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Cited by 1,086Bone Metabolism and DiseasesWnt/β-catenin signaling in development and cancerBone health and osteoporosis research

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Cbfa1-independent decrease in osteoblast proliferation, osteopenia, and persistent embryonic eye vascularization in mice deficient in Lrp5, a Wnt coreceptor

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Kato, Masaki, Patel, Millan S., Levasseur, Regis, Lobov, Ivan, Chang, Benny H.-J., Glass, Donald A., Hartmann, Christine, Li, Lan, Hwang, Tae-Ho, Brayton, Cory F., Lang, Richard A., Karsenty, Gerard, Chan, Lawrence·2002·The Journal of Cell Biology
DOI 10.1083/jcb.200201089PMC PMC2199263
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Abstract

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.

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Figure 1. Targeted disruption of Lrp5. (A) Structure of the Lrp5 protein and location of targeted disruption of Lrp5 (vertical arrow). (B) Northern blot analysis showing a broad pattern of Lrp5 expression. Gapdh was used as a control for RNA integrity. (C) Northern blot analysis showing no Lrp5 transcript in Lrp52/2 animals using a 39 probe (left) and the presence of a truncated transcript when using a 59 probe (right).
Figure 1. Targeted disruption of Lrp5. (A) Structure of the Lrp5 protein and location of targeted disruption of Lrp5 (vertical arrow). (B) Northern blot analysis showing a broad pattern of Lrp5 expression. Gapdh was used as a control for RNA integrity. (C) Northern blot analysis showing no Lrp5 transcript in Lrp52/2 animals using a 39 probe (left) and the presence of a truncated transcript when using a 59 probe (right).CC BY-NC-SA · PubMed Central
Figure 2. Lrp5 expression in osteoblasts and ocular macrophages. (A–C) Lrp5 expression in osteoblasts and bone lining cells in the primary spongiosa of the femur in wild-type mice. (A) Hoechst nuclear staining is shown in blue; (B) Anti-Lrp5 immunoreactivity in red. (C) A merge of the red and blue channels. (D) DIC image of distal femur from wild-type with a box showing the region examined in A–C. (E–G) Lrp5 expression in the primary spongiosa of the femur in Lrp5 − / − mice. (E) Hoechst nuclear staining. (F) Anti-Lrp5 immunoreactivity. (G) Merge. (H–M) Whole-mount preparations of hyaloid vessels from wild-type (H–J) and Lrp5 − / − (K–M) mice. (H and K) Immunoreactivity for Lrp5. (I and L) Immunoreactivity with the macrophage-specific antibody F4/80, both with Hoechst nuclear staining. (J and M) Merge of the red, blue, and green channels; yellow cells indicate that macrophages are positive for Lrp5.
Figure 2. Lrp5 expression in osteoblasts and ocular macrophages. (A–C) Lrp5 expression in osteoblasts and bone lining cells in the primary spongiosa of the femur in wild-type mice. (A) Hoechst nuclear staining is shown in blue; (B) Anti-Lrp5 immunoreactivity in red. (C) A merge of the red and blue channels. (D) DIC image of distal femur from wild-type with a box showing the region examined in A–C. (E–G) Lrp5 expression in the primary spongiosa of the femur in Lrp5 − / − mice. (E) Hoechst nuclear staining. (F) Anti-Lrp5 immunoreactivity. (G) Merge. (H–M) Whole-mount preparations of hyaloid vessels from wild-type (H–J) and Lrp5 − / − (K–M) mice. (H and K) Immunoreactivity for Lrp5. (I and L) Immunoreactivity with the macrophage-specific antibody F4/80, both with Hoechst nuclear staining. (J and M) Merge of the red, blue, and green channels; yellow cells indicate that macrophages are positive for Lrp5.CC BY-NC-SA · PubMed Central
Figure 3. Low bone mass in Lrp5 − / − mice. (A) Radiograph of the whole body of 2 mo-old wild-type and Lrp5 − / − mice showing decreased lucency of the entire Lrp5 − / − skeleton. (B) Histological analysis of vertebrae from 2-mo-old wild-type and Lrp5 − / − mice showing low bone mass in the mutant mice. (C) Histological analysis of vertebrae from 2-wk-old wild-type and Lrp5 − / − mice showing a decreased number of trabeculae in the primary spongiosa of Lrp5 − / − mice. (D) Lrp5 − / − mice have a lower bone mass throughout life. Green triangle indicates a significantly lower bone mass in Lrp5 +/− mice. Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
Figure 3. Low bone mass in Lrp5 − / − mice. (A) Radiograph of the whole body of 2 mo-old wild-type and Lrp5 − / − mice showing decreased lucency of the entire Lrp5 − / − skeleton. (B) Histological analysis of vertebrae from 2-mo-old wild-type and Lrp5 − / − mice showing low bone mass in the mutant mice. (C) Histological analysis of vertebrae from 2-wk-old wild-type and Lrp5 − / − mice showing a decreased number of trabeculae in the primary spongiosa of Lrp5 − / − mice. (D) Lrp5 − / − mice have a lower bone mass throughout life. Green triangle indicates a significantly lower bone mass in Lrp5 +/− mice. Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.CC BY-NC-SA · PubMed Central
Figure 4. Functional defect in Lrp5 − / − osteoblasts. (A) Calcein double labeling in 6-mo-old wild-type and Lrp5 − / − mice. The distance between the two labels (double-headed arrows) represents the MAR which is decreased in Lrp5 − / − mice. As a result, the BFR is significantly lower in Lrp5 − / − mice. (B) Decreased osteoblast number per bone area in the primary (left) and secondary (right) spongiosa of Lrp5 − / − mice compared with wild-type. (C) Delayed mineralization in primary Lrp5 − / − osteoblast cultures compared with wild-type cultures at day 10 of mineralization. (D) Normal tibial growth plate in 2-mo-old Lrp5 − / − mice (von Kossa stain). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
Figure 4. Functional defect in Lrp5 − / − osteoblasts. (A) Calcein double labeling in 6-mo-old wild-type and Lrp5 − / − mice. The distance between the two labels (double-headed arrows) represents the MAR which is decreased in Lrp5 − / − mice. As a result, the BFR is significantly lower in Lrp5 − / − mice. (B) Decreased osteoblast number per bone area in the primary (left) and secondary (right) spongiosa of Lrp5 − / − mice compared with wild-type. (C) Delayed mineralization in primary Lrp5 − / − osteoblast cultures compared with wild-type cultures at day 10 of mineralization. (D) Normal tibial growth plate in 2-mo-old Lrp5 − / − mice (von Kossa stain). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.CC BY-NC-SA · PubMed Central
Figure 5. Postnatal onset of the bone phenotype in Lrp5 − / − mice. (A) Subtle delay of fifth middle phalanx ossification in newborn Lrp5 − / − mice. (B) Delayed ossification of metacarpals. (C) Carpals (bracket). (D) Humeri in 4-d-old Lrp5 − / − mice. Arrows in A, B, and D indicate ossification centers. A subtle delay was also evident in 44% (8/18) of heterozygote mice (D, middle). (E) Sections of calvaria from 4-d-old mice showing a decreased number of BrdU-positive cells (arrowhead) in Lrp5 − / − mice compared with wild-type littermates. Double-headed arrows indicate the greater thickness of wild-type calvarium. (F) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in wild-type and Lrp5 − / − mice (n = 8 per genotype). (G) Quantification of alkaline phosphatase positive stromal cell progenitors (CFU-F) in the bone marrow of wild-type and Lrp5 − / − mice (n = 9 mice per genotype). (H) Quantification of apoptotic cells by TUNEL assay in wild-type and Lrp5 − / − mice (n = 5 per genotype). (I) Northern blot analysis of Cbfa1 expression in wild-type and Lrp5 − / − calvaria, long bones and primary osteoblasts. (J) Northern blot analysis of Osteocalcin expression in wild-type and Lrp5 − / − calvaria and long bones. (I and J) Gapdh expression level was used as a loading control; results are representative of three samples (n = 3 mice per sample). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.
Figure 5. Postnatal onset of the bone phenotype in Lrp5 − / − mice. (A) Subtle delay of fifth middle phalanx ossification in newborn Lrp5 − / − mice. (B) Delayed ossification of metacarpals. (C) Carpals (bracket). (D) Humeri in 4-d-old Lrp5 − / − mice. Arrows in A, B, and D indicate ossification centers. A subtle delay was also evident in 44% (8/18) of heterozygote mice (D, middle). (E) Sections of calvaria from 4-d-old mice showing a decreased number of BrdU-positive cells (arrowhead) in Lrp5 − / − mice compared with wild-type littermates. Double-headed arrows indicate the greater thickness of wild-type calvarium. (F) Osteoblast mitotic index (percentage of BrdU-positive cells per total cell number) in wild-type and Lrp5 − / − mice (n = 8 per genotype). (G) Quantification of alkaline phosphatase positive stromal cell progenitors (CFU-F) in the bone marrow of wild-type and Lrp5 − / − mice (n = 9 mice per genotype). (H) Quantification of apoptotic cells by TUNEL assay in wild-type and Lrp5 − / − mice (n = 5 per genotype). (I) Northern blot analysis of Cbfa1 expression in wild-type and Lrp5 − / − calvaria, long bones and primary osteoblasts. (J) Northern blot analysis of Osteocalcin expression in wild-type and Lrp5 − / − calvaria and long bones. (I and J) Gapdh expression level was used as a loading control; results are representative of three samples (n = 3 mice per sample). Asterisks indicate a statistically significant difference between two groups of mice (P < 0.05). Error bars represent SD.CC BY-NC-SA · PubMed Central
Figure 6. LRP5-dependent activation of gene expression by Wnt proteins. (A) Wnt1 activates Lef1-dependent gene expression in wild-type but poorly in Lrp5 − / − primary osteoblasts. (B) Cotransfection of LRP5 in Lrp5 − / − osteoblasts allows Wnt1 to activate Lef1-dependent gene expression to wild-type levels. (C) Cotransfection of LRP5 with Wnt3a-conditioned medium or an expression vector for Wnt4 in wild-type (white bars) and Lrp5 − / − primary osteoblasts (black bars). (D) Western blot analysis to confirm expression of transfected plasmids and to demonstrate the ability of Wnt1-LRP5 cotransfection to stimulate cytoplasmic β-catenin accumulation. (E) Direct binding of LRP5 to Wnt1 or Wnt4. COS-7 cells were transfected with Flag-LRP5 and HA-Wnt1 or HA-Wnt4 as indicated. LRP5 or Wnts were immunoprecipitated from the cell lysate using anti-Flag or anti-HA antibody, subjected to SDS-PAGE and immunoblotted with anti-HA and anti-Flag antibody, respectively.
Figure 6. LRP5-dependent activation of gene expression by Wnt proteins. (A) Wnt1 activates Lef1-dependent gene expression in wild-type but poorly in Lrp5 − / − primary osteoblasts. (B) Cotransfection of LRP5 in Lrp5 − / − osteoblasts allows Wnt1 to activate Lef1-dependent gene expression to wild-type levels. (C) Cotransfection of LRP5 with Wnt3a-conditioned medium or an expression vector for Wnt4 in wild-type (white bars) and Lrp5 − / − primary osteoblasts (black bars). (D) Western blot analysis to confirm expression of transfected plasmids and to demonstrate the ability of Wnt1-LRP5 cotransfection to stimulate cytoplasmic β-catenin accumulation. (E) Direct binding of LRP5 to Wnt1 or Wnt4. COS-7 cells were transfected with Flag-LRP5 and HA-Wnt1 or HA-Wnt4 as indicated. LRP5 or Wnts were immunoprecipitated from the cell lysate using anti-Flag or anti-HA antibody, subjected to SDS-PAGE and immunoblotted with anti-HA and anti-Flag antibody, respectively.CC BY-NC-SA · PubMed Central
Figure 7. Expression of Wnts and Wnt signaling molecules in bone. (A) Wnt1 is highly expressed in calvaria compared with day 11 (E11) embryos or primary osteoblasts (24 cycle RT-PCR, top). Wnt1 expression is not present in proliferating (D0) Lrp5 − / − osteoblasts (30 cycle RT-PCR, middle). (B) Wnt4, Wnt14, Frizzled2, and Frizzled6 expression in calvaria and primary osteoblasts (24 cycle RT-PCR). (C) Decreased expression of Lef1, but not Tcf4, in Lrp5 − / − osteoblasts determined by RNase protection assay.
Figure 7. Expression of Wnts and Wnt signaling molecules in bone. (A) Wnt1 is highly expressed in calvaria compared with day 11 (E11) embryos or primary osteoblasts (24 cycle RT-PCR, top). Wnt1 expression is not present in proliferating (D0) Lrp5 − / − osteoblasts (30 cycle RT-PCR, middle). (B) Wnt4, Wnt14, Frizzled2, and Frizzled6 expression in calvaria and primary osteoblasts (24 cycle RT-PCR). (C) Decreased expression of Lef1, but not Tcf4, in Lrp5 − / − osteoblasts determined by RNase protection assay.CC BY-NC-SA · PubMed Central
Figure 8. Regression of hyaloid vessels is defective in Lrp5−/− mice. (A) Schematic indicating the location of the temporary ocular capillary networks including the hyaloid vessels (vasa hyaloidia propria), the TVL and PM. (B) Quantification of the number of hyaloid vessels during a developmental timecourse for wild-type (red line) and Lrp5 − / − mice (green line). (C) Quantification of the apoptotic index (apoptotic capillary segments per total capillary segments) within the hyaloid vessels during a developmental time course for wild-type (red line) and Lrp5 − / − mice (green line). Whole-mount preparations of hyaloid vessels for wild-type (D–F) and Lrp5 − / − mice (G–I). Hyaloid vessel preparations are shown as pairs of brightfield and fluorescence images. Brightfield images are shown in the upper panel of each pair. In the lower panel, DAPI labeling of nuclei is shown in blue and apoptotic cell detection in red. The normal process of regression in wild-type hyaloid vessels is apparent (D to F) as is the lack of regression and a lower number of apoptotic capillary segments in Lrp5 − / − hyaloid vessels (G to I). D–I are at 50× magnification.
Figure 8. Regression of hyaloid vessels is defective in Lrp5−/− mice. (A) Schematic indicating the location of the temporary ocular capillary networks including the hyaloid vessels (vasa hyaloidia propria), the TVL and PM. (B) Quantification of the number of hyaloid vessels during a developmental timecourse for wild-type (red line) and Lrp5 − / − mice (green line). (C) Quantification of the apoptotic index (apoptotic capillary segments per total capillary segments) within the hyaloid vessels during a developmental time course for wild-type (red line) and Lrp5 − / − mice (green line). Whole-mount preparations of hyaloid vessels for wild-type (D–F) and Lrp5 − / − mice (G–I). Hyaloid vessel preparations are shown as pairs of brightfield and fluorescence images. Brightfield images are shown in the upper panel of each pair. In the lower panel, DAPI labeling of nuclei is shown in blue and apoptotic cell detection in red. The normal process of regression in wild-type hyaloid vessels is apparent (D to F) as is the lack of regression and a lower number of apoptotic capillary segments in Lrp5 − / − hyaloid vessels (G to I). D–I are at 50× magnification.CC BY-NC-SA · PubMed Central

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  1. 1996, Am. J. Hum. Genet., 59, 146
  2. 2001, Curr. Opin. Genet. Dev., 5, 543
  3. 1993, Osteoporos. Int., 1, S54
  4. 1987, J. Bone Miner. Res., 6, 595
  5. 1998, Nature., 95, 707
  6. 1998, Cell., 97, 707
  7. 1986, Clin. Genet., 2, 185
  8. 1999, J. Mol. Med., 77, 306, 10.1007/s001090050356 · DOI

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Metadata

Authors
Kato, M., Patel, M. S., Levasseur, R., Lobov, I., Chang, B. H.-J., Glass, D. A., Hartmann, C., Li, L., Hwang, T.-H., Brayton, C. F., Lang, R. A., Karsenty, G., & Chan, L.
Affiliations
  • Baylor College of Medicine (US)
  • 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030
  • University of Cincinnati (US)
  • Harvard University (US)
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2002-04-15
Volume
157
Issue
2
Pages
303-314
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.200201089
PMC
PMC2199263
OpenAlex
W2116610925
Cited by
1,086
References
46
Citations by year
2026: 8 · 2025: 10 · 2024: 18 · 2023: 13 · 2022: 21 · 2021: 24 · 2020: 33 · 2019: 27 · 2018: 26 · 2017: 40 · 2016: 27 · 2015: 39 · 2014: 56 · 2013: 65 · 2012: 61
FWCI
22.08
Type
journal article
Language
en
Open access
Locations
  • The Journal of Cell Biology (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Funders
  • March of Dimes Foundation
  • Universiteit Utrecht
  • National Institutes of Health (5r01dk058882-03, AR42919)
  • Canadian Institutes of Health Research
  • National Heart, Lung, and Blood Institute (HL16512, R01HL016512, HL-51586, R01HL051586)
  • National Institute of Diabetes and Digestive and Kidney Diseases (DK 58882)
  • National Institute of Arthritis and Musculoskeletal and Skin Diseases (P01AR042919, AR42919)
  • National Institute of Dental and Craniofacial Research (DE11290, R01DE011290)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Bone Metabolism and Diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
  • Health Sciences › Medicine › Orthopedics and Sports Medicine › Bone health and osteoporosis research
Keywords
LRP5, Wnt signaling, osteoblast proliferation, osteopenia, vascular regression, endothelial cell apoptosis, macrophages, osteoporosis-pseudoglioma syndrome, bone formation, postnatal development
MeSH terms
Animals, Bone and Bones, Bone Development, Bone Diseases, Metabolic, Calcium, Cell Division, Eye, Eye Abnormalities, Immunohistochemistry, Macrophages, Neoplasm Proteins, Organ Size, Osteoblasts, Phenotype, Proto-Oncogene Proteins, Receptors, LDL, Transcription Factors, Signal Transduction, Mutagenesis, Site-Directed, Apoptosis, Mice, Knockout, Gene Expression Regulation, Developmental, In Situ Nick-End Labeling, LDL-Receptor Related Proteins, Zebrafish Proteins, Core Binding Factor Alpha 1 Subunit, Wnt Proteins, Mice, Low Density Lipoprotein Receptor-Related Protein-5

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