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J Appl Physiol 96: 793-801, 2004. First published October 24, 2003; doi:10.1152/japplphysiol.00797.2003
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HIGHLIGHTED TOPICS
Oxygen Sensing in Health and Disease

Enhanced survival effect of pyruvate correlates MAPK and NF-{kappa}B activation in hydrogen peroxide-treated human endothelial cells

Yong-Jin Lee,1 Il-Jun Kang,1 Rolf Bünger,2 and Young-Hee Kang1

1Division of Life Sciences and Silver Biotechnology Research Center, Hallym University, Chuncheon 200-702, Korea; and 2Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814

Submitted 29 July 2003 ; accepted in final form 8 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We recently reported that pyruvate inhibited translocation and activation of p53 caused by DNA damage due to oxidant injury (Lee YJ, Kang IJ, Bünger R, and Kang YH. Microvasc Res 66: 91-101, 2003); this was associated with increased expression of apoptosis-related bcl-2 and decreased expression of bax gene. This study attempted to delineate possible regulatory sites and mechanisms of antiapoptotic pyruvate, focusing on reactive oxygen species-mediated signaling in a human umbilical vein endothelial cell model. We compared the effects of the cytosolic reductant L-lactate and malate-aspartate shuttle blocker aminooxyacetate, both of which increase cytosolic NADH, on the downstream signaling pathway. Hydrogen peroxide (0.5 mM H2O2) depleted intracellular total glutathione that was prevented by pyruvate but not by L-lactate or aminooxyacetate. Activation of caspase-3 and the cleavage of procaspase-6 and procaspase-7 were strongly inhibited by pyruvate but markedly enhanced by L-lactate and aminooxyacetate, implicating redox-related antiapoptotic mechanisms of pyruvate. Western blot analysis and immunochemical data revealed that H2O2-induced transactivation of nuclear factor-{kappa}B (NF-{kappa}B) was also inhibited by pyruvate but not by L-lactate or aminooxyacetate. In addition, H2O2 downregulated extracellular signal-regulated kinase (ERK1/2) and phosphorylated p38 mitogen-activated protein kinase (MAPK), effects that were fully reversed by pyruvate within 2 h. Collectively, these findings indicate that pyruvate can protect cellular glutathione, thus enhancing cellular antioxidant potential, and that enhanced antioxidant potential can desensitize NF-{kappa}B transactivation due to reactive oxygen species, suggesting possible metabolic redox relations to NF-{kappa}B. Furthermore, pyruvate blocked the p38 MAPK pathway and activated the ERK pathway in an apparently redox-sensitive manner, which may regulate expression of genes believed to prevent apoptosis and promote cell survival. Thus pyruvate may have therapeutic potential for reducing endothelial dysfunction and improving survival during oxidative stress.

apoptosis; caspase-3; reactive oxygen species

OXIDATIVE INJURY FOLLOWING diverse insults, including ischemia-reperfusion and cytokine-induced inflammation, can induce cardiac and endothelial cell death by apoptosis (1, 20, 40). Antioxidants or agents capable of inhibiting production of reactive oxygen species (ROS) have been shown to prevent apoptosis (1, 30, 39). Pyruvate can detoxify hydrogen peroxide (H2O2) and other ROS and inhibit transition metal-induced LDL oxidation (3, 15, 18, 19), implying that pyruvate acts as an antioxidant that may inhibit production of ROS. In previous studies from our laboratory, pyruvate prevented H2O2-induced endothelial apoptosis (18, 26) by disabling the mitochondrial death pathway involving bcl-2 and bax genes by acting as an anaplerotic precursor and an indispensable mitochondrial redox substrate (41). Similarly, ceramide induces cytochrome c release from mitochondria; this process is mediated via changes of the mitochondrial redox state (11). Nevertheless, direct cytosolic mechanisms of pyruvate protection against apoptosis cannot be ruled out. A possible cytosolic mechanism for rendering pyruvate an antioxidant includes the pentose phosphate pathway-linked NADPH/GSH system and the cytosolic NADPH-linked isocitrate dehydrogenase (15, 24, 37).

The transcription factor p53 affects the mitochondrial membrane potential through ROS (29) and induces oxidative stress-induced apoptosis (8, 29, 43). It has recently been reported that p53 is activated by nuclear factor-{kappa}B (NF-{kappa}B) and is essential for H2O2-induced apoptosis in glioma cell lines (8). It is tempting to speculate that pyruvate could modulate the transcription factors involved in apoptotic pathways. Indeed, anti-oxidative and antiapoptotic pyruvate inhibited translocation and transactivation of p53 during cellular oxidant generation caused by H2O2 injury, thereby modulating expression of the downstream target gene proteins of bcl-2 and bax in apoptotic death cascade (26). This implies that p53 could play a pivotal role in H2O2 toxicity and pyruvate cytoprotection.

It is well established that redox state-related signaling of ROS may activate the transduction pathways leading to apoptosis (5, 6, 34). Alternatively, ROS may also induce apoptosis through redox-unrelated signaling pathways via protein tyrosine kinase- and/or protein kinase C mediation. It has been shown that H2O2 activates the death signaling pathway of Fas, which is mediated by tyrosine kinase (42). On the other hand, there is growing evidence that ROS may cause cell death via a mediation of mitogen-activated protein kinase (MAPK) under various pathological conditions (7, 9, 10, 47). Hyperoxia generates ROS through activation of NADPH oxidase, which mediates lung epithelial cell death via an activation of extracellular signal-regulated kinase (ERK)1/2, upstream of caspase activation (47). Activation of p38 MAPK primarily mediates ROS-induced apoptosis, whereas concomitant activation of c-Jun NH2-terminal kinase (JNK) represents a scavenger pathway for inhibiting apoptosis (7). In addition, H2O2, by virtue of synergistically acting with NO, induces neuronal apoptosis via activation of p38 MAPK and caspase-3 (45). Antioxidant N-acetylcysteine inhibits activation of JNK and p38 MAPK and activity of redox-sensitive activating protein-1 and NF-{kappa}B, which regulates expression of apoptotic genes (23, 46). N-acetylcysteine, a metabolic precursor of GSH, can also prevent apoptosis and promote cell survival by activating the ERK pathway. Considering that antioxidant pyruvate inhibits the NAD(P)H oxidase and enhances the cytosolic GSH potential (3), we hypothesized that pyruvate-related redox manipulations could block apoptosis by influencing MAPK signaling pathways.

To test this hypothesis, we examined the effect of pyruvate on transactivation of NF-{kappa}B in the H2O2 injury model of human umbilical vein endothelial cells (HUVECs). We also tested whether pyruvate may modulate activation of MAPK and downstream target genes of caspases involved in signaling pathways of H2O2 toxicity. Possible redox relations to NF-{kappa}B and MAPK were examined with the use of the cytosolic reductant L-lactate, aminooxyacetate, a transaminase inhibitor blocking NADH-reducing equivalent transfer via the malateaspartate cycle, and cytosolic redox-neutral acetate.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HUVEC culture. We prepared HUVECs from human umbilical cords using collagenase (Worthington Biochemical, Lakewood, NJ) as previously described elsewhere (17) and maintained cultures at 37°C humidified atmosphere of 5% CO2 in air. Cells were incubated in 25 mM HEPES-buffered M199 containing 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin supplemented with 0.75 mg/ml human epidermal growth factor, and 0.075 mg/ml hydrocortisone (Clonetics, San Diego, CA). HUVECs were used within five to seven passages and were confirmed by their cobblestone morphology and uptake of fluorescently 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled acetylated LDL (Molecular Probes, Eugene, OR; Ref. 44).

H2O2 incubation and oxidant stress. HUVECs were plated at 90-95% confluence and incubated in 25 mM HEPES-buffered M199 containing 10% FBS and growth supplements for 24 h. The cells were pretreated for 30 min with 10 mM pyruvate, 10 mM L-lactate, 10 mM acetate, or 0.5 mM aminooxyacetate and then exposed to 0.5 mM H2O2 for another 30 min. Non-H2O2-treated cells were also incubated under the same conditions as those used for the H2O2 protocols. H2O2 was detoxified by adding 100 U/ml catalase (Sigma Chemical, St. Louis, MO) at the end of the 30-min incubation period to inactivate extracellular H2O2. Cells were washed and resupplied with fresh medium containing the test concentration of pyruvate, L-lactate, acetate, or aminooxyacetate. Incubations were continued for another 24 h before biochemical and molecular analyses were performed.

Measurement of cellular total glutathione. To determine the cellular antioxidant potentials, we measured cellular total glutathione using a 5,5'-dithiobis(2-nitrobenzoic acid) assay with a minor modification (2). After culture protocols were completed, cells were washed with PBS and extracted with ice-cold 2.25% (wt/vol) 5-sulfosalicylic acid. Cell extracts were centrifuged at 12,000 g for 20 min at 4°C. The supernatant was used for subsequent determination of total glutathione, and the remaining pellet was dissolved in a lysis buffer of 0.1 M NaOH plus 0.1% SDS for the determination of cell protein content. Total glutathione in acid-soluble extracts was determined by enzymatic recycling assay in 100 mM sodium phosphate buffer (pH 7.4) containing 0.75 mM 5,5'-dithiobis(2-nitrobenzoic acid), 5.5 mM EDTA, 1 U/ml glutathione reductase, and 0.26 mM NADPH. The linear changes in absorbance at 405 nm were recorded for 6 min. Parallel measurements of glutathione standards were performed to quantify total glutathione (expressed as nmol/mg cell protein).

Intracellular oxidant generation. Oxidant generation of HUVECs was measured as a previously described (26, 38). After the challenge with H2O2 was completed, cells were loaded for 30 min with 10 µM 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; Sigma Chemical). The dye solution was freshly prepared in prewarmed M199 (+2% FBS). After we completed dye loading at 37°C, the cells were rinsed twice with PBS, and the cultures were photographed with a fluorescence microscope.

Western blot analysis. Whole cell extracts from HUVECs were prepared in a lysis buffer (1% {beta}-mercaptoenthanol, 1% {beta}-glycero-phosphate, 0.1 M Na3VO4, 0.5 M NaF, and protease inhibitor cocktail). Cell lysates were electrophoresed on 8-15% SDS-PAGE and transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotechnology, Buckinghamshire, UK). Nonspecific binding of the membrane was blocked in TBS-T [20 mM Tris base (pH 7.5), 137 mM NaCl, and 0.1% Tween 20] containing 5% skim milk. The membrane was incubated with polyclonal rabbit anti-human caspase-3, caspase-6, and caspase-7 (1:1,000 dilution; Cell Signaling Technology, Beverly, MA), monoclonal mouse antibody for phospho-p38 MAPK (Thr180/Tyr182, 1:1,000 dilution; Cell Signaling Technology), and monoclonal mouse antibody for phospho-p44/42 MAPK (ERK1/2 MAPK, Thr202/Tyr204, 1:1,000 dilution; Cell Signaling Technology). After the membrane was washed in TBS-T, it was incubated with a goat anti-rabbit IgG or anti-mouse IgG conjugated to horseradish peroxidase (1:10,000 dilution; Jackson ImmunoResearch Laboratory, West Grove, PA); this was followed by a washing in TBS-T. The protein expression levels were determined with Super-signal West pico chemiluminescence detection reagents (Pierce Biotechnology, Rockford, IL) and Konica X-ray film (Konica, Tokyo, Japan). Polyclonal rabbit anti-{beta}-actin (1:1,000 dilution; Santa Cruz Biotechnology) was used for comparative control expression.

Caspase-3-like protease activity. The cell extracts were washed with ice-cold PBS and suspended in 100 mM HEPES buffer (pH 7.4) containing 0.5 mM phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin and pepstatin, and 10 µg/ml leupeptin. The cell suspension was lysed by three freeze-thaw cycles, and the cytosolic fraction was obtained by centrifugation at 12,000 g for 20 min 4°C. The Asp-Glu-Val-Asp (DEVDase) activity was determined by measuring proteolytic cleavage of a chromogenic substrate for caspase-3-like protease, Ac-DEVD-p-nitroanilide (pNA). Cell lysate protein fraction (50 µg) was added to the buffer, which contained 150 µM Ac-DEVD-pNA in a final volume of 150 µl. The reaction mixture was incubated at 37°C for 1 h. The increase in absorbance of enzymatically released pNA was measured at 405 nm every 20 min.

NF-{kappa}B protein localization. To determine translocation of NF-{kappa}B protein to the nucleus, immunocytochemical analysis was performed with anti-human NF-{kappa}B. After HUVECs were washed with PBS containing 0.05% Tween 20 (PBS/T), HUVECs (on a glass chamber slide) were incubated with 10% normal goat serum to block any nonspecific binding. Cells were fixed with 4% ice-cold formaldehyde for 30 min and washed with PBS/T. Polyclonal rabbit anti-human NF-{kappa}B p65 (1:50 dilution; Santa Cruz Biotechnology) was then added to cells and incubated overnight at 4°C. Cells were washed with PBS/T and incubated with a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:200 dilution, Jackson ImmunoResearch Laboratory). Fluorescent images were obtained by an Olympus BX50 fluorescent microscope with differential interference contrast and reflected light fluorescence.

Preparation of nuclear protein extract. Nuclear protein extracts were prepared by a detergent lysis procedure from HUVECs to assay the DNA binding activity of NF-{kappa}B (35). Cells were washed with PBS; lysed in a buffer of 20 mM HEPES (pH 7.9) containing 1 mM EDTA, 10 mM NaCl, 1 mM dithiothreitol, 0.1% Nonidet P-40, 0.4 mM phenylmethylsulfonyl fluoride, 0.1 ng/ml leupeptin, and 5 µg/ml apotronin; and incubated on ice for 10 min. Nuclei were spun down at 3,000 g for 20 min. Cytosolic fraction was collected, and proteins of nucleus pellets were extracted with vigorous shaking in a high-salt buffer containing 20 mM HEPES (pH 7.9) with 420 mM NaCl, 1 mM EDTA, 25% glycerol, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, 0.1 ng/ml leupeptin, and 5 µg/ml apotronin at 4°C for 20 min. The nuclear debris was pelleted at 3,000 g for 20 min, and the supernatant was stored at -70°C.

To determine NF-{kappa}B localization, we conducted Western blot analyses with nuclear protein extracts and cytosolic protein fraction using anti-human NF-{kappa}B (1:1,000 dilution). The Western blot analytical procedures were as described above.

Electrophoretic mobility shift assay. Double-stranded oligonucleotide containing the consensus sequence of the binding site for NF-{kappa}B (5'-AGTTGAGGGGACTTTCCCAGGC-3') was purchased from Promega (Madison, WI) and used as the electrophoretic mobility shift assay (EMSA) probe. EMSA was carried out as previously described (25). Briefly, 0.35 pmol of NF-{kappa}B probe was labeled with 30 µCi of [{gamma}-32P]ATP (Amersham Pharmacia Biotechnology) and T4 polynucelotide kinase (Promega) and then incubated for 30 min at 37°C. The DNA binding reaction was performed for 10 min in a 25-µl reaction mixture containing 50 mM Tris·HCl (pH 7.5), 250 mM NaCl, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM dithiothreitol, 25% glycerol, 0.25 mg/ml poly(dl-dC)·poly(dI-dC), 32P-labeled-specific oligonucleotide probe, and 4 µg of nuclear extract. The oligonucleotide-DNA complex was separated from free oligonucleotide probe by an electrophoresis on 5% nondenaturing polyacrylamide gel in 0.25 x TAE buffer (40 mM Tris-acetate and 1.0 mM EDTA, pH 8.0). Gels were dried, and radiolabeled probe was detected autoradiographically on Konica X-ray films. For competition assays to determine binding specificity, 40x molar excess of unlabeled NF-{kappa}B oligonucleotide (3.5 pmol) was added to NF-{kappa}B binding reactions.

Data analysis. The results are presented as means ± SE. Statistical analyses were conducted using Statistical Analysis Systems statistical software package version 6.12 (SAS Institute, Cary, NC). One-way ANOVA was used to determine effects of pyruvate on apoptotic parameters. The differences among metabolite treatment groups were analyzed with Duncan's multiple range test and were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Glutathione pool depletion in H2O2 toxicity and pyruvate cytoprotection. In an attempt to study the interaction between thiol-redox and pyruvate in H2O2 toxicity, we measured intracellular total glutathione using an enzymatic recycling assay. Cellular glutathione loss has been interpreted to indicate GSH peroxidase activity due to oxidative stress (16). As shown in Fig. 1A, total glutathione was depleted in cells exposed to 0.5 mM H2O2 within 24 h. The glutathione pool depletion was associated with ~50% cell killing, reflecting substantial oxidant injury (Fig. 1B). Pretreatment of endothelial cells with 10 mM pyruvate significantly preserved the glutathione level during H2O2 injury. In contrast, 10 mM L-lactate, acetate, or aminooxyacetate did not prevent the depletion of cellular glutathione. The massive cell death induced by H2O2 was inhibited by pyruvate but not by L-lactate or aminooxyacetate (Fig. 1B), suggesting NADH oxidase-dependent ROS formation possibly stimulated by accumulated cytosolic NADH.



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  		Fig. 1. Total glutathione content (A) and cell viability (B) in H2O2-pyruvate-treated human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated with 10 mM pyruvate, 10 mM L-lactate, 10 mM acetate, or 0.5 mM aminooxyacetate, challenged with 0.5 mM H2O2 for 30 min, and then continuously incubated for 24 h. Total glutathione levels were determined in cell pellets. Total glutathione data represent means ± SE from 7 independent experiments with multiple estimations. HUVEC viability was assessed by 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazolium bromide (MTT) within 24 h. *P < 0.05, relative to H2O2-untreated control cells. +P < 0.05, relative to H2O2-untreated control cells and H2O2-alone-treated cells.

 

Pyruvate inhibition of intracellular oxidant generation and caspase activation. We subsequently attempted to determine whether, in H2O2-exposed cells, pyruvate affected intracellular oxidant formation, as evidenced by oxidation of DCF-DA. Figure 2 shows the expected lack of staining in the H2O2-free controls. The H2O2-alone-exposed cells showed heavy fluorescence, indicative of marked oxidant generation. These fluoromicroscopic observation data imply that H2O2-induced oxidants appeared to be responsible for apoptosis. The pyruvate-H2O2-exposed cells revealed no increase in DCF fluorescence, demonstrating that pyruvate blocked an accumulation of intracellular oxidants in endothelial cells due to H2O2 and that the mitochondrial H2O2 effect and intracellular oxidant burden possibly resulting from the pyruvate treatment appear to be below detection limit. In contrast, H2O2-exposed cells treated with equimolar L-lactate or acetate or submillimolar aminooxyacetate remained DCF positive (Fig. 2).



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  		Fig. 2. Pyruvate inhibition of H2O2-induced intracellular DCF fluorescence. Confluent HUVECs were loaded with 2',7'-dichlorodi-hydrofluorescein diacetate (DCF-DA) and were left untreated or stimulated with 0.5 mM H2O2 for 30 min in the presence of 10 mM pyruvate, 10 mM L-lactate, 10 mM acetate, or 0.5 mM aminooxyacetate. Oxidant generation was measured by DCF fluorescence. Fluorescent images (2 separate experiments) of representative H2O2-free controls and H2O2-treated cells were measured with a fluorescence microscopy. Magnification = x200.

 

ROS can trigger the translocation of cytosolic bax into mitochondria, which activates bax and induces the release of cytochrome c from mitochondria to the cytosol, stimulating proteolytic caspases in particular (5). There was a marked increase in DEVDase activity in H2O2-alone-treated HUVECs (Fig. 3A), indicating increased cytosolic proteolytic activity due to H2O2 toxicity. Pyruvate inhibited the caspase-3-like activity, whereas preincubation with L-lactate, acetate, or aminooxyacetate did not ameliorate the activity of the caspase-3-like enzyme in response to H2O2. In addition, Western blot data showed that H2O2 caused caspase-3 activation in HUVECs within 24 h. This activation was strongly inhibited by pyruvate but not by L-lactate, acetate, or aminooxyacetate (Fig. 3B). On the other hand, Western blot analysis also clearly showed that H2O2 caused cleavage of procaspase-6 and procaspase-7, other proapoptotic caspases, in HUVECs within 24 h, which was also strongly inhibited by pyruvate (Fig. 4). In addition, pyruvate appeared to further inhibit activation of both caspase-6 and caspase-7 when partially blocked by 50 µM zVAD-fmk, a broad inhibitor of caspases. In contrast, L-lactate and aminooxyacetate failed to prevent the H2O2-induced cleavage of caspase-6 and caspase-7 (Fig. 4).



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  		Fig. 3. Caspase-3-like activity (A) and caspase-3 activation (B) during H2O2-induced oxidative stress. HUVECs were pretreated with 10 mM pyruvate, 10 mM L-lactate, 10 mM acetate, or 0.5 mM aminooxyacetate, exposed to 0.5 mM H2O2 for 30 min, and then continuously incubated for 24 h. DEVDase, Asp-Glu-Val-ASP. A: cytosolic extracts were prepared from cells, and caspase-3-like activity was determined by measuring the cleavage of the substrate DEVD-p-nitroanilide. *P < 0.05 for H2O2-untreated control cells. Bar graph data are from 6 independent experiments. B: total cell extract protein was subjected to 15% SDS-PAGE and Western blot analysis. {beta}-Actin protein was used as an internal control. Bands are representative of 4 independent experiments.

 


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  		Fig. 4. Cleavage of procaspase-6 and procaspase-7 in H2O2-exposed and/or pyruvate-treated endothelial cells. Cells were pretreated with 10 mM pyruvate, 10 mM L-lactate, 10 mM acetate, or 0.5 mM aminooxyacetate and then exposed to 0.5 mM H2O2 for 30 min. Total cell extract protein was subjected to 12% SDS-PAGE and Western blot analysis by using anti-human full-length caspase-6 or full-length caspase-7. Addition of 50 µM zVAD-fmk, a broad inhibitor of caspases, resulted in a substantial inhibition of activation of both caspases. Cotreatment of zVAD-fmk with pyruvate caused further inhibition of activation (cleavage) of both caspases. Results are representative of 4 respective and independent experiments.

 

Effect of pyruvate on NF-{kappa}B translocation and DNA binding activity. It has been demonstrated that nuclear translocation and transactivation of NF-{kappa}B is followed by ROS formation and that these processes are inhibited by antioxidants (31, 46). We hypothesized that antioxidant pyruvate might inhibit oxidant-induced apoptosis by interfering with DNA binding and transactivation of NF-{kappa}B transcription factor. To test this hypothesis, we explored the effects of pyruvate on cellular localization of NF-{kappa}B p65 in H2O2-exposed HUVECs by using Western blot analysis. After exposure to H2O2, the nuclear NF-{kappa}B p65 protein level increased; this change was accompanied by a decrease in the cytosolic p65 protein level (Fig. 5A). When cells were pretreated with pyruvate, a substantial increase in cytosolic p65 protein level was detected, whereas the p65 level in nuclear protein extract apparently decreased. In contrast, L-lactate did not reverse the translocation of NF-{kappa}B induced by H2O2 (Fig. 5A).



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  		Fig. 5. Western blot data (A), microphotographs (B), and electrophoretic mobility shift assay (EMSA; C) data showing effect of pyruvate on translocation and DNA binding activity of nuclear factor-{kappa}B (NF-{kappa}B) in H2O2-exposed HUVECs. HUVEC extracts were prepared from cells pretreated with 10 mM pyruvate, 10 mM L-lactate, 10 mM acetate, or 0.5 mM aminooxyacetate, followed by exposure to 0.5 mM H2O2 for 30 min. A: cytosolic and nuclear protein extracts were electrophoresed on 8% SDS-PAGE gel and then subjected to Western blot analysis with anti-human NF-{kappa}B p65. B: HUVECs incubated on a chamber slide were pretreated with 10 mM pyruvate, 10 mM L-lactate, 10 mM acetate, or 0.5 mM aminooxyacetate and then exposed to 0.5 mM H2O2 for 30 min. Fluorescent images for the NF-{kappa}B localization were obtained by binding with a FITC-conjugated IgG. Microphotographs are representative of 3 independent experiments. Magnification = x200. C: DNA binding activity of NF-{kappa}B was measured by EMSA. Nuclear extracts were incubated with double-stranded 32P-labeled NF-{kappa}B probe, size-fractionated on 5% nondenaturing polyacrylamide gel, and exposed to X-ray film. Competition EMSA with unlabeled NF-{kappa}B consensus sequence at 40x molar excess confirmed specificity of NF-{kappa}B protein binding. Assay was repeated 4 times with representative autoradiogram shown.

 

We used a fluorescent microscope to evaluate the intracellular localization of NF-{kappa}B p65 in HUVECs after H2O2 injury using specific NF-{kappa}B p65 antibody (Fig. 5B). Cytoplasmic immunofluorescence staining was observed in the untreated control, whereas an all-heavy nuclear staining in H2O2-alone-exposed cells was observed, indicative of nuclear localization of H2O2-activated NF-{kappa}B p65 at single cell level. Pyruvatetreated cells reduced the staining of nuclear p65 induced by H2O2. However, L-lactate, acetate, or aminooxyacetate did not inhibit the H2O2-induced nuclear translocation of NF-{kappa}B (Fig. 5B).

EMSA was carried out to examine DNA binding activity of nuclear NF-{kappa}B in HUVECs pretreated with pyruvate, L-lactate, acetate, or aminooxyacetate and followed by H2O2 injury. As shown in Fig. 5C, a significant increase in NF-{kappa}B binding activity was detected in nuclear extracts of H2O2-exposed cells, demonstrating that the level of protein-NF-{kappa}B DNA complex was increased due to H2O2. The specificity of the NF-{kappa}B binding was confirmed by a binding with excess unlabeled oligonucleotide containing NF-{kappa}B binding site (lane 7). The activation of NF-{kappa}B binding was blunted by pretreatment with pyruvate, suggesting that pyruvate may protect against H2O2 toxicity by specifically preventing the formation of NF-{kappa}B-dependent DNA-protein complex in the nucleosol. In contrast, L-lactate, acetate, and aminooxyacetate had no effect on NF-{kappa}B binding stimulated by H2O2 (Fig. 5C).

Antiapoptotic pyruvate and MAPK activation. We attempted to find out whether, in H2O2-exposed cells, pyruvate might inhibit apoptosis through blocking MAPK signaling cascades. We examined the effects of pyruvate on the phosphorylation of ERK1/2 or of p38 MAPK by using antibodies specific for the phosphorylated MAPK in HUVECs. Western blot analysis revealed that treatment with 0.5 mM H2O2 for 30 min did not induce the phosphorylation of ERK1/2 MAPK but elicited the phosphorylation of p38 MAPK (Fig. 6). Pretreatment of HUVECs with 10 mM pyruvate resulted in the phosphorylation of ERK1/2, which occurred rapidly and remained high for up to 60 min. In contrast, the phosphorylation of p38 MAPK by H2O2 was almost completely and immediately blocked by pyruvate (Fig. 6). These results indicate that both ERK1/2 and p38 MAPK are involved in pyruvate protection against H2O2-induced apoptotic toxicity.



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  		Fig. 6. Phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in H2O2-exposed and/or pyruvate-treated HUVECs. Total cell extracts of HUVECs were immunoblotted with an antibody to phosphorylated ERK1/2 or phosphorylated p38 MAPK. Representative blots are typical of 4 respective and independent experiments. p, the phosphorylated form of MAPK.

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We (26) have previously shown that endothelial cell survival under oxidative stress may be a function of the expression ratio of antiapoptotic Bcl-2 and proapoptotic Bax proteins and that exogenous pyruvate but not L-lactate, acetate, or aminooxyacetate can modulate their expression possibly through inhibiting activation of p53 caused by DNA damage, thereby protecting human vascular endothelium against H2O2-induced apoptosis. On the other hand, pyruvate controls cytosolic NADH redox states, being a key substrate of the near-equilibrium lactate dehydrogenase, acts as an anaplerotic precursor of citrate, and provides mitochondrial NADH via the pyruvate dehydrogenase. These metabolic mechanisms, alone or combined, may improve the intrinsic cytosolic and mitochondrial tolerance against oxidative stress and prevent apoptosis.

Pyruvate strongly inhibits glycolysis and stimulates glycogen synthesis via allosteric citrate inhibition of phosphofructokinase and hence metabolically stabilizes cytosolic NAD+ and pentose phosphate-dependent GSH systems. The mitochondrial pathway for the pyruvate protection might be linked to mitochondrial anaplerosis that indirectly improves pentose phosphate pathway activity and also the NADPH-linked cytosolic isocitrate dehydrogenase, thus bolstering cytosolic GSH potentials (15, 24, 33, 37). Metabolic models suggested that the glucose-6-phosphate dehydrogenase, the first and rate-limiting enzyme of the oxidative pentose phosphate pathway, may be the most significant NADPH producer in hepatocytes (24). In vascular smooth muscle, pharmacological inhibition of glucose-6-phosphate dehydrogenase by dehydroepiandrosterone decreased GSH contents, and, in bovine aortic endothelium, overexpression of glucose-6-phosphate dehydrogenase was associated with increased NADPH levels and preservation of glutathione stores during oxidative stress (27, 28). Indeed, our study demonstrates that pyruvate significantly maintained cellular total glutathione and full viability during the H2O2 challenge (Fig. 1). On the other hand, L-lactate and aminooxyacetate, both of which greatly increase cytosolic NADH, were clearly less effective in protecting cellular glutathione pool and cell viability during the H2O2 injury. In addition, acetate did not prevent depletion of cellular glutathione and loss of viability by H2O2 (Fig. 1 and Refs. 18 and 26), although it readily enters the mitochondria as acetyl-CoA and inhibits phosphofructokinase via citrate; however, unlike pyruvate, it is not an anaplorotic metabolite and also has no direct effects on the cytosolic NADH system.

A direct cytosolic mechanism contributing to the depletion of total glutathione and ROS loading in the incubation with L-lactate or aminooxyacetate might be related to intracellular ROS loading due to accumulation of cytosolic NADH that donates electrons to NADH oxidase and stimulates superoxide formation (3, 32). Consistent with this interpretation, the pyruvate-H2O2-exposed cells that likely operated at greatly decreased cytosolic NADH revealed a substantial disappearance of ROS as evidenced by DCF fluorescent staining, suggesting that pyruvate minimized accumulation of intracellular oxidants in HUVECs. However, the quantitative importance of such antioxidant effects for the H2O2 injury is far from clear.

Cellular and molecular antiapoptotic features of pyruvate can be inferred from its redox effects via both cytosolic and mitochondrial mechanisms. It is well established that the signaling of ROS may activate the transduction pathways leading to apoptosis (6, 34). Alternatively, ROS may induce apoptosis through redox-unrelated signaling pathways involving mediation of tyrosine kinase and/or protein kinase C. Redox status-related effects of pyruvate may function in the turn-off phase of signal transduction by ROS and determine the outcome in terms of survival by downregulating activation of redox-sensitive caspases as well. Indeed, unlike NADH stimulating L-lactate and aminooxyacetate or redox-neutral acetate, pyruvate inhibited activation of caspase-6, caspase-7, and caspase-3, all of which are key effectors in apoptotic signaling pathways.

The activation of caspases in the presence of H2O2 could be due to direct oxidative stress or could be mediated by mitochondria or by Fas; any of these mechanisms could be inhibited by pyruvate. It has been demonstrated that the death signaling pathway of Fas ligand is activated by H2O2 through a mediation of tyrosine kinase (42). This study did not examine activation of caspases, i.e., caspase-8, mediated via Fas ligand in the presence of H2O2. Generation of intracellular ROS elicits the translocation of cytosolic bax to mitochondria, which activates bax to induce the dissociation of cytochrome c from the inner mitochondrial membrane, resulting in increased activities of cytosolic caspases (5). We have previously shown that H2O2 downregulated antiapoptotic bcl-2 and upregulated proapoptotic bax in HUVECs, thereby rendering caspase-3 cleaved (26). These H2O2 effects were inhibited by preincubation with pyruvate but not with L-lactate, acetate, or aminooxyacetate.

MAPK cascades belong to the class of protein kinase signal transduction pathways that are differentially used to relay numerous extracellular signals within cells and have been reported to be involved in various cellular functions, including stress responses and apoptosis (7, 9-11, 31). In particular, many protein kinases and transcription regulatory factors are activated under the conditions of oxidative stress due to ROS (7, 10, 23, 31, 34, 45-47). It is not known whether pyruvate-related redox or anaplerotic manipulations could stabilize or activate the expression of these kinases and transcription factors (48). We observed that pyruvate inhibited p38 MAPK phosphorylation and promoted ERK1/2 activation, two MAPKs that are believed to be components of death or survival pathways triggered by oxidative stress, respectively. It is tempting to speculate that L-lactate and aminooxyacetate, by elevating intracellular ROS due to accumulation of cytosolic NADH, cause endothelial cell death via p38 MAPK activation, an effect upstream of caspase activation. Indeed, H2O2-induced apoptosis, if at least mediated in part by ROS-activated p38 MAPK and ROS-inhibited ERK1/2 followed by caspase activation in HUVECs, was clearly inhibited by pyruvate but not by L-lactate or by aminooxyacetate.

Experimental manipulations of cellular antioxidant systems or intracellular redox states can directly or indirectly influence induction of transcription factors and expression of related genes, as suggested by other investigators (13, 21, 46). The transcription factor p53 may affect the mitochondrial membrane potential through ROS generation (29) and may induce apoptosis due to mitochondrial depolarization (8, 43). On exposure to DNA-damaging agents, the tumor-suppressor gene p53 transactivates the expression of bax (4), whereas it down-regulates the expression of bcl-2 (22). We (26) recently demonstrated that antioxidant pyruvate inhibits activation and translocation of p53 transcription factor following DNA damage caused by H2O2 injury; it also modulates expression of its downstream genes for Bcl-2 and Bax family proteins at mitochondrial levels and perhaps other components of apoptotic death cascades.

Whether H2O2-induced activation of p53 and apoptosis result from mitochondrial deenergization and depolarization, as suggested by other investigators (14, 36), was not investigated. It was recently shown that activation of p53 by NF-{kappa}B is essential for H2O2-induced apoptosis in glioma cell lines (8). Our findings showed that the pyruvate inhibition of H2O2-induced cleavage of caspases was associated with the blockade of translocation and activation of NF-{kappa}B, possibly implying that the pyruvate cytoprotection may be regulated by a redox-sensitive NF-{kappa}B-dependent signaling mechanism in cytochrome c-dependent or -independent pathways. Preservation of H2O2-depleted intracellular total glutathione after a treatment with pyruvate but not with L-lactate, acetate, or aminooxyacetate most likely reflected reduced oxidative stress due to increased cellular antioxidant potential (25), and such enhanced metabolic status could attenuate ROS-dependent death signaling cascades. It has also been reported that increased intracellular glutathione disulfide is a primary signal that is required directly or indirectly for NF-{kappa}B activation during immune stimulation (21). In addition, glutathione depletion was shown to be associated with the augmentation of oxidative stress-mediated inflammatory state in a NF-{kappa}B/ROS-dependent mechanism (13).

In summary, our findings provide new insights into the relative contributions of ERK1/2, p38 MAPK, NF-{kappa}B transcription factor, and downstream effectors of caspases responsible for mediating their effects on cell survival after oxidant injury (Fig. 7). H2O2-induced apoptosis appeared to be mediated by ROS-activated p38 MAPK and NF-{kappa}B followed by caspase activation in HUVECs. Unlike cytosolic reductants such as L-lactate and aminooxyacetate, antioxidant pyruvate activated redox-sensitive ERK1/2 and inhibited phosphorylation of p38 MAPK as well as nuclear transactivation of NF-{kappa}B following H2O2 injury. These survival-signaling pathways are not obligatorily coupled but clearly related to cellular redox systems; our data show that they are readily responsive to metabolic antioxidants and anaplerotic precursors such as pyruvate. Consequently, interventions with metabolic antioxidants could be promising in the design and development of new treatment strategies aimed at limiting cellular oxidative damage.



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  		Fig. 7. Schematic diagram showing antiapoptotic feature of pyruvate in the oxidant-induced endothelial apoptosis and the effects of pyruvate/NAD+ on modulation of these gene proteins. As depicted, pyruvate inhibits the direct apoptotic signaling cascades induced by H2O2. Arrows indicate activation or induction; {perp} indicates inhibition or blockade. Bottom table summarizes the principal effects of pyruvate/NAD+ on the various signaling gene proteins explored on the basis of the final hypothetical upper graph in Fig. 7. {uparrow}, Increase; {downarrow}, decrease.

 


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This research was supported by a research grant from Hallym University, Korea, and by Grant R12-2001-007202-0 from Korea Science and Engineering Foundation through the Silver Biotechnology Research Center at Hallym University.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-H. Kang, Division of Life Sciences, Hallym Univ., #1 Ockchon-dong, Chuncheon, Kangwon-do 200-702, South Korea (E-mail: yhkang{at}hallym.ac.kr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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