Rapid Phosphorylation of Heterogeneous Nuclear Ribonucleoprotein C1/C2 in Response to Physiologic Levels of Hydrogen Peroxide in Human Endothelial Cells (2002)

Hydrogen peroxide (H2O2) has been implicated as a signaling agent in numerous signal transduction pathways in mammalian cells. However, to date, no sensor for low concentrations (<10 μm) of H2O2 has been identified. Using a functional proteomic approach, nuclear extracts from human umbilical vein endothelial cells were analyzed by two-dimensional PAGE with or without prior treatment with a low concentration of H2O2. A protein doublet with a molecular mass of 39–41 kDa and a pI of ∼5.0 was observed to be consistently altered by the treatment. Using proteolytic peptide mass fingerprinting, the protein was identified as heterogeneous nuclear ribonucleoprotein C1/C2, a nuclear restricted, pre-mRNA-binding protein. Upon two-dimensional PAGE, each heterogeneous nuclear ribonucleoprotein-C splice form was present as multiple spots because of differing levels of phosphorylation. Upon treatment with H2O2, there was an increase in phosphorylation at 10–20 min, which partially reversed by 30 min. Subsequently, at 60 min after treatment, a population of unphosphorylated protein was transiently present. The effects were observed with as little as 1 μmH2O2 and were maximal with 5–8 μm H2O2. The H2O2-stimulated phosphorylation was inhibited by catalase, but not by the transcriptional inhibitor actinomycin D. Hydrogen peroxide (H2O2) has been implicated as a signaling agent in numerous signal transduction pathways in mammalian cells. However, to date, no sensor for low concentrations (<10 μm) of H2O2 has been identified. Using a functional proteomic approach, nuclear extracts from human umbilical vein endothelial cells were analyzed by two-dimensional PAGE with or without prior treatment with a low concentration of H2O2. A protein doublet with a molecular mass of 39–41 kDa and a pI of ∼5.0 was observed to be consistently altered by the treatment. Using proteolytic peptide mass fingerprinting, the protein was identified as heterogeneous nuclear ribonucleoprotein C1/C2, a nuclear restricted, pre-mRNA-binding protein. Upon two-dimensional PAGE, each heterogeneous nuclear ribonucleoprotein-C splice form was present as multiple spots because of differing levels of phosphorylation. Upon treatment with H2O2, there was an increase in phosphorylation at 10–20 min, which partially reversed by 30 min. Subsequently, at 60 min after treatment, a population of unphosphorylated protein was transiently present. The effects were observed with as little as 1 μmH2O2 and were maximal with 5–8 μm H2O2. The H2O2-stimulated phosphorylation was inhibited by catalase, but not by the transcriptional inhibitor actinomycin D. heterogeneous nuclear ribonucleoprotein human umbilical vein endothelial cell isoelectric focusing 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid matrix-assisted laser desorption ionization Partially reduced oxygen species, often referred to as reactive oxygen species, include superoxide (O2·¯), hydrogen peroxide (H2O2), and hydroxyl radical (·OH). Of these species, H2O2 is the only one with the chemical stability required to establish significant steady-state concentrations in vivo (1Cadenas E. Davies K.J.A. Free Radic. Biol. Med. 2000; 29: 222-230Crossref PubMed Scopus (2370) Google Scholar). H2O2 has the added advantage of being small and uncharged, allowing it to freely diffuse across plasma membranes (2Ohno Y. Gallin J.I. J. Biol. Chem. 1985; 260: 8438-8446Abstract Full Text PDF PubMed Google Scholar). These properties of relative stability, neutrality, and small size make H2O2 an ideal candidate for a signaling molecule analogous to nitric oxide (·NO). It has been generally accepted that small quantities of H2O2 “leak” out of the mitochondria during oxidative phosphorylation. It has been estimated that 1–2% of all oxygen consumed in humans is transformed into H2O2 rather than water (3Boveris A. Chance B. Biochem. J. 1973; 134: 707-716Crossref PubMed Scopus (2096) Google Scholar). It has also been known for some time that in leukocytes, NADPH oxidase enzymatically produces O2·¯, which is then rapidly converted to H2O2 by both enzymatic and nonenzymatic routes (for review, see Ref. 4Segal A.W. Abo A. Trends Biochem. Sci. 1993; 18: 43-47Abstract Full Text PDF PubMed Scopus (567) Google Scholar). The activity of this enzyme is responsible for the respiratory burst as part of the inflammatory response. The catalytic component of this multisubunit complex is a heme-containing glycoprotein (gp91phox). An important recent advance in biology has been the demonstration that there are multiple isoforms of this hemoprotein and that some of these isoforms are expressed in human endothelial cells and in human vascular smooth muscle (for reviews, see Refs. 5Lambeth J.D. Cheng G. Arnold R.S. Edens W.A. Trends Biochem. Sci. 2000; 25: 459-461Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar and 6Griendling K.K. Sorescu D. Ushio-Fukai M. Circ. Res. 2000; 86: 494-501Crossref PubMed Scopus (2604) Google Scholar). The amount of O2·¯ and H2O2 produced by endothelial cells and vascular smooth muscle is considerably less than that produced by leukocytes, suggesting that the low-output enzymes in the vessel wall produce H2O2 for signaling rather than for cytostasis. This would be analogous to the ·NO systems in which an inducible high-output enzyme in macrophages generates ·NO for cytostasis and a constitutive low-output enzyme in endothelial cells generates·NO for signaling (for review, see Ref. 7Moncada S. Palmer R.M.J. Higgs E.A. Pharmacol. Rev. 1991; 43: 109-142PubMed Google Scholar). There is a large and growing list of growth factors, cytokines, and vasoactive modulators that stimulate the production of H2O2 and other reactive oxygen species within mammalian cells (8Bae Y.S. Kang S.W. Seo M.S. Baines I.C. Tekle E. Chock P.B. Rhee S.G. J. Biol. Chem. 1997; 272: 217-221Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar, 9Chae H.J. Kang J.S. Han J.I. Bang B.G. Chae S.W. Kim K.W. Kim H.M. Kim H.R. Immunopharmacol. Immunotoxicol. 2000; 22: 317-337Crossref PubMed Scopus (11) Google Scholar, 10Chen Q. Olashaw N. Wu J. J. Biol. 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Furthermore, the presence of a low concentration of H2O2 appears to be required for the proper functioning of numerous signal transduction pathways. In the absence of additional factors, low concentrations of H2O2 appear to mediate a “pro-life” signal to mammalian cells in culture. Studies in which H2O2 was applied to mammalian cells have shown that low concentrations of H2O2 (<10 μm) stimulate mitogenesis and/or promote survival in a wide variety of cell types, including endothelial cells (18Ohguro N. Fukuda M. Sasabe T. Tano Y. Br. J. Ophthalmol. 1999; 83: 1064-1068Crossref PubMed Scopus (62) Google Scholar, 19Ostrovidov S. Franck P. Capiaumont J. Dousset B. Belleville F. In Vitro Cell. Dev. Biol. Anim. 1998; 34: 259-264Crossref PubMed Scopus (15) Google Scholar, 20Yasuda M. Ohzeki Y. Shimizu S. Naito S. Ohtsuru A. Yamamoto T. Kuroiwa Y. Life Sci. 1999; 64: 249-258Crossref PubMed Scopus (200) Google Scholar, 21Burdon R.H. Free Radic. Biol. 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Mozena J.D. Chatterjee P. Engelhardt J.F. Zwacka R.M. Oberley L.W. Fang X. Spector A.A. Weintraub N.L. Circ. Res. 1999; 85: 524-533Crossref PubMed Scopus (188) Google Scholar). In contrast to the effects seen with low concentrations of H2O2, higher concentrations of H2O2 (50 μm to 1 mm) typically cause cells in culture to undergo growth arrest, apoptosis, and/or necrosis (for review, see Ref. 26Davies K.J.A. IUBMB Life. 1999; 48: 41-47Crossref PubMed Google Scholar). Currently, the molecular mechanisms by which low concentrations of H2O2 function in mammalian cells are poorly understood. In Escherichia coli and Salmonella, there is a transcription factor named Oxy-R that serves as a sensor for H2O2 (27Zheng M. Aslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (970) Google Scholar). Cysteines 199 and 208 of Oxy-R react with H2O2 to from a disulfide bond, resulting in the activation of the transcription factor. The reaction is fast, with a second order rate constant of 105m−1 s−1, allowing the transcription factor to be activated by low concentrations of H2O2 (28Aslund F. Zheng M. Beckwith J. Storz G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6161-6165Crossref PubMed Scopus (434) Google Scholar). Once activated by H2O2, Oxy-R stimulates transcription of the bacterial catalase gene. To date, no definitive Oxy-R-like H2O2 sensor has been identified in humans. In fact, there is virtually no information on how human cells respond biochemically to concentrations of H2O2 below 10 μm. Several signal transduction pathways in cultured mammalian cells have been reported to be activated by the application of H2O2, including tyrosine kinases (29Abe J. Berk B.C. J. Biol. Chem. 1999; 274: 21003-21010Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 30Hardwick J.S. Sefton B.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4527-4531Crossref PubMed Scopus (168) Google Scholar, 31Konishi H. 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Chem. 1998; 273: 32670-32678Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) and nuclear factor-κB (38Schreck R. Rieber P. Baeuerle P.A. EMBO J. 1991; 10: 2247-2258Crossref PubMed Scopus (3415) Google Scholar). However, in these circumstances, the concentration of H2O2 required for activation typically ranged from 50 μm to 1 mm, well above the concentrations that would expected to be routinely generated for signaling physiologically. These higher concentrations are best considered as oxidative stress. Thus, although low concentrations of H2O2 (<10 μm) have been shown to be involved in numerous signal transduction pathways and to independently stimulate mitogenesis and survival, there is currently no information on precisely how human cells respond biochemically to these low concentrations of H2O2. Here we have employed a functional proteomic approach to identify signaling pathways for sensing low physiologic levels of H2O2 in human endothelial cells. With this approach, it was determined that heterogeneous nuclear ribonucleoprotein (hnRNP)1C1/C2, a nuclear restricted, pre-mRNA-binding protein (for review, see Ref. 39Dreyfuss G. Matunis M.J. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1332) Google Scholar), is rapidly and reversibly phosphorylated in response to low concentrations of H2O2. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh human umbilical cords and cultured as described previously (40Gimbrone M.A. Prog. Hemostasis Thromb. 1976; 3: 1-28PubMed Google Scholar). HUVECs were prepared confluent in 10-cm dishes at passage 3 in Medium 199 (BioWhittaker, Inc.) containing 20% fetal calf serum, 100 μg/ml heparin (Sigma), and 50 μg/ml endothelial cell growth supplement (Biomedical Technologies, Inc.). Confluent endothelial cells were washed twice with Dulbecco’s modified Eagle’s medium (without phenol red; BioWhittaker, Inc.) and incubated in this medium in the absence of serum for 1 h at 37 °C. The cells were then incubated with the same medium in the absence or presence of varying concentrations of H2O2 (0–21 μm) for 60 min. The cells were washed twice with ice-cold phosphate-buffered saline (67 mm phosphate and 150 mm NaCl, pH 7.0) and harvested by scraping in Buffer A (10 mm Tris, 140 mm NaCl, and 1 mm EDTA, pH 8.0). The cells were then pelleted by spinning at 500 × g for 15 min. Nuclear extracts were prepared as described previously (41Slomiany B.A. Kelly M.M. Kurtz D.T. BioTechniques. 2000; 28: 938-942Crossref PubMed Scopus (20) Google Scholar). Briefly, the cells were resuspended in Buffer B (10 mm HEPES, 10 mm KCl, 750 μm spermidine, 150 μm spermine, 0.2 mm EDTA, 1 mmdithiothreitol, and 20 μg/ml phenylmethylsulfonyl fluoride, pH 7.9) containing 0.1% Nonidet P-40 and incubated on ice for 10 min. The crude nuclei were isolated by centrifugation at 16,000 ×g for 5 min. The crude nuclear pellet was resuspended in 600 μl of Buffer B and then underlaid with 300 μl of Buffer B containing 30% sucrose. The sample was centrifuged at 1300 ×g for 10 min. The nuclear pellet was resuspended in 60 μl of Buffer C (20 mm Tris, 75 mm NaCl, 0.5 mm EDTA, and 50% glycerol, pH 8.0) to which was added an equal volume of cold 0.8 m ammonium sulfate. The nuclear slurry was incubated on ice for 20 min and then centrifuged at 16,000 × g for 5 min. The supernatant was applied to a Bio-Spin 6 chromatography column (Bio-Rad) equilibrated with isoelectric focusing (IEF) sample buffer (9 m urea, 65 mm dithiothreitol, 1% CHAPS, and 0.1% Bio-Lyte 3/10 ampholyte (Bio-Rad)). Confluent HUVECs in Medium 199 containing fetal calf serum, heparin, and endothelial cell growth supplement were incubated in the absence or presence of varying concentrations of H2O2 (0–9 μm) for 0–90 min at 37 °C. In some experiments, the cells were treated with catalase (1000 units/ml) or actinomycin D (5 μg/ml), either without H2O2 or added 1 min prior to H2O2. The nuclear pellet was obtained as described for cell treatment Procedure 1. The nuclear pellet was resuspended in 100 μl of IEF sample buffer and then centrifuged at 16,000 × g for 15 min. The supernatant was applied to a Bio-Spin 6 chromatography column equilibrated with IEF sample buffer. HUVEC nuclear extracts were subjected to IEF using a PROTEAN IEF cell and 17-cm IPG Ready-Strips (pH 3–10 and 4–7; Bio-Rad). Upon completion of IEF, the IPG Ready-Strips were incubated first with equilibration buffer (375 mm Tris, 6 m urea, 2% SDS, and 20% glycerol, pH 8.8) containing 130 mmdithiothreitol for 10 min and then with equilibration buffer containing 135 mm iodoacetamide for 10 min. The strips were placed on 10% polyacrylamide gels and electrophoresed with a constant current of 20 mA/gel. In some experiments, the gels were then fixed and stained using Silver-Stain Plus (Bio-Rad) following the procedure supplied by the manufacturer. Stained gels were analyzed using the program PD-Quest (Bio-Rad) to detect changes in the protein spots upon treatment. The portion of the gel containing a protein spot of interest was excised using a razor blade, cutting as close to the spot as possible. The silver-stained spot was then destained by washing the gel spot with 15 mm potassium ferricyanide and 50 mm sodium thiosulfate until clear. The gel portion was washed three times with H2O, followed by two washes with 200 mmammonium bicarbonate and 50% acetonitrile. The gel portion was then dehydrated in a SpeedVac concentrator (Savant Instruments, Inc.) and rehydrated using 200 mm ammonium bicarbonate containing 0.5 μg of trypsin (Promega). The gel portion was incubated at 37 °C overnight. The reaction was quenched with 1% trifluoroacetic acid, and then the gel spot was washed twice with 60% acetonitrile in 0.1% trifluoroacetic acid. The quench and the two washes were pooled and concentrated to 30% of the initial volume in the SpeedVac concentrator. The sample was then absorbed onto a reverse-phase Zip-Tip (Millipore Corp.). The resin was washed with 0.1% trifluoroacetic acid, and the peptides were eluted with 60% acetonitrile in 0.1% trifluoroacetic acid. The eluate was then concentrated to 30% of the initial volume in the SpeedVac concentrator. The masses of the tryptic peptides were determined at the University of Michigan Protein Core Facility by matrix-assisted laser desorption ionization (MALDI) mass spectrometry using a VESTEC-2000 mass spectrometer. The peptide masses together with the apparent molecular mass and pI of the protein were used to search the Human Protein using the program Chem. 2000; PubMed Scopus Google Scholar). at polyacrylamide gels were onto at 0.2 A for h at °C. were with in buffer 150 mm NaCl, and 1% pH and then incubated with at and the membranes were incubated with Inc.) at In some experiments, the membranes were with serum in and at was used as the followed by Inc.). were using an Plus was using an Nuclear extracts from endothelial cells by cell treatment Procedure were applied to (Bio-Rad) equilibrated with 50 mm Tris, 100 10 mm and 1 mmdithiothreitol, pH The extracts were then incubated for h at 37 °C in the presence of either Inc.) or the sodium (20 mm) and sodium the were applied to equilibrated with IEF sample buffer and then subjected to two-dimensional PAGE and H2O2 concentrations of were determined using an of at E. Y. J. Immunol. PubMed Scopus Google Scholar). were on a 50 not were obtained from A functional proteomic approach was used to for nuclear that are altered by the application of low concentrations of H2O2 to In the procedure treatment Procedure confluent HUVECs were for 1 h and then treated with a low concentration of H2O2 for an additional Nuclear extracts were prepared from isolated nuclei by ammonium With this nuclear from HUVECs typically protein spots upon two-dimensional PAGE at pH 3–10 The of these spots were by the of low concentrations of H2O2. However, one of these protein spots was consistently altered by the application of H2O2 A and This spot as a doublet with molecular masses of and kDa and a pI of on the protein was present at three times the concentration of the protein the protein doublet as spots with a pI of after the application of H2O2, each protein the doublet as spots with apparent pI from to shown in there was significant of the with 1 μm H2O2, and the to be with The was with a portion of the protein present in the sample being altered to a higher pI 60 min after treatment with low concentrations of H2O2. peptide mass was on the portion of the two spots and present after cells with μmH2O2 for 1 h were pooled from a of a of × endothelial cells. mass are in Using the program the peptide masses were used to search the Human Protein spots with significant of S. J. Cell. Biol. 2000; PubMed Scopus Google Scholar). is a nuclear restricted, protein that is present as a in which and are splice differing by the presence of an additional in C.G. M.S. M. G. Proc. Natl. Acad. Sci. U. S. A. 86: PubMed Scopus Google Scholar, Cell. Biol. PubMed Scopus Google Scholar). This the doublet observed upon two-dimensional of the portion of the protein be for by the observed tryptic peptides However, of the portion of the protein was not suggesting that this portion of the protein be the of peptide spot peptide peptide in peptide masses are With in a masses are To the of H2O2 on the time was In initial it was observed that serum as used in the in the spot not all were in medium In addition, because has been reported to be to G. Choi Cell. Biol. PubMed Scopus Google Scholar), the protein was from the nuclei with by the two-dimensional in prior to treatment, as to three spots with pI of and The presence of the form at pI was and only the spots at pI and were present 10–20 min after treatment with μm H2O2, the spots were and a spot at pI This partially reversed by 30 and by min, a protein spot at pI This reversed by min. Thus, the apparent of is with at present in human endothelial cells. concentrations of H2O2 stimulate the of a form at 10–20 min, followed by the of a form at min. the of the form at pI min after was in the the concentration of this was determined by in there was significant formation of the form at pI with μm H2O2, and the to be with 5 Thus, the concentration for the of the form at pI min after is in with that observed for the of the form at pI min after To that the observed at 20 min was to H2O2 and not to the presence of the of catalase on the was the of catalase to the cells in the absence of H2O2 a in the of the of This that levels of H2O2 present during cell culture are to the pI of some of the added together with H2O2, catalase inhibited the to pI and at 20 min. Thus, the of H2O2 on the pI of is inhibited by a specific H2O2 The of is to as it is generated in the G. Matunis M.J. Pinol-Roma S. Burd C.G. Annu. Rev. Biochem. 1993; 62: 289-321Crossref PubMed Scopus (1332) Google Scholar). Thus, the of this protein seen 20 min after treatment be a or be the of rapidly To transcription is required for the 20 min after treatment, the of the transcriptional inhibitor actinomycin D was shown in the of the form at pI (20 min after H2O2 was not inhibited by actinomycin D. This that the production of is not required for the and that H2O2 stimulate the of It has been that is phosphorylated in cells J. Biol. Chem. Full Text PDF PubMed Google Scholar). The and of phosphorylation have not been The of spots present on the two-dimensional gels would be with containing to phosphate It would be expected that the spots on the would than the spots on the To that the of spots was to in