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J. Biol. Chem., Vol. 279, Issue 47, 49199-49205, November 19, 2004

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Cystathionine -Lyase Overexpression Inhibits Cell Proliferation via a H₂S-dependent Modulation of ERK1/2 Phosphorylation and p21^Cip/WAK-1*

Guangdong Yang, Kun Cao¶, Lingyun Wu||**, and Rui Wang, Supported by an Investigator Award from CIHR

From the Department of Physiology, College of Medicine, The Cardiovascular Research Group and the ^||Department of Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada

Received for publication, August 6, 2004 , and in revised form, September 2, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cystathionine -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 p21^Cip/WAK-1. Blocking the action of ERK with U0126 inhibited the induction of p21^Cip/WAK-1, suggesting that ERK activation functions upstream of p21^Cip/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 p21^Cip/WAK-1 activation. These findings support the hypothesis that endogenously produced H₂S may play a fundamental role in cell proliferation and survival.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (1, 2). Two pyridoxal-5'-phosphate-dependent enzymes, cystathionine -synthase (EC 4.2.1.22 [EC] ) and cystathionine -lyase (CSE)¹ (EC 4.4.1.1 [EC] ), are responsible for the endogenous production of H₂S in mammalian tissues, which use L-cysteine as the main substrate (3–5). Cystathionine -synthase is a predominant H₂S-generating enzyme in the brain and nervous system (6), and CSE is mainly expressed in liver, kidney, and vascular smooth muscles (7, 8). Cystathionine -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 (9) and induces cell death predominantly by an apoptotic mechanism in polymorphonuclear cells (10). H₂S treatment has also been shown to lead to nasal lesions and olfactory epithelial necrosis (11). 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 (12, 13). 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 p21^Cip/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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (8).

Cloning of CSE cDNA and Stable Transfection—PCR was used to amplify the open reading frame of CSE (GenBank^TM accession number AY032875 [GenBank] ) 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 (14). 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 x 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 x 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 x g for 15 min at 4 °C. SDS-PAGE and Western blot analysis were performed as described previously (15). The primary antibody dilutions were 1:1000 for phosphorylated or total ERK, p38 MAPK, and c-Jun NH -terminal kinase, 1:500 for p21^Cip/WAF-1 and cyclin D1, and 1:5000 for -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 -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 -actin. The primers of CSE (GenBank^TM accession number AY032875 [GenBank] ) were 5'-AGCGATCACACCACAGACCAAG-3' (sense, position 432–453) and 5'-ATCAGCACCCAGAGCCAAAGG-3' (antisense, position 589–609). These primers produced a product of 178 bp. The primers of -actin (Ambion) produced a product of 295 bp. A standard curve was constructed with a series of dilutions of total RNA (Ambion) transcribed to cDNA using the protocol outlined above to confirm the same amplifying efficiency in the PCR. A standard melting curve analysis was performed using a thermal cycling profile that began at 95 °C for 10 s, increased to 55 °C for 15 s, and then ramped to 95 °C in one-degree increments to confirm the absence of primer dimers. Product size was determined by running PCR products on a 1.8% agarose gel. Relative mRNA quantification was calculated by using the arithmatic formula "2^-CT" (16, 17), where CT is the difference between the threshold cycle of a given target cDNA and an endogenous reference cDNA. Thus, this value yields the amount of the target normalized to an endogenous reference.

Measurement of Cellular Proliferation—Cell count was performed first to assay the cell growth curve in CSE-transfected HEK-293 cells. Briefly, an equal number of cells (2 x 10⁵) were seeded in 35-mm Petri dishes, and then cells were counted daily using a hemocytometer. The medium was changed every 3 days.

DNA synthesis was assessed by the level of [³H]thymidine incorporation. An equal number of cells seeded in the 24-well plates (2 x 10⁴ cells/well) were cultured in growth medium for 48 h. They were washed three times with serum-free medium and then incubated in the same medium for 24 h for synchronization. After the cells were stimulated with 10% FBS in minimal essential medium for 6 h, 0.1 µCi/ml [³H]thymidine was added to each well in growth medium for another 6 h. The medium was discarded, and the cells were washed three times with 0.5 ml of ice-cold phosphate-buffered saline containing 1 mM MgCl₂ and 1 mM CaCl₂. The cells were precipitated with 0.5 ml of 5% ice-cold trichloroacetic acid for 10 min, and then the precipitates were lysed with 0.5 ml of 0.1% SDS, 0.1 N NaOH. Four-hundred microliters of cell solution were put into scintillation vials and mixed with 5 ml of scintillation fluid. Radioactivity was determined using a liquid scintillation counter (Beckman LS3801).

Reagents and Chemicals—H₂S stock solution was freshly prepared by directly bubbling distilled water with pure H₂S gas (Praxair) to make the saturated H₂S solution (0.09 M at 30 °C) (8). H₂S stock solution was diluted to different concentrations in cell culture medium, and the pH of the medium was adjusted to 7.4.

The CSE antibody was kindly provided by Dr. N. Nishi (Kagawa Medical School). The MAPK antibody, ERK/MEK (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase) inhibitor U0126, and p21^Cip/WAK-1 antibody were obtained from New England Biolabs (Camarillo, CA). Cyclin D1 antibody was from Lab Vision Corporation (Fremont, CA). [³H]Thymidine was purchased from Amersham Biosciences. Methemoglobin, ammonium hydroxide, and sodium pyruvate were from Sigma. Horseradish peroxidase-conjugated goat anti-rabbit IgG antibody was from Bio-Rad.

Statistical Analysis—All data are expressed as means ± S.E. and represent at least three independent experiments. Statistical comparisons were made using the Student's t test or one-way analysis of variance followed by a post hoc analysis (Tukey test) where applicable. The level of significance was set at p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overexpression of CSE cDNA in HEK-293 Cells—HEK-293 cells were transfected with a CSE cDNA/pIRES2-EGFP construct or an identical empty vector lacking a cDNA insert as a control (mock). The transfection of HEK-293 cells with CSE cDNA or the control empty vector did not change the morphological characteristics of the cell. In HEK-293 cells transfected with CSE cDNA or the control empty vector, green fluorescence emitted by green fluorescent protein was observed. In contrast, green fluorescence was not detectable in wild-type HEK-293 cells (data not shown). The expression of CSE was verified by Western blot analysis, real time PCR analysis, and the H₂S production rate. Western blot and real time PCR analysis revealed that transfection of CSE cDNA, but not of the mock cDNA, resulted in significant increases in both CSE protein expression and CSE mRNA levels compared with wild-type HEK-293 cells (Fig. 1, A and B). Transfection of HEK-293 cells with CSE cDNA also resulted in a marked increase in the H₂S production rate (1.51 ± 0.12 nmol/g/min) compared with mock-transfected cells (0.41 ± 0.06 nmol/g/min, p < 0.05) (Fig. 1C).

View larger version (13K): [in this window] [in a new window]   FIG. 1. Overexpression of CSE cDNA in HEK-293 cells. A, CSE protein expression was increased in CSE stably transfected cells. After being cultured for 48 h, the cells (3 x 106) were lysed, and 30 µg of protein were subjected to Western blot analysis using a monoclonal anti-CSE antibody. Results are representative of three individual experiments. B, the CSE mRNA level was higher in CSE stably transfected cells. The CSE mRNA level was normalized to that of an internal standard gene (-actin) using real time PCR. Experiments were repeated three times. *, p < 0.05 versus mock or wild-type cells. AU, arbitrary unit. C, the H2S production rate was significantly increased in CSE stably transfected cells. After being cultured for 48 h, the cells (3 x 106) were collected and homogenized to measure the H2S production rate. Data represent four independent experiments. *, p < 0.05 versus mock or wild-type cells.

View larger version (13K): [in this window] [in a new window]   FIG. 2. CSE overexpression inhibited cell growth and DNA synthesis. A, cell growth curves of different types of cells. An equal number of cells (2 x 105) were plated, and growth rates were analyzed by cell counting for 5 days. Data represent four independent experiments. *, p < 0.05 versus mock or wild-type cells. B, DNA synthesis measured by [3H]thymidine incorporation. Quiescent cells were stimulated with 10% FBS for 12 h and [3H]thymidine (0.1 µCi/ml) was added in the last 6 h and measured by scintillation counting. cpm, counts per minute. *, p < 0.05 versus mock or wild-type HEK-293 cells. All data are from at least three independent experiments.

Sustained Activation of MAPK and Up-regulation of p21^Cip/WAK-1 by CSE Overexpression—The MAPK family, including ERK, p38 MAPK, and c-Jun NH₂-terminal kinase, plays an important regulatory role in the proliferation and differentiation of cells. Transfection of HEK-293 cells with CSE cDNA, but not the mock cDNA, resulted in a marked accumulation of phosphorylated ERK and p38 MAPK (Fig. 3A). Neither the phosphorylation of c-Jun NH₂-terminal kinase nor total amounts of MAPK were affected. Incubating cells with 10 µM U0126 or 20 µM SB203580 (the p38 MAPK inhibitor) for 24 h significantly decreased the expression of phosphorylated ERK and p38 MAPK (Fig. 3B).

View larger version (39K): [in this window] [in a new window]   FIG. 3. CSE overexpression induced sustained increases in MAPK activity. A, a sustained phosphorylation of ERK and p38 MAPK induced by CSE overexpression. After being cultured for 48 h, the cells (3 x 106) were collected and subjected to Western blot analysis using specific antibodies. B, inhibition of ERK and p38 MAPK. After being plated for 24 h, the cells were cultured in the presence or absence of U0126 (10 µM) or SB203580 (20 µM) for another 24 h and lysed to be subjected to Western blot analysis. The data in A and B represent one typical experiment from three independent experiments.

View larger version (36K): [in this window] [in a new window]   FIG. 4. Up-regulation of p21Cip/WAK-1 but not of cyclin D1 by CSE overexpression and their correlation with MAPK. After being plated for 24 h, the cells were cultured in the presence or absence of U0126 (10 µM) or SB203580 (20 µM) for 24 h and lysed to be subjected to Western blot analysis. The graphs represent the optical density of the bands of p21Cip/WAK-1 or cyclin D1 generated from three independent experiments that were normalized with the expression of -actin.

Inhibitory Effects of CSE Overexpression on Cell Proliferation Mainly Due to Endogenously Produced H₂S—To determine the mechanism by which CSE overexpression inhibited cell growth, we assessed the effects of H₂S, ammonium, and pyruvate, three products of CSE-catalyzed cysteine degradation, on cell growth. When confluent serum-starved wild-type HEK-293 cells were stimulated with 10% FBS in the presence of H₂S (100 µM), DNA synthesis decreased significantly. However, 100 µM ammonium hydroxide and 100 µM sodium pyruvate failed to inhibit DNA synthesis (Fig. 5A). Furthermore, we tested the effect of the H₂S scavenger methemoglobin (1, 25) on CSE overexpression-mediated cell growth inhibition. As shown in Fig. 5B, methemoglobin at 10 µM partly but significantly reversed the antiproliferative effect of CSE (p < 0.05). Pretreating wild-type HEK-293 cells with 10 µM methemoglobin for 1 h prior to adding 100 µM H₂S significantly abolished the antiproliferative effect of H₂S (Fig. 5A). Decreased H₂S production in CSE-overexpressed cells by methemoglobin also provided evidence that methemoglobin scavenged the endogenous H₂S (Fig. 5C). To assess the role of exogenous H₂S on MAPK and cell cycle protein expression, we tested the status of ERK and p21^Cip/WAF-1 in wild-type HEK-293 cells after exposure to 100 µM H₂S. The expression of ERK and p21^Cip/WAF-1 increased after incubating wild-type HEK-293 cells with H₂S for 2 h (p < 0.05) (Fig. 6), indicating that H₂S likely mediates the antiproliferative effect of CSE.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Effect of exogenous or endogenous H2S on cell proliferation. A, exogenous H2S inhibited cell proliferation induced by serum. After the quiescent wild-type HEK-293 cells were precultured with or without methemoglobin (10 µM) for 1 h, H2S (100 µM), ammonium hydroxide (NH3, 100 µM), or sodium pyruvate (100 µM) was added for another 12 h. [3H]Thymidine (0.1 µCi/ml) was added for the last 6 h, and its incorporation was measured by scintillation counting. *, p < 0.05. B, methemoglobin reversed the inhibitory effect of CSE overexpression on cell proliferation. The quiescent cells were cultured in the presence or absence of methemoglobin (10 µM) for 12 h, and [3H]thymidine (0.1 µCi/ml) was added for the last 6 h. Results in A and B are calculated from triplicate wells of three independent experiments. *, p < 0.05. C, methemoglobin-scavenged endogenous H2S produced in CSE stably transfected HEK-293 cells. After being cultured for 48 h, the cells (3 x 106) were collected and homogenized to measure the H2S production rate. Data represent three independent experiments. *, p < 0.05 versus mock-transfected cells.

View larger version (19K): [in this window] [in a new window]   FIG. 6. Exogenous H2S increases the ERK and p21Cip/WAK-1 activity. The quiescent wild-type HEK-293 cells were cultured in the presence or absence of H2S (100 µM) for the indicated times, and Western blot analysis was used to determine the activation of ERK (A) and p21Cip/WAK-1 (B). The histograms represent the ratio of phosphorylated ERK optical density to total ERK (A)orp21Cip/WAK-1 optical density to -actin (B) from three independent experiments. *, p < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CSE is a key enzyme of the trans-sulfuration pathway, which interconverts L-methionine and L-cysteine. It also uses L-cysteine as an alternative substrate to form H₂S (4). As a pyridoxal phosphate-dependent enzyme, CSE is expressed in a range of mammalian cells and tissues, and it seems to be the main H₂S-forming enzyme in the liver, kidney, and cardiovascular system (7, 8). Deficiency of H₂S-producing enzymes results in some disorders such as homocystinuria, which are characterized by mental retardation, skeletal abnormalities, increased urine homocysteine, increased risks of thromboembolism, and early onset of atherosclerosis (9, 25, 26).

Many previous studies on H₂S focused on the toxicological profile rather than the physiological function of the gas. Recent studies, however, have demonstrated the biological functions of H₂S, including hyperpolarization of cell membranes, relaxation of smooth muscle cells, and decreased neuronal excitability (1, 2, 6, 8, 27, 28). Little is known about the modulatory 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 results indicate that the CSE overexpression increased intracellular H₂S production, inhibition of cell proliferation, and DNA synthesis (Figs. 1 and 2). CSE induced the expression of cdk inhibitor p21^Cip/WAF-1 following sustained ERK activity, suggesting a cell cycle arrest. The antiproliferative effect of CSE is likely mediated via the release of H₂S because the H₂S scavenger methemoglobin (1, 25) partly and significantly reversed the antiproliferative effect of CSE (Fig. 5B). Exogenous H₂S alone also inhibited cell proliferation, evidenced by decreased [³H]thymidine incorporation. The other CSE products, ammonium and pyruvate, failed to inhibit cell proliferation. Pretreatment of wild-type HEK-293 cells with methemoglobin for 1 h prior to the addition of H₂S abolished the antiproliferative effect of H₂S (Fig. 5A). Moreover, exogenous H₂S induced sustained ERK and p21^Cip/WAF-1 activation (Fig. 6). These findings support the hypothesis that endogenously produced H₂S plays an important role in cell proliferation and survival.

The mitogen-activated protein kinase cascade plays a crucial role in transducing extracellular signals into responses governing growth and differentiation. The ERK pathway is involved in the stimulation of cellular proliferation (29–31), although MAPK-induced growth inhibition has also been reported (32, 33). Here, we provide evidence that a sustained increase in ERK activity induced by CSE overexpression leads to inhibited cell growth (Fig. 3A). The seemingly conflicting results can partially be explained by the fact that the MAPK pathway plays roles in both progression and inhibition of cell proliferation (21, 34). The final cellular response (i.e. cell cycle arrest or cellular proliferation) to activation of the ERK/MAPK pathway depends on the strength and duration of the MAPK signal. Transient or cyclical activation may contribute to cell cycle progression, whereas sustained high levels of ERK may lead to cell growth inhibition. Because active MAPK accumulates in the nucleus, it has been suggested that the duration and magnitude of MAPK activation will direct qualitative changes in gene expression, which in turn will determine whether a cell re-enters the cell cycle, undergoes cell cycle arrest, or remains quiescent. Although p38 MAPK was also increased in CSE-transfected cells, it may not be involved in CSE overexpression-induced p21^Cip/WAF-1 up-regulation (Figs. 3B and 4) because the inhibition of p38 activity by SB203580 did not change the expression of p21^Cip/WAF-1.

p21^Cip/WAF-1 is one of the cdk inhibitory proteins, and it plays an important role in growth arrest, cellular differentiation, DNA repair, cell senescence, and apoptosis (35, 36). Increased p21^Cip/WAF-1 expression is also an important indicator for MAPK-dependent cell growth arrest (20, 21, 37, 38). Our data provide evidence that the inhibition of growth by CSE overexpression in HEK-293 cells was accompanied by an induction of the cdk inhibitor p21^Cip/WAF-1, which was dependent on the ERK activation (Fig. 4). Correlation between p21^Cip/WAF-1 induction, prolonged ERK activation, and the inhibition of p21^Cip/WAF-1 by U0126 confirmed that p21^Cip/WAF-1 induction occurs after prolonged ERK activation. In the progression of the cell cycle, activation of cdks has been demonstrated (23). The kinase activity of cdks is negatively regulated by cdk inhibitors such as p21^Cip/WAF-1. p21^Cip/WAF-1 has been shown to directly bind with cyclin, cdk complexes, and DNA polymerase cofactor, a proliferating cell nuclear antigen (18). Blocking the activation of ERK blocked the induction of p21^Cip/WAF-1 expression, suggesting that ERK activation is correlated with CSE overexpression-induced up-regulation of p21^Cip/WAF-1 and cell growth inhibition. Several studies indicate that sustained ERK activation allows for the induction of cyclin D1 (20, 21). p21^Cip/WAF-1 can inhibit cyclin D1 activation and thereby prevent cyclin D1/CRM1 association (24). However, our study demonstrated that cyclin D1 was not involved in CSE overexpression-induced cell growth inhibition. Although ERK and p21^Cip/WAF-1 likely play roles in CSE overexpression-mediated cell growth inhibition, it is also possible that some other molecules that were not tested here are involved. Furthermore, the linkage between sustained ERK activation and expression of p21^Cip/WAF-1 needs to be identified.

In conclusion, this study provides evidence for the first time that CSE regulates cell proliferation through a H₂S-mediated phosphorylation of ERK and p21^Cip/WAK-1. Collectively, the ability of the CSE/H₂S system to inhibit cell growth suggests that genetic approaches to manipulate CSE expression and H₂S production may provide a novel therapeutic avenue in the treatment of CSE/H₂S disorder-related diseases.

	FOOTNOTES

 
^* This study was supported in part by the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by a post-doctoral fellowship award from the Saskatchewan Health Research Foundation, Canada.

^¶ Supported by a post-doctoral fellowship award from the Canadian Institutes of Health Research (CIHR) and Canadian Hypertension Society.

^** Supported by a New Investigator Award from CIHR.

To whom correspondence should be addressed: Dept. of Physiology, University of Saskatchewan, 107 Wiggins Rd., Saskatoon, Saskatchewan S7N 5E5, Canada. Tel.: 306-966-6592; Fax: 306-966-6532; E-mail: wangrui{at}duke.usask.ca.

¹ The abbreviations used are: CSE, cystathionine -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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Wang, R. (2002) FASEB J. 16, 1792-1798[Abstract/Free Full Text]
2. Wang, R. (2003) Antioxid. Redox Signal. 5, 493-501[CrossRef][Medline] [Order article via Infotrieve]
3. Kery, V., Bukovska, G., and Kraus, J. P. (1994) J. Biol. Chem. 269, 25283-25288[Abstract/Free Full Text]
4. Erickson, P. F., Maxwell, I. H., Su, L. J., Baumann, M., and Glode, L. M. (1990) Biochem. J. 269, 335-340[Medline] [Order article via Infotrieve]
5. Stipanuk, M. H., and Beck, P. W. (1982) Biochem. J. 206, 267-277[Medline] [Order article via Infotrieve]
6. Eto, K., and Kimura, H. (2002) J. Neurochem. 83, 80-86[CrossRef][Medline] [Order article via Infotrieve]
7. Barber, T., Triguero, A., Martinez-Lopez, I., Torres, L., Garcia, C., Miralles, V. J., and Vina, J. R. (1999) J. Nutr. 129, 928-933[Abstract/Free Full Text]
8. Zhao, W., Zhang, J., Lu, Y., and Wang, R. (2001) EMBO J. 20, 6008-6016[CrossRef][Medline] [Order article via Infotrieve]
9. Valitutti, S., Castellino, F., and Musiani, P. (1990) Ann. Allergy 65, 463-468[Medline] [Order article via Infotrieve]
10. Mariggio, M. A., Minunno, V., Riccardi, S., Santacroce, R., De Rinaldis, P., and Fumarulo, R. (1998) Immunopharmacol. Immunotoxicol. 20, 399-408[Medline] [Order article via Infotrieve]
11. Brenneman, K. A., Meleason, D. F., Sar, M., Marshall, M. W., James, R. A., Gross, E. A., Martin, J. T., and Dorman, D. C. (2002) Toxicol. Pathol. 30, 200-208[CrossRef][Medline] [Order article via Infotrieve]
12. Eplancke, B., and Gaskins, H. R. (2003) FASEB J. 17, 1310-1312[Abstract/Free Full Text]
13. Hristl, S. U., Eisner, H. D., Dusel, G., Kasper, H., and Scheppach, W. (1996) Dig. Dis. Sci. 41, 2477-2481[CrossRef][Medline] [Order article via Infotrieve]
14. Cao, K., Tang, G., Hu, D., and Wang, R. (2002) Biochem. Biophys. Res. Commun. 296, 463-469[CrossRef][Medline] [Order article via Infotrieve]
15. Sun, X., Cao, K., Yang, G., Huang, Y., Hanna, S. T., and Wang, R. (2004) Biochem. Pharmacol. 67, 147-156[CrossRef][Medline] [Order article via Infotrieve]
16. Saussoy, P., Vaerman, J. L., Straetmans, N., Deneys, V., Cornu, G., Ferrant, A., and Latinne, D. (2004) Clin. Chem. 50, 1165-1173[Abstract/Free Full Text]
17. Shu, P. Y., Chang, S. F., Kuo, Y. C., Yueh, Y. Y., Chien, L. J., Sue, C. L., Lin, T. H., and Huang, J. H. (2003) J. Clin. Microbiol. 41, 2408-2416[Abstract/Free Full Text]
18. Waga, S., Hannon, G. J., Beach, D., and Stillman, B. (1994) Nature 369, 574-578[CrossRef][Medline] [Order article via Infotrieve]
19. Chen, J., Jackson, P. K., Kirschner, M. W., and Dutta, A. (1995) Nature 374, 386-388[CrossRef][Medline] [Order article via Infotrieve]
20. Sewing, A., Wiseman, B., Lloyd, A. C., and Land, H. (1997) Mol. Cell. Biol. 17, 5588-5597[Abstract]
21. Woods, D., Parry, D., Cherwinski, H., Bosch, E., Lees, E., and McMahon, M. (1997) Mol. Cell. Biol. 17, 5598-5611[Abstract]
22. Hu, P. P., Shen, X., Huang, D., Liu, Y., Counter, C., and Wang, X. F. (1999) J. Biol. Chem. 274, 35381-35387[Abstract/Free Full Text]
23. Ullmannova, V., Stockbauer, P., Hradcova, M., Soucek, J., and Haskovec, C. (2003) Leuk. Res. 27, 1115-11123[CrossRef][Medline] [Order article via Infotrieve]
24. Kobayashi, T., Ikeno, S., Hosokawa, N., Uehara, Y., Hori, M., and Destruxin, E. (2004) Biol. Pharm. Bull. 27, 587-590[CrossRef][Medline] [Order article via Infotrieve]
25. Baumbach, G. L., Sigmund, C. D., Bottiglieri, T., and Lentz, S. R. (2002) Circ. Res. 91, 931-937[Abstract/Free Full Text]
26. Wang, H., Jiang, X., Yang, F., Gaubatz, J. W., Ma, L., Magera, M. J., Yang, X., Berger, P. B., Durante, W., Pownall, H. J., and Schafer, A. I. (2003) Blood 101, 3901-3907[Abstract/Free Full Text]
27. Dombkowski, R. A., Russell, M. J., and Olson, K. R. (2004) Am. J. Physiol. 286, R678-R685
28. Zhao, W., Ndisang, J. F., and Wang, R. (2003) Can. J. Physiol. Pharmacol. 81, 848-853[CrossRef][Medline] [Order article via Infotrieve]
29. Blenis, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 5889-5892[Abstract/Free Full Text]
30. Leung, D. W., Tompkins, C., Brewer, J., Ball, A., Coon, M., Morris, V., Waggoner, D., and Singer, J. W. (2004) Mol. Cancer 3, 15-24[CrossRef][Medline] [Order article via Infotrieve]
31. Taniguchi, F., Harada, T., Sakamoto, Y., Yamauchi, N., Yoshida, S., Iwabe, T., and Terakawa, N. (2003) J. Clin. Endocrinol. Metab. 88, 773-780[Abstract/Free Full Text]
32. Hirakawa, T., and Ruley, H. E. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 1519-1523[Abstract/Free Full Text]
33. Martin, S., Favot, L., Matz, R., Lugnier, C., and Andriantsitohaina, R. (2003) Biochem. Pharmacol. 65, 669-675[CrossRef][Medline] [Order article via Infotrieve]
34. Pumiglia, K. M., and Decker, S. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 448-452[Abstract/Free Full Text]
35. Dotto, G. P. (2000) Biochim. Biophys. Acta 1471, 43-56
36. Sherr, C. J., and Roberts, J. M. (1999) Genes Dev. 13, 1501-1512[Free Full Text]
37. Zhu, H., Zhang, L., Wu, S., Teraishi, F., Davis, J. J., Jacob, D., and Fang, B. (2004) Oncogene 23, 4984-4992[CrossRef][Medline] [Order article via Infotrieve]
38. Chang, M. W., Barr, E., Lu, M. M., Barton, K., and Leiden, J. M. (1995) J. Clin. Investig. 96, 2260-2268[Medline] [Order article via Infotrieve]

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