Mitogenic effect of oxidized low-density lipoprotein on vascular smooth muscle cells mediated by activation of Ras/Raf/MEK/MAPK pathway

Abstract

1. It has been demonstrated that oxidized low-density lipoprotein (OX-LDL) is a risk factor in atherosclerosis by stimulating vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms of OX-LDL-induced cell proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with mitogen-activated protein kinase (MAPK) activation in rat cultured VSMCs. 2. Both native-LDL (N-LDL) and OX-LDL induced a time- and concentration-dependent incorporation of [(3)H]-thymidine in VSMCs. 3. OX-LDL induced time- and concentration-dependent phosphorylation of p42/p44 MAPK. Pretreatment of these cells with pertussis toxin or U73122 attenuated the OX-LDL-induced responses. 4. Pretreatment with PMA for 24 h, preincubation with a PKC inhibitor staurosporine or the tyrosine kinase inhibitors, genistein and herbimycin A for 1 h, substantially reduced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation induced by OX-LDL. 5. Removal of Ca(2+) by BAPTA/AM or depletion of the internal Ca(2+) pool by thapsigargin significantly inhibited OX-LDL-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation. 6. OX-LDL-induced [(3)H]-thymidine incorporation and p42/p44 MAPK phosphorylation was inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK) in a concentration-dependent manner. 7. Overexpression of dominant negative mutants of Ras (H-Ras-15A) and Raf (Raf-N4) significantly suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. 8. These results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G protein-coupled receptor that involves the activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB in rat cultured VSMCs.

Figure 1

[³H]-thymidine incorporation induced by N-LDL or OX-LDL in VSMCs. (A) Time course, after 24 h in serum-free medium, the cells were stimulated with vehicle, N-LDL or OX-LDL at a concentration of 100 μg ml⁻¹. The cells were labelled with 1 μCi ml⁻¹ [³H]-thymidine for the times indicated in the continuous presence of N-LDL or OX-LDL. (B) Concentration-dependence, the cells were stimulated with various concentrations of N-LDL or OX-LDL. After stimulation for 8 h, VSMCs were labelled with 1 μCi ml⁻¹ [³H]-thymidine for another 16 h in the continued presence of N-LDL or OX-LDL. The incorporation of [³H]-thymidine was determined as described in Methods. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate.

Figure 2

Time course of OX-LDL-stimulated p42/p44 MAPK phosphorylation in VSMCs. The cells were grown to confluency, made quiescent by serum-deprivation for 24 h and incubated with 100 μg ml⁻¹ OX-LDL for 2 – 60 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with anti-phospho-p42/p44 MAPK polyclonal antibody for activated p42/p44 MAPK and anti-p42 MAPK antibody for the total p42 MAPK as an indicator of protein loading in each well. Bands were visualized by an ECL method. Data are expressed as the mean±s.e.mean of three separate experiments. ^*P<0.01, compared with the respective basal level.

Figure 3

Concentration-dependence of OX-LDL-stimulated p42/p44 MAPK phosphorylation in VSMCs. The cells were grown to confluency, made quiescent by serum-deprivation for 24 h and incubated with various concentrations of OX-LDL for 5 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Bands were visualized by an ECL method as described in Figure 2. Data are expressed as the mean±s.e.mean of three separate experiments. ^*P<0.01, compared with the respective basal level.

Figure 4

Effects of pertussis toxin and U73122 on DNA synthesis and p42/p44 MAPK phosphorylation induced by OX-LDL in VSMCs. (A) The cells were grown to confluency, made quiescent by serum-deprivation and preincubated with pertussis toxin (PTX, 100 ng ml⁻¹) for 24 h or U73122 (10 μM) for 1 h, and then stimulated with vehicle or 100 μg ml⁻¹ OX-LDL. After 8 h incubation, cells were labelled with 1 μCi ml⁻¹ [³H]-thymidine for another 16 h in the continuous presence of OX-LDL. The incorporation of [³H]-thymidine was determined as described in Methods. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. ^*P<0.01, compared with the cells exposed to OX-LDL. (B) For MAPK experiment, after incubation with PTX for 24 h or U73122 for 1 h, the cells were stimulated with 100 μg ml⁻¹ OX-LDL for 5 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Bands were visualized by an ECL method as described in Figure 2. Data are expressed as the mean±s.e.mean of three separate experiments. ^*P<0.01, compared with the cells exposed to OX-LDL.

Figure 5

Involvement of PKC in DNA synthesis and p42/p44 MAPK phosphorylation induced by OX-LDL in VSMCs. (A) The cells were grown to confluence, made quiescent by serum-deprivation for 24 h. The cells were preincubated with 1 μM staurosporine (STA) for 1 h, or 1 μM PMA for 24 h before stimulated with vehicle or 100 μg ml⁻¹ OX-LDL. The incorporation of [³H]-thymidine was determined as described in Figure 4. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. ^*P<0.01, compared with the cells exposed to OX-LDL. (B) For MAPK experiment, after incubation with STA for 1 h, or PMA for 24 h, the cells were stimulated with 100 μg ml⁻¹ OX-LDL for 5 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Bands were visualized by an ECL method as described in Figure 2. Data are expressed as the mean±s.e.mean of three separate experiments. ^*P<0.05, compared with the cells exposed to OX-LDL.

Figure 6

Effect of BAPTA plus EGTA on OX-LDL-induced [³H]-thymidine incorporation and p42/p44 MAPKphosphorylation in VSMCs. (A) The cells were grown to confluence, made quiescent by serum-deprivation for 24 h. The cells were preincubated with 30 μM BAPTA and 5 mM EGTA for 1 h and then stimulated with vehicle or 100 μg ml⁻¹ OX-LDL. The incorporation of [³H]-thymidine was determined as described in Figure 4. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. ^*P<0.01, compared with the cells exposed to OX-LDL. (B) For MAPK experiment, after incubation with BAPTA and EGTA for 1 h, the cells were stimulated with 100 μg ml⁻¹ OX-LDL for 5 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Bands were visualized by an ECL method as described in Figure 2. Data are expressed as the mean±s.e.mean of three separate experiments. ^*P<0.01, compared with the cells exposed to OX-LDL.

Figure 7

Effects of diltiazem, ryanodine, and thapsigargin on OX-LDL-induced [³H]-thymidine incorporation in VSMCs. The cells were grown to confluence, made quiescent by serum-deprivation for 24 h. The cells were preincubated with 10 μM diltiazem (Dil), ryanodine or thapsigargin (TG) for 1 h and then stimulated with vehicle or 100 μg ml⁻¹ OX-LDL. The incorporation of [³H]-thymidine was determined as described in Figure 4. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. ^*P<0.001, compared with the cells exposed to OX-LDL.

Figure 8

Involvement of tyrosine kinase in DNA synthesis and p42/p44 MAPK phosphorylation induced by OX-LDL in VSMCs. (A) The cells were grown to confluence, made quiescent by serum-deprivation for 24 h. The cells were preincubated with 10 μM genistein and 10 μM herbimycin A for 1 h, and then stimulated with vehicle or 100 μg ml⁻¹ OX-LDL. The incorporation of [³H]-thymidine was determined as described in Figure 4. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. ^*P<0.01, compared with the cells exposed to OX-LDL. (B) For MAPK experiment, after incubation with genistein (GEN) and herbimycin A (HER) for 1 h, the cells were stimulated with 100 μg ml⁻¹ OX-LDL for 5 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Bands were visualized by an ECL method as described in Figure 2. Similar results were obtained in three independent experiments.

Figure 9

Effects of MAPK kinase inhibitors on OX-LDL-stimulated DNA synthesis and p42/p44 MAPK phosphorylation in VSMCs. (A) The cells were grown to confluence, made quiescent by serum-deprivation for 24 h. The cells were preincubated with 10 μM PD98059 and 10 μM SB203580 for 1 h, and then stimulated with vehicle or 100 μg ml⁻¹ OX-LDL. The incorporation of [³H]-thymidine was determined as described in Figure 4. Data are expressed as the mean±s.e.mean of three separate experiments determined in triplicate. ^*P<0.01, compared with the cells exposed to OX-LDL. (B) For MAPK experiment, after incubation with increasing concentrations of PD98059 and SB203580 for 1 h, the cells were stimulated with 100 μg ml⁻¹ OX-LDL for 5 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Bands were visualized by an ECL method as described in Figure 2. Data are expressed as the mean±s.e.mean of three separate experiments. ^*P<0.01, compared with the cells exposed to OX-LDL.

Figure 10

Requirement of Ras and Raf for OX-LDL- and PDGF-BB-induced activation of MEK1/2 and p42/p44 MAPK in VSMCs. Cells were transfected with plasmids encoding pZIP-NeoSV, H-Ras-15A, pCGN, or Raf-N4, and then stimulated with OX-LDL (100 μg ml⁻¹) or PDGF-BB (20 ng ml⁻¹) for 10 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Western blot analysis was performed using an antiserum reactive with the anti-phospho-MEK1/2, anti-phospho-p42/p44 MAPK, and total p42 MAPK (as a control) polyclonal antibody. Bands were visualized by an ECL method. ^*P<0.05; #P<0.001, compared with respective control.

Figure 11

Effects of BQ-123 and losartan on OX-LDL-stimulated p42/p44 MAPK phosphorylation in VSMCs. The cells were grown to confluence, made quiescent by serum-deprivation for 24 h. The cells were preincubated with 10 μM BQ-123 or 10 μM losartan for 1 h, and then stimulated with vehicle or 100 μg ml⁻¹ OX-LDL for 5 min. The cell lysates were subjected to 10% SDS – PAGE and transferred to nitrocellulose membrane. Bands were visualized by an ECL method as described in Figure 2. Data are expressed as the mean±s.e.m. of three separate experiments.