ARTICLE |
Correspondence to: Christopher P. Austin, Dept. of Pharmacology, Merck Research Laboratories, WP26A-3000, PO Box 4, Sumneytown Pike, West Point, PA 19486. E-mail: christopher_austin@merck.com
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Summary |
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LRP5 is a novel member of the low-density lipoprotein receptor family that is genetically associated with Type 1 diabetes. As a start to defining the normal function of LRP5 and to generate testable hypotheses of its potential role in Type 1 diabetes pathogenesis, we carried out an extensive expression analysis of this gene at the mRNA and protein levels in normal human, monkey, and mouse, as well as in non-obese diabetic (NOD) mice at several stages of diabetes development. In all species, expression of LRP5 was found in four functionally important cell types: the distributed mononuclear phagocyte system, the islets of Langerhans, vitamin A-metabolizing cells, and CNS neurons. Given the critical role of macrophages in the onset and progression of islet cell destruction in Type 1 diabetes and the hypothesized role of retinoids as modifiers of diabetes progression, these findings suggest that LRP5 may confer Type 1 diabetes risk by altering the normal functioning of one or more of these regulatory systems. Specifically, given that the LRP5 polymorphisms associated with diabetes are in the promoter region of the gene, alterations in LRP5 expression may be responsible for diabetes susceptibility and therefore may be potential targets for therapeutic intervention. (J Histochem Cytochem 48:13571368, 2000)
Key Words: Type I diabetes, low-density lipoprotein, LRP5, NOD mice, macrophages, retinoids
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Introduction |
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TYPE I DIABETES (previously called insulin-dependent diabetes mellitus, or IDDM) is a common disease characterized by autoimmune destruction of the insulin-producing ß-cells of the pancreas. The disease develops as a consequence of the interaction of environmental and genetic risk factors (
The lipoprotein receptor family is made up of a rapidly expanding number of structurally related proteins which serve a variety of functions in lipid metabolism and signal transduction (2-macroglobulin, and has been shown to bind over 25 other ligands. LRP1 therefore appears to be a general scavenger receptor (
To begin to understand the biology and function of LRP5, we have examined in detail its expression in non-diabetic human and monkey tissues as well as in non-diabetic mice and in the Type I diabetes model, the non-obese diabetic (NOD) mouse. We show that, in all species, LRP5 is expressed in the distributed phagocytic system, in the pancreatic islets, in retinoid-storing cells, and in neurons of the CNS. These patterns suggest that LRP5 may play a role in several physiologically important cell signaling systems and suggest several potential pathogenic mechanisms by which LRP5 may contribute to Type I diabetes susceptibility.
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Materials and Methods |
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Tissues
Human tissues were obtained from the National Disease Research Interchange (NDRI; Philadelphia, PA). Human thymoma samples were a generous gift of Dr. I. Roxanis (John Radcliffe Hospital; Oxford, UK). African Green monkey (Chlorocebus aethiops) and non-diabetic SwissWebster mouse tissues were obtained though Laboratory Animal Resources at Merck Research Laboratories. NOD mice (
Antibodies
Anti-human Antibody.
Peptide SYRHLRPPPPSPCTDSS from the C-terminus of LRP5 was synthesized by Research Genetics (Huntsville, AL) as an eight-branch multiple antigenic peptide (MAP) with a molecular weight of 16,008. Antibody to the peptide was produced in two rabbits according to established protocols by Cocalico Biologicals (Reamstown, PA). The antibody was used either directly in crude serum or after affinity-purification. Affinity-purification was performed on a 1 ml BioRad Affi-Gel 15 column (Hercules, CA) to which 2 mg of the MAP peptide was bound covalently. The antibody was eluted with 100 mM Glycine-HCl, pH 2.5, and the eluant was neutralized with 1 M phosphate, pH 8.0. The affinity purification was monitored by ELISA.
Anti-mouse Antibody. A polypeptide corresponding to amino acids 281533 of the extracellular domain of mouse LRP5 was produced as follows: PCR primers 5'-AAGCTCAGCTTCATCCACCG-3' and 5'-GAGCTCCCGTCTATGTTGATCACCTCG-3' were used to amplify a 965-nucleotide product using plasmid DNA containing the full-length LRP5 cDNA as the template. The PCR fragment was digested with the restriction endonucleases Nco I and Sac I and subcloned into the NcoI and SacI sites of pET-32b (Novagen; Madison, WI). The plasmid construct was transformed into AD494(DE3)pLysS (Novagen). The resulting thioredoxin:LRP5 fusion protein was expressed and purified using a His-Bind Resin (Novagen) according to the manufacturer's instructions. Imidizole elution from the His-Bind resin yielded approximately 0.6 mg of purified protein per 100 ml bacterial culture. The purified protein was used to immunize two rabbits from CoVance (Denver, PA).
Recombinant Adenoviruses
Adenoviruses containing the mouse and human LRP5 genes were constructed to provide cells expressing high levels of recombinant protein. The cDNAs for hLRP5 and mRP5 were fused to the human CMV promoter and the bovine growth hormone polyadenylation signal and cloned into the adenovirus shuttle vector pE1sp1A (
Western Blotting Analysis
Proteins were separated by SDS-PAGE on a 10% acrylamide Tris-glycine gel (Novex; San Diego CA). Proteins were transferred to a PVDF nylon membrane by electrophoresis. The membrane was blocked for 1 hr with 5% milk in Tris-buffered (pH 7.4) saline containing 0.1% Triton X-100. Primary antibody incubations were performed using a 1:500 dilution of crude serum for 1 hr at room temperature (RT). A goat anti-rabbit horseradish peroxidase conjugate (Amersham; Piscataway NJ) was used as the second antibody. Bands were detected using ECL reagents (Amersham) and exposing the blot to Hyperfilm MP (Amersham).
Immunohistochemistry
Cryostat sections of 8-week-old SwissWebster mouse pancreases and NOD mouse were thaw-mounted on Superfrost Plus glass slides (Fisher Scientific; Pittsburgh, PA) and fixed for 15 min in 4% paraformaldehyde. Sections were then blocked with 10% normal donkey serum in PBS and incubated with anti-LRP5 AB #884 for 2 hr at RT. Bound antibody was detected with Texas Red-conjugated donkey anti-rabbit (Jackson Immunoresearch; Avondale, PA). Sections were counterstained with 4'6-diamidino-2-phenylindole (DAPI; Molecular Probes, Eugene, OR) to visualize nuclei. LRP5-positive sections were then rinsed, blocked again with 10% normal donkey serum, and then incubated with either guinea pig anti-glucagon antisera (Linco Research; St. Charles, MO), or guinea pig anti-insulin (Dako; Carpinteria, CA). Signal was visualized with FITC-conjugated anti-guinea pig IgG (Jackson Immunoresearch). Fluorescent images were captured using a CCD Spot Cam (Phase 3 Imaging Systems; Milford, MA), recompiled in Adobe Photoshop (San Jose, CA), and co-localization was indicated by yellow fluorescent signal. Sections of mouse spleen and thymus were similarly prepared, stained, and analyzed using the same double immunofluorescence staining technique with anti-LRP5 AB #884 and the anti-pan-macrophage marker BM8 (BMA Biomedicals; Augst, Switzerland).
Fresh frozen human liver specimens (NDRI) were cryosectioned at 8 µm and fixed in 4% paraformaldehyde in PBS for 1 hr. Antigen retrieval was performed on the sections by immersion in AR-10 antigen retrieval solution (Biogenex; San Ramon, CA) and pulse-microwaved to 100C for 10 min. Sections were rinsed, blocked in 10% normal donkey serum in PBS, and stained with a mixture of anti-LRP5 antibody #171 and either macrophage or dendritic marker clones RFD7 (Serotec; Oxford, UK), RFD1 (Serotec), or 19.2 mannose receptor (Pharmingen; San Diego, CA), for 18 hr at 4C in PBS with 1% normal donkey serum. Secondary antibody was FITC- or Texas Red-conjugated donkey anti-mouse or donkey anti-rabbit (Jackson Immunoresearch).
In Situ Hybridization
The plasmid Bluescript SK+ containing a 181-bp cDNA insert coding for LRP5 was linearized with either HindIII or BamHI (Life Technologies; Lexington, KY) to create template DNA. T7 and T3 polymerases were used with Biotin RNA Labeling Mix (Boehringer Mannheim; Mannheim, Germany) to in vitro-transcribe antisense and sense riboprobes, respectively. In situ probes specific for mouse LRP5 were prepared in the same manner, linearizing the Bluescript plasmid containing a 178-bp specific fragment for mouse LRP5. All chromogenic in situ hybridization was carried out as previously described (
Combined In Situ Hybridization/Immunohistochemistry
Immunohistochemistry was performed sequentially on sections that showed optimal fluorescent signal for LRP5 mRNA. After in situ hybridization, sections were rinsed in PBS, blocked in 10% normal donkey serum, and incubated with appropriate profiling antibody (see Table 1) according to the manufacturer's recommended conditions. Antibody binding was detected with either the appropriate FITC-conjugated antibody (Jackson Immunoresearch) or FITC-conjugated streptavidin (Vector Labs). Sections were coverslipped with Prolong anti-fade mounting medium (Molecular Probes). Images were digitally acquired and reassembled using a Nikon E1000 epifluorescence microscope and a Spot Cam CCD camera (Phase 3 Imaging Systems).
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Results |
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Characterization of Anti-LRP5 Antibodies
Antibodies directed against either the C-terminal 17 amino acids of human LRP5 (AS171), or a 253-amino-acid portion of the extracellular domain of mouse LRP5 (AS884), were characterized by Western blotting analysis (Fig 1). Cell membranes were prepared from HEK-293 cells infected with an adenovirus containing either the human LRP5 cDNA, the mouse Lrp5 cDNA, or a control insert. The mature LRP5 protein has a predicted molecular weight of 176,666. A band of approximately MW 175,000 is detected by the AS884 antibody in membranes prepared from cells infected with the mouse LRP5 cDNA but not from cells infected with either the human LRP5 cDNA or a control adenovirus. Therefore, it appears that the AS884 antibody is specific for mouse LRP5. In contrast, AS171 antibody recognizes a band of approximately 175 kD in both human and mouse and therefore is not selective for the species of the receptor. This result is not unanticipated because there is only a single amino acid difference between mouse and human in the 17-amino-acid peptide used for immunization.
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LRP5 Is Expressed in Pancreatic Islets
Because LRP5 was cloned as a susceptibility gene for Type I diabetes (
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In SwissWebster and C57BL/10SnJ mice, pancreatic LRP5 expression was also found mainly in the islets but, in contrast to the human and monkey pancreas, immunolocalization studies demonstrated the LRP5-expressing cells in the mouse to be glucagon-expressing -cells (Fig 2a2c). In the 1-month-old NOD mouse, expression of LRP5 was indistinguishable from that in non-diabetic SwissWebster mice, showing localization to the
-cells. Fig 2h shows the typical location of the
-cells in the periphery of the islet. In 3-month-old NOD mice, there was severe leukocytic infiltration of the islets characteristic of NOD insulitis. No insulin immunoreactivity was seen in these islets, indicating that the expected ß-cell destruction had indeed occurred. In these islets, LRP5-expressing cells were still present and still co-expressed glucagon (Fig 2i). No LRP5 expression was seen in the cells infiltrating these NOD islets, although isolated LRP5-positive macrophage-like cells similar to those seen in the human and monkey pancreas were also seen in the mouse pancreatic parenchyma (Fig 2m). Immunohistochemical analysis revealed that these cells were immunoreactive for the pan-macrophage marker BM-8 (Table 1; and data not shown).
LRP5 Is Expressed in Macrophages of Human and Mouse Spleen and Thymus, Human Liver, and in a Macrophage Cell Line
Because LRP5 has been implicated in an autoimmune disease (Type 1 diabetes) and expression was seen in macrophages of the pancreas, LRP5 expression was examined in other tissues with larger populations of macrophages. In human and mouse spleen, LRP5-expressing cells were seen in the non-filtering areas of the red pulp, the location characteristic of macrophages (
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Renal Tubules Express LRP5
Northern blotting analysis had shown that LRP5 is most highly expressed in the liver and kidney (
Vitamin A-storing Stellate Cells of the Liver Express LRP5
Given the prominent LRP5 expression in the liver by Northern blotting (
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LRP5 Is Expressed in the Retinal Pigmented Epithelium
Macrophages of the pancreas, spleen, and thymus, the THP.1 macrophage cell line, Kupffer cells of the liver, and hepatic stellate cells all express LRP5, and all perform phagocytic functions. Furthermore, the stellate cells of the liver are the main site of uptake and storage of vitamin A. We therefore asked if the other major phagocytic cell type that metabolizes and stores vitamin A, the cells of the retinal pigmented epithelium (RPE), also express LRP5. In situ hybridization on Rhesus monkey (Fig 4i) and SwissWebster mouse retina (not shown) demonstrated strong LRP5 expression in the RPE of both species. In addition, there was weaker expression in the inner segments of the photoreceptor layer (Fig 4i), which is the location of the retinal rods and cones, which utilize vitamin A-derived retinoids as their visual pigment.
LRP5 Is Expressed in CNS Neurons
In addition to its expression in the RPE and photoreceptors, LRP5 was seen to be expressed in the ganglion cells of the retina (Fig 4i). These are the projection neurons of the retina, which share many properties with other CNS neurons (
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Discussion |
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LRP5, a novel member of the LDL receptor superfamily, was originally cloned on the basis of its genetic association with Type I diabetes in humans (
In the distributed phagocytic system, LRP5 expression was observed in tissue macrophages of the pancreas, spleen, and thymus, the macrophage cell line THP.1, the Kupffer cells of the liver, and the pigmented epithelium of the retina. Macrophages and dendritic cells are the first cells to infiltrate the islets of NOD mice (
High-level LRP5 expression was seen in pancreatic islets, the target of immune destruction in Type I diabetes. Islet expression was seen in all species examined but, intriguingly, the islet cell type that expresses LRP5 was seen to differ between primates and rodents. In human and African green monkey pancreas, LRP5 was expressed in insulin-expressing ß-cells, the cells destroyed in Type I diabetes. In contrast, in wild-type SwissWebster mice and in NOD mice at various stages of diabetes development, LRP5 was clearly expressed in the glucagon-expressing -cells. Consistent with this finding, the number of LRP5-expressing cells was maintained at a normal level even in fully diabetic NOD mice. The reason for this species difference is not clear, but it suggests a difference in the role of LRP5 in the pathogenesis of Type I diabetes in the two species. The fact that ß-cells are normally antigen-presenting, whereas
-cells are not, supports this contention (
Several cell types that transport, store, and metabolize retinoids expressed LRP5, including the stellate (Ito) cells of the liver, the tubule epithelium of the kidney, and the pigmented epithelium and photoreceptors of the retina. Vitamin A has been implicated in the development and/or progression of autoimmune diabetes in the BB rat (
LRP5 expression was detected in CNS neurons, and several data suggest the possibility of a role for LRP5 in Alzheimer's disease pathogenesis. In situ hybridization studies showed that in human, monkey, and mouse, LRP5 is highly expressed in the cerebral cortex, hippocampus, hypothalamus, and retina, all of which are preferentially affected in Alzheimer's disease. Alzheimer's disease has been genetically associated with the LRP1 ligands 2-macroglobulin and APOE. APOE has been reported to also bind LRP5 (
Comparison of LRP5 expression with that of other members of the LRP gene family reveals several similarities that suggest further clues to the function of LRP5 and other members of this gene family. Cellular localization data are available for only four of the eight currently known LRPs. LRP1, like LRP5, is expressed selectively in macrophages, CNS neurons, hepatocytes, and smooth muscle cells (2-macroglobulin, tissue and urinary plasminogen activator, lipoproteins, including APOE-enriched lipoproteins and lipoprotein lipase, and extracellular matrix proteins, such as thrombospondin (
Our knowledge of the biology of the LRPs is certainly incomplete, but the data currently suggest that this gene family is important in multiple regulatory networks, including lipoprotein metabolism, immune regulation, and vitamin homeostasis. It is likely that the LRPs, already genetically associated with Type 1 diabetes and with Alzheimer's disease (
Received for publication June 9, 2000; accepted June 14, 2000.
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