Utta Berchner-Pfannschmidt

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.

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Two‐photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580–1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two‐photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two‐photon and the one‐photon emission spectra. However, a blue‐shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non‐linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression

It has been proposed that hydroxyl radicals (·OH) generated in a perinuclear iron-dependent Fenton reaction are involved in O2-dependent gene expression. Thus, it was the aim of this study to localize the cellular compartment in which the Fenton reaction takes place and to determine whether scavenging of ·OH can modulate hypoxia-inducible factor 1 (HIF-1)-dependent gene expression. The Fenton reaction was localized by using the nonfluorescent dihydrorhodamine (DHR) 123 that is irreversibly oxidized to fluorescent rhodamine 123 while scavenging ·OH together with gene constructs allowing fluorescent labeling of mitochondria, endoplasmic reticulum (ER), Golgi apparatus, peroxisomes, or lysosomes. A 3D two-photon confocal laser scanning microscopy showed ·OH generation in distinct hot spots of perinuclear ER pockets. This ER-based Fenton reaction was strictly pO2-dependent. Further colocalization experiments showed that the O2-sensitive transcription factor HIF-1α was present at the ER under normoxia, whereas HIF-1α was present only in the nucleus under hypoxia. Inhibition of the Fenton reaction by the ·OH scavenger DHR attenuated HIF-prolyl hydroxylase activity and interaction with von Hippel–Lindau protein, leading to enhanced HIF-1α levels, HIF-1α transactivation, and activated expression of the HIF-1 target genes plasminogen activator inhibitor 1 and heme oxygenase 1. Further, ·OH scavenging appeared to enhance redox factor 1 (Ref-1) binding and, thus, recruitment of p300 to the transactivation domain C because mutation of the Ref-1 binding site cysteine 800 abolished DHR-induced transactivation. Thus, the localized Fenton reaction appears to impact the expression of hypoxia-regulated genes by means of HIF-1α stabilization and coactivator recruitment.

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.

Reactive oxygen species modulate HIF-1 mediated PAI-1 expression: involvement of the GTPase Rac1

The hypoxia-inducible transcription factor HIF-1 mediates upregulation of plasminogen activator inhibitor-1 (PAI-1) expression under hypoxia. Reactive oxygen species (ROS) have also been implicated in PAI-1 gene expression. However, the role of ROS in HIF-1-mediated regulation of PAI-1 is not clear. We therefore investigated the role of the GTPase Rac1 which modulates ROS production in the pathway leading to HIF-1 and PAI-1 induction. Overexpression of constitutively activated (RacG12V) or dominant-negative (RacT17N) Rac1 increased or decreased, respectively, ROS production. In RacG12V-expressing cells, PAI-1 mRNA levels as well as HIF-alpha nuclear presence were reduced under normoxia and hypoxia whereas expression of RacT17N resulted in opposite effects. Treatment with the antioxidant pyr-rolidinedithiocarbamate or coexpression of the redox factor-1 restored HIF-1 and PAI-1 promoter activity in RacG12V-cells. In contrast, NFkappaB activation was enhanced in RacG12V-cells, but abolished by RacT17N. Thus, these findings suggest a mechanism explaining modified fibrinolysis and tissue remodeling in an oxidized environment.

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.

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.

Nuclear Oxygen Sensing: Induction of Endogenous Prolyl-hydroxylase 2 Activity by Hypoxia and Nitric Oxide

The abundance of the transcription factor hypoxia-inducible factor is regulated through hydroxylation of its α-subunits by a family of prolyl-hydroxylases (PHD1–3). Enzymatic activity of these PHDs is O2-dependent, which enables PHDs to act as cellular O2 sensor enzymes. Herein we studied endogenous PHD activity that was induced in cells grown under hypoxia or in the presence of nitric oxide. Under such conditions nuclear extracts contained much higher PHD activity than the respective cytoplasmic extracts. Although PHD1–3 were abundant in both compartments, knockdown experiments for each isoenzyme revealed that nuclear PHD activity was only due to PHD2. Maximal PHD2 activity was found between 120 and 210 μm O2. PHD2 activity was strongly decreased below 100 μm O2 with a half-maximum activity at 53 ± 13 μm O2 for the cytosolic and 54 ± 10 μm O2 for nuclear PHD2 matching the physiological O2 concentration within most cells. Our data suggest a role for PHD2 as a decisive oxygen sensor of the hypoxia-inducible factor degradation pathway within the cell nucleus.