Nitric Oxide Modulates Oxygen Sensing by Hypoxia-inducible Factor 1-dependent Induction of Prolyl Hydroxylase 2
2006; Elsevier BV; Volume: 282; Issue: 3 Linguagem: Inglês
10.1074/jbc.m607065200
ISSN1083-351X
AutoresUtta Berchner‐Pfannschmidt, Hatice Yamac, Buena Trinidad, Joachim Fandrey,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoThe 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. 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. Shortage of oxygen, hypoxia, requires the initiation of adaptive mechanisms of cells to either improve transport of oxygen to tissues or to ensure sufficient ATP generation through glycolytic pathways (1Wenger R.H. Stiehl D.P. Camenisch G. Sci. STKE 2005. 2005; : re12Google Scholar). Central for the adaptation to hypoxia is the transcription factor hypoxia-inducible factor-1 (HIF-1), 2The abbreviations used are: HIF, hypoxia-inducible factor; E3, ubiquitin-protein isopeptide ligase; NO, nitric oxide; PHD, prolyl hydroxylase; GSNO, S-nitrosoglutathione; Hyp-564, hydroxylated proline 564 of HIF-1α; siRNA, short interfering RNA; CREB, cAMP-response element-binding protein. which up-regulates the expression of genes in control of angiogenesis, erythropoiesis, and glycolysis. HIF-1 is composed of an O2-regulated α-subunit (HIF-1α) and a constitutive β-subunit (HIF-1β). 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Alternatively, the inhibitory effect of NO on hypoxia-induced HIF-1α was explained by increased NO-derived species and/or reactive oxygen species that contribute to destabilization of HIF-1α by reactivation of PHD activity (36Wellman T.L. Jenkins J. Penar P.L. Tranmer B. Zahr R. Lounsbury K.M. FASEB J. 2003; 18: 379-381PubMed Google Scholar, 37Callapina M. Zhou J. Schnitzer S. Metzen E. Lohr C. Deitmer J.W. Brune B. Exp. Cell Res. 2005; 306: 274-284Crossref PubMed Scopus (59) Google Scholar, 38Kohl R. Zhou J. Brune B. Free Radical Biol. Med. 2006; 40: 1430-1442Crossref PubMed Scopus (64) Google Scholar). In addition, calcium and calpain were found to contribute to the degradation of HIF-1α by NO under hypoxia (39Zhou J. Kohl R. Herr B. Frank R. Brune B. Mol. Biol. Cell. 2006; 17: 1549-1558Crossref PubMed Scopus (52) Google Scholar). Interestingly, NO increased the activity of PHDs that were inhibited by hypoxia-mimicking agents like CoCl2 (40Wang F. Sekine H. Kikuchi Y. Takasaki C. 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Cell Biol. 2002; 80: 421-426Crossref PubMed Scopus (159) Google Scholar), we studied the role of NO in regulating PHD expression and activity of HIF-1α. Herein we report a biphasic effect of NO on HIF-1α by (i) early induction through inhibition of PHD activity and (ii) later reduction by increased PHD2 protein amounts. Independently from other proposed mechanisms, we identified the induction of PHD2 as an underlying mechanism of NO-induced degradation of HIF-1α. Reagents—S-Nitrosoglutathione (GSNO) was synthesized as described previously (24Sandau K.B. Fandrey J. Brune B. Blood. 2001; 97: 1009-1015Crossref PubMed Scopus (226) Google Scholar). Cell Culture—The human osteosarcoma cells (U2OS) were a kind gift from J. M. Gleadle and P. Ratcliffe (Oxford, UK). U2OS cells 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, 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. In case of experiments where GSNO or MG132 were added under hypoxic conditions (1% O2, 94% N2,5%CO2)a hypoxic work station (Invivo2 400, Ruskinn, Leicester, UK) was used. Western Blot Analysis—Whole cell protein extracts were prepared from 35-mm dishes of cells that were about 80% confluent. 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. The protein concentration was determined with a commercial protein assay reagent (Bio-Rad). 70 μg of protein/lane were loaded onto a 7.5% or 10% SDS-polyacrylamide gel and after electrophoresis blotted onto nitrocellulose membranes. As primary antibodies, a mouse monoclonal anti-HIF-1α (diluted 1:750; Transduction Laboratories, San Diego, CA), a mouse monoclonal anti-α-tubulin (diluted 1:750; Santa Cruz Biotechnology, Heidelberg, Germany), a rabbit polyclonal anti-PHD2 (diluted 1:3000; Abcam, Cambridge, UK), and a rabbit polyclonal anti-PHD1 (diluted 1:1000; Abcam, Cambridge, UK) were used. The mouse monoclonal anti-PHD3 antibody (diluted 1:20) and the rabbit polyclonal antibody against hydroxylated proline 564 of HIF-1α (Hyp-564; diluted 1:1000) were a kind gift from P. Ratcliffe and have been described and characterized previously (14Appelhoff 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 (824) Google Scholar, 42Kageyama Y. Koshiji M. To K.K.W. Tian Y.M. Ratcliffe P.J. Huang L.E. FASEB J. 2004; 18: 1028-1030Crossref PubMed Scopus (62) Google Scholar). Horseradish peroxidase-conjugated goat anti-mouse IgG or goat anti-rabbit IgG (1:1,000,000 dilution; Sigma) were used as secondary antibodies. ECL Western blotting system (Amersham Biosciences) was used for detection. Reverse Transcription and Quantitative Real Time PCR— Total RNA was extracted using the guanidinium isothiocyanate method from 6-well dishes as described previously (43Stolze I. Berchner-Pfannschmidt U. Freitag P. Wotzlaw C. Rossler J. Frede S. Acker H. Fandrey J. Blood. 2002; 100: 2623-2628Crossref PubMed Scopus (64) Google Scholar). 1 μg total RNA was reverse transcribed with oligo(dT) and Moloney murine leukemia virus reverse transcriptase (Promega). Human PHD1, -2, and -3 cDNAs and the cDNA of the housekeeping gene 60 S acidic ribosomal protein was quantified by real time PCR using the qPCR™ master mix for SYBR Green I (Eurogentec, Verviers, Belgium) and the GeneAmp5700 sequence Detection System (PerkinElmer Life Sciences). The PCRs were set up in a final volume of 25 μl with 0.5 μl of cDNA in 1× reaction buffer with SYBR Green I, 10 pmol of forward primer, and 10 pmol of reverse primer. The primers used were as follows: human PHD2, forward (5′-GCACGACACCGGGAAGTT-3′) and reverse (5′-CCAGCTTCCCGTTACAGT-3′); human PHD3, forward (5′-GGCCATCAGCTTCCTCCTG-3′) and reverse (5′-GGTGATGCAGCGACCATCA-3′); human PHD1, forward (5′-GGCGATCCCGCCGCGC-3′) and reverse (5′-CCTGGGTAACACGCCCCAGCTTCCCGTACAGT-3′), and human 60 S acidic ribosomal protein, forward (5′-ACGAGGTGTGCAAGGAGGGC-3′) and reverse (5′-GCAAGTCGTCTCCCATCTGC-3′). The PCR amplification profile was as follows: 10 min at 95 °C followed by 30 cycles 15 s at 95 °C and 1 min at 60 °C. Agarose gel electrophoresis, purification, and DNA sequencing confirmed the identity of the PCR products. 10-Fold dilutions of purified PCR products starting at 1 pg to 0.1 fg were used as standards. The quantified cDNA of the PHDs were normalized to cDNA of the 60 S acidic ribosomal protein and expressed as normalized PHD mRNA level. All reverse transcription-PCRs were done in triplicate from RNA from three separate culture dishes. Short Interfering RNA (siRNA) Treatment—For siRNA experiments, cells were 30–50% confluent and transfected with siRNAs using Oligofectamine (Invitrogen) according to the manufacturer's instructions. siRNA sequence targeting HIF-1α has been described previously (44Berchner-Pfannschmidt U. Petrat F. Doege K. Trinidad B. Freitag P. Metzen E. de Groot H. Fandrey J. J. Biol. Chem. 2004; 279: 44976-44986Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Sequences designed to suppress expression of 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), and luciferase (siControl nontargeting siRNA) were purchased from Dharmacon. Cells were transfected once with siRNA directed against HIF-1α (100 nm) at 24 h or twice with siRNA directed against PHD2 (10 nm) or PHD3 (2.5 nm) at 24 and 48 h. Mock control cells were subjected to transfection procedure without oligonucleotides under the same conditions. After transfection, cells were grown for 24 h and then exposed to normoxic or hypoxic atmosphere for the indicated time. Cells were lysed, and whole cell lysates were extracted as described above. Immunofluorescence and Microscopy—U2OS cells were grown on poly-d-lysine-coated glass coverslips in 24-well dishes overnight. Subconfluent cells were transfected with siRNA as described above and 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 PBS. As primary antibody, the mouse monoclonal anti-HIF-1α (diluted 1:50; Transduction Laboratories, San Diego, CA), and as secondary antibody an Alexa-568-conjugated goat anti-mouse IgG (1:400, Molecular Probes, Inc., Eugene, OR) antibody was used as described previously (44Berchner-Pfannschmidt U. Petrat F. Doege K. Trinidad B. Freitag P. Metzen E. de Groot H. Fandrey J. J. Biol. Chem. 2004; 279: 44976-44986Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Coverslips were mounted on the slides with Mowiol (Calbiochem). Visualization was performed with a laser-scanning microscope (LSM 510, Zeiss, Oberkochen, Germany) equipped with a helium/neon laser. Red fluorescence of Alexa-568 was collected through a 585-nm long pass filter. The objective lens was a ×63 numerical aperture 1.40 Plan-Apochromat. Fluorescence intensities were visualized in false colors as indicated by the color table. Cell Viability—Toxicity of GSNO was excluded for 250 μm GSNO as judged from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (45Hansen M.B. Nielsen S.E. Berg K. J. Immunol. Methods. 1989; 119: 203-210Crossref PubMed Scopus (3353) Google Scholar). NO Induces HIF-1α Accumulation and Expression of PHD2 and -3—Considerable cell-specific and NO donor-dependent differences have been reported with respect to the effects on HIF-1α accumulation and PHD activity. We therefore first determined how 250 μm GSNO affects HIF-1α and PHD protein levels in U2OS cells. NO maximally induced HIF-1α accumulation under normoxic or hypoxic conditions within 2 h (Fig. 1, A and B). This effect appeared to be transient, since HIF-1α returned to basal levels within 4 h (Fig. 1A, 4 h). HIF-1α was maximally induced by hypoxia alone after 4 h and remained elevated during the course of the experiment. The addition of NO led to a decrease of HIF-1α, most prominently after 6 h of incubation (Fig. 1B, 6 h). Thus, NO had a biphasic effect on HIF-1α induction, with an early increase of HIF-1α followed by a later inhibition of its accumulation. PHD1 and PHD2 protein were already found in substantial amounts under normoxia, whereas PHD3 was hardly detectable (Fig. 1, A and B, 0 h). PHD2 and PHD3 protein levels were induced by NO under normoxia, particularly at 4 and 6 h for PHD2 and at 4 h for PHD3 (Fig. 1A), respectively. Under hypoxia, NO induced PHD2 and PHD3 at 2, 4, and 6 h, whereas under hypoxia alone induction of PHD2 and PHD3 was observed at 6 and 8 h (Fig. 1B). PHD1 protein levels were unaffected both by NO or hypoxia (Fig. 1, A and B). Thus, the observed decrease in HIF-1α accumulation during the late phase of NO treatment was correlated with an induction of PHD2 and PHD3 levels (Fig. 1, A and B, 4–8 h). The same effects on HIF-1α and PHD2 levels were observed in a neuroblastoma cell line, SH-SY5Y (data not shown). In addition, similar effects were obtained with the NO donor spermine-NONOate and DETA-NO (data not shown) but with slightly slower kinetics according to the longer lasting release of NO (46Dehne N. Li T. Petrat F. Rauen U. de Groot H. Biol. Chem. 2004; 385: 341-349Crossref PubMed Scopus (9) Google Scholar). PHD2 and PHD3 are known HIF-1 target genes (16Metzen E. Stiehl D.P. Doege K. Marxsen J.H. Hellwig-Burgel T. Jelkmann W. Biochem. J. 2005; 387: 711-717Crossref PubMed Scopus (162) Google Scholar, 17Pescador N. Cuevas Y. Naranjo S. Alcaide M. Villar D. Landazuri M.O. Del Peso L. Biochem. J. 2005; 390: 189-197Crossref PubMed Scopus (167) Google Scholar). We therefore studied if the NO-dependent induction of HIF-1α in the early phase of NO treatment is reflected by corresponding changes of the PHD2 and -3 mRNA levels using quantitative real time PCR. Transcript levels of both genes were increased after 2 and 4 h of NO treatment under normoxia or in addition to hypoxia (Fig. 2, A–D). After 8 h of NO treatment, however, PHD2 and PHD3 mRNA levels returned to the levels of the normoxic or hypoxic controls. As with PHD1 protein, PHD1 mRNA levels were neither affected by NO nor by hypoxia. NO-induced PHD2 and -3 Expression Is Strictly HIF-1α-dependent—The early increase and late decrease in PHD2 and -3 mRNA levels reflect the time course of the HIF-1α regulation by NO (see Fig. 1). We therefore studied the role of HIF-1α in the induction of PHD2 and -3 under NO treatment by suppression of HIF-1α using siRNA. siRNA directed against HIF-1α completely prevented the induced accumulation of HIF-1α protein by NO or hypoxia (Fig. 3A), whereas both the nontarget control siRNA directed against luciferase and the mock control showed normal induction of HIF-1α. Suppression of HIF-1α by using siRNA abolished the induction of PHD2 and -3 protein by NO, which indicates that NO induced PHD2 and -3 expression, is strictly HIF-1α-dependent in U2OS cells (Fig. 3B). PHD Activity is Transiently Inhibited by NO—NO has been found to inhibit the activity of PHD enzymes in vitro (28Metzen E. Zhou J. Jelkmann W. Fandrey J. Brune B. Mol. Biol. Cell. 2003; 14: 3470-3481Crossref PubMed Scopus (362) Google Scholar). Indeed, HIF-1α accumulated within the first 2 h of NO treatment, but this effect disappeared at 4 h under normoxia and hypoxia (Fig. 1, A and B). To prove a transient inhibition of PHD activity by NO, the hydroxylation status of HIF-1α was assessed by Western blot using an antibody against Hyp-564 (42Kageyama Y. Koshiji M. To K.K.W. Tian Y.M. Ratcliffe P.J. Huang L.E. FASEB J. 2004; 18: 1028-1030Crossref PubMed Scopus (62) Google Scholar). For that purpose, proteasomal degradation of hydroxylated HIF-1α was inhibited by MG132. Treatment with dimethyloxalylglycine, a well known inhibitor of PHD activity (4Jaakkola P. Mole D.R. Tian Y.M. Wilson M.I. Gielbert J. Gaskell S.J. Kriegsheim A. Hebestreit H.F. Mukherji M. Schofield C.J. Maxwell P.H. Pugh C.W. Ratcliffe P.J. Science. 2001; 292: 468-472Crossref PubMed Scopus (4489) Google Scholar, 12Epstein A.C. 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 (2749) Google Scholar), abolished accumulation of Hyp-564 under normoxia, as expected (Fig. 4A). Likewise, NO decreased Hyp-564 compared with untreated controls under normoxia (Fig. 4B) or inhibited hydroxylation of HIF-1α in addition to hypoxia (Fig. 4C) after 2 h of incubation. In contrast, after 4 h of incubation with NO, Hyp-564 levels were induced under normoxia or at least returned to control levels under hypoxic conditions. Thus, PHD activity had returned after 4 h of NO treatment, indicating that inhibition of PHDs was a transient process. Under normoxia, PHD activity was apparently higher after 4 h of NO treatment, as indicated by increased amounts of Hyp-564. The transient inhibition of PHDs reflects the release kinetics of NO from GSNO as previously reported (24Sandau K.B. Fandrey J. Brune B. Blood. 2001; 97: 1009-1015Crossref PubMed Scopus (226) Google Scholar). We assumed that inhibition of PHDs requires the presence of NO and that the return of PHD activity was most likely due to exhausted NO release from GSNO. To test this hypothesis, we preincubated GSNO in complete medium on cells for 2 h and applied this medium to fresh cells for a further 2-h incubation. In comparison with "fresh" GSNO (plus sign in Fig. 5A), preincubated medium (plus sign with asterisk in Fig. 5A) no longer induced HIF-1α, neither under normoxia nor under hypoxia. Likewise, HIF-1α was reinduced during NO treatment for 4 h (Fig. 5B, 4 h), when fresh GSNO was reapplied for the last 2 h (Fig. 5B, 2 + 2), both under normoxia and hypoxia. The by-products of the NO release from GSNO, GSH and GSSG, did not affect accumulation of HIF-1α, and the NO scavenger carboxyl-PTIO prevented the induction of HIF-1α by NO (data not shown). Thus, the NO-dependent inhibition of PHDs was limited to the time when NO was released from the donor. NO-induced PHD Protein Levels Promote HIF-1α Degradation—To address the functional consequences of NO-induced PHD2 and -3 expression on HIF-1α, U2OS cells were pretreated with NO for 6 h. At this time point, direct inhibition of PHD activity by NO has already ceased (see Figs. 4 and 5); thus, effects of increased PHD2 and PHD3 protein levels may be expected. Pretreatment with NO under normoxia increased PHD2 and -3 levels and led to delayed accumulation of HIF-1α during a subsequent 2-h hypoxic incubation (Fig. 6A). This effect of PHD2 and -3 induction can also be seen in Fig. 1B by comparing HIF-1α accumulation with or without NO treatment under hypoxia for 6 h. To analyze the effect of PHD2 and -3 induction on the degr
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