Type I cell ROS kinetics under hypoxia in the intact mouse carotid body ex vivo: a FRET-based study.

Reactive oxygen species (ROS) mainly originating from NADPH oxidases have been shown to be involved in the carotid body (CB) oxygen-sensing cascade. For measuring ROS kinetics, type I cells of the mouse CB in an ex vivo preparation were transfected with the ROS sensor construct FRET-HSP33. After 2 days of tissue culture, type I cells expressed FRET-HSP33 as shown by immunohistochemistry. In one population of CBs, 5 min of hypoxia induced a significant and reversible decrease of type I cell ROS levels (n = 9 CBs; P < 0.015), which could be inhibited by 4-(2-aminoethyl)benzensulfonylfluorid (AEBSF), a highly specific inhibitor of the NADPH oxidase subunits p47(phox) and p67(phox). In another population of CBs, however, 5 min of hypoxia induced a significant and reversible increase of ROS levels in type I cells (n = 8 CBs; P < 0.05), which was slightly enhanced by administration of 3 mM AEBSF. These different ROS kinetics seemed to coincide with different mice breeding conditions. Type I cells of both populations showed a typical hypoxia-induced membrane potential (MP) depolarization, which could be inhibited by 3 mM AEBSF. ROS and MP closely followed the hypoxic decrease in CB tissue oxygen as measured with an O2-sensitive dye. We conclude that attenuated p47(phox) subunit activity of the NADPH oxidase under hypoxia is the physiological trigger for type I cell MP depolarization probably due to ROS decrease, whereas the observed ROS increase has no influence on type I cell MP kinetics under hypoxia.

PHD3 regulates differentiation, tumour growth and angiogenesis in pancreatic cancer.

PURPOSE: Tumour hypoxia activates hypoxia-inducible factor-1 (HIF-1) and indluences angiogenesis, cell survival and invasion. Prolyl hydroxylase-3 (PHD3) regulates degradation of HIF-1α. The effects of PHD3 in tumour growth are largely unknown. EXPERIMENTAL DESIGN: PHD3 expression was analysed in human pancreatic cancer tissues and cancer cell lines by real-time quantitative PCR and immunohistochemistry. PHD3 overexpression was established by stable transfection and downregulation by short interfering RNA technology. VEGF was quantified by enzyme-linked immunosorbent assay. Matrigel invasion assays were performed to examine tumour cell invasion. Apoptosis was measured by annexin-V staining and caspase-3 assays. The effect of PHD3 on tumour growth in vivo was evaluated in an established orthotopic murine model. RESULTS: PHD3 was upregulated in well-differentiated human tumours and cell lines, and regulated hypoxic VEGF secretion. PHD3 overexpression mediated tumour cell growth and invasion by induction of apoptosis in a nerve growth factor-dependent manner by the activation of caspase-3 and phosphorylation of focal adhesion kinase HIF-1 independently. In vivo, PHD3 inhibited tumour growth by abrogation of tumour angiogenesis. CONCLUSION: Our results indicate essential functions of PHD3 in tumour growth, apoptosis and angiogenesis and through HIF-1-dependent and HIF-1-independent pathways.

Hydroxylation of hypoxia-inducible transcription factors and chemical compounds targeting the HIF-alpha hydroxylases.

The hypoxia-inducible transcription factors (HIFs) are central components in the cellular responses to a lack of O(2), i.e. hypoxia. Homologs of the HIF system (HIF-1, -2 and -3) are detectable in all nucleated cells of multicellular organisms. Active HIFs are heterodimers (HIF-alpha/ beta). In hypoxia the O(2)-labile alpha-subunit is translocated to the nucleus where it binds HIF-beta. Over 100 HIF target genes have already been identified. The translational products of these genes increase O(2) delivery to hypoxic tissues, such as erythropoietin which stimulates the production of red blood cells, and they adapt cellular metabolism to hypoxia, such as glycolytic enzymes. HIFs are inactive in normoxia because of O(2)-dependent enzymatic hydroxylation and subsequent degradation of their alpha-subunit. Three HIF-alpha prolyl hydroxylases (PHD1, 2 and 3) initiate proteasomal degradation while an asparaginyl hydroxylase (factor inhibiting HIF-1, FIH-1) inhibits the function of the C-terminal transactivation domain of HIF-alpha. In addition to O(2) and 2-oxoglutarate, the HIF-alpha hydroxylases require Fe(2+) and ascorbate as co-factors. Products of glycolysis can act as endogenous inhibitors of HIF hydroxylases which may lead to sustained activation of HIFs in cancer cells. The cofactor requirements define the routes to inhibition of the enzymes when HIF activation is desirable. In particular, 2-oxoglutarate analogues have emerged as promising tools for stimulation of erythropoiesis and angiogenesis ("HIF-stabilizers"). However, as the HIF system promotes the transcription of many genes, and other 2-oxoglutarate dependent dioxygenases are likely to be inhibited by the same analogues, careful evaluation of the inhibitors seems mandatory prior to their clinical use.

Interleukin 4 and prolonged hypoxia induce a higher gene expression of lysyl hydroxylase 2 and an altered cross-link pattern: important pathogenetic steps in early and late stage of systemic scleroderma?

The major pathological processes of systemic scleroderma (SSc) comprise inflammation and microvascular damage in the early or acute progressive stage as well as tissue fibrosis and hypoxia in the chronic end stage. Fibrosis seems to be a general phenomenon characterized by an increase of hydroxylysine aldehyde derived collagen cross-links which has been shown in vitro for systemic scleroderma fibroblasts. In the present study, we analyzed the cross-link pattern and the gene expression of lysyl hydroxylase 2 (LH2) in the skin of SSc. Furthermore, we determined the modulatory impact of inflammatory cytokines (interleukin 4, TNF- alphaand interleukin 1alpha/beta) and prolonged hypoxia on the cross-link profile and the gene expression of LH2, respectively. The concentration of hydroxylysine aldehyde derived cross-links was significantly increased in SSc, while the level of lysine aldehyde derived cross-links was not changed. Accordingly, a marked increase of the transcriptional level of LH2 was found. In long term dermal fibroblast cultures, only interleukin 4 induced an increase of hydroxylysine aldehyde derived cross-links accompanied by a higher gene expression of LH2. Furthermore, prolonged hypoxia induced a marked increase of the mRNA level of LH2 in relation to collagen I. The skin of SSc is characterized by an increase of the transcriptional activity of LH2 leading to an altered cross-link pattern. The changes in the quality of the collagenous matrix can also be obtained in cell culture by the exposure of fibroblasts to interleukin 4 or prolonged hypoxia emphasizing the role of this mediator in the acute and the low oxygen tension in the chronic phase of the disease.

Erythropoietin in the control of red cell production.

The understanding of the endocrine regulation of red cell production has been extended greatly since the erythropoietin gene was cloned and recombinant human erythropoietin has become available for experimental and clinical applications. Human erythropoietin is a 30 kDa glycoprotein. It is composed of 165 amino acids and 4 carbohydrate side chains. Studies in rodents have shown that blood-borne erythropoietin originates from peritubular cells, possibly fibroblasts, in the renal cortex and from parenchymal cells in the liver. In addition, erythropoietin mRNA has been demonstrated in spleen, lung and brain. Tissue hypoxia is the main stimulus for erythropoietin synthesis. Erythropoietin gene expression is controlled by DNA-binding proteins, primarily by hypoxia-inducible factor 1. Erythropoietin maintains red cell production by inhibiting apoptosis of erythrocytic progenitors, and by stimulating their proliferation and differentiation into normoblasts. The functional human erythropoietin receptor, a 484-amino acid glycoprotein, is member of the class I cytokine receptor superfamily. Lack of erythropoietin results in anaemia. Recombinant human erythropoietin is efficient for treatment of the anaemia of chronic renal failure. In addition, the drug is increasingly administrated to persons suffering from anaemia of chronic diseases and to surgical patients, thus abolishing the need for homologous red cell transfusion.

Vascular endothelial growth factor gene expression in the human breast cancer cell line MX-1 is controlled by O2 availability in vitro and in vivo.

The vascular endothelial growth factor (VEGF) plays an important role in angiogenesis. Mediated by the hypoxia-inducible transcription factor HIF-1alpha/beta, a reduction in O2 tension (pO2) leads to increased VEGF gene expression in nonmalignant tissues. In tumor cells VEGF mRNA levels are often constitutively elevated. We examined pO2-dependent VEGF mRNA expression and VEGF protein formation in the human breast cancer cell line MX-1 in vitro and in vivo. For in vitro study MX-1 cultures were grown on dishes with a gas-permeable bottom to expose the cells to defined O2 concentrations (from 95% to 0%) for 4 h. Northern blot analysis showed significant VEGF mRNA in MX-1 cultures under normoxic conditions which was further increased by hypoxia. The amount of secreted VEGF was also elevated in hypoxic cultures. Western blot analysis revealed a correlation between the severity of hypoxia and HIF-1alpha protein amounts in the nucleus. Furthermore, DNA-binding activity of HIF-1 could be demonstrated by gel-shift assays. For in vivo study immunodeficient nude mice bearing MX-1 tumor transplants were exposed to inspiratory hypoxia (10% O2). Northern blot and immunohistochemical analyses of MX-1 tumor transplants showed that VEGF mRNA and VEGF protein levels were increased in mice 17 h after the induction of inspiratory hypoxia. Thus, pO2-dependence of VEGF gene expression can be maintained in cancer cells, even in vivo, which may be relevant in regard to therapeutic attempts to inhibit tumor angiogenesis by increasing tumor oxygenation.

Evidence against a major role for Ca2+ in hypoxia-induced gene expression in human hepatoma cells (Hep3B).

1. The human hepatoma cell line Hep3B is a widely used model for studies of hypoxia-related gene expression. Cytosolic free calcium concentration ([Ca2+]i) has been implicated in cellular oxygen-sensing processes. We investigated whether calcium ions have a significant impact on the production of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). 2. We found that the calcium ionophore ionomycin induced a rapid and sustained increase of [Ca2+]i while thapsigargin, an inhibitor of endoplasmic reticulum calcium ATPase, only caused a 20 % elevation of [Ca2+]i within 10 min after application. However, the calcium content of intracellular stores was considerably reduced by thapsigargin after an incubation period of 24 h. 3. Variations in [Ca2+]o did not result in altered EPO or VEGF secretion rates. Ionomycin decreased EPO production while the lowering of VEGF production was not statistically significant. In the presence of extracellular Ca2+ the membrane permeant calcium chelator BAPTA-AM stimulated the production of EPO (P < 0.05) but not of VEGF while EGTA-AM, a closely related agent, affected neither EPO nor VEGF formation under these conditions. Incubation with thapsigargin resulted in decreased EPO synthesis (P < 0.05) but stimulated VEGF secretion (P < 0.05). 4. In the absence of extracellular calcium, EGTA-AM led to an accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5. Our data suggest that the calcium transient and the cytosolic Ca2+ concentration do not play a key role in hypoxia-induced EPO and VEGF production in Hep3B cells.

Pericellular PO2 and O2 consumption in monolayer cell cultures.

The current study was based on the uncertainty as to how well monolayer cell cultures growing in customary polystyrene dishes are supplied with O2. For dishes maintained in an air-5% CO2 atmosphere at 37 degrees C, microelectrode measurements revealed that the pericellular steady-state PO2 was 78 mm Hg in confluent bovine endothelial, 110 mm Hg in rat renal mesangial, and 0 (< 0.2) mm Hg in renal (LLC-PK1 and LLC-MK2) or hepatic (HepG2, Hep3B) epithelial cell cultures. These measured PO2 values were in good agreement with those calculated from Fick's law of gas diffusion, applied for the present culture conditions (one-dimensional O2 diffusion, 0.52 cm medium height), the individual cell layer density and the tissue-specific rate of O2 utilization. Our results provide reasons to speculate that conventional monolayer cultures are often hypoxic when incubated in an air-5% CO2 atmosphere. Diffusion-limitations of cellular O2 availability are to be taken into consideration when tissue cultures are used to study PO2-dependent processes.

Cytosolic Ca(2+) levels and DNA synthesis of human umbilical vein endothelial cell cultures are unresponsive to thrombopoietin treatment.

The hormone thrombopoietin (TPO) induces proliferation of megakaryocytic progenitors and augments agonist-induced mobilization of Ca(2+) in platelets. Because the action of TPO is not restricted to the megakaryocytic lineage, we studied the occurrence of TPO receptor mRNA and protein, and effects of TPO on cytosolic Ca(2+) levels and DNA synthesis in human umbilical vein endothelial cell cultures (HUVECs). Polymerase chain reaction following reverse transcription (RT-PCR) of total mRNA revealed that TPO receptor (MPL) mRNA was expressed only at low level in our samples. TPO receptor protein was not detectable in HUVEC lysates investigated by immunoprecipitation and immunoblotting. In contrast to vascular endothelial growth factor (VEGF), TPO did neither alter fura2 fluorescence as a measure of cytosolic Ca(2+) levels nor increase 5-bromo-2'-deoxyuridine incorporation into DNA of HUVECs. In conclusion, our data demonstrate that HUVECs are neither structurally nor functionally responsive to TPO.

Regulation of hypoxia-inducible factor is preserved in the absence of a functioning mitochondrial respiratory chain.

Hypoxia-inducible factor (HIF) mediates a large number of transcriptional responses to hypoxia and has an important role in processes that include angiogenesis and erythropoiesis. The HIF DNA binding complex consists of 2 basic-helix-loop-helix PAS proteins designated alpha and beta subunits. Regulation occurs principally through the alpha subunits, which are stabilized and activated in hypoxia. Although substantial evidence implicates reactive oxygen species (ROS) in the regulatory process, the precise mechanisms remain unclear. Mitochondria are an important source of ROS, and in one model it has been proposed that hypoxia increases the generation of ROS at complex III in the mitochondrion and that this signal acts through a transduction pathway to stabilize HIF-1alpha and to activate HIF. To test this model the induction of the HIF-1alpha subunit and the HIF target gene, glucose-transporter-1, was examined in a variety of mutant cells that lacked mitochondrial DNA (rho0) or had other genetic defects in mitochondrial respiration. HIF induction by hypoxia was essentially normal in all cells tested. Hydrogen peroxide production was measured by the luminol/peroxidase method and found to be reduced in rho0 versus wild-type cells and reduced by hypoxia in both rho0 and wild-type cells. Furthermore, concentrations of rotenone that maximally inhibited respiration did not affect HIF activation by hypoxia. These data do not support the model outlined above and indicate that a functional respiratory chain is not necessary for the regulation of HIF by oxygen.

Interleukin-1beta and tumor necrosis factor-alpha stimulate DNA binding of hypoxia-inducible factor-1.

The rate of transcription of several genes encoding proteins involved in O(2) and energy homeostasis is controlled by hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of alpha and beta subunits. HIF-1 is considered the primary trans-acting factor for the erythropoietin (EPO) and vascular endothelial growth factor (VEGF) genes. Since EPO gene expression is inhibited by the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), while no such effect has been reported with respect to the VEGF gene, we investigated the effects of IL-1beta and TNF-alpha on the activation of the HIF-1 DNA-binding complex and the amount of HIF-1alpha protein in human hepatoma cells in culture. Under normoxic conditions, both cytokines caused a moderate activation of HIF-1 DNA binding. In hypoxia, cytokines strongly increased HIF-1 activity compared with the effect of hypoxia alone. Only IL-1beta increased HIF-1alpha protein levels. In transient transfection experiments, HIF-1-driven reporter gene expression was augmented by cytokines only under hypoxic conditions. In contrast to their effect on EPO synthesis, neither IL-1beta nor TNF-alpha decreased VEGF production. The mRNA levels of HIF-1alpha and VEGF were unaffected. Thus, cytokine-induced inhibition of EPO production is not mediated by impairment of HIF-1 function. We propose that HIF-1 may be involved in modulating gene expression during inflammation.

Hypoxia and interleukin-1beta stimulate vascular endothelial growth factor production in human proximal tubular cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) promotes angiogenesis and inflammatory reactions. VEGF mRNA is detectable in the proximal tubules of inflamed kidneys but not in normals. In other organs VEGF gene expression is induced by hypoxia and cytokines such as interleukin 1 (IL-1). To identify the cellular mechanisms in control of tubular VEGF production, we studied effects of hypoxia and IL-1beta in VEGF mRNA levels, VEGF secretion, and activity of the hypoxia-inducible dimeric transcription factor 1 (HIF-1alpha/beta) in human proximal tubular epithelial cells (PTECs) in primary culture. METHODS: PTECs were grown in monolayers from human kidneys. Hypoxia was induced by incubation at 3% O2. VEGF mRNA was quantitated by competitive polymerase chain reaction following reverse transcription. VEGF was measured by enzyme-linked immunoassay. HIF-1alpha was demonstrated by Western blot analysis and HIF-1 DNA binding by gel shift assay. RESULTS: Significant amounts of VEGF mRNA and VEGF protein were measured in PTEC extracts and culture media, respectively. Stimulation of VEGF synthesis at low O2 tension and following IL-1beta treatment was detectable at the protein level only. Nuclear HIF-1alpha protein levels and HIF-1 binding to DNA were also increased under these conditions. CONCLUSIONS: PTECs in culture produce VEGF. One mechanism of induction appears to be increased DNA binding of HIF-1 to hypoxia-responsive elements in the VEGF gene promoter. In inflammatory diseases of the kidney, tubular cell-derived VEGF may contribute to microvascular leakage and monocyte extravasation.

C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation.

HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. Recent studies have defined posttranslational modification by prolyl hydroxylation as a key regulatory event that targets HIF-alpha subunits for proteasomal destruction via the von Hippel-Lindau ubiquitylation complex. Here, we define a conserved HIF-VHL-prolyl hydroxylase pathway in C. elegans, and use a genetic approach to identify EGL-9 as a dioxygenase that regulates HIF by prolyl hydroxylation. In mammalian cells, we show that the HIF-prolyl hydroxylases are represented by a series of isoforms bearing a conserved 2-histidine-1-carboxylate iron coordination motif at the catalytic site. Direct modulation of recombinant enzyme activity by graded hypoxia, iron chelation, and cobaltous ions mirrors the characteristics of HIF induction in vivo, fulfilling requirements for these enzymes being oxygen sensors that regulate HIF.