Lactosylceramide stimulates Ras-GTP loading, kinases (MEK, Raf), p44 mitogen-activated protein kinase, and c-fos expression in human aortic smooth muscle cells.

Previously, our laboratory has shown that lactosylceramide (LacCer) can serve as a mitogenic agent in the proliferation of aortic smooth muscle cells "a hallmark in the pathogenesis of atherosclerosis" (Chatterjee, S. (1991) Biochem. Biophys. Res. Commun. 181, 554-561). Here we report a novel aspect of LacCer-mediated signal transduction. We demonstrate that LacCer (10 microM) can stimulate the phosphorylation of mitogen-activated protein (MAP) kinase p44MAPK to phosphorylated p44MAPK in aortic smooth muscle cells from rabbit or human origin. Western immunoblot assays and direct measurement of activity in immunoprecipitated MAP kinase revealed that within 5 min of incubation of cells with LacCer there was a 3.5-fold increase in the activity of p44MAPK. This continued up to 10 min of incubation; thereafter, the MAP kinase activity decreased in these cells. Phosphoamino acid analysis revealed that the tyrosine and threonine moieties of p44MAPK was phosphorylated by LacCer. Incubation of cells with ceramide and glucosylceramide did not significantly stimulate p44MAPK activity. Preincubation with tyrphostin (20 microM; a potent and specific inhibitor of tyrosine kinase) markedly inhibited the LacCer mediated stimulation in p44MAPK activity. Next we investigated the upstream and downstream parameters in MAP kinase signaling pathways. We found that lactosylceramide stimulated (7-fold) the loading of GTP on Ras. Concomitantly, LacCer stimulated the phosphorylation of MAP kinase kinases (MEK) and Raf within 2.5 min. Lactosylceramide specifically induced c-fos mRNA expression (3-fold) in these cells as compared to control. In summary, one of the biochemical mechanisms in LacCer mediated induction in the proliferation of aortic smooth muscle cells may involve Ras-GTP loading, activation of the kinase cascade (MEK, Raf, p44MAPK), and c-fos expression.

Previously, our laboratory has shown that lactosylceramide (LacCer) can serve as a mitogenic agent in the proliferation of aortic smooth muscle cells "a hallmark in the pathogenesis of atherosclerosis" (Chatterjee, S. (1991) Biochem. Biophys. Res. Commun. 181, 554 -561). Here we report a novel aspect of LacCer-mediated signal transduction. We demonstrate that LacCer (10 M) can stimulate the phosphorylation of mitogen-activated protein (MAP) kinase p44 MAPK to phosphorylated p44 MAPK in aortic smooth muscle cells from rabbit or human origin. Western immunoblot assays and direct measurement of activity in immunoprecipitated MAP kinase revealed that within 5 min of incubation of cells with LacCer there was a 3.5-fold increase in the activity of p44 MAPK . This continued up to 10 min of incubation; thereafter, the MAP kinase activity decreased in these cells. Phosphoamino acid analysis revealed that the tyrosine and threonine moieties of p44 MAPK was phosphorylated by LacCer. Incubation of cells with ceramide and glucosylceramide did not significantly stimulate p44 MAPK activity. Preincubation with tyrphostin (20 M; a potent and specific inhibitor of tyrosine kinase) markedly inhibited the LacCer mediated stimulation in p44 MAPK activity. Next we investigated the upstream and downstream parameters in MAP kinase signaling pathways. We found that lactosylceramide stimulated (7-fold) the loading of GTP on Ras. Concomitantly, Lac-Cer stimulated the phosphorylation of MAP kinase kinases (MEK) and Raf within 2.5 min. Lactosylceramide specifically induced c-fos mRNA expression (3-fold) in these cells as compared to control. In summary, one of the biochemical mechanisms in LacCer mediated induction in the proliferation of aortic smooth muscle cells may involve Ras-GTP loading, activation of the kinase cascade (MEK, Raf, p44 MAPK ), and c-fos expression.
Glycosphingolipids are minor, yet integral, constituents of cell membranes (1). Glycosphingolipids and its metabolic products have been recently shown to play critical roles as bioregulators of a variety of processes such as cell proliferation (2,3), cell mobility (4), and programmed cell death (apoptosis) (5). Previously a ganglioside, GM 3 , 1 was shown to inhibit the phos-phorylation of epidermal growth factor. That may in part, contribute to the inhibition of cell proliferation by this compound (3). We found that lactosylceramide (LacCer) exhibited a time and concentration-dependent proliferation of aortic smooth muscle cells (2). Since proliferation of smooth muscle cells is considered a hallmark in the pathogenesis in atherosclerosis, we measured the level of LacCer and other glycosphingolipids in human subjects who had this disease. We found that the level of glucosylceramide (GlcCer) and LacCer were markedly elevated in the plaque and calcified plaque compared to unaffected aorta from patients who died from atherosclerosis at The Johns Hopkins Hospital. 2 Moreover, LacCer derived from plaque was at least 2-fold more potent in stimulating the proliferation of smooth muscle cells compared to LacCer from unaffected aorta tissues. Although our findings may suggest an important role for LacCer in cell proliferation, the mitogenic signaling events predicted by this lipid is not clear.
The best known mitogen activated protein (MAP) kinases are: p44 MAPK (extra cellular signal regulated kinase, ERK1), and p42 MAPK (ERK2). These are a group of serine/threonine protein kinases that constitute an activation process triggered by a variety of growth stimuli (6 -10). Such protein kinases have been suggested to phosphorylate and activate transcriptional factors such as c-myc (11)(12)(13), c-fos (14), and p62 TCF (15,16), which regulates the expression of genes essential for cell proliferation (17). MAP kinase kinase (MAPK-K) identified as a dual-specificity kinase, are involved in the activation and phosphorylation of the tyrosine and threonine residues in ERKs (MAP kinases) within the TEY motif in the conserved domain VIII (18). MAPK-K is a substrate for Rous sarcoma associated factor (Raf-1), a serine-threonine kinase (19). The latter has been shown to integrate the signaling of various receptor tyrosine kinases (9): G-protein coupled receptors (20,21), upstream serine/threonine kinase, for example, protein kinase C, and other kinases (9).
In this paper, we present evidence that LacCer specifically activates the phosphorylation of p44 MAPK in cultured human aortic smooth muscle cells. Upstream activators, Ras/Raf/ MEK, are involved in this signal kinase cascade. Concomitantly, LacCer specifically stimulated c-fos proto-oncogene expression. Such a combination of biochemical pathways may delineate signaling events involved in LacCer mediated induction in aortic smooth muscle cell proliferation. 32  Chemicals-All standard cultured reagents were supplied by Life Technologies, Inc. Tyrphostin AG-126 was obtained from Calbiochem (San Diego, CA). Myelin basic protein substrate peptide (APR TPGG RR), specific for MAP kinase, anti-MAP kinase (ERK-T) polyclonal antibody, specific for p44 MAPK and p42 MAPK (for mouse, rat, and human systems), anti-Raf-1, anti-MEK, and anti-p21 ras antibody were obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Glycosphingolipids and other standard reagents were obtained from Sigma. cDNAs for c-fos and c-jun were a generous gift from Prof. Daniel Nathans and c-myc was a gift from Dr. Chi Van Dang at The Johns Hopkins University. cDNA for MAP kinase phosphatase-I, 3CH134 was a gift from Dr. Jennifer L. Duff (University of Washington, Seattle, WA). cDNA for GAPDH was a gift from Dr. D. Dewitt, Dept. of Biochemistry, Michigan State University. Human aortic smooth muscle cells were a gift from Dr. Cecila Giachelli. The polyethyleneimine TLC plates were purchased from E.M. Separations, Gibbstown, NJ.

Isotopes-[␥-
Cells-Human aortic smooth muscle cells (A-SMC) were prepared and cultured in minimum essential medium supplemented with 10% fetal calf serum/penicillin/streptomycin, 100 units/ml, and 50 g/ml glutamine according to the procedure of Ross (22).
Incubation of Cells with LacCer and Other Lipids-Cells (ϫ10 5 ) were seeded in 100-mm 2 plastic Petri dishes in the above growth medium. Fresh medium was added every 3 days. On the seventh day of cell growth when cells were confluent, the medium was replaced and washed with sterile phosphate-buffered saline. Next, 8 ml of Ham's F-10 medium was added to each plate. After priming the cells for 2 h in this medium, various agonists/antagonists were added. Vehicle alone was added to control dishes. After incubation for a certain time (described separately in individual experiments), cells were washed and harvested in sterile phosphate-buffered saline containing 1 mM sodium vanadate (Na 3 VO 4 ) to inhibit phosphatase activity, and lysed in RIPA lysis buffer. The cell lysate was immunoprecipitated with anti-MAP kinase antibody conjugated with protein A-agarose.
Incubation of Cells with Tyrphostin-Confluent culture of cells were incubated with 20 M tryphostin for 30 min. Next, LacCer was added and incubation continued for various time periods and cells were harvested (detailed in legends to Figures).
Immunoprecipitation of MAP Kinase-A-SMC were lysed in 100 l of modified RIPA buffer containing 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 10 mM sodium fluoride, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 1 mM pepstatin, 25 mM Tris-HCl, pH 7.4, 1% Triton X-100, and 0.5% Nonidet P-40 (23). The lysate was centrifuged and the supernatant was incubated with 4 g of anti-MAP kinase antibody conjugated with protein A-agarose overnight at 4°C. The immunoprecipitates were washed twice with 25 mM Tris buffer, pH 7.4, containing 2 mM EDTA and 150 mM NaCl. A portion of the immunoprecipitate was boiled for 5 min in Laemmli sample buffer, rapidly sedimented, and the supernatant was analyzed by SDS-PAGE. The immunocomplex was used for MAP kinase activity assays.
Measurement of MAP Kinase Activity-MAP kinase activity was determined by the phosphorylation of MAP kinase-specific myelin basic protein (peptide APRTPGGRR) as described previously (24). The assay was performed with 2-3 g of protein in a final volume of 25 l containing 1 mg/ml myelin basic protein, 50 M [␥-32 P]ATP (1800 cpm/ pmol), 0.5 mM adenosine 3Ј-5Ј-cyclic monophosphate-dependent protein kinase inhibitor, and assay dilution buffer containing 30 mM ␤-glycerophosphate, 20 mM MOPS, pH 7.2, 20 mM MgCl 2 , 5 mM EGTA, 1 mM dithiothreitol, and 0.5 mM Na 3 VO 4 . The kinase reaction was initiated upon the addition of [␥-32 P]ATP for 15 min at 30°C. The reaction was terminated with the addition of 10 l of ice-cold 40% trichloroacetic acid and spotted onto a 2.4-cm 2 piece of Whatman P-81 phosphocellulose paper. Free ␥-32 P was removed by five washes (5 min each) with 1% phosphoric acid and one wash in 95% ethanol. Radioactivity was measured by liquid scintillation counting. The activity of MAPK was expressed as picomole/min/mg protein. Protein was determined according to the method of Lowry et al. (25) with the use of bovine serum albumin as standard.
Western Blot Analysis-Following incubation Ϯ agonists/antagonists, A-SMC were washed in situ with ice-cold phosphate-buffered saline containing 1 mM Na 3 VO 4 , then lysed in RIPA lysis buffer as described earlier. The detergent insoluble material was separated by centrifugation (10,000 ϫ g, 15 min, 4°C), and the soluble supernatant fraction was used as enzyme source. The enzyme preparation (40 g) was subjected to 12.5% SDS-PAGE. The protein was then transferred electrophoretically onto a polyvinylidine difluoride membrane for 1 h at 50 V and 52 mA/gel at room temperature (26). Next, the membrane was blocked for 1 h with 3% non-fat dry milk in TBS-T (10 mM Tris-HCl and 50 mM NaCl with 0.005% Tween 20, pH 8.0), and then incubated overnight at 4°C with the appropriate primary antibody. Next, the membrane was incubated for 2 h with the secondary antibody (alkaline phosphatase-conjugated anti-rabbit IgG) after washing four times with TBS-T. Membrane was then washed and incubated in the dark for 3-6 min with the substrate 5-bromo-4-chloro-4-indolylphosphate AP buffer (100 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl 2 , pH 9.5), and nitro blue tetrazolium. The reaction was terminated by rinsing the membrane with distilled water.
Phosphoamino Acid Analysis of p44 MAPK -A-SMC were metabolically labeled with [ 32 P]orthophosphoric acid (H 3 PO 4 ) as described previously (27). Briefly, cells were incubated for 90 min in phosphate-free Dulbecco's minimal essential medium, and subsequently labeled by incubation for 16 h with 3 mCi/ml carrier-free O-[ 32 P]phosphate. The labeled cells were stimulated by incubation with LacCer (10 M) for 5 min. Cell lysis and immunoprecipitation of p44 MAPK were performed as described above. 32 P-Labeled p44 MAPK immunoprecipitates were digested with 6 N HCl at 110°C under vacuum for 1 h. Phosphoamino acids were separated by two-dimensional thin layer cellulose chromatography together with unlabeled DL-O-phosphoserine, DL-O-phosphothreonine, and DL-O-phosphotyrosine (1 mg/ml). The solvents used for the first and second dimensions were butanol:pyridine:acetic acid:water (13:10:2:8) (v/v), and butanol:pyridine:acetic acid:water (15:10:3:12) (v/ v), respectively (28). Following development, the chromatograms were stained with ninhydrin reagent and subjected to autoradiography at Ϫ70°C for 6 days to identify the labeled phosphoamino acid.
Raf-1 and MEK Assay-A-SMC were incubated with 10 M LacCer and harvested at different time periods. Whole cell lysates were prepared as described above. Fourty micrograms of total cell protein were separated by 10% SDS-PAGE and electrophoretically transferred onto polyvinylidine difluoride membrane. Next, the membrane was incubated with Raf-1 antibody overnight at 4°C. For the MEK assay, total cell proteins were separated by 12.5% SDS-PAGE and blotted with MEK antibody overnight at 4°C.
Ras Activation Assay-A-SMC were labeled with [ 32 P]orthophosphate in phosphate-free media for 16 h as described and incubated with 10 M LacCer. At various time points, cells were lysed in RIPA lysis buffer (23). The whole cell lysates were immunoprecipitated with antihuman p21 ras antibody. Immunoprecipitates were washed with 8 ϫ 1 ml of 50 mM HEPES, pH 7.4, 500 mM NaCl, 5 mM MgCl 2 , 0.1% Triton X-100, and 0.005% SDS. Nucleotide (GTP and GDP) associated with ras were eluted with 2 mM EDTA, 2 mM dithiothreitol, 0.2% SDS, 0.5 mM GTP, 0.5 mM GDP at 68°C for 20 min (29). The eluted nucleotides were separated on polyethyleneimine TLC plates using 0.75 M KH 2 PO 4 (pH 3.4) as a solvent and exposed to x-ray film.
Northern Blot Analysis of c-fos, c-jun, and c-myc Gene Expression-A-SMC were grown in a p100 ϫ 20-mm dish in 8 ml of complete Dulbecco's minimum essential medium supplemented with 10% serum with antibiotics. When the cells were sparsely confluent (ϳ80%) fresh medium containing 0.5% serum was added and incubation continued for 72 h. Then the fresh F-10 medium was added and 2 h later cells were incubated for 1 h with and without linoleic acid (20 M) and LacCer (10 M). The total RNA was isolated by the modified acid guanidinium thiocycanate-phenol-choroform extraction method (30) and 20 g of total RNA were separated by electrophoresis on 1% formaldehyde gel (25 mM MOPS, pH 7.8, 1 mM EDTA, 1% (w/v) formaldehyde), transferred to Zeta Probe blotting membrane (Bio-Rad) by alkaline blotting in transfer buffer (pH 12.0) containing 3 M NaCl, 8 mM NaOH, 2 mM sodium lauryl sarcosine overnight. 1.25 Kilobases of HindII and EcoRI fragment of human c-fos cDNA, 2.6 kilobases of EcoRI fragment of mouse c-jun cDNA, and 1.8 kilobases of EcoRI fragment of human c-myc cDNAs were labeled with [␣-32 P]dCTP using a random labeling method according to the instructions provided by the manufacturer (Bethesda Research Laboratories). The membranes were prehybridized in prehybridization buffer (50% formamide, 5 ϫ SSC, 5 ϫ Denhardt's reagent, 50 mM sodium phosphate, 250 g/ml salmon sperm DNA) at 42°C for 2 h and hybridized in hybridization buffer (prehybridization buffer with 10% (w/v) dextran sulfate and appropriate probe, 2 ϫ 10 6 cpm/ml) overnight at 42°C. The blots were washed with wash buffer I (2 ϫ SSC, 0.2% SDS) twice at room temperature, 15 min each time. Then the blots were washed with buffer-II (0.1 [times ]SSC, 0.1% SDS) twice at 65°C, 20 min each time prior to autoradiography. As a control, the blot was stripped off and reprobed with labeled cDNA for GAPDH and photographed.

Effects of Concentration of LacCer, GlcCer, and Ceramide on
the Activity of p44 MAPK -LacCer exerted a concentration-dependent stimulation in the phosphorylation/activation of p44 MAPK (Fig. 1). Maximum stimulation ϳ3.5-fold in the activity of MAPK occurred with 10 M LacCer. At a higher concentration of LacCer (50 M) the induction of phosphorylation of MAPK was on the order of ϳ2-fold compared to control. In contrast, incubation of cells with similar concentrations of either GlcCer or Cer did not appreciably stimulate the phosphorylation of p44 MAPK (Fig. 1).
Effects of Time of Incubation with LacCer on MAPKs-Western immunoblot assays ( Fig. 2A) followed by densitometric analysis (Fig. 2B) revealed that incubation of cells with LacCer (10 M) exerted a nearly 3-fold increase in the phosphorylation of p44 MAPK . In contrast, LacCer did not phosphorylate p42 MAPK . Immunocomplex kinase assay showed that within 2.5 min of incubation of cells with LacCer (10 M) a significant increase (2.5-fold) in the activation of p44 MAPK occurred (Fig.  2C). Maximum stimulation, 3.5-fold compared to control, occurred 5 min after the incubation of cells with LacCer. Thereafter, phosphorylation decreased continually up to 60 min when it was near normal levels.
Phosphoamino Acid Analysis of p44 MAPK -To determine which amino acids in p44 MAPK were phosphorylated as a consequence of incubation of cells with LacCer, we pursued the experiments in the presence of [ 32 P]orthophosphate. Following immunoprecipitaton with MAPK antibodies and washing, the immunoprecipitates were digested with acid, neutralized, and subjected to two-dimensional TLC. We found that LacCer markedly increased the phosphorylation of tyrosine and threonine residues in p44 MAPK , but not the serine residue (Fig. 3). The ratio of phosphorylation of tryrosine versus threonine residues in p44 MAPK in control was 1.6:1. Whereas, the labeling ratio of tyrosine versus threonine residues in p44 MAPK in Lac-Cer treated cells was on the order of 1.8:1. Moreover, we ob-served a 8.5-fold and 7.5-fold increase in the phosphorylation in tyrosine and threonine residues, respectively, in p44 MAPK in LacCer-treated cells as compared to control.
Effects of LacCer on MAPK Phosphatase-To ascertain that the increase in the phosphorylation of p44 MAPK in cells incubated with LacCer was not due to the inhibition of MAPK phosphatase, we pursued Northern assays in cells incubated with and without LacCer. The level of mRNA for MAPK phosphatase remained unchanged in cells incubated with or without LacCer (data not shown).
Effects of Tyrphostin on MAPK Activity-When cells were incubated with tyrphostin, a potent inhibitor of tyrosine kinase upstream of MAPK, it inhibited the LacCer (10 -20 M) induced phosphorylation of MAPK (Fig. 4). Such findings suggest that tyrosine kinase is involved in LacCer-mediated signal transduction cascade.
Effects of LacCer on Ras-GTP Loading-Cells prelabeled with 32 P were incubated with LacCer as described earlier. At various time points, the cell lysates were prepared and immu- noprecipitated with p21 ras antibody. The nucleotides eluted from immunoprecipitates were subjected to TLC analysis (Fig.  5A). We found that within 1 min there was a 6-fold increase in Ras-GTP loading that decreased to 3-fold in 2.5 min as compared to control (Fig. 5B). After 30 min of incubation of cells with LacCer, significant GTP radioactivity was still associated with Ras.
Effects of LacCer on the Phosphorylation of Raf-The effects of time of incubation of cells, with LacCer on the phosphorylation of Raf-1 was assessed by Western immunoblot assay. We observed that 2.5 min after incubation of cells with LacCer, there was an appearance of a band with reduced mobility (presumably the phosphorylated form of Raf) compared to Raf (Fig. 6). A similar observation was made at 5 min after incubation of cells with LacCer thereafter, the phosphorylated form of Raf was not observed.
Effects of LacCer on the Phosphorylation of MEK-Western immunoblot assays employing MEK2 antibody revealed that LacCer exerted a time-dependent increase in the phosphorylation of MEK2 (Fig. 7). This was evidenced by a shift in the mobility of a MEK2 antibody recognizable band (Fig. 7A). Densitometric analysis of the gel revealed that after 2.5 min of incubation of cells with LacCer an increase in MEK2 phosphorylation on the order of 3-fold occurred. Maximum increase in MEK2 (4-fold) occurred 5 min after incubation of cells with LacCer as compared to control (Fig. 7B). Ten minutes after incubation of cells with LacCer there was ϳ2-fold higher activity of MEK kinase as compared to control Effects of LacCer on the Expression of Proto-oncogenes: c-fos, c-myc, and c-jun-Northern assays revealed that after 1 h of incubation of cells with 10 M LacCer, the level of c-fos but not c-myc and c-jun was increased by 3-fold (Fig. 8B) as compared to control. In contrast, preincubation of cells with linoleic acid (20 M) (Fig. 8) increased the level of c-fos, c-jun, and c-myc on the order of 6-fold, 4-fold, and 1.5-fold, respectively, as compared to control. The level of GAPDH remained unchanged upon incubation of cells with LacCer or linoleic acid. DISCUSSION Our studies generated several novel findings. First, we found that LacCer specifically stimulated the phosphorylation of p44 MAPK , whereas GlcCer and Cer did not. Second, tyrphostin (tyrosine kinase inhibitor) markedly abrogated LacCer medi- ated induction in the phosphorylation of p44 MAPK . Third, Lac-Cer stimulated Ras-GTP loading and the phosphorylation of MEK2 and Raf. Fourth, LacCer specifically stimulated the mRNA level of c-fos proto-oncogene in A-SMC.
To delineate the signal transduction events in LacCer-mediated proliferation in human A-SMC, we examined its effects on various parameters in the signal transduction cascade under carefully controlled yet, varying conditions. The cells were grown to confluence, washed, and incubated with Ham's F-10 medium without serum for 2 h. Next, various agonists and antagonists were added. We were concerned that contamination of any reagents or cell cultures with bacteria or lipopolysaccharide may also effect the phosphorylation of MAPK (31). Accordingly, appropriate control experiments were pursued to demonstrate that lipopolysaccharide (10 g/ml) did not alter the phosphorylation of either p44 MAPK or p42 MAPK in cultured human A-SMC (data not shown). Next, we analyzed the effects of LacCer and other glycosphingolipids simultaneously employing two methods, these were: 1) the measurement of MAPK activity of immunoprecipitated MAPK employing myelin basic protein fragment (APRTPGGRR) as substrate and [ 32 P]ATP as the phosphate donor; and 2) Western immunoblot assay followed by densitometric scanning of individual gel bands. As shown in various figures, both of these approaches yielded similar results confirming the validity of our experimental techniques and the interpretation of results.
Our studies clearly revealed that LacCer induced the phosphorylation of tyrosine and threonine residues in p44 MAPK . This phenomenon was abrogated by an inhibitor of tyrosine kinase, e.g. tyrphostin (32). However, staurosporine, an inhibitor of protein kinase C, failed to impair this process 3 (data not shown). These findings reveal that LacCer mediated p44 MAPK phosphorylation and cell proliferation are independent of protein kinase C. This is in contrast to a previous study in which GlcCer mediated proliferation in a continuous renal cell line, Madin-Darby canine kidney, was shown to be abrogated by inhibitors of protein kinase C (33).
Recent studies from several laboratories reveal that the induction of MAPKs may also be due to inactivation of MAPK phosphatases (34). In particular, generation of free oxygen radicals and hydrogen peroxide were shown to inhibit MAPK phosphatase and consequently increase the level of p44 MAPK in neutrophils (35). To rule out this possibility, we pursued Northern blot assays in cells incubated Ϯ LacCer and found that the mRNA level of MAPK phosphatase was similar (data not shown). Thus, our findings indicate that LacCer mediated induction of p44 MAPK is not due to the inhibition of phosphatase activity.
The catabolic products of LacCer are GlcCer, Cer, sphingosine, and stearic acid. In order to determine the specificity of LacCer mediated stimulation of p44 MAPK phosphorylation, we also investigated the effects of GlcCer and Cer that are highly enriched in atherosclerotic plaque intima and media, but not in unaffected intima and media. 2 We found that GlcCer and Cer did not alter p44 MAPK phosphorylation. The effects of LacCer on upstream regulators, for example, Ras, Raf, and MEK of MAPK, were investigated. The protooncogene p21 ras has been identified as a key molecular switch involved in regulating cell activation triggered by various mitogens (29,36,37). In its resting state, p21 ras is in a GDPbound state. Upon in vivo activation p21 ras releases GDP and binds GTP (38,39). Immunoprecipitation of p21 ras from stimulated and nonstimulated cells followed by nucleotide (both GTP and GDP) elution revealed that a substantial increase in GTP bound p21 ras occurred upon LacCer treatment. The maximal effect was observed at 1 min after LacCer addition. Thereafter, GTP bound p21 ras was decreased. This time dependent increase of p21 ras GTP loading suggests that LacCer activates p44 MAPK via the activation of p21 ras . We speculate that the relatively short duration of LacCer induced p21 ras GTP loading may be due to the interaction of the latter with its effector or GTPase activating protein. An investigation is required whether the LacCer mediated activation occurs by direct interaction with p21 ras or indirectly through secondary factors.
Raf-1, the product of the c-Raf 1 proto-oncogene is pivotal in transmitting signals from Ras in the plasma membrane to cytosolic nuclear compartments of the cells (40,41). The stimulation of Raf-1 activity depends on the activation of the small G-protein p21 ras (42). Active GTP-ras binds to the NH 2 -terminal domain of Raf-1 and recruits Raf-1 to the plasma membranes, which is sufficient to cause its activation (40,41,43). We investigated whether Raf-1 is phosphorylated/activated after stimulation of p21 ras (measured by p21 ras ⅐GTP loading) by LacCer. Significant phosphorylation of Raf-1 was observed within 2.5-5 min of incubation of cells with LacCer. Thus the activation of Raf-1 by LacCer was transient in A-SMC. Since Raf-1 activity is known to participate in the G 0 /G 1 transition of cells (42), we may speculate that LacCer mediated Raf-1 activation may similarly help in the transition of A-SMC from the G 0 phase to the G 1 phase of the cells. Next, we measured the phosphorylation of MEK by LacCer to assess whether the Raf-1, a serine-threonine kinase, transmits proliferative signals to downstream effectors, e.g. p44 MAPK via the activation of putative MAPK activator MEK. MEK2 phosphorylation was stimulated within 2.5-5 min after the addition of 10 M LacCer, and then it was dissipated at 10 min. Taken together, these findings lead us to speculate that LacCer induced activation of p44 MAPK may be mediated by a kinase cascade that includes Ras, Raf, and MEK2 in a descending order.
Induced expression of proto-oncogene c-fos, c-jun, and c-myc mRNA is an early response to various growth stimuli (44 -46). These proto-oncogenes encode for nuclear binding transcriptional factors (44,45) and play a crucial role in mitogen-induced cell proliferation (47). To determine whether these protooncogenes are also involved in LacCer induced A-SMC proliferation, subsequent to p44 MAPK activation, we measured mRNA levels of these proto-oncogenes. Among the three protooncogenes tested, only the c-fos mRNA level increased after 1 h of LacCer incubation as compared to control. In contrast, cellular levels of c-jun or c-myc mRNA in LacCer treated and control cells were similar. Equal amounts of RNA loading was confirmed by GAPDH analysis. This data suggested that Lac-Cer specifically induced c-fos proto-oncogene expression via activation of p44 MAPK .
Why LacCer specifically induced the phosphorylation of p44 MAPK and c-fos mRNA expression is not clearly understood from our studies. Previous studies with smooth muscle cells have revealed that fatty acids, e.g. linoleic acid and arachidonic acid, stimulate the phosphorylation of both p42 MAPK and p44 MAPK (48). In addition, linoleic acid stimulated the mRNA levels of c-fos, c-myc, and c-jun in rat vascular smooth muscle cells (49) and also in our studies with human A-SMC. Furthermore, recent studies have shown that tumor necrosis factor-␣ specifically stimulate MEK1, that in turn induce the activation of p42 MAPK (50). Our preliminary studies indicate that LacCer did not stimulate MEK1, instead LacCer stimulated MEK2. Thus there may be a clear dichotomy in regard to agonist specific induction of MEK2 and activation/phosphorylation of p44 MAPK (ERK1). Since other studies have shown that the ERK group of MAP kinases phosphorylate Elk-1 and increases ternary complex formation (15,16) and activation of c-fos downstream in the signaling pathway, it is possible that LacCer, like other growth factors, specifically mediates this process. This may ultimately increase transcriptional activity and DNA synthesis, subsequently cell proliferation. Further work in this area is warranted to explain this phenomena to understand the pathophysiology of LacCer mediated A-SMC proliferation in atherosclerosis.