Hypoxia-inducible factor-1α and poly [ADP ribose] polymerase 1 cooperatively regulate Notch3 expression under hypoxia via a noncanonical mechanism.

Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.

Epidermal sensing of oxygen is essential for systemic hypoxic response.

Skin plays an essential role, mediated in part by its remarkable vascular plasticity, in adaptation to environmental stimuli. Certain vertebrates, such as amphibians, respond to hypoxia in part through the skin; but it is unknown whether this tissue can influence mammalian systemic adaptation to low oxygen levels. We have found that epidermal deletion of the hypoxia-responsive transcription factor HIF-1alpha inhibits renal erythropoietin (EPO) synthesis in response to hypoxia. Conversely, mice with an epidermal deletion of the von Hippel-Lindau (VHL) factor, a negative regulator of HIF, have increased EPO synthesis and polycythemia. We show that nitric oxide release induced by the HIF pathway acts on cutaneous vascular flow to increase systemic erythropoietin expression. These results demonstrate that in mice the skin is a critical mediator of systemic responses to environmental oxygen.

S-2-hydroxyglutarate regulates CD8+ T-lymphocyte fate.

R-2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R-2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R- and S-2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8+ T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S-2-hydroxyglutarate predominates over R-2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8+ T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S-2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8+ T cells. Thus, S-2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic-epigenetic axis, to immune fate and function.

A genetic and developmental pathway from STAT3 to the OCT4-NANOG circuit is essential for maintenance of ICM lineages in vivo.

Although it is known that OCT4-NANOG are required for maintenance of pluripotent cells in vitro, the upstream signals that regulate this circuit during early development in vivo have not been identified. Here we demonstrate, for the first time, signal transducers and activators of transcription 3 (STAT3)-dependent regulation of the OCT4-NANOG circuitry necessary to maintain the pluripotent inner cell mass (ICM), the source of in vitro-derived embryonic stem cells (ESCs). We show that STAT3 is highly expressed in mouse oocytes and becomes phosphorylated and translocates to the nucleus in the four-cell and later stage embryos. Using leukemia inhibitory factor (Lif)-null embryos, we found that STAT3 phosphorylation is dependent on LIF in four-cell stage embryos. In blastocysts, interleukin 6 (IL-6) acts in an autocrine fashion to ensure STAT3 phosphorylation, mediated by janus kinase 1 (JAK1), a LIF- and IL-6-dependent kinase. Using genetically engineered mouse strains to eliminate Stat3 in oocytes and embryos, we firmly establish that STAT3 is essential for maintenance of ICM lineages but not for ICM and trophectoderm formation. Indeed, STAT3 directly binds to the Oct4 and Nanog distal enhancers, modulating their expression to maintain pluripotency of mouse embryonic and induced pluripotent stem cells. These results provide a novel genetic model of cell fate determination operating through STAT3 in the preimplantation embryo and pluripotent stem cells in vivo.

HIF-dependent and reversible nucleosome disassembly in hypoxia-inducible gene promoters.

Hypoxia causes dramatic changes in gene expression profiles, and the mechanism of hypoxia-inducible transcription has been analyzed for use as a model system of stress-inducible gene regulation. In this study, changes in chromatin organization in promoters of hypoxia-inducible genes were investigated during hypoxia-reoxygenation conditions. Most of the hypoxia-inducible gene promoters were hypersensitive to DNase I under both normal and hypoxic conditions, and our data indicate an immediate recruitment of transcription factors under hypoxic conditions. In some of the hypoxia-inducible promoters, nucleosome-free DNA regions (NFRs) were established in parallel with hypoxia-induced transcription. We also show that the hypoxia-inducible formation of NFRs requires that hypoxia-inducible transcription factors (HIFs) bind to the promoters together with the transcriptional coactivator CBP. Within 1 h after the hypoxia exposure was ended (reoxygenation), HIF complexes were dissociated from the promoter regions. Within 24 h of reoxygenation, the hypoxia-induced transcription returned to basal levels and the nucleosome structure was reassembled in the hypoxia-inducible NFRs. Nucleosome reassembly required the function of the transcriptional coregulator SIN3A. Thus, reversible changes in nucleosome organization mediated by transcription factors are notable features of stress-inducible gene regulation.

Regulation of hypoxia-inducible gene expression after HIF activation.

Hypoxia causes dramatic changes in the expression profiles of genes that encode glycolytic enzymes, vascular endothelial growth factors, erythropoietin, and other factors in a tissue-specific manner through activating hypoxia-inducible transcription factors (HIFs) such as HIF1α and HIF2α. It has been elucidated that the activity of HIFs is fundamentally regulated by their protein stability in an oxygen-dependent manner. However, little is known about how stabilized HIFs regulate transcription of their target genes in hypoxic cells. Additionally, the roles of HIF3α, the third member of the HIFs, are still enigma due to its various splicing variants and the complicated phenotypes of Hif3a-gene modified mouse lines. Here, we summarize how molecular systems fine-tune hypoxia-inducible transcription with the cooperation of HIFs and their negative regulators, including IPAS, one of the HIF3α splicing variants. Since epigenetic mechanisms contribute to stress-inducible and cell-type specific gene regulation, the HIF-dependent reorganization of nucleosome structures in hypoxia-inducible gene promoters is also discussed.

Hypoxia regulates microRNA expression in the human carotid body.

The carotid body (CB) is the key sensing organ for physiological oxygen levels in the body. Under conditions of low oxygen (hypoxia), the CB plays crucial roles in signaling to the cardiorespiratory center in the medulla oblongata for the restoration of oxygen homeostasis. How hypoxia regulates gene expression in the human CB remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the identity and impact of important post-transcriptional regulators such as non-coding RNAs, and in particular miRNAs are not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentially regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins.

A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.

Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPß1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPß1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.

Hypoxia Regulates MicroRNA Expression in the Human Carotid Body.

How hypoxia regulates gene expression in the human carotid body (CB) remains poorly understood. While limited information on transcriptional regulation in animal CBs is available, the impact of important post-transcriptional regulators, such as non-coding RNAs, and in particular miRNAs is not known. Here we show using ex vivo experiments that indeed a number of miRNAs are differentially regulated in surgically removed human CB slices when acute hypoxic conditions were applied. Analysis of the hypoxia-regulated miRNAs shows that they target biological pathways with upregulation of functions related to cell proliferation and immune response and downregulation of cell differentiation and cell death functions. Comparative analysis of the human CB miRNAome with the global miRNA expression patterns of a large number of different human tissues showed that the CB miRNAome had a unique profile which reflects its highly specialized functional status. Nevertheless, the human CB miRNAome is most closely related to the miRNA expression pattern of brain tissues indicating that they may have the most similar developmental origins.

Hypoxia-induced and calpain-dependent cleavage of filamin A regulates the hypoxic response.

The cellular response to hypoxia is regulated by hypoxia-inducible factor-1α and -2α (HIF-1α and -2α). We have discovered that filamin A (FLNA), a large cytoskeletal actin-binding protein, physically interacts with HIF-1α and promotes tumor growth and angiogenesis. Hypoxia induces a calpain-dependent cleavage of FLNA to generate a naturally occurring C-terminal fragment that accumulates in the cell nucleus. This fragment interacts with the N-terminal portion of HIF-1α spanning amino acid residues 1-390 but not with HIF-2α. In hypoxia this fragment facilitates the nuclear localization of HIF-1α, is recruited to HIF-1α target gene promoters, and enhances HIF-1α function, resulting in up-regulation of HIF-1α target gene expression in a hypoxia-dependent fashion. These results unravel an important mechanism that selectively regulates the nuclear accumulation and function of HIF-1α and potentiates angiogenesis and tumor progression.

Increased Serine-Arginine (SR) Protein Phosphorylation Changes Pre-mRNA Splicing in Hypoxia.

The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Inhibitory PAS domain protein (IPAS), a dominant negative regulator of hypoxia-inducible gene expression, is generated from hypoxia inducible transcription factor-3α (HIF-3α) pre-mRNA by an alternative splicing mechanism. Inactivation of the IPAS transcript in mice leads to the neo-vascularization of the cornea, suggesting that IPAS is an important regulator of anti-angiogenesis in this tissue. For the first time we demonstrate that serine-arginine (SR) proteins are involved in oxygen tension-dependent changes in pre-mRNA splicing. SR proteins isolated from hypoxic cells differentially interact with RNA (compared with proteins isolated from cells cultured under normoxic conditions). They possess the differential ability to activate hypoxia-dependent splice sites, and they are more phosphorylated than those isolated from normoxic HeLa cells. We also show that expression of SR protein kinases (CLK1, SRPK1, SRPK2) in hypoxic cells is elevated at mRNA and protein levels. Increased expression of CLK1 kinase is regulated by HIFs. Reduction of CLK1 cellular expression levels reduces hypoxia-dependent full-length carbonic anhydrase IX (CAIX) mRNA and CAIX protein formation and changes hypoxia-dependent cysteine-rich angiogenic inducer 61 (Cyr61) mRNA isoform formation profiles.

Hypoxia-inducible factor-3α promotes angiogenic activity of pulmonary endothelial cells by repressing the expression of the VE-cadherin gene.

The variants of the hypoxia-inducible factor-3α gene HIF-3α and NEPAS are known to repress the transcriptional activities driven by HIF-1α and HIF-2α. Although NEPAS has been shown to play an important role in vascular remodeling during lung development, little is known about the roles of HIF-3α in adult lung function. Here, we examined pulmonary endothelial cells (ECs) isolated from wild-type (WT) and HIF-3α functional knockout (KO) mice. The expression levels of angiogenic factors (Flk1, Ang2 and Tie2) were significantly greater in the HIF-3α KO ECs than those in the WT ECs irrespective of oxygen tension. However, the HIF-3α KO ECs showed impaired proliferative and angiogenic activities. The impaired EC function was likely due to the excess vascular endothelial (VE)-cadherin, an inhibitor of Flk1/PI3 kinase/Akt signaling, as treatment of the cells to a neutralizing antibody partly restored the phenotype of the HIF-3α KO ECs. Importantly, we found that the mRNA levels of HIF-2α and Ets-1 were significantly increased by HIF-3α ablation. Given that both factors are known to activate the VE-cadherin gene, the transcriptional repression of these factors by HIF-3α might be important for silencing the irrelevant expression of the VE-cadherin gene. Collectively, these data show novel and unique roles of HIF-3α for angiogenic gene regulation in pulmonary ECs.

Physiologic hypoxia promotes maintenance of CML stem cells despite effective BCR-ABL1 inhibition.

C-abl oncogene 1, nonreceptor tyrosine kinase (ABL1) kinase inhibitors such as imatinib mesylate (imatinib) are effective in managing chronic myeloid leukemia (CML) but incapable of eliminating leukemia stem cells (LSCs), suggesting that kinase-independent pathways support LSC survival. Given that the bone marrow (BM) hypoxic microenvironment supports hematopoietic stem cells, we investigated whether hypoxia similarly contributes to LSC persistence. Importantly, we found that although breakpoint cluster region (BCR)-ABL1 kinase remained effectively inhibited by imatinib under hypoxia, apoptosis became partially suppressed. Furthermore, hypoxia enhanced the clonogenicity of CML cells, as well as their efficiency in repopulating immunodeficient mice, both in the presence and absence of imatinib. Hypoxia-inducible factor 1 α (HIF1-α), which is the master regulator of the hypoxia transcriptional response, is expressed in the BM specimens of CML individuals. In vitro, HIF1-α is stabilized during hypoxia, and its expression and transcriptional activity can be partially attenuated by concurrent imatinib treatment. Expression analysis demonstrates at the whole-transcriptome level that hypoxia and imatinib regulate distinct subsets of genes. Functionally, knockdown of HIF1-α abolished the enhanced clonogenicity during hypoxia. Taken together, our results suggest that in the hypoxic microenvironment, HIF1-α signaling supports LSC persistence independent of BCR-ABL1 kinase activity. Thus, targeting HIF1-α and its pathway components may be therapeutically important for the complete eradication of LSCs.

Consensus toxicity factors for polychlorinated dibenzo-p-dioxins, dibenzofurans, and biphenyls combining in silico models and extensive in vitro screening of AhR-mediated effects in human and rodent cells.

Consensus toxicity factors (CTFs) were developed as a novel approach to establish toxicity factors for risk assessment of dioxin-like compounds (DLCs). Eighteen polychlorinated dibenzo-p-dioxins, dibenzofurans (PCDD/Fs), and biphenyls (PCBs) with assigned World Health Organization toxic equivalency factors (WHO-TEFs) and two additional PCBs were screened in 17 human and rodent bioassays to assess their induction of aryl hydrocarbon receptor-related responses. For each bioassay and compound, relative effect potency values (REPs) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin were calculated and analyzed. The responses in the human and rodent cell bioassays generally differed. Most notably, the human cell models responded only weakly to PCBs, with 3,3',4,4',5-pentachlorobiphenyl (PCB126) being the only PCB that frequently evoked sufficiently strong responses in human cells to permit us to calculate REP values. Calculated REPs for PCB126 were more than 30 times lower than the WHO-TEF value for PCB126. CTFs were calculated using score and loading vectors from a principal component analysis to establish the ranking of the compounds and, by rescaling, also to provide numerical differences between the different congeners corresponding to the TEF scheme. The CTFs were based on rat and human bioassay data and indicated a significant deviation for PCBs but also for certain PCDD/Fs from the WHO-TEF values. The human CTFs for 2,3,4,7,8-pentachlorodibenzofuran, 1,2,3,4,7,8-hexachlorodibenzofuran, 1,2,3,4,6,7,8-heptachlorodibenzo-p-dioxin, and 1,2,3,4,7,8,9-heptachlorodibenzofuran were up to 10 times greater than their WHO-TEF values. Quantitative structure-activity relationship models were used to predict CTFs for untested WHO-TEF compounds, suggesting that the WHO-TEF value for 1,2,3,7,8-pentachlorodibenzofuran could be underestimated by an order of magnitude for both human and rodent models. Our results indicate that the CTF approach provides a powerful tool for condensing data from batteries of screening tests using compounds with similar mechanisms of action, which can be used to improve risk assessment of DLCs.

Generating specificity and diversity in the transcriptional response to hypoxia.

The sensing of oxygen levels and maintenance of oxygen homeostasis is crucial for cells. The hypoxic-sensitive regulation of gene expression allows information about the oxygen status to be converted into appropriate cellular responses. Although there is a core transcriptional pathway, the signalling cascade can be modified to allow diversity and specificity in the transcriptional output. In this Review, we discuss recent advances in our understanding of the mechanisms and factors that contribute to the observed diversity and specificity. A deeper knowledge about how hypoxic signalling is tuned will further our understanding of the cellular hypoxic response in normal physiology and how it becomes derailed in disease.

Regulation of CDKN2B expression by interaction of Arnt with Miz-1--a basis for functional integration between the HIF and Myc gene regulatory pathways.

BACKGROUND: Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear. METHODS: The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student's t-test and ANOVA. RESULTS: In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells. CONCLUSIONS: Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.

HIF-1α and HIF-2α induce angiogenesis and improve muscle energy recovery.

BACKGROUND: Cardiovascular patients suffer from reduced blood flow leading to ischaemia and impaired tissue metabolism. Unfortunately, an increasing group of elderly patients cannot be treated with current revascularization methods. Thus, new treatment strategies are urgently needed. Hypoxia-inducible factors (HIFs) upregulate the expression of angiogenic mediators together with genes involved in energy metabolism and recovery of ischaemic tissues. Especially, HIF-2α is a novel factor, and only limited information is available about its therapeutic potential. METHODS: Gene transfers with adenoviral HIF-1α and HIF-2α were performed into the mouse heart and rabbit ischaemic hindlimbs. Angiogenesis was evaluated by histology. Left ventricle function was analysed with echocardiography. Perfusion in rabbit skeletal muscles and energy recovery after electrical stimulation-induced exercise were measured with ultrasound and (31)P-magnetic resonance spectroscopy ((31)P-MRS), respectively. RESULTS: HIF-1α and HIF-2α gene transfers increased capillary size up to fivefold in myocardium and ischaemic skeletal muscles. Perfusion in skeletal muscles was increased by fourfold without oedema. Especially, AdHIF-1α enhanced the recovery of ischaemic muscles from electrical stimulation-induced energy depletion. Special characteristic of HIF-2α gene transfer was a strong capillary growth in muscle connective tissue and that HIF-2α gene transfer maintained left ventricle function. CONCLUSIONS: We conclude that both AdHIF-1α and AdHIF-2α gene transfers induced beneficial angiogenesis in vivo. Transient moderate increases in angiogenesis improved energy recovery after exercise in ischaemic muscles. This study shows for the first time that a moderate increase in angiogenesis is enough to improve tissue energy metabolism, which is potentially a very useful feature for cardiovascular gene therapy.

In vitro and in silico derived relative effect potencies of ah-receptor-mediated effects by PCDD/Fs and PCBs in rat, mouse, and guinea pig CALUX cell lines.

For a better understanding of species-specific relative effect potencies (REPs), responses of dioxin-like compounds (DLCs) were assessed. REPs were calculated using chemical-activated luciferase gene expression assays (CALUX) derived from guinea pig, rat, and mouse cell lines. Almost all 20 congeners tested in the rodent cell lines were partial agonists and less efficacious than 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). For this reason, REPs were calculated for each congener using concentrations at which 20% of the maximal TCDD response was reached (REP20TCDD). REP20TCDD values obtained for PCDD/Fs were comparable with their toxic equivalency factors assigned by the World Health Organization (WHO-TEF), while those for PCBs were in general lower than the WHO-TEF values. Moreover, the guinea pig cell line was the most sensitive as indicated by the 20% effect concentrations of TCDD of 1.5, 5.6, and 11.0 pM for guinea pig, rat, and mouse cells, respectively. A similar response pattern was observed using multivariate statistical analysis between the three CALUX assays and the WHO-TEFs. The mouse assay showed minor deviation due to higher relative induction potential for 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,6,7,8-hexachlorodibenzofuran and lower for 1,2,3,4,6,7,8-heptachlorodibenzofuran and 3,3',4,4',5-pentachlorobiphenyl (PCB126). 2,3,7,8-Tetrachlorodibenzofuran was more than two times more potent in the mouse assay as compared with that of rat and guinea pig cells, while measured REP20TCDD for PCB126 was lower in mouse cells (0.05) as compared with that of the guinea pig (0.2) and rat (0.07). In order to provide REP20TCDD values for all WHO-TEF assigned compounds, quantitative structure-activity relationship (QSAR) models were developed. The QSAR models showed that specific electronic properties and molecular surface characteristics play important roles in the AhR-mediated response. In silico derived REP20TCDD values were generally consistent with the WHO-TEFs with a few exceptions. The QSAR models indicated that, e.g., 1,2,3,7,8-pentachlorodibenzofuran and 1,2,3,7,8,9-hexachlorodibenzofuran were more potent than given by their assigned WHO-TEF values, and the non-ortho PCB 81 was predicted, based on the guinea-pig model, to be 1 order of magnitude above its WHO-TEF value. By combining in vitro and in silico approaches, REPs were established for all WHO-TEF assigned compounds (except OCDD), which will provide future guidance in testing AhR-mediated responses of DLCs and to increase our understanding of species variation in AhR-mediated effects.

The human carotid body releases acetylcholine, ATP and cytokines during hypoxia.

Studies on experimental animals established that the carotid bodies are sensory organs for detecting arterial blood O2 levels and that the ensuing chemosensory reflex is a major regulator of cardiorespiratory functions during hypoxia. However, little information is available on the human carotid body responses to hypoxia. The present study was performed on human carotid bodies obtained from surgical patients undergoing elective head and neck cancer surgery. Our results show that exposing carotid body slices to hypoxia for a period as brief as 5 min markedly facilitates the release of ACh and ATP. Furthermore, prolonged hypoxia for 1 h induces an increased release of interleukin (IL)-1ß, IL-4, IL-6, IL-8 and IL-10. Immunohistochemical analysis revealed that type 1 cells of the human carotid body express an array of cytokine receptors as well as hypoxia-inducible factor-1α and hypoxia-inducible factor-2α. Taken together, these results demonstrate that ACh and ATP are released from the human carotid body in response to hypoxia, suggesting that these neurotransmitters, as in several experimental animal models, play a role in hypoxic signalling also in the human carotid body. The finding that the human carotid body releases cytokines in response to hypoxia adds to the growing body of information suggesting that the carotid body may play a role in detecting inflammation, providing a link between the immune system and the nervous system.

Recruitment of HIF-1alpha and HIF-2alpha to common target genes is differentially regulated in neuroblastoma: HIF-2alpha promotes an aggressive phenotype.

In neuroblastoma specimens, HIF-2alpha but not HIF-1alpha is strongly expressed in well-vascularized areas. In vitro, HIF-2alpha protein was stabilized at 5% O2 (resembling end capillary oxygen conditions) and, in contrast to the low HIF-1alpha activity at this oxygen level, actively transcribed genes like VEGF. Under hypoxia (1% O2), HIF-1alpha was transiently stabilized and primarily mediated acute responses, whereas HIF-2alpha protein gradually accumulated and governed prolonged hypoxic gene activation. Knockdown of HIF-2alpha reduced growth of neuroblastoma tumors in athymic mice. Furthermore, high HIF-2alpha protein levels were correlated with advanced clinical stage and high VEGF expression and predicted poor prognosis in a clinical neuroblastoma material. Our results demonstrate the relevance of HIF-2alpha in neuroblastoma progression and have general tumor biological implications.