Joachim Fandrey

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 α-subunit of HIF-1 is hydroxylated on Pro564 and Pro402 and thus targeted for proteasomal degradation. Three human oxygen-dependent HIF-1α 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-1α 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.

Bacterial lipopolysaccharide induces HIF-1 activation in human monocytes via p44/42 MAPK and NF-κB

Inflammatory mediators activate the transcriptional complex HIF-1 (hypoxia-inducible factor-1), the key regulator of hypoxia-induced gene expression. Here we report that bacterial LPS (lipopolysaccharide) induces HIF-1alpha mRNA expression and HIF-1alpha protein accumulation in human monocytes as well as in non-differentiated and differentiated cells of the human monocytic cell line THP-1 under normoxic conditions. LPS and hypoxia synergistically activated HIF-1. Whereas LPS increased HIF-1alpha mRNA expression through activation of a NF-kappaB (nuclear factor kappaB) site in the promoter of the HIF-1alpha gene, hypoxia post-translationally stabilized HIF-1alpha protein. HIF-1alpha activation was followed by increased expression of the HIF-1 target gene encoding ADM (adrenomedullin). Knocking down HIF-1alpha by RNA interference significantly decreased ADM expression, which underlines the importance of HIF-1 for the LPS-induced ADM expression in normoxia. Simultaneously with HIF-1 activation, an increase in p44/42 MAPK (mitogen-activated protein kinase) phosphorylation was observed after incubation with LPS. In cells pretreated with the p44/42 MAPK inhibitor PD 98059 or with RNAi (interfering RNA) directed against p44/42 MAPK, LPS-induced HIF-1alpha accumulation and ADM expression were significantly decreased. From these results we conclude that LPS critically involves the p44/42 MAPK and NF-kappaB pathway in the activation of HIF-1, which is an important transcription factor for LPS-induced ADM expression.

Nitric Oxide Modulates Oxygen Sensing by Hypoxia-inducible Factor 1-dependent Induction of Prolyl Hydroxylase 2

The transcription factor complex hypoxia-inducible factor 1 (HIF-1) plays a crucial role in cellular adaptation to low oxygen availability. O2-dependent HIF prolyl hydroxylases (PHDs) modify HIF-1α, which is sent to proteasomal degradation under normoxia. Reduced activity of PHDs under hypoxia allows stabilization of HIF-1α and induction of HIF-1 target gene expression. Like hypoxia, nitric oxide (NO) was found to inhibit normoxic PHD activity leading to HIF-1α accumulation. In contrast under hypoxia, NO reduced HIF-1α levels due to enhanced PHD activity. Herein, we studied the role of NO in regulating PHD expression and the consequences thereof for HIF-1α degradation. We report a biphasic response of HIF-1α and PHDs to NO treatment both under normoxia and hypoxia. In the early phase, NO inhibits PHD activity that leads to HIF-1α accumulation, whereas in the late phase, increased PHD levels reduce HIF-1α. NO induces expression of PHD2 and -3 mRNA and protein under normoxia and hypoxia in a strictly HIF-1-dependent manner. NO-treated cells with elevated PHD levels displayed delayed HIF-1α accumulation and accelerated degradation of HIF-1α upon reoxygenation. Subsequent suppression of PHD2 and -3 expression using small interfering RNA revealed that PHD2 was exclusively responsible for regulating HIF-1α degradation under NO treatment. In conclusion, we identified the induction of PHD2 as an underlying mechanism of NO-induced degradation of HIF-1α.

Regulation of hypoxia-inducible factors during inflammation.

The microenvironment of inflamed and injured tissue is characterized by low levels of oxygen and glucose and high levels of inflammatory cytokines, reactive oxygen, and nitrogen species and metabolites. The transcription factor complex hypoxia-inducible factor (HIF)-1 is regulated by hypoxia as well as by a broad variety of inflammatory mediators. In cells of the innate and adaptive immune system, HIF-1 is upregulated by bacterial and viral compounds, even under normoxic conditions. This upregulation prepares these cells to migrate to and to function in hypoxic and inflamed tissues. Once extravasated from the vasculature, the activity of cells is further enhanced by stimulation of HIF-1 by proinflammatory cytokines like interleukin (IL)-1beta (beta) and tumor necrosis factor (TNF) alpha (alpha), and locally expressed tissue factors. Crosstalk between hypoxic induction of HIF-1 and other signaling pathways activated by inflammation ensures a cell type-specific and stimulus-adequate cellular response. Prolonged activation of HIF-1 under conditions of inflammation, however, may contribute to the survival of damaged tissue and cells, thus promoting the development of tumors.

The Proinflammatory Cytokine Interleukin 1β and Hypoxia Cooperatively Induce the Expression of Adrenomedullin in Ovarian Carcinoma Cells through Hypoxia Inducible Factor 1 Activation

Adrenomedullin (ADM) is a potent hypotensive peptide produced by macrophages and endothelial cells during ischemia and sepsis. The molecular mechanisms that control ADM gene expression in tumor cells are still poorly defined. It is known, however, that hypoxia potently increases ADM expression by activation of the transcription factor complex hypoxia inducible factor 1 (HIF-1). Proinflammatory cytokines produced by tumor invading macrophages likewise activate expression of ADM. Herein, we show that apart from hypoxia, the proinflammatory cytokine interleukin 1beta (IL-1beta) induced the expression of ADM mRNA through activation of HIF-1 under normoxic conditions and enhanced the hypoxia-induced expression in the human ovarian carcinoma cell line OVCAR-3. IL-1beta significantly increased accumulation and nuclear translocation of HIF-1alpha under normoxic conditions and amplified hypoxic HIF-1 activation. IL-1beta treatment affected neither HIF-1alpha mRNA levels nor the hydroxylation status of HIF-1alpha and, thus, stability of the protein. Instead cycloheximide effectively prevented the increase in HIF-1alpha protein, indicating a stimulatory effect of IL-1beta on HIF-1alpha translation. Finally, treatment of HIF-1alpha with short interfering RNA revealed a significant role for HIF-1 in the IL-1beta-dependent stimulation of ADM expression.

Erythropoietin gene expression in different areas of the developing human central nervous system

UNLABELLEDnEvidence from cell culture and animal experiments suggests a neuroprotective and neurotrophic function of erythropoietin (EPO). We have quantitated the distribution of EPO mRNA expression in the developing human central nervous system (CNS).nnnPATIENTS AND METHODSnUp to seven biopsies from different areas of the CNS of four preterm fetuses (gestational age 23-37 weeks) were obtained at routine postmortem examinations. EPO mRNA was quantitated by competitive PCR in samples from the CNS, the kidneys, and the liver where the EPO gene is predominantly expressed at this gestational age.nnnRESULTSnEPO mRNA was most abundant in one sample from the cerebellum (0.29 amol/microg total RNA [amol=10(-18)mol]) and two from the pituitary gland (0.23 amol/microg total RNA), but levels varied considerably. EPO mRNA in the cortex cerebri (median 0.12 amol/microg total RNA; n=4) dominated over the expression in the corpora amygdala (median 0.05 amol/microg total RNA; n=4), the hippocampus (median 0.03 amol/microg total RNA; n=4), or the basal ganglia (median 0.01 amol/microg total RNA; n=3). Only little EPO mRNA (<0.01 and 0.06 amol/microg total RNA) was found in the spinal cord. EPO mRNA levels in the cerebellum, pituitary gland, or the cerebral cortex were within the same range as in the liver (0.03-1.67 amol/microg total RNA; n=4), or the kidneys (0.06-0.79 amol/microg total RNA; n=4).nnnCONCLUSIONnWe found the EPO gene expressed throughout the fetal human CNS. Our data provide the basis to discuss a function for EPO in the brain of humans as well.

Hypoxia-inducible Factor Prolyl-4-hydroxylase PHD2 Protein Abundance Depends on Integral Membrane Anchoring of FKBP38

Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor α subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor α regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidyl-prolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a non-linear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn2+ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well as regulation of PHD2 protein abundance by FKBP38, is dependent on membrane- anchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically our data indicate that PHD2 protein stability is regulated by a ubiquitin-independent proteasomal pathway involving FKBP38 as adaptor protein that mediates proteasomal interaction. We hypothesize that FKBP38-bound PHD2 is constantly degraded whereas cytosolic PHD2 is stable and able to function as an active prolyl-4-hydroxylase.

Hypoxia-induced erythropoietin expression in human neuroblastoma requires a methylation free HIF-1 binding site.

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH‐SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth‐associated protein‐43 (GAP‐43), and neuron‐specific enolase (ENO). NB cells with a more epithelial phenotype like SH‐SHEP and LAN‐5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up‐regulation of hypoxia‐inducible factor‐1 (HIF‐1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3′ enhancer was only present in the SH‐SHEP and LAN‐5 cells but not in SH‐SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.

Role of reactive oxygen species in the regulation of HIF-1 by prolyl hydroxylase 2 under mild hypoxia.

Abstract The function and survival of eukaryotic cells depends on a constant and sufficient oxygen supply. Cells recognize and respond to hypoxia by accumulation of the transcription factor hypoxia-inducible factor 1 (HIF-1), composed of an oxygen-sensitive HIF-1α and a constitutive HIF-1β subunit. Besides physiology, HIF-1 induction is involved in major pathological processes such as cardiovascular disease, inflammation and cancer, which are associated with the formation of reactive oxygen species (ROS). ROS have been reported to affect HIF-1 activity but the role for ROS in regulating HIF-1 has not been definitely settled. In order to shed light on the redox-regulation of HIF-1 by ROS, we studied the impact of exogenous ROS treatment (H2O2) on HIF-1α and HIF-1 regulatory protein prolyl hydroxylase 2 (PHD2) in the human osteosarcoma cell line U2OS. At early reaction periods, H2O2 induced HIF-1α but at prolonged observation phases the opposite occurred. Herein, modulation of PHD activity appeared to be the key element, because knockdown and inhibition of the PHD2 prevented reduction of HIF-1α. However, H2O2 treatment constantly suppressed HIF-1 transactivation at all time-points. Our data indicate a dual redox regulation of HIF-1α protein amount with a constant suppression of HIF-1 target gene expression by ROS.

Oxygen-sensing under the influence of nitric oxide

The transcription factor complex Hypoxia inducible factor 1 (HIF-1) controls the expression of most genes involved in adaptation to hypoxic conditions. Oxygen-dependency is maintained by prolyl- and asparagyl-4-hydroxylases (PHDs/FIH-1) belonging to the superfamily of iron(II) and 2-oxoglutarate dependent dioxygenases. Hydroxylation of the HIF-1alpha subunit by PHDs and FIH-1 leads to its degradation and inactivation. By hydroxylating HIF-1alpha in an oxygen-dependent manner PHDs and FIH-1 function as oxygen-sensing enzymes of HIF signalling. Besides molecular oxygen nitric oxide (NO), a mediator of the inflammatory response, can regulate HIF-1alpha accumulation, HIF-1 activity and HIF-1 dependent target gene expression. Recent studies addressing regulation of HIF-1 by NO revealed a complex and paradoxical picture. Acute exposure of cells to high doses of NO increased HIF-1alpha levels irrespective of the residing oxygen concentration whereas prolonged exposure to NO or low doses of this radical reduced HIF-1alpha accumulation even under hypoxic conditions. Several mechanisms were found to contribute to this paradoxical role of NO in regulating HIF-1. More recent studies support the view that NO regulates HIF-1 by modulating the activity of the oxygen-sensor enzymes PHDs and FIH-1. NO dependent HIF-1alpha accumulation under normoxia was due to direct inhibition of PHDs and FIH-1 most likely by competitive binding of NO to the ferrous iron in the catalytically active center of the enzymes. In contrast, reduced HIF-1alpha accumulation by NO under hypoxia was mainly due to enhanced HIF-1alpha degradation by induction of PHD activity. Three major mechanisms are discussed to be involved in enhancing the PHD activity despite the lack of oxygen: (1) NO mediated induction of a HIF-1 dependent feedback loop leading to newly expressed PHD2 and enhanced nuclear localization, (2) O2-redistribution towards PHDs after inhibition of mitochondrial respiration by NO, (3) reactivation of PHD activity by a NO mediated increase of iron and 2-oxoglutarate and/or involvement of reactive oxygen and/or nitrogen species.