Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5) impair retinal angiogenesis in patients with familial exudative vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. The defective retinal vasculature phenotype in human FEVR patients is recapitulated in Lrp5 knockout (Lrp5−/−) mouse with delayed and incomplete development of retinal vessels. In this study we examined gene expression changes in the developing Lrp5−/− mouse retina to gain insight into the molecular mechanisms that underlie the pathology of FEVR in humans. Gene expression levels were assessed with an Illumina microarray on total RNA from Lrp5−/− and WT retinas isolated on postnatal day (P) 8. Regulated genes were confirmed using RT-qPCR analysis. Consistent with a role in vascular development, we identified expression changes in genes involved in cell-cell adhesion, blood vessel morphogenesis and membrane transport in Lrp5−/− retina compared to WT retina. In particular, tight junction protein claudin5 and amino acid transporter slc38a5 are both highly down-regulated in Lrp5−/− retina. Similarly, several Wnt ligands including Wnt7b show decreased expression levels. Plasmalemma vesicle associated protein (plvap), an endothelial permeability marker, in contrast, is up-regulated consistent with increased permeability in Lrp5−/− retinas. Together these data suggest that Lrp5 regulates multiple groups of genes that influence retinal angiogenesis and may contribute to the pathogenesis of FEVR.
LRP1b and the closely related LRP1 are large members of the low-density lipoprotein receptor family. At the protein level LRP1b is 55% identical to LRP1, a multifunctional and developmentally essential receptor with roles in cargo transport and cellular signaling. Somatic LRP1b mutations frequently occur in non-small cell lung cancer and urothelial cancers, suggesting a role in the modulation of cellular growth. In contrast to LRP1, LRP1b-deficient mice develop normally, most likely due to its restricted expression pattern and functional compensation by LRP1 or other receptors. LRP1b is expressed predominantly in the brain, and a differentially spliced form is present in the adrenal gland and in the testis. Despite the presence of a potential furin cleavage site and in contrast to LRP1, immunoblotting for LRP1b reveals the presence of a single 600-kDa polypeptide species. Using a yeast two-hybrid approach, we have identified two intracellular proteins, the postsynaptic density protein 95 and the aryl hydrocarbon receptor-interacting protein, that bind to the intracellular domain of LRP1b. In addition, we have found several potential ligands that bind to the extracellular domain. Analysis of LRP1b knockout mice may provide further insights into the role of LRP1b as a tumor suppressor and into the mechanisms of cancer development.
The low-density lipoprotein receptor–related protein (Lrp)-5 functions as a Wnt coreceptor. Here we show that mice with a targeted disruption of Lrp5 develop a low bone mass phenotype. In vivo and in vitro analyses indicate that this phenotype becomes evident postnatally, and demonstrate that it is secondary to decreased osteoblast proliferation and function in a Cbfa1-independent manner. Lrp5 is expressed in osteoblasts and is required for optimal Wnt signaling in osteoblasts. In addition, Lrp5-deficient mice display persistent embryonic eye vascularization due to a failure of macrophage-induced endothelial cell apoptosis. These results implicate Wnt proteins in the postnatal control of vascular regression and bone formation, two functions affected in many diseases. Moreover, these features recapitulate human osteoporosis-pseudoglioma syndrome, caused by LRP5 inactivation.
low bone mass; blindness; Wnt; osteoblast function; vascular regression
The immunolocalization of the low density lipoprotein receptor-related protein 1 (LRP1) and its ligand alpha 2-Macroglobulin (α2M) was examined in tissues from human donor eyes of normal, diabetic and sickle cell disease subjects. Streptavidin alkaline phosphatase immunohistochemistry was performed with a mouse anti-human LRP1 and rabbit anti-human α2M antibodies. Retinal and choroidal blood vessels were labeled with mouse anti-human CD34 antibody in adjacent tissue sections. Mean scores for immunostaining from the pathological and control eyes were statistically compared.
LRP1 immunoreactivity was very weak to negative in the neural retina of normal subjects except in scattered astrocytes. LRP1 expression in diabetic eyes was detected in the inner limiting membrane (ILM), astrocytes, inner photoreceptor matrix, choriocapillaris and choroidal stroma. The ligand α2M, however, was limited mainly to blood vessel walls, some areas of the inner nuclear layer (INL), photoreceptors, RPE-Bruch’s membrane–choriocapillaris complex, intercapillary septa, and choroidal stroma. In sickle cell eyes, avascular and vascular retina as well as choroidal neovascularization (CNV) were analyzed. In avascular areas, LRP1 immunoreactivity was in innermost retina (presumably ILM, astrocytes, and Muller cells) and INL as well as RPE–Bruch’s membrane–choriocapillaris complex and choroidal stroma. α2M was very weak in avascular peripheral retina compared to vascularized areas and limited to stroma in choroid. In contrast, in areas with CNV, LRP1 immunoreactivity was significantly decreased in overlying retina and in RPE–Bruch’s membrane and choroidal stroma compared to the controls, while α2M was elevated in RPE–Bruch’s membrane near CNV compared to normal areas in sickle cell choroid. The mean scores revealed that LRP1 and α2M in neural retina were significantly elevated in astrocytes and ILM in diabetic eyes (p ≤ 0.05), whereas in sickle cell eyes scores were elevated in ILM and INL (p ≤ 0.05). In addition, α2M immunoreactivity was in photoreceptors in both ischemic retinopathies. In choroid, the patterns of LRP1 and α2M expression were different and not coincident.
This is the first demonstration of the presence of LRP1 and α2M in human proliferative retinopathies. Elevated LRP1 expression in sickle cell neural retina and diabetic inner retina and choroid suggests that LRP1 plays an important role in ischemic neovascular diseases.
α2-Macroglobulin; LRP1; Diabetes Mellitus; Sickle cell disease; Ischemia
Disorders of vascular structure and function play a central role in a wide variety of CNS diseases. Mutations in the Frizzled4 (Fz4) receptor, Lrp5 co-receptor, or Norrin ligand cause retinal hypovascularization, but the role of Norrin/Fz4/Lrp signaling in vascular development has not been defined. Using mouse genetic and cell culture models, we show that loss of Fz4 signaling in endothelial cells causes defective vascular growth, which leads to chronic but reversible silencing of retinal neurons. Loss of Fz4 in all endothelial cells disrupts the blood brain barrier in the cerebellum, while excessive Fz4 signaling disrupts embryonic angiogenesis. Sox17, a transcription factor that is up-regulated by Norrin/Fz4/Lrp signaling, plays a central role in inducing the angiogenic program controlled by Norrin/Fz4/Lrp. These experiments establish a cellular basis for retinal hypovascularization diseases due to insufficient Frizzled signaling, and they suggest a broader role for Frizzled signaling in vascular growth, remodeling, maintenance, and disease.
Wnt7b is a Wnt ligand that has been demonstrated to play critical roles in several developmental processes, including lung airway and vascular development and chorion-allantois fusion during placental development. Wnt signaling involves the binding of Wnt ligands to cell surface receptors of the frizzled family and coreceptors of the LRP5/6 family. However, little is known of the ligand-receptor specificity exhibited by different Wnts, Fzds, and LRPs in Wnt signaling. Expression analysis of Fzds and LRP5/6 in the developing lung and vasculature showed that Fzd1, -4, -7, and -10 and LRP5/6 are expressed in tissue-specific patterns during lung development. Fzd1, -4, and -7 are expressed primarily in the developing lung mesenchyme, and Fzd10 is expressed in airway epithelium. LRP5 and LRP6 are expressed in airway epithelium during lung development, whereas LRP5 but not LRP6 expression is observed in the muscular component of large blood vessels, including the aorta. Cell transfection studies demonstrate that Wnt7b can activate the canonical Wnt pathway but not the noncanonical Wnt pathway in a cell-specific manner. Biochemical analysis demonstrates that Wnt7b can bind to Fzd1 and -10 on the cell surface and cooperatively activate canonical Wnt signaling with these receptors in the presence of LRP5. Together, these data demonstrate that Wnt7b signals through Fzd1 and -10 and LRP5 and implicate these Wnt coreceptors in the regulation of lung airway and vascular development.
The low-density lipoprotein (LDL) receptor-related protein (originally called LRP, but now referred to as LRP1) is a large endocytic receptor that is widely expressed in several tissues. LRP1 is a member of the LDL receptor family that plays diverse roles in various biological processes including lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. Deletion of the LRP1 gene leads to lethality in mice, revealing a critical, but as of yet, undefined role in development. Tissue-specific gene deletion studies reveal an important contribution of LRP1 in the vasculature, central nervous system, in macrophages and in adipocytes. Three important properties of LRP1 dictate its diverse role in physiology: first, its ability to recognize more than thirty distinct ligands; second, its ability to bind a large number of cytoplasmic adaptor proteins via determinants located on its cytoplasmic domain in a phosphorylation-specific manner; and third, its ability to associate with and modulate the activity of other transmembrane receptors such as integrins and receptor tyrosine kinases.
Defective smooth muscle expression of LDL receptor-related protein-1 (Lrp1) increases atherosclerosis in hypercholesterolemic mice. This study explored the importance of smooth muscle Lrp1 expression under normolipidemic conditions.
Methods and Results
Smooth muscle cells isolated from control (smLrp1+/+) and smooth muscle-specific Lrp1 knockout (smLrp1−/−) mice were characterized based on morphology, smooth muscle marker protein expression levels, and growth rates in vitro. Vascular functions were assessed by aortic constrictive response to agonist stimulation in situ and neointimal hyperplasia to carotid arterial injury in vivo. The smLrp1−/− smooth muscle cells displayed reduced α-actin and calponin expression and an accelerated growth rate due to sustained phosphorylation of platelet derived growth factor receptor (PRGFR) and protein kinase B/Akt. Vasoconstrictive response to agonist stimulation was impaired in aortic rings isolated from smLrp1−/− mice. Injury-induced neointimal hyperplasia was significantly increased in smLrp1−/−mice. The increase in neointima was associated with corresponding elevated activation of PDGFR signaling pathway.
Smooth muscle expression of Lrp1 is important in maintaining normal vascular functions under normolipidemic conditions. The absence of Lrp1 expression results in greater smooth muscle cell proliferation, deficient contractile protein expression, impairment of vascular contractility, and promotion of denudation-induced neointimal hyperplasia.
Lipoprotein receptors; Smooth muscle cell phenotype; Vasocontractility; Growth factor receptor signaling
The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor involved in the metabolism of various extracellular ligands including proteinases that play critical roles in tumor invasion. Although several studies have demonstrated an increased expression of LRP1 in cancer cells, its function in tumor development and progression remains largely unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and MMP9 and thereby promotes the migration and invasion of human glioblastoma U87 cells. Knockdown of LRP1 expression greatly decreased U87 cell migration and invasion, which was rescued by forced expression of a functional LRP1 minireceptor. Inhibition of ligand binding to LRP1 by a specific antagonist RAP also led to reduced cancer cell migration and invasion. Because MMPs play critical roles in cancer cell migration and invasion, we examined the expression of several MMPs and found that the expression of functional MMP2 and MMP9 was selectively decreased in LRP1-knockdown cells. More importantly, decreased cell migration and invasion of LRP1-knockdown cells were completely rescued by exogenous expression of MMP2 or MMP9, suggesting that these MMPs are likely downstream targets of LRP1-mediated signaling. We further show that the level of phosphorylated ERK was significantly decreased in LRP1-silenced cells, suggesting that ERK is a potential mediator of LRP1-regulated MMP2 and MMP9 expression in U87 cells. Together, our data strongly suggest that LRP1 promotes glioblastoma cell migration and invasion by regulating the expression and function of MMP2 and MMP9 perhaps via an ERK-dependent signaling pathway.
LRP1; U87 glioblastoma; invasion; MMP2; MMP9
Mutation in the EGFP domain of LDL receptor-related protein 6 (LRP6R611C) is associated with hypercholesterolemia and early-onset atherosclerosis, but the mechanism by which it causes disease is not known.
Cholesterol uptake was examined in cells from LRP6 +/− mice and LRP6R611C mutation carriers. Splenic B cells of LRP6 +/− mice have significantly lower LRP6 expression and LDL uptake than those of the wildtype (WT) littermates. Although similar levels of total LRP6 were found in lymphoblastoid cells (LCLs) of LRP6R611C mutation carriers and those of the unaffected family member, LDL uptake was significantly lower in the mutant cells. Mutant and wildtype receptors show similar affinities for apoB at neutral pH. LRP6 colocalized with LDL and was coimmunoprecipitated with NPC1, an endocytic regulator of LDL trafficking. However, the cellular localization of LRP6 in the mutant cells shifted from cell surface to late endosomes/lysosomes. Plasma membrane expression levels of LRP6R611C was lower compared to WT receptor and declined to a greater extent in LDL-rich medium. Further examinations revealed lower efficacy of apoB dissociation from LRP6R611C compared to WT receptor at an acidic pH. These studies identify LRP6 as a receptor for LDL endocytosis and imply that R611C mutation results in reduced LRP6 membrane expression and decreased LDL clearance. Based on our findings, we conclude that the increased affinity of the mutant receptor for LDL in acidic pH leads to their impaired dissociation in late endosomes.which compromises their recycling to the plasma membrane.
Atherosclerosis; cholesterol; hypercholesterolemia; receptors; genetics
Loss- and gain-of-function mutations in the broadly expressed gene Lrp5 affect bone formation causing osteoporosis and high bone mass, respectively. Although Lrp5 is viewed as a Wnt coreceptor osteoblast-specific disruption of β-Catenin does not affect bone formation. Instead, we show here that Lrp5 inhibits expression of Tph1, the rate-limiting biosynthetic enzyme for serotonin in enterochromaffin cells of the duodenum. Accordingly, decreasing serotonin blood levels normalizes bone formation and bone mass in Lrp5-deficient mice and gut- but not osteoblast-specific Lrp5 inactivation decreases bone formation in a β-Catenin–independent manner. Moreover, gut-specific activation of Lrp5, or inactivation of Tph1, increases bone mass and prevents ovariectomy-induced bone loss. Serotonin acts on osteoblasts through the Htr1b receptor and CREB to inhibit their proliferation. By identifying duodenum-derived serotonin as a hormone inhibiting bone formation in an Lrp5-dependent manner this study broadens our understanding of bone remodeling and suggests novel therapies to increase bone mass.
We have identified a mouse recessive mutation that leads to attenuated and hyperpermeable retinal vessels, recapitulating some pathological features of familial exudative vitreoretinopathy (FEVR) in human patients. DNA sequencing reveals a single nucleotide insertion in the gene encoding the low-density lipoprotein receptor-related protein 5 (LRP5), causing a frame shift and resulting in the replacement of the C-terminal 39 amino acid residues by 20 new amino acids. This change eliminates the last three PPP(S/T)P repeats in the LRP5 cytoplasmic domain that are important for mediating Wnt/β-catenin signaling. Thus, mutant LRP5 protein is probably unable to mediate its downstream signaling. Immunostaining and three-dimensional reconstructions of retinal vasculature confirm attenuated retinal vessels. Ultrastructural data further reveal that some capillaries lack lumen structure in the mutant retina. We have also verified that LRP5 null mice develop similar alterations in the retinal vasculature. This study provides direct evidence that LRP5 is essential for the development of retinal vasculature, and suggests a novel role played by LRP5 in capillary maturation. LRP5 mutant mice can be a useful model to explore the clinical manifestations of FEVR.
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.
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-β2 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-β signaling pathway in macLRP1-/- mice. Further, we observed that LRP1 binds TGF-β2 and macrophages lacking LRP1 accumulate twice as much TGF-β2 in conditioned media. Finally, TNF-α modulation of the TGF-β2 gene in macrophages is attenuated when LRP1 is expressed. Together, the data reveal that LRP1 modulates both the expression and protein levels of TGF-β2 in macrophages.
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-β2 protein levels and to attenuate expression of the TGF-β2 gene resulting in suppression of the TGF-β signaling pathway.
The human skeleton is affected by mutations in Low-density lipoprotein Receptor-related Protein 5 (LRP5). To understand how LRP5 influences bone properties, we generated mice with inducible Lrp5 mutations that cause high bone mass and low bone mass phenotypes in humans. We conditionally-induced Lrp5 mutations in osteocytes and found that bone properties in these mice were comparable to bone properties in mice with inherited mutations. We also conditionally-induced an Lrp5 mutation in cells that contribute to the appendicular skeleton, and not to the axial skeleton, and we observed bone properties were altered in the limbs, and not in the spine. These data indicate that Lrp5 signaling functions locally and suggest increasing LRP5 signaling in mature bone cells as a strategy to treat human low bone mass disorders, such as osteoporosis.
Low-density lipoprotein receptor–related proteins 5 and 6 (Lrp5 and Lrp6) are co-receptors of Wnt ligands and play important roles in Wnt/β-catenin signal transduction. Mice homozygous for a germline deletion of Lrp6 die at birth with several associated defects, while Lrp5-deficient mice are viable. Here we conditionally deleted Lrp5 and/or Lrp6 in the mouse gut (gut−/−) by crossing mice carrying floxed alleles of Lrp5 and Lrp6 to a strain expressing Cre recombinase from the villin promoter (villin-Cre). The changes in morphology, differentiation and Wnt signal transduction were validated using immunohistochemistry and other staining. Consistent with observations in mice carrying a homozygous germline deletion in Lrp5, intestinal development in Lrp5gut−/− mice was normal. In addition, mice homozygous for villin-Cre–induced deletion of Lrp6 (Lrp6gut−/−) were viable with apparently normal intestinal differentiation and function. However, mice homozygous for villin-Cre inactivated alleles of both genes (Lrp5gut−/−;Lrp6gut−/−) died within one day of birth. Analysis of embryonic Lrp5gut−/−;Lrp6gut−/− intestinal epithelium showed a progressive loss of cells, an absence of proliferation, and a premature differentiation of crypt stem/precursor cells; no notable change in differentiation was observed in the embryos lacking either gene alone. Further immunohistochemical studies showed that expression of the Wnt/β-catenin target, cyclin D1, was specifically reduced in the intestinal epithelium of Lrp5gut−/−;Lrp6gut−/− embryos. Our data demonstrate that Lrp5 and Lrp6 play redundant roles in intestinal epithelium development, and that Lrp5/6 might regulate intestinal stem/precursor cell maintenance by regulating Wnt/β-catenin signaling.
Lrp5; Lrp6; Wnt/β-catenin signaling; intestine; epithelium; embryonic development
The canonical wingless and Int1 (Wnt) signaling pathway plays key roles in multiple biologic events. The pathway co-receptor, low density lipoprotein receptor-related protein 5 (LRP5), is involved in the pathogenesis of retinal diseases and has been implicated in glaucoma. We studied whether a disease-associated polymorphism LRP5.Q89R, which is located in the second blade of the first β-propeller domain, directly alters Wnt signaling activity with cell-based assays.
The LRP5.Q89R polymorphism was evaluated by transfection of HEK293T or GTM3 cells with expression vectors. LRP5 expression and interaction with the molecular chaperone mesoderm development (MESD) were determined by western immunoblotting and co-immunoprecipitation analyses. To compare membrane-associated LRP5 proteins, surface proteins were labeled with biotin and pulled down with avidin beads followed by western immunoblotting. TCF-reporter plasmid-based luciferase assays were used to determine whether LRP5.Q89R affects the canonical Wnt signaling, or has altered efficacy to suppression by Dickkopf-1 (DKK-1).
Cell-based assays showed that this polymorphism did not change protein expression, interaction with the molecular chaperone MESD, protein trafficking, Wnt signaling transduction, or its efficacy in DKK1-mediated inhibition.
Our data suggest that this specific polymorphism does not appear to alter the canonical Wnt signaling pathway. Further studies of LRP5 polymorphisms are needed to elucidate their roles in various associated diseases.
Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by regulating the activation of platelet-derived growth factor receptor β (PDGFRβ) in vascular smooth muscle cells (SMCs). Activated PDGFRβ undergoes tyrosine phosphorylation and subsequently interacts with various signaling molecules, including phosphatidylinositol 3-kinase (PI3K), which binds to the phosphorylated tyrosine 739/750 residues in mice, and thus regulates actin polymerization and cell movement.
Methods and Principal Findings
In this study, we found disorganized actin in the form of membrane ruffling and enhanced cell migration in LRP1-deficient (LRP1−/−) SMCs. Marfan syndrome-like phenotypes such as tortuous aortas, disrupted elastic layers and abnormally activated transforming growth factor β (TGFβ) signaling are present in smooth muscle-specific LRP1 knockout (smLRP1−/−) mice. To investigate the role of LRP1-regulated PI3K activation by PDGFRβ in atherogenesis, we generated a strain of smLRP1−/− mice in which tyrosine 739/750 of the PDGFRβ had been mutated to phenylalanines (PDGFRβ F2/F2). Spontaneous atherosclerosis was significantly reduced in the absence of hypercholesterolemia in these mice compared to smLRP1−/− animals that express wild type PDGFR. Normal actin organization was restored and spontaneous SMC migration as well as PDGF-BB-induced chemotaxis was dramatically reduced, despite continued overactivation of TGFβ signaling, as indicated by high levels of nuclear phospho-Smad2.
Conclusions and Significance
Our data suggest that LRP1 regulates actin organization and cell migration by controlling PDGFRβ-dependent activation of PI3K. TGFβ activation alone is not sufficient for the expression of the Marfan-like vascular phenotype. Thus, regulation of PI3 Kinase by PDGFRβ is essential for maintaining vascular integrity, and for the prevention of atherosclerosis as well as Marfan syndrome.
Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC).
LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens.
Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set.
LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.
Ischemic proliferative retinopathy, characterized by pathologic retinal neovascularization, is a major cause of blindness in working age adults and children. Defining the molecular pathways distinguishing pathological neovascularization from normal vessels is critical to controlling these blinding diseases with targeted therapy. Because mutations in Wnt signaling cause defective retinal vasculature in humans with some characteristics of the pathologic vessels in retinopathy, we investigated the potential role of Wnt signaling in pathologic retinal vascular growth in proliferative retinopathy.
Methods and Results
In this study we show that Wnt receptors (Frizzled4 and Lrp5) and activity are significantly increased in pathologic neovascularization in a mouse model of oxygen-induced proliferative retinopathy. Loss of Wnt co-receptor Lrp5 and downstream signaling molecule disheveled2 significantly decreases the formation of pathologic retinal neovascularization in retinopathy. Loss of Lrp5 also affects retinal angiogenesis during development and formation of the blood retinal barrier, which is linked to significant down-regulation of tight junction protein claudin5 (Cln5) in Lrp5−/− vessels. Blocking Cln5 significantly suppresses Wnt-pathway driven endothelial cell sprouting in vitro and developmental and pathologic vascular growth in retinopathy in vivo.
These results demonstrate an important role of Wnt signaling in pathologic vascular development in retinopathy and show a novel function of Cln5 in promoting angiogenesis.
angiogenesis; vessels; retinopathy; Wnt
Deletion of the Mesd gene region blocks gastrulation and mesoderm differentiation in mice. MESD is a chaperone for the Wnt co-receptors: low-density lipoprotein receptor-related protein (LRP) 5 and 6 (LRP5/6). We hypothesized that loss of Wnt signaling is responsible for the polarity defects observed in Mesd deficient embryos. However, because the Mesd deficient embryo is considerably smaller than Lrp5/6 or Wnt3 mutants, we predicted that MESD function extends more broadly to the LRP family of receptors. Consistent with this prediction, we demonstrated that MESD function in vitro was essential for maturation of the β-propeller/EGF domain common to LRPs. To begin to understand the role of MESD in LRP maturation in vivo, we generated a targeted Mesd knockout and verified that loss of Mesd blocks WNT signaling in vivo. Mesd mutants continue to express pluripotency markers, Oct4, Nanog, and Sox2, suggesting that Wnt signaling is essential for differentiation of the epiblast. Moreover, we demonstrated that MESD was essential for the apical localization of the related LRP2 (Megalin/MEG) in the visceral endoderm, resulting in impaired endocytic function. Combined, our results provide evidence that MESD functions as a general LRP chaperone, and suggest that the Mesd phenotype results from both signaling and endocytic defects resulting from mis-folding of multiple LRP receptors.
mesd; lrp; low-density lipoprotein receptor related protein; visceral endoderm; megalin; lrp2; lysosome; chaperone; wnt
Disorders of retinal vascular growth and function are responsible for vision loss in a variety of diseases, including diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and retinal artery or vein occlusion. Over the past decade, a new signaling pathway that controls retinal vascular development has emerged from the study of inherited disorders - in both humans and mice - that are characterized by retinal hypovascularization. This pathway utilizes a glial-derived extracellular ligand, Norrin, that acts on a transmembrane receptor, Frizzled4, a coreceptor, Lrp5, and an auxiliary membrane protein, Tspan12, on the surface of developing endothelial cells. The resulting signal controls a transcriptional program that regulates endothelial growth and maturation. It will be of great interest to determine whether modulating this pathway could represent a therapeutic approach to human retinal vascular disease.
The low density lipoprotein receptor–related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1–deficient MEFs demonstrated increased Rac1 activation compared with LRP-1–expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR−/− MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK− cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR−/− MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal–regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.
urokinase-type plasminogen activator; uPAR; extracellular signal–regulated kinase; cell migration; VLDL receptor
Formation of the neuromuscular junction (NMJ) requires agrin, a factor released from motoneurons, and MuSK, a transmembrane tyrosine kinase that is activated by agrin. However, how signal is transduced from agrin to MuSK remains unclear. Here we report that low-density lipoprotein receptor (LDLR)-related protein (LRP) 4 (LRP4) functions as a co-receptor of agrin. LRP4 is specifically expressed in myotubes and is concentrated at the NMJ. The extracellular domain of LRP4 interacts with neuronal, but not muscle, agrin. Expression of LRP4 enables agrin binding activity and MuSK signaling in cells that otherwise does not respond to agrin. Suppression of LRP4 expression attenuates agrin binding activity, agrin-induced MuSK tyrosine phosphorylation and AChR clustering in muscle cells. LRP4 also interacts with MuSK in a manner that is stimulated by agrin. Finally, we showed that LRP4 becomes tyrosine-phosphorylated in agrin-stimulated muscle cells. These observations identify LRP4 as a functional co-receptor of agrin that is necessary for agrin-induced MuSK signaling and AChR clustering.
Low-density lipoprotein receptor-related protein 2 (LRP2) is a multifunctional cell surface receptor conserved from nematodes to humans. In mammals, it acts as regulator of sonic hedgehog and bone morphogenetic protein pathways in patterning of the embryonic forebrain and as a clearance receptor in the adult kidney. Little is known about activities of this LRP in other phyla. Here, we extend the functional elucidation of LRP2 to zebrafish as model organism of receptor (dys)function. We demonstrate that expression of Lrp2 in embryonic and larval fish recapitulates the patterns seen in mammalian brain and kidney. Furthermore, we studied the consequence of receptor deficiencies in lrp2 and in lrp2b, a homologue unique to fish, using ENU mutagenesis or morpholino knockdown. While receptor-deficient zebrafish suffer from overt renal resorption deficiency, their brain development proceeds normally, suggesting evolutionary conservation of receptor functions in pronephric duct clearance but not in patterning of the teleost forebrain.
megalin; forebrain development; LDL receptor-related proteins; pronephros
Although Lrp5 is known to be an important contributor to the mechanisms regulating bone mass, its precise role remains unclear. The aim of this study was to establish whether mutations in Lrp5 are associated with differences in the growth and/or apoptosis of osteoblast-like cells and their proliferative response to mechanical strain in vitro. Primary osteoblast-like cells were derived from cortical bone of adult mice lacking functional Lrp5 (Lrp5−/−), those heterozygous for the human G171V High Bone Mass (HBM) mutation (LRP5G171V) and their WT littermates (WTLrp5, WTHBM). Osteoblast proliferation over time was significantly higher in cultures of cells from LRP5G171V mice compared to their WTHBM littermates, and lower in Lrp5−/− cells. Cells from female LRP5G171V mice grew more rapidly than those from males, whereas cells from female Lrp5−/− mice grew more slowly than those from males. Apoptosis induced by serum withdrawal was significantly higher in cultures from Lrp5−/− mice than in those from WTHBM or LRP5G171V mice. Exposure to a single short period of dynamic mechanical strain was associated with a significant increase in cell number but this response was unaffected by genotype which also did not change the ‘threshold’ at which cells responded to strain. In conclusion, the data presented here suggest that Lrp5 loss and gain of function mutations result in cell-autonomous alterations in osteoblast proliferation and apoptosis but do not alter the proliferative response of osteoblasts to mechanical strain in vitro.