Deletion of a single β-catenin allele in osteocytes abolishes the bone anabolic response to loading

Abstract

The Wnt/β-catenin signaling pathway is essential for bone cell viability and function and for skeletal integrity. To determine if β-catenin in osteocytes plays a role in the bone anabolic response to mechanical loading, 18- to 24-week-old osteocyte β-catenin haploinsufficient mice (Dmp1-Cre × β-catenin fl/ + ; HET cKO) were compared with their β-catenin fl/fl (control) littermates. Trabecular bone volume (BV/TV) was significantly less (58.3%) in HET cKO females versus controls, whereas male HET cKO and control mice were not significantly different. Trabecular number was significantly less in HET cKO mice compared with controls for both genders, and trabecular separation was greater in female HET cKO mice. Osteoclast surface was significantly greater in female HET cKO mice. Cortical bone parameters in males and females showed subtle or no differences between HET cKO and controls. The right ulnas were loaded in vivo at 100 cycles, 2 Hz, 2500 µϵ, 3 days per week for 3 weeks, and the left ulnas served as nonloaded controls. Calcein and alizarin complexone dihydrate were injected 10 days and 3 days before euthanization, respectively. Micro-computed tomography (µCT) analysis detected an 8.7% and 7.1% increase in cortical thickness in the loaded right ulnas of male and female control mice, respectively, compared with their nonloaded left ulnas. No significant increase in new cortical bone formation was observed in the HET cKO mice. Histomorphometric analysis of control mice showed a significant increase in endocortical and periosteal mineral apposition rate (MAR), bone-formation rate/bone surface (BFR/BS), BFR/BV, and BFR/TV in response to loading, but no significant increases were detected in the loaded HET cKO mice. These data show that deleting a single copy of β-catenin in osteocytes abolishes the anabolic response to loading, that trabecular bone in females is more severely affected and suggest that a critical threshold of β-catenin is required for bone formation in response to mechanical loading.

Keywords: BONE FORMATION; MECHANICAL LOADING; OSTEOCYTE; β-CATENIN.

Figure 1

Bone phenotype of control and HET cKO femurs. A) Representative 3D μCT images of femoral trabecular bone. Ex-vivo high-resolution analyses of distal femurs to determine B) trabecular Bone Mineral Density (BMD), C) trabecular bone volume / total volume (BV/TV) D) trabecular number, E) trabecular thickness, F) trabecular separation G) percentage ash content. H) Cortical BMD, I) cortical BV / TV and J) cortical thickness in control (white bars) and HET cKO mice (gray bars). (B–G) Bar graphs represent means plus SD. Group size n=7 for male and female controls and male HET cKO mice; n=9 for HET cKO mice. Statistical comparisons; a: p<0.05 between male and female of same genotype b: p<0.05 comparing control to HET cKO.

Figure 2

Female β–catenin heterozygous deleted mice showed increase osteoclast activity compared to their controls. A) Shows representative images of fixed sections from femurs. Images were taken using 40X objective bar = 50μm. B) Histomorphometric quantitation of osteoclast number per bone perimeter. C) Histomorphometric quantitation of osteoclast surface per bone perimeter. Group size n=3 for controls, male and female, and male HET cKO mice. Statistical comparison a: p<0.05 between male and female of same genotype b: p<0.05 comparing control to HET cKO.

Figure 3

Biomechanical properties of control femurs as compared to HET cKO. A) Representative 3D images of entire femur and cortical cross section using the BoneJ plug-in (ImageJ). B) Table listing the biomechanical parameters: Stiffness, Ultimate Force and Young's Modulus. Group size n=8 for male controls and HET cKO; n=6 for and female controls; n=10 for female HET cKO mice. Statistical comparisons; a: p<0.05 between male and female of same genotype.

Figure 3

Biomechanical properties of control femurs as compared to HET cKO. A) Representative 3D images of entire femur and cortical cross section using the BoneJ plug-in (ImageJ). B) Table listing the biomechanical parameters: Stiffness, Ultimate Force and Young's Modulus. Group size n=8 for male controls and HET cKO; n=6 for and female controls; n=10 for female HET cKO mice. Statistical comparisons; a: p<0.05 between male and female of same genotype.

Figure 4

Strain gage analysis of male (Panel A) and female (Panel B) control and HET cKO ulnae to determine the load:strain relationship. Group size n=4 for female HET cKO, n=4 for males HET cKO, female controls n=3 and male controls n=3.

Figure 5

β-catenin heterozygous mice have decreased response to loading. Representative images of double fluorochrome labeling using calcein and alizarin red of loaded and non-loaded ulnae 3 mm distal to the midshaft region. Panel A: Male control and HET cKO mice. Panel B: Female control and HET cKO mice. Right top corner shows a magnification of similar regions to better visualize double labeling. Sections were viewed under fluorescent light to observe mineral deposition due to loading. Control females and males showed clear double labeling after loading; this response was absent in β-catenin heterozygous mice.

Figure 6

Histomorphometric analysis of mineral apposition rates on periosteal and endosteal surfaces of loaded and non-loaded ulnae. A) endocortical and B) periosteal surface analysis. Group size was n=5 for males and n=4 for females. C) Representative percent change in cortical thickness comparing loaded to non-loaded ulnae using μCT analysis. Statistical comparisons; b: p<0.05 comparing control to HET cKO; c: p<0.05 comparing non-loaded versus loaded.