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Originally published In Press as doi:10.1074/jbc.M303171200 on July 11, 2003

J. Biol. Chem., Vol. 278, Issue 38, 36257-36263, September 19, 2003
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Apolipoprotein E Binding to Low Density Lipoprotein Receptor-related Protein-1 Inhibits Cell Migration via Activation of cAMP-dependent Protein Kinase A*

Yanjuan Zhu and David Y. Hui {ddagger}

From the Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0529

Received for publication, March 27, 2003 , and in revised form, June 30, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The importance of apolipoprotein E (apoE)1 in protecting against atherosclerosis was convincingly demonstrated by studies with experimental animals. Transgenic mice overexpressing rat apoE displayed marked resistance to diet-induced hypercholesterolemia and did not develop atherosclerosis (1, 2). In contrast, mice with targeted disruption of the apoE gene developed spontaneous atherosclerosis even under basal low fat/low cholesterol dietary conditions (3, 4). Atherosclerosis in apoE-null mice could be prevented by increasing circulating apoE levels through recombinant adenovirus-mediated apoE gene transfer to the liver (5). The decrease in atherosclerosis in this model was accompanied by decreased total cholesterol, very low density lipoprotein, and intermediate density lipoprotein levels (5). Although these results seemed to suggest that apoE prevents atherosclerosis by lowering plasma cholesterol levels, atherosclerosis was found to be more severe in cholesterol-fed apoE+/- mice than in cholesterol-fed apoE+/+ mice despite the relative similar plasma cholesterol level in the two groups (4, 6). Additionally, transgenic expression of apoE in the arterial wall inhibited atheroma formation and severity without affecting plasma cholesterol level in cholesterol-fed C57BL/6 mice (1). Subsequent studies showed that low levels of apoE secreted by the adrenal gland also inhibited atherosclerosis without correcting for hypercholesterolemia in apoE-deficient mice (7). Taken together, this lack of correlation between total cholesterol and atherosclerosis lesion suggests that apoE may have direct impact on atherosclerosis by mechanisms independent of its ability to modulate plasma lipid levels.

The precise mechanism by which apoE protects against vascular diseases independent of its effects on cholesterol metabolism is not completely understood. The migration of vascular smooth muscle cells from the media to the intima followed by their proliferation are hallmarks of atherosclerotic lesions and vascular stenosis after balloon angioplasty and/or stent implantation. Our laboratory has shown previously that apoE inhibits PDGF-induced smooth muscle cell migration and proliferation (8, 9). Our recent data also revealed that apoE inhibition of smooth muscle cell proliferation is mediated through nitric-oxide synthase activation as a consequence of its binding to cell surface proteoglycans (9, 10). In contrast, apoE inhibition of smooth muscle cell migration appears to be mediated by low density lipoprotein receptor-related protein-1 (LRP-1) (10, 11) and is independent of inducible nitric-oxide synthase activation (9). The mechanism by which LRP-1 modulates apoE inhibition of cell migration has not been identified to date. In view of recent reports of LRP-1 association with a stimulatory heterotrimeric G-protein and activation of downstream protein kinase A (PKA)-dependent pathways (12), this study was undertaken to test the hypothesis that apoE inhibition of cell migration is mediated through the activation of cAMP/PKA pathways.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, and recombinant human platelet-derived growth factor-BB (PDGF-BB) were purchased from Invitrogen. The cell-permeable myristoylated protein kinase A inhibitory peptide (PKI) was obtained from Calbiochem. Forskolin was bought from Sigma. Direct cAMP enzyme immunoassay kit was obtained from Assay Designs Inc. (Ann Arbor, MI). The PKA assay kit was obtained from MBL International. Transwell polycarbonate membrane filters were purchased from Corning Costar Corp. (Cambridge, MA). Wild type murine embryonic fibroblasts (MEF-1), the heterozygous LRP-deficient fibroblasts PEA-10, and the homozygous LRP-negative fibroblasts PEA-13 were obtained from the American Type Culture Collection (Manassas, VA). The SilencerTM siRNA construction kit was obtained from Ambion Inc. (Austin, TX), and the NucleofectorTM transfection device and AoSMC NucleofectorTM reagent kit were purchased from Amaxa Biosystems (Gaithersburg, MD).

Apolipoprotein E Isolation—Human apoE was isolated from fresh plasma from healthy volunteers by the method of Rall et al. (13). The purity of apoE was assessed by SDS-polyacrylamide gel electrophoresis, and samples containing only a single band with Mr = 34,000 were used. In most experiments, purified apoE was resuspended in phosphate-buffered saline and added directly to the culture medium without reconstitution with lipid. In selected experiments, lipid-reconstituted apoE was prepared by the cholate dialysis method before use. The apoE-lipid emulsion complex was prepared by drying 3 mg of egg phosphatidylcholine and 60 µg of cholesterol in chloroform:methanol (2:1 v:v) under a stream of nitrogen and then dissolving the thin lipid film in a 300-µl solution containing 10 mM Tris-HCl, pH 7.4, 140 mM NaCl, 1 mM EDTA, 32 mg of cholate, and 300 µg of apoE. The lipid-protein mixture was incubated for 1 h at ambient temperature and then dialyzed exhaustively against 3 changes of buffered saline before use.

Isolation of Mouse Aortic Smooth Muscle Cells—Aortic smooth muscle cells were isolated from C57BL/6 wild type mice using a modification of Mimura's procedure (14). Briefly, thoracic aortas were dissected from mice, and the adventitial tissue was trimmed away. The aortas were then incubated in Hanks' solution containing 1 mg/ml collagenase and 3.3 units/ml elastase for 30 min at 37 °C. The adventitial tissue was dissected away, and the remaining tissue was incubated in Hanks' solution containing collagenase (1 mg/ml) and elastase (3.3 units/ml) for 1 h at 37 °C. Cell clumps were dissociated by aspiration through a 10-ml pipette. The cell suspension was centrifuged at 150 x g for 5 min at room temperature and resuspended in 10 ml of DMEM containing 10% fetal bovine serum. The isolated cells were characterized as smooth muscle cells based on positive immunohistochemistry staining with smooth muscle {alpha}-actin-specific antibodies and by morphological characteristics similar to that observed with human smooth muscle cells. The primary smooth muscle cells were cultured in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. Cells between passages 1 and 4 were used for experiments.

Fibroblasts Cell Culture—The wild type MEF-1, heterozygous LRP-deficient PEA-10, and the homozygous LRP-negative PEA-13 fibroblasts were grown in monolayer cultures on plastic tissue culture-treated dishes or multiple well cluster plates in high glucose DMEM containing 10% fetal bovine serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM glutamine. Cells were incubated at 37 °C in 90% air, 10% CO2 atmosphere. Cells between passages 3–12 were made quiescent by incubation for 48 h in the presence of 0.4% fetal bovine serum before use.

Cell Migration Assay—Migration of mouse vascular smooth muscle cells and embryonic fibroblasts toward a PDGF-BB gradient was examined according to the procedure of Law et al. (15), as described previously (8, 9). Briefly, 3 x 105 cells were made quiescent by incubation with DMEM and 0.4% fetal bovine serum for 48 h before and then incubated for 30 min at 37 °C in a 1-ml medium solution with or without apoE. A 0.1-ml aliquot of the cell suspension was added to the top chamber of tissue culture-treated Transwell polycarbonate membrane with 8-µm pores in 24-well plates. The lower Transwell compartment contained 0.6 ml of DMEM, 0.4% fetal bovine serum, and 0.2% bovine serum albumin with or without 10 ng/ml PDGF-BB. After incubating for 4 h at 37 °C, the upper surface of the filters was washed with phosphate-buffered saline. The cells were then fixed with methanol for 10 min at 4 °C followed by hematoxylin staining. The number of cells that migrated to the lower surface of each filter was counted in different high power fields at a magnification of 320. Experiments were performed in triplicate.

Direct cAMP Measurement—Smooth muscle cells and fibroblasts were seeded into 6-well plates. After 24 h of incubation at 37 °C, the cells were made quiescent by incubation with DMEM and 0.4% fetal bovine serum for 48 h. Cells were incubated in culture medium with or without apoE. The reaction was stopped by aspiration of the incubation medium followed by the addition of 0.5 ml of 0.1 M HCl. Cell lysis was achieved after incubation at 37 °C for an additional 10 min. The lysed cells were scraped into Eppendorf tubes. The samples were centrifuged at 1300 x g for 10 min at 4 °C. The supernatants were used to determine cAMP concentration using a direct enzyme immunoassay kit by the procedure as described by the manufacturer. Total protein concentration in each sample was determined by Bio-Rad protein assay. The cAMP level in each sample was normalized to the protein concentration and expressed as pmol/mg of protein.

Protein Kinase A Activity Assay—Protein kinase A activity was determined using the PKA assay system according to the manufacturer's instructions. Briefly, cells were grown to monolayers in 6-well plates. After 48 h of incubation in DMEM containing 0.4% fetal bovine serum, the cells were incubated with or without apoE for 30 min at 37 °C. At the end of the incubation period, the cells were rinsed twice with phosphate-buffered saline, scraped, and lysed in radioimmunoprecipitation assay buffer containing a complete proteinase inhibitor mixture. Cellular debris was removed by centrifugation, and the supernatant was divided into aliquots in triplicate on a 96-well microtiter plate coated with the immobilized PKA substrate peptide (RFARKGSLRQKNV) for 10 min. This reaction was stopped by adding 20% H3PO4. Phosphorylation was quantified by adding a biotinylated anti-phosphorylated PKA substrate peptide antibody 2B9 followed by peroxidase-conjugated streptavidin. Color development was achieved by O-phenylenediamine and hydrogen peroxide, and optical density was measured at 492 nm in a spectrophotometer.

Generation of LRP-1-deficient Smooth Muscle Cells—Knockdown of LRP-1 gene expression in smooth muscle cells was accomplished by transfecting aortic smooth muscle cells with LRP-1-specific siRNA. The siRNA was synthesized with the aid of the SilencerTM siRNA construction kit (Ambion Inc) using oligonucleotide primers corresponding to the T7 promoter sequence, 5'-TAATACGACTCACTATAG-3' and the sense and antisense sequence to residues 557–577 of LRP-1, 5'-GAGACCAAATCACCTGTATCTCTATAGTGAGTCGTATTA-3' and 5'-TTGAGATACAGGTGATTTGGTCTATAGTGAGTCGTATTA-3', respectively. Five µg of the siRNA was transfected into primary smooth muscle cells using the Nucleofector kit specific for transfecting smooth muscle cells. The cells were made quiescent 24 h after transfection by incubating for 48 h with DMEM and 0.4% fetal bovine serum. Half of the transfected cells were used for experiments to determine apoE effects on intracellular cAMP. The remaining cells were lysed in radioimmunoprecipitation assay buffer, and 30 µg of the cell lysate was analyzed for LRP-1 expression by Western blot with the 11H4 monoclonal antibody, which recognizes the 85-kDa subunit of LRP-1 as described (11).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of ApoE on Intracellular cAMP Level and PKA Activity in Primary Smooth Muscle Cells—Previous studies show that LRP-1 ligands such as urokinase-type plasminogen activator and lactoferrin increased cAMP levels and induced PKA activity in cultured melanoma and hepatoma cells (12). However, whether apoE binding to LRP-1 also elicits cAMP accumulation and PKA induction is unknown. More importantly, the physiological significance of the LRP-1 ligand-induced cAMP accumulation and PKA activation has not been established. Because the cAMP/PKA pathway has been shown to regulate smooth muscle cells migration (16–18) and apoE binding to LRP-1 inhibits smooth muscle cell migration toward PDGF (11), we tested the hypothesis that the inhibitory effect of apoE on smooth muscle cell migration is mediated through its stimulation of the cAMP/PKA pathway. The first set of experiments examined the effects of apoE on intracellular cAMP levels and PKA activity in primary smooth muscle cells in culture. The treatment of smooth muscle cells with apoE resulted in a dose-dependent increase in intracellular cAMP levels with a 4-fold increase observed at 50 µg/ml (Fig. 1A). The increase in cellular cAMP accumulation was observed within 5 min after apoE addition and was sustained for at least 1 h (Fig. 1B). The apoE-induced intracellular cAMP accumulation resulted in the activation of PKA activity in the smooth muscle cells, with a 2-fold induction observed when cells were incubated with 25 µg/ml apoE (Fig. 2).



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  		FIG. 1.
Effect of apo E on intracellular cAMP level in primary smooth muscle cells. Mouse aortic smooth muscle cells cultured in 6-well plates at a density 2.5 x 105 cells/well were made quiescent by incubation for 48 h in DMEM containing 0.4% fatal bovine serum. The cells were incubated for 30 min with varying concentrations of apoE (panel A) or with 25 µg/ml apoE for 0–60 min (panel B). At the end of the incubation period, cells were washed with phosphate-buffered saline 3 times and lysed in 0.1 M HCl. The cell lysates were used immediately for cAMP determinations by enzyme immunoassay. Data are expressed as pmol/mg of protein. Values are the means ± S.D. of three separate experiments. The asterisks denotes significant difference from samples incubated without apoE (p < 0.001).

 


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  		FIG. 2.
Effect of apoE on protein kinase A activity in smooth muscle cells. Quiescent primary culture of mouse aortic smooth muscle cells (passage 3) was incubated for 30 min with or without 25 µg/ml apoE. At the end of the incubation period, the cells were rinsed with phosphate-buffered saline, scraped, and lysed in radioimmunoprecipitation buffer. The cell lysates were used to determine protein kinase A activity based on the phosphorylation of the PKA substrate peptide (RFARKGSLRQKNV). Results are expressed as the mean ± S.D. of three separate experiments. The asterisk denotes significant difference from samples incubated without apoE (p < 0.001).

 

ApoE Inhibition of Smooth Muscle Cell Migration Requires PKA Activation—The intracellular cAMP accumulation and increased PKA activity in smooth muscle cells after apoE incubation suggested the possibility that apoE may inhibit smooth muscle cell migration via activation of cAMP-dependent PKA. This possibility was examined by determining the effect of a selective PKA inhibitor on apoE inhibition of PDGF-directed smooth muscle cell migration. Results showed that the cell-permeable peptide inhibitor of PKA, myristoylated PKI, abolished the anti-migratory property of apoE in a dose-dependent manner (Fig. 3). These findings provided strong support for the hypothesis that the anti-migratory effect of apoE is mediated through the cAMP/PKA pathway.



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  		FIG. 3.
Effect of PKA inhibition on the ability of apoE to inhibit smooth muscle cell migration. Quiescent mouse smooth muscle cells were incubated with apoE at 37 °C for 30 min in the presence or absence of the cell-permeable PKA inhibitor-myristoylated PKI as indicated. At the end of the incubation period the cells were placed into the top chamber of Transwell membrane at a density 3x104 cells/well in a 24-well plate. The lower chamber of the plate contained basal medium and 10 ng/ml PDGF-BB. Cells were incubated in the Transwell for 4 h at 37 °C. The number of cells that migrated to the lower surface was counted in different high power fields (HPF). Data are expressed as the mean ± S.D. (n = 6). The asterisks indicate significant difference from PDGF-stimulated migration at p < 0.001.

 

ApoE Induced cAMP Accumulation via Its Interaction with LRP-1—Previous studies have already shown that apoE binding to LRP-1 is required, and heparan sulfate proteoglycans are not involved, for its inhibition of PDGF-directed smooth muscle cell migration (11). These observations along with the results reported above suggested that the apoE-induced cAMP accumulation requires the presence of LRP-1 but not heparan sulfate proteoglycans on the cell surface. This hypothesis was examined by comparing the effect of apoE on the cAMP level in normal and LRP-1-deficient smooth muscle cells. The results showed that siRNA knockdown of LRP-1 expression in smooth muscle cells abolished apoE-induced intracellular cAMP accumulation (Fig. 4). In contrast, mock-transfected smooth muscle cells, which retained a normal level of LRP-1 expression, were responsive to apoE-induced cAMP accumulation (Fig. 4). The prior incubation of smooth muscle cells with heparanase to remove cell surface heparan sulfate proteoglycans also had no effect on the ability of apoE to increase intracellular cAMP level (Fig. 5). These results documented that apoE induction of cAMP accumulation in smooth muscle cells is mediated via LRP-1.



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  		FIG. 4.
Role of LRP-1-specific siRNA on the ability of apoE to induce cAMP accumulation in smooth muscle cells. 1 x 106 smooth muscle cells were transfected with 5 µg of LRP-1-specific siRNA before plating into 6-mm dishes in DMEM containing 10% fetal bovine serum. Cell quiescence was induced by incubation for 48 h in DMEM containing 0.4% fetal bovine serum. The inset shows the results of immunoblotting with antibodies that recognized the 85-kDa subunit of LRP-1 in lysates isolated from nontransfected cells (lane 1) and cells transfected with vehicle (lane 2) or with siRNA (lane 3). Lysates obtained from cells incubated for 30 min with or without 25 µg/ml apoE were used to determine intracellular cAMP levels. Data are expressed as pmol of cAMP/mg of cell protein. Values represent the mean ± S.D. from three separate experiments of duplicate determinations. The asterisk denotes a significant difference from control at p < 0.01.

 


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  		FIG. 5.
Effect of apoE on intracellular cAMP levels in heparanase-treated smooth muscle cells. Quiescent smooth muscle cells were incubated with 25 µg/ml apoE in the presence or absence of heparanase III (1 units/ml) for 30 min. Cells were washed 3 times with phosphate-buffered saline and lysed in 0.1 M HCl. The cell lysates were used immediately for cAMP determinations by enzyme immunoassay. Data are expressed as pmol of cAMP/mg of cell protein. Values are the means ± S.D. from three separate experiments. The asterisks denote significant difference from control cells at p < 0.01.

 

We also took advantage of the availability of murine embryonic fibroblasts expressing different levels of LRP-1 to test the hypothesis that LRP-1 present on the surface of non-smooth muscle cells may also modulate apoE induction of intracellular cAMP accumulation and cell migration. The initial experiments were performed to determine whether apoE also inhibits migration of fibroblasts. Results showed that apoE inhibited the migration of wild type MEF-1 fibroblasts and the heterozygous LRP-1 deficient PEA-10 cells in a dose-dependent manner (Fig. 6). In contrast, apoE was unable to inhibit the migration of PEA-13 cells, which are homozygous LRP-1-negative cells (Fig. 6). These results documented that apoE also inhibits fibroblast migration and the mechanism of inhibition is mediated via its binding to LRP-1, similar to that observed in smooth muscle cells (11).



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  		FIG. 6.
Effect of apoE on PDGF-directed migration of mouse embryonic fibroblasts. Quiescent wild type mouse embryonic fibroblasts MEF-1 (open bars), heterozygous LRP-deficient fibroblasts PEA-10 (hatched bars), and homozygous LRP-1 negative PEA-13 fibroblasts (filled bars) were incubated with or without apoE for 30 min before their placement into the top chamber of the Transwell in which the bottom chamber contained 10 ng/ml PDGF-BB. Cell migration was determined after 4 additional hours of incubation at 37 °C. The number of cells that migrated to the lower surface was counted in different high power fields (HPF). Data are expressed as the mean ± S.D. (n = 6). The asterisks indicate significant difference from PDGF-stimulated cell migration in the absence of apoE (p < 0.001).

 

The next series of experiments compared wild type MEF-1 and LRP-1-deficient PEA-13 cells for their response to apoE-induced cAMP accumulation. The results showed that apoE increased intracellular cAMP level in the MEF-1 cells in a time-dependent manner. A 4-fold increase in intracellular cAMP level was observed after incubating MEF-1 cells with apoE for 15 min, and the increase persisted for at least 1 h (Fig. 7). In contrast, apoE has a minimal effect on intracellular cAMP level in the LRP-1-deficient PEA-13 cells (Fig. 7). Importantly, apoE inhibition of MEF-1 fibroblast migration was also abolished by the protein kinase A inhibitor PKI (Fig. 8). These results indicated that apoE inhibition of fibroblast migration is also mediated via induction of cAMP accumulation and the consequential activation of PKA. Finally, to verify that the lack of apoE-induced cAMP accumulation in LRP-deficient cells is not a generalized phenomenon of abnormal cyclic nucleotide metabolism due to LRP-1 deficiency, we tested the effect of forskolin, a direct activator of adenylyl cyclase and inhibitor of smooth muscle cell migration (17, 19), on the migration of MEF-1, PEA-10, and PEA-13 cells. Results, as shown in Fig. 9, clearly demonstrated that forskolin inhibited PDGF-stimulated migration of MEF-1, PEA-10, and PEA-13 with similar efficiency. Taken together, these results documented that the pathway of apoE inhibition of cell migration is a general phenomenon in LRP-1-expressing cells, and mechanism involves induction of intracellular cAMP accumulation and the subsequent activation of PKA activity.



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  		FIG. 7.
ApoE induced intracellular cAMP accumulation in mouse embryonic fibroblasts. Quiescent wild type MEF-1 fibroblasts (filled circles) and LRP-negative PEA-13 fibroblasts (open circles), plated in 6-well plates at a density 2.5 x 105 cells/well, were incubated with 25 µg/ml apoE for the indicated times. At the end of the incubation period, cells were washed with phosphate-buffered saline and lysed in 0.1 M HCl. The cell lysates were used immediately for cAMP immunoassay. Data are expressed as pmol/mg of protein. Values are the mean ± S.D. from three separate experiments. The asterisks indicate significant difference from samples incubated without apoE (p < 0.001).

 


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  		FIG. 8.
Effect of protein kinase A inhibition on apoE inhibition of fibroblast migration. Quiescent wild type MEF-1 fibroblasts were incubated with apoE at 37 °C for 30 min in the presence or absence of the protein kinase A inhibitor-myristoylated PKI. At the end of the incubation period the cells were placed into the top chamber of Transwell membrane at a density of 3 x 104 cells/well in a 24-well plate. The lower chamber contained basal medium and 10 ng/ml PDGF-BB. Cells were incubated in the Transwell for 4 h at 37 °C. The number of cells that migrated to the lower surface was counted in different high power fields (HPF). Data are expressed as the mean ± S.D. (n = 6). The asterisks denote significant difference from PDGF-stimulated migration at p < 0.01.

 


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  		FIG. 9.
Effect of forskolin on PDGF-directed migration of mouse embryonic fibroblasts. Quiescent wild type mouse embryonic fibroblasts MEF-1 (open bars), heterozygous LRP-deficient fibroblasts PEA-10 (hatched bars), and homozygous LRP-1 negative PEA-13 fibroblasts (filled bars) were incubated with or without forskolin at the indicated concentrations for 30 min before their addition to the top chamber of the Transwell in which the bottom chamber contained PDGF-BB at the concentration as indicated. Cell migration was determined after 4 additional hours of incubation at 37 °C. The number of cells that migrated to the lower surface was counted in different high power fields (HPF). Data are expressed as the mean ± S.D. (n = 6). The asterisks indicate significant difference from PDGF-stimulated migration at p < 0.01.

 

Although our experiments used delipidated apoE to test for its mechanism in inhibition of cell migration, apoE exists in vivo predominantly in the lipidated form in association with lipoprotein particles. Thus, additional experiments were performed to determine whether lipidated apoE can also inhibit cell migration via the cAMP-dependent mechanism similar to that observed with delipidated apoE. Using wild type and LRP-1-deficient fibroblasts as a model for these experiments, results showed that both lipid-bound and lipid-free apoE were similarly effective in inhibiting PDGF-directed migration of the wild type MEF-1 fibroblasts (Fig. 10). Both lipidated and delipidated apoE were also capable of inducing intracellular cAMP accumulation in the wild type MEF-1 cells (Fig. 11). In contrast, neither form of apoE was capable of suppressing PDGF-directed migration (Fig. 10) or the induction of intracellular cAMP accumulation (Fig. 11) in LRP-1-deficient PEA-13 fibroblasts.



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  		FIG. 10.
Effects of lipidated and delipidated apoE on PDGF-directed cell migration. Quiescent wild type MEF-1 fibroblasts (open bars) and LRP-1 negative PEA-13 fibroblasts (filled bars) were incubated with or without 25 µg of delipidated or lipidated apoE for 30 min at 37 °C before addition to the top chamber of Transwell membranes at a density of 3 x 104 cells/well in 24-well dishes. The lower chamber of each well contained basal medium only (Basal) or medium containing 10 ng/ml PDGF-BB. Cells were incubated for 4 additional hours at 37 °C. The number of cells that migrated to the lower surface was counted in different high power fields (HPF). Data are expressed as the mean ± S.D. (n = 6). The asterisks denote significant difference from PDGF-stimulated migration at p < 0.01.

 


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  		FIG. 11.
Effects of lipidated and delipidated apoE on intracellular cAMP levels. Quiescent wild type MEF-1 fibroblasts (open bars) and LRP-1 negative PEA-13 fibroblasts (filled bars) were incubated for 30 min with 25 µg/ml delipidated or lipidated apoE. The cells were washed with buffered saline and lysed in 0.1 M HCl. The cell lysates were used immediately for cAMP determinations by enzyme immunoassay. Data are expressed as pmol/mg of cell protein. Values are reported as means ± S.D. from three separate experiments. Asterisks denote significant difference from cells incubated without apoE at p < 0.05.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous results demonstrate that apoE inhibits smooth muscle cell migration and proliferation via distinct mechanisms (9, 10). The mechanism for apoE inhibition of cell proliferation is mediated via its binding to cell surface proteoglycans (10), and the subsequent activation of inducible nitric-oxide synthase activity (9). However, apoE binding to cell surface proteoglycans has no effect on its ability to inhibit cell migration (10). The latter process is mediated via LRP-1 (11). The mechanism by which apoE binding to LRP-1 results in inhibition of cell migration has not been delineated. Results of the current study documented that apoE inhibition of PDGF-induced smooth muscle cell migration is related to the activation of cAMP/PKA-signaling pathway. This conclusion is based on the observation that apoE increased intracellular cAMP level in a dose-dependent manner and the resulting increase of PKA activity in primary smooth muscle cells. Importantly, the inhibitory effect of apoE on cell migration was abolished by the specific PKA inhibitor PKI and in cells lacking a functional LRP-1.

The causative effect of apoE-induced cAMP/PKA on inhibition of cell migration is consistent with the effects of other known cAMP/PKA activators on cell migration. For example, the cAMP activator forskolin as well as stable cAMP analogues such as 8-bromo-cAMP are effective inducers of PKA activity and inhibitors of smooth muscle cell migration (17, 19). In addition, adrenomedullin has also been shown to inhibit the migration of rat aortic smooth muscle cells by inducing intracellular cAMP accumulation (17). Cytochrome P450 epoxygenase-derived eicosanoids have also been demonstrated to inhibit smooth muscle cell migration by increasing intracellular cAMP level and PKA activity (19). Both adrenomedullin and the P450 epoxygenase-derived eicosanoids induced cAMP accumulation by stimulation of the heterotrimeric G protein G{alpha}s (20, 21). Because LRP-1 also interacts with G{alpha}s (12), it is possible that apoE induces intracellular cAMP accumulation and PKA activation in smooth muscle cells through similar mechanisms. Alternatively, apoE may induce cAMP accumulation and PKA activation by an indirect mechanism, possibly through the activation of protein kinase C. Previous studies demonstrated apoE induced protein kinase C{alpha} translocation and activation in neuroblastoma cells (22). Protein kinase C activation also resulted in inhibition of smooth muscle cell migration in a manner that is dependent on cAMP accumulation and PKA activity (16). Whether the apoE-induced cAMP/PKA signaling and its inhibition of cell migration is mediated through G{alpha}s or protein kinase C activation or both remains to be determined.

The results of the current study showing the inability of apoE to inhibit migration of LRP-1-negative cells are consistent with our previous observation that LRP-1 is the apoE receptor responsible for mediating its anti-migratory function (11). These results are also consistent with results showing that other LRP-1 ligands, including anti-LRP and the receptor-associated protein RAP, are also potent inhibitors of cell migration and invasion (23–25). Although LRP-1 has been thought to be an endocytic receptor highlighted by its distribution within clathrin-coated pits on the cell surface (26–31), recent data clearly documented that LRP-1 is also located in caveolae (32, 33), interacts with cytoplasmic adaptor proteins (34, 35), and serves as a signal transduction receptor (36, 37). This study showed that in smooth muscle cells and embryonic fibroblasts, LRP-1 is a signal transduction receptor mediating apoE inhibition of cell migration via activation of the cAMP/PKA pathway. The importance of LRP-1 function in limiting vascular cell activation is also evident from recent studies showing abnormal PDGF receptor signaling and increased atherosclerosis in smooth muscle-specific LRP-1 knockout mice (38).

It is interesting to note that delipidated apoE was equally effective as apoE in lipid emulsions in the induction of cAMP/PKA pathways in LRP-positive cells but not in LRP-negative cells. These results suggest that apoE reconstitution into lipoproteins is not required for its interaction with LRP-1. This is in contrast to the requirement of lipid reconstitution for apoE interaction with the low density lipoprotein receptor. Although we cannot exclude the possibility that the delipidated apoE became lipidated during its incubation with the tissue culture cells before its interaction with LRP-1, apoE prepared similarly and added to human fibroblast cell culture using an identical procedure did not interact with low density lipoprotein receptors on these cells.2 Taken together, these observations suggest that minimally lipidated apoE is sufficient to interact with LRP-1 on the cell surface. This hypothesis is consistent with results reported recently by Narita et al. (39) demonstrating the direct binding of lipid-poor apoE to LRP-1. The ability of lipid-poor apoE to interact with LRP-1 further supports our hypothesis that apoE-LRP interaction has additional cell signaling function in addition to its well documented role in lipid transport.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant HL61332. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0529. Tel.: 513-558-9152; Fax: 513-558-2141; E-mail: huidy{at}email.uc.edu.

1 The abbreviations used are: apo, apolipoprotein; LRP-1, low density lipoprotein receptor-related protein-1; PKA, protein kinase A; PDGF-BB, platelet-derived growth factor-BB; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interfering RNA; PKI, PKA inhibitory peptide; MEF, murine embryonic fibroblast. Back

2 Y. Zhu and D. Y. Hui, unpublished observation. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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