Effect of sclerostin inactivation in a mouse model of severe dominant osteogenesis imperfecta

Abstract

Osteogenesis imperfecta (OI) is a rare bone disease that is associated with fractures and low bone mass. Sclerostin inhibition is being evaluated as a potential approach to increase bone mass in OI. We had previously found that in Col1a1^Jrt/+ mice, a model of severe OI, treatment with an anti-sclerostin antibody had a minor effect on the skeletal phenotype. In the present study, we assessed the effect of genetic sclerostin inactivation in the Col1a1^Jrt/+ mouse. We crossed Col1a1^Jrt/+ mice with Sost knockout mice to generate Sost-deficient Col1a1^Jrt/+ mice and assessed differences between Col1a1^Jrt/+ mice with homozygous Sost deficiency and Col1a1^Jrt/+ mice with heterozygous Sost deficiency. We found that Col1a1^Jrt/+ mice with homozygous Sost deficiency had higher body mass, femur length, trabecular bone volume, cortical thickness and periosteal diameter as well as increased biomechanical parameters of bone strength. Differences between genotypes were larger at the age of 14 weeks than at 8 weeks of age. Transcriptome analysis of RNA extracted from the tibial diaphysis revealed only 5 differentially regulated genes. Thus, genetic inactivation of Sost increased bone mass and strength in the Col1a1^Jrt/+ mouse. It appears from these observations that the degree of Sost suppression that is required for eliciting a beneficial response can vary with the genetic cause of OI.

Figure 1

Chromosomal location of Col1a1 and Sost and breeding strategy. (A) Col1a1 and Sost are both located on murine chromosome 11, separated by about 7 MB (assembly GRCm38.p6). (B) Col1a1^Jrt/+ (FVB background) and Sost^−/− (C57BL6 background) mice were bred (1) to generate Col1a1^Jrt/+;Sost^+/− and Col1a1^+/+;Sost^+/− mice of mixed background (2). These mice were then crossed until a crossover event was observed that resulted in the generation of a Col1a1^Jrt/+;Sost^−/− mouse (3). (C) Col1a1^Jrt/+;Sost^−/− mice were bred with Col1a1^Jrt/+;Sost^+/− mice to generate three of the four genotypes that were used for phenotype analyses. Mice that are homozygous for the Jrt allele (Col1a1^Jrt/Jrt) are not viable and die in utero (and are therefore crossed out in the schematic). (D) A separate colony was maintained to generate mice of mixed FVB/C57BL6 background that were wild type for both the Col1a1 and the Sost locus. These mice were used to compare results of the three viable genotypic groups shown in (C).

Figure 2

Body mass, femur length and serum markers of bone metabolism. Variation with genotype and age in body mass, femur length, and serum markers of bone metabolism. Error bars represent standard errors. Significance levels for differences to WT mice (ANOVA) are indicated above each bar: *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (p ≥ 0.05). Significance levels for differences between Jrt;Sost-het and Jrt;Sost-ko are indicated above the horizontal lines. n = 8–15 mice per group for body mass and femur length. n = 6–11 mice per group for serum markers.

Figure 3

Trabecular bone characterization. Trabecular bone analyses at the distal femur (A–C by microCT) and at lumbar vertebra 4 (D–I by histomorphometry). Error bars represent standard errors. Significance levels for differences to WT mice (ANOVA) are indicated above each bar: * p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (p ≥ 0.05). Significance levels for differences between Jrt;Sost-het and Jrt;Sost-ko are indicated above the horizontal lines. n = 6–12 mice per group for femur analyses and 11–15 mice per group for vertebra analyses.

Figure 4

Histology of vertebral bones. Histological longitudinal sections of lumbar vertebra stained with Goldner’s trichrome staining at 8 and 14 weeks of age. Mineralized bone appears blue, unmineralized bone pink and bone marrow purple.

Figure 5

Cortical bone characterization. Femur midshaft microCT and biomechanics. (A) MicroCT images of midshaft femur scans. (B–D) MicroCT data for bone structure. (E–G) Results of three-point bending tests. Error bars represent standard errors. Significance levels for differences to WT mice (ANOVA) are indicated above each bar: *p < 0.05, **p < 0.01, ***p < 0.001, ns: not significant (p ≥ 0.05). Significance levels for differences between Jrt;Sost-het and Jrt;Sost-ko are indicated above the horizontal lines. n = 6–15 mice per group.