Christoph Wotzlaw

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.

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.

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.

Two-photon excitation and emission spectra of the green fluorescent protein variants ECFP, EGFP and EYFP

Two‐photon (TP) excitation (820–1150 nm) and emission (280–700 nm) spectra for the fluorescent proteins (FPs) ECFP 3 , EGFP 3 and EYFP 3 produced in human tumour cells were recorded. TP excitation spectra of pure and highly enriched samples were found to be more differentiated in comparison with their one‐photon (OP) spectra. They exhibited more pronounced main and local maxima, which coincided among different purity grades within small limits. TP and OP emission spectra of pure and enriched samples were identical. However, in crude samples, excitation was slightly blue‐shifted and emission red‐shifted. The data indicate that both OP and TP excitation routes led to the same excited states of these molecules. The emission intensity is dependent on the pH of the environment for both types of excitation; the emission intensity maximum can be recorded in the alkaline range. Reconstitution of emission intensity after pH quenching was incomplete, albeit that the respective spectral profiles were identical to those prequenching. When emission data were averaged over the whole range of excitation, the resulting emission profile and maximum coincided with the data generated by optimal excitation. Therefore, out‐of‐maximum excitation, common practice in TP excitation microscopy, can be used for routine application.

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‐1a and its constitutive β subunit also known as ARNT is the key event of the cellular genetic response to hyp‐oxia. 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‐1α than of HIF‐1β, indicating that each subunit can move independently. We applied fluorescence resonance energy transfer to calculate the nanometer distance between α and β subunits of the transcriptionally active HIF‐1 complex bound to DNA. Both N termini of the fluorophore‐labeled HIF‐1 sub‐units 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.—Wotzlaw, C., Otto, T., Berchner‐Pfannschmidt, U., Metzen, E., Acker, H., Fandrey, J. Optical analysis of the HIF‐1 complex in living cells by FRET and FRAP. FASEB J. 21, 700–707 (2007)

Visualization of the three-dimensional organization of hypoxia-inducible factor-1α and interacting cofactors in subnuclear structures

Abstract Cells need oxygen (O2) to meet their metabolic demands. Highly efficient systems of O2-sensing have evolved to initiate responses enabling cells to adapt their metabolism to reduced O2 availability. Of central importance is the activation of hypoxiainducible factor-1 (HIF-1), a transcription factor complex that controls the expression of genes the products of which regulate glucose uptake and metabolism, vasotonus and angiogenesis, oxygen capacity of the blood as well as cell growth and death. Activation of HIF-1 requires the accumulation and nuclear translocation of the HIF-1α subunit, its dimerization with HIF-1β and the binding of coactivator proteins such as p300. In this study we investigated the threedimensional (3D) distribution of HIF-1α within the nucleus and assigned its localization to known nuclear compartments. Using twophoton microscopy we determined the colocalization of HIF-1α and -β subunits within nuclear domains as well as overlaps between HIF-1α and p300. Our data provide information on the nuclear distribution of HIF-1α with respect to subnuclear domains that could serve as specific locations for hypoxiainduced gene expression.

Multifocal animated imaging of changes in cellular oxygen and calcium concentrations and membrane potential within the intact adult mouse carotid body ex vivo

Carotid body (CB) type I cell hypoxia-sensing function is assumed to be based on potassium channel inhibition. Subsequent membrane depolarization initiates an intracellular calcium increase followed by transmitter release for excitation of synapses with linked nerve endings. Several reports, however, contradict this generally accepted concept by showing that type I cell oxygen-sensing properties vary significantly depending on the method of their isolation. We report therefore for the first time noninvasive mapping of the oxygen-sensing properties of type I cells within the intact adult mouse CB ex vivo by using multifocal Nipkow disk-based imaging of oxygen-, calcium- and potential-sensitive cellular dyes. Characteristic type I cell clusters were identified in the compact tissue by immunohistochemistry because of their large cell nuclei combined with positive tyrosine hydroxylase staining. The cellular calcium concentrations in these cell clusters either increased or decreased in response to reduced tissue oxygen concentrations. Under control conditions, cellular potential oscillations were uniform at ∼0.02 Hz. Under hypoxia-induced membrane depolarization, these oscillations ceased. Simultaneous increases and decreases in potential of these cell clusters resulted from spontaneous burstlike activities lasting ∼1.5 s. type I cells, identified during the experiments by cluster formation in combination with large cell nuclei, seem to respond to hypoxia with heterogeneous kinetics.

Nanoscopy of the cellular response to hypoxia by means of fluorescence resonance energy transfer (FRET) and new FRET software

Background Cellular oxygen sensing is fundamental to all mammalian cells to adequately respond to a shortage of oxygen by increasing the expression of genes that will ensure energy homeostasis. The transcription factor Hypoxia-Inducible-Factor-1 (HIF-1) is the key regulator of the response because it coordinates the expression of hypoxia inducible genes. The abundance and activity of HIF-1 are controlled through posttranslational modification by hydroxylases, the cellular oxygen sensors, of which the activity is oxygen dependent. Methods Fluorescence resonance energy transfer (FRET) was established to determine the assembly of the HIF-1 complex and to study the interaction of the α-subunit of HIF-1 with the O2-sensing hydroxylase. New software was developed to improve the quality and reliability of FRET measurements. Results FRET revealed close proximity between the HIF-1 subunits in multiple cells. Data obtained by sensitized FRET in this study were fully compatible with previous work using acceptor bleaching FRET. Interaction between the O2-sensing hydroxylase PHD1 and HIF-1α was demonstrated and revealed exclusive localization of O2-sensing in the nucleus. The new software FRET significantly improved the quality and speed of FRET measurements. Conclusion FRET measurements do not only allow following the assembly of the HIF-1 complex under hypoxic conditions but can also provide important information about the process of O2-sensing and its localisation within a cell. MCS codes: 92C30, 92C05, 92C40