Cystathionine γ-Lyase Overexpression Inhibits Cell Proliferation via a H2S-dependent Modulation of ERK1/2 Phosphorylation and p21Cip/WAK-1

Cystathionine γ-lyase (CSE) is a key enzyme in the trans-sulfuration pathway. CSE uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we used CSE stably overexpressed HEK-293 cells to explore the effect of the CSE/H2S system on cell growth and proliferation. The overexpression of CSE resulted in increases in CSE mRNA levels, CSE proteins, and intracellular H2S production rates, as well as the inhibition of cell proliferation and DNA synthesis. These effects were accompanied by a sustained ERK activation and up-regulation of the cyclin-dependent kinase inhibitor p21Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21Cip/WAK-1, suggesting that ERK activation functions upstream of p21Cip/WAK-1 activation to initiate the CSE overexpression-induced cell growth inhibition. The antiproliferative effect of CSE is likely mediated by endogenously produced H2S because the H2S scavenger methemoglobin (10 μm) significantly decreased the H2S production rate and reversed the antiproliferative effect afforded by CSE. Exogenous H2S (100 μm) also inhibited cell proliferation. However, the other CSE-catalyzed products, ammonium and pyruvate, failed to inhibit cell proliferation. Methemoglobin also abolished the inhibitory effect of exogenous H2S on cell proliferation. Moreover, exogenous H2S induced a sustained ERK and p21Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival. Cystathionine γ-lyase (CSE) is a key enzyme in the trans-sulfuration pathway. CSE uses L-cysteine as a substrate to produce hydrogen sulfide (H2S). The CSE/H2S system has been shown to play an important role in regulating cellular functions in different systems. In the present study, we used CSE stably overexpressed HEK-293 cells to explore the effect of the CSE/H2S system on cell growth and proliferation. The overexpression of CSE resulted in increases in CSE mRNA levels, CSE proteins, and intracellular H2S production rates, as well as the inhibition of cell proliferation and DNA synthesis. These effects were accompanied by a sustained ERK activation and up-regulation of the cyclin-dependent kinase inhibitor p21Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21Cip/WAK-1, suggesting that ERK activation functions upstream of p21Cip/WAK-1 activation to initiate the CSE overexpression-induced cell growth inhibition. The antiproliferative effect of CSE is likely mediated by endogenously produced H2S because the H2S scavenger methemoglobin (10 μm) significantly decreased the H2S production rate and reversed the antiproliferative effect afforded by CSE. Exogenous H2S (100 μm) also inhibited cell proliferation. However, the other CSE-catalyzed products, ammonium and pyruvate, failed to inhibit cell proliferation. Methemoglobin also abolished the inhibitory effect of exogenous H2S on cell proliferation. Moreover, exogenous H2S induced a sustained ERK and p21Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival. The endogenous production of hydrogen sulfide (H2S) and its physiological functions, including membrane hyperpolarization and smooth muscle cell relaxation, place this gas in the family of gas transmitters, together with nitric oxide and carbon monoxide (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, 2Wang R. Antioxid. Redox Signal. 2003; 5: 493-501Crossref PubMed Scopus (429) Google Scholar). Two pyridoxal-5′-phosphate-dependent enzymes, cystathionine β-synthase (EC 4.2.1.22) and cystathionine γ-lyase (CSE) 1The abbreviations used are: CSE, cystathionine γ-lyase; cdk, cyclin-dependent kinase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; EGFP, enhanced green fluorescent protein; HEK, human embryonic kidney; MAPK, mitogen-activated protein kinase; PCR, polymerase chain reaction. (EC 4.4.1.1), are responsible for the endogenous production of H2S in mammalian tissues, which use L-cysteine as the main substrate (3Kery V. Bukovska G. Kraus J.P. J. Biol. Chem. 1994; 269: 25283-25288Abstract Full Text PDF PubMed Google Scholar, 4Erickson P.F. Maxwell I.H. Su L.J. Baumann M. Glode L.M. Biochem. J. 1990; 269: 335-340Crossref PubMed Scopus (112) Google Scholar, 5Stipanuk M.H. Beck P.W. Biochem. J. 1982; 206: 267-277Crossref PubMed Scopus (616) Google Scholar). Cystathionine β-synthase is a predominant H2S-generating enzyme in the brain and nervous system (6Eto K. Kimura H. J. Neurochem. 2002; 83: 80-86Crossref PubMed Scopus (71) Google Scholar), and CSE is mainly expressed in liver, kidney, and vascular smooth muscles (7Barber T. Triguero A. Martinez-Lopez I. Torres L. Garcia C. Miralles V.J. Vina J.R. J. Nutr. 1999; 129: 928-933Crossref PubMed Scopus (29) Google Scholar, 8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). Cystathionine β-synthase and CSE are important for the metabolism of sulfur-containing amino acids (e.g. cystathionine), as well as the production of H2S, ammonium, and pyruvate from L-cysteine. H2S inhibits cell proliferation (9Valitutti S. Castellino F. Musiani P. Ann. Allergy. 1990; 65: 463-468PubMed Google Scholar) and induces cell death predominantly by an apoptotic mechanism in polymorphonuclear cells (10Mariggio M.A. Minunno V. Riccardi S. Santacroce R. De Rinaldis P. Fumarulo R. Immunopharmacol. Immunotoxicol. 1998; 20: 399-408Crossref PubMed Scopus (57) Google Scholar). H2S treatment has also been shown to lead to nasal lesions and olfactory epithelial necrosis (11Brenneman K.A. Meleason D.F. Sar M. Marshall M.W. James R.A. Gross E.A. Martin J.T. Dorman D.C. Toxicol. Pathol. 2002; 30: 200-208Crossref PubMed Scopus (37) Google Scholar). On the other hand, H2S induces serum-independent cell cycle entry in rat intestinal epithelial cells and increases the fraction of colonic mucosa cells in the S phase (12Eplancke B. Gaskins H.R. FASEB J. 2003; 17: 1310-1312Crossref PubMed Scopus (149) Google Scholar, 13Hristl S.U. Eisner H.D. Dusel G. Kasper H. Scheppach W. Dig. Dis. Sci. 1996; 41: 2477-2481Crossref PubMed Google Scholar). However, little is known about the cellular consequences of an elevated CSE expression or about the associated increase in endogenously produced H2S. In the present study, we overexpressed the CSE gene using a highly effective expression system. The successful overexpression of CSE was confirmed by measuring the CSE protein contents, CSE mRNA expression level, and endogenous H2S production rate. Proliferation of the transfected HEK-293 cells was monitored. We determined whether the CSE overexpression-induced cellular changes were because of overproduced H2S. Finally, the effect of both the cyclin-dependent kinase (cdk) inhibitor p21Cip/WAK-1 and the ERK/mitogen-activated protein kinase (MAPK) pathway on the CSE/H2S system was examined. Our findings support the hypothesis that endogenously produced H2S may play a fundamental role in cell proliferation and survival. Cell Culture and Measurement of H2S Production—HEK-293 cells (American Type Culture Collection, Manassas, VA) were cultured at 37 °C in a humidified incubator with 95% air and 5% CO2 in minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Invitrogen), 100 units of penicillin, and 100 μg of streptomycin/ml. The cultured cells were subjected to gene transfection when they had grown to 70–80% confluence. The H2S production rate in CSE stably transfected HEK-293 cells was measured as described previously (8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). Cloning of CSE cDNA and Stable Transfection—PCR was used to amplify the open reading frame of CSE (GenBank™ accession number AY032875) from rat vascular tissues using the primers 5′-CGTCCCAGCATGCAGAAGAA-3′ and 5′-CAGTTATTCAGAAGGTCTGGCCC-3′. The amplified open reading frame of CSE was subcloned into TA cloning vector (PCR4-TOPO). Positive clone containing CSE open reading frame insert was sequenced to confirm the accuracy of the inserted CSE sequence. The constructs containing CSE cDNA were cleaved and subcloned into the mammalian expression vector pIRES2-EGFP (Clontech), which contained the human cytomegalovirus immediate early promoter/enhancers and the SV40 poly(A) signal. For stable transfection, the constructs were linearized with KpnI (MBI Fermentas) and then subjected to phenol-chloroform extraction and ethanol precipitation. Linearized constructs were mixed with a FuGENE 6 transfection reagent (Roche Applied Science) in a ratio of 1 μg to 3 μl in 100 μl of FBS-free minimal essential medium (14Cao K. Tang G. Hu D. Wang R. Biochem. Biophys. Res. Commun. 2002; 296: 463-469Crossref PubMed Scopus (43) Google Scholar). After incubating for 45 min at room temperature (20–22 °C), the mixture was added to HEK-293 cells in 2 ml of FBS-free minimal essential medium. After 48 h of transfection, the cells were trypsinized, counted, and replated at 1 × 105 cells/plate in 35-mm plates, which contained 500 μg/ml G418 for antibiotic selection. Mock (empty vector) transfection was also performed. In the pIRES2-EGFP vector, the EGFP gene (which encodes the enhanced green fluorescent protein) was expressed separately from the gene of interest and was used as a transfection marker. Non-transfected HEK-293 cells were included as the negative control for antibiotic selection. After 5 weeks of the antibiotic selective culturing, survival-transfected cells were harvested and grown to establish the sublines, which were subsequently examined for the presence of the CSE gene. Stable transfectants were used at passage numbers <15. Wild-type HEK-293 cells were maintained in identical conditions but without selection antibiotic G418 treatment. Western Immunoblotting—Cultured cells (3 × 106) were harvested and lysed in a lysis buffer (0.5 m EDTA, 1 m Tris-Cl, pH 7.4, 0.3 m sucrose, 1 μg/ml antipain hydrochloride, 1 mm benzamidine hydrochloride hydrate, 1 μg/ml leupeptin hemisulfate, 1 mm 1,10-phenanthroline monohydrate, 1 μm pepstatin A, 0.1 mm phenylmethylsulfonyl fluoride, and 1 mm iodoacetamide). The extracts were clarified by centrifugation at 14,000 × g for 15 min at 4 °C. SDS-PAGE and Western blot analysis were performed as described previously (15Sun X. Cao K. Yang G. Huang Y. Hanna S.T. Wang R. Biochem. Pharmacol. 2004; 67: 147-156Crossref PubMed Scopus (33) Google Scholar). The primary antibody dilutions were 1:1000 for phosphorylated or total ERK, p38 MAPK, and c-Jun NH -terminal kinase, 1:500 for p21Cip/WAF-1 and cyclin D1, and 1:5000 for β-actin. Horseradish peroxidase-conjugated secondary antibody was used at 1:5000. The immunoreactions were visualized by ECL and exposed to x-ray film (Kodak Scientific Imaging film, X-Omat Blue XB-1). Membranes were stripped by incubation in a buffer containing 100 mm β-mercaptoethanol, 2% SDS, and 62.5 mm Tris-HCl, pH 6.8. mRNA Collection and Reverse Transcription—Total cellular RNA from wild-type, mock, or CSE-transfected cells was harvested after the cells were plated for 48 h. Monolayers were rinsed twice with phosphate-buffered saline (pH 7.6), and RNA was collected using TRIzol reagent (Molecular Research Center, Inc., Cincinnati, OH). Contaminated DNA was removed using the DNA-free kit (Ambion, Austin, TX), and total RNA (2 μg) was reverse transcribed into cDNA with avian myeloblastosis virus reverse transcriptase using random hexamer primers according to the manufacturer's protocol (Roche Applied Science). Controls without reverse transcriptase were used to check for genomic DNA contamination in each sample. Real Time Quantitative PCR—Real time PCR was performed in an iCycler iQ apparatus (Bio-Rad) associated with the iCycler optical system software (version 3.1) using SYBR Green PCR Master Mix. All PCRs were performed in a 20-μl volume using 96-well optical grade PCR plates and optical sealing tape. Negative controls for this experiment were samples without a template. The cycling conditions were 95 °C for 90 s followed by 38 cycles of 95 °C for 10 s and 60 °C for 20 s. For quantification, the target gene was normalized to the internal standard gene β-actin. The primers of CSE (GenBank™ accession number AY032875) were 5′-AGCGATCACACCACAGACCAAG-3′ (sense, position and position These primers produced a of The primers of produced a of standard was with a of dilutions of total RNA transcribed to cDNA using the protocol to confirm the in the standard analysis was performed using a cycling that at 95 °C for 10 to °C for 15 and then to 95 °C in to confirm the of was determined by PCR on a mRNA was by using the P. V. G. A. D. Chem. 2004; PubMed Scopus Google Scholar, L.J. Huang J. 2003; 41: PubMed Scopus Google Scholar), is the the cycle of a target cDNA and an endogenous this the of the target normalized to an endogenous Measurement of was performed to the cell growth in CSE-transfected HEK-293 an number of cells (2 × were in 35-mm and then cells were using a The medium was 3 DNA was by the of number of cells in the plates (2 × were cultured in growth medium for 48 h. were with medium and then in the medium for h for After the cells were with 10% in minimal essential medium for 6 0.1 was added to each well in growth medium for 6 h. The medium was and the cells were with ml of phosphate-buffered saline containing 1 mm and 1 mm The cells were with ml of 5% for 10 and then the were lysed with ml of SDS, 0.1 of cell were into and mixed with 5 ml of was determined using a and was by with H2S gas to the H2S m at (8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). H2S was to different in cell and the pH of the medium was to The CSE antibody was by The protein signal-regulated kinase inhibitor and p21Cip/WAK-1 antibody were from antibody was from was from ammonium and pyruvate were from Horseradish peroxidase-conjugated antibody was from are expressed as and at were using the or analysis of followed by a analysis The of was at of CSE cDNA in HEK-293 cells were transfected with a CSE or an identical vector a cDNA insert as a control The transfection of HEK-293 cells with CSE cDNA or the control vector the of the In HEK-293 cells transfected with CSE cDNA or the control vector, green by green fluorescent protein was In green was in HEK-293 cells The expression of CSE was by Western blot time PCR and the H2S production rate. Western blot and time PCR analysis that transfection of CSE but of the resulted in increases in both CSE protein expression and CSE mRNA with HEK-293 cells and of HEK-293 cells with CSE cDNA also resulted in a increase in the H2S production rate with cells Proliferation and DNA in CSE-transfected HEK-293 whether overexpression of the CSE gene proliferation and DNA of HEK-293 cell growth were with or HEK-293 cells transfected with CSE cDNA a significantly cell growth rate on the the the number of cells was of the cells antiproliferative effect of CSE overexpression was also by the of shown in HEK-293 cells transfected with CSE cDNA had cells transfected with or cells of and of p21Cip/WAK-1 by CSE including ERK, p38 MAPK, and c-Jun kinase, an important role in the proliferation and of of HEK-293 cells with CSE but the resulted in a of phosphorylated ERK and p38 the of c-Jun kinase total of were cells with 10 μm U0126 or 20 μm p38 for h significantly decreased the expression of phosphorylated ERK and p38 p21Cip/WAF-1 is a inhibitory and cell cycle by with cyclin and or to a cell S. D. B. 1994; PubMed Scopus Google Scholar, J. M.W. A. PubMed Scopus Google Scholar). p21Cip/WAF-1 expression is an important for cell growth A. B. H. Biol. 17: PubMed Scopus Google Scholar, D. D. H. M. Biol. 17: PubMed Scopus Google Scholar, X. Huang D. Y. C. Wang J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). shown in the expression of p21Cip/WAF-1 significantly in CSE-transfected HEK-293 cells that in or the of activation with CSE p21Cip/WAF-1 we the effects of U0126 and on the expression of After incubating cells with 10 μm the expression of p21Cip/WAF-1 in CSE-transfected HEK-293 cells decreased to the level, μm) had little that p21Cip/WAF-1 induction by CSE overexpression may from activation of the ERK pathway. is an of and which the rate of cell cycle V. P. M. J. C. Res. 2003; PubMed Scopus (29) Google Scholar). p21Cip/WAF-1 inhibit cyclin by to cyclin D1, cyclin T. S. Y. M. Biol. 2004; PubMed Scopus Google Scholar). Our that cyclin expression was in of and U0126 and had little effect on cyclin expression of CSE on Cell Proliferation to the mechanism by which CSE overexpression inhibited cell we the effects of H2S, ammonium, and pyruvate, of CSE-catalyzed on cell HEK-293 cells were with 10% in the presence of H2S (100 DNA decreased However, 100 μm ammonium and 100 μm pyruvate failed to inhibit DNA we the effect of the H2S scavenger methemoglobin (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, T. Res. 2002; PubMed Scopus Google Scholar) on CSE cell growth inhibition. shown in methemoglobin at 10 μm but significantly reversed the antiproliferative effect of CSE HEK-293 cells with 10 μm methemoglobin for 1 h to 100 μm H2S significantly abolished the antiproliferative effect of H2S H2S production in cells by methemoglobin also that methemoglobin the endogenous H2S the role of exogenous H2S on and cell cycle protein we the of ERK and p21Cip/WAF-1 in HEK-293 cells after to 100 μm H2S. The expression of ERK and p21Cip/WAF-1 after incubating HEK-293 cells with H2S for 2 h that H2S likely the antiproliferative effect of H2S increases the ERK and p21Cip/WAK-1 The HEK-293 cells were cultured in the presence or of H2S (100 μm) for the and Western blot analysis was used to the activation of ERK and p21Cip/WAK-1 The the ratio of phosphorylated ERK optical to total ERK optical to from Our present that the CSE expression is an important for cell the endogenous production of H2S, the overexpression of CSE significantly inhibited cell proliferation. In this of ERK and p21Cip/WAF-1 were by H2S, to cell growth inhibition. CSE is a key enzyme of the trans-sulfuration which and L-cysteine. also uses L-cysteine as an substrate to H2S P.F. Maxwell I.H. Su L.J. Baumann M. Glode L.M. Biochem. J. 1990; 269: 335-340Crossref PubMed Scopus (112) Google Scholar). a CSE is expressed in a of mammalian cells and tissues, and to the main enzyme in the liver, kidney, and system (7Barber T. Triguero A. Martinez-Lopez I. Torres L. Garcia C. Miralles V.J. Vina J.R. J. Nutr. 1999; 129: 928-933Crossref PubMed Scopus (29) Google Scholar, 8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar). of in as which are by of and early of (9Valitutti S. Castellino F. Musiani P. Ann. Allergy. 1990; 65: 463-468PubMed Google Scholar, T. Res. 2002; PubMed Scopus Google Scholar, H. X. Yang F. L. Yang X. W. 2003; PubMed Scopus Google Scholar). on H2S on the the physiological of the the functions of H2S, including hyperpolarization of cell of smooth muscle and decreased (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, 2Wang R. Antioxid. Redox Signal. 2003; 5: 493-501Crossref PubMed Scopus (429) Google Scholar, K. Kimura H. J. Neurochem. 2002; 83: 80-86Crossref PubMed Scopus (71) Google Scholar, 8Zhao W. Zhang J. Lu Y. Wang R. EMBO J. 2001; 20: 6008-6016Crossref PubMed Scopus (1646) Google Scholar, R.A. J. 2004; PubMed Scopus Google Scholar, W. Wang R. J. Pharmacol. 2003; PubMed Scopus Google Scholar). is known about the effect of endogenous CSE/H2S on cell growth and proliferation. In the present study, we used CSE stably transfected HEK-293 cells to explore the effects of CSE overexpression on cell growth and proliferation. Our that the CSE overexpression intracellular H2S inhibition of cell and DNA 1 and CSE induced the expression of inhibitor p21Cip/WAF-1 sustained ERK suggesting a cell cycle The antiproliferative effect of CSE is likely mediated the of H2S because the H2S scavenger methemoglobin (1Wang R. FASEB J. 2002; 16: 1792-1798Crossref PubMed Scopus (1549) Google Scholar, T. Res. 2002; PubMed Scopus Google Scholar) and significantly reversed the antiproliferative effect of CSE Exogenous H2S also inhibited cell by decreased The other CSE products, ammonium and pyruvate, failed to inhibit cell proliferation. of HEK-293 cells with methemoglobin for 1 h to the of H2S abolished the antiproliferative effect of H2S Moreover, exogenous H2S induced sustained ERK and p21Cip/WAF-1 activation These findings support the hypothesis that endogenously produced H2S an important role in cell proliferation and survival. The mitogen-activated protein kinase a role in extracellular into growth and The ERK pathway is in the of cellular proliferation J. Sci. S. A. PubMed Scopus Google Scholar, C. J. A. M. V. D. 2004; PubMed Scopus Google Scholar, F. T. Y. S. T. J. 2003; PubMed Scopus Google Scholar), growth inhibition has also been T. Sci. S. A. PubMed Scopus Google Scholar, S. L. R. C. R. Biochem. 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Biol. 17: PubMed Scopus Google Scholar, D. D. H. M. Biol. 17: PubMed Scopus Google Scholar). p21Cip/WAF-1 inhibit cyclin activation and cyclin T. S. Y. M. Biol. 2004; PubMed Scopus Google Scholar). However, that cyclin was in CSE overexpression-induced cell growth inhibition. ERK and p21Cip/WAF-1 likely play in CSE cell growth is also that other that were are the sustained ERK activation and expression of p21Cip/WAF-1 to In this for the time that CSE cell proliferation a of ERK and p21Cip/WAK-1. the of the CSE/H2S system to inhibit cell growth that to CSE expression and H2S production may a in the treatment of CSE/H2S

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