Activation of p38 Mitogen-Activated Protein Kinase by Oxidized LDL in Vascular Smooth Muscle Cells
Mediation via Pertussis Toxin–Sensitive G Proteins and Association With Oxidized LDL-Induced Cytotoxicity
Abstract—Oxidized low-density lipoproteins (oxLDL) have been shown to play a crucial role in atherosclerosis, but the underlying molecular mechanisms have not been fully understood. The present study showed that oxLDL strongly evoked phosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) in rat vascular smooth muscle cells (VSMCs) in concentration- and time-dependent manners, reaching the maximal activation at 100 μg/mL within 5 minutes. The results from immunofluorescence staining also revealed that p38 MAPK was activated by oxLDL in 5 minutes, and the activated p38 MAPK was translocated from cytoplasm to nucleus of VSMCs in 15 minutes. Activation of p38 MAPK by oxLDL was apparently not mediated by their classical scavenger receptors and was not affected by tyrosine kinase inhibitors. However, activation of p38 MAPK was effectively blocked by pretreatment with pertussis toxin and was significantly reduced by phospholipase C inhibitor U-73122. OxLDL also inhibited forskolin-stimulated cAMP accumulation and increased inositol phosphate formation. More interestingly, inhibition of p38 MAPK by its specific inhibitor SB203580 significantly blocked oxLDL-induced cytotoxicity (increased leakage of cytoplasmic lactate dehydrogenase to the culture medium, reduced [3H]thymidine incorporation, and attenuated mitochondrial metabolism of tetrazolium salt, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), MTS) in VSMCs, and pretreatment with pertussis toxin also inhibited oxLDL-induced cytotoxicity. Taken together, our data clearly demonstrated that oxLDL effectively activated p38 MAPK in VSMCs, which was likely mediated via pertussis toxin–sensitive G proteins, and the p38 activation was functionally associated with oxLDL-induced cytotoxicity in VSMCs.
Atherosclerosis and its complications, namely heart attack, stroke, and peripheral vascular diseases, are the most prevalent causes of death in developed countries1 and become one of the most rapidly increased risk factors for mortality in several developing countries. Oxidized low-density lipoproteins (oxLDL), produced by oxidative modification of LDL, have been demonstrated to induce multiple functional alterations that are potentially involved in atherogenesis.1 2 3 During an early stage of atherosclerosis, oxLDL stimulate transformation of macrophages and vascular smooth muscle cells (VSMCs) to foam cells, induce proliferation and migration of vascular cells,4 and retard endothelial regeneration.5 At molecular level, oxLDL are also shown to promote expression of adhesion molecules, heat shock proteins, and coagulation proteins, suppress production of endothelium-derived relaxing factor, nitric oxide, and prostacyclin, and induce various proinflammatory cytokines and growth factors in almost all vascular cells. During a late stage of atherosclerosis, oxLDL, because of their cytotoxicity to various vascular cells, are also reported to trigger a series of cellular responses in the arterial wall that result in the progression of fatty-streak lesions to complicated atheromatous lesions and lead to plaque rupture.6 However, molecular mechanisms underlying the cytotoxicity of oxLDL are still under investigation.
It is commonly believed that oxLDL directly interact on the cell surface with scavenger receptors instead of LDL receptors.3 7 Interaction of oxLDL with scavenger receptors leads to uptake of oxLDL into the cells. It has been recently demonstrated that in monocyte/macrophage cells, oxLDL activate the nuclear peroxisome proliferator-activated receptor gamma (PPARγ)-dependent transcription and promote cell differentiation through a signaling pathway involving scavenger receptor-mediated particle uptake.8 However, VSMCs normally express few scavenger receptors,9 although the receptor expression could be induced by oxidative stress via activation of protein kinase C (PKC)10 or by cytomegalovirus. It has been more recently reported that in VSMCs, oxLDL elicit tyrosine phosphorylation of epidermal growth factor (EGF) receptor and activation of its signaling pathway.11 There is evidence available that in macrophages, oxLDL induce a rapid and transient rise in [Ca2+]i and suppress activation of nuclear factor kappa B (NF-κB),12 which are both sensitive to pertussis toxin (PTX), suggesting that in VSMCs, oxLDL may also function via G protein–mediated signaling pathway.
Mitogen-activated protein kinase (MAPK) is serine-threonine kinase that performs important functions as mediators of cellular responses to a variety of extracellular stimuli. Four major subfamilies of structurally related MAPK have been identified in mammalian cells13 14 15 16 : the extracellular signal–regulated kinases (ERK1/2, also termed p42/44 MAPK), the c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPK), BMK1 (also termed ERK5), and p38 MAPK (also termed CSBP), a more recently described member of the MAPK family. p42/44 MAPK is characteristically activated by various growth factors and associated with cell proliferation and hypertrophy. p38 MAPK subfamily, containing at least 4 members,14 16 is strongly activated in response to stress stimuli such as UV radiation, heat shock, hyperosmolarity, and to proinflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1). Activation of p38 MAPK cascades can trigger apoptosis or programmed cell death,17 which has been implicated to play an important role in the development of atherosclerosis.13 14 18 Previous investigations have shown that oxLDL activate p42/44 MAPK in VSMCs and macrophages19 20 21 ; as yet, it is unclear whether oxLDL can stimulate p38 MAPK, which was also present in VSMCs.22 23 The present study, therefore, was undertaken to investigate the potential effect of oxLDL on p38 MAPK and the possible signaling pathway and biological effects.
Materials and Methods
Chemicals and Reagents
DMEM, FCS, genistein, and horseradish peroxidase–conjugated goat anti-rabbit secondary antibody were purchased from GIBCO-BRL. FITC-conjugated goat anti-rabbit antibody was from TAGO Inc. The rabbit polyclonal phospho-p38 MAPK antibody specific for dual-phosphorylated 180Thr and 182Tyr of p38 MAPK, total-p38 MAPK antibody detecting phosphorylation-state independent p38 MAPK, and p38 MAPK assay kit were purchased from New England Biolabs Inc. Nitrocellulose membranes (Hybond) and enhanced chemiluminescence detection system were obtained from Amersham Pharmacia Biotech. TNF-α and IL-1α were from Pharmingen. SB203580, U-73122, and U-73343 were from Calbiochem Inc. All other reagents, unless indicated, were from Sigma Chemical Co.
Rat thoracic aortic VSMCs from male Sprague-Dawley rats weighing ≈200 g were isolated using collagenase and elastase and cultured by a modification of the procedures described elsewhere.24 Immunohistochemical characterization of VSMC isolates was positive for smooth muscle–specific α-actin and negative for factor VIII. Cells were incubated at 37°C in a humidified atmosphere of 5% CO2/95% air. Cells from passages 5 to 10 were used for the present experiments.
Lipoprotein Isolation and Oxidation
LDL (density=1.019 to 1.063 g/mL) were separated from freshly drawn normal human plasma by sequential ultracentrifugation, as described previously.12 The obtained LDL were extensively dialyzed at 4°C against 0.15 mol/L NaCl and 0.01% EDTA (pH 8.0) and quantified by determination of their protein portion using Bradford protein assay kit (Bio-Rad, Hercules, Calif). After the EDTA was removed, LDL were undertaken to oxidative modification by Cu2+ incubation (5 μmol/L CuSO4, 20 hours at 37°C). Then the Cu2+ was removed by extensive dialysis. A control experiment showed that vehicle with addition of Cu2+ (up to 10 μmol/L) had no effect on p38 MAPK phosphorylation. The extent of modification was assessed by the measurement of thiobarbituric acid-reactive substances (TBARS) and by determination of electrophoretic mobility on agarose gels in barbital buffer at pH 8.6.25 The obtained oxLDL possessed a TBARS value of 25 nmol/mg of protein, whereas the LDL showed no detectable TBARS. The oxLDL moved 2 to 3 times faster on agarose gel electrophoresis than the LDL did. As a control, oxidized albumin was prepared as described previously.26
Western Blot Analysis
Cells were cultured in 12-well plates to ≈90% confluence. The growth medium was removed and replaced with DMEM containing 0.1% serum for 48 hours. Two hours before use, the cultures were incubated in serum-free DMEM. After treated with different agents at 37°C in serum-free DMEM, the cells were lysed with the SDS sample buffer containing 62.5 mmol/L Tris (pH 6.8), 2% SDS (wt/vol), and 10% glycerol. Aliquots of cell lysates were used for determination of protein. Samples were heated at 95°C for 5 minutes and centrifuged (13 000g, 5 minutes) at 4°C, and the supernatant (equal amounts of protein, 20 μg/lane) was analyzed by SDS-PAGE in a 10% acrylamide gel. Proteins were transferred to nitrocellulose membranes, and the membranes were blocked with 5% nonfat dry milk in TBST (20 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, and 0.1% Tween-20). The membranes were blotted with the primary antibody (phospho-p38 or total-p38 MAPK) then horseradish peroxidase–conjugated antibody and detected by enhanced chemiluminescence according to the manufacturer’s instructions. Molecular weights of proteins were estimated by using prestained markers (Bio-Rad). For repeated immunoblotting, membranes were stripped in 62.5 mmol/L Tris (pH 6.7), 2% SDS, and 0.1 mol/L 2-mercaptoethanol for 30 minutes at 50°C.
Immunofluorescence Staining of p38 MAPK
Cells established in gelatin-coated cover glasses were stimulated with oxLDL for 0, 5, 15, or 60 minutes and washed twice in PBS (pH 7.4). Cells were then fixed for 10 minutes in 3% paraformaldehyde in PBS at room temperature and permeabilized in 100% methanol for 10 minutes at −20°C. After a blocking step with 1% BSA in PBS, cells were incubated with phospho-p38 MAPK antibody for 16 hours at 4°C. Excess primary antibody was removed by wash with PBS, followed by incubation with FITC-conjugated goat anti-rabbit antibody prepared in PBS-BSA solution for 1 hour. Cells were washed free of unbound second antibody by washing with PBS for 5 minutes with gentle shaking. Images were captured using a Nikon fluorescent microscope.
Immunoprecipitation and p38 MAPK Activity Assay
After stimulation with oxLDL (100 μg/mL, 5 minutes, 37°C) in serum-free DMEM without or with pretreatment with PTX (100 ng/mL, 24 hours) or SB203580 (10 μmol/L, 15 minutes), the cells were lysed with lysis buffer. Cell lysates were incubated with total-p38 MAPK antibody (1:100 dilution) overnight at 4°C and then incubated with protein A–Sepharose beads for 2 hours at 4°C with gentle rocking. The beads were washed 4 times with lysis buffer and 2 times with kinase buffer. p38 MAPK activity in immunoprecipitates was measured using the p38 MAPK assay kit according to the manufacturer’s instructions. This protocol measures p38 MAPK-induced phosphorylation of recombinant activating transcription factor-2 (ATF-2) fusion protein, as assessed by Western blotting using phospho-ATF-2 antibody.
cAMP Assay and Inositol Phosphate Formation Assay
Cells were challenged with agonist in the presence of 10 μmol/L forskolin and 500 μmol/L 3-isobutyl-1-methylxanthine at 37°C for 10 minutes. The reactions were terminated with 1 N perchloric acid and neutralized with 2 N K2CO3. The cAMP level of each sample was determined using radioimmunoassay, as described previously.27 Protein content of each samples was determined using the modified Bradford-Pierce assay (Pierce Chemicals, Rockford, Ill).
Cells were plated in 12-well plates and incubated in 0.75 mL of inositol-free DMEM containing 2.5 μCi/mL myo-[2-3H]inositol for 20 hours before assay. Labeled cells were washed and preincubated with assay medium (DMEM containing 20 mmol/L HEPES, 20 mmol/L LiCl) for 10 minutes. The cells were then incubated in assay medium containing oxLDL for the indicated time. Inositol phosphate formation was estimated by determining the ratio of [3H]inositol phosphate to [3H]inositol plus [3H]inositol phosphate.28
Lactate Dehydrogenase (LDH) Leakage Measurements
Cytotoxicity was evaluated by a colorimetric assay for quantification of cell death and cell lysis, on the basis of measurement of cytoplasmic LDH activity released by damaged cells into the culture supernatant. The assay was conducted with the cytotoxicity detection kit (Boehringer Mannheim) according to the manufacturer’s instructions. Briefly, cells were cultured in 96-well microtiter plates with DMEM containing 0.1% serum for 48 hours and treated with indicated concentrations of oxLDL for 24 hours in serum-free DMEM. After incubation, the cell-free supernatants were transferred into an optically clear 96-well flat-bottom microtiter plate. The reaction mixture was added to each well and then the absorbance of the samples at 490 nm was recorded using a microtiter plate reader (BioTek Instruments).
Determination of DNA Synthesis in VSMCs
Cells were cultured in 96-well microtiter plates with DMEM containing 0.1% serum for 48 hours and treated with indicated concentrations of oxLDL for 24 hours in serum-free DMEM. Cells were pulsed in 100 μL with 1 μCi/well of [3H]thymidine (22 Ci/mmol, Shanghai Institute of Nuclear Sciences) 4 hours before harvest and finally transferred onto strips of GF/B filters with a multiple automated sample harvester. Radioactivity of individual samples was measured by liquid scintillation counting.
Cell Proliferation Assay
Cells were cultured in 96-well microtiter plates with DMEM containing 0.1% serum for 48 hours and treated with indicated concentrations of oxLDL for 24 hours in serum-free DMEM. The number of viable cells in each well was estimated by measurement of mitochondrial metabolism rate of tetrazolium salt, (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium), MTS, using the CellTiter cell proliferation assay kit (Promega) according to the manufacturer’s instructions. Briefly, 20 μL of MTS was added to each well during the last 4 hours of stimulation with oxLDL; the absorbance of formazan at 490 nm was then recorded using a microtiter plate reader.
Results of the experiments were expressed as mean±SD. Student t test was used for the statistical analysis of the results. Values of P<0.05 were considered to be significant.
OxLDL-Induced Phosphorylation and Activation of p38 MAPK in VSMCs
It has been established that p38 MAPK is activated by dual phosphorylation of 180Thr and 182Tyr residues,29 and the phosphorylation of p38 MAPK has been widely used to represent its activation. As shown in Figure 1A⇓ and 1C⇓, exposure of VSMCs to oxLDL for 5 minutes strongly stimulated phosphorylation of p38 MAPK in a dose-dependent manner, with the maximal response (6.7±0.8-fold versus control) at 100 μg/mL and the half-maximal response at 20 μg/mL. In contrast, LDL displayed a weaker ability to stimulate phosphorylation of p38 MAPK (3.1±0.4-fold versus control at 100 μg/mL) (Figure 1B⇓ and 1C⇓). Direct measurement of p38 MAPK activity in p38 MAPK immunoprecipitates showed that oxLDL clearly stimulated p38 MAPK activation, which was consistent with p38 MAPK phosphorylation (see Figure 6⇓). The results from the same blot reprobed with total-p38 MAPK antibody demonstrated that the treatment of VSMCs with oxLDL or LDL did not change the total amount of p38 MAPK (Figure 1⇓).
The time course of oxLDL-induced phosphorylation of p38 MAPK showed that a significant stimulation of p38 MAPK phosphorylation occurred rapidly within 1 to 3 minutes of exposure to oxLDL, and the maximal stimulation of p38 MAPK was achieved at 5 minutes (Figure 2A⇓ and 2C⇓). Phosphorylation of p38 MAPK slowly declined but remained at a detectable level at 60 minutes. Again, LDL stimulated p38 MAPK phosphorylation at a lower level compared with oxLDL (Figure 2B⇓ and 2C⇓).
The next experiment was to investigate which components of oxLDL were responsible for the p38 MAPK activation. The results revealed that lysophosphatidylcholine (LPC) could effectively activate p38 MAPK. In contrast, neither phosphatidylcholine (PC), cholesterol, 25-hydroxycholesterol, nor oxidized albumin could activate p38 MAPK (Figure 3B⇓ and 3C⇓). Figure 3⇓ also demonstrated that oxLDL stimulated phosphorylation of p38 MAPK as effectively as IL-1α, H2O2, and TNF-α.
OxLDL Prompted Nuclear Translocation of p38 MAPK
Members of the MAPK family are translocated on activation to the nucleus where they are thought to elicit their effects on transcription. With immunofluorescence staining using phospho-p38 MAPK antibody, it was found that, in the absence of stimulation, very low background of phosphorylated p38 MAPK was obtained in VSMCs (Figure 4A⇓). Stimulation of the cells with oxLDL for 5 minutes resulted in significantly enhanced immunoreactivity to phosphorylated p38 MAPK primarily in the cytoplasm with apparently weaker nucleus staining (Figure 4B⇓). After a 15-minute stimulation of the cells with oxLDL, however, the predominant immunoreactivity to phosphorylated p38 MAPK was detected in the nucleus (Figure 4C⇓), indicating the translocation of activated p38 MAPK from the cytoplasm to the nucleus. By 60 minutes, the immunofluorescence staining of phosphorylated p38 MAPK in the cytoplasm was reduced to the background, but it still remained at detectable levels in the nucleus (Figure 4D⇓).
Effects of oxLDL Were Mediated by PTX-Sensitive G Proteins
Pretreatment of cells with dextran sulfate or polyinosinic acid (200 μg/mL, data not shown), the LDL receptor/scavenger receptor inhibitors, exerted no effect on the stimulation of p38 MAPK phosphorylation by oxLDL (Figure 5⇓). In addition, preincubation with tyrphostin 51 or genistein (25 μmol/L, data not shown), the tyrosine kinase inhibitors, which effectively inhibited EGF-induced p38 MAPK phosphorylation (data not shown), did not affect the oxLDL stimulation of p38 MAPK phosphorylation (Figure 5B⇓ and 5C⇓). However, p38 MAPK phosphorylation (Figure 5⇓) and activity (Figure 6⇓) induced by oxLDL were strongly inhibited when cells were preincubated with PTX. In contrast, pretreatment of cells with cholera toxin (CTX) hardly affected oxLDL-induced phosphorylation of p38 MAPK (Figure 5⇓). Moreover, oxLDL could significantly inhibit forskolin-stimulated accumulation of cAMP, which was also PTX sensitive (Figure 6C⇓). Taken together, these data indicated the effects of oxLDL were likely mediated by PTX-sensitive Gi/Go proteins but unlikely by the LDL receptors, scavenger receptors, or tyrosine kinase receptors.
Phospholipase C (PLC) Was Involved in the OxLDL-Induced Effects
PLC has been shown as one component of the signaling pathways for activation of p38 MAPK.30 As presented in Figure 7⇓, U-73122, a PLC inhibitor, which has been shown to specifically inhibit phosphoinositide hydrolysis, considerably blocked the p38 MAPK phosphorylation induced by oxLDL. As a control, U-73343, the inactive structural analogue of U-73122, failed to block the oxLDL-induced p38 MAPK activation. Neither compound displayed any significant effect on basal p38 MAPK phosphorylation (Figure 7⇓). In addition, hydrolysis of phosphatidylinositol in response to oxLDL stimulation was observed within 2 to 5 minutes by inositol phosphate formation assay (Figure 7C⇓). And this effect of oxLDL was blocked by U-73122 but not by U-73343 (data not shown). However, the ability of oxLDL to stimulate p38 MAPK was affected neither by pretreatment with specific PKC inhibitors, calphostin C, or Gö6976 nor by chelators of [Ca2+]i, BAPTA/AM, or dantrolene (data not shown), indicating the effects of oxLDL were independent of PKC or [Ca2+]i. Furthermore, a phosphatidyl inositol 3-kinase inhibitor, wortmannin (50 nmol/L), significantly reduced (about 50%) oxLDL-stimulated p38 MAPK activation (data not shown), suggesting that phosphatidyl inositol 3-kinase might be involved in the effects of oxLDL.
Inhibition of p38 MAPK Reduced OxLDL-Induced Cytotoxicity
To test the probable association of oxLDL activation of p38 MAPK with its cytotoxic effects, the present study applied 3 independent assays: leakage of cytoplasmic LDH to the culture medium, [3H]thymidine incorporation, and MTS cell proliferation. Treatment of cells with oxLDL (30 and 100 μg/mL) produced a significant LDH leakage from cytoplasm (Figure 8A⇓), a profound decrease in the [3H]thymidine incorporation into DNA (Figure 8B⇓), and a remarkable reduction in viable cell numbers (Figure 8C⇓). A specific p38 MAPK inhibitor, the pyridinyl imidazole compound SB203580, has been popularly used to block p38 MAPK activation.31 The coapplication of SB203580 along with oxLDL greatly inhibited oxLDL-induced cytotoxic effects in all of 3 measurements used in the present study (Figure 8⇓). Furthermore, preincubation of cells with PTX also effectively reversed the cytotoxic effects of oxLDL, as measured by 3 assays (Figure 8⇓). Treatment with either SB203580 or PTX individually did not affect viability of VSMCs, as determined in the control experiments (data not shown).
Additional experiments were carried out to measure the dose responses of SB203580, and the results (Figure 9⇓) showed that the inhibitor dose dependently blocked p38 MAPK phosphorylation and oxLDL-induced cytotoxicity at similar potency (IC50 ≈1 μmol/L). Our results also showed (Figure 6⇑) that SB203580 effectively blocked in vitro phosphorylation of recombinant ATF-2, a downstream target of p38 MAPK.
In the present study, our data showed for the first time that in VSMCs, oxLDL could strongly stimulate phosphorylation and activation of p38 MAPK and significantly induce translocation of activated p38 MAPK into the nucleus. Our results also revealed the possible signaling pathways for oxLDL to exert their effects: it was apparently not mediated by their classical scavenger receptors or by the general tyrosine kinase receptors but was likely mediated by PTX-sensitive G proteins and at least partially via PLC. Of more interest is that inhibition of p38 MAPK by its specific inhibitor SB203580 significantly reduced oxLDL-induced cytotoxicity in VSMCs, and pretreatment of VSMCs with PTX also inhibited oxLDL-induced cytotoxicity. Thus, the present study not only identified p38 MAPK as an important effector that mediated rapid cellular responses to oxLDL but also demonstrated that activation of p38 MAPK by oxLDL was crucial in oxLDL-induced cytotoxicity in VSMCs.
It has been shown that oxLDL can activate p42/44 MAPK in VSMCs,19 20 and this may be attributed to the lipid moiety of oxLDL, such as LPC, which is present in oxLDL at levels up to 40 μg/mg.19 32 Our preliminary results demonstrated that LPC could also effectively activate p38 MAPK. In contrast, neither PC, cholesterol, nor 25-hydroxycholesterol could activate p38 MAPK, suggesting that the cholesterol moiety may not be directly involved in the oxLDL-induced activation of p38 MAPK. Although the protein moiety of oxLDL was unable to activate p42/44 MAPK,19 and oxidized albumin was also not responsible for p38 MAPK activation, whether apolipoprotein B can activate p38 MAPK remains to be investigated. According to several reviews,3 7 oxLDL can be divided with some different physical and chemical properties into minimally oxidized LDL and oxLDL. The oxLDL used in the present study can be correspondingly defined as highly oxidized LDL. On the basis of the activities of native LDL and highly oxidized LDL, minimally oxidized LDL should possess moderate activity to stimulate p38 MAPK. Additional studies are needed to investigate the relationship between the extent of LDL oxidation and activation of p38 MAPK.
Scavenger receptors, a family of trimeric membrane glycoproteins at the surface of vascular cells, can specifically mediate the uptake of oxLDL.7 So far, several different receptors for oxLDL have been identified.3 33 Until now, none of these receptors with a single membrane–spanning domain is reported to couple to any kind of heterotrimeric G proteins. Moreover, the rapidity of p38 MAPK response (within 5 minutes) suggested that oxLDL internalization by scavenger receptors was unlikely to mediate oxLDL-induced p38 MAPK activation. Our data that dextran sulfate or polyinosinic acid had no effect on oxLDL-induced p38 MAPK phosphorylation further supported the notion that activation of p38 MAPK by oxLDL was not mediated by scavenger receptors. Although oxLDL are recently reported11 to activate EGF receptor, which is consequently able to activate p38 MAPK, the inability of tyrosine kinase inhibitors to block oxLDL-induced p38 activation in the present study suggested that EGF receptor was unlikely responsible for the rapid effect of oxLDL.
Our results showed that in VSMCs, p38 MAPK activation by oxLDL was completely inhibited by PTX. As a control, basic fibroblast growth factor –stimulated p38 MAPK phosphorylation was not inhibited by PTX pretreatment (data not shown). Our data also showed that oxLDL could significantly inhibit the forskolin-stimulated cAMP accumulation, which could be blocked by PTX pretreatment, confirming the ability of oxLDL to activate Gi/Go proteins in transmembrane signal transduction. In macrophages, oxLDL have been shown to suppress activation of NF-κB via PTX-sensitive G proteins.12 Our preliminary results showed that in U937 macrophage-like cells, oxLDL also strongly and dose dependently stimulated p38 MAPK activation (data not shown). It is likely that the same PTX-sensitive G protein–coupled receptor(s) is responsible for the observed effects of oxLDL in macrophages12 and VSMCs. However, more studies are needed to identify the receptor molecule. In addition, our results also indicated that oxLDL-induced p38 MAPK activation involved PLC and phosphatidyl inositol 3-kinase and was independent of PKC or [Ca2+]i, as previously reported in the case of p38 MAPK activation mediated by PTX-sensitive formyl peptide receptors.30
It has been established that p38 MAPK can be activated by a variety of environmental stresses such as osmotic shock, ultraviolet radiation, heat shock, and proinflammatory cytokines, and activation of p38 MAPK cascades can trigger apoptosis or programmed cell death,17 cardiac hypertrophy,34 inflammation, and cell cycle arrest at the G1/S transition. Apoptosis and inflammation have been implicated to play a causative role in atherosclerosis.18 The present study provided direct evidence that the cytotoxicity induced by oxLDL in VSMCs was associated with activation of p38 MAPK, because inhibition of p38 MAPK attenuated oxLDL-induced cytotoxicity, as measured by 3 independent assays. Given that SB203580 could not completely block oxLDL-induced cytotoxicity, it is likely that other pathways, such as endocytosis of oxLDL via scavenger receptors and subsequent oxidative damage of lysosomal membranes by several oxLDL peroxidation products, may also contribute to the effects of oxLDL. In addition, p38 MAPK activated by oxLDL in VSMCs may belong to 2 isoforms of p38 MAPK (p38α and p38β but not p38γ and p38δ), because of p38 MAPK isoform inhibitory selectivity35 36 of SB203580 used in the study. However, which isoform of p38 MAPK is functionally expressed in VSMCs and which one is activated by oxLDL remains to be investigated. In addition, whether p38 MAPK can be activated directly or indirectly via uptake of oxLDL into the cells through the scavenger receptors must be determined. Also, in VSMCs, it is not clear whether JNK/SAPK is activated by oxLDL and involved in oxLDL-induced cytotoxicity, because high concentrations of SB203580 also inhibit JNK.37 38 It has been well accepted that exposure of vascular cells to cytotoxic doses of oxLDL leads to biological consequences that are thought to collectively contribute to the progression of atherosclerotic plaque.3 We therefore speculated that p38 MAPK activation by oxLDL might be relevant to plaque rupture and thrombosis. To specifically interrupt the p38 MAPK activation by oxLDL might thus develop into a therapeutic strategy to alter the progression of atherosclerosis.
This work was supported by research grants from the National Natural Science Foundation of China (39600063, 39625015, and 39630130), Chinese Academy of Sciences (KY951-A1-301 and KJ951-B1-608), and German Max-Planck Society. The authors wish to thank Zhe Zhang, Ping Wang, Xu-Min Zhang, and Pei-Hua Wu for their kind assistance.
- Received September 23, 1998.
- Accepted January 24, 1999.
- © 1999 American Heart Association, Inc.
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