles in allowing non-stem glioma cells to acquire stem-like traits, as the expression of both HIFs increase upon temozolomide therapy and knockdown of HIFs expression inhibits the interconversion between non-stem glioma cells and GSCs post-therapy. On the basis of our results, we propose that anti-glioma chemotherapy promotes the accumulation of HIFs in the glioblastoma multiforme cells that induces the formation of therapy-resistant GSCs responsible for recurrence. Mol Cancer Ther; 15(12); 3064-76. ©2016 AACR.

Selective Estrogen Receptor Modulators Suppress Hif1α Protein Accumulation in Mouse Osteoclasts.

Anti-bone resorptive drugs such as bisphosphonates, the anti-RANKL antibody (denosumab), or selective estrogen receptor modulators (SERMs) have been developed to treat osteoporosis. Mechanisms underlying activity of bisphosphonates or denosumab in this context are understood, while it is less clear how SERMs like tamoxifen, raloxifene, or bazedoxifene inhibit bone resorption. Recently, accumulation of hypoxia inducible factor 1 alpha (Hif1α) in osteoclasts was shown to be suppressed by estrogen in normal cells. In addition, osteoclast activation and decreased bone mass seen in estrogen-deficient conditions was found to require Hif1α. Here, we used western blot analysis of cultured osteoclast precursor cells to show that tamoxifen, raloxifene, or bazedoxifene all suppress Hif1α protein accumulation. The effects of each SERM on osteoclast differentiation differed in vitro. Our results suggest that interventions such as the SERMs evaluated here could be useful to inhibit Hif1α and osteoclast activity under estrogen-deficient conditions.

Characterization of VHL missense mutations in sporadic clear cell renal cell carcinoma: hotspots, affected binding domains, functional impact on pVHL and therapeutic relevance.

BackgroundThe VHL protein (pVHL) is a multiadaptor protein that interacts with more than 30 different binding partners involved in many oncogenic processes. About 70 % of clear cell renal cell carcinoma (ccRCC) have VHL mutations with varying impact on pVHL function. Loss of pVHL function leads to the accumulation of Hypoxia Inducible Factor (HIF), which is targeted by current targeted treatments. In contrast to nonsense and frameshift mutations that highly likely nullify pVHL multipurpose functions, missense mutations may rather specifically influence the binding capability of pVHL to its partners. The affected pathways may offer predictive clues to therapy and response to treatment. In this study we focused on the VHL missense mutation pattern in ccRCC, and studied their potential effects on pVHL protein stability and binding partners and discussed treatment options.MethodsWe sequenced VHL in 360 sporadic ccRCC FFPE samples and compared observed and expected frequency of missense mutations in 32 different binding domains. The prediction of the impact of those mutations on protein stability and function was assessed in silico. The response to HIF-related, anti-angiogenic treatment of 30 patients with known VHL mutation status was also investigated.ResultsWe identified 254 VHL mutations (68.3 % of the cases) including 89 missense mutations (35 %). Codons Ser65, Asn78, Ser80, Trp117 and Leu184 represented hotspots and missense mutations in Trp117 and Leu 184 were predicted to highly destabilize pVHL. About 40 % of VHL missense mutations were predicted to cause severe protein malfunction. The pVHL binding domains for HIF1AN, BCL2L11, HIF1/2α, RPB1, PRKCZ, aPKC-λ/ι, EEF1A1, CCT-ζ-2, and Cullin2 were preferentially affected. These binding partners are mainly acting in transcriptional regulation, apoptosis and ubiquitin ligation. There was no correlation between VHL mutation status and response to treatment.ConclusionsVHL missense mutations may exert mild, moderate or strong impact on pVHL stability. Besides the HIF binding domain, other pVHL binding sites seem to be non-randomly altered by missense mutations. In contrast to LOF mutations that affect all the different pathways normally controlled by pVHL, missense mutations may be rather appropriate for designing tailor-made treatment strategies for ccRCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2688-0) contains supplementary material, which is available to authorized users.

Abstract B09: Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine

Abstract Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media (DFSM). The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping by single nucleotide polymorphism (SNP) arrays revealed that the majority of cultures in FBS (6 of 8), and all of the cultures in DFSM (8 of 8), contained normal, not cancer cells. To distinguish cancer vs. normal cells in subsequent experiments, we sequenced the von Hippel Lindau (VHL) gene, which is mutated in a large percentage of patients, in a cohort of samples for which cryopreserved viable single cell suspensions were available. Once patients with sequence-detectable mutations were identified, the cells were thawed and cultured as before. Established cultures were then sequenced for the patient-specific VHL mutations. Once again, the majority of cultures in FBS (1 of 7), and all of the cultures in DSFM (7 of 7) contained VHL-wild type cells. The loss of VHL leads to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Therefore, in an attempt to select for VHL-mutant cancer cells, we used fluorescence activated cell sorting (FACS) to isolate cells expressing Carbonic Anhydrase IX (CA9), a cell surface HIF target. Isolated CA9+ cells were cultured in FBS-containing media, and upon genotyping, we found that VHL-mutant ccRCC cell cultures were established with an efficiency of ~85%. Parallel cultures of bulk single cell suspensions in serum-free conditions again selected for growth of normal (VHL-wild type) cells. The normal cells were verified to be renal proximal tubule epithelial cells based on their expression of renal proximal tubule markers Aquaporin-1 and Alkaline Phosphatase. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. The ability to establish patient-derived cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients. Citation Format: Nazleen Lobo, Craig Gedye, Kevin R. Brown, Joshua Paterson, Natalie Stickle, Jason Moffat, Michael A.S. Jewett, Laurie E. Ailles. Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr B09.

Macrophage NOS2 in Tumor Leukocytes.

Leukocytes and especially macrophages are a major cellular constituent of the tumor mass. The tumor microenvironment not only determines their activity but in turn these cells also contribute to tumor initiation and progression. Recent Advances: Proinflammatory stimulated macrophages upregulate inducible nitric oxide synthase (NOS2) and produce high steady-state NO concentrations. NO provokes tumor cell death by initiating apoptosis and/or necrosis. Mechanisms may comprise p53 accumulation, immunestimulatory activities, and an increased efficacy of chemo- and/or radiotherapy. However, the potential cytotoxic activity of macrophages often is compromised in the tumor microenvironment and instead a protumor activity of macrophages dominates. Contributing factors are signals generated by viable and dying tumor cells, attraction and activation of myeloid-derived suppressor cells, and hypoxia. Limited oxygen availability not only attenuates NOS2 activity but also causes accumulation of hypoxia-inducible factors 1 and 2 (HIF-1/HIF-2). Activation of the HIF system is tightly linked to NO formation and affects the expression of macrophage phenotype markers that in turn add to tumor progression. To make use of the cytotoxic arsenal of activated macrophages directed against tumor cells, it will be critical to understand how, when, and where these innate immune responses are blocked and whether it will be possible to reinstall their full capacity to kill tumor cells. Low-dose irradiation or proinflammatory activation of macrophages in the tumor microenvironment may open options to boost NOS2 expression and activity and to initiate immunestimulatory features of NO that may help to restrict tumor growth. Antioxid. Redox Signal. 26, 1023-1043.

Hypoxia and hypoxia-inducible factors in chronic kidney disease

It is generally accepted that renal hypoxia plays an important role in the progression of chronic kidney disease (CKD). This review focuses on renal hypoxia and hypoxia-inducible factor (HIF), a master regulator of cellular adaptation to hypoxia, during CKD progression. The kidney, which is physiologically hypoxic, is exposed to increased levels of hypoxia in CKD; insufficient oxygenation in the tubulointerstitium triggers injury and accelerates the deterioration of renal function, culminating in end-stage kidney disease (ESKD). HIF accumulates during specific stages of pathological progression; however, adaptation to hypoxia usually fails. In such cases, decreased vascular endothelial growth factor expression and upregulated antiangiogenic factors result in sustained capillary rarefaction. In addition, oxygen consumption in tubules is primarily increased by enhanced oxidative stress, and the transcriptional activity of HIF becomes suboptimal, which is partly mediated by methylglyoxal in diabetic kidney disease and by indoxyl sulfate, a representative uremic toxin, in advanced CKD. Oxygen-dependent canonical regulators of HIF involve prolyl hydroxylase domain-containing protein (PHD) and factor inhibiting HIF-1 (FIH-1), whereas recent studies have revealed noncanonical and oxygen-independent HIF regulation in the kidney. As a consequence, HIF accumulation usually fails to protect the kidney against hypoxia, which is likely to accelerate progression to ESKD, via several maladaptation mechanisms. The precise roles of HIF and its regulation in CKD warrant further investigation in light of promising data demonstrating that HIF stabilization by PHD inhibitors may be a new therapeutic approach for CKD.

RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

Selective enhancement of hypoxic cell killing by tempol-regulated suicide gene expression

The presence of hypoxic regions within solid tumors is caused by an imbalance between cell proliferation and angiogenesis. Such regions may facilitate the onset of recurrence after radiation therapy and chemotherapy, as hypoxic cells show resistance to these treatments. We found that tempol, a nitroxide, strongly induces the accumulation of hypoxia-inducible factor (HIF)-1α, particularly under conditions of hypoxia. We, therefore, evaluated whether tempol enhances the gene expression via HIF-1α, potentially leading to various applications for cancer gene therapy targeting hypoxic cells. Consequently, following treatment with tempol under hypoxia, the luciferase (Luc) activity in the cells transfected with the plasmid containing the luc gene with the oxygen-dependent degradation domain and a promoter composed of hypoxia-responsive elements increased up to approximately 10-fold compared to that observed in cells treated identically with the exception of tempol. The plasmid constructed by replacing the luc gene with the fcy::fur fusion gene as a suicide gene, strongly induced the accumulation of the Fcy::Fur fusion protein, only when incubated in the presence of the hypoxic mimic CoCl2 and tempol. The transfected cells were successfully killed with the addition of 5-fluorocytosine to the cell culture according to the fcy::fur fusion gene expression. As similar but lesser enhancement of the Luc activity was also observed in solid tumor tissues in nude mice, this strategy may be applied for hypoxic cancer eradication.

Potential contributions of intimal and plaque hypoxia to atherosclerosis.

Injury of arterial endothelium by abnormal shear stress and other insults induces migration and proliferation of vascular smooth muscle cells (VSMCs), which in turn leads to intimal thickening, hypoxia, and vasa vasorum angiogenesis. The resultant new blood vessels extend from the tunica media into the outer intima, allowing blood-borne oxidized low-density lipoprotein (oxLDL) particles to accumulate in outer intimal tissues by extravasation through local capillaries. In response to oxLDL accumulation, monocytes infiltrate into arterial wall tissues, where they differentiate into macrophages and subsequently evolve into foam cells by uptaking large quantities of oxLDL particles, the latter process being stimulated by hypoxia. Increased oxygen demand due to expanding macrophage and foam cell populations contributes to persistent hypoxia in plaque lesions, whereas hypoxia further promotes plaque growth by stimulating angiogenesis, monocyte infiltration, and oxLDL uptake into macrophages. Molecularly, the accumulation of hypoxia-inducible factor (HIF)-1α and the expression of its target genes mediate many of the hypoxia-induced processes during plaque initiation and growth. It is hoped that further understanding of the underlying mechanisms may lead to novel therapies for effective intervention of atherosclerosis.

Hypoxia and the pharmaceutical diclofenac influence the circadian responses of three-spined stickleback

Pollution with low concentrations of pharmaceuticals, especially when combined with low-oxygen conditions (hypoxia), is a threat to aquatic ecosystems worldwide. The non-steroidal anti-inflammatory drug diclofenac is commonly detected in wastewater effluents, and has potential to accumulate in the bile of fish. Diclofenac has been shown to activate aryl hydrocarbon receptor (AHR), which induces transcription in the metabolic enzyme cytochrome P450 1a (cyp1a). Previously, crosstalk has been shown to occur between AHR and hypoxia inducible factor 1 (HIF-1). In addition, both of these transcription factors interact with the proteins regulating circadian (24-h) rhythms in vertebrates. Yet little is known about the significance of these interactions during simultaneous exposure to chemicals and hypoxia in fish in vivo. We exposed wild-caught three-spined sticklebacks (Gasterosteus aculeatus) to diclofenac (1μg/L, 14 days), hypoxia (2.0mg/L, up to 24h) and the combination of both. We then analyzed markers of chemical biotransformation (EROD activity, cyp1a and ahr mRNA levels), glycolysis (lactate dehydrogenase (LDH) enzyme activity, ldh and enolase 1a mRNA levels), and the transcription of core circadian clock genes clock and period 1 in liver tissue. Samples were taken at three time points during the light period in order to address disturbances in the circadian variation of metabolic processes. The results show that mRNA levels and LDH activity tended to be lowest before the dark period, but this pattern was disturbed by hypoxia and diclofenac. Diclofenac and hypoxia co-exposure induced EROD activity more strongly than diclofenac exposure alone, while cyp1a mRNA level was increased also by hypoxia and diclofenac alone. LDH activity and mRNA expression showed a clear time-dependent response during hypoxia, which is consistent with the previously suggested decreased accumulation of HIF-1 during the dark period. Furthermore, LDH activity and transcription was disturbed by diclofenac, indicating important effects of environmental pollutants in disturbing natural acclimation. This study demonstrates the need for more studies to understand the potential disturbances in endogenous rhythms caused by environmental pollution in natural populations.

The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway

It has been well documented that activation of the ErbB3–PI3K–Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K–Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.

Abstract 727: Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis

Abstract Humans are unable to synthesise ascorbate (vitamin C) due to a mutation in the gene for gulono-lactone oxidase (Gulo), the terminal enzyme in the synthesis pathway. Ascorbate is a vital micronutrient required for many biological functions, including its action as a vital cofactor for Cu- and Fe-containing enzymes. As enzyme cofactor for HIF-hydroxylases, it is important for the regulation of hypoxia-inducible factor-1 (HIF-1), which governs cancer growth and spread. A lack of ascorbate increases both the level and activity of HIF-1 in cell culture. This study aimed to restore ascorbate synthesis to human cells and to determine the effect of intracellular ascorbate biosynthesis on HIF-1 levels. HepG2 cells were gene modified using Lipofectamine LTX with a plasmid encoding the mouse Gulo cDNA and a control plasmid encoding the green fluorescent protein (GFP). Modified HepG2 cells were tested for genomic incorporation by PCR, for Gulo enzyme production by Western blotting, and ascorbate synthesis by high performance liquid chromatography with electrochemical detection (HPLC-ECD). Protein levels of HIF-1alpha in hypoxic cells were measured by Western blot analysis. Parental HepG2 cells accumulated a mean of 120 nmol ascorbate per 1 million cells when loaded for 24h with 0.5 mM ascorbate. Gene-modified HepG2Gulo cells demonstrated a change in morphology and an increase in adherence, compared to HepG2GFP and parental HepG2 cells. A PCR-positive stable clone was able to synthesise ascorbate but only when the Gulo substrate, L-gulono-1,4-lactone, was supplied. Intracellular ascorbate levels then reached 5% of saturation levels. Hypoxic HIF-1 accumulation was reduced by addition of gulonolactone to this clone. However, gulonolactone also reduced HIF-1 levels in parental HepG2 cells, suggesting that it may similarly act as cofactor for the HIF-hydroxlases. This study indicated that the ascorbate biosynthesis pathway in human cells is significantly modified and may contain numerous non-functional members besides gulono-lactone oxidase. Future biochemical studies will determine which other enzymes are non-functional. Citation Format: Teresa Flett, Elizabeth Campbell, Elisabeth Phillips, Margreet CM Vissers, Gabi U. Dachs. Does gene transfer of gulono-lactone oxidase into human hepatocellular carcinoma cells restore ascorbate biosynthesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 727. doi:10.1158/1538-7445.AM2014-727