SORL1 is a receptor for tau that promotes tau seeding.

Sortilin-related receptor 1 (SORL1) is an intracellular sorting receptor genetically implicated in Alzheimer's disease (AD) that impacts amyloid precursor protein trafficking. The objective of these studies was to test the hypothesis that SORL1 binds tau, modulates its cellular trafficking and impacts the aggregation of cytoplasmic tau induced by pathological forms of tau. Using surface plasmon resonance measurements, we observed high-affinity binding of tau to SORL1 and the vacuolar protein sorting 10 domain of SORL1. Interestingly, unlike LDL receptor-related protein 1, SORL1 binds tau at both pH 7.4 and pH 5.5, revealing its ability to bind tau at endosomal pH. Immunofluorescence studies confirmed that exogenously added tau colocalized with SORL1 in H4 neuroglioma cells, while overexpression of SORL1 in LDL receptor-related protein 1-deficient Chinese hamster ovary (CHO) cells resulted in a marked increase in the internalization of tau, indicating that SORL1 can bind and mediate the internalization of monomeric forms of tau. We further demonstrated that SORL1 mediates tau seeding when tau RD P301S FRET biosensor cells expressing SORL1 were incubated with high molecular weight forms of tau isolated from the brains of patients with AD. Seeding in H4 neuroglioma cells is significantly reduced when SORL1 is knocked down with siRNA. Finally, we demonstrate that the N1358S mutant of SORL1 significantly increases tau seeding when compared to WT SORL1, identifying for the first time a potential mechanism that connects this specific SORL1 mutation to Alzheimer's disease. Together, these studies identify SORL1 as a receptor that contributes to trafficking and seeding of pathogenic tau.

LRP1 protects against excessive superior mesenteric artery remodeling by modulating angiotensin II-mediated signaling.

Vascular smooth muscle cells (vSMCs) exert a critical role in sensing and maintaining vascular integrity. These cells abundantly express the low-density lipoprotein receptor-related protein 1 (LRP1), a large endocytic signaling receptor that recognizes numerous ligands, including apolipoprotein E-rich lipoproteins, proteases, and protease-inhibitor complexes. We observed the spontaneous formation of aneurysms in the superior mesenteric artery (SMA) of both male and female mice in which LRP1 was genetically deleted in vSMCs (smLRP1-/- mice). Quantitative proteomics revealed elevated abundance of several proteins in smLRP1-/- mice that are known to be induced by angiotensin II-mediated (AngII-mediated) signaling, suggesting that this pathway was dysregulated. Administration of losartan, an AngII type I receptor antagonist, or an angiotensinogen antisense oligonucleotide to reduce plasma angiotensinogen concentrations restored the normal SMA phenotype in smLRP1-/- mice and prevented aneurysm formation. Additionally, using a vascular injury model, we noted excessive vascular remodeling and neointima formation in smLRP1-/- mice that was restored by losartan administration. Together, these findings reveal that LRP1 regulates vascular integrity and remodeling of the SMA by attenuating excessive AngII-mediated signaling.

Characterization of interaction between blood coagulation factor VIII and LRP1 suggests dynamic binding by alternating complex contacts.

BACKGROUND: Deficiency in blood coagulation factor VIII (FVIII) results in life-threating bleeding (hemophilia A) treated by infusions of FVIII concentrates. To improve disease treatment, FVIII has been modified to increase its plasma half-life, which requires understanding mechanisms of FVIII catabolism. An important catabolic actor is hepatic low density lipoprotein receptor-related protein 1 (LRP1), which also regulates many other clinically significant processes. Previous studies showed complexity of FVIII site for binding LRP1. OBJECTIVES: To characterize binding sites between FVIII and LRP1 and suggest a model of the interaction. METHODS: A series of recombinant ligand-binding complement-type repeat (CR) fragments of LRP1 including mutated variants was generated in a baculovirus system and tested for FVIII interaction using surface plasmon resonance, tissue culture model, hydrogen-deuterium exchange mass spectrometry, and in silico. RESULTS: Multiple CR doublets within LRP1 clusters II and IV were identified as alternative FVIII-binding sites. These interactions follow the canonical binding mode providing major binding energy, and additional weak interactions are contributed by adjacent CR domains. A representative CR doublet was shown to have multiple contact sites on FVIII. CONCLUSIONS: FVIII and LRP1 interact via formation of multiple complex contacts involving both canonical and non-canonical binding combinations. We propose that FVIII-LRP1 interaction occurs via switching such alternative binding combinations in a dynamic mode, and that this mechanism is relevant to other ligand interactions of the low-density lipoprotein receptor family members including LRP1.

Regulation of tau internalization, degradation, and seeding by LRP1 reveals multiple pathways for tau catabolism.

In Alzheimer's disease (AD), pathological forms of tau are transferred from cell to cell and "seed" aggregation of cytoplasmic tau. Phosphorylation of tau plays a key role in neurodegenerative tauopathies. In addition, apolipoprotein E (apoE), a major component of lipoproteins in the brain, is a genetic risk determinant for AD. The identification of the apoE receptor, low-density lipoprotein receptor-related protein 1 (LRP1), as an endocytic receptor for tau raises several questions about the role of LRP1 in tauopathies: is internalized tau, like other LRP1 ligands, delivered to lysosomes for degradation, and does LRP1 internalize pathological tau leading to cytosolic seeding? We found that LRP1 rapidly internalizes 125I-labeled tau, which is then efficiently degraded in lysosomal compartments. Surface plasmon resonance experiments confirm high affinity binding of tau and the tau microtubule-binding domain to LRP1. Interestingly, phosphorylated forms of recombinant tau bind weakly to LRP1 and are less efficiently internalized by LRP1. LRP1-mediated uptake of tau is inhibited by apoE, with the apoE4 isoform being the most potent inhibitor, likely because of its higher affinity for LRP1. Employing post-translationally-modified tau derived from brain lysates of human AD brain tissue, we found that LRP1-expressing cells, but not LRP1-deficient cells, promote cytosolic tau seeding in a process enhanced by apoE. These studies identify LRP1 as an endocytic receptor that binds and processes monomeric forms of tau leading to its degradation and promotes seeding by pathological forms of tau. The balance of these processes may be fundamental to the spread of neuropathology across the brain in AD.

High-Affinity Binding of LDL Receptor-Related Protein 1 to Matrix Metalloprotease 1 Requires Protease:Inhibitor Complex Formation.

Matrix metalloprotease (MMP) activation contributes to the degradation of the extracellular matrix (ECM), resulting in a multitude of pathologies. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted endocytic and signaling receptor that is responsible for internalization and lysosomal degradation of diverse proteases, protease inhibitors, and lipoproteins along with numerous other proteins. In this study, we identified MMP-1 as a novel LRP1 ligand. Binding studies employing surface plasmon resonance revealed that both proMMP-1 and active MMP-1 bind to purified LRP1 with equilibrium dissociation constants (KD) of 19 and 25 nM, respectively. We observed that human aortic smooth muscle cells readily internalize and degrade 125I-labeled proMMP-1 in an LRP1-mediated process. Our binding data also revealed that all tissue inhibitors of metalloproteases (TIMPs) bind to LRP1 with KD values ranging from 23 to 33 nM. Interestingly, the MMP-1/TIMP-1 complex bound to LRP1 with an affinity (KD = 0.6 nM) that was 30-fold higher than that of either component alone, revealing that LRP1 prefers the protease:inhibitor complex as a ligand. Of note, modification of lysine residues on either proMMP-1 or TIMP-1 ablated the ability of the MMP-1/TIMP-1 complex to bind to LRP1. LRP1's preferential binding to enzyme:inhibitor complexes was further supported by the higher binding affinity for proMMP-9/TIMP-1 complexes than for either of these two components alone. LRP1 has four clusters of ligand-binding repeats, and MMP-1, TIMP-1, and MMP-1/TIMP-1 complexes bound to cluster III most avidly. Our results reveal an important role for LRP1 in controlling ECM homeostasis by regulating MMP-1 and MMP-9 levels.

High-affinity binding of plasminogen-activator inhibitor 1 complexes to LDL receptor-related protein 1 requires lysines 80, 88, and 207.

It is well-established that complexes of plasminogen-activator inhibitor 1 (PAI-1) with its target enzymes bind tightly to low-density lipoprotein (LDL) receptor-related protein 1 (LRP1), but the molecular details of this interaction are not well-defined. Furthermore, considerable controversy exists in the literature regarding the nature of the interaction of free PAI-1 with LRP1. In this study, we examined the binding of free PAI-1 and complexes of PAI-1 with low-molecular-weight urokinase-type plasminogen activator to LRP1. Our results confirmed that uPA:PAI-1 complexes bind LRP1 with ∼100-fold increased affinity over PAI-1 alone. Chemical modification of PAI-1 confirmed an essential requirement of lysine residues in PAI-1 for the interactions of both PAI-1 and uPA:PAI-1 complexes with LRP1. Results of surface plasmon resonance measurements supported a bivalent binding model in which multiple sites on PAI-1 and uPA:PAI-1 complexes interact with complementary sites on LRP1. An ionic-strength dependence of binding suggested the critical involvement of two charged residues for the interaction of PAI-1 with LRP1 and three charged residues for the interaction of uPA:PAI-1 complexes with LRP1. An enhanced affinity resulting from the interaction of three regions of the uPA:PAI-1 complex with LDLa repeats on LRP1 provided an explanation for the increased affinity of uPA:PAI-1 complexes for LRP1. Mutational analysis revealed an overlap between LRP1 binding and binding of a small-molecule inhibitor of PAI-1, CDE-096, confirming an important role for Lys-207 in the interaction of PAI-1 with LRP1 and of the orientations of Lys-207, -88, and -80 for the interaction of uPA:PAI-1 complexes with LRP1.

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis.

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.

Macrophage LRP1 Promotes Diet-Induced Hepatic Inflammation and Metabolic Dysfunction by Modulating Wnt Signaling.

Hepatic inflammation is associated with the development of insulin resistance, which can perpetuate the disease state and may increase the risk of metabolic syndrome and diabetes. Despite recent advances, mechanisms linking hepatic inflammation and insulin resistance are still unclear. The low-density lipoprotein receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is highly expressed in macrophages, adipocytes, hepatocytes, and vascular smooth muscle cells. To investigate the potential role of macrophage LRP1 in hepatic inflammation and insulin resistance, we conducted experiments using macrophage-specific LRP1-deficient mice (macLRP1-/- ) generated on a low-density lipoprotein receptor knockout (LDLR-/- ) background and fed a Western diet. LDLR-/-; macLRP1-/- mice gained less body weight and had improved glucose tolerance compared to LDLR-/- mice. Livers from LDLR-/-; macLRP1-/- mice displayed lower levels of gene expression for several inflammatory cytokines, including Ccl3, Ccl4, Ccl8, Ccr1, Ccr2, Cxcl9, and Tnf, and reduced phosphorylation of GSK3α and p38 MAPK proteins. Furthermore, LRP1-deficient peritoneal macrophages displayed altered cholesterol metabolism. Finally, circulating levels of sFRP-5, a potent anti-inflammatory adipokine that functions as a decoy receptor for Wnt5a, were elevated in LDLR-/-; macLRP1-/- mice. Surface plasmon resonance experiments revealed that sFRP-5 is a novel high affinity ligand for LRP1, revealing that LRP1 regulates levels of this inhibitor of Wnt5a-mediated signaling. Collectively, our results suggest that LRP1 expression in macrophages promotes hepatic inflammation and the development of glucose intolerance and insulin resistance by modulating Wnt signaling.

LRP1 (Low-Density Lipoprotein Receptor-Related Protein 1) Regulates Smooth Muscle Contractility by Modulating Ca2+ Signaling and Expression of Cytoskeleton-Related Proteins.

Objective- Mutations affecting contractile-related proteins in the ECM (extracellular matrix), microfibrils, or vascular smooth muscle cells can predispose the aorta to aneurysms. We reported previously that the LRP1 (low-density lipoprotein receptor-related protein 1) maintains vessel wall integrity, and smLRP1-/- mice exhibited aortic dilatation. The current study focused on defining the mechanisms by which LRP1 regulates vessel wall function and integrity. Approach and Results- Isometric contraction assays demonstrated that vasoreactivity of LRP1-deficient aortic rings was significantly attenuated when stimulated with vasoconstrictors, including phenylephrine, thromboxane receptor agonist U-46619, increased potassium, and L-type Ca2+ channel ligand FPL-64176. Quantitative proteomics revealed proteins involved in actin polymerization and contraction were significantly downregulated in aortas of smLRP1-/- mice. However, studies with calyculin A indicated that although aortic muscle from smLRP1-/- mice can contract in response to calyculin A, a role for LRP1 in regulating the contractile machinery is not revealed. Furthermore, intracellular calcium imaging experiments identified defects in calcium release in response to a RyR (ryanodine receptor) agonist in smLRP1-/- aortic rings and cultured vascular smooth muscle cells. Conclusions- These results identify a critical role for LRP1 in modulating vascular smooth muscle cell contraction by regulating calcium signaling events that potentially protect against aneurysm development.

Evidence That Factor VIII Forms a Bivalent Complex with the Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1): IDENTIFICATION OF CLUSTER IV ON LRP1 AS THE MAJOR BINDING SITE.

Hemophilia A is a bleeding disorder caused by a deficiency in coagulation factor VIII (fVIII) that affects 1 in 5,000 males. Current prophylactic replacement therapy, although effective, is difficult to maintain due to the cost and frequency of injections. Hepatic clearance of fVIII is mediated by the LDL receptor-related protein 1 (LRP1), a member of the LDL receptor family. Although it is well established that fVIII binds LRP1, the molecular details of this interaction are unclear as most of the studies have been performed using fragments of fVIII and LRP1. In the current investigation, we examine the binding of intact fVIII to full-length LRP1 to gain insight into the molecular interaction. Chemical modification studies confirm the requirement for lysine residues in the interaction of fVIII with LRP1. Examination of the ionic strength dependence of the interaction of fVIII with LRP1 resulted in a Debye-Hückel plot with a slope of 1.8 ± 0.5, suggesting the involvement of two critical charged residues in the interaction of fVIII with LRP1. Kinetic studies utilizing surface plasmon resonance techniques reveal that the high affinity of fVIII for LRP1 results from avidity effects mediated by the interactions of two sites in fVIII with complementary sites on LRP1 to form a bivalent fVIII·LRP1 complex. Furthermore, although fVIII bound avidly to soluble forms of clusters II and IV from LRP1, only soluble cluster IV competed with the binding of fVIII to full-length LRP1, revealing that cluster IV represents the major fVIII binding site in LRP1.

Regulation of Itch and Nedd4 E3 Ligase Activity and Degradation by LRAD3.

Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a number of biological processes. Precise regulation of their enzymatic activity is required for normal physiological function. Nedd4-like E3 ligases exist in an inactive form resulting from intramolecular interactions of their catalytic HECT domain with their WW domains. We identified the low-density-lipoprotein receptor class A domain containing 3 (LRAD3), a member of the LDL receptor family, as a potent activator of Itch and Nedd4 as evidenced by their increased auto-ubiquitination when bound to LRAD3. LRAD3 contains two PPxY motifs within its intracellular domain, both of which can bind to the WW domains on Itch and other Nedd4 family members with high affinity. Mutational analysis revealed that binding of Itch to the terminal LRAD3 PPxY motif is required to promote its auto-ubiquitination. We also determined that association of Itch and Nedd4 with LRAD3 leads to increased auto-ubiquitination and subsequent degradation through proteasome-mediated processes. Our findings reveal that LRAD3 is a component of pathways that function effectively to modulate Itch and Nedd4 auto-ubiquitination and levels. The identification of potential ligands for LRAD3 that may modulate LRAD3-induced activation of Itch and Nedd4 is likely to identify additional novel substrates and cellular functions for these important E3 ligases.

Mac-1 Regulates IL-13 Activity in Macrophages by Directly Interacting with IL-13Rα1.

Mac-1 exhibits a unique inhibitory activity toward IL-13-induced JAK/STAT activation and thereby regulates macrophage to foam cell transformation. However, the underlying molecular mechanism is unknown. In this study, we report the identification of IL-13Rα1, a component of the IL-13 receptor (IL-13R), as a novel ligand of integrin Mac-1, using a co-evolution-based algorithm. Biochemical analyses demonstrated that recombinant IL-13Rα1 binds Mac-1 in a purified system and supports Mac-1-mediated cell adhesion. Co-immunoprecipitation experiments revealed that endogenous Mac-1 forms a complex with IL-13Rα1 in solution, and confocal fluorescence microscopy demonstrated that these two receptors co-localize with each other on the surface of macrophages. Moreover, we found that genetic inactivation of Mac-1 promotes IL-13-induced JAK/STAT activation in macrophages, resulting in enhanced polarization along the alternative activation pathway. Importantly, we observed that Mac-1(-/-) macrophages exhibit increased expression of foam cell differentiation markers including 15-lipoxygenase and lectin-type oxidized LDL receptor-1 both in vitro and in vivo. Indeed, we found that Mac-1(-/-)LDLR(-/-) mice develop significantly more foam cells than control LDLR(-/-) mice, using an in vivo model of foam cell formation. Together, our data establish for the first time a molecular mechanism by which Mac-1 regulates the signaling activity of IL-13 in macrophages. This newly identified IL-13Rα1/Mac-1-dependent pathway may offer novel targets for therapeutic intervention in the future.

LDL Receptor-Related Protein-1 (LRP1) Regulates Cholesterol Accumulation in Macrophages.

Within the circulation, cholesterol is transported by lipoprotein particles and is taken up by cells when these particles associate with cellular receptors. In macrophages, excessive lipoprotein particle uptake leads to foam cell formation, which is an early event in the development of atherosclerosis. Currently, mechanisms responsible for foam cell formation are incompletely understood. To date, several macrophage receptors have been identified that contribute to the uptake of modified forms of lipoproteins leading to foam cell formation, but the in vivo contribution of the LDL receptor-related protein 1 (LRP1) to this process is not known [corrected]. To investigate the role of LRP1 in cholesterol accumulation in macrophages, we generated mice with a selective deletion of LRP1 in macrophages on an LDL receptor (LDLR)-deficient background (macLRP1-/-). After feeding mice a high fat diet for 11 weeks, peritoneal macrophages isolated from Lrp+/+ mice contained significantly higher levels of total cholesterol than those from macLRP1-/- mice. Further analysis revealed that this was due to increased levels of cholesterol esters. Interestingly, macLRP1-/- mice displayed elevated plasma cholesterol and triglyceride levels resulting from accumulation of large, triglyceride-rich lipoprotein particles in the circulation. This increase did not result from an increase in hepatic VLDL biosynthesis, but rather results from a defect in catabolism of triglyceride-rich lipoprotein particles in macLRP1-/- mice. These studies reveal an important in vivo contribution of macrophage LRP1 to cholesterol homeostasis.

Generation of a Potent Low Density Lipoprotein Receptor-related Protein 1 (LRP1) Antagonist by Engineering a Stable Form of the Receptor-associated Protein (RAP) D3 Domain.

The low density lipoprotein receptor-related protein 1 (LRP1) is a member of the low density lipoprotein receptor family and plays important roles in a number of physiological and pathological processes. Expression of LRP1 requires the receptor-associated protein (RAP), a molecular chaperone that binds LRP1 and other low density lipoprotein receptor family members in the endoplasmic reticulum and traffics with them to the Golgi where the acidic environment causes its dissociation. Exogenously added RAP is a potent LRP1 antagonist and binds to LRP1 on the cell surface, preventing ligands from binding. Following endocytosis, RAP dissociates in the acidic endosome, allowing LRP1 to recycle back to the cell surface. The acid-induced dissociation of RAP is mediated by its D3 domain, a relatively unstable three-helical bundle that denatures at pH <6.2 due to protonation of key histidine residues on helices 2 and 3. To develop an LRP1 inhibitor that does not dissociate at low pH, we introduced a disulfide bond between the second and third helices in the RAP D3 domain. By combining this disulfide bond with elimination of key histidine residues, we generated a stable RAP molecule that is resistant to both pH- and heat-induced denaturation. This molecule bound to LRP1 with high affinity at both neutral and acidic pH and proved to be a potent inhibitor of LRP1 function both in vitro and in vivo, suggesting that our stable RAP molecule may be useful in multiple pathological settings where LRP1 blockade has been shown to be effective.

Development of human serine protease-based therapeutics targeting Fn14 and identification of Fn14 as a new target overexpressed in TNBC.

The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)-containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome c-related proapoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared with vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. The Cancer Genome Atlas analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P < 0.0001) compared with hormone receptor-positive breast cancer, and in basal-like 2 tumors (P = 0.01) compared with other TNBC molecular subtypes. IHC analysis of a 101 patient TNBC tumor microarray showed that 55 of 101 (54%) of tumors stained positive for Fn14, suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully human, GrB-containing fusion constructs may form the basis for a new class of novel, potent, and highly effective constructs for targeted therapeutic applications.

The LDL receptor-related protein 1 (LRP1) regulates the PDGF signaling pathway by binding the protein phosphatase SHP-2 and modulating SHP-2- mediated PDGF signaling events.

BACKGROUND: The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor ß (PDGFRß) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRß pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRß signaling pathway by binding SHP-2 and competing with the PDGFRß for this molecule. METHODOLOGY/PRINCIPAL FINDINGS: To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRß kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRß. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that phosphorylated forms of LRP1 and PDGFRß compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.

LRP1 protects the vasculature by regulating levels of connective tissue growth factor and HtrA1.

OBJECTIVE: Low-density lipoprotein receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is abundant in vascular smooth muscle cells. Mice in which the lrp1 gene is deleted in smooth muscle cells (smLRP1(-/-)) on a low-density lipoprotein receptor-deficient background display excessive platelet derived growth factor-signaling, smooth muscle cell proliferation, aneurysm formation, and increased susceptibility to atherosclerosis. The objectives of the current study were to examine the potential of LRP1 to modulate vascular physiology under nonatherogenic conditions. APPROACH AND RESULTS: We found smLRP1(-/-) mice to have extensive in vivo aortic dilatation accompanied by disorganized and degraded elastic lamina along with medial thickening of the arterial vessels resulting from excess matrix deposition. Surprisingly, this was not attributable to excessive platelet derived growth factor-signaling. Rather, quantitative differential proteomic analysis revealed that smLRP1(-/-) vessels contain a 4-fold increase in protein levels of high-temperature requirement factor A1 (HtrA1), which is a secreted serine protease that is known to degrade matrix components and to impair elastogenesis, resulting in fragmentation of elastic fibers. Importantly, our study discovered that HtrA1 is a novel LRP1 ligand. Proteomics analysis also identified excessive accumulation of connective tissue growth factor, an LRP1 ligand and a key mediator of fibrosis. CONCLUSIONS: Our findings suggest a critical role for LRP1 in maintaining the integrity of vessels by regulating protease activity as well as matrix deposition by modulating HtrA1 and connective tissue growth factor protein levels. This study highlights 2 new molecules, connective tissue growth factor and HtrA1, which contribute to detrimental changes in the vasculature and, therefore, represent new target molecules for potential therapeutic intervention to maintain vessel wall homeostasis.

Macrophage LRP1 suppresses neo-intima formation during vascular remodeling by modulating the TGF-ß signaling pathway.

BACKGROUND: Vascular remodeling in response to alterations in blood flow has been shown to modulate the formation of neo-intima. This process results from a proliferative response of vascular smooth muscle cells and is influenced by macrophages, which potentiate the development of the intima. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that recognizes a number of ligands including apoE-containing lipoproteins, proteases and protease-inhibitor complexes. Macrophage LRP1 is known to influence the development of atherosclerosis, but its role in vascular remodeling has not been investigated. METHODOLOGY/PRINCIPAL FINDINGS: To define the contribution of macrophage LRP1 to vascular remodeling, we generated macrophage specific LRP1-deficient mice (macLRP1-/-) on an LDL receptor (LDLr) knock-out background. Using a carotid ligation model, we detected a 2-fold increase in neointimal thickening and a 2-fold increase in the intima/media ratio in macLRP1-/- mice. Quantitative RT-PCR arrays of the remodeled vessel wall identified increases in mRNA levels of the TGF-ß2 gene as well as the Pdgfa gene in macLRP1-/- mice which could account for the alterations in vascular remodeling. Immunohistochemistry analysis revealed increased activation of the TGF-ß signaling pathway in macLRP1-/- mice. Further, we observed that LRP1 binds TGF-ß2 and macrophages lacking LRP1 accumulate twice as much TGF-ß2 in conditioned media. Finally, TNF-α modulation of the TGF-ß2 gene in macrophages is attenuated when LRP1 is expressed. Together, the data reveal that LRP1 modulates both the expression and protein levels of TGF-ß2 in macrophages. CONCLUSIONS/SIGNIFICANCE: Our data demonstrate that macrophage LRP1 protects the vasculature by limiting remodeling events associated with flow. This appears to occur by the ability of macrophage LRP1 to reduce TGF-ß2 protein levels and to attenuate expression of the TGF-ß2 gene resulting in suppression of the TGF-ß signaling pathway.

LRAD3, a novel low-density lipoprotein receptor family member that modulates amyloid precursor protein trafficking.

We have identified a novel low-density lipoprotein (LDL) receptor family member, termed LDL receptor class A domain containing 3 (LRAD3), which is expressed in neurons. The LRAD3 gene encodes an ∼50 kDa type I transmembrane receptor with an ectodomain containing three LDLa repeats, a transmembrane domain, and a cytoplasmic domain containing a conserved dileucine internalization motif and two polyproline motifs with potential to interact with WW-domain-containing proteins. Immunohistochemical analysis of mouse brain reveals LRAD3 expression in the cortex and hippocampus. In the mouse hippocampal-derived cell line HT22, LRAD3 partially colocalizes with amyloid precursor protein (APP) and interacts with APP as revealed by coimmunoprecipitation experiments. To identify the portion of APP that interacts with LRAD3, we used solid-phase binding assays that demonstrated that LRAD3 failed to bind to a soluble APP fragment (sAPPα) released after α-secretase cleavage. In contrast, C99, the ß-secretase product that remains cell associated, coprecipitated with LRAD3, confirming that regions within this portion of APP are important for associating with LRAD3. The association of LRAD3 with APP increases the amyloidogenic pathway of APP processing, resulting in a decrease in sAPPα production and increased Aß peptide production. Pulse-chase experiments confirm that LRAD3 expression significantly decreases the cellular half-life of mature APP. These results reveal that LRAD3 influences APP processing and raises the possibility that LRAD3 alters APP function in neurons, including its downstream signaling.