Ablation of cathepsin k activity in the young mouse causes hypermineralization of long bone and growth plates

Abstract

Cathepsin K deficiency in humans causes pycnodysostosis, which is characterized by dwarfism and osteosclerosis. Earlier studies of 10-week-old male cathepsin K-deficient (knockout, KO) mice showed their bones were mechanically more brittle, while histomorphometry showed that both osteoclasts and osteoblasts had impaired activity relative to the wild type (WT). Here, we report detailed mineral and matrix analyses of the tibia of these animals based on Fourier transform infrared microspectroscopy and imaging. At 10 weeks, there was significant hypercalcification of the calcified cartilage and cortices in the KO. Carbonate content was elevated in the KO calcified cartilage as well as cortical and cancellous bone areas. These data suggest that cathepsin K does not affect mineral deposition but has a significant effect on mineralized tissue remodeling. Since growth plate abnormalities were extensive despite reported low levels of cathepsin K expression in the calcified cartilage, we used a differentiating chick limb-bud mesenchymal cell system that mimics endochondral ossification but does not contain osteoclasts, to show that cathepsin K inhibition during initial stages of mineral deposition retards the mineralization process while general inhibition of cathepsins can increase mineralization. These data suggest that the hypercalcification of the cathepsin K-deficient growth plate is due to persistence of calcified cartilage and point to a role of cathepsin K in bone tissue development as well as skeletal remodeling.

Figure 1

Histology of the growth plate in WT, HET, and KO mouse tibias. a) Typical sections from WT and b) KO growth plates. Original magnification 20X; scale bar = 100 um. c) Growth plate widths within the epiphyseal and metaphyseal boundaries (defined by the last intact hypertrophic chondrocyte) expressed as mean ±SD. * p< 0.05 relative to WT.

Figure 1

Histology of the growth plate in WT, HET, and KO mouse tibias. a) Typical sections from WT and b) KO growth plates. Original magnification 20X; scale bar = 100 um. c) Growth plate widths within the epiphyseal and metaphyseal boundaries (defined by the last intact hypertrophic chondrocyte) expressed as mean ±SD. * p< 0.05 relative to WT.

Figure 2

FTIR Images of the growth plate in cathepsin K-KO and WT tibias: a) mineral/matrix ratio and b) crystal size and perfection (crystallinity) in the same sections. The color bars represent the scales for each of the parameters. The axes are in pixels, where 1 pixel ∼6.3 um. The adjacent pixel histograms show the distribution of pixels with each value in the corresponding image. The pixel histograms were expanded to be of comparable value distributions to facilitate visual comparison.

Figure 3

Mean and standard deviation of FTIR imaging analysis of bone sections from growth plate (GP), metaphyseal cancellous bone (TB), and midshaft cortical bone (CB). Data is shown for WT (n=6) and KO (n=5). Parameters shown are: M /M (mineral/matrix ratio), XLR (collagen cross-link ratio), XST (crystallinity = crystal size/perfection), and carbonate/phosphate ratio (C/P). C/P multiplied by 1000. *p<0.05 relative to WT for same parameter.

Figure 4

FTIR Images of midshaft cortical and metaphyseal cancellous bone in KO and WT tibias. Typical images from the same sample are shown for mineral/matrix ratio, carbonate/phosphate ratio, collagen maturity, and crystallinity. The images for each parameter in each bone type are based on the same color scale. In these images one pixel =6.25 um.

Figure 5

a) Distribution of cathepsin-K in: (A) the rat growth plate, (B) a micromass cultures of differentiating chick limb-bud mesenchymal cells at day 21 in mineralizing media (4mM Pi), (C) a negative control of day 21 chick cells in mineralizing media without the antibody. b) Kinetics of ⁴⁵Ca uptake in differentiating micromass cultures in the presence and absence of the specific cathepsin K inhibitor. The y axis shows the differential uptake (mineralizing-control) for each treatment condition, normalized to the uptake at day 21. Error bars are SD for three independent experiments, run at different times. The day at which addition of the cathepsin K specific inhibitor (1.4 uM) started is shown. The lines are the best fits to the mineralizing control data (heavy solid line), the data at day 9 (dashed line, overlays day 5 and 7 data), and the data at day 14 (dashed-dotted line; overlays day 11 data). c) Mean differential ⁴⁵Ca uptake in micromass cultures in the presence and absence of the general cathepsin B/S/L inhibitor added from day 9 at day 16, 19, and 21 (n=3). * p<0.05 significantly different from control mineralizing cultures without the inhibitor.

Figure 5

a) Distribution of cathepsin-K in: (A) the rat growth plate, (B) a micromass cultures of differentiating chick limb-bud mesenchymal cells at day 21 in mineralizing media (4mM Pi), (C) a negative control of day 21 chick cells in mineralizing media without the antibody. b) Kinetics of ⁴⁵Ca uptake in differentiating micromass cultures in the presence and absence of the specific cathepsin K inhibitor. The y axis shows the differential uptake (mineralizing-control) for each treatment condition, normalized to the uptake at day 21. Error bars are SD for three independent experiments, run at different times. The day at which addition of the cathepsin K specific inhibitor (1.4 uM) started is shown. The lines are the best fits to the mineralizing control data (heavy solid line), the data at day 9 (dashed line, overlays day 5 and 7 data), and the data at day 14 (dashed-dotted line; overlays day 11 data). c) Mean differential ⁴⁵Ca uptake in micromass cultures in the presence and absence of the general cathepsin B/S/L inhibitor added from day 9 at day 16, 19, and 21 (n=3). * p<0.05 significantly different from control mineralizing cultures without the inhibitor.

Figure 5

a) Distribution of cathepsin-K in: (A) the rat growth plate, (B) a micromass cultures of differentiating chick limb-bud mesenchymal cells at day 21 in mineralizing media (4mM Pi), (C) a negative control of day 21 chick cells in mineralizing media without the antibody. b) Kinetics of ⁴⁵Ca uptake in differentiating micromass cultures in the presence and absence of the specific cathepsin K inhibitor. The y axis shows the differential uptake (mineralizing-control) for each treatment condition, normalized to the uptake at day 21. Error bars are SD for three independent experiments, run at different times. The day at which addition of the cathepsin K specific inhibitor (1.4 uM) started is shown. The lines are the best fits to the mineralizing control data (heavy solid line), the data at day 9 (dashed line, overlays day 5 and 7 data), and the data at day 14 (dashed-dotted line; overlays day 11 data). c) Mean differential ⁴⁵Ca uptake in micromass cultures in the presence and absence of the general cathepsin B/S/L inhibitor added from day 9 at day 16, 19, and 21 (n=3). * p<0.05 significantly different from control mineralizing cultures without the inhibitor.