Targeting the LRP5 pathway improves bone properties in a mouse model of osteogenesis imperfecta

Abstract

The cell surface receptor low-density lipoprotein receptor-related protein 5 (LRP5) is a key regulator of bone mass and bone strength. Heterozygous missense mutations in LRP5 cause autosomal dominant high bone mass (HBM) in humans by reducing binding to LRP5 by endogenous inhibitors, such as sclerostin (SOST). Mice heterozygous for a knockin allele (Lrp5(p.A214V) ) that is orthologous to a human HBM-causing mutation have increased bone mass and strength. Osteogenesis imperfecta (OI) is a skeletal fragility disorder predominantly caused by mutations that affect type I collagen. We tested whether the LRP5 pathway can be used to improve bone properties in animal models of OI. First, we mated Lrp5(+/p.A214V) mice to Col1a2(+/p.G610C) mice, which model human type IV OI. We found that Col1a2(+/p.G610C) ;Lrp5(+/p.A214V) offspring had significantly increased bone mass and strength compared to Col1a2(+/p.G610C) ;Lrp5(+/+) littermates. The improved bone properties were not a result of altered mRNA expression of type I collagen or its chaperones, nor were they due to changes in mutant type I collagen secretion. Second, we treated Col1a2(+/p.G610C) mice with a monoclonal antibody that inhibits sclerostin activity (Scl-Ab). We found that antibody-treated mice had significantly increased bone mass and strength compared to vehicle-treated littermates. These findings indicate increasing bone formation, even without altering bone collagen composition, may benefit patients with OI.

Keywords: ANABOLICS; CELL/TISSUE SIGNALING; DISEASES AND DISORDERS OF BONE; GENETIC ANIMAL MODELS; OSTEOGENESIS IMPERFECTA; PARACRINE PATHWAYS; WNT/BETA-CATENIN/LRPS.

Figure 1

The Lrp5^p.A214V allele improves bone properties in wild-type and in OI mice. (left panels) 3-D μCT reconstructions of the distal and midshaft femurs from male 12-week-old wild-type (Col1a2^+/+;Lrp5^+/+), OI (Col1a2^+/p.G610C;Lrp5^+/+), OI with HBM (Col1a2^+/p.G610C;Lrp5^+/p.A214V), and HBM (Col1a2^+/+;Lrp5^+/p.A214V) mice. Small brackets in the coronal section of the distal femurs indicate the location of the transverse sections depicted immediately below. (right panels) Graphs depicting mean (± SD) measures of bone mineral density (BMD), distal femur trabecular bone volume/total volume (BV/TV), midshaft femur cortical thickness, ultimate force to failure, and energy to ultimate force in female (open bars) and male (shaded bars) 12-week-old wild-type, OI, OI with HBM, and HBM mice. Genotypes, wild-type (WT) or heterozygous knockin (OI or HBM, with respect to the Lrp5 and Col1a2) are indicated, as is the number (N) of animals with each genotype that were studied. Brackets indicate comparisons and p values between OI and wild-type mice, between OI with HBM and OI mice, and between OI with HBM and wild-type mice. NS – not significant.

Figure 2

The Lrp5^p.A214V allele increases bone formation and does not alter mutant collagen deposition in Col1a2^+/p.G610C mice. (top panels) Fluorescence micrographs of dual fluorochrome (calcein green and alizarin complexone) labeled trabecular femur bone from male 12-week-old wild-type (Col1a2^+/+;Lrp5^+/+), OI (Col1a2^+/p.G610C;Lrp5^+/+), OI with HBM (Col1a2^+/p.G610C;Lrp5^+/p.A214V), and HBM (Col1a2^+/+;Lrp5^+/p.A214V) mice. (top graphs) Graphs depicting mean (± SD) trabecular mineralizing surface/total bone surface (MS/BS), trabecular mineral apposition rate (MAR), and trabecular bone formation rates (BFR) in male (shaded bars) 12-week-old mice, and osteoblast surface and osteoclast surface in male (shaded bars) 12-week-old mice. (middle graphs) Graphs depicting mean (± SD) periosteal mineralizing surface/total bone surface (MS/BS), periosteal mineral apposition rate (MAR), and periosteal bone formation rates (BFR) in male (shaded bars) 12-week-old mice, and serum P1NP and CTX (mean ± SE) in female (open bars) and male (shaded bars) 12-week-old mice. CTX data for female mice are similar to those of males, but not shown. P1NP and CTX graphs show values (mean ± SE) from wild-type mice reported from other papers (41, 42). Genotypes, wild-type (WT) or heterozygous knockin (OI or HBM, with respect to the Lrp5 and Col1a2) are indicated, as is the number (N) of animals with each genotype that were studied. Brackets indicate comparisons and p values between OI and wild-type mice, between OI with HBM and OI mice, and between OI with HBM and wild-type mice. NS – not significant. (bottom left panel) Coomassie blue stained SDS-PAGE gel containing α₁(I) and α₂(I) polypeptide chains (arrows) recovered from OI with HBM, OI, and WT bone. Representative mass spectroscopy data for the OI with HBM bone show the mutant α₂(I) polypeptide is present and comparable in abundance to wild-type α₂(I) polypeptide, which was also observed in OI bone (data not shown). (bottom right panel) Graph depicting mean fold-changes in gene expression between OI with HBM bone and OI bone (y-axis) and the average number of mapped reads/gene (x-axis). Circles represent individual genes. The 9 genes whose expression differed significantly between the two genotypes are indicated and shaded red. The Col1a1 and Col1a2 genes, whose expression did not differ significantly between the two genotypes, are indicated and shaded blue.

Figure 3

Sclerostin inhibiting antibody treatment improves bone properties in wild-type and in OI mice. (left panels) 3-D μCT reconstructions of the distal and midshaft femurs from male 12-week-old wild-type (Col1a2^+/+) and OI (Col1a2^+/p.G610C) mice that received 6 weeks of vehicle or Scl-Ab. (right panels) Graphs depicting mean (± SD) measures of bone mineral density (BMD), distal femur trabecular bone volume/total volume (BV/TV), midshaft femur cortical thickness, ultimate force to failure, and energy to ultimate force in female (open bars) and male (shaded bars) 12-week-old wild-type and OI mice that received 6 weeks of vehicle or Scl-Ab. Col1a2 genotypes, wild-type (WT) or heterozygous knockin (OI) are indicated, as is the number (N) of animals with each genotype that were studied. Brackets indicate comparisons and p values between vehicle treated OI and vehicle treated wild-type mice, between Scl-Ab treated OI and vehicle treated OI mice, and between Scl-Ab treated OI and vehicle treated wild-type mice. NS – not significant.

Figure 4

Sclerostin inhibiting antibody therapy increases bone formation in wild-type and in OI mice. (top panels) Fluorescence micrographs of dual fluorochrome labeled midshaft femur bone from male 12-week-old wild-type and OI (Col1a2^+/p.G610C) mice that received 6 weeks of vehicle or Scl-Ab. Higher magnification images represent the areas indicated with white rectangles. The red arrows indicate the fluorochrome (democlocycline and calcein green/alizarin complexone) labels. Images of dual flurochrome labeled trabecular bone were similar to those seen in Figure 2 (data not shown). (middle graphs) Graphs depicting mean (± SD) periosteal mineralizing surface/total bone surface (MS/BS), periosteal mineral apposition rate (MAR), and periosteal bone formation rates (BFR) in male (shaded bars) 12-week-old mice, and serum P1NP and CTX (mean ± SE) in female (open bars) and male (shaded bars) 12-week-old mice. CTX data for female mice are similar to those of males but not shown. P1NP and CTX graphs show values (mean ± SE) from wild-type mice reported from other papers (41, 42). (bottom graphs) Graphs depicting mean (± SD) trabecular mineralizing surface/total bone surface (MS/BS), trabecular mineral apposition rate (MAR), and trabecular bone formation rates (BFR) in male (shaded bars) 12-week-old mice, and osteoblast surface and osteoclast surface in male (shaded bars) 12-week-old mice.Col1a2 genotypes, wild-type (WT) or heterozygous knockin (OI) are indicated, as is the number (N) of animals with each genotype that were studied. Brackets indicate comparisons and p values between vehicle treated OI and vehicle treated wild-type mice, between Scl-Ab treated OI and vehicle treated OI mice, and between Scl-Ab treated OI and vehicle treated wild-type mice. NS – not significant.