Differential turnover of cortical and trabecular bone in transgenic mice overexpressing cathepsin K

Bone 36 (2005) 854 – 865 www.elsevier.com/locate/bone

Differential turnover of cortical and trabecular bone in transgenic mice overexpressing cathepsin K Jukka Morko, Riku Kiviranta, Sara Hurme, Juho Rantakokko, Eero VuorioT Department of Medical Biochemistry and Molecular Biology, University of Turku, FI-20520 Turku, Finland Received 1 October 2004; revised 18 January 2005; accepted 8 February 2005

Abstract Cathepsin K is a major osteoclastic protease. We have recently shown that overexpression of mouse cathepsin K gene in transgenic UTU17 mouse model results in high turnover osteopenia of metaphyseal trabecular bone at the age of 7 months. The present report extends these studies to a systematic analysis of cortical bone in growing and adult mice overexpressing cathepsin K. Mice homozygous for the transgene locus (UTU17+/+) and their control littermates were studied at the age of 1, 3, 7, and 12 months. Bone properties were analyzed using peripheral quantitative computed tomography (pQCT), histomorphometry, histochemistry, radiography, and biomechanical testing. In addition, the levels of biochemical markers of bone turnover were measured in the sera. Unexpectedly, cortical thickness and cortical bone mineral density were increased in the diaphyseal region of growing and adult UTU17+/+ mice. This was associated with an increased number of vascular canals leading to increased cortical porosity in UTU17+/+ mice without changes in the ultimate bending force or stiffness of the bone. In UTU17+/+ mice, osteopenia of metaphyseal trabecular bone was observed already at the age of 1 month. In sera of 1-month-old UTU17+/+ mice, the activity of tartrate-resistant acid phosphatase 5b was decreased and the levels of osteocalcin increased. Our results support the role of cathepsin K as a major proteinase in osteoclastic bone resorption. Excessive production of cathepsin K induced osteopenia of metaphyseal trabecular bone and increased the porosity of diaphyseal cortical bone. The increased cortical thickness and bone mineral density observed in diaphyses of UTU17+/+ mice demonstrate the different nature and reactivity of trabecular and cortical bone in mice. These results suggest that the biomechanical properties of cortical bone are preserved through adaptation as outlined in Wolff’s law. D 2005 Elsevier Inc. All rights reserved. Keywords: Biochemical markers; Bone turnover; Cathepsin K; Computed tomography; Histomorphometry – animal

Introduction Bone modeling/remodeling is a complex process consisting of differential bone resorption and formation to which biomechanical forces contribute through an interplay of different cell types [1,2]. Bone resorption by osteoclasts consists of several steps, e.g., cell attachment, polarization, demineralization of bone matrix, degradation of organic bone matrix, and cell detachment, followed by osteoclast apoptosis or by initiation of a new resorption cycle [3]. Several lines of evidence suggest that cathepsin K, a lysosomal cysteine proteinase, is a key enzyme in the degradation of organic bone matrix. It is highly and quite T Corresponding author. Fax: +358 2 333 7229. E-mail address: [email protected] (E. Vuorio). 8756-3282/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2005.02.006

specifically expressed by osteoclasts and secreted into the resorption lacunae [4 –6] where it can efficiently degrade matrix proteins, including native type I collagen [7– 9]. Expression of the cathepsin K gene (Ctsk) is highly upregulated under conditions of enhanced bone resorption such as mouse immobilization osteopenia [10]. Inhibition of cathepsin K activity in vitro and in vivo reduces bone resorption [11,12]. Ctsk-deficient mice develop an osteopetrosis-like phenotype due to their inability to degrade organic bone matrix [13 –15]. In humans, mutations in the Ctsk gene cause pycnodysostosis, an osteopetrotic disease characterized by increased bone mass, short stature, and increased bone fragility [16]. We have recently produced a transgenic UTU17 mouse model harboring extra copies of the Ctsk gene that result in overexpression of cathepsin K [17]. Histomorphometric

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analyses demonstrated decreased trabecular bone volume in mice heterozygous for the transgene locus (UTU17+/ ) at the age of 7 months, indicating that excessive cathepsin K is alone sufficient to enhance osteoclastic bone resorption and result in osteopenia of metaphyseal trabecular bone. As no changes were observed in the number of osteoclasts between UTU17+/ and control mice, the enhanced bone resorption was accomplished by increased resorption capacity of osteoclasts due to overexpression of a single protease. Histomorphometric analyses also revealed an increased rate of bone turnover in 7-month-old UTU17+/ mice. Due to tight coupling of bone resorption and formation during the bone remodeling cycle, the accelerated bone turnover also stimulated differentiation and/or function of osteoblasts as the mRNA levels of core binding factor a1 (Cbfa1), the number of osteoblasts, and the amount of mineralizing surface were increased in 7-month-old UTU17+/ mice. The aim of the present study was to extend our studies on the transgenic UTU17 mouse model to cortical bone. The study was performed on mice homozygous for the transgene locus (UTU17+/+) exhibiting the highest increase in Ctsk mRNA levels. Analyses of these mice by peripheral quantitative computed tomography (pQCT), histomorphometry, histochemistry, radiography, biomechanical testing, and by measurement of bone turnover markers revealed increased thickness, density, and porosity of diaphyseal cortical bone in UTU17+/+ mice and confirmed the osteopenic phenotype of their metaphyseal trabecular bone.

Materials and methods The experimental protocol was approved by the Institutional Committee for Animal Welfare, University of Turku, Turku, Finland. Experimental animals Three lines of transgenic UTU17 mice overexpressing cathepsin K gene in FVB/N genetic background were produced as described earlier [17]. As there were no systematic differences in bone phenotype of different UTU17 mouse lines, one line (UTU17.2) harboring the highest cathepsin K mRNA levels in bone (approximately 8fold higher than controls) was selected for further studies. This study was conducted on hind limb and serum samples collected from female mice homozygous (UTU17+/+) for the transgene locus and from their non-transgenic littermates, which served as controls. The homozygous mice harbor six copies of an engineered 14-kb cathepsin K transgene with a silent mutation introducing a novel KpnI restriction site in exon 6. The litters were genotyped by Southern analysis as described earlier [17]. Samples were collected at the age of 1, 3, 7, and 12 months. Mice were anesthetized by an

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intraperitoneal injection (0.015 – 0.017 ml/g of body weight) of 2.5% (w/v) Avertin (tribromoethyl alcohol and tertiary amyl alcohol) [18] and weighted. Blood for serum samples was collected by cardiac puncture, after which the mice were sacrificed by cervical dislocation. Left hind limbs were used for peripheral quantitative computed tomography (pQCT) analysis, radiography, and biomechanical testing. Right hind limbs were collected for histology, histomorphometry, and protein extraction (for Western analysis). Peripheral quantitative computed tomography The mineral densities and cross-sectional dimensions of the left hind limbs from transgenic UTU17+/+ and control mice (n = 11 –15 per genotype at each time point) were measured using Stratec XCT Research M device with a software version 5.40 (Norland Stratec Medizintechnik, Birkenfeld, Germany). For metaphyseal analysis, the scan lines were adjusted to 1 –4 mm with 0.5-mm intervals, and for diaphyseal analysis to 6 and 6.5 mm from the proximal end of the tibia using the scout view given by the pQCT device. All measurements were made using a voxel size of 0.070  0.070  0.5 mm3. A threshold value of 710 mg/ cm3 was used in the measurements of cortical bone and that of 350 mg/cm3 for trabecular bone. Calculations of cortical thickness were made using the ring model supplied by the software. The reproducibility of the pQCT measurements was defined as a coefficient of variation (SD/mean), which was calculated from 10 repeated measurements of the same sample with repositioning before each measurement. The coefficient values were 2.0%, 0.5%, 2.8%, and 1.2% for cortical thickness (Ct.Th), cortical bone mineral density (Ct.BMD), trabecular bone mineral density (Tb.BMD), and for total cross-sectional area (Tt.CSA), respectively. Histomorphometry For histomorphometry, the samples were fixed overnight in ice-cold 40% ethanol and processed as undecalcified hard-tissue sections [19]. For cortical histomorphometry, samples from 1-month-old UTU17+/+ (n = 7) and control mice (n = 7) were brought into 70% ethanol, and cortical cylinders were harvested with a diamond saw from right proximal femurs, dehydrated, and embedded in Tecknovit 7200 VLC (Heraeus Kulzer, Armonk, NY, USA). Transversal sections of 20 Am were cut using the Exakt cuttinggrinding method (Exakt Apparatebau, Norderstedt, Germany), and unstained sections were evaluated using a light/ epifluorescent microscope. Images were captured from the microscope with a MCID camera and analyzed with M5 image analysis software (Imaging Research, St. Catharines, Canada). Calculations of periosteal and endocortical diameters were made using periosteal and endocortical perimeters and the ring model. For trabecular histomorphometry, right femurs from 3month-old UTU17+/+ (n = 9) and control mice (n = 7) were

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dehydrated in an increasing series of alcohol, defatted in xylene, embedded into methyl methacrylate (Merck-Schuchardt, Hohenbrunn, Germany), and cut longitudinally into mid-sagittal sections. For quantitative histomorphometry, 5Am sections were stained with Masson – Goldner trichrome method and 10-Am sections were analyzed without counterstaining for dynamic parameters. All measurement fields were adjusted immediately proximal to the growth plate. The sections were evaluated using a light/epifluorescent microscope interfaced to a microcomputer through a digitizing tablet, and the histomorphometric data was analyzed with OsteoMeasure software version 2.2 (Osteometrics, Atlanta, GA, USA). All histomorphometric analyses were performed following the instructions of the American Society of Bone and Mineral Research histomorphometry nomenclature committee [20]. Histochemistry Tissue distribution of cathepsin K in paraffin-embedded decalcified sections of right tibias was studied using polyclonal antibodies raised against mouse cathepsin K [21]. The specificity of the affinity-purified antibodies to mouse cathepsin K in protein extracts of knee joints (containing proximal tibial and distal femoral ends) was confirmed by Western analysis (Fig. 1), as described earlier [15]. Immunohistochemistry was performed using the biotin-streptavidin complex method following the protocol recommended by the supplier (Histomouse -SP Bulk kit; Zymed Laboratories, South San Francisco, CA, USA). Enzyme digestion (Digest-All 1; Zymed Laboratories) was used to enhance the immunohistochemical staining. Streptavidin/Biotin Blocking kit (Vector Laboratories, Burlingame, CA, USA) was used to block non-specific binding of streptavidin. Diaminobenzidine (Liquid DAB -Plus Sub-

strate kit; Zymed Laboratories) was used as a substrate to horseradish peroxidase, and the sections were counterstained with hematoxylin (Merck KGaA, Darmstadt, Germany). The specificity of the reactions was controlled by omitting the primary antibody. Tissue distribution of osteoclasts in paraffin-embedded decalcified sections of right tibias was studied by enzyme histochemistry. Cells with multiple nuclei, an intimate contact to bone and positive for tartrate-resistant acid phosphatase (TRACP), were considered as osteoclasts. TRACP staining was performed according to the protocol recommended by the supplier (Leukocyte Acid Phosphatase kit; Sigma-Aldrich, St. Louis, MO, USA). The sections were counterstained with hematoxylin (Merck KGaA). Radiography Left tibias from 3-month-old UTU17+/+ (n = 9) and control mice (n = 14) were subjected to radiographic analysis. The tibias were imaged in anterior – posterior projection in a Faxitron cabinet X-ray system (HewlettPackard, McMinneville, OR, USA) on Min-R diagnostic film (Kodak, Windsor, CO, USA) at 19 kV. Radiographs were digitized with a MCID camera and bone lengths were measured from these images using the M5 image analysis software (Imaging Research). Biomechanical testing Left tibias from 3-month-old UTU17+/+ (n = 9) and control mice (n = 14) were subjected to a standardized three-point bending test [22]. The tibias were placed horizontally with the anterior surface upward into a universal testing device (Avalon Technologies, Rochester, MN, USA) with a constant span length of 6 mm. The tibias were loaded vertically at a constant rate of 1.0 mm/min until failure. The force – displacement curve was measured using a force transducer, and the analog signal was recorded with computed A/D conversion and a strip-chart recorder (LKBProducter, Bromma, Sweden). Ultimate bending force to failure and bending stiffness were calculated from the force –displacement curves. Serum analyses

Fig. 1. The specificity of polyclonal rabbit anti-mouse cathepsin K antibodies to detect cathepsin K in extracts of knee joints by Western analysis. The extracts were electrophoresed on 12.5% (w/v) SDS – polyacrylamide gels and stained with coomassie blue (shown in the middle). Molecular weight standards are shown on the left. The polyclonal cathepsin K antibodies specifically detected proteins with sizes corresponding to the pro form and the mature form of cathepsin K (38 and 27 kDa, respectively), as shown on the right.

Levels of (cathepsin K-derived) cross-linked carboxyterminal telopeptides of type I collagen (CTX) and the activity of TRACP 5b were used as biochemical markers of bone resorption, and levels of osteocalcin as a biochemical marker of bone formation in the sera of 7 –12 transgenic UTU17+/+ and control mice at each time point [23,24]. The levels of CTX were measured using an enzyme-linked immunosorbent assay (ELISA) according to the protocol recommended by the supplier (RatLaps ELISA; Nordic Bioscience Diagnostics, Herlev, Denmark). For the measurement of TRACP 5b activity, polyclonal anti-TRACP 5b

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antibodies were used to capture the enzyme, followed by activity determination as described earlier [25]. The levels of osteocalcin were determined using an immunoradiometric assay (IRMA) following the protocol suggested by the supplier (Mouse Osteocalcin IRMA kit; Immutopics, San Clemente, CA, USA). Statistical analyses All data are expressed as mean T standard deviation. Statistical analyses were performed with statistical software, SAS System for Windows (SAS Institute, Cary, NC, USA). Since normal distribution of the data could not be assumed for the small sample size, evaluation of the data was based on nonparametric tests. Mann – Whitney U test was used to evaluate the data between UTU17+/+ and control mice. Kruskall – Wallis test followed by Bonferroni-adjusted Mann –Whitney U test was performed to evaluate the data between time points. P values less than 0.05 were considered statistically significant.

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studied (Fig. 2). In other words, both the thickness of mineralized cortex and the amount of mineral per cortical volume were increased in diaphyses of growing and adult mice overexpressing cathepsin K. Detailed analysis of Ct.Th and Ct.BMD at individual time points demonstrated that the thickness and density of cortical bone in UTU17+/+ and control mice increased from proximal metaphysis towards diaphysis (Fig. 3). Especially in 1-month-old mice, the cortex in proximal metaphysis was extremely thin but thickened substantially towards middiaphysis (Fig. 3A). In the metaphyseal area of UTU17+/+ mice, the cortices were thicker than controls (0.11 T 0.015 mm versus 0.10 T 0.007 mm, P < 0.01) and more mineralized (852 T 19.8 mg/cm3 versus 827 T 11.2 mg/ cm3, P < 0.05) during rapid growth, at the age of 1 month (Figs. 3A and E). Later, in 3- and 7-month-old mice, no differences were seen between transgenic and control mice in these parameters in metaphyseal cortical bone (Figs. 3B, C, F, and G). However, metaphyseal Ct.BMD was again significantly increased in 12-month-old UTU17+/+ mice (1210 T 31.0 mg/cm3) when compared with control mice (1182 T 17.9 mg/cm3, P < 0.05; Fig. 3H).

Results Histomorphometry of diaphyseal cortical bone In vitro pQCT measurements of cortical bone in growing and adult mice pQCT analysis of cortical thickness (Ct.Th) and cortical bone mineral density (Ct.BMD) in the diaphyseal region of UTU17+/+ and control mice revealed that both parameters increased markedly from the age of 1 to 3 months (Fig. 2). Thereafter, the rate of increase slowed down, but in both UTU17+/+ and control mice both parameters continued to exhibit statistically significant increases from 3 to 7 months of age ( P < 0.001; Fig. 2). Although the changes in Ct.Th and Ct.BMD in the diaphyses of both UTU17+/+ and control mice occurred in the same direction, both parameters were systematically higher in UTU17+/+ mice at all time points

Cross-sectional histomorphometry of diaphyseal cortical bone was carried out at the age of 1 month, when cortical thickening in mice overexpressing Ctsk was detectable by pQCT. The analyses verified the observation of increased cortical thickness in UTU17+/+ mice (Table 1). Analysis of periosteal and endocortical perimeters and diameters did not demonstrate any statistically significant differences between UTU17+/+ and control mice. However, on the endocortical surface of UTU17+/+ mice, the diameter of bone marrow cavity was decreased to 95% ( P = 0.2468) of that in control mice, indicating that the thickening was more prominent on this side of the cortex. There were no statistically significant changes in dynamic parameters, i.e., mineral apposition rate

Fig. 2. Increased cortical thickness (A) and cortical bone mineral density (B) in diaphyses of UTU17+/+ mice, measured at the age of 1, 3, 7, and 12 months. The results represent the mean T standard deviation. Open symbols denote UTU17+/+ mice and black symbols control animals. Statistically significant differences between UTU17+/+ and control mice are marked: **P < 0.01, ***P < 0.001 (Mann – Whitney U test; n = 11 – 15 for each symbol).

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Fig. 3. Positional differences in cortical thickness (A – D) and cortical bone mineral density (E – H) in metaphyseal and diaphyseal regions of UTU17+/+ and control mice at the age of 1 (A and E), 3 (B and F), 7 (C and G), and 12 months (D and H). For metaphyseal analysis, the scan lines were adjusted to 1 – 4 mm and for diaphyseal analysis to 6 – 6.5 mm from the proximal end of the tibia. The results represent the mean T standard deviation. Open symbols indicate UTU17+/+ mice and black symbols control animals. *P < 0.05, **P < 0.01, ***P < 0.001 (Mann – Whitney U test; n = 11 – 15 for each symbol).

or bone formation rate, between UTU17+/+ and control mice. Histomorphometry also revealed the existence of intracortical canals, defined as cavities larger than osteocyte lacunae which were embedded within cortical bone [20]. When treated as individual structures, their number and total area were found to be increased in UTU17+/+ mice (Table 1). In addition, there was a trend towards increased mean canal area in UTU17+/+ mice, but the difference did not reach statistical significance ( P = 0.0530). The increased number of canals resulted in a 1.7-fold increase in cortical bone porosity in UTU17+/+ mice. Dynamic histomorphometry revealed active formation of new bone on the surface of the intracortical canals both in UTU17+/+ and control mice (Figs. 4A, B, F, and G). Structures resembling cement lines and newly formed bone lamellae were seen around osteons (Figs. 4C and H). Intracortical canals were also present in longitudinal sections of diaphyseal cortical bone, as shown for transgenic UTU17+/+ mice (Figs. 4D, E, and J). Cells positive for cathepsin K and TRACP were observed inside these canals, suggesting the presence of resorbing osteoclasts (Figs. 4D and E). As the intracortical canals were also occupied by

blood vessels, indicated by the presence of erythrocytes, the canals were considered as vascular canals resembling those of the Haversian system. Biomechanical testing of diaphyseal cortical bone A standardized three-point bending test was carried out to evaluate the functional effect of cathepsin K overexpression on the biomechanical properties of diaphyseal cortical bone. The three-point bending test did not demonstrate statistically significant differences in ultimate bending force or in bone stiffness between 3-month-old UTU17+/+ and control mice (Table 2). In addition, there were no differences in body weights, bone lengths, or in total crosssectional area of diaphyses between UTU17+/+ and control mice either. In vitro pQCT measurements of metaphyseal trabecular bone in growing and adult mice pQCT analyses of UTU17+/+ and control mouse bones in vitro confirmed our earlier observations of reduced amount of trabecular bone in mice overexpressing cathepsin K. In

J. Morko et al. / Bone 36 (2005) 854 – 865 Table 1 Histomorphometry of the diaphyseal cortical bone of the 1-month-old transgenic UTU17+/+ and control mice Parameter Cortical thickness (Ct.Th; Am) Periosteal Perimeter (Ps.Pm; mm) Bone diameter (B.Dm; mm) Mineral apposition rate (Ps.MAR; Am/day) Bone formation rate (Ps.BFR/BS; Am3/Am2 per day) Endocortical Perimeter (Ec.Pm; mm) Marrow diameter (Ma.Dm; mm) Mineral apposition rate (Ec.MAR; Am/day) Bone formation rate (Ec.BFR/BS; Am3/Am2 per day) Canal number (N.Ca) Canal area (Ca.Ar; Am2) Mean canal area (MCa.Ar; Am2) Cortical porosity (Ct.Po; %)

Control (n = 7)

180 T 11T

4.03 T 0.18 1.28 T 0.06 5.68 T 1.79

4.02 T 0.12 1.28 T 0.04 5.46 T 1.39

5.15 T 1.52

5.08 T 1.47

2.87 T 0.21 0.91 T 0.07 1.77 T 0.40

2.71 T 0.16 0.86 T 0.05 1.61 T 0.15

1.41 T 0.54

1.36 T 0.19

38.4 12471 330 1.92

T T T T

8.5 3784 83 0.47

Table 2 Biomechanical testing of the diaphyseal cortical bone of the 3-month-old transgenic UTU17+/+ and control mice

UTU17+/+ (n = 7)

164 T 15

46.7 20709 453 3.18

T T T T

5.5T 5465T 122 0.47TT

All data are expressed as the mean T standard deviation. Statistical significance of the results was analyzed by nonparametric Mann – Whitney U test. T P < 0.05. TT P < 0.01.

control mice, trabecular bone mineral density (Tb.BMD) reached its maximum at the age of 3 months, after which it started to decline until the age of 7 months ( P < 0.001; Fig. 5A). In UTU17+/+ mice, a similar pattern of statistically significant changes in Tb.BMD was observed. However, in transgenic mice overexpressing cathepsin K, Tb.BMD was significantly lower at the age of 1 and 3 months (Fig. 5A). Tb.BMD was highest in proximal metaphysis and declined towards diaphysis both in UTU17+/+ and control mice (Figs. 5B and C). Thus, the reduction in Tb.BMD in UTU17+/+ mice was greatest in proximal metaphysis and gradually

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Control (n = 14) Three-point bending Ultimate failure load (N) Stiffness (N/mm) Body weight (g) Bone length (mm) Total cross-sectional area (mm2)

14.3 24.0 22.4 17.8 1.49

T T T T T

1.6 5.3 1.8 0.3 0.15

UTU17+/+ (n = 9) 15.3 23.3 21.3 17.5 1.51

T T T T T

1.2 3.4 1.6 0.4 0.06

All data are expressed as the mean T standard deviation. Statistical significance of the results was analyzed by nonparametric Mann – Whitney U test.

disappeared towards diaphysis. Analysis of the intramedullary area of diaphyseal bone at the age of 1, 3, 7, and 12 months demonstrated that background value for the Tb.BMD measurements at the threshold values used was 140 mg/cm3 (data not shown). The same Tb.BMD value was observed in metaphyseal region of 7- and 12-month-old UTU17+/+ and control mice (Fig. 5A). Histomorphometry of metaphyseal trabecular bone Since the Tb.BMD peaked at the age of 3 months, histomorphometric analysis of metaphyseal trabecular bone focused at this time point. The analyses confirmed the observation made by pQCT. In addition to decreased density of mineral in bone marrow cavity (Tb.BMD) of UTU17+/+ mice, the actual amount of trabecular bone was also decreased in 3-month-old mice overexpressing Ctsk (Table 3). The decreased trabecular bone volume in UTU17+/+ mice was due to both decreased trabecular number and decreased trabecular thickness. This was also observed as increased trabecular separation. There were no statistically significant differences in the number or surface of either osteoclasts or osteoblasts between 3-month-old UTU17+/+ and control mice. The dynamic parameters, namely miner-

Fig. 4. Bone formation and resorption in intracortical canals. Cortical cross-sectional (A – C and F – H) and longitudinal sections (D, E, and J) are shown from 1month-old control (A – C) and UTU17+/+ (D – J) mice. Low-power fluorescence microscopic images (A and F) and higher magnifications of intracortical canals by fluorescence (B and G) and phase-contrast microscopy (C and H) are shown. Cortical longitudinal sections from UTU17+/+ mice were stained with cathepsin K antibody (D), and for tartrate-resistant acid phosphatase (TRACP) activity (E); control sections were stained without the primary antibody (J). The scale bar in panel A corresponds to 150 Am in panels A and F, 50 Am in panels B, C, G, and H, and 20 Am in panels D, E, and J.

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Fig. 5. Decreased trabecular bone mineral density (Tb.BMD) in metaphyses of UTU17+/+ mice. Metaphyseal Tb.BMD was measured at the age of 1, 3, 7, and 12 months (A), and Tb.BMD in metaphyseal region at the age of 1 month (B) and at the age of 3 months (C). The results represent the mean T standard deviation. Open symbols denote UTU17+/+ mice and black symbols control animals. *P < 0.05, **P < 0.01, ***P < 0.001 (Mann – Whitney U test; n = 11 – 15 for each symbol).

alizing surface, mineral apposition rate, bone formation rate, and bone turnover rate, were not altered at this time point either. Serum bone resorption and formation markers In growing and adult UTU17+/+ and control mice, serum CTX levels and serum TRACP 5b activity were used as biochemical markers of total bone resorption. Both in UTU17+/+ and control mice, the levels of CTX and the activity of TRACP 5b decreased rapidly from the age of 1 to 3 months (Figs. 6A and B). Thereafter, the CTX levels continued to decrease slightly up to 7 months of age in both UTU17+/+ and control mice ( P < 0.001). Although the levels of CTX were higher in UTU17+/+ mice compared to

age-matched littermate controls at every time point measured, the increases did not reach statistical significance ( P > 0.0745; Fig. 6A). However, the activity of TRACP 5b was significantly decreased in UTU17+/+ mice at the age of 1 month (Fig. 6B). Serum osteocalcin was used as a biochemical marker of total bone formation in growing and adult UTU17+/+ and control mice. Both in UTU17+/+ and control mice, the levels of osteocalcin declined rapidly from the age of 1 to 3 months followed by a slight decrease up to 7 months of age ( P < 0.001; Fig. 6C). Serum levels of osteocalcin were significantly increased in 1-month-old UTU17+/+ mice compared to age-matched littermate controls.

Discussion Table 3 Histomorphometry of the metaphyseal trabecular bone of the 3-month-old transgenic UTU17+/+ and control mice Parameter

Control (n = 7) UTU17+/+ (n = 9)

Bone volume (BV/TV; %) 21.6 T Trabecular number (Tb.N; /mm) 5.59 T Trabecular thickness (Tb.Th; mcm) 38.5 T Trabecular separation (Tb.Sp; mcm) 142 T Osteoclast number (N.Oc/B.Pm; 0.77 T mm 1) Osteoclast surface (Oc.S/BS; %) 1.63 T Osteoblast number (N.Ob/B.Pm; 10.1 T mm 1) Osteoblast surface (Ob.S/BS; %) 11.1 T Mineralizing surface (MS/BS; %) 35.6 T Mineral apposition rate (MAR; 1.71 T Am/day) Bone formation rate (BFR/BS; 222 T Am3/Am2 per year) Bone turnover rate (BFR/BV; 1249 T %/year)

T T T T T

4.9 0.57 8.9 19 0.23

14.0 4.58 30.0 197 0.85

4.6T 0.92T 5.9T 51T 0.47

0.51 3.3

1.95 T 1.16 12.0 T 1.8

3.5 7.1 0.09

14.0 T 2.2 32.9 T 7.5 1.91 T 0.47

47

234 T 85

473

1606 T 678

All data are expressed as the mean T standard deviation. Statistical significance of the results was analyzed by nonparametric Mann – Whitney U test. T P < 0.05.

The most interesting findings of this study were the increased thickness and mineral density of diaphyseal cortical bone in association with increased porosity of diaphyseal cortex and decreased amount of metaphyseal trabecular bone in transgenic UTU17+/+ mice overexpressing cathepsin K. As several studies have demonstrated the essential role of cathepsin K in resorption of both trabecular and cortical bone [13 –15], the observation was unexpected and suggests fundamental differences in turnover between these two bone compartments in mice. To interpret this unexpected observation, we have compared the phenotype of UTU17+/+ mice with those of other gene-modified mouse models exhibiting enhanced bone resorption. Increased osteoclastogenesis resulting in enhanced bone resorption has been observed in osteoprotegerin (OPG)-deficient mice [26,27] and in mice overexpressing soluble receptor activator of nuclear factor nB ligand (sRANKL) [28], soluble macrophage colony-stimulating factor (sCSF-1) [29], and granulocyte colony-stimulating factor (G-CSF) [30]. Enhanced resorption of trabecular bone, due to increased resorption capacity of osteoclasts, has also been

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Fig. 6. Levels of CTX (A), activity of TRACP 5b (B), and levels of osteocalcin (C) in the sera of UTU17+/+ and control mice, measured at the age of 1, 3, 7, and 12 months. The results represent the mean T standard deviation. Open symbols denote UTU17+/+ mice and black symbols control animals. *P < 0.05, **P < 0.01 (Mann – Whitney U test; n = 7 – 12 for each symbol).

observed in mice overexpressing TRACP [31]. Phenotypes of these gene-modified mouse models exhibiting enhanced bone resorption are summarized in Table 4. As noticed, none of the phenotypes exhibit the same combination of findings as UTU17+/+ mice. We interpret the 1.7-fold increase in cortical porosity to be a clear indication of enhanced intracortical bone resorption in UTU17+/+ mice due to cathepsin K overexpression. An increase in the number of vascular canals rather than in the mean canal area suggests increased intracortical bone turnover in these mice. Similarly, in sCSF-1 overexpression and OPG deficient mice, increased cortical porosity has been associated with high intracortical bone turnover [26,29,32]. However, there are also two major differences in cortical bone phenotype between these and UTU17+/+ mice. Although increased or unaltered cortical thickness was also observed in sCSF-1 over-

expression and OPG-deficient mice, it was associated with increased bone formation rate on endocortical surface [26,29,32], which was not observed in UTU17+/+ mice. Furthermore, in neither of these mouse models, increased volumetric mineral density of cortical bone has been reported, as was observed in diaphyses of UTU17+/+ mice. This indicates differential endocortical bone turnover and mineralization of cortical bone between mouse models exhibiting Ctsk overexpression and enhanced osteoclastogenesis. In addition to UTU17+/+ mice, differential reactivity of intra- and endocortical bone has also been observed in postmenopausal osteoporosis [33 – 36], but the association of increased cortical porosity with increased mineral density seems to be unique for transgenic mice overexpressing cathepsin K. Unaltered mineral apposition rates in diaphyses of UTU17+/+ mice indicate unaltered osteoblastic activity.

Table 4 Comparison of the phenotypes of gene-modified mouse models exhibiting enhanced bone resorption

Metaphyseal trabecular bone Amount of bone Number of osteoclasts Bone turnover Number of osteoblasts Osteoblastic activity Diaphyseal cortical bone Thickness Porosity Osteoblastic activity Volumetric mineral density Biomechanical properties Serum biochemical markers Bone degradation products Bone formation markers

UTU17

OPG KO

sRANKL OE

sCSF-1 OE

G-CSF OE

TRACP OE

0 +c +c +c

+ + + +

+ Nr 0 Nr

0 0/+b Nr Nr Nr

+a Nr 0a 0a

0 + Nr +

+ + 0 + 0

0 + +

Nr Nr Nr Nr

+ + + Nr Nr

Nr Nr Nr Nr

Nr Nr Nr Nr Nr

0 +

Nr +

Nr +

Nr Nr

Nr +

Nr Nr

a

Abbreviations: KO, knockout; OE, overexpression; +, increase; , decrease; 0, no change; Nr, not reported. References: UTU17 [17], OPG KO [26,27,32], sRANKL OE [28], sCSF-1 OE [29], G-CSF OE [30], TRACP OE [31]. a Vertebral bone. b On the surface of cortical bone. c During aging.

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Therefore, the accumulation of mineral in diaphyseal cortex of UTU17+/+ mice most likely occurs after the initial mineralization phase during the maturation of the mineral component. This conclusion is further supported by the observation that the increase in cortical bone mineral density in UTU17+/+ mice was most prominent in mid-diaphyses, in the region of the highest degree of mineralization. The organic component of bone matrix is known to be important for both initiation of mineralization and maturation of the mineral component [37]. The importance of an intact network of type I collagen fibrils is exemplified by abnormal mineral deposition in osteogenesis imperfecta (OI) and in a mouse model for OI [38 – 41]. Both exhibit an increased volumetric mineral density of cortical bone, analogous to UTU17+/+ mice. Noncollagenous proteins are also important for mineral deposition as demonstrated by the increased mineral content in bone matrix of mice deficient for osteocalcin [42,43], osteonectin [44,45], and osteopontin [46 – 48]. Since cathepsin K is capable of degrading various organic components of bone matrix, including type I collagen, osteocalcin, and osteonectin [7– 9,49], the enhanced degradation of matrix components in UTU17+/+ mice may affect matrix composition, and thus contribute to the increase in cortical bone mineral density observed in diaphyses of these mice. An expected consequence of the increased cortical porosity in UTU17+/+ mice would be a reduction in the biomechanical properties of cortical bone, which was not seen in these mice. This is probably explained by the increased thickness and mineralization of cortical bone in UTU17+/+ mice. These observations are in line with the concept of biomechanical adaptation [50], known as Wolff’s law, to explain preservation of bone strength and stiffness under conditions of altered bone morphology, i.e., porosity in UTU17+/+ mice. However, even this hypothesis may not be valid for all transgenic models, as impaired biomechanical properties have been observed under enhanced osteoclastogenesis in bones of OPG-deficient and sRANKL overexpression mice [27,28]. The present study confirms our recent report on osteopenia of metaphyseal trabecular bone in the UTU17 mouse model [17]. High-turnover osteopenia has also been observed in OPG-deficient and TRACP overexpression mice [26,29,31,32]. In the present study, the trabecular osteopenia in UTU17+/+ mice was confirmed by pQCT at the age of 1 and 3 months, and by histomorphometry at the age of 3 months. Although dynamic histomorphometry demonstrated elevated rate of bone turnover in mice overexpressing Ctsk at the age of 3 months, the increase was not statistically significant ( P = 0.4079) as we have previously observed at 7 months of age [17]. This can probably be explained by the marked age-dependent reduction in bone turnover that takes place in mice upon aging [51]. Closer analysis of histomorphometric parameters of UTU17 and control mice between 3 and 7 months of age revealed that, except for osteoclast number per bone

perimeter, the levels of all bone parameters in 7-month-old mice were systematically lower than those in 3-month-old mice of the same genotype. Such analyses further demonstrated smaller reduction in bone turnover rate, osteoblast number, mineralizing surface, and in bone formation rate in UTU17 mice than in control mice. This may explain why some of the effects of cathepsin K overexpression only become obvious later in life. We suggest that the highturnover component of the metaphyseal osteopenia in UTU17 mouse model only becomes detectable after cessation of growth, when bone turnover is normally reduced to a much lower level. Differential reactivity of trabecular and cortical bone has also been detected in OPG-deficient mice and in human studies on the effects of fluoride, bisphosphonate, and parathyroid hormone, suggesting fundamental differences in bone turnover between these two compartments [26,32,52– 54]. The conclusion is further supported by various structural and functional differences observed between trabecular and cortical bone, namely in matrix composition and organization, mineralization, turnover activity, and in the orientation of bone remodeling units [55 – 57]. The differential reactivity of trabecular and cortical bone has been suggested to be due to the different environments of bone cells in these two bone compartments [57]. In trabecular bone, bone cells are in intimate contact with cells of the marrow cavity, and subsequently bone turnover may be controlled primarily by local osteotropic cytokines produced by adjacent bone marrow cells. In cortical bone, the cells are more distant from the influences of local cytokines produced by marrow cells, and bone turnover may be controlled more by systemic osteotropic hormones [57]. The differential regulation of bone turnover in these bone compartments most likely also contributes to their differential reactivity in UTU17+/+ mice. The differential responses to cathepsin K overexpression seen in trabecular and cortical bones prompted further analyses of bone turnover using biochemical serum markers. Although the levels of cathepsin K-derived CTX fragment of type I collagen telopeptide [23] were elevated in the sera of growing and adult UTU17+/+ mice, the increase did not reach statistical significance. In humans, bone turnover markers are most useful for monitoring short-term effects of different therapies, whereas their use in predicting bone mass in longterm is limited [24]. This appears to be the case also in UTU17+/+ mice. On the other hand, TRACP 5b activity was decreased in 1-month-old UTU17+/+ mice. TRACP 5b activity has been suggested to correlate with total number of osteoclasts both in an in vitro differentiation assay of osteoclasts [25] and in vivo following orchiectomy in rats [58]. Decreased TRACP 5b activity in the sera of 1-monthold UTU17+/+ mice thus suggests a decrease in total number of osteoclasts in these mice. However, the number of osteoclasts per trabecular bone perimeter (N.Oc/B.Pm) was unaltered in 3-month-old UTU17+/+ mice (Table 3). This may be explained by the reduction in the amount of trabecular

J. Morko et al. / Bone 36 (2005) 854 – 865

bone in UTU17+/+ mice. Interestingly, serum levels of osteocalcin, a bone formation marker, were increased in 1month-old UTU17+/+ mice. Increased levels of bone formation markers have also been observed in sera of OPGdeficient mice [26,32] and of mice overexpressing sRANKL and G-CSF [28,30]. As most of the osteocalcin synthesized is incorporated into bone matrix and released into circulation during bone resorption [24], increased osteoblastic activity and increased bone resorption may both contribute to the elevated osteocalcin levels in UTU17+/+ mice. Analysis of the present gene-modified mouse model suggests that the entire developmental history of bone contributes to the skeletal phenotype of adult mice. In UTU17+/+ mice, increased thickness and density of diaphyseal cortex were observed already at the age of 1 month. This is in line with earlier observations of cathepsin K expression already during embryonic skeletal development in osteoclasts, chondroclasts, and in hypertrophic chondrocytes [5,59]. Overexpression of cathepsin K in UTU17+/+ mice may thus influence the development and growth of endochondral bones at an early stage. This justifies further studies focusing on early endochondral bone development to clarify the mechanisms underlying the skeletal phenotype of UTU17+/+ mice. Together with our previous study, the present study supports the role of cathepsin K as a major proteinase in osteoclastic bone resorption. In transgenic UTU17+/+ mice, excessive cathepsin K alone (i.e., without increased osteoclastogenesis) is sufficient to enhance osteoclastic bone resorption and results in osteopenia of metaphyseal trabecular bone and in increased porosity of diaphyseal cortical bone. However, our results also demonstrate the different nature and reactivity of trabecular and cortical bone in mice. In addition to increased porosity, overexpression of cathepsin K results in increased thickness and mineral density of diaphyseal cortical bone. This was associated with preserved biomechanical properties. The different responses seen in UTU17+/+ mice underline the complexities of skeletal biology, in which a diverse interplay of different cell types, biomechanical forces, and the entire developmental history of the bone contribute to the phenotype. This emphasizes the necessity to evaluate gene functions in living organism, as demonstrated by the unexpected skeletal phenotypes in several transgenic and knockout mouse models.

Acknowledgments The authors are grateful to Dr. Dieter Bro¨mme for providing us with mouse cathepsin K antibodies and to Dr. H. Kalervo Va¨a¨na¨nen for his expert advice. The expert technical help of Ms. Merja Lakkisto, Ms. Tuula Oivanen, Ms. Pirkko Rauhama¨ki, and Ms. Maria Stro¨m is also gratefully acknowledged. Funding: J.M. and R.K. have been recipients of training grants from TULES and TuBS graduate schools. J.M. has

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received personal research grant from the Research and Science Foundation of Farmos and R.K. from the Research and Science Foundation of Farmos and the Finnish Cultural Foundation. This study has been financially supported by research grants from Schering Oy and the Academy of Finland (project #205346).

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