Low-Density Lipoproteins Induce Heat Shock Protein 27 Dephosphorylation, Oligomerization, and Subcellular Relocalization in Human Vascular Smooth Muscle Cells
Objective— High levels of circulating low-density lipoproteins (LDL) are a major atherosclerotic risk factor. The effects of intimal LDL on vascular smooth muscle cell (VSMC) phenotype and function during plaque remodeling and vascular repair are not fully understood. We have investigated whether exposure of VSMC to LDL induces changes on the proteomic profile of the heat shock protein (HSP) family–molecular chaperones involved in atherosclerosis.
Methods & Results— 2D electrophoresis demonstrates that LDL modifies the proteomic profile of HSP27 (HSPB1). Western blot analysis evidenced a significant HSP27 dephosphorylation after exposure of cells to native LDL (nLDL) and aggregated-LDL (agLDL) for 24 hours (P<0.05). Dephosphorylation of HSP27 was not related to changes in p38MAPK phosphorylation. Both nLDL and agLDL induced relocalization of unphosphorylated HSP27 to the tip of actin stress fibers and focal adhesion structures in VSMC. During cell adhesion, phospho-HSP27 was located at the cell surface contact region in LDL-treated cells, whereas it remained cytosolic in control cells. Immunohistochemistry studies showed that phosphorylated HSP27 is rarely found in lipid-rich areas of atherosclerotic plaques in human coronary arteries.
Conclusion— Our results indicate that in VSMC, LDL modulate HSP27 phosphorylation and subcellular localization, affecting actin polymerization and cytoskeleton dynamics.
Low-density lipoproteins (LDL) are responsible for the transport and internalization of cholesterol in the arterial vessel wall. High levels of LDL in plasma have a causal role in atherosclerosis.1 Increasing amounts of evidence indicate that circulating lipoproteins may exert cellular effects that are not exclusively related to their lipid transport function. Infiltrated LDL within the arterial wall become modified (oxidation, glycosylation, aggregation). Modified LDL have been associated with changes in endothelial function,2 as well as with migration, proliferation, and apoptosis of vascular smooth muscle cells (VSMCs).3 Migration and proliferation of VSMCs, as well as extracellular matrix production and catabolism, are important events in the development of atherosclerosis and arterial remodeling.4 VSMCs acquire or lose various functions to be able to fulfill the requirements for intimal remodeling.5 Previous studies from our group demonstrated that LDL induce changes on VSMC gene expression and phenotype, leading to alterations in vascular function.6–8 We have recently demonstrated that aggregated LDL (agLDL) impair VSMC migration and wound repair after injury.9
In response to stress, cells activate cytoprotective pathways. Heat shock proteins (HSP) are molecular chaperones that have the ability to protect proteins from damage induced by factors such as free radicals, heat, or ischemia. It has been demonstrated that HSP are highly expressed in atherosclerotic lesions of humans, rabbits, and apolipoprotein E–deficient mice.10 Interestingly, HSP90 (HSP90AA1) expression is induced in VSMC by oxidative stress11 and plays a role in the maintenance of endothelial barrier integrity by binding endothelial nitric oxide synthase and guanylyl cyclase (reviewed in Antonova et al12). Overexpression of HSP70 (HSPA1A) in mouse heart increases resistance to ischemic injury.13 Small HSP can also protect against ischemic injury in cardiomyocytes.14
Among small HSP, HSP27 (HSPB1) acts as a molecular chaperone in vitro15 and is also involved in F-actin assembly.16,17 In smooth muscle, HSP27 is constitutively expressed and, when phosphorylated, colocalizes with contractile proteins.18 In the cell, HSP27 phosphorylation (pHSP27) is necessary to perform most of its functional activity. Indeed, pHSP27 is required for cell migration,19 and HSP27 is rapidly phosphorylated20 on cellular stress and growth. pHSP27 stabilizes the actin cytoskeleton,21 whereas inhibition of the p38MAPK pathway leads to dephosphorylation of HSP27 and impairment of actin filament stabilization.22 In atherosclerosis, HSP27 expression has been reported to be increased in normal-appearing vessels adjacent to atherosclerotic plaques; however, levels are reduced in the plaque itself, with decreased pHSP27 in both plaque and adjacent artery segments.23 On the other hand, de Souza et al have shown increased pHSP27 in patients with cardiac graft vasculopathy.24 A major difference between spontaneous atherosclerosis and accelerated atherosclerosis occurring in vascular grafts is the absence of infiltrated lipids in later regions. On the basis of our previous finding that agLDL impair VSMC migration and wound repair after injury,9 we hypothesized that LDL, infiltrated and aggregated by binding to proteoglycans in the vascular wall, induce changes in the protein distribution pattern (proteomic profile) of the HSP family. Those changes might have a modulating role in the response of neointima VSMC to stress and contribute to the development and progression of spontaneous atherosclerosis. This study reveals that LDL induce a significant alteration in the proteomic profile of HSP27 in VSMC, decreasing pHSP27 and modulating VSMC cytoskeleton dynamics.
Materials and Methods
For expanded methods, see supplemental materials, available online at http://atvb.ahajournals.org.
Human Coronary VSMC Culture and LDL Preparation
Primary human VSMC were obtained by the explant technique from human nonatherosclerotic coronary arteries of hearts obtained from heart transplantation surgery at the Hospital de la Santa Creu i Sant Pau.25 Cells were arrested at subconfluence and incubated for 24 hours in serum-free M199 medium in the presence or absence of native LDL (nLDL) or agLDL (100 μg/mL). Thereafter, cells were harvested in PBS or directly extracted with 0.15 mmol/L NaCl/0.05 mmol/L Tris-HCl, pH 7.2/1% Triton X-100/1% sodium deoxycholate/0.1% SDS (RIPA) buffer and stored at −80°C until being used. We have specifically investigated whether the HSP family in human VSMC is affected by 24 hours of incubation with LDL because at this time period, the accumulation of intracellular cholesteryl ester derived from LDL, native or aggregated, has reached a plateau in human VSMC,8 and therefore, LDL internalization is not dependent on the turnover expression of LDL receptor-related protein (LRP) and LDL receptors in the cells.8
Human LDL (density, 1.019 to 1.063 g/mL) were obtained from pooled sera of normocholesterolemic volunteers and were isolated and prepared by ultracentrifugation as described previously.26
Cells were treated as described with LDL and were harvested with PBS containing 5mmol/L EDTA to maintain the integrity of adhesion molecules. Cells were replated on coverslips and allowed to adhere for 1 hour. Then cells were washed and fixed for analysis of HSP27 subcellular localization by confocal microscopy as described below.
Protein Extraction and Proteomic Analysis
Frozen cell pellets were sequentially extracted in tris and urea buffers.9 To prepare total cell lysates, cells were extracted in RIPA buffer or 50 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L EDTA, 10% glycerol, 0.1% Triton X-100, and 1 mmol/L phenylmethylsulfonyl fluoride.
Protein extracts of control and LDL-treated cells were separated by 2D gel electrophoresis, after sample contaminants (salts, nucleic acids, lipids) were removed. Spot patterns were analyzed for differences (PDQuest Software 8.0.1, BioRad), and protein spots of interest were identified by matrix-assisted laser desorption ionization–time of flight analysis.
Western blot analysis, size exclusion chromatography, immunohistochemistry, and confocal microscopy were performed as previously described.7,6,27
Results are presented as mean±SD, and the number of experiments is shown in every case. Statistical differences between control and treated groups were analyzed by the nonparametric Mann-Whitney test for paired data. A probability value of 0.05 or less was considered significant.
Proteomic Analysis Showed a Differential Pattern for HSP27 After LDL Treatment
Differential proteomic analysis was used to identify changes induced by nLDL and agLDL in the proteomic profile of the HSP family in human VSMC. We performed a sequential extraction of hydrophilic (mainly cytosolic) and hydrophobic cellular proteins, including cytoskeleton and integral-membrane proteins. Average gels were obtained from at least 3 independent experiments. Analysis of the gels showed a total of 880±176 protein spots; 16% of the total protein spots were detected only in the tris-soluble fraction, whereas 48% were extracted with the urea-detergent buffer. Six protein spots corresponding to the HSP 78-kDa glucose-regulated protein (BiP), HSP70, HSP60, and HSP27 were identified by fingerprinting analysis in the subproteomes of control and LDL-treated VSMC (Table and Supplemental Figure I). All the identified protein spots showed good correlation, within the experimental error range of determined molecular mass and isoelectric point (pI) values, with those predicted from their amino acid sequences. Among the group of HSP, LDL induced significant changes in the proteomic pattern of 2 spots identified by matrix-assisted laser desorption ionization–time of flight analysis as HSP27 (Supplemental Figure I and Figure 1A, spots 4137 and 5139) in the VSMC tris-soluble fraction. Spots 4137 and 5139 differed in their pI (6.17 versus 6.55, respectively), presumably corresponding to different phosphorylated forms. The most basic HSP27 spot (5139) was the predominant form both in the control group and in the LDL-treated groups. Thus, the intensity of spot 5139 was approximately 5-fold that of spot 4137 in control cells. The difference in the relative intensity between the two spots was increased in cells treated with nLDL or agLDL (mean±SD for ratio of intensity level of spot 5139 to that of spot 4137: control, 3.8±3.4; nLDL, 11.0±6.6; agLDL, 9.9±3.0) (Figure 1A). Besides, in the urea-soluble fraction, HSP27 was identified as a single spot of pI 6.55 with a detection level one fourth that of the corresponding spot in the tris-soluble fraction.
Total levels of HSP27 partitioned in the tris and urea/detergent fractions of control and LDL-treated VSMC were compared by Western blot analysis, using an antibody against total HSP27. As shown in Figure 1B, cellular HSP27 was mainly extracted by the tris buffer. Total levels of HSP27 were not significantly altered by the LDL treatment, neither in the tris fraction nor in the urea/detergent fraction. Taken together, our results suggest that LDL induce a relevant change in the proteomic profile of HSP27 in VSMC, without significantly affecting its cellular content. Furthermore, culture supernatants of control and LDL-treated VSMC were negative for HSP27 (data not shown).
LDL Induced Dephosphorylation of HSP27
The phosphorylation state of HSP27 partitioned in the tris-soluble fraction from VSMC treated without and with LDL (nLDL or agLDL) for 24 hours was analyzed by immunoblotting after one-dimensional electrophoresis (n=3). As shown in Figure 2A, LDL induced a decrease in pHSP27, evident with antibodies against 3 phosphorylation sites (Ser82, Ser78, and Ser15) The effect was highly significant, as shown in Figure 2B for Ser82 (control, 0.42±0.09; nLDL, 0.22±0.1; agLDL, 0.17±0.18 arbitrary units [AU]; P<0.05; Figure 2A and 2B). Changes at Ser78 and Ser15 showed a similar pattern (Ser78: control, 0.65±0.3; nLDL, 0.39±0.21; agLDL, 0.44±0.27 AU; Ser15: control, 0.66±0.29; nLDL, 0.47±0.25; agLDL, 0.42±0.20 AU).
Changes induced by LDL in pHSP27 at Ser82 were further characterized by 2D electrophoresis (Supplemental Figure II). In control cells, immunoblotting for total HSP27 revealed 5 spots with similar molecular mass (27 kDa) and pI between 5.5 and 7.5. The 3 most acidic spots were also detected by the anti-pSer82-HSP27 antibody, indicating a high phosphorylation rate in these spots. VSMC treated with LDL depicted a decrease in the intensity of the positive acidic spots for total HSP27 with an increase in the intensity of the spots with more basic pI. In addition, the signal intensity of the spots labeled with the anti-pSer82-HSP27 antibody was decreased (compare merge images for C and agLDL in Supplemental Figure II). An additional spot with pI 7.5 was detected for total HSP27 in protein extracts of cells treated with agLDL (Supplemental Figure II, spot 6 for total HSP27), indicating a shift to more basic forms of HSP27 and confirming dephosphorylation of the protein.
LDL Induced Changes in HSP27 Oligomerization
HSP27 can form oligomers of high molecular weight. Oligomerization depends on dephosphorylation of HSP27 dimers. In a set of experiments, by size exclusion chromatography, we investigated whether LDL could induce changes in HSP27 oligomerization. Control VSMC showed a broad range of oligomers, going from 27 to 800 kDa. In contrast, LDL-treated cells show a displacement toward higher molecular mass (>250 KDa) and absence of the lower molecular mass forms (<55KDa). The HSP27 dimer, which plays a critical role in actin polymerization, is present in control cells but not in LDL-treated cells. In addition, a displacement is observed for high molecular mass bands in the LDL groups and more specifically for the agLDL-treated cells. (Figure 2C; molecular masses [kDa] stated on the figure correspond to the beginning of each fraction).
Localization of HSP27 in VSMC
HSP27 biological activity in the cell is dependent on its phosphorylation and on its subcellular localization. The distribution pattern of total HSP27, its phosphorylated form (Ser82), and the colocalization with actin stress fibers (F-actin) was analyzed by confocal microscopy in control and LDL-treated VSMC. Control cells showed a broad labeling signal for HSP27 throughout the cytoplasm, with some colocalization with actin stress fibers (Figure 3Aa). Labeling for pHSP27 (Ser82) showed a labeling distribution similar to that of total HSP27 throughout the cytosol of the cells, but with a dotted pattern. Colocalization between pHSP27 and stress fibers was not apparent (Figure 3Bd).
In LDL-treated cells, signal for total HSP27-fluorescence labeling was distributed over the cytosol, as it was in the controls. Moreover, in the presence of LDL, HSP27 was also strongly evident at the cell borders, in areas containing adhesion structures (Figure 3Ab and 3Ac, arrow). pHSP27 labeling was much lower, although it had a subcellular dotted pattern distribution similar to that in control VSMC throughout the cytosol (Figure 3Be and 3Bf). Very weak or no labeling for pHSP27 was detected at the cell borders of VSMC treated with nLDL or agLDL (compare Figure 3Ab and 3Ac with Figure 3Ae and 3Af, respectively).
To determine whether LDL might affect the subcellular distribution of HSP27 in relation to the actin cytoskeleton, we analyzed the colocalization of HSP27 with gelsolin, an F-actin severing protein that also caps free plus ends of F-actin.28 Double labeling with total HSP27 and gelsolin antibodies showed overlapping signals (Supplemental Figure III, yellow) in both controls and LDL-treated cells. The percentage of colocalization, measured by computer-assisted confocal analysis, was significantly higher in agLDL-treated cells (80.0±2.7) than in nLDL-treated cells (60.8±2.4, P=0.007 versus agLDL) or control cells (56.3±4.4, P<0.0001 versus agLDL). In addition, at the cell borders, the control cells showed the lowest HSP27/gelsolin overlapping (23%) compared with nLDL-treated (86%) or agLDL-treated cells (73%). The increase in gelsolin/HSP27 colocalization was particularly evident at lamellipodia in both nLDL- and agLDL-treated VSMC (Supplemental Figure IIIa through IIIc and zoomed borders IIIa′ through IIIc′). When immunofluorescence was performed using antibodies against pSer82-HSP27, a low pHSP27/gelsolin colocalization was observed at cell borders (control, 34%; nLDL, 24%; agLDL, 18%), in both control cells and LDL-treated cells (Supplemental Figure IIId through IIIf, zooms IIId′ through IIIf′). These results indicated that in control cell borders, the HSP27 form colocalizing with gelsolin is mostly phosphorylated, whereas incubation with LDL increased the relative concentration of the unphosphorylated HSP27 form in these regions. Indeed, upon phosphorylation, HSP27 liberates the barbed ends to allow actin polymerization to take place.
In an independent set of experiments, we further investigated whether long-term exposure to LDL affected subcellular localization of pHSP27 in VSMC during early stages of cell adhesion. To this end, VSMC incubated with and without LDL for 24 hours were harvested, plated, and allowed to attach and spread for 60 minutes. At this time point, analysis by confocal microscopy showed a wide and homogeneous labeling for pHSP27 throughout the cytosol of the control cells, whereas cells that had been exposed to LDL depicted an heterogeneous distribution pattern. Thus, in addition to a concentrated location around the depolymerized actin, positive signals were consistently detected at the cell border, where labeling for actin was very weak or was not detected (Supplemental Figure IVA, borders indicated by arrows). Serial stack analysis demonstrated that pHSP27 labeling at the border of the cells was mainly located at cell-substratum contact regions (Supplemental Figure IVB, compare 1 with 6 in nLDL and agLDL).
HSP27 Distribution Pattern in Human Coronary Atherosclerotic Lesions
HSP27 (total and phosphorylated) and apolipoprotein B (ApoB) were localized by immunohistochemistry in representative segments of human coronary arteries, with different degrees of spontaneous atherosclerotic lesions. Strong staining for total HSP27 was evident in the neointima of early lesions and fibrous plaques (Figure 4A and 4E). In the fibrous plaques, HSP27 was detected in neointima regions also positive for α-actin and for HAM-56 as specific markers for smooth muscle cell (brown signal in Figure 4H) and macrophages (Figure 4I) respectively. Compared with total HSP27, immunostaining for pHSP27, in adjacent sections of the early lesions, depicted a similar positive distribution pattern, specially in regions located at the intimal thickenings (compare Figure 4B and 4C) that did not show ApoB infiltration (Figure 4D). In contrast, pHSP27 positive signals were weak and sparsely distributed throughout the neointima of fibrous plaques (compare Figure 4E and 4F) rich in ApoB staining (Figure 4G). A low pHSP27 signal was consistently found in areas with otherwise positive staining for ApoB (Figure 4G). ApoB-rich areas with low or no staining signal for pHSP27 were also evident in advanced atherosclerotic lesions (Figure 4L through 4N).
As lipid-laden regions of the plaque are highly populated by macrophages, we tested by immunoblot the level of phosphorylation induced by LDL in monocyte-derived macrophages. Interestingly, LDL reduced expression levels of pHSP27 in macrophages with respect to control cells (Supplemental Figure V), as they do in VSMC. Levels of pHSP27 were lower than in VSMC.
Protein Phosphatase A2 Was Involved in HSP27 Dephosphorylation by LDL
To investigate whether the LDL effect on HSP27 was mediated through the p38MAPK pathway, we analyzed the rate of p38MAPK phosphorylation by Western blot in VSMC treated without and with nLDL and agLDL for 24 hours. Results shown in Figure 5A revealed that the relative level of phosphorylated p38MAPK (referred to as total p38MAPK level) did not differ significantly between nLDL- or agLDL-treated VSMC and controls (ratio of p-p38MAPK to total p38MAPK: control, 0.88±0.04; nLDL, 0.85±0.41; agLDL, 1.26±0.46; P=not significant), suggesting that the lower levels of pHSP27 in the presence of LDL are not due to reduced or inhibited p38MAPK activity. In addition, Western blot analysis of total and phosphorylated MAPKAPK2 (p-MAPKAPK2), a kinase that directly phosphorylates HSP27, did not show any significant difference between controls and LDL-treated groups (ratio of p-MAPKAPK2 to total MAPKAPK2: control, 0.210±0.109; nLDL, 0.1±0.097; agLDL, 0.104±0.070; P=not significant), indicating that activation of MAPKAPK2 was not involved in the process (Figure 5A).
Next, we investigated whether LDL effects were mediated by protein phosphatase A2 (PPA2). To this aim, the effect of the PPA2 inhibitor okadaic acid (OA) on pHSP27 was analyzed in VSMC treated without (control) or with nLDL or agLDL for 24 hours. Preincubation with 20 nmol/L OA for 2 hours before addition of LDL and maintenance of 20 nmol/L OA during LDL incubation, to ensure the total inhibition of PPA2, induced a 45-fold increase in the phosphorylation of HSP27, as demonstrated by Western blot analysis with specific antibodies against pSer82 and total HSP27 (Supplemental Figure VI). However, the phosphorylation rates did not differ between control and LDL-treated groups (Supplemental Figure VI). When OA (20 nmol/L) was added only during the last 2 hours of LDL incubation, the pHSP27 rate was increased 2- to 4-fold compared with those in VSMC incubated in the absence of OA (ratio of pHSP27 in OA-treated versus non–OA-treated VSMC: control, 4.04±2.02; nLDL, 2.06±0.15; agLDL, 2.32±0.58-fold increase). As shown in Supplemental Figure VI, pHSP27 rates in the nLDL and agLDL groups treated with OA were 51% and 57% of the control group, respectively, indicating that the LDL effects persisted after 22 hours. Because OA might induce inhibition of other phosphatases, in addition to PPA2, cells were treated with fostriecin, which has shown specific inhibition of PPA2 at low concentrations.29 Fostriecin (200 nmol/L, for 30 minutes before harvesting), caused the level of pHSP27 to increase 2-fold in control cells (0.55±0.09 versus 1.17±0.02 arbitrary units) and up to 4-fold in LDL-treated cells (nLDL: 0.37±0.09 versus 1.69±0.37; agLDL: 0.35±0.13 versus 1.67±0.23; P<0.005, Figure 5B). Taken together, these results suggested that LDL modulate pHSP27 levels via PPA2.
Dephosphorylation of HSP27 by LDL Impaired Stress Fiber Formation in Migrating Cells
To investigate whether dephosphorylation of HSP27 was implicated in the inhibition of VSMC migration induced by LDL, cells were processed to perform the wound healing assay, and the PPA2 inhibitor fostriecin (200 nmol/L) was added 2 hours before the cell monolayer was injured. At the migration border, LDL-treated VSMC showed weaker signal for F-actin than control cells (Supplemental Figure VIIA through VII, A through C). This effect was not observed when cells were pretreated with fostriecin. Indeed, cells treated with fostriecin not only presented an enhanced signal for pHSP27, as shown by immunofluorescence, but also showed a stronger signal for F-actin and thicker stress fibers (Supplemental Figure VIIA, D through F). VSMC migration kinetics were reduced by nLDL and agLDL, as previously shown by our group,9 and were not affected by the exposure to the PPA2 inhibitor at this time and PPA2 inhibitor concentration (Supplemental Figure VIIB). On the other hand, suppression of HSP27 expression by specific short interfering RNAs reduced migration in control VSMC (short interfering RNA, random, 55.5%±2.6, versus short interfering RNA, HSP27, 11.5%±3.7, after 8 hours of migration; P<0.05; Supplemental Figure VIII).
HSPs have been implicated in a wide variety of processes, both physiological and pathological, including atherosclerosis.10 Available data appear to indicate that HSP27 may be an atheroprotective molecule and novel biomarker. However, the mechanisms involved are not elucidated. A recent study, based on an HSP27-overexpressing/ApoE−/− mouse model, suggests that HSP27 competitively inhibits the uptake of acetylated LDL by macrophages.30
The present study, using a proteomic approach and confocal microscopy, addresses the early changes induced by atherogenic LDL levels in human VSMC. Effects induced by agLDL are of special significance in the development of spontaneous atherosclerosis, because the earliest pathogenic event in atherogenesis is the entry and intimal retention of LDL that become aggregated by binding extracellular matrix proteoglycans.31 Here, we show that in VSMC, LDL induce HSP27 dephosphorylation, oligomerization, and subcellular relocalization after 24 hours of incubation, a time point in which the maximal incorporation of cholesteryl esters has been reached.
Many protective roles of HSP27, including regulation of cell motility and coordination of actin dynamics, are modulated by its phosphorylated forms.22 Therefore, the downregulation of pHSP27 induced in VSMC by LDL may represent a protective response of the cell to the massive entry of cholesterol and hence play an important mechanistic role in the progression of the atherosclerotic plaques. In agreement with the in vitro studies, human advanced atherosclerotic lesions, particularly in areas with a high content of ApoB, show weak or undetectable pHSP27 staining. On the contrary, clear positive signals for total HSP27 are observed usually localized in smooth muscle cell–rich areas of the neointima. Indeed, pHSP27 has been reported to increase in patients with cardiac allograft vasculopathy,24 strongly supporting the findings of our study, because graft-related accelerated atherosclerotic lesions are restenotic, with little participation of lipids.
It has been suggested that both the protective and the chaperone properties of HSP27 correlate with the ability of HSP27 to form large oligomers. Phosphorylation of Ser15 has been proposed as the mechanism to dissociate large oligomers.32 In our study, concomitant to HSP27 dephosphorylation, LDL induce formation of large oligomers. The results presented here are in line with those available in the literature,33 which describes phospho-HSP27 being found mainly in monomer to tetramer forms, whereas dephosphorylated HSP27 tends to form large oligomers (12-mer to 35-mer). LDL may then have a dual effect on HSP27; that is, it initiates protective or chaperone activities and impairs cytoskeletal dynamics to reduce shape changes and migration.
HSP27 has been detected in serum of individuals free of atherosclerosis, and low plasma levels have been proposed as potential biomarkers for atheroma plaque formation and rupture.34 We have not detected any HSP27 in VSMC culture medium, in either the absence or the presence of LDL. Therefore, although in atherosclerotic plaques HSP27 is expressed by both VSMC and macrophages, plasma HSP27 is not released by VSMC. Interestingly, it has recently been proposed that extracellular release of HSP27 by macrophages is mediated by estrogen and competitively inhibits acetylated LDL (acLDL) binding to scavenger receptor-A.30
Phosphorylation of HSP27 induces changes in the actin cytoskeleton and actin-dependent events. Indeed, it has been proposed that HSP27 is an actin capping protein that on phosphorylation liberates the barbed ends, facilitating actin polymerization. We have previously demonstrated that LDL impair VSMC migration,9 a process that requires actin polymerization, supporting the novel concept of LDL interference in actin dynamics. Here, we demonstrate that LDL induce translocation of the nonphosphorylated HSP27 toward the cell borders and more specifically toward the lamellipodia. The increased colocalization of HSP27, but not of its phosphorylated forms, with gelsolin at the cell borders of LDL-treated groups strongly reinforces the relevance of the HSP27 as actin-capping protein in the presence of LDL. Blockade of pHSP27 by fostriecin stimulates actin polymerization in the front line of migrating cells, as we could observe by an increase in F-actin content. Moreover, silencing HSP27 with specific short interfering RNAs reduced migration in control cells (Supplemental Figure VIII), showing the contribution of HSP27 to cell functional dynamics.
In addition, we show that pHSP27 locates at cell-surface contact regions at early cell adhesion stages in LDL-treated VSMC. This process occurs shortly after cell plating, at time points when actin cytoskeleton is still disorganized9 and stress fibers are not yet formed. The translocation of pHSP27 from the cytosol to the border of the cells, at the substrate boundary layer, together with the localization of the nonphosphorylated HSP27 at the barbed ends indicates changes in focal adhesion formation and actin polymerization machineries in the LDL-treated VSMC that impair cell functions.9
The regulation of pHSP27 is mediated by a well-known signaling pathway involving MAPK.20 HSP27 is a downstream target of the p38MAPK cascade; p38MAPK activates MAPKAPK2/3, which directly phosphorylates HSP2722 and induces actin polymerization.20 It has been reported that modified LDL (oxidated, glycated) can induce p38MAPK transient activation in different cell types.35–38 However, the significant decrease in pHSP27, below control levels, after 24 hours of treatment with LDL is neither related to p38MAPK activity nor to MAPKAPK2. LDL-related dephosphorylation of HSP27 is not due to the switch-off of MAPK but to a counterregulation by PPA2,39 because inhibition of PPA2 by either OA or fostriecin blocked the effect of LDL on pHSP27. From this study, we cannot exclude a short-term inhibitory effect of LDL on p38MAPK-induced pHSP27, because our work has been centered on a 24-hour effect of LDL on human VSMC, when LDL internalization is not dependent on the turnover expression of its surface receptors8 and the accumulation of intracellular cholesteryl ester derived from LDL has reached a plateau.8
pHSP27 plays a key role in the regulation of actin polymerization dynamics, and here we show that in VSMC, LDL modulates pHSP27 patterns, HSP27 oligomerization, and HSP27/pHSP27 subcellular localization. This seems to be a new mechanism by which vessel wall–infiltrated LDL may affect VSMC function. Our results may help to establish a hypothesis to explain the low VSMC cellularity of soft atheromas that makes them susceptible to rupture. Vessel wall infiltrated–LDL, such as agLDL, may modify VSMC function by inducing changes in HSP27/pHSP27, modulating cytoskeletal dynamics, and reducing adhesion and migration kinetics in VSMC.
The authors are indebted to Dr Oriol Juan and Dr Esther Peña for their help in immunohistochemistry and confocal microscopy, to Ilaria Ramaiola for her support in size exclusion chromatography, to Montse Gómez-Pardo for technical assistance, and to Professor John McGregor for helpful discussion.
Sources of Funding
This work was made possible by funds from Centro de Investigación Biomédica en Red-Obesidad y Nutrición (CIBER OBENU) Instituto de Salud Carlos III CB06/03 (L.B.), SAF 2006/10091 (L.B.), Lilly Foundation (L.B.), Fondo de Investigación Sanitaria (FIS)-Instituto de Salud Carlos III FIS-PI071070 (T.P.), Red de Investigación Clínica y Básica en Insuficiencia Cardiaca (REDINSCOR) RD06/0003/0015 (V.L.-C.), Red de Terapia Celular (TERCEL) RD06/0010/0017, and “Fundación Jesus Serra.”
Received on: October 6, 2008; final version accepted on: March 29, 2010.
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