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

2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês

10.1074/jbc.m804390200

ISSN

1083-351X

Autores

Utta Berchner‐Pfannschmidt, Suzan Tug, Buena Trinidad, Felix Oehme, Hatice Yamac, Christoph Wotzlaw, Ingo Flamme, Joachim Fandrey,

Tópico(s)

Mitochondrial Function and Pathology

Resumo

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. 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. The capability of mammalian cells to sense oxygen and adapt gene expression according to O2 availability is critical for the maintenance of oxygen homoeostasis within the tissue. The heterodimeric transcriptional regulator hypoxia-inducible factor (HIF) 2The abbreviations used are: HIF, hypoxia-inducible factor; PHD, prolyl-hydroxylase; pVHL, von Hippel-Lindau protein; GFP, green fluorescent protein; GSNO, S-nitrosoglutathione; siRNA, short interfering RNA; VBC, pVHL in complex with elongins B and C; HOX, hypoxia; NOX, normoxia; E3, ubiquitin-protein isopeptide ligase; wt, wild type; HEK, human embryonic kidney; cO2, oxygen concentration. 2The abbreviations used are: HIF, hypoxia-inducible factor; PHD, prolyl-hydroxylase; pVHL, von Hippel-Lindau protein; GFP, green fluorescent protein; GSNO, S-nitrosoglutathione; siRNA, short interfering RNA; VBC, pVHL in complex with elongins B and C; HOX, hypoxia; NOX, normoxia; E3, ubiquitin-protein isopeptide ligase; wt, wild type; HEK, human embryonic kidney; cO2, oxygen concentration. is central to the regulation of gene expression in response to decreased oxygen levels, i.e. hypoxia (1Semenza G.L. Curr. Opin. Genet. Dev. 1998; 8: 588-594Crossref PubMed Scopus (918) Google Scholar). HIFs are composed of the constitutive β-subunit and one of three O2-labile α-subunits HIF-1, -2, or -3α (2Wang G.L. Jiang B.H. Rue E.A. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5510-5514Crossref PubMed Scopus (4974) Google Scholar, 3Giaccia A.J. Simon M.C. Johnson R. Genes Dev. 2004; 18: 2183-2194Crossref PubMed Scopus (293) Google Scholar). Both the stability and the activity of the HIF α-subunits are regulated by oxygen-dependent post-translational modifications.Under normoxic conditions HIFαs are hydroxylated by a family of prolyl-4-hydroxylases (PHD1–3) at two conserved proline residues in HIF-1α and -2α (Pro402/564 or Pro405/531, respectively) or a single proline residue in HIF-3α (Pro490) (4Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. von Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. 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Hypoxia reduces prolyl-hydroxylation by PHDs, resulting in stabilization of the α-subunit. This allows dimerization with the β-subunit and induces expression of about 100 genes involved in adaptation to hypoxia (10Wenger R.H. Stiehl D.P. Camenisch G. Sci. STKE. 2005; 306: re12Google Scholar). Transcriptional activity of the HIF complex is regulated by hydroxylation of an asparagine residue within the C-terminal trans-activating domain of HIF-1α and -2α by an asparagyl-hydroxylase termed factor-inhibiting HIF; active factor-inhibiting HIF hydroxylates Asp803 in HIF-1α under normoxia, which impedes binding of the transcriptional coactivator p300/CBP (11Mahon P.C. Hirota K. Semenza G.L. Genes Dev. 2001; 15: 2675-2686Crossref PubMed Scopus (1100) Google Scholar,12Lando D. Peet D.J. Gorman J.J. Whelan D.A. Whitelaw M.L. Bruick R.K. Genes Dev. 2002; 16: 1466-1471Crossref PubMed Scopus (1201) Google Scholar). By hydroxylating HIF-αs in an oxygen-dependent manner, PHDs and factor-inhibiting HIF function as oxygen-sensing enzymes of HIF activation.The three PHD isoenzymes belong to the superfamily of iron and 2-oxoglutarate-dependent dioxygenases. These enzymes need O2 as cosubstrate, which provides the molecular basis for their O2-sensing function. Oxygen dependence of PHDs is reflected by their Km(O2) values, which are in the range of 85–250 μm depending on the source of recombinant enzyme, substrate length and assay condition (13Hirsila M. Koivunen P. Gunzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 14Koivunen P. Hirsila M. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2006; 281: 28712-28720Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 15Ehrismann D. Flashman E. Genn D.N. Mathioudakis N. Hewitson K.S. Ratcliffe P.J. Schofield C.J. Biochem. J. 2007; 401: 227-234Crossref PubMed Scopus (169) Google Scholar). In general all three PHDs preferentially hydroxylate Pro564 in HIF-1α and Pro531 in HIF-2α, whereas Pro402 and Pro405 in HIF-1α and HIF-2α are less or in the case of PHD3 are not at all hydroxylated (16Huang J.H. Zhao Q. Mooney S.M. Lee F.S. J. Biol. Chem. 2002; 277: 39792-39800Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 17Chan D.A. Sutphin P.D. Yen S.E. Giaccia A.J. Mol. Cell. Biol. 2005; 25: 6415-6426Crossref PubMed Scopus (188) Google Scholar, 18Epstein A.C.R. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2697) Google Scholar). Although the availability of O2 serves as a general determinant of PHD activity, the cellular capacity for HIFα hydroxylation by PHDs is also affected by their abundance under the respective conditions. Importantly PHD2 and PHD3 expression, but not that of PHD1, is induced by hypoxia in an HIF-1-dependent manner (18Epstein A.C.R. Gleadle J.M. McNeill L.A. Hewitson K.S. O'Rourke J. Mole D.R. Mukherji M. Metzen E. Wilson M.I. Dhanda A. Tian Y.M. Masson N. Hamilton D.L. Jaakkola P. Barstead R. Hodgkin J. Maxwell P.H. Pugh C.W. Schofield C.J. Ratcliffe P.J. Cell. 2001; 107: 43-54Abstract Full Text Full Text PDF PubMed Scopus (2697) Google Scholar, 19Metzen E. Stiehl D.P. Doege K. Marxsen J.H. Hellwig-Burgel T. Jelkmann W. Biochem. J. 2005; 387: 711-717Crossref PubMed Scopus (161) Google Scholar, 20Pescador N. Cuevas Y. Naranjo S. Alcaide M. Villar D. Landazuri M. Del Peso L. Biochem. J. 2005; 390: 189-197Crossref PubMed Scopus (163) Google Scholar). This HIF-1-dependent induction of the cellular O2-sensors generates an auto-regulatory loop controlling HIF-1α stability under hypoxia and reoxygenation (21Berra E. Benizri E. Ginouves A. Volmat V. Roux D. Pouyssegur J. EMBO J. 2003; 22: 4082-4090Crossref PubMed Scopus (1070) Google Scholar, 22Marxsen J.H. Stengel P. Doege K. Heikkinen P. Jokilehto T. Wagner T. Jelkmann W. Jaakkola P. Metzen E. Biochem. J. 2004; 381: 761-767Crossref PubMed Scopus (273) Google Scholar, 23D'Angelo G. Duplan E. Boyer N. Vigne P. Frelin C. J. Biol. Chem. 2003; 278: 38183-38187Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). PHD enzyme abundance is of such importance that increased levels of PHD can compensate for decreased oxygen availability (24Stiehl D.P. Wirthner R. Koditz J. Spielmann P. Camenisch G. Wenger R.H. J. Biol. Chem. 2006; 281: 23482-23491Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar).Differential expression and regulation of PHD isoenzymes may enable fine tuning of hypoxic responses in different tissues and under different conditions. In cultured cells, all three PHDs can regulate HIFαs, but PHD2 was found to be the most active isoform in a number of cell lines. PHD2 has a preference for HIF-1α, whereas PHD1 and PHD3 prefer HIF-2α (25Appelhoff R.J. Tian Y.M. Raval R.R. Turley H. Harris A.L. Pugh C.W. Ratcliffe P.J. Gleadle J.M. J. Biol. Chem. 2004; 279: 38458-38465Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar). Although PHD2 plays a dominant role under normoxia and hypoxia (21Berra E. Benizri E. Ginouves A. Volmat V. Roux D. Pouyssegur J. EMBO J. 2003; 22: 4082-4090Crossref PubMed Scopus (1070) Google Scholar, 26Berchner-Pfannschmidt U. Yamac H. Trinidad B. Fandrey J. J. Biol. Chem. 2007; 282: 1788-1796Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), all three PHDs can contribute to PHD activity under prolonged hypoxic stress (27Ginouves A. Ilc K. Macias N. Pouyssegur J. Berra E. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 4745-4750Crossref PubMed Scopus (190) Google Scholar). In addition, all three PHD enzymes exhibit different tissue distribution and distinct patterns of subcellular localization. We reported that overexpressed PHDs fused to green fluorescence protein (GFP) were differentially localized in cells with PHD1 solely found in the nucleus, PHD2 mainly in the cytoplasm, and PHD3 evenly distributed between both compartments (28Metzen E. Berchner-Pfannschmidt U. Stengel P. Marxsen J.H. Stolze I. Klinger M. Huang W.Q. Wotzlaw C. Hellwig-Burgel T. Jelkmann W. Acker H. Fandrey J. J. Cell Sci. 2003; 116: 1319-1326Crossref PubMed Scopus (361) Google Scholar). Interestingly, endogenous PHDs were mainly located in the cytoplasm (29Soilleux E.J. Turley H. Tian Y.M. Pugh C.W. Gatter K.C. Harris A.L. Histopathology. 2005; 47: 602-610Crossref PubMed Scopus (76) Google Scholar), but increased PHD2 protein expression in tumor tissues was located in the cell nuclei (30Jokilehto T. Rantanen K. Luukkaa M. Heikkinen P. Grenman R. Minn H. Kronqvist P. Jaakkola P.M. Clin. Cancer Res. 2006; 12: 1080-1087Crossref PubMed Scopus (77) Google Scholar).Recently we reported that inhibition of PHDs by NO results in the up-regulation of PHD2 and 3 expression because of HIF-1α accumulation in addition to hypoxia (26Berchner-Pfannschmidt U. Yamac H. Trinidad B. Fandrey J. J. Biol. Chem. 2007; 282: 1788-1796Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Subsequently, hypoxic HIFα induction was reduced by increased PHD levels, suggesting a role for NO as an inducer of PHD activity. Here we highlight the unexpected finding that the O2-dependent activity of endogenous PHD2 is enhanced in cells preincubated under hypoxia or NO. In contrast to the proposed cytosolic localization of PHD2, we observed that nuclear PHD2 activity was higher than cytoplasmic PHD2 activity. Moreover, nuclear PHD2 activity was steeply decreased at O2 concentration below 100 μm (half-maximum activity at 54 μm O2), which matches the physiological range of O2 concentration of most cells. Our data support the notion that PHD2 acts as the prominent oxygen sensor enzyme of the HIF pathway, but it is particularly active in the cell nucleus. We suggest a role for nuclear oxygen sensing via PHD2 under conditions of hypoxia or NO when HIF-1α is present in the nucleus.EXPERIMENTAL PROCEDURESReagents—S-Nitrosoglutathione (GSNO) was synthesized as described previously (31Sonnenschein K. de Groot H. Kirsch M. J. Biol. Chem. 2004; 279: 45433-45440Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Cell Culture—The human osteosarcoma cells (U-2OS), renal clear carcinoma cells (RCC4), embryonal kidney cells (HEK293), and colon carcinoma cells (DLD1) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 units/ml streptomycin in a normoxic atmosphere of 21% O2, 74% N2 and 5% CO2 (by volume). For cell culture experiments, the cells were either exposed to normoxia or placed in a hypoxic incubator with 1% O2, 94% N2, 5% CO2 (Heraeus incubator, Hanau, Germany) for variable periods of time.Protein Extraction—Whole cell lysates were prepared from 35-mm dishes of cells that were about 80% confluent. The cells were lysed in 50 μl of extraction buffer (300 mm sodium chloride, 10 mm Tris, pH 7.9, 1 mm EDTA, 0.1% Nonidet P-40, 1× protease inhibitor mixture; Roche Applied Science) for 20 min on ice and centrifuged (3600 × g at 4 °C for 5 min). The supernatant was used as whole cell extract. Cytoplasmic and nuclear extracts were obtained from 60-mm dishes of cells that were about 80% confluent by using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce). The cell membranes were lysed in 100 μl of ice-cold cytoplasmic extraction reagent I (1× protease inhibitor mixture; Roche Applied Science) for 10 min on ice. 5.5 μl of ice-cold cytoplasmic extraction reagent II was added, incubated for 1 min, and centrifuged (16,000 × g at 4 °C for 10 min). The supernatant was used as cytoplasmic extract fraction. The insoluble pellet that contains nuclei was washed two times with phosphate-buffered saline to remove cytoplasmic remains. The washed pellet was resuspended in 50 μl of ice-cold nuclear extraction reagent (1× protease inhibitor mixture; Roche Applied Science), incubated for 40 min on ice, and centrifuged (16,000 × g at 4 °C for 10 min). The supernatant was used as nuclear extract. Extracts obtained by this procedure generally had less than 10% contamination between nuclear and cytoplasmic fractions. The protein concentration was determined with a commercial protein assay reagent (Bio-Rad).Western Blot Analysis—70 μg of whole cell lysate or 25 μg of cytoplasmic and nuclear extracts were loaded per lane onto a 7.5% or 10% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes after electrophoresis. The membranes were probed for detection of HIF-1α (or HIF-2α), PHD2, α-tubulin, and Lamin A or PHD3, PHD1, and endoplasmatic reticulum marker Grp78 (glucose-regulated protein 78), and Lamin A, respectively. As primary antibodies the following mouse monoclonal antibodies were used: anti-HIF-1α (diluted 1:750; Transduction Laboratories, San Diego, CA), anti-α-tubulin (diluted 1:750; Santa Cruz Biotechnology, Heidelberg, Germany), anti-Grp78 (diluted 1:250; Abcam, Cambridge, UK), and anti-Lamin A (diluted 1:750; Abcam). The mouse monoclonal anti-PHD3 antibody (diluted 1:20) was a kind gift from P. Ratcliffe (Oxford, UK) and has been characterized previously (25Appelhoff R.J. Tian Y.M. Raval R.R. Turley H. Harris A.L. Pugh C.W. Ratcliffe P.J. Gleadle J.M. J. Biol. Chem. 2004; 279: 38458-38465Abstract Full Text Full Text PDF PubMed Scopus (798) Google Scholar). Rabbit polyclonal antibodies used as primary antibody were anti-HIF-2α (diluted 1:1000; Abcam), anti-PHD2 (diluted 1:3000; Abcam), and anti-PHD1 (diluted 1:1000; Abcam). Horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:1,000,000 dilution; Sigma) were used as secondary antibodies. The ECL Western blotting system (Amersham Biosciences) was used for detection.To obtain the relative PHD2 protein amounts, the immunoblot signals were quantified by densitometry. PHD2 protein amounts of whole cell lysates and cytosolic extracts were normalized to the respective α-tubulin signal, whereas PHD2 amounts of nuclear extracts were normalized to the nuclear envelope marker Lamin A.PHD Activity Assay—The enzymatic activity of whole cell lysate or of cytoplasmic and nuclear extracts was determined by an in vitro hydroxylation assay as described before for recombinant PHDs (32Oehme F. Jonghaus W. Narouz-Ott L. Huetter J. Flamme I. Anal. Biochem. 2004; 330: 74-80Crossref PubMed Scopus (34) Google Scholar). Whole cell lysates, cytosolic, and nuclear extracts were dialyzed using the Slide-A-Lyzer dialysis cassettes (Pierce) to exclude effects on enzyme activity caused by buffer components. Briefly, 10 ng of biotinylated HIF-1α-derived peptides (amino acids 556–574) either wild type (wt), the P564A mutant, or the corresponding HIF-2α-derived peptide (amino acids 523–542, including the target proline 531) were bound to NeutrAvidin-coated 96-well plates (Pierce). Hydroxylase reactions were carried out by using 1 μg/μl protein of whole cell lysate, cytoplasmic, or nuclear extracts in the presence of 100 μm 2-oxoglutarate, 1 μm FeSO4, and 2 mm ascorbate for 1 h at room temperature. After washing, 0.022 μg/μl thioredoxin-tagged pVHL in complex with elongins B and C (VBC) was allowed to bind to the hydroxylated peptide. Recombinant VBC was expressed in Escherichia coli and purified as described (33Tan S. Protein Expression Purif. 2001; 21: 224-234Crossref PubMed Scopus (177) Google Scholar). Bound VBC complex was detected by rabbit anti-thioredoxin antibodies and secondary horseradish peroxidase-coupled anti-rabbit antibodies (Sigma) using the 3,3′, 5,5′-tetramethyl-benzidine substrate kit (Pierce). The peroxidase reaction was stopped by adding H2SO4, and absorbance was determined at 450 nm in a microplate reader. Each experiment was calibrated to an internal standard curve using hydroxyproline (Pro564)-containing HIF-1α peptide (amino acids 556–574) and VBC complex. The assay allowed the detection of a hydroxylated HIF-1α substrate peptide in a linear concentration range between 1 and 40 nm. For oxygen titration experiments, the hydroxylation reaction was performed in a hypoxic work station (Invivo2 400; Ruskinn, Leicester, UK). All of the reagents and solutions were allowed to equilibrate to the indicated oxygen concentration. PHD activity is given as the amount of HIFα peptide that was hydroxylated by 1 μg protein of the respective cell extract (fmol/μg). Normalized PHD2 activity was determined by dividing the PHD activity to the relative PHD2 protein amounts obtained by densitometry of immunoblots as described above.Short Interfering RNA (siRNA) Treatment—For siRNA experiments, the cells were seeded at 30–50% confluence and transfected with double-stranded antisense oligonucleotides specific for PHD2 (sense, 5′-CAAGGUAAGUGGAGGUAUAUU-3′; antisense, 5′-UAUACCUCCACUUACCUUGUU-3′; GenBank™ accession number EGLN1 NM_022051), PHD3 (sense, 5′-GUACUUUGAUGCUGAAGAAUU-3′; antisense, 5′-UUCUUCAGCAUCAAAGUACUU-3′; GenBank™ accession number EGLN3 NM_022073), or PHD1 (sense, 5′-ACAGAAAGGUGUCCAAGUAUU-3′; antisense, 5′-UACUUGGACACCUUUCUGUUU-3′). The cells were transfected twice with siRNA directed against PHD2 (10 nm) or PHD3 (2.5 nm) or PHD1 (2.5 nm) at 24 and 48 h using Oligofectamine (Invitrogen) according to the manufacturer's instructions. siRNA against Luciferase was used as a nontargeting control, and mock control cells were transfected without oligonucleotides under the same conditions. After transfection the cells were grown for 24 h and then exposed to normoxic or hypoxic atmosphere with or without GSNO for the indicated time. The cells were lysed and whole cell lysates or cytosolic and nuclear extracts were obtained as described above.Immunofluorescence and Microscopy—The cells were grown on poly-d-lysine-coated glass coverslips in 24-well dishes overnight. Subconfluent cells were subjected to hypoxia for 6 h, fixed by ice-cold methanol/acetone (1:1) for 10 min on ice, and blocked with 3% bovine serum albumin in phosphate-buffered saline. As primary antibody the rabbit polyclonal anti-PHD2 (diluted 1:200; Abcam) was used, and as secondary antibody an Alexa-532-conjugated goat anti-mouse IgG (1:400; Molecular Probes) antibody was used. The coverslips were mounted on the slides with Mowiol (Calbiochem, Bad Soden, Germany). Three-dimensional visualization was performed with laser scanning microscope (PCM2000; Nikon) equipped with a two photon Coherent Mira 900M laser as described before (34Liu Q. Berchner-Pfannschmidt U. Moller U. Brecht M. Wotzlaw C. Acker H. Jungermann K. Kietzmann T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4302-4307Crossref PubMed Scopus (141) Google Scholar, 35Berchner-Pfannschmidt U. Wotzlaw C. Merten E. Acker H. Fandrey J. Biol. Chem. 2004; 385: 231-237Crossref PubMed Scopus (23) Google Scholar). Yellow fluorescence of Alexa-532 was collected through a 585-nm long pass filter. The objective lens was a 63× NA 1.40 Plan-Apochromat. Fluorescence intensities were visualized in false colors as indicated.RESULTSInduction of Endogenous PHD Activity under Hypoxia or NO Is Specifically Caused by PHD2—Incubation of U-2OS cells under hypoxia or NO lead to induction of PHD2 protein levels (1.8-, 2.0-, and 2.2-fold compared with controls; Fig. 1A). We have previously shown that this induction of PHD2 protein accumulation is a result of HIF-1-dependent gene expression of PHD2 in response to hypoxia and NO (26Berchner-Pfannschmidt U. Yamac H. Trinidad B. Fandrey J. J. Biol. Chem. 2007; 282: 1788-1796Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In consequence, HIF-1α was reduced by increased PHD2 under hypoxia and NO compared with hypoxia alone (Fig. 1A). To test for PHD activity under these conditions, a PHD activity assay (32Oehme F. Jonghaus W. Narouz-Ott L. Huetter J. Flamme I. Anal. Biochem. 2004; 330: 74-80Crossref PubMed Scopus (34) Google Scholar) was established for cell extracts using a HIF-1α(556–574) peptide containing the target proline 564 as substrate. As shown in Fig. 1B, PHD activity was low in normoxic cells but induced in hypoxic cells (2.6-fold induction) or cells that were treated with NO both under normoxia (3.6-fold induction) or hypoxia (5.8-fold induction). Because this PHD activity assay was not fully established for cell extracts, we tested a P564A mutated HIF-1α peptide to show that PHD activity measured in cell extracts was completely dependent on the presence of the target proline (Fig. 1B, mut).The induction of PHD activity in response to hypoxia or NO closely correlated with the induction of the respective PHD2 protein amounts (Fig. 1A). To verify that these changes were responsible for the increase in PHD activity, PHD2 expression was suppressed by specific siRNA. siRNA against PHD2 completely abolished the PHD activity in extracts from cells incubated under normoxia, hypoxia, or NO (Fig. 1C). It has previously been shown that the HIF-1α peptide used in our PHD activity assay can be hydroxylated by all recombinant PHD isoenzymes in an O2-dependent manner (13Hirsila M. Koivunen P. Gunzler V. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2003; 278: 30772-30780Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 14Koivunen P. Hirsila M. Kivirikko K.I. Myllyharju J. J. Biol. Chem. 2006; 281: 28712-28720Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 15Ehrismann D. Flashman E. Genn D.N. Mathioudakis N. Hewitson K.S. Ratcliffe P.J. Schofield C.J. Biochem. J. 2007; 401: 227-234Crossref PubMed Scopus (169) Google Scholar). We therefore tested whether endogenous PHD3 or PHD1 contributed to the overall PHD activity of U-2OS cells by silencing PHD3 or PHD1 expression using siRNA. Although PHD3 protein levels were induced by hypoxia or NO treatment, the suppression of PHD3 had no impact on PHD activity (Fig. 1D). Furthermore PHD1 protein levels were neither affected by hypoxia nor NO, and the suppression of PHD1 had no major effect on total PHD activity (Fig. 1E). U-2OS cells were found to hardly express HIF-2α (supplemental Fig. S1). Nevertheless, we wondered whether endogenous PHDs would hydroxylate a HIF-2α peptide in our PHD activity assay. Suppression of PHD2 by siRNA completely abolishes PHD activity, whereas suppression of PHD3 or PHD1 had no impact on hydroxylation of HIF-2α peptide (supplemental Fig. S2). In summary, the data indicate that PHD2 is the key oxygen sensor enzyme for HIFα regulation in U-2OS cells.Because only PHD2 was responsible for total PHD activity, we further analyzed PHD2 activity by normalizing PHD activity to PHD2 protein amounts. Normalized PHD2 activity was induced by hypoxia (1.5-fold) and NO (2.0-fold under normoxia, 2.6-fold under hypoxia) when compared with normoxic cells (Fig. 1F). Taken together, hypoxia and NO induce PHD activity by induction of PHD2 protein. The additional induction of PHD2 activity observed after normalization indicate that the induction of activity cannot be explained by changes in protein amounts alone and suggest the action of an additional mechanism.PHD2 Activity Is Prominently Induced in Cell Nuclei—To test for the subcellular distribution of PHD2 in U-2OS cells, we performed immunostaining and fluorescence microscopy. We found that PHD2 was more present in the nucleus than in the cytoplasm under normoxia, hypoxia, or NO (Fig. 2A). To further analyze whether the nuclear PHD2 contributes to the cellular PHD activity, we isolated cytoplasmic and nuclear extracts. Western blot analysis of these extracts confirmed that PHD2 was more abundant in the nucleus than in the cytoplasm under normoxia as well as after induction by hypoxia or NO (1.4–2.4-fold induction in the nuclear fractions compared with 1.0–1.7-fold induction in cytosolic fractions; see Fig. 2B). Likewise PHD3 was found in both compartments with increased protein levels upon hypoxia and NO treatment. PHD1 was localized mainly in the nucleus and was affected neither by hypoxia nor by NO (Fig. 2B). HIF-1α was not detectable under normoxic conditions but strongly accumulated in cell nuclei under hypoxia; this hypoxic induction was reduced by after NO treatment (Fig. 2B), corroborating previous data (26Berchner-Pfannschmidt U. Yamac H. Trinidad B. Fandrey J. J. Biol. Chem. 2007; 282: 1788-1796Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).FIGURE 2PHD2 activity is prominently induced in cell nuclei. U-2OS cells were incubated in the presence (+) or absence (-) of 250 μm GSNO under NOX or under HOX conditions for 6 h. A, cells were immunostained and imaged by fluorescence microscopy to show subcellular localization of PHD2 (upper row). Three-dimensional visualization of PHD2 distribution within a single cell is shown in false color as indicated by the color bar (lower row). B, cytoplasmic or nuclear extracts were analyzed for expression of HIF-1α, PHD1, 2, and 3.α-Tubulin (α-Tub) serves as a cytoplasma marker, Grp78 serves as a marker for the endoplasmatic reticulum, and Lamin A serves as a nuclear envelope marker protein. The numbers indicate fold induction of PHD2 protein amounts compared with cytosolic extracts of cells incubated under normoxia. Immunoblots shown are representative of at least three independent experiments. C, cytosolic or nuclear extracts were subjected to PHD activity assay using the HIF-1α(556–574) peptide in the presence of 210 μm O2. The numbers above the bars indicate fold induction of PHD activity compared with cytosolic extracts of normoxic cells. Data are the means of two independent experiments. D and E, PHD2 was suppressed by transfecting U-2OS cells with the specific siRNA duplexes as described in the method section. siRNA directed against luciferase (Luc) serves as nontarget control. To induce PHD activity maximally, the cells were incubated in the presence of 250 μm GSNO under hypoxia for 6 h, and cytosolic and nuclear extracts were obtained. D, cytosolic or nuclear extracts were subjected to PHD activity assay using either the wild type (proline(564); wt) or mutated (alanine 564; mut) HIF-1α(556–574) peptide. E, cytoplasmic (C) or nuclear (N) extracts were analyzed for expression of HIF-1α and PHD2 by Western blotting. α-Tubulin serves as a cytoplasma marker, and Lamin A is a nuclear envelope marker protein. F, normalized PHD2 activity was obtained by dividing the PHD activity to the PHD2 protein amounts quantified by densitometry of immunoblots. Normalized PHD2 activity is given as fold induction compared with cytosolic extracts of cells incubated under normoxia. The data are the means of two independent experiments

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