Low-density Lipoprotein Receptor Family Research Articles

Triglyceride Rich Lipoprotein -LPL-VLDL Receptor and Lp(a)-VLDL Receptor Pathways for Macrophage Foam Cell Formation.

Very low-density lipoprotein (VLDL) receptor is a member of the low-density lipoprotein (LDL) receptor family. It binds triglyceride rich lipoprotein (TGRL) but not LDL, because it recognizes apolipoprotein (apo)E only but not apoB. The VLDL receptor functions as a peripheral lipoprotein receptor in concert with lipoprotein lipase (LPL) in heart, muscle, adipose tissue and macrophages. In contrast to the LDL receptor, VLDL receptor binds apo E2/2 VLDL and apoE3/3 VLDL particles, and its expression is not down-regulated by intracellular lipoproteins. It has been reported that both LDL-cholesterol (LDL-C) and postprandial triglyceride (chyromicron and VLDL remnants) are risk factors for human atherosclerotic cardiovascular disease (ASCVD). True ligands such as lipoprotein particles of the VLDL receptor are chyromicron remnant (CMR) and VLDL remnant (postprandial hyperlipidemia). Although the oxidized LDL (oxLDL)-scavenger receptors pathway is considered to be the main mechanism for macrophage foam cell formation, it seems that the TGRL-LPL-VLDL receptor pathway is also involved. Since Lp(a) is one of the ligands for the VLDL receptor, the Lp(a)- VLDL receptor pathway is another potential alternative. The expression of VLDL receptor protein in mouse macrophages is modest compared to that in rabbit and human macrophages, both in vitro and in vivo. Therefore, we need to elucidate the mechanism of human ASCVD not by using the mouse model and scavenger receptors pathway but instead using the rabbit model and VLDL receptor pathway, respectively.

The LDL Receptor-Related Protein 1: At the Crossroads of Lipoprotein Metabolism and Insulin Signaling.

The metabolic syndrome is an escalating worldwide public health concern. Defined by a combination of physiological, metabolic, and biochemical factors, the metabolic syndrome is used as a clinical guideline to identify individuals with a higher risk for type 2 diabetes and cardiovascular disease. Although risk factors for type 2 diabetes and cardiovascular disease have been known for decades, the molecular mechanisms involved in the pathophysiology of these diseases and their interrelationship remain unclear. The LDL receptor-related protein 1 (LRP1) is a large endocytic and signaling receptor that is widely expressed in several tissues. As a member of the LDL receptor family, LRP1 is involved in the clearance of chylomicron remnants from the circulation and has been demonstrated to be atheroprotective. Recently, studies have shown that LRP1 is involved in insulin receptor trafficking and regulation and glucose metabolism. This review summarizes the role of tissue-specific LRP1 in insulin signaling and its potential role as a link between lipoprotein and glucose metabolism in diabetes.

Molecular Heterogeneity in Peripheral T-Cell Lymphoma Not Otherwise Specified Revealed By Comprehensive Mutational Profiling

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of lymphoproliferative disorders arising from mature T-cells. Among them, PTCL-not otherwise specified (PTCL-NOS) is a diagnosis of exclusion, comprising the largest fraction of PTCL with a diverse underlying pathogenesis. Recently, the concept of nodal T-cell lymphomas with T-follicular helper (TFH) phenotype, including angioimmunoblastic T-cell lymphoma (AITL) and PTCL-NOS that manifests a TFH phenotype, has been proposed, a distinguishing feature of which is the high frequency of TET2, IDH2, DNMT3A, and RHOA(G17V) mutations. Although recent large-scale genetic studies have uncovered mutational landscapes of several other subtypes of PTCLs, such as cutaneous T-cell lymphoma and adult T-cell leukemia/lymphoma (ATL), the entire picture of somatic alterations in PTCL-NOS still remains elusive. In addition, their similarities and differences among various histological subtypes in PTCLs have not been fully elucidated.To address this issue, we initially analyzed our and publicly available whole-exome/genome as well as transcriptome sequencing data from PTCL-NOS and other related PTCLs. Then, we carried out an extensive investigation of somatic mutations and structural variations (SVs) in PTCL-NOS using targeted-capture sequencing of 118 PTCL-NOS samples. Consistent with previous reports, TET2 (35%) was the most frequently mutated gene in PTCL-NOS with the majority (78%) affected by multiple mutations, followed by RHOA (25%), TP53 (16%), KMT2C (12%), PLCG1 (12%), and HLA-B (11%). Besides them, a considerable proportion of patients harbored mutations in components of T-cell receptor (TCR) /NF-κB pathway (such as PRKCB, CARD11, IRF4, and PRDM1), other signal transduction molecules (STAT3, NOTCH1, and SOCS1), chemokine receptors (CCR4 and CCR7), epigenetic modifiers (CREBBP, KDM6A, IDH2, and DNMT3A), transcriptional regulators (GATA3 and TBL1XR1), and molecules associated with immune evasion (HLA-A, HLA-B, FAS, B2M, and CD58). In addition to deteriorating SVs involving frequently affected genes (TP53, FAS, GATA3, and TBL1XR1), we discovered several genes almost exclusively affected by SVs, including TP73, IKZF2, and NFKB2, and CD274.Novel targets of recurrent mutation were also identified, including PDCD1, YTHDF2, and LRP1B, which were frequently targeted by nonsense and frameshift mutations distributed throughout the entire genes. Among them, PDCD1encodes PD-1 receptor transmitting an inhibitory signal from PD-L1 and PD-L2 ligands in T cells, and its loss of function seems to enable tumor cells to escape from the suppression by this negative signal. Although the roles of YTHDF2, a reader protein of N6-methyladenosine, and LRP1B, a member of the low density lipoprotein receptor family, in T cells are not immediately apparent, these findings shed light on a new biological function of these genes.Next, we investigated the co-existence relationship between frequently altered genes in PTCL-NOS. Interestingly, mutations characteristic of TFH lymphomas (TET2, RHOA, IDH2, and DNMT3A) tended to co-occur in a subset of PTCL-NOS cases, whereas they were almost mutually exclusive with mutations in TP53 and TCR/NF-κB pathway genes. This observation reveals the molecular distinction between TFH and non-TFH lymphomas in PTCL-NOS: the former is similar to AITL, although TET2 mutations did not show higher allelic burden than RHOA and IDH2mutations. In contrast, the latter is at least partly characterized by the genetic alterations shared with ATL.In summary, our findings illuminate the landscape of somatic alterations in PTCL-NOS and provide a novel insight into their genetic and molecular heterogeneity, which would help us to exploit a new therapeutic strategy to combat this disease. DisclosuresOhshima:CHUGAI PHARMACEUTICAL CO.,LTD.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Research Funding, Speakers Bureau. Ogawa:Kan research institute: Consultancy, Research Funding; Sumitomo Dainippon Pharma: Research Funding; Takeda Pharmaceuticals: Consultancy, Research Funding. Kataoka:Kyowa Hakko Kirin: Honoraria; Yakult: Honoraria; Boehringer Ingelheim: Honoraria.

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.

Role of LRP-1 in cancer cell migration in 3-dimensional collagen matrix

ABSTRACTThe low-density lipoprotein receptor-related protein-1 (LRP-1) is a member of Low Density Lipoprotein Receptor (LDLR) family, which is ubiquitously expressed and which is described as a multifunctional endocytic receptor which mediates the clearance of various extracellular matrix molecules including serine proteinases, proteinase-inhibitor complexes, and matricellular proteins. Several studies showed that high LRP-1 expression promotes breast cancer cell invasiveness, and LRP-1 invalidation leads to cell motility abrogation in both tumor and non-tumor cells. Furthermore, our group has reported that LRP-1 silencing prevents the invasion of a follicular thyroid carcinoma despite increased pericellular proteolytic activities from MMP2 and uPA using a 2D-cell culture model. As the use of 3D culture systems is becoming more and more popular due to their promise as enhanced models of tissue physiology, the aim of the present work is to characterize for the first time how the 3D collagen type I matrix may impact the ability of LRP-1 to regulate the migratory properties of thyroid carcinoma using as a model FTC-133 cells. Our results show that inhibition of LRP-1 activity or expression leads to morphological changes affecting cell-matrix interactions, reorganizations of the actin-cytoskeleton especially by inhibiting FAK activation and increasing RhoA activity and MLC-2 phosphorylation, thus preventing cell migration. Taken together, our results suggest that LRP-1 silencing leads to a decrease of cell migratory capacity in a 3D configuration.

Rapid hepatic clearance of full length CCN-2/CTGF: a putative role for LRP1-mediated endocytosis

CCN-2 (connective tissue growth factor; CTGF) is a key factor in fibrosis. Plasma CCN-2 has biomarker potential in numerous fibrotic disorders, but it is unknown which pathophysiological factors determine plasma CCN-2 levels. The proteolytic amino-terminal fragment of CCN-2 is primarily eliminated by the kidney. Here, we investigated elimination and distribution profiles of full length CCN-2 by intravenous administration of recombinant CCN-2 to rodents. After bolus injection in mice, we observed a large initial distribution volume (454 mL/kg) and a fast initial clearance (120 mL/kg/min). Immunosorbent assay and immunostaining showed that CCN-2 distributed mainly to the liver and was taken up by hepatocytes. Steady state clearance in rats, determined by continuous infusion of CCN-2, was fast (45 mL/kg/min). Renal CCN-2 clearance, determined by arterial and renal vein sampling, accounted for only 12 % of total clearance. Co-infusion of CCN-2 with receptor-associated protein (RAP), an antagonist of LDL-receptor family proteins, showed that RAP prolonged CCN-2 half-life and completely prevented CCN-2 internalization by hepatocytes. This suggests that hepatic uptake of CCN-2 is mediated by a RAP-sensitive mechanism most likely involving LRP1, a member of the LDL-receptor family involved in hepatic clearance of various plasma proteins. Surface plasmon resonance binding studies confirmed that CCN-2 is an LRP1 ligand. Co-infusion of CCN-2 with an excess of the heparan sulphate-binding protamine lowered the large initial distribution volume of CCN-2 by 88 % and reduced interstitial staining of CCN-2, suggesting binding of CCN-2 to heparan sulphate proteoglycans (HSPGs). Protamine did not affect clearance rate, indicating that RAP-sensitive clearance of CCN-2 is HSPG independent. In conclusion, unlike its amino-terminal fragment which is cleared by the kidney, full length CCN-2 is primarily eliminated by the liver via a fast RAP-sensitive, probably LRP1-dependent pathway.

Expression of a recombinant full-length LRP1B receptor in human non-small cell lung cancer cells confirms the postulated growth-suppressing function of this large LDL receptor family member.

Low-density lipoprotein (LDL) receptor-related protein 1B (LRP1B), a member of the LDL receptor family, is frequently inactivated in multiple malignancies including lung cancer. LRP1B is therefore considered as a putative tumor suppressor. Due to its large size (4599 amino acids), until now only minireceptors or receptor fragments have been successfully cloned. To assess the effect of LRP1B on the proliferation of non-small cell lung cancer cells, we constructed and expressed a transfection vector containing the 13.800 bp full-length murine Lrp1b cDNA using a PCR-based cloning strategy. Expression of LRP1B was analyzed by quantitative RT-PCR (qRT-PCR) using primers specific for human LRP1B or mouse Lrp1b. Effective expression of the full length receptor was demonstrated by the appearance of a single 600 kDa band on Western Blots of HEK 293 cells. Overexpression of Lrp1b in non-small cell lung cancer cells with low or absent endogenous LRP1B expression significantly reduced cellular proliferation compared to empty vector-transfected control cells. Conversely, in Calu-1 cells, which express higher endogenous levels of the receptor, siRNA-mediated LRP1B knockdown significantly enhanced cellular proliferation. Taken together, these findings demonstrate that, consistent with the postulated tumor suppressor function, overexpression of full-length Lrp1b leads to impaired cellular proliferation, while LRP1B knockdown has the opposite effect. The recombinant Lrp1b construct represents a valuable tool to unravel the largely unknown physiological role of LRP1B and its potential functions in cancer pathogenesis.

Mechanism of human rhinovirus infections.

About 150 human rhinovirus serotypes are responsible for more than 50 % of recurrent upper respiratory infections. Despite having similar 3D structures, some bind members of the low-density lipoprotein receptor family, some ICAM-1, and some use CDHR3 for host cell infection. This is also reflected in the pathways exploited for cellular entry. We found that even rhinovirus serotypes binding the same receptor can travel along different endocytic pathways and release their RNA genome into the cytosol at different locations. How this may account for distinct immune responses elicited by various rhinoviruses and the observed symptoms of the common cold is briefly discussed.

Molecular Characterization and Function Analysis of the Vitellogenin Receptor from the Cotton Bollworm, Helicoverpa armigera (Hübner) (Lepidoptera, Noctuidae).

Developing oocytes accumulate plentiful yolk protein during oogenesis through receptor-mediated endocytosis. The vitellogenin receptor (VgR), belonging to the low-density lipoprotein receptor (LDLR) family, regulates the absorption of yolk protein. In this work, the full-length vitellogenin receptor (HaVgR) in the cotton bollworm Helicoverpa armigera was identified, encoding a 1817 residue protein. Sequence alignment revealed that the sequence of HaVgR contained all of the conservative structural motifs of LDLR family members, and phylogenetic analysis indicated that HaVgR had a high identity among Lepidoptera and was distinct from that of other insects. Consistent with other insects, HaVgR was specifically expressed in ovarian tissue. The developmental expression pattern showed that HaVgR was first transcribed in the newly metamorphosed female adults, reached a peak in 2-day-old adults and then declined. Western blot analysis also revealed an ovarian-specific and developing expression pattern, which was consistent with the HaVgR mRNA transcription. Moreover, RNAi-mediated HaVgR knockdown strongly reduced the VgR expression in both the mRNA and protein levels, which inhibited the yolk protein deposition in the ovaries, led to the dramatic accumulation of vitellogenin and the up-regulation of HaVg expression in hemolymph, and eventually resulted in a declined fecundity. Together, all of these findings demonstrate that HaVgR is a specific receptor in uptake and transportation of yolk protein for the maturation of oocytes and that it plays a critical role in female reproduction.

Abstract 411: Obesity Alters the Expression of Very Low Density Lipoprotein Receptor (vldlr) Expression in Peripheral Blood Monocytes: Possible Links Between Vldlr Expression and Monocyte Phenotypes

Objective: Very low density lipoprotein receptor (VLDLR) is a member of the low-density lipoprotein receptor family that binds multiple ligands including TG-rich lipoproteins. The role of VLDLR in lipid uptake had been demonstrated in cell culture and animal models, but little is known about the effects of obesity on VLDLR expression in human. Accordingly, we examined the impact of obesity on VLDLR expression in peripheral blood monocytes and explored possible links with monocyte inflammatory markers and circulating lipids. Subjects and measurements: Blood was collected from a total of 69 subjects including 23 normal-weight (body mass index: BMI < 25 kg/m 2 ) controls and 46 obese (BMI > 30 kg/m 2 ) subjects. Blood lipids, glucose and hormones were measured. Monocytes were isolated from buffy coats using Ficoll density centrifugation followed by magnetic separation of CD14-positive cells with magnetic beads. Expression of VLDLR, and pro- and ant-inflammatory markers of monocytes was examined by western blotting and quantitative qPCR. The uptake of labeled VLDL (DiI-VLDL) was also assessed in monocyte cultures. Results: Insulin, and total and VLDL triglycerides were increased in plasma of obese subjects. In addition, VLDLR protein and mRNA levels were significantly (p< 0.05) higher (~ 40%) in obese compared to lean subjects. Monocytes of obese subjects exhibited higher expression of IL-6 and TNF-α (+29 to 43%), but lower expression of IL-10 and CD163 (-30 to 47%). The levels of monocyte VLDLR was positively correlated with blood triglycerides and insulin, and negatively with adiponectin. The uptake of labeled VLDL, assessed by fluorescence microscope, was higher in monocytes of obese subjects. Conclusions: Our findings indicate that obesity upregulates the expression of VLDLR and pro-inflammatory markers in peripheral blood monocytes. The uptake VLDL was also increased in monocyte of obese subjects suggesting that obesity-induced VLDLR expression may explain the changes of VLDL uptake and monocyte phenotype.

Abstract 98: Antiphospholipid Antibodies Induce Thrombosis by Activating Endothelial PP2A via ApoER2-Dab2-PSD95 Complex Formation

In the antiphospholipid syndrome (APS), patients generate antiphospholipid antibodies (aPL) that promote thrombosis. We previous showed that aPL recognition of the endothelial cell surface protein β2-Glycoprotein I (β2GPI) causes β2GPI to interact with the LDL receptor family member ApoER2 and thereby antagonize endothelial NO synthase (eNOS). The decrease in bioavailable NO then leads to exaggerated thrombus formation. In the present work we sought to determine how aPL and the β2GPI-ApoER2 tandem antagonize eNOS. Initial experiments employed co-immunoprecipitation, RNAi-based gene silencing and adenoviral introduction of mutant proteins into human aortic endothelial cells. We discovered that in contrast to normal human IgG from healthy subjects (NHIgG), aPL invoke the formation of a complex between the cytoplasmic tail of ApoER2 and Dab-2 and PSD95, and that the formation of the complex is required for eNOS antagonism. We also found that ApoER2-Dab2-PSD95 complex formation in response to aPL potently activates the serine/threonine phosphatase PP2A, which in turn dephosphorylates eNOS-S1179, thereby extinguishing eNOS enzymatic activity. Furthermore, we found that upon aPL treatment, the PP2A catalytic and regulatory subunits are recruited to the ApoER2-Dab2-PSD95 complex. To test if these processes are operative in APS-related thrombosis in vivo, intravital microscopy of the mesenteric microcirculation was employed to evaluate thrombus formation in mice. In wild-type mice, aPL administration caused exaggerated thrombus formation compared to treatment with NHIgG. In contrast, ApoER2 did not promote thrombosis in knock-in ApoER2-EIG mice harboring a mutant receptor incapable of interacting with Dab-2. Moreover, in wild-type mice aPL treatment caused a dramatic increase in PP2A activity in the aorta, and administration of the PP2A inhibitor Endothall fully prevented thrombus formation induced by aPL. Having revealed the molecular underpinnings of the disorder, current treatment of APS with anticoagulation, which is often ineffective and fraught with complications, can potentially be replaced by new mechanism-based therapies targeting ApoER2 complex formation or PP2A.

Abstract 142: Characterizing the Mechanisms of Factor VIII Binding to Low-density Lipoprotein Receptor-related Protein 1

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, while effective, is difficult to maintain due to 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. While it is well established that fVIII binds LRP1, the molecular details of this interaction are unclear. The goal of this project is to better define the molecular interactions between fVIII and LRP1, particularly to determine the regions of LRP1 that are responsible for binding and clearance and if activation of fVIII is required to bind LRP1. We mapped the binding of fVIII to the Cluster II and IV regions of LRP1, with higher affinity for cluster IV. This is in contrast to many LRP1 ligands that bind to multiple regions with similar affinity. The canonical model of LDL receptor family member binding involves lysine residues on the ligand docking in acidic pockets on the receptor. We examined the binding of fVIII to LRP1 using surface plasmon resonance and found that increasing ionic strength, which disrupts the charged interactions required between lysine residues and the acidic pockets, did not alter the affinity of fVIII to LRP1. These results suggest that fVIII may bind to LRP1 via a different mode of binding than the canonical model. A recent publication raised the possibility that LRP1-mediated clearance of fVIII may first require its activation. To test this hypothesis, we compared the clearance of wild-type fVIII with a mutant fVIII in which the thrombin cleavage sites were converted to alanine. Both fVIII molecules were cleared from the circulation with a fast and slow phase, with no significant difference between WT and mutant fVIII. These results suggest that activation of fVIII is not essential for clearance in vivo . Together these data reveal important insights into the binding mechanisms between fVIII and LRP1 and provide insight into ways to extend fVIII half-life in vivo and improve prophylactic replacement therapy.

Quercetin stabilizes apolipoprotein E and reduces brain Aβ levels in amyloid model mice

Apolipoprotein E (apoE) is a major cholesterol carrier that regulates lipid homeostasis by mediating lipid transport from one tissue or cell type to another. In the central neural system (CNS), apoE is mainly produced by astrocytes, and transports cholesterol to neurons via apoE receptors, which are members of the low-density lipoprotein receptor family. The APOEε4 gene is a strong genetic risk factor for late-onset sporadic Alzheimer's disease (AD), likely through its strong effect on the accumulation of amyloid-β (Aβ) peptide. ApoE protein levels in cerebrospinal fluid (CSF) and plasma are reduced in APOEε4 carriers and in patients with AD. Furthermore, altered cholesterol levels are also associated with the risk of AD. Aβ accumulation, oligomerization and deposition in the brain are central to the pathogenesis of AD. Mounting evidence demonstrates that apoE and apoE receptors play important roles in these processes. Astrocyte-derived apoE is pivotal for cerebral cholesterol metabolism and clearance of Aβ. Thus, we hypothesized that increased apoE in the brain may be an effective therapeutic strategy for AD. We report here that quercetin can significantly increase apoE levels by inhibiting apoE degradation in immortalized astrocytes. Importantly, we show that oral administration of quercetin significantly increased brain apoE and reduced insoluble Aβ levels in the cortex of 5xFAD amyloid model mice. Our results demonstrate that quercetin increases apoE levels through a novel mechanism and can be explored as a novel class of drug for AD therapy.

Human extravillous trophoblasts bind but do not internalize antiphospholipid antibodies

IntroductionObstetric morbidity in women with antiphospholipid antibodies (aPLs) may reflect the adverse effects of aPLs on placental cells such as extravillous trophoblasts and the syncytiotrophoblast. Antiphospholipid antibodies may affect the syncytiotrophoblast after being internalised by members of the Low-density lipoprotein receptor (LDLR) family and the antigen of aPLs, β2 glycoprotein I. AimThis study aimed to determine whether aPL internalization was a mechanism by which aPLs adversely affect extravillous trophoblasts. Methodof Study: Fluorescently-labelled monoclonal aPLs IIC5 or ID2 were incubated with first trimester extravillous trophoblast outgrowths and visualized by microscopy. The subcellular expression of β2 glycoprotein I and LDLR family members was investigated by live/permeabilised immunocytochemistry. ResultsUnlike the syncytiotrophoblast of anchoring villi, monoclonal aPLs were not internalised by extravillous trophoblasts, which expressed LDLR family members intracellularly. The aPL IIC5 bound to the surface of extravillous trophoblasts in a pattern similar to the extracellular expression of β2 glycoprotein I. ConclusionsThe mechanisms of action of aPLs are different in extravillous trophoblasts and the syncytiotrophoblast. The interaction of aPLs with the extravillous trophoblast surface, which may involve β2 glycoprotein I, is consistent with reports that aPLs trigger intracellular signaling cascades through cell-surface receptors.

Rhinoviruses and Their Receptors: Implications for Allergic Disease.

Human rhinoviruses (RVs) are picornaviruses that can cause a variety of illnesses including the common cold, lower respiratory tract illnesses such as bronchitis and pneumonia, and exacerbations of asthma. RVs are classified into three species, RV-A, B, and C, which include over 160 types. They utilize three major types of cellular membrane glycoproteins to gain entry into the host cell: intercellular adhesion molecule 1 (ICAM-1) (the majority of RV-A and all RV-B), low-density lipoprotein receptor (LDLR) family members (12 RV-A types), and cadherin-related family member 3 (CDHR3) (RV-C). CDHR3 is a member of cadherin superfamily of transmembrane proteins with yet unknown biological function, and there is relatively little information available about the mechanisms of RV-C interaction with CDHR3. A coding single nucleotide polymorphism (rs6967330) in CDHR3 could promote RV-C infections and illnesses in infancy, which could in turn adversely affect the developing lung to increase the risk of asthma. Further studies are needed to determine how RV infections contribute to pathogenesis of asthma and to develop the optimal treatment approach to control asthma exacerbations.

ApoE Receptor 2 Mediation of Trophoblast Dysfunction and Pregnancy Complications Induced by Antiphospholipid Antibodies in Mice.

Pregnancies in women with the antiphospholipid syndrome (APS) are frequently complicated by fetal loss and intrauterine growth restriction (IUGR). How circulating antiphospholipid antibodies (aPL) cause pregnancy complications in APS is poorly understood. We sought to determine whether the low-density lipoprotein receptor family member apolipoprotein E receptor 2 (ApoER2) mediates trophoblast dysfunction and pregnancy complications induced by aPL. Placental and trophoblast ApoER2 expression was evaluated by immunohistochemistry and immunoblotting. Normal human IgG and aPL were purified from healthy individuals and APS patients, respectively. The role of ApoER2 in aPL-induced changes in trophoblast proliferation and migration and in kinase activation was assessed using RNA interference in HTR-8/SVneo cells. The participation of ApoER2 in aPL-induced pregnancy loss and IUGR was evaluated in pregnant ApoER2(+/+) and ApoER2(-/-) mice injected with aPL or normal human IgG. We found that ApoER2 is abundant in human and mouse placental trophoblasts and in multiple trophoblast-derived cell lines, including HTR-8/SVneo cells. ApoER2 and its interaction with the cell surface protein β2 -glycoprotein I were required for aPL-induced inhibition of cultured trophoblast proliferation and migration. In parallel, aPL antagonism of Akt kinase activation by epidermal growth factor in trophoblasts was mediated by ApoER2. Furthermore, in a murine passive-transfer model of pregnancy complications of APS, ApoER2(-/-) mice were protected from both aPL-induced fetal loss and aPL-induced IUGR. ApoER2 plays a major role in the attenuation of trophoblast function by aPL, and the receptor mediates aPL-induced pregnancy complications in vivo in mice. ApoER2-directed interventions can now potentially be developed to combat the pregnancy complications associated with APS.

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.

Multifarious effects of 17-β-estradiol on apolipoprotein E receptors gene expression during osteoblast differentiation <i>in vitro </i>

Apolipoprotein E (ApoE) regulated bone metabolism in mice might mediate uptake of lipid particles into target cells such as osteoblasts via receptor-mediated endocytosis by apoE receptors, which includes the low-density lipoprotein receptor (LDLR) family and heparan sulfate proteoglycans (HSPGs). There is no report regarding the expression of ApoE receptors mRNA induced by estrogen during osteoblast differentiation in vitro. Primary osteoblasts were collected from the calvaria of newborn mice and were subjected to osteoblast mineralization culture with serial concentrations of 17-β-estradiol (E2) in vitro. RNA was isolated at days 0, 5 and 25 of differentiation. Real-time PCR was conducted to analyze apoE receptors mRNA levels. We found that most LDLR family members genes were induced during osteoblast differentiation in vitro. The effect of E2 on apoE receptors gene expression during osteoblast differentiation was multifarious. The most noted members of the LDLR family involved in the maintenance of bone metabolism were LRP5, LRP6, LRP4, and Apoer2. LRP6 was up-regulated, while LRP5, LRP4, and Apoer2 were down-regulated by E2. Given that LRP6 is required for early stages of differentiation, we speculate E2 promotes osteoblast differentiation mainly in the early stage.

Low Density Lipoprotein Receptor Related Proteins as Regulators of Neural Stem and Progenitor Cell Function.

The central nervous system (CNS) is a highly organised structure. Many signalling systems work in concert to ensure that neural stem cells are appropriately directed to generate progenitor cells, which in turn mature into functional cell types including projection neurons, interneurons, astrocytes, and oligodendrocytes. Herein we explore the role of the low density lipoprotein (LDL) receptor family, in particular family members LRP1 and LRP2, in regulating the behaviour of neural stem and progenitor cells during development and adulthood. The ability of LRP1 and LRP2 to bind a diverse and extensive range of ligands, regulate ligand endocytosis, recruit nonreceptor tyrosine kinases for direct signal transduction and signal in conjunction with other receptors, enables them to modulate many crucial neural cell functions.

GRP94 Regulates Circulating Cholesterol Levels through Blockade of PCSK9-Induced LDLR Degradation.

Clearance of circulating low-density lipoprotein cholesterol (LDLc) by hepatic LDL receptors (LDLR) is central for vascular health. Secreted by hepatocytes, PCSK9 induces the degradation of LDLR, resulting in higher plasma LDLc levels. Still, it remains unknown why LDLR and PCSK9 co-exist within the secretory pathway of hepatocytes without leading to complete degradation of LDLR. Herein, we identified the ER-resident GRP94, and more precisely its client-binding C-terminal domain, as a PCSK9-LDLR inhibitory binding protein. Depletion of GRP94 did not affect calcium homeostasis, induce ER stress, nor did it alter PCSK9 processing or its secretion but greatly increased its capacity to induce LDLR degradation. Accordingly, we found that hepatocyte-specific Grp94-deficient mice have higher plasma LDLc levels correlated with ∼ 80% reduction in hepatic LDLR protein levels. Thus, we provide evidence that, in physiological conditions, binding of PCSK9 to GRP94 protects LDLR from degradation likely by preventing early binding of PCSK9 to LDLR within the ER.