LDL-Activated p38 in Endothelial Cells Is Mediated by Ras
Abstract—Endothelial dysfunction is a major atherogenic proinflammatory event. LDL causes the activation and phenotypic changes of cultured vascular endothelial cells (ECs). We previously reported that LDL activates c-Jun and AP-1 in ECs. In this study, we demonstrated that p38–ATF-2 is activated by LDL in human ECs and that this activation is mediated by Ras. When ECs are incubated with LDL in pathophysiological concentrations, the p38-mediated ATF-2 phosphorylation and ATF-2 transactivation are increased in a time- and dose-dependent manner. To elucidate the upstream mechanism in LDL-activated p38 in ECs, we demonstrate that LDL increases Ras translocation from the cytoplasm to the cellular membrane, with concurrent increases in Ras binding activity to GST–Raf-1. Overexpression of RasN17, a dominant negative mutant of Ras, attenuates the LDL-induced increases in (1) phosphorylation of ATF-2, (2) phosphorylation of c-Jun, (3) AP-1 binding, and (4) AP-1–driven luciferase activity. To study the effect of p38 in the regulation of an LDL targeting gene, we show that a specific p38 inhibitor attenuates LDL-induced E-selectin at the mRNA level. Thus, LDL activates both p38 and JNK signaling pathways through Ras activation, and furthermore, these events may play an important role in LDL-induced endothelial activation.
- Received March 15, 2001.
- Accepted April 27, 2001.
Endothelial dysfunction is one of the earliest proinflammatory vascular events leading to atherosclerosis.1 Native LDL has been implicated in initiating endothelial cell (EC) dysfunction.2 When incubated with LDL, ECs in culture are activated, and several genes involved in atherogenesis, including E-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1, are upregulated.3 4 5 6 EC activation by LDL involves mobilization of calcium, activation of protein kinases, and an increase in the transactivation of AP-1 transcription factor.3 4 7 8 9 The effects of LDL on intracellular signal transduction leading to EC activation, however, have been studied only to a limited extent.
In response to stimulation by mitogens, cytokines, UV irradiation, and other environmental stresses, membrane-associated small GTPase molecules, eg, p21-Ras, are activated. Ras cycles between an active GTP-bound and an inactive GDP-bound state, functioning as a molecular switch in response to cell-activating stimuli. Ras can trigger at least 3 diverging mitogen-activated protein kinase
(MAPK) cascades. The first cascade is mediated by Raf-1 activation and transmits signals through MAPK/extracellular signal-regulated kinase (ERK) kinase
(MEK)1/2 to activate ERK. The second cascade operates through MEKK1 and JNKK to activate c-Jun NH2-terminal kinases (JNKs). The third cascade leads to p38 activation. Efficient activation of p38 requires phosphorylation of Thr-180 and Tyr-182. At least 3 Thr/Thy kinases (MKK3, MKK4/SEK1, and MKK6) phosphorylate and activate p38. This leads to the activation of multiple transcription factors, such as ATF-2 and CHOP, that induce the expression of proinflammatory genes, such as E-selectin.10
ATF-2 can form heterodimers with c-Jun, which positively regulate c-Jun promoter activity.11 Recently, we found that LDL increases AP-1 activity and activates the JNK–c-Jun pathway but not the ERK/c-Fos pathway in human ECs.8 9 This report was designed to study whether LDL activates the p38–ATF-2 pathway in ECs and to elucidate the upstream signaling involved. Our study demonstrates that LDL enhances Ras translocation to membrane and Ras activation in human umbilical vein ECs (HUVECs) and that these events activate both p38–ATF-2 and JNK–c-Jun signaling pathways. Furthermore, we show that LDL-induced E-selectin is blocked by a specific p38 inhibitor.
Cell Culture and LDL Isolation
HUVECs were isolated and maintained as described previously.8 All experiments were performed with cells up to passage 3 in EC medium and cultured to confluence before LDL treatment. LDL was isolated from nonfrozen human plasma as described.8 9 The LDL preparations contained <0.0005 U endotoxin/mg cholesterol as determined by the chromogenic Limulus test (BioWhittaker). For all studies, LDL was used at a final cholesterol culture concentration of 240 mg/dL (6.24 mmol/L).
Western Blotting Analysis
Cellular membrane and cytosolic proteins from whole-cell lysate were isolated as previously described.12 Western analyses with antibodies against H-Ras (Transduction Laboratories) or Anti-ACTIVE MAPK pAb antibody (Promega) were performed as previously described.9 12
Affinity Precipitation/Immunoblot of Activated Ras
GST fusion protein, corresponding to the human Ras-binding domain (RBD, residues 1 to 149) of Raf-1 bound to glutathione agarose, was from Upstate Biotechnology. The procedure used to measure Ras binding on GST–Raf-1 RBD was described by company protocol (Upstate Biotechnology).
ATF-2 and c-Jun Phosphorylation Assay
The bacterial expression vector GST–ATF-2 (residues 1 to 109)13 was provided by R.J. Davis (University of Massachusetts Medical School). A GST–ATF-2 fusion protein was isolated and purified. The assay for c-Jun phosphorylation was performed as described.9 The procedures for the ATF-2 phosphorylation assay were similar to those for c-Jun, except that the cell extracts were incubated with GST–ATF-2. In the study of the role of p38 on ATF-2 phosphorylation, p38 protein was immunoprecipitated with an antibody against p38. Then, the immunoprecipitated p38 was incubated with agarose-bound GST–ATF-2 in a kinase buffer.14
The recombinant adenovirus Ad-RasN17 encoding for RasN17 was constructed as described previously.15 The adenoviruses were plaque-purified, expanded and titrated in 293 cells, and purified by cesium chloride methods.16 For adenoviral infection, confluent HUVECs were exposed to adenoviral vectors (Ad-RasN17 or Ad-β-gal as control) at a multiplicity of infection of 100 to 500 for 2 hours. After the viruses had been washed out, HUVECs were continuously incubated for 18 to 24 hours before the treatment.16
Plasmids and Transfection
For transactivation experiments, we used the Targefect transfection method (Targeting Systems). The in vivo trans-reporting system was purchased from Stratagene. This system includes a pFA–c-Jun, pFC–ATF-2, pFA2-Elk, or pFA-CHOP (CHOP is a transcription factor specific response to p38 activation) as an activator plasmid, and a reporter plasmid (pFR-Luc). pFC-dbd plasmid and pFC-MEKK plasmid were used as a negative and positive control, respectively. pRSV-β-gal was cotransfected as a transfection control. After 24 hours of LDL or phorbol 12-myristate 13-acetate (PMA) exposure, samples were collected and assayed for luciferase activity. The results were normalized against β-galactosidase.9
Electrophoretic Mobility Shift Assay
After infection by the adenoviral construct Ad-RasN17, HUVEC monolayers on 100-mm dishes were exposed to 240 mg/dL of LDL for 6 hours or to 50 ng/mL PMA for 2 hours.8 Nuclear extracts were prepared, and an electrophoretic mobility shift assay was performed with consensus sequences for AP-1 and nuclear factor (NF)-κB as described.8
Total RNA isolation and Northern analysis for hABC1 and von Willebrand factor (vWF) expression were performed.8 The probes of E-selectin and vWF cDNA were labeled with [α-32P]dCTP by DECApriming (Ambion) as previously described.6
Quantitative data were expressed as mean±SEM. Statistical significance of the data was evaluated by Student’s t test. Probability values of P<0.05 were considered significant. For nonquantitative data, the results were expressed as representative of ≥3 independent experiments.
LDL Increases p38 Activity in ECs
We have previously shown that LDL activates JNK/c-Jun, but not ERK/c-Fos, in ECs and proposed that Ras may play an important role in LDL-induced EC activation.8 9 As another major MAPK pathway induced by Ras, p38/ATF-2 activation by LDL in ECs was investigated in this study. Confluent HUVECs were exposed to LDL in concentrations up to 240 mg/dL for various times. Cell lysates were collected from various samples for ATF-2 phosphorylation assay. As shown in Figure 1A⇓, LDL increases ATF-2 phosphorylation 30 minutes after LDL exposure, reaching a peak at 2 hours. To confirm the involvement of p38 in ATF-2 phosphorylation, we immunoprecipitated p38 proteins with anti-p38 antibody and then used the precipitated p38 to perform the kinase activity assay. A similar pattern of ATF-2 phosphorylation was obtained with the p38-immunoprecipitated samples compared with whole-cell lysates. Exposure of ECs to different concentrations of LDL caused increased p38 activity in a dose-dependent fashion, with incremental increases over the concentration range of 160 to 240 mg/dL (Figure 1B⇓). Therefore, LDL at a concentration of 240 mg/dL was used in the rest of the experiments.
LDL Increases ATF-2 and c-Jun Transactivation in ECs
To further investigate whether LDL activates ATF-2 transactivation in ECs as a consequence of p38 activation, ECs were cotransfected with activator plasmids pFC–ATF-2, pFA-CHOP, pFA–c-Jun, or pFA2-Elk, and the reporter plasmid pFR-Luc. pFC-MEKK plasmid was used as a positive control in the groups of pFC–ATF-2, pFA-CHOP, and pFA–c-Jun transfection. PMA was a positive control for pFA2-Elk transfection. pFC-dbd plasmid containing the DNA binding domain of the yeast GAL4 but lacking any activation domain served as negative control. Eighteen hours after transfection, cells were exposed to LDL for 24 hours, and samples were collected for luciferase activity assay. LDL increased ATF-2 transactivation activity by 2.8-fold, CHOP by 2.9-fold, and c-Jun by 3-fold. LDL failed to activate pFA2-Elk, however, whereas PMA induced activity >6-fold (see Figure 2⇓). The cotransfection of pFC-dbd and pFC-luc had only minimum luciferase activity, and LDL exposure caused no change (data not shown).9 Thus, LDL promotes both ATF-2 and c-Jun activation, but not Elk activation, in human ECs. These data, together with our previous reports showing that LDL activates JNK/c-Jun,8 9 indicate that LDL activates ECs through both p38 and JNK pathways.
LDL Increases Ras Membrane Translocation and Activity in HUVECs
We studied whether Ras is an upstream molecule for the activation of p38 and JNK cascades in ECs by LDL. We first examined the membrane translocation of Ras by determining Ras abundance in whole-cell lysates and the cell membrane. In whole-cell lysates, LDL did not increase the amount of Ras up to 6 hours. Ras was increased by LDL exposure in 1 to 2 hours, however, in the membrane fraction of the cells (Figure 3A⇓). Thus, LDL promotes Ras translocation to the cytoplasmic membrane. To test Ras activation by LDL, we used GST–Raf-1 RBD conjugated with agarose to pull down the GTP-bound form of Ras.17 The Raf-1–bound Ras was increased by LDL exposure (see Figure 3B⇓). The LDL-increased Ras activation began at 30 minutes, reached a peak at 2 hours, and returned to basal level at 6 hours. These data demonstrate LDL activation of Ras in human ECs.
RasN17 is a dominant negative mutant of Ras in which the Lys-17 in the wild type has been replaced by an Asn. RasN17 competes with the endogenous Ras and thus inhibits the Ras-mediated signaling pathway.15 We overexpressed the wild type of Ras (wtRas) or RasN17 in HUVECs, which were then treated with LDL for 2 hours. The cell lysates were collected for the GST–Raf-1 binding assay. Raf-1–bound Ras in cells with wtRas overexpression was increased by LDL exposure. In contrast, RasN17 overexpression blocked this Ras activation in the LDL-treated samples as well as untreated controls (Figure 3C⇑, bottom). Furthermore, LDL caused Ras translocation to the cell membrane, as detected by immunoblotting, in wtRas-transfected cells (data not shown). Therefore, LDL activates both endogenous and overexpressed wtRas, but not the Ras mutant with low GTP affinity.
Dominant Negative Mutant of Ras Attenuates LDL Activation of P38 and JNK
To elucidate the role of Ras in LDL activation of P38, we infected HUVECs with an adenoviral construct, Ad-RasN17, to overexpress RasN17. These cells, as well as control cells (infected with adRSV-β-gal), were then exposed to LDL. Cell lysates were collected for ATF-2 and c-Jun phosphorylation assays. In parallel experiments, cells were treated with PMA, thus serving as a positive control. As shown in Figure 4⇓, both LDL and PMA increased ATF-2 and c-Jun phosphorylation in cells infected with Ad-β-gal (top 2 panels). These effects were attenuated in cells infected with Ad-RasN17. The results suggest that Ras plays an important role in the signaling of LDL activation of p38 and JNK. Because PMA, but not LDL, induced ERK1/2 phosphorylation in ECs,9 the inhibitory effect of RasN17 on PMA-induced ERK1/2 phosphorylation is shown in Figure 4⇓, bottom.
Dominant Negative Mutant of Ras Attenuates the LDL-Induced AP-1 Activation
Because mutant Ras blocks p38/ATF-2 and JNK/c-Jun pathways and Jun/ATF-2 heterodimers bind to the AP-1 site in the promoters of many genes (eg, the gene encoding c-Jun),11 RasN17 should attenuate LDL-induced AP-1 activation. To test this, we investigated the modulation of AP-1 binding by LDL using electrophoretic mobility shift assay in Ad-RasN17–infected ECs. As shown in Figure 5⇓, RasN17 abolished the LDL-induced increase in AP-1 binding and reduced the PMA-induced increase in AP-1 binding. Many inducible molecules in ECs, eg, VCAM-1 and ICAM-1, contain a number of AP-1–like and NF-κB–like binding motifs within their 5′ promoter regions.4 18 19 Because LDL activated AP-1 but not NF-κB,8 LDL-induced Ras-p38 and Ras-JNK pathways should have little effect on NF-κB activation. PMA increased the binding activity of NF-κB, and this effect was not blocked by Ad-RasN17 in ECs (Figure 5⇓).
To test whether the blockage of p38 and JNK by RasN17 leads to a similar outcome in LDL-induced AP-1 activation, we cotransfected pAP-1-luc and RasN17 plasmids into HUVECs. The transfected cells were then exposed to LDL. As shown in Figure 6⇓, after RasN17 transfection, LDL no longer induced AP-1 activation (P<0.01). The basal level of AP-1 activity was also decreased in RasN17 transfection samples compared with controls. Consequently, a Ras-dependent pathway appears to be involved in the AP-1 activation by LDL.
A Specific p38 Kinase Inhibitor Blocks LDL-Induced E-Selectin
We previously reported that LDL increased endothelial cell adhesiveness by inducing adhesion molecules, including E-selectin.6 To elucidate the role of p38 in the induction of LDL-induced targeting genes, we chose E-selectin as an example in ECs, because it is an ATF-2–regulatory gene.10 We exposed HUVECs to LDL with or without SB203580, a specific p38 kinase inhibitor, for 48 to 72 hours. Total RNA was collected from various samples for Northern analysis. As shown in Figure 7⇓, SB203580 blocked LDL-increased E-selectin mRNA in both LDL exposure times. vWF mRNA was also detected as an endothelial internal control.
Hypercholesterolemia is a well-known risk factor for the development of atherosclerosis. The elevated level of LDL is associated with endothelial dysfunction and lesion formation.20 21 LDL activates JNK,9 promotes AP-1 binding,8 and induces adhesion molecules in cultured human vascular ECs.4 6 The present study was designed to determine whether LDL activates ECs by promoting p38, another MAPK family member, and whether Ras mediates LDL activation of the p38 pathway. Our findings demonstrate that (1) LDL activates the p38-mediated signaling pathway, (2) LDL increases Ras membrane translocation and activation, (3) a dominant negative mutant of Ras attenuates LDL-induced ATF-2 and c-Jun phosphorylation and AP-1 activation, and (4) a specific p38 kinase inhibitor attenuates LDL-induced induction of E-selectin at the mRNA level.
Originally identified as a proto-oncogene, Ras has been broadly studied for its function and regulation. There is little information, however, on the physiological and pathophysiological roles of Ras in cardiovascular cells, especially in endothelium exposed to lipoprotein. As a small GTPase molecule, Ras moves to the plasma membrane after the synthesis in free cytoplasmic ribosomes and the farnesylation of its posttranslational C-terminal. Ras activation can trigger at least 3 diverging MAPK cascades, ie, p38, JNK, and ERK. Distinct intracellular MAPK signaling cascades are differentially activated to orchestrate transcriptional activation in response to various extracellular stimuli. LDL activates the membrane-associated Ras in human ECs, which in turn preferentially activates p38 and JNK, the 2 MAPKs particularly involved in stress responses. Both native LDL and oxidized LDL have been found to stimulate p44/42 and p38 MAPKs in smooth muscle cells and microphages.22 23 24 25 In contrast, neither native LDL nor Cu2+-oxidized LDL stimulates ERK activity in bovine aortic ECs.25 It is not clear why LDL causes this differential activation of MAPKs in ECs. Fluid shearing of vascular ECs increases the JNK activity by >10-fold but activates ERK to a much lesser degree.26 Osmotic pressure and UV irradiation also selectively activate the JNK pathway but not the ERK pathway.27 28 Ras can activate both Raf-1 and MEKKs. Raf-1 activates ERK but not JNK/p38, whereas MEKKs activate JNK/p38 but not ERK.29 Thus, LDL-stimulated Ras may activate MEKKs to a greater extent than Raf-1. Alternatively, other LDL-induced signaling pathways may inhibit Raf-1 signaling. cAMP inhibition of ERK activation by preventing Ras-dependent activation of Raf-1 has been reported.30 Indeed, LDL increased cAMP-responsive element–binding protein binding and protein kinase A activation in ECs.8 Recently, the phosphatidylinositol 3-kinase (PI3K)–Akt pathway was shown to inhibit the Raf-MEK-ERK pathway through Akt phosphorylation of Raf-1.31 32 The involvement of the PI3K-Akt pathway in the LDL-mediated EC activation remains to be studied. Regarding upstream signaling, Cdc42 and Rac-1, in addition to Ras, can also selectively activate JNK and p38.33 34 Thus, LDL may also activate JNK/p38 through Rho family GTPase-mediated signal transduction.
The guanine nucleotide exchange factors are important in Ras activation in response to growth factors, shear stress, and cytokines. In many instances, docking proteins such as Grb2/Sos are necessary for Ras activation. Sos is a cytoplasmic Ras-GEF that is constitutively associated with the adaptor protein Grb2. When Grb2 interacts with a tyrosine-phosphorylated membrane receptor, it positions Sos at the plasma membrane where it can promote activation of Ras. Alternatively, a tyrosine-phosphorylated Shc may serve as a bait for Grb2 docking and Sos activation. We detected no membrane translocation of these docking proteins in HUVECs at up to 6 hours of LDL exposure (Zhu et al, unpublished observation). Thus, LDL may activate Ras in ECs through a process independent of growth factor activation. Conversely, the elevated cellular cholesterol level resulting from LDL exposure could promote the membrane translocation of caveolin-1 and Ras, which is a caveolin-binding signaling molecule, into caveolae in human ECs.12 Thus, caveolin may play a role in LDL-mediated Ras activation.
E-selectin is an important adhesion molecule that is transiently and specifically expressed in ECs on stimulation with cytokines and LDL.6 35 E-selectin can serve as a marker for endothelial activation. ATF2 was reported to play an important role in the activation of the E-selectin promoter.10 LDL increases E-selectin at both the mRNA and protein levels6 8 and can increase the E-selectin promoter activity (Zhu et al, unpublished observation). Here, we report that a p38 kinase inhibitor attenuates LDL-induced E-selectin at the mRNA level. Thus, it appears likely that LDL activates the E-selectin promoter in ECs through the ATF-2–dependent mechanism. The blocking effects of RasN17 on ATF-2 and c-Jun phosphorylation and AP-1 transactivation provide further evidence for the involvement of Ras in this transcriptional activation.
Collectively, the results of the present and previous studies show that LDL activates ECs predominantly through Ras-JNK/p38 signaling pathways. This supports our working hypothesis that LDL perturbs the cell membrane and activates membrane-associated proteins (eg, Ras), which, in turn, activate the JNK/p38 pathways. Subsequently, c-Jun and ATF-2 are activated and form a c-Jun/ATF-2 dimer to induce c-Jun expression via the TRE site at the Jun promoter. Accumulation of c-Jun by de novo synthesis consistently elevates the level of AP-1, which further chronically induces other specific cellular genes regulated by AP-1 (eg, ICAM-1 and E-selectin), leading to EC activation.
This study was supported in part by NIH grant HL-43023 (to M.B.S.), HL-60789 (to J.Y.-J.S.), and American Heart Association, Western States Affiliate grant 98-252 (to Y.Z.). J.Y.-J.S. is an Established Investigator of the American Heart Association.
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