(Received for publication, October 22, 1996, and in revised form, March 19, 1997)
From The Johns Hopkins University School of Medicine, Lipid Research Atherosclerosis Unit, Department of Pediatrics, Baltimore, Maryland 21287-3654
Previously, our laboratory reported that lactosylceramide (LacCer) stimulated human aortic smooth muscle cell proliferation via specific activation of p44 mitogen-activated protein kinase (MAPK) in the p21ras/Raf-1/MEK2 pathway and induced expression of the transcription factor c-fos downstream to the p44 MAPK signaling cascade (Bhunia A. K., Han, H., Snowden, A., and Chatterjee S. (1996) J. Biol. Chem. 271, 10660-10666). In the present study, we explored the role of free oxygen radicals in LacCer-mediated induction of cell proliferation. Superoxide levels were measured by the lucigenin chemiluminescence method, MAPK activity was measured by immunocomplex kinase assays, and Western blot analysis and c-fos expression were measured by Northern blot assay. We found that LacCer (10 µM) stimulates endogenous superoxide production (7-fold compared with control) in human aortic smooth muscle cells specifically by activating membrane-associated NADPH oxidase, but not NADH or xanthine oxidase. This process was inhibited by an inhibitor of NADPH oxidase, diphenylene iodonium (DPI), and by antioxidants, N-acetyl-L-cysteine (NAC) or pyrrolidine dithiocarbamate. NAC and DPI both abrogated individual steps in the signaling pathway leading to cell proliferation. For example, the p21ras·GTP loading, p44 MAPK activity, and induction of transcription factor c-fos all were inhibited by NAC and DPI as well as an antioxidant pyrrolidine dithiocarbamate or reduced glutathione (GSH). In contrast, depletion of GSH by L-buthionine (S,R)-sulfoximine up-regulated the above described signaling cascade.
In sum, LacCer, by virtue of activating NADPH oxidase, produces superoxide (a redox stress signaling molecule), which mediates cell proliferation via activation of the kinase cascade. Our findings may explain the potential role of LacCer in the pathogenesis of atherosclerosis involving the proliferation of aortic smooth muscle cells.
Glycosphingolipid (GSL)1 and its metabolic products have been shown to play critical roles as bioregulators of a variety of processes such as cell proliferation (1, 2), cell mobility (3), and programmed cell death (apoptosis) (4). Lactosylceramide (LacCer), a ubiquitous GSL, has been implicated in diverse biological functions (5). For example, we have found that LacCer exhibits a time- and concentration-dependent proliferation of aortic smooth muscle cells (ASMC) (1). Because proliferation of smooth muscle cells is considered a hallmark in the pathogenesis of atherosclerosis, we previously measured the levels of LacCer and other GSLs in human subjects who had this disease. We found that the levels of LacCer and glucosylceramide were markedly higher in the plaque and calcified plaque than in unaffected aorta from patients who died from atherosclerosis at The Johns Hopkins Hospital (6). Recently, we observed that LacCer stimulated the activation of p44 mitogen-activated protein kinase (p44 MAPK) and the expression of the transcription factor c-fos (7), which perhaps regulates the genes essential for cell proliferation. Moreover, upstream activators of p44 MAPK; p21ras, Raf, and MEK2, but not MEK1, are involved in the activation of p44 MAPK by LacCer. Although our findings suggested that LacCer stimulated this kinase cascade, it is unclear whether LacCer itself or second messengers generated by LacCer are responsible for the activation of this signaling cascade.
A class of highly diffusible and ubiquitous molecules, termed reactive
oxygen species, has recently been recognized to act as signaling
intermediates for cytokines including interleukin-1 and tumor necrosis
factor- (8, 9). The reactive oxygen species (ROS) encompass species
such as superoxide (O
2), hydrogen peroxide, nitric oxide, and
hydroxyl radicals (10). Oxidative stress, which is an excess production
of ROS, plays a role in different pathological conditions such as
atherosclerosis and cancer (11, 12). In addition, O
2 has
numerous effects on cell function including induction of growth,
regulation of kinase activity, and inactivation of endothelial derived
relaxation factor, nitric oxide (13, 14). Thus superoxide and its
metabolites can function as intracellular and intercellular second
messengers, transducing receptor stimulation into biochemical response.
Because they are very small, rapidly diffusible, and highly reactive, free radical and redox stress are now thought to participate in cellular signaling (13, 15, 16). Current evidence indicate that the
different stimuli use reactive oxygen species as signaling messengers
to activate transcription factors and induce gene expression (17, 18).
The functional role of GSLs, particularly LacCer, in generating free
oxygen radicals has not been reported to the best of our knowledge. In
the present study, we found that LacCer stimulated the generation of
O
2 via activation of NADPH oxidase in H-ASMC. Alteration in
the redox status by the LacCer-dependent production of
O
2 stimulated the loading of GTP to Ras, p44 MAPK activation,
c-fos expression, and cell proliferation.
[-32P]ATP (6000 mCi/mmol), [
-32P]dCTP (3000 Ci/mmol), and
[32P]orthophosphoric acid (H3PO4)
(carrier-free) and [3H]thymidine were purchased from
Amersham Life Science Inc. Diphenylene iodonium (DPI) obtained from LC
Laboratories. Glycosphingolipids and all other chemicals were purchased
from Sigma. Bovine erythrocyte membrane-derived LacCer was a gift from
Prof. T. Taki (Department of Biochemistry, Tokyo Medical and Dental
University, Japan). Human plaque intima-derived LacCer was prepared in
our laboratories. The purity of glycosphingolipids (>99%) was
assessed by high pressure liquid chromatography and/or high pressure
thin layer chromatography. Anti-p21ras antibody and anti-MAPK
antibody (Specific for p44 MAPK and p42 MAPK) were obtained from
Upstate Biotechnology, Inc. (Lake placid, NY). cDNAs for
c-fos and glyceraldehyde-3-phosphate dehydrogenase were a
generous gift from Prof. Daniel Nathans (The Johns Hopkins University)
and Dr. D. Dewitt (Department of Biochemistry, Michigan State
University), respectively. The polyethyleneimine TLC plates were
purchased from E. M. Separations (Gibbstown, NJ).
H-ASMC were prepared and cultured in minimum essential medium supplemented with 10% fetal calf serum; penicillin (100 µg/ml), streptomycin (100 µg/ml), and glutamine (50 µg/ml), according to the procedure of Ross (19).
Isolation of LacCer from Human Atherosclerotic PlaqueIsolation, purification, characterization, and quantitation of LacCer from human atherosclerotic plaque intima was pursued according to the standard published protocol in our laboratory (6).
Vehicle for GlycosphingolipidsStock solution of LacCer
were prepared in chloroform-methanol (1:2, v/v), dried under a stream
of nitrogen, dissolved in dimethyl sulfoxide (Me2SO) and
added to culture medium to give the desired concentrations of LacCer.
Cells incubated with 0.01% Me2SO served as a control. PMA,
staurosporine, or DPI stock solutions were prepared in
Me2SO and stored at 20 °C until use. Aqueous solutions of NAC and allopurinol were prepared either in medium or in buffer.
Lucigenin, an acridylium compound (Sigma) that emits light
upon reduction and interaction with O2, was used to measure
O
2 production (20). Briefly, cultured H-ASMC were harvested,
and cell pellets were suspended in a balanced salt solution (130 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 35 mM phosphoric acid, and 20 mM HEPES, pH 7.4).
The viability of the suspended cells as determined by the trypan blue
exclusion principle was >90%. To measure O
2 production,
intact cells preincubated at room temperature were added to a 96-well
plate containing dark-adapted lucigenin (500 µM) in
balanced salt solution. Next, LacCer was added to it as a stimulant,
and photon emission was measured every 20 s for 10 min in a
scintillation counter (Packard TOP counter). The GSL solutions
(dissolved in Me2SO) were added to cells to reach a final
concentration of Me2SO of 0.01%. Moreover, vehicle (0.01% Me2SO) served as a control in most experiments. The amount
of O
2 produced at each time point was calculated by comparison
with a standard curve generated using xanthine/xanthine oxidase.
H-ASMC were incubated with 10 µM LacCer, and control cells were incubated with Me2SO (final concentration 0.01%). In some experiments, cells were preincubated with 100 µg/ml BSO, an inhibitor of de novo GSH synthesis, followed by incubation with 10 µM LacCer. At various time intervals cells were harvested, and cell pellets were suspended in a solution of 25% metaphosphoric acid and 100 mM potassium phosphate buffer (pH 8.0), sonicated for 10 min, and centrifuged at 30,000 × g for 30 min at 4 °C. The supernatant was used for the GSH assay fluorometrically at 420 nm (excitation wave length 350 nm) using o-phthalialdehyde (Sigma) as a fluorescence reagent (21).
Cell Fractionation and NADH/NADPH Oxidase AssayConfluent H-ASMC were incubated with 10 µM LacCer. At different time intervals, cells were washed with ice-cold phosphate- buffered saline, harvested, and homogenized in lysis buffer containing 20 mM potassium phosphate buffer, pH 7.0, 1 mM EGTA, 10 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 0.5 mM phenylmethylsulfonyl fluoride. The membrane (both plasma membrane and mitochondrial membrane) and cytosol was prepared by centrifugation of cell homogenate at 29,100 × g for 20 min at 4 °C (22). Membrane was resuspended in the original volume of lysis buffer. NADH and NADPH oxidase activity was measured in both cytosolic and membrane fraction as described previously by the lucigenin chemiluminescence method (22). Briefly, the reaction mixture contained 50 mM phosphate buffer (pH 7.0), 1 mM EGTA, 150 mM sucrose, and 500 µM lucigenin as the electron acceptor and either 100 µM NADPH or 100 µM NADH as an electron donor. The reaction was initiated by the addition of membrane homogenate (150-200 µg of protein). Luminescence was monitored as described above. In some experiments, NADPH oxidase activity was measured in the membrane preparations in the presence of 1 mM KCN (mitochondrial poison). Protein content was measured by the method of Lowry et al. (23) with bovine serum albumin serving as a standard.
Immunoprecipitation and MAPK Activity AssayASMC were lysed
in 100 µl of radioimmune precipitation lysis buffer containing 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 10 mM sodium fluoride, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
pepstatin, 25 mM Tris·HCl (pH 7.4), 1% Triton X-100, and
Nonidet P-40. The lysate was centrifuged and immunoprecipitated with
anti-MAP kinase antibody conjugated with protein A-agarose as described
earlier (7). Part of the immunocomplex was directly used for the MAP
kinase activity assay (24). Briefly, 25 µl of total reaction mixture
contained 1 mg/ml myelin basic protein (peptide APRTPGGRR), 50 µM [-32P]ATP (1800 cpm/pmol), 0.5 mM adenosine 3
-5
-cyclic monophosphate-dependent protein kinase inhibitor, and assay dilution buffer containing 30 mM
-glycerophosphate, 20 mM MOPS (pH 7.2),
20 mM MgCl2, 5 mM EGTA, 1 mM dithiothreitol, 0.5 mM
Na3VO4, and 2-3 µg of immunoprecipitated protein. The reaction was initiated upon the addition of
[
-32P]ATP for 15 min at 30 °C and terminated with
the addition of 10 µl of ice-cold 40% trichloroacetic acid and
spotted onto p81 phosphocellulose paper. Free
[
-32P]ATP was removed by five washes (5 min each) with
1% phosphoric acid. Radioactivity was measured by liquid scintillation
counting.
Immunocomplexes, prepared as described above, were subjected to electrophoresis in 12.5% SDS-polyacrylamide gel electrophoresis. The protein was then transferred onto a polyvinylidine membrane and blotted with anti-MAP kinase antibody as described previously (7).
Ras·GTP Loading AssayH-ASMC were metabolically labeled with [32P]orthophosphate in phosphate-free media for 16 h as described previously (7) and incubated with 10 µM LacCer with or without antagonists. At different time points, cells were lysed in radioimmune precipitation lysis buffer and cell lysates were immunoprecipitated with human p21ras antibody (Upstate Biotechnology). Immunoprecipitates were washed, and bound nucleotides (GTP and GDP) associated with p21ras were eluted with 2 mM EDTA, 2 mM dithiothreitol, 0.2% SDS, 0.5 mM GTP, and 0.5 mM GDP at 68 °C for 20 min as described (25). The eluted nucleotides were separated on polyethyleneimine TLC plates using 0.75 M KH2PO4 (pH 3.4) as a solvent and then exposed to an x-ray film.
Northern Blot Analysis of c-fos ExpressionTotal cellular
RNA was isolated from H-ASMC that had been preincubated with 15 mM NAC for 30 min, 5 µM diphenylene iodonium for 30 min, 100 µg/ml L-buthionine
(S,R)-sulfoximine (BSO) for 4 h, followed by
stimulation with 10 µM LacCer for 1 h prior to RNA
extraction (26). Twenty micrograms of total RNA were separated by
electrophoresis on 1% agarose gel (25 mM MOPS, pH 7.8, 1 mM EDTA, 1% (w/v) formaldehyde), transferred to a
-probe blotting membrane (Bio-Rad), and hybridized with
32P-labeled c-fos cDNA probes as described.
As a control, the blot was stripped off and reprobed with
32P-labeled cDNA for glyceraldehyde-3-phosphate
dehydrogenase analysis and photographed.
H-ASMC were incubated with different antagonists with or without LacCer (10 µM) for 24 h. Next, [3H]thymidine (5 µCi/ml media) was added. After incubation for 2 h, cells were washed with phosphate-buffered saline. The incorporation of [3H]thymidine in H-ASMC was measured as described previously (1).
Lactate Dehydrogenase AssayLactate dehydrogenase activity in the culture medium was measured as described (1).
Intact H-ASMC were incubated with different GSLs and their
constituent sugars as indicated. Only LacCer induced the generation of
O2 (Fig. 1). Other GSLs and constituents did
not stimulate the O
2 generation in ASMC (Fig. 1) up to a
concentration of 200 µM (data not shown). LacCer
stimulated O
2 production in a
concentration-dependent manner (Fig.
2A) as measured by the lucigenin
chemiluminescence assay. Maximal generation of O
2 (7-fold
increase as compared with control) was observed with 10 µM LacCer. Kinetic analysis revealed that the lucigenin
chemiluminescence signal was maximal (7-fold) at 2.5 min and then
decreased to about 4.5-fold at 30 min (Fig. 2B). To examine
the specific effect of LacCer in the stimulation of O
2
generation, we incubated H-ASMC with two additional sources of LacCer.
Bovine erythrocyte membrane LacCer (LacCer(E)) or LacCer
derived from human atherosclerotic plaque intima (LacCer(P)) (Fig. 1) both exerted a concentration- (Fig. 2C) and time-
(Fig. 2D) dependent stimulation of O
2 production
with similar kinetics as stearoyl LacCer (Fig. 2, A and
B). To test whether the membrane integrity of cells was lost
upon incubation of cells with LacCer, we measured LDH activity in the
culture medium. No increase of LDH activity was observed in culture
medium up to 50 µM LacCer in the incubation mixture (Fig.
2E). These data indicate that membrane integrity was intact
during LacCer- (10 µM) induced O
2 generation.
Effect of Superoxide Dismutase (SOD) and Antioxidants on LacCer-induced Superoxide Generation
The addition of SOD (100 units/ml) to intact cells did not inhibit LacCer-mediated generation of
O2 (Fig. 3A). But NAC or PDTC, both
cell-permeable antioxidants (28), abrogated lucigenin signals
(O
2 production) in control cells and in cells incubated with
LacCer (Fig. 3A). The addition of LacCer (10 µM) to cell homogenate (before subcellular fractionation)
also generated O
2 (Fig. 3B). This phenomenon was
blocked by the addition of 100 units/ml SOD (Fig. 3B) or 15 mM NAC or 100 µM PDTC (Fig.
3C).
Effect of LacCer and L-Buthionine (S,R)-Sulfoximine on the Intracellular Level of GSH in H-ASMC
Intracellular level of
GSH was decreased in a time-dependent manner by LacCer
(Fig. 4A). A low level of GSH was observed
upon incubation of cells with BSO (100 µg/ml) and also with BSO plus LacCer (Fig. 4A). The level of GSH was also decreased in a
concentration-dependent manner in cells incubated with BSO
only (Fig. 4B). Concomitantly, the basal level of
O2 was high (1.1 nmol/mg of protein) (Fig. 4C) in
cells preincubated with 100 µg/ml BSO, an inhibitor of de
novo GSH synthesis, (15, 29) for 24 h. Moreover,
chemiluminescence signals (O
2 production) were further
increased (about 8-fold compared with control) upon incubation of
BSO-preincubated cell with LacCer (Fig. 4C).
LacCer Activates NADPH Oxidase
At various time points,
following stimulation of H-ASMC with LacCer, NADPH oxidase activity was
measured in a plasma membrane preparation with NADPH as a co-factor.
NADPH oxidase activity in control cells (vehicle only) was 2.9 ± 0.03 nmol/min/mg of protein but was increased 4-fold within 2.5 min, as
compared with control (Fig. 5A). NADPH
oxidase activity was 3-fold higher in cells incubated with LacCer at 10 min compared with control. The mitochondrial poison KCN did not inhibit
LacCer-induced NADPH oxidase activity (data not shown). Preincubation
of LacCer-stimulated plasma membrane preparations with 5 µM DPI, an inhibitor of NADPH oxidase (30, 31),
completely abrogated LacCer stimulated NADPH oxidase activity (Fig.
5A). No NADPH oxidase activity was observed in cytosolic
fraction (Fig. 5B). No stimulation of NADH oxidase activity
was observed in LacCer-stimulated cytosol or in membrane preparation
(Fig. 5C). An addition of exogenous LacCer after the isolation of plasma membrane preparations of nonstimulated cells did
not alter NADPH oxidase activity (data not shown). These findings suggest that LacCer induced generation of O2 due to the
activation of NADPH oxidase.
Contribution of NADH Oxidase, Xanthine Oxidase, and Protein Kinase C in LacCer-mediated O
To ascertain that
LacCer-mediated O2 production is due to the activation of
NADPH oxidase and not NADH oxidase, we measured O
2 generation
in intact cells preincubated with DPI (5 µM) prior to
stimulation with LacCer. DPI completely inhibited LacCer-mediated O
2 generation (Fig. 6A). A moderate
inhibition of O
2 production also occurred in control cells
incubated with DPI (Fig. 6A). The addition of staurosporine
(STP), a potent inhibitor of protein kinase C, or depletion
of protein kinase C by treatment of cells with PMA (100 nM)
for 24 h (32, 33) failed to impair the LacCer-mediated stimulation
of O
2 production (Fig. 6B). The contribution of
xanthine oxidase in O
2 generation in LacCer-stimulated H-ASMC
was examined next. Incubation of cells with 100-200 µM
allopurinol, a specific inhibitor of xanthine oxidase (34, 35), did not
inhibit O
2 production by LacCer (Fig. 6C). These
observations suggested that LacCer-induced O
2 generation
was dependent on NADPH oxidase but independent of PKC or xanthine
oxidase.
Effect of LacCer on MAPK Activity/Phosphorylation in Cells Preincubated with NAC, GSE, BSO, and DPI
Western blot assay
employing antibody against p42 MAPK and p44 MAPK followed by
densitometric scan (scan data not shown) revealed that LacCer
specifically stimulated the phosphorylation of p44 MAPK approximately
3.5-fold as compared with control (Fig. 7A). H-ASMC pretreated with 15 mM NAC for 30 min or 100 µM PDTC for 1 h or 15 mM GSE for 4 h abrogated LacCer-induced p44 MAPK phosphorylation. However, BSO, an
inhibitor of glutathione synthesis (by inhibiting GSH-synthesizing
enzyme, -glutamyl cysteine synthetase) (15, 29), stimulated the
phosphorylation of p44 MAPK approximately 2-fold. Co-incubation of
cells with BSO plus LacCer had an additive effect on the
phosphorylation of p44 MAPK. LacCer also stimulated the activity of
MAPK approximately 3.5-fold as compared with control (Fig.
7B). In contrast, NAC, DPI, PDTC, GSE, and GSH abrogated LacCer-induced MAPK activity. Incubation of cells with BSO stimulated MAPK activity about 2-fold compared with control. Co-incubation of
cells with BSO and LacCer only moderately stimulated MAPK activity (4-fold) further, as compared with LacCer alone (3.5-fold) (Fig. 7B).
Effect of LacCer on Ras·GTP Loading in Cells Preincubated with NAC, BSO, and DPI
LacCer exerted a time-dependent
stimulation of Ras·GTP loading compared with control. Maximum
stimulation of Ras·GTP loading occurred 1 min after incubation of
cells with LacCer (Fig. 8A). NAC and DPI both
abrogated LacCer-induced Ras·GTP loading (Fig. 8, B and
C). In contrast, cells preincubated with BSO alone showed significant (above base-line value) (Fig. 8D) Ras·GTP
loading. Co-incubation of cells with BSO and LacCer exerted further
effects on Ras·GTP loading within 1 min, and loading was sustained at this high level for 2.5 min (Fig. 8E). This observation
indicates that free radicals are involved in LacCer-mediated Ras
activation.
Effect of NAC, BSO, and DPI on LacCer-induced c-fos Expression
Northern blot assay revealed that LacCer increased the
c-fos mRNA levels approximately 4-fold (densitometric
scan not shown) compared with control (Fig. 9). This
phenomenon was abrogated by NAC and DPI. In contrast, BSO had a
moderately additive effect on the LacCer-mediated induction of
c-fos mRNA levels in H-ASMC. Interestingly, LacCer did
not alter the mRNA levels of c-jun and c-myc
in H-ASMC (7).
Inhibition of LacCer-mediated Cell Proliferation by NAC and DPI
We found that LacCer stimulated the proliferation of H-ASMC
approximately 5-fold as compared with control (Fig.
10). NAC and DPI abrogated the LacCer-mediated
induction in cell proliferation. In contrast, BSO and BSO plus LacCer
stimulated cell proliferation approximately 5.5-fold.
LacCer, a ubiquitous GSL, plays a pivotal role in the biosynthesis
of complex GSL (5). However, its biological function is not well
understood. Our laboratory has reported a close relationship between
increased levels of LacCer and hyperproliferation in diverse human
diseases. For example, in human atherosclerotic plaque (6), familial
hypercholesterolemia (36, 37), and human polycystic kidney disease, an
increased cellular/tissue level of LacCer was accompanied by cell
hyperproliferation (38). Among several GSLs investigated, we found that
LacCer exerts the highest stimulation in H-ASMC proliferation (1), and
LacCer from human atherosclerotic plaque tissue was significantly more
effective in stimulating H-ASMC proliferation than LacCer from the
unaffected aorta (6). Such studies indicated that one of the biological
functions of LacCer may involve cell proliferation. Next, we showed
that LacCer specifically stimulated the activation of Ras·GTP
loading, Raf-1, and MEK2 upstream to p44 MAPK, and the expression of
growth response early gene c-fos, downstream to the p44 MAPK
signaling pathway (7). However, these studies did not elucidate whether
LacCer itself or second messengers generated by LacCer regulated this pathway. In this report we provide evidence that NADPH oxidase dependent O2 generation was increased in H-ASMC upon LacCer
addition in a time- and concentration-dependent manner.
This, in turn, activated the p21ras·GTP loading, activation
of the kinase cascade, and induction of c-fos mRNA that
finally led to cell proliferation. Our hypothetical model depicting
LacCer-mediated redox signaling leading to the proliferation of aortic
smooth muscle cells is summarized in Fig. 11.
The generation of O2 in our study was measured by the
lucigenin chemiluminescence method. Lucigenin chemiluminescence is sensitive to detect both intracellular and extracellular O
2, because diacridinium nitrate (lucigenin) can enter into the cells and
upon reaction with O
2 emits light. This light was detected using a single photon counter (Packard Top counter). To determine the
specificity of LacCer-mediated stimulation of O
2 generation, we incubated cells with three different sources of LacCer; stearoyl LacCer, bovine erythrocyte membrane-derived LacCer, and LacCer-derived from human atherosclerotic plaque intima and catabolic products of
LacCer. Only LacCer stimulated the generation of O
2 with
similar kinetics (Fig. 2, A-D). The integrity of membrane
was not compromised in cells incubated with LacCer as evidenced by the
absence of leakage of cytosolic lactate dehydrogenase in the
medium.
The following observations support the tenet that O2 was
generated intracellularly. First, preincubation of cells with SOD (a
scavenger of superoxide radicals) did not abrogate LacCer-induced O
2 production. This observation may be due to the inability of SOD to penetrate the cell membrane. Second, NAC and PDTC (both are
membrane-permeable molecules) both inhibited LacCer-induced O
2
generation. We were concerned that the hydrolysis product of NAC,
acetate, might contribute to the NAC-mediated inhibition of
LacCer-induced O
2 generation. Accordingly, the effects of acetate in LacCer-mediated O
2 generation were measured. We
found that sodium acetate (15 mM) did not inhibit
O
2 generation in control cells (vehicle only) or in
LacCer-incubated cells (data not shown). Taken together, our data
suggest that LacCer-mediated O
2 generation in intact cells is
endogenous.
To determine which reactive oxygen species-generating enzymes are
involved in LacCer-induced O2 generation, we took advantage of
inhibitors known specifically to inhibit NADPH oxidase/NADH oxidase and
xanthine oxidase. We found that LacCer specifically stimulated NADPH
oxidase activity in a time-dependent manner, but not NADH
oxidase. Moreover, O
2 production in LacCer-stimulated intact cells was completely blocked by DPI, a flavoprotein containing NADPH oxidase inhibitor. Previously, DPI has been used to demonstrate its specific effect on the inhibition of NADPH oxidase (30, 31). The
nonspecific effects of DPI on other flavoproteins by direct binding
have also appeared. Since mitochondrial poison KCN did not inhibit
LacCer-induced NADPH oxidase activation (data not shown) it appears
that the LacCer-induced O
2 production was due to plasma
membrane-bound NADPH oxidase. Allopurinol has been shown to
specifically inhibit xanthine oxidase (34, 35). We found that
allopurinol did not inhibit LacCer-induced O
2 production. Staurosporine is a well known potent inhibitor of PKC (32, 33). Similarly, preincubation of cells with PMA (100 nM) for
24 h depletes PKC activity (32, 33). Under these conditions,
LacCer-mediated O
2 generation was not impaired. Such studies
indicate that PKC may not be involved in LacCer-induced O
2
production. This finding confirms our previous studies in which we
showed that staurosporine failed to impair the p44 MAPK activation and
cell proliferation by LacCer (7). Taken together, our data suggest that
LacCer-induced O
2 production in H-ASMC occurs predominantly
due to PKC-independent activation of NADPH oxidase. Previously, high
concentrations of O
2 were shown to have a growth-inhibitory
effect that may lead to apoptosis (39). By contrast, in our study,
LacCer induced the production of a relatively small amount of
O
2 (3.5 nmol/mg of protein) in H-ASMC via the activation of
NADPH oxidase. This small amount of O
2 generated by LacCer may
be responsible for the growth-promoting effect of LacCer. Thus,
O
2 may serve as integral signaling molecules that exert a
concentration-dependent effect on cell proliferation and
apoptosis.
To prevent oxidative damage and allow survival in an oxygen
environment, mammalian cells have developed an elaborate antioxidant defense system that includes nonenzymatic antioxidants. The major nonprotein thiol and potent antioxidant in the cells is GSH, which principally buffers the intracellular redox state. We found that LacCer
decreased the intracellular GSH level. BSO, an inhibitor of de
novo GSH-synthesizing enzyme -glutamyl cysteine synthetase, decreased basal GSH level. GSH level was further decreased upon the
addition of LacCer to cells preincubated with BSO. The decrease in
intracellular GSH may lead to alterations in the activity of redox-sensitive enzymes, including protein-tyrosine kinases,
p21ras, and MAP kinases (15, 41). Moreover, it was reported
earlier that the generation of O
2 produced an increase in
intracellular pH (40). Since p21ras is one of the redox-sensing
proteins (41), alteration of redox status by LacCer via production of
endogenous O
2 caused the activation of p21ras by
loading GTP. We have previously shown that activation of p21ras
transmits its activation signal to p44 MAPK via the Raf and MEK2 pathway (7). Moreover, it is evident from our study that depletion of
GSH increased the susceptibility of p21ras to oxidative stress
generated by LacCer, whereas inhibition of O
2 production with
DPI and reduction of O
2 level by antioxidant PDTC (data not
shown) or NAC abrogated LacCer-induced p21ras·GTP loading.
This observation is consistent with a previous report that O
2
caused activation of p21ras in Jurkat cell lines (41).
Previously, we reported that p44 MAPK activation was the target of
LacCer-mediated signal transduction (7). In the present study,
phosphorylation/activation of p44 MAPK was abrogated by PDTC, NAC, and
DPI. Therefore, it is possible that LacCer-mediated O2
production via the activation of NADPH oxidase specifically phosphorylates p44 MAPK but not p42 MAPK. We were interested in whether
GSH supplementation caused inhibition of p44 MAPK phosphorylation and
activation. Since GSH is not transported across the membrane, we
incubated cells with GSE, a readily transported derivative of GSH (42).
Preincubation of GSE blocked the p44 MAPK phosphorylation/activation by
LacCer. In contrast, in vivo depletion of GSH by BSO
increased phosphorylation/activation of p44 MAPK by LacCer as compared
with control. Thus, decreased levels of GSH potentiate the sensitivity of cells to LacCer and enhance mitogenic signaling via activation of
p44 MAPK. In contrast, phosphorylation/activation of p44 MAPK but not
p42 MAPK was abrogated by inhibitors of superoxide-generating enzyme
DPI and antioxidant PDTC or NAC. Although the precise
MAPK-dependent cellular alterations engendering a modified
response to oxidants remain to be defined, the present study provides
strong support for a crucial role for the p44 MAPK signaling pathway in
regulating cell proliferation in response to oxidative stress induced
by LacCer.
The protooncogene c-fos functions as an inducible transcription factor in signal transduction processes (43). Elevated expression of the c-fos gene via alteration of redox state has been previously shown to accompany cell proliferation. Also, in our previous study, LacCer specifically induced the expression of the c-fos transcriptional factor (7, 43) downstream to p44 MAPK. As expected, DPI and NAC both abrogated LacCer- induced c-fos mRNA expression. In contrast, BSO stimulated c-fos mRNA expression. These results support the notion that oxidative stress generated by LacCer induced expression of c-fos mRNA as well as cell proliferation.
Exogenously added GSLs, e.g. LacCer, to the culture medium
may be incorporated into the plasma membrane, the lipophillic ceramide moiety being inserted into the lipid bilayer and thus increasing the
proportion of this lipid in cell membrane (44). An increased level of
LacCer also occurs in patients with hypercholesterolemia (36), perhaps
contributing to an increase in the production of O2 in
endothelial cells (45). In fact, recently, we have observed that LacCer
can stimulate O
2 production in human arterial endothelial
cells via the activation of NADPH oxidase.2
How LacCer activates NADPH oxidase is not clear from our study and
requires further investigation. In summary, LacCer-mediated generation
of low levels of superoxide may constitute a novel biochemical
signaling pathway in H-ASMC proliferation. Our findings may explain the
potential role of LacCer in the pathogenesis of atherosclerosis
involving the proliferation of aortic smooth muscle cells.
We are grateful to Prof. Daniel Nathans for providing c-fos cDNA probes.