Biomedical subjects

Eric Metzen

Publications and source records attributed to Eric Metzen.

12 recordsLinked to original sources

PubMed2006

Optical analysis of the HIF-1 complex in living cells by FRET and FRAP.

Hypoxia-inducible factor-1 (HIF-1) coordinates the cellular response to a lack of oxygen by controlling the expression of hypoxia-inducible genes that ensure an adequate energy supply. Assembly of the HIF-1 complex by its oxygen-regulated subunit HIF-1alpha and its constitutive beta subunit also known as ARNT is the key event of the cellular genetic response to hypoxia. By two-photon microscopy, we studied HIF-1 assembly in living cells and the mobility of fluorophore-labeled HIF-1 subunits by fluorescence recovery after photobleaching. We found a significantly slower nuclear migration of HIF-1alpha than of HIF-1beta, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between alpha and beta subunits of the transcriptionally active HIF-1 complex bound to DNA. Both N termini of the fluorophore-labeled HIF-1 subunits were localized as close as 6.2 nm, but even the N and C terminus of the HIF-1 complex were not further apart than 7.4 nm. Our data suggest a more compact 3-dimensional organization of the HIF complex than described so far by 2-dimensional models.

Fluorescence Recovery After Photobleaching↗

PubMed2006

Gonadotropin-releasing hormone antagonists do not influence the secretion of steroid hormones but affect the secretion of vascular endothelial growth factor from human granulosa luteinized cell cultures.

OBJECTIVE: To evaluate the secretion of E(2), P, and vascular endothelial growth factor (VEGF) in human granulosa luteinized cell cultures with the presence of a gonadotropin-releasing hormone (GnRH) agonist or antagonist. DESIGN: In vitro cell culture study. SETTING: Research laboratory of a university hospital. PATIENTS: Granulosa luteinized cells were obtained from 24 patients undergoing ovarian stimulation for IVF treatment. INTERVENTIONS: Granulosa cells were cultured for 48 hours with 1 nM of cetrorelix or leuprorelide. For a further 48 hours, granulosa cells were cultured with or without the combination of cetrorelix plus leuprorelide. MAIN OUTCOMES: At the end of each culturing period, the concentrations of E(2), P, and VEGF were measured in culture supernatants by immunoassays. RESULTS: Estradiol and P concentrations were similar between the culture supernatants from controls and treatment groups. The VEGF concentrations in supernatants from cultures with cetrorelix (2,315.1 +/- 1,565.5 pg/mL) were moderately, but significantly, lower than in controls (2,604.3 +/- 1,907.1 pg/mL) or cultures with leuprorelide (2,558.8 +/- 1,403.1 pg/mL). CONCLUSIONS: The GnRH analogues do not affect steroidogenesis in human granulosa luteinized cell cultures. The GnRH antagonists moderately affect the secretion of VEGF from human granulosa luteinized cells.

Adult↗

PubMed2005

Inhibition of mitochondrial respiration elevates oxygen concentration but leaves regulation of hypoxia-inducible factor (HIF) intact.

The transcription factor hypoxia-inducible factor-1 (HIF-1) is critical for erythropoietin and other factors involved in the adaptation of the organism to hypoxic stress. Conflicting results have been published regarding the role of the mitochondrial electron transport chain (ETC) in the regulation of HIF-1alpha. We assessed cellular hypoxia by pimonidazole staining and blotting of the O2-labile HIF-1 alpha-subunit in human osteosarcoma cell cultures (U2OS and 143B). In conventional, gas-impermeable cell culture dishes, ETC inhibitors had no effect on pimonidazole staining or HIF-1alpha abundance in a 20% O2 atmosphere; both parameters were undetectable. Pimonidazole staining and HIF activity were substantial in 0.1% O2 irrespective of ETC inhibition. At an intermediate oxygen concentration (3% O2) pimonidazole staining and HIF-alpha expression were detectable but strongly reduced after ETC inhibition in conventional cell cultures. All effects of ETC inhibition on HIF-1alpha regulation were eliminated in gas-permeable dishes. As shown in a 143B subclone deficient in mitochondrial DNA (206rho0), genetic inactivation of the ETC led to similar responses with respect to HIF-1alpha regulation as ETC inhibitors. Our data demonstrate that reduction of oxygen consumption reduces the O2 gradient in conventional cell cultures, causing elevation of the cellular O2 concentration, which leads to degradation of HIF-alpha.

Active Transport, Cell Nucleus↗

PubMed2005

Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element.

The HIFs (hypoxia-inducible factors) are a family of heterodimeric transcription factors essential for the adaptation of cells to reduced oxygen supply. Three human PHDs (prolyl hydroxylase domain proteins, PHD1-PHD3) initiate oxygen-dependent degradation of HIF-alpha-subunits in normoxia. RNA interference directed against PHD2, but not PHD1 or PHD3, is sufficient to stabilize HIF-1alpha in normoxia. Therefore PHD2 is regarded as the main cellular oxygen sensor. PHD2 itself is up-regulated by hypoxia and may thus limit hypoxic signalling. By sequence analysis, we predicted a promoter approx. 3.5 kb 5' of the translation start codon and a second promoter located in a CpG island immediately upstream of the coding sequence. A consensus HIF-1-binding site that is conserved in the murine phd2 gene was detected in the CpG island. By electrophoretic mobility-shift assay, we demonstrated binding of HIF-1 to the putative HIF-1-binding site. In luciferase reporter vectors, the isolated upstream promoter was inactive in all cell lines tested unless 200 bp were deleted at the 3'-end. The downstream promoter was active and induced by hypoxia. In reporter vectors containing both promoter sequences, luciferase activity was equal to vectors containing only the downstream promoter. In cells transfected with a vector containing both promoters, a single luciferase transcript was detectable. This transcript had the same length as transcripts from a vector containing the downstream promoter only. We conclude that the phd2 gene is transcribed exclusively from the downstream promoter that contains a functional hypoxia-responsive, cis-regulatory element. Our results establish that PHD2 is a direct HIF target gene.

Base Sequence↗

PubMed2005

Nitric oxide reverses desferrioxamine- and hypoxia-evoked HIF-1alpha accumulation--implications for prolyl hydroxylase activity and iron.

Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)-HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.

Amino Acids, Dicarboxylic↗

PubMed2005

Review: hypoxia-inducible factor-1 (HIF-1): a novel transcription factor in immune reactions.

Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional complex that has been recognized primarily for its role in the maintenance of oxygen and energy homoeostasis. The HIF-1alpha subunit is O(2) labile and is degraded by the proteasome following prolyl-hydroxylation and ubiquitination in normoxic cells. The present review summarizes evidence that HIF-1 is also involved in immune reactions. Immunomodulatory peptides, including interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha), stimulate HIF-1 dependent gene expression even in normoxic cells. Both the hypoxic and the cytokine-induced activation of HIF-1 involve the phosphatidylinositol- 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK) signaling pathways. In addition, heat shock proteins (HSP) and other cofactors interact with HIF-1 subunits. HIF-1 increases the transcription of several genes for proteins that promote blood flow and inflammation, including vascular endothelial growth factor (VEGF), heme oxygenase-1, endothelial and inducible nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2). The pharmacologic activation of the HIF-1 complex can be desirable in ischemic and inflammatory disorders. In contrast, HIF-1 blockade may be beneficial to prevent tumor angiogenesis and tumor growth.

Animals↗

PubMed2004

Chelation of cellular calcium modulates hypoxia-inducible gene expression through activation of hypoxia-inducible factor-1alpha.

Hypoxia-Inducible Factor-1 (HIF-1) is the key transcription factor in control of the expression of hypoxia-inducible genes needed by cells to adapt to decreased oxygen availability. Herein, we investigated the HIF-1alpha-mediated gene expression of carbonic anhydrase 9 (CA9) in response to hypoxia and changes of intracellular calcium levels in the neuroblastoma cell line SH-SY5Y. Decreasing the intracellular calcium level by BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) induced HIF-1alpha nuclear accumulation and enhanced HIF-1 DNA binding within 1 h of incubation. Like hypoxia, BAPTA stimulated HIF-1-dependent transcription by increasing the activity of the C-terminal transactivation domain of HIF-1alpha and greatly enhanced expression of the HIF-1 target gene CA9. Detailed analysis of HIF-1alpha accumulation revealed that BAPTA attenuated the interaction of HIF-1alpha with von-Hippel-Lindau protein thus decreasing proteasomal degradation of HIF-1alpha. Knock down of HIF-1alpha mRNA and protein by small interference RNA for HIF-1alpha revealed that both hypoxia and the BAPTA-induced gene expression of CA9 were strictly dependent on HIF-1alpha. In contrast, elevation of cytosolic calcium level by thapsigargin reduced the BAPTA-mediated effects. Measurements of intracellular calcium under hypoxia revealed a change in the cellular calcium distribution. BAPTA-dependent induction of HIF-1 activity was not caused by its in vitro capability to chelate iron. Instead, effective chelation of cellular calcium caused the accumulation of HIF-1alpha protein through inhibition of HIF-prolyl hydroxylases and activated HIF-1-dependent gene expression under normoxic conditions.

Blotting, Western↗

PubMed2004

Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-alpha-prolyl-4-hydroxylases.

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive alpha-subunit (HIF-1alpha or HIF-2alpha) and a constitutively expressed beta-subunit. In normoxia, HIF-1alpha is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1alpha. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1alpha expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-alpha. Using a VHL capture assay, we demonstrate that HIF-alpha prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1alpha after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1alpha in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia.

Adenocarcinoma, Clear Cell↗

PubMed2003

Nitric oxide impairs normoxic degradation of HIF-1alpha by inhibition of prolyl hydroxylases.

Hypoxia inducible factor-1 (HIF-1) is the master regulator of metabolic adaptation to hypoxia. It is appreciated that HIF-1alpha accumulation is achieved under normoxic conditions by e.g., nitric oxide. We determined molecular mechanisms of HIF-1alpha accumulation under the impact of S-nitrosoglutathione (GSNO). In human embryonic kidney cells GSNO provoked nuclear accumulation of HIF-1alpha. This appeared unrelated to gene transcription and protein translation, thus pointing to inhibition of HIF-1alpha degradation. Indeed, GSNO as well as the hypoxia mimic CoCl2 decreased ubiquitination of HIF-1alpha and GSNO-induced HIF-1alpha failed to coimmunoprecipitate with pVHL (von Hippel Lindau protein). Considering that HIF-1alpha-pVHL interactions require prolyl hydroxylation of HIF-1alpha, we went on to demonstrate inhibition of HIF-1alpha prolyl hydroxylases (PHDs) by GSNO. In vitro HIF-1alpha-pVHL interactions revealed that GSNO dose-dependently inhibits PHD activity but not the interaction of a synthetic peptide resembling the hydroxylated oxygen-dependent degradation domain of HIF-1alpha with pVHL. We conclude that GSNO-attenuated prolyl hydroxylase activity accounts for HIF-1alpha accumulation under conditions of NO formation during normoxia and that PHD activity is subject to regulation by NO.

Cell Hypoxia↗

PubMed2003

Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing.

Hypoxia-inducible factor1 (HIF-1) is an essential transcription factor for cellular adaptation to decreased oxygen availability. In normoxia the oxygen-sensitive alpha-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1 alpha prolyl hydroxylases (PHD1, PHD2, and PHD3) function as oxygen sensors in vivo. Furthermore, the asparagine hydroxylase FIH-1 (factor inhibiting HIF) has been found to hydroxylate Asp803 of the HIF-1 C-terminal transactivation domain, which results in the decreased ability of HIF-1 to bind to the transcriptional coactivator p300/CBP. We have fused these enzymes to the N-terminus of fluorescent proteins and transiently transfected the fusion proteins into human osteosarcoma cells (U2OS). Three-dimensional 2-photon confocal fluorescence microscopy showed that PHD1 was exclusively present in the nucleus, PHD2 and FIH-1 were mainly located in the cytoplasm and PHD3 was homogeneously distributed in cytoplasm and nucleus. Hypoxia did not influence the localisation of any enzyme under investigation. In contrast to FIH-1, each PHD inhibited nuclear HIF-1 alpha accumulation in hypoxia. All hydroxylases suppressed activation of a cotransfected hypoxia-responsive luciferase reporter gene. Endogenous PHD2mRNA and PHD3mRNA were hypoxia-inducible, whereas expression of PHD1mRNA and FIH-1mRNA was oxygen independent. We propose that PHDs and FIH-1 form an oxygen sensor cascade of distinct subcellular localisation.

Cell Compartmentation↗

PubMed2002

The interaction between primate blood and mouse islets induces accelerated clotting with islet destruction.

BACKGROUND: Mouse islets transplanted under the renal subcapsular space of cynomolgus monkeys are subject to a form of hyperacute rejection, the mechanism of which is unclear. As islets are in contact with whole blood at the time of transplantation, the effect of platelets and the coagulation cascade on islet destruction was assessed. METHODS: Coagulation was assessed using thromboelastography on citrated/recalcified human blood samples with freshly isolated C57/Bl6 mouse islets. A dynamic islet perifusion system was used to assess the effect of islets on blood cells and coagulation factors. Cytotoxicity was evaluated using (51)Cr labelled islets incubated with human blood and islet destruction was also evaluated using a histological grading system. Continuous PO(2) measurements were made in a static incubation system to assess the role of hypoxia in islet destruction. RESULTS: Mouse islets incubated in human blood induced accelerated coagulation and rapid consumption of platelets within 15 min. Within 1 h of incubation, 52% of mouse islets exposed to xenogeneic human blood showed features of severe damage with necrosis when compared with islets incubated in syngeneic blood. Specific lysis of the xenogeneic islets was demonstrable (Mean percentage lysis: 48%, P < 0.05 vs. control) after 4 h incubation in human blood. Oxygen levels remained constant at a level adequate to maintain islet viability in separate experiments. CONCLUSION: Mouse islets induce rapid activation of the clotting cascade and platelet consumption in vitro when exposed to human blood, which correlated with histological evidence of significant destruction demonstrable within 1 h of exposure to human or non-human primate blood. This in vitro model has features which appear to correlate with the islet destruction seen in vivo and could be a useful model for the study of the mechanisms underlying the rapid destruction of xenogeneic islets in primate recipients.

Animals↗

PubMedDate not supplied

HIF hydroxylation and cellular oxygen sensing.

Hypoxia-inducible factor (HIF) is a transcriptional complex that mediates a broad range of cellular and systemic responses to hypoxia. Analysis of HIF-alpha subunits has demonstrated that its activity is regulated by a series of oxygen-dependent enzymatic hydroxylations at specific prolyl and asparaginyl residues. Combined structural/genetic approaches have identified the relevant enzymes as members of the 2-oxoglutarate-dependent dioxygenase superfamily, possessing a beta-barrel 'jelly-roll' conformation that aligns a 2-histidine/1-carboxylate iron co-ordination motif at the catalytic centre. HIF prolyl hydroxylation is performed by a closely related set of isoenzymes (PHD1-3) that differ in abundance and subcellular localisation. Hydroxylation of either human HIF-1alpha Pro402 or Pro564 promotes interaction with the von Hippel-Lindau tumour suppressor protein (pVHL). In oxygenated cells this process targets HIF-alpha for rapid proteasomal destruction. HIF asparaginyl hydroxylation is performed by a protein termed factor inhibiting HIF (FIH). In oxygenated cells hydroxylation of human HIF-1alpha Asn803 prevents interaction with the p300 transcriptional co-activator, providing a second mechanism by which HIF-mediated transcription is inactivated. Genetic studies demonstrate a critical function for both types of enzyme in regulating the HIF transcriptional cascade. Limitation of activity in hypoxia supports a central role of these hydroxylases in cellular oxygen sensing. Regulation of the amount of hydroxylase protein, and the supply of other co-substrates and co-factors, particularly the cellular availability of iron, also contribute to tuning the physiological response to hypoxia.

Animals↗