Modulation of Smooth Muscle Cell Migration by Members of the Low-Density Lipoprotein Receptor Family

【摘要】  Low-density lipoprotein receptor family members (LRs) play a key role in the catabolism of many membrane-associated proteins, such as complexes between proteinases and their receptors, in addition to being involved in lipoprotein metabolism as suspected by the hitherto well-established functions of low-density lipoprotein receptor, in a variety of tissues. Recent studies using receptor-deficient or -overexpressing animals and cells have suggested that certain LRs are important regulators of the migration (and proliferation) of vascular smooth muscle cells (SMCs). LR expression is markedly induced in intimal or medial SMCs during the formation of atherosclerotic lesions. Because LRs can modulate the activity of the urokinase-type plasminogen activator (uPA) receptor and possibly of the platelet-derived growth factor (PDGF) receptor, LRs may influence the migration of SMCs through functional modulation of these membrane receptors. Therefore, SMC migration may be regulated by time-restricted expression of LRs. In agreement with the concept of functional interaction between LRs and membrane signaling receptors, a negative regulator of uPA receptor protein catabolism, LR11, has been identified. Statins modulate the PDGF-induced migration of intimal SMCs via the LR11/uPA receptor cascade. Selective modification of the LRs/uPA receptor/PDGF receptor systems in SMCs may be important for suppression of atherosclerotic plaque formation as well as for preventing intimal thickening after angioplasty.

LDL receptor family members (LRs) regulate the catabolism of membrane-associated proteins and are expressed in SMCs of atherosclerostic lesions. LRs modulate the activity of the urokinase-type plasminogen activator (uPA) receptor and possibly of the PDGF receptor. Selective modification of the LRs/membrane receptor system may be important for suppression of atherosclerosis.

【关键词】  LDL receptor family smooth muscle cells migration LR urokinasetype plasminogen activator receptor PDGF receptor

Introduction

The members of the low-density lipoprotein receptor family (LRs) are characterized by distinct functional domains present in characteristic numbers and arrangements ( Figure 1 ). The common structural domains in most LRs are the so-called low-density lipoprotein (LDL) receptor ligand binding repeats (type A), epidermal growth factor precursor homology repeats (type B1 and B2), epidermal growth factor precursor homology repeats with a consensus tetrapeptide, Tyr-Trp-Thr-Asp, and in the cytoplasmic region, signals for receptor internalization via coated pits. These LRs discovered to date are the LDL receptor, LDL receptor-related protein-1 (LRP-1), megalin, the very low-density lipoprotein (VLDL) receptor/LR8, apolipoprotein E receptor 2/LR8B, LR11, and, most recently, LRPs 3 through 7. 1-3 LRP-1 and megalin are giant LRs in which the amino acid sequence contains multiple repeats of each functional component of the LDL receptor. 4,5 The domain structures of VLDL receptor/LR8 and apolipoprotein E receptor 2/LR8B are most similar to that of the LDL receptor. 6-8 LRs indeed show considerable sequence identity (70% to 100%) between molecules harboring common structures and among a wide range of species. Such sequence conservation is thought to indicate evolution from an ancestral gene by duplication or exon shuffling. The avian VLDL receptor/LR8 is essential for reproduction as a receptor for the yolk accumulation. 8,9

Figure 1. Schematic presentation of the LRs. The common structural modules in most of LRs are: (1) the so-called "LDL receptor ligand binding repeats (type A)," complement-type domains consisting of 40 residues displaying a triple-disulfide-bond-stabilized negatively charged surface; (2) epidermal growth factor (EGF) precursor homology repeats (type B1 and B2), also containing 6 cysteins each; (3) EGF precursor homology repeats consisting of 50 residues each, most often in groups of 5, with a consensus tetrapeptide, Tyr-Trp-Thr-Asp (YWTD); and (4) in the cytoplasmic region, signals for receptor internalization via coated pits, containing the consensus tetrapeptide Asn-Pro-Xaa-Tyr (NPXY).

LRs play a key role in lipoprotein metabolism, as demonstrated by the well-established actions of the LDL receptor in a variety of tissues. 1 Extensive functional analyses have also revealed that LRs play an important role in the catabolism of many membrane-associated proteins such as complexes between proteinases and their receptors. 1-3 Recent studies using receptor-deficient or -overexpressing animals and cells have suggested that certain LRs are also important as regulators of the migration (and proliferation) of various cells such as fibroblasts, neurons, and vascular smooth muscle cells (SMCs). 10-17

Histochemical studies have revealed that the expression of LRs, as well as scavenger receptors, is markedly induced during the development of atherosclerotic lesions. 1,18 For instance, the VLDL receptor/LR8 is highly expressed by SMCs, macrophages, and endothelial cells in rabbit atherosclerotic lesions, whereas the LDL receptor is not abundant in arterial walls. 18,19 LRP-1 expression is also induced in atheromatous plaques. 18-20 We identified strong LR11 expression inside plaques, particularly by intimal SMCs located at the interface between intima and media. 21,22 In addition, LRP-1B is expressed by SMCs of the medial layer and in thickened intimal regions. 23 Thus, changes in the expression of LRs by vascular cells, particularly SMCs, may play a role in the development of atherosclerosis.

The migration and proliferation of SMCs, as well as extracellular matrix (ECM) production and catabolism by these cells, are important events in the development of atherosclerosis and intimal thickening after coronary angioplasty. 24 When thickening of the intima occurs, SMCs migrate from the media into the intima. During migration, SMCs acquire or lose various functions to perform the above-mentioned activities in the intima. 25,26 However, the mechanisms that control the migration of intimal SMCs have not been clarified because of the complex intracellular machinery and the interactions of numerous internal or external factors and signaling pathways. There is conclusive evidence that migration of SMCs from the media into the intima contributes to the formation of stable plaque. 27,28 Here, we focus on the role of LRs in regulating membrane receptor functions related to the migration of SMCs associated with atherosclerosis.

Platelet-Derived Growth Factor-Mediated Migratory Activity of Intimal SMCs

There is a distinct difference in migratory activity between cultured SMCs isolated from the intimal and medial layers of atherosclerotic aortas. 29 Cultured intimal SMCs differ from medial SMCs in many ways, including their morphology, proliferative potential, and gene expression. 29-31 The phenotypic modifications of SMCs that migrate to the intimal layer seem to contribute to an enhanced synthetic capacity, representing a mechanism that influences plaque stability. In fact, cultured intimal SMCs exhibit a phenotype resembling that of fetal or dedifferentiated SMCs. 25,26 Among the many genes involved in the process of phenotypic modification that occurs in the intima, 32,33 the expression of myosin heavy chain isoforms, such as SM1, SM2, and SMemb/nonmuscle myosic heavy chain-B (NMHC-B), has been well characterized. 25,26,34

Many factors may contribute to altering the migratory potential of SMCs in the intima, including changes of contact with the ECM and exposure to growth factors. Cultured SMCs tend to mimic these changes because primary cultured cells rapidly lose their differentiation markers and develop a synthetic phenotype. Conversely, SMCs grown in 3D cultures, such as a honeycomb structure, are able to retain the contractile phenotype. 35 Thus, various cell culture models have provided information about factors that influence the migration of intimal SMCs. Among them, sensitivity to growth factors (including platelet-derived growth factor ) is known to be important for inducing SMC migration. 25 PDGF-BB-mediated intracellular signals induce migration, which is commonly observed using a migration assay system such as Boyden?s chamber. The influence of PDGF-BB on the migration of SMCs is mediated by a specific membrane receptor: PDGF ß-receptor. 36 During the process of migration of SMCs from the media into the intima, one of the strongly expressed genes is PDGF ß-receptor, 37 which contributes to the migratory capacity of intimal SMCs. 38,39 The PDGF ß-receptor is highly expressed even in the media of diabetic models, which show accelerated plaque formation. 40,41 PDGF-BB negatively regulates the transcription of multiple genes in SMCs and thus modulates differentiation. 42 Accordingly, the switch that induces PDGF ß-receptor gene expression seems to be closely related to increasing the migratory capacity of intimal SMCs.

Urokinase and Its Receptor System Are Activated During SMC Migration

In addition to chemoattractants, several proteases and their inhibitors are involved in the migration of SMCs through the process of matrix degradation. 24 Local protease activation is important for enhancing the mobility of migrating cells, particularly for SMCs to migrate through the ECM to target sites in plaque or thickened intima. Thus, matrix metalloproteinases (MMPs) are integral for SMC migration into the intima. 24 Conversion of pro-MMPs to active MMPs, as well as MMP-9 expression, is mediated by urokinase-type plasminogen activator (uPA)-generated plasmin. 43,44 The resulting matrix degradation releases growth factors such as fibroblast growth factor-2 and latent transforming growth factor-ß, and these chemoattractants further promote the migration of SMCs. Thus, urokinase appears to be necessary for migration of SMCs through the surrounding ECM.

Both tissue-type plasminogen activator and uPA cleave plasminogen to release plasmin. Expression of tissue-type plasminogen activator and uPA is increased in atherosclerotic plaque, 45-47 and a study using knockout mice has revealed a role of uPA in the development of intimal hyperplasia. 48 Accordingly, uPA is thought to play an important role in the target-oriented movement of SMCs because its activation can be localized via binding to its receptor (the uPA receptor) on the cell surface. The receptor-mediated potentiation of protease activity for plasminogen also causes an increase of plasmin activation around cell surface receptors. Subsequent production of plasmin leads to the degradation of ECM components and also has the potential to activate some MMPs. The essential role of this process in enhancing cell mobility has been intensively studied with regard to tumor invasion and neuronal migration. 49,50

Expression of uPA by medial SMCs increases rapidly and significantly after balloon catheter injury to a vessel, corresponding with the time course of SMC migration. 51 Virally mediated overexpression of uPA by the endothelial cells of the carotid arteries promotes lesion growth in cholesterol-fed rabbits. 52 After arterial injury, intimal thickening is significantly reduced in uPA-deficient mice. 48,53 Thus, uPA itself seems to promote intimal thickening after vascular injury. However, despite the ability of uPA to influence the migration of cultured SMCs, 54-56 intimal formation is unaffected in uPA receptor knockout mice. 57 The specific proteolytic activity of uPA plays a role in the processes of arterial repair after injury, although the details of the mechanism regulating association with its receptor have not been clarified in the setting of atherosclerosis.

In addition to the proteolytic cascade initiated by binding of uPA to its cell surface receptor, uPA possibly facilitates cell migration by inducing intracellular signaling pathways. 58 The uPA receptor is a glycosylphosphatidylinositol-anchored protein, and therefore signaling activity is mediated by its interaction with other membrane molecules. Binding of uPA to its receptor on the cell surface influences the migratory activity through the formation of a complex involving the uPA receptor, vitronectin, and integrin. 50,58 These interactions at the cell membrane stimulate intracellular signaling cascades, as well as uPA receptor-mediated activation of extracellular proteolysis. 50,58 uPA stimulates the migration of SMCs via its receptor signaling cascade containing the Janus kinase, Tyk2, and phosphatidylinositol 3-kinase. Active GTP-bound forms of small GTPases (RhoA and Rac1) are the downstream targets for Tyk2 and phosphatidylinositol 3-kinase activation. Phosphorylation of myosin light chain is one of the end points of the uPA receptor-mediated signaling pathways. Observations suggesting a possible role of uPA (independent of ECM degradation) in cell migration have been reported so the uPA receptor may also modulate migration/invasion in a protease-independent manner. These findings, together with the results obtained in uPA receptor knockout mice, 57 have led to the conception that the uPA receptor modulates SMC migration through cooperation between extracellular proteolysis and intracellular signaling. Proteolysis of the ECM accelerates migration and is coordinated with adhesive and structural changes that promote cell motility, with both processes leading the cells to their targets in the plaques.

LRs Are Novel Modulators of uPA Receptor Function During PDGF-Mediated Migration of SMCs

Functional modulation of the uPA receptor through the pathways with participation of LRs has been established. 59 LRs are known to play an integral role in the catabolism of lipoproteins and of complexes between proteinases and their receptors. 2,3 A large member of the family, LRP-1, is involved in the intake of uPA receptors and uPA/uPA receptor complexes by cells for subsequent degradation or recycling. 60 Extensive studies have revealed that other LRs, such as VLDL receptor/LR8 12 and LRP-1B, 61 also have the capacity to catabolize uPA/uPA receptor complexes.

LRP-1 is involved in the internalization of the uPA/uPA receptor complex, in which formation is induced by plasminogen activator inhibitor-1, and this process is dependent on LRP-1. 10,11,62,63 LRP-1 is a large molecule composed of 2 subunits. Two NP X Y motifs exist in the intracellular domain of LRP-1, and these motifs are not only important for endocytosis but also for intracellular signaling through molecules such as Shc. 64-66 Inhibition of uPA receptor internalization increases cell surface uPA receptor expression and enhances cell motility. 10,16,63,67

Deficiency of LRP-1 in SMCs causes atherosclerosis, which is mediated by the modulation of intracellular PDGF signaling. 17 This is attributable to the influence of LRP-1 on PDGF ß-receptor signaling or metabolism, possibly because of a molecular interaction at the cell surface. 17,68-70 LRP-1B is the giant family member that is most similar to LRP-1; it also binds to the PDGF ß-receptor and modulates receptor-mediated signaling in SMCs. 23 These findings suggest that SMC migration might be regulated by the time-restricted expression of LRs, which determines the outcome of PDGF ß-receptor- and uPA receptor-mediated signaling. In accordance with the concept of functional interaction between LRs and membrane signaling receptors, LR11 has been identified by us and others as a negative regulator of protein catabolism for uPA receptor. 71,72 Previous histochemical studies have revealed that LRs are markedly induced during the development of atherosclerotic lesions. 1,18 Altered expression of LRP-1 and the uPA receptor possibly reflects the vascular response to injury. Upregulation of LRP-1 mRNA has been detected in the aortas of rabbits fed a high-cholesterol diet. 1,18 Both LRP-1 mRNA and protein are expressed in normal and atherosclerotic human arteries. 19,20 Increased vascular expression of the uPA receptor is observed in cholesterol-fed rabbits and human atherosclerotic arteries. 73 Because LRs are able to modulate uPA receptor activity and possibly PDGF receptor activity, LRs are expected to regulate the migration of SMCs through the functional modulation of these membrane receptors ( Figure 2 ).

Figure 2. Proposed model for the regulation of SMC migration by LRs through the uPA/uPA receptor system. The uPA/uPA receptor system induces cell migration through both increased degradation of the ECM and receptor-mediated intracellular signaling that promotes motility. uPA receptor expression is regulated by LRs such as LRP-1, VLDL receptor/LR8, and LRP-1B. SMCs in plaques produce LR11, which is localized on the cell surface and also secreted by the cells. LR11 binds to and interacts with the uPA receptor on the cell surface or on neighboring cells. Formation of this complex inhibits internalization of the uPA receptor via other LRs (LRP-1, LRP-1B, etc.) and thereby prevents its degradation and relocation, resulting in the enhanced uPA receptor expression on the cell surface. Finally, SMCs expressing LR11 gain an increased migratory capacity that is mediated by activation of the uPA/uPA receptor system. LR11 gene transcription is induced by PDGF-BB and mediated by the PDGF ß-receptor. LRP-1 (and LRP-1B) interacts with the PDGF ß-receptor and modulates receptor-mediated intracellular signaling by PDGF-BB, which promotes migratory activity. Thus, LRs possibly regulate the migration of intimal SMCs in atherosclerotic plaques via modulation of PDGF receptor-meditated signaling, which is also linked with the uPA/uPA receptor system. Statins inhibit the migration of intimal SMCs by decreasing uPA receptor expression via the downregulation of LR11 gene expression.

Involvement of LRs in Regulating SMC Migration in the Intima

Recent functional studies using genetically altered animals or cells revealed that LRs are important regulators of the migration of various cells via modulation of cytokine signaling or protease activation. 13,16 SMC-specific inactivation of LRP-1 in mice has revealed a novel role of LRP-1, which forms a complex with the PDGF receptor. 17 LRP-1 ablation results in a decrease of vascular wall integrity and causes marked susceptibility to cholesterol-induced atherosclerosis in mice. 17 In murine embryonic fibroblasts and fibrosarcoma cells, loss of LRP-1 expression is associated with increased cell surface expression of the uPA receptor and is correlated with increased cell migration in vitro. 10 Similar changes were reported to occur when VLDL receptor/LR8 activity was neutralized in cultured breast cancer cells. 12 LR-mediated regulation of cell migration appears to depend partly on modulation of the uPA/uPA receptor system involved in the degradation of the ECM or modulation of uPA receptor-mediated intracellular signaling through activation of extracellular signal-regulated kinase and Rac1.

A negative regulator of receptor catabolism, LR11, controls uPA receptor localization on the plasma membrane because both the membrane-spanning and secreted forms of LR11 bind to and colocalize with the uPA receptor on the cell surface. 21,74 Expression of LR11 is induced by stimulation of PDGF-BB in SMCs and is observed in intimal SMCs localized at the intima/media border in the atherosclerotic plaques of experimental animals. 21 Overexpression of LR11 by SMCs enhances their migration by elevating uPA receptor expression. 21 Contrarily, neutralization of LR11 reduces the intimal thickening after cuff injury in mice. 21

Modulation of the LR11/uPAR Pathway for Prevention of Atherosclerosis

Statins are potent inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase that are known to be effective for preventing atherosclerosis. Statins have recently been shown to perform a multitude of activities that are involved in the functional modulation of vascular cells such as influences on cell proliferation and secretion. 75,76 One of the major effects of statins on SMCs is modulation of migration. However, the mechanism involved and clinical significance of such inhibition of migration, which has been observed in vitro, have not been elucidated. PDGF-induced migration of SMCs is suppressed by statins in vitro. 77,78 Statins reduce protease expression in atheromatous plaques, and hydrophilic statins decrease SMC numbers and collagen gene expression in vivo. 79 However, phenotypic modulation of intimal SMCs by statins has not yet been investigated. LR11 plays an important role in the induction of migration after enhancement by PDGF-BB in vitro. A potent 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, pitavastatin, reduced the expression of both LR11 and SMemb/NMHC-B in atherosclerotic plaques (unpublished data, 2006). In fact, the enhanced expression of LR11, uPA receptor, and SMemb/NMHC-B by cultured intimal SMCs is reduced by pitavastatin to the levels seen in cells from the media. When expression of the uPA receptor, SMemb/NMHC-B, and endogenous LR11 is increased by PDGF-BB, the enhanced migratory activity of SMCs is blocked by pitavastatin via suppression of endogenous LR11 production. Thus, modulation of the LR11/uPA receptor system plays a role in PDGF-induced migration of intimal SMCs ( Figure 2 ).

It has not yet been clarified whether inhibition of the migration of intimal SMCs leads to the regression of atherosclerotic plaque or prevents restenosis after coronary angioplasty. Activation of pathways mediated by the uPA receptor and the PDGF receptor that increase the migration of intimal SMCs is thought to be essential for the formation of mature plaque after endothelial injury leads to the initiation of atherosclerosis. Unregulated expression of these membrane receptors may reduce the stability of plaque because the programmed migration of SMCs from the media to target regions in the intima would be disturbed. LRs are a possible candidate for modulating SMC migration to control the process of atherosclerosis. Selective modification of the LRs/uPA receptor/PDGF receptor system in SMCs, associated with the change to a dedifferentiated phenotype, appears to be important for the occurrence of intimal thickening after angioplasty as well as plaque formation in atherosclerosis.

Acknowledgments

This work was supported by grants from the Japanese Ministry of Education, Science, and Culture to Y.S. and H.B. We acknowledge that our research in this review was able to be performed because of help from numerous collaborators and colleagues. Finally, we thank Dr W.J. Schneider (University and Biocenter of Vienna) for his collaborative assistance over many years on this topic.

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作者单位：Departments of Genome Research and Clinical Application (H.B.), and Clinical Cell Biology (Y.S.), Chiba University Graduate School of Medicine, Japan.