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Endocrinology, doi:10.1210/en.2006-1475
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Endocrinology Vol. 148, No. 6 2778-2787
Copyright © 2007 by The Endocrine Society

Pretreatment with Anticatabolic Agents Blunts But Does Not Eliminate the Skeletal Anabolic Response to Parathyroid Hormone in Oophorectomized Mice

Rana Samadfam, Qingwen Xia and David Goltzman

The Calcium Research Laboratory and the Department of Medicine, McGill University Health Centre and McGill University, Montréal, Québec, Canada H3A 1A1

Address all correspondence and requests for reprints to: David Goltzman, M.D., Royal Victoria Hospital, Room H467, 687 Pine Avenue, Montréal, Québec, Canada H3A 1A1. E-mail: david.goltzman{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have indicated that bisphosphonate pretreatment can inhibit the anabolic actions of PTH. We examined the capacity of two anticatabolic agents with different mechanisms of action, alendronate and osteoprotegerin (OPG), to influence the anabolic activity of PTH. Oophorectomized mice were pretreated for 30 d with alendronate or OPG and then treated for 30 d with the respective anticatabolic alone or the respective anticatabolic plus PTH(1–34). Bones were analyzed by bone mineral density (BMD), microcomputed tomography, histology and histomorphometry, and biochemical bone markers. OPG pretreatment produced a greater inhibition of bone turnover and a greater increase in bone than alendronate. Increases in bone were sustained during subsequent treatment with vehicle or continued administration of the anticatabolic. Pretreatment with each anticatabolic blunted the capacity of PTH to increase BMD and bone volume and continued treatment with each anticatabolic agent also reduced the effectiveness of PTH. Although both anticatabolics decreased the maximal PTH effect, BMD and bone volume increased more when PTH was added than when only anticatabolics were used. These results demonstrate that mechanistically distinct anticatabolics may reduce PTH efficacy, that the characteristics of this inhibition may reflect the different modes of action of the anticatabolics, but that the addition of PTH still provides a skeletal benefit even if the anabolic effect is submaximal.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ANTICATABOLIC AGENTS (1), also referred to as antiresorptive agents, have been the most commonly used osteoporosis therapy for several decades, and among these, bisphosphonates have been the most frequently used. Bisphosphonates are known to localize quite selectively to bone by binding avidly to the mineral phase of bone matrix. This facilitates their incorporation into osteoclasts during the resorptive phase of bone turnover during which they inhibit the enzyme, farnesyl pyrophosphate synthase, thereby preventing signaling of small GTPases and inducing apoptosis in these actively resorbing osteoclasts (2). A novel class of anticatabolic agents has recently been developed based on the discovery of the TNF-related cytokine, receptor activator of nuclear factor-{kappa}B ligand (RANKL) (3, 4). This cytokine can be produced by marrow mesenchymal cells of the osteoblastic lineage and bind to its cognate receptor on osteoclast precursors, maturing osteoclasts, and actively resorbing osteoclasts. It can therefore facilitate the entire sequence of development, activation, and survival of mature osteoclasts. Antibodies to RANKL have recently found utility in the clinic in preventing RANKL activity and inhibiting bone turnover in diseases characterized by excess bone resorption (5). Another component of the RANKL-receptor activator of nuclear factor-{kappa}B pathway is the naturally occurring RANKL antagonist, osteoprotegerin (OPG), a soluble decoy receptor that can potently inhibit bone resorption and indeed when over- expressed in vivo can cause osteopetrosis (6).

Only one anabolic agent is currently available for human use, i.e. PTH (7). The use of PTH in conjunction with anticatabolics has been reported to inhibit the anabolic activity of PTH (8, 9, 10), have no deleterious effect (11, 12), or have accentuating effects (13, 14, 15, 16), in both animal and human studies. In humans two large studies demonstrated that either pretreatment with a bisphosphonate followed by treatment with an anabolic (11) or cotreatment with a bisphosphonate together with an anabolic (10) reduced the efficacy of the anabolic. Nevertheless, although the same bisphosphonate was used, the paradigms may not have been completely comparable inasmuch as the pretreatment study used PTH(1–34), whereas the cotreatment study used PTH(1–84), and it is not clear that the properties of these two anabolic agents are identical. We recently reported the effect of cotreating ovariectomized (OVX) mice with the bioactive amino-terminal fragment of PTH, PTH(1–34) plus either OPG or alendronate (one of the most commonly used bisphosphonates) and demonstrated that cotreatment enhanced the anabolic effect of PTH(1–34) on the skeleton (17). In the present study, we examined the effect of pretreatment of OVX mice with either OPG or alendronate on the anabolic activity of PTH(1–34) to compare the efficacy of the newer class of anticatabolic agent with that of the commonly used bisphosphonate. This pretreatment paradigm more closely represents the more common situation in which these agents will likely be used clinically in view of the widespread use of anticatabolic agents and the current practice of generally initiating osteoporosis therapy with an anticatabolic.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice and materials
Three-month-old oophorectomized and sham-operated C57/BL6 mice were obtained from Harlan Animal Labs (Indianapolis, IN). Recombinant human OPG, recombinant human PTH(1–34), and alendronate were generously provided by Amgen Inc. (Thousand Oaks, CA). All the compounds were freshly prepared in sterile PBS, and PBS alone was administered to animals as the vehicle control group.

In vivo experiments
All animal experiments were carried out in compliance with and approved by the Institutional Animal Care and Use Committee. Mice were maintained in a virus- and parasite-free barrier facility and exposed to a 12-h light, 12-h dark cycle. Fifteen-week-old C57/BL6 mice, oophorectomized at 10 wk of age of age were assigned to three different groups and each pretreated sc for 30 d with PBS, alendronate (100 µg/kg·wk), or OPG (10 mg/kg twice a week). The doses selected were consistent with those that had been reported to reduce bone resorption in mice or rats in vivo (18, 19) and that have been previously used by us (17).

The PBS-pretreated group then received either PBS (10 mice/group) or PTH (80 µg/kg·d; 10 mice/group) for a period of 30 d. The alendronate- and OPG-pretreated groups were each assigned to four different groups (10 mice/group), which received PBS, PTH, or alendronate (if they received pretreatment with alendronate); OPG (if they received pretreatment with OPG); PTH in combination with alendronate (if pretreated with alendronate); or PTH in combination with OPG (if pretreated with OPG). The animals were then killed and blood samples were collected for serum analysis. Tibias and femurs were also obtained for histology and microcomputed tomography (microCT), and humeri for real-time PCR analyses. Bone mineral density (BMD) was measured at time 0, 1, and 2 months after initiating the experiment.

BMD analysis
Mice were anesthetized with a mixture of ketamine (100 mg/kg) and xylazine (0.1 mg/kg) in PBS and placed prone on the platform of a PIXImus densitometer (software version 1.46.007; Lunar Corp., Madison, WI) for BMD and bone mineral content measurements of the whole specimen according to the manufacturer’s instruction. In some experiments, the variability in measurements was examined by repeating scans after repositioning the animals. Percent coefficient of variation of BMD for the repeated scans was 1–3% at all skeletal sites examined.

MicroCT
MicroCT was performed on the left femur after removal of soft tissues and overnight fixation in 4% paraformaldehyde. The distal metaphysis was scanned with a Skyscan 1072 microCT instrument (Skyscan, Antwerp, Belgium) (20). Image acquisition was performed at 100 kV and 98 µA with a 0.9° rotation between frames. Thresholding was applied to the images to segment the bone from the background. The distal 3.5 mm of the femora was reconstructed using two-dimensional data from scanned slices with the 3D Creator software supplied with the instrument. The trabecular bone region of interest was drawn to include cancellous bone in the lower metaphysis, and three-dimensional analysis was performed to calculate bone volume per tissue volume (BV/TV). The resolution of the microCT images is 18.2 µm.

Histology
Lumbar spine and tibias were removed and fixed in a fixative of 2% paraformaldehyde containing 0.075 M lysine and 0.01 M sodium periodate overnight at 4 C and processed histologically as previously described (20). The samples were decalcified in EDTA glycerol solution for 5–7 d at 4 C. Decalcified tibias and other tissues were dehydrated and embedded in paraffin, after which 5-µm sections were cut on a rotary microtome. The sections were stained with hematoxylin and eosin or histochemically for tartrate-resistant acid phosphatase (TRAP) activity (21) or immunohistochemically as described below. Alternatively, undecalcified bone was embedded in LR white acrylic resin (London Resin Co., London, UK), and 1-µm sections were cut on an ultramicrotome. These sections were stained for mineral with the von Kossa staining procedure and counterstained with toluidine blue.

Immunohistochemical staining
Mouse monoclonal antibody against the type 1 PTH receptor (Upstate Cell Signaling Solutions, Charlottesville, VA) was applied to dewaxed paraffin sections overnight at room temperature. As a negative control, preimmune serum was substituted for the primary antibody. After washing with high salt buffer [50 mM Tris-HCl, 2.5% NaCl, 0.05% Tween 20 (pH 7.6)] for 10 min at room temperature followed by two 10-min washes with Tris-buffered saline, the sections were incubated with secondary antibody (biotinylated goat antimouse IgG; Sigma, St. Louis, MO), washed as before, and incubated with the Vectastain ABC-AP kit (Vector Laboratories, Burlington, Ontario, Canada) for 45 min. After washing as before, red pigmentation was produced by incubating with a substrate of alkaline phosphatase, i.e. fast red TR/Naphthol AS-MX phosphate (Sigma), containing 1 mM levamisole as endogenous alkaline phosphatase inhibitor, for 10–15 min. After washing with distilled water, the sections were counterstained with methyl green and mounted with Kaiser’s glycerol jelly.

Histochemical staining for TRAP
Enzyme histochemistry for TRAP was performed as previously described (21). Dewaxed sections were preincubated for 20 min in buffer containing 50 mM sodium acetate and 40 mM sodium tartrate (pH 5.0). Sections were incubated for 15 min at room temperature in the same buffer containing 2.5 mg/ml naphthol AS-MX phosphate (Sigma) in dimethylformamide as substrate and 0.5 mg/ml fast garnet GBC (Sigma) as a color indicator for the reaction product. After washing with distilled water, the sections were counterstained with methyl green and mounted in Kaiser’s glycerol jelly.

Double-calcein labeling
Double-calcein labeling was performed by ip injection of mice with 10 µg calcein/g body weight (C-0875; Sigma) 10 and 3 d before the animals were killed. Bones were harvested and embedded in methyl methacrylate matrix without decalcification. Serial sections were cut, and the freshly cut surface of each section was viewed and imaged using fluorescence microscopy. The double-calcein interlabel width was measured in the secondary spongiosa using Northern Eclipse image analysis software (version 6.0; Empix Imaging Inc., Mississauga, Ontario, Canada) and the mineral apposition rate (MAR; MAR = interlabel width/labeling period) was calculated.

Histomorphometry
Histomorphometric measurements were performed below the growth plate at the proximal tibial metaphysis. The parameter measured for bone structure was the total BV/TV (percent). The parameter measured for bone resorption was the number of osteoclasts per bone perimeter (millimeters). The parameters obtained for bone formation were the osteoblast perimeter per bone perimeter (percent), MAR (per day per year), and mineralizing surface (mineralizing surface/bone surface, percent).

Serum osteocalcin and TRAP-5b levels
A mouse osteocalcin two-site immunoradiometric assay (Immutopics Inc., San Clemente, CA) was used for the measurement of serum osteocalcin levels according to the manufacturer’s specifications. A mouse TRAP-5b assay (IDS Inc., Fountain Hills, AZ) was used for the determination of osteoclast-derived TRAP-5b in mouse serum samples. The assay was performed according to the manufacturer’s specifications.

Computer-assisted image analysis
After hematoxylin and eosin staining or histochemical or immunohistochemical staining of sections from four mice of each group, images of fields were photographed with a digital camera (Sony, New York, NY). Images of micrographs from single sections were digitally recorded using a rectangular template, and recordings were processed and analyzed using Northern Eclipse image analysis software (Empix Imaging Inc.).

Quantitative real-time PCR
RNA was isolated from mouse humerus, using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The RNeasy plus mini kit (QIAGEN, Valencia, CA) was then used to purify RNA from any genomic DNA contamination. Reverse transcription reactions were performed using the SuperScript first-strand synthesis system (Invitrogen). To determine the number of cDNA molecules in the reverse transcription samples, real-time PCR was performed using the LightCycler system (Roche Molecular Biochemicals, Indianapolis, IN). The conditions were 2 µl of LightCycler DNA master SYBR Green I (Roche), 0.25 µM of each 5' and 3' primer (see Table 1Go), and 2 µl of sample and/or H2O to a final volume of 20 µl. The MgCl2 concentration was adjusted to 3 mM. Samples were amplified for 35 cycles with a temperature transition rate of 20 C/sec for all three steps, which were denaturation at 95 C for 10 sec, annealing for 5 sec, and extension at 72 C for 20 sec. SYBR green fluorescence was measured to determine the amount of double-stranded DNA. To discriminate specific from nonspecific cDNA products, a melting curve was obtained at the end of each run. Products were denatured at 95 C for 3 sec; the temperature was decreased to 58 C for 15 sec and raised slowly from 58 to 95 C using a temperature transition rate of 0.1 C/sec. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as the ratio of osteocalcin mRNA to GAPDH mRNA and RANKL mRNA to GAPDH mRNA.


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  		TABLE 1. RT-PCR primers used with their name, orientation, sense (S), and antisense (AS) sequence, annealing temperature (Tm), and length of PCR product

 
Statistical analysis
Statistical comparisons, using Graph-Pad Prism (GraphPad Software Inc., San Diego, CA) were made using ANOVA, with a Bonferroni adjustment. P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of alendronate or OPG pretreatment on the anabolic response to PTH
Pretreatment with alendronate did not significantly increase the BMD in oophorectomized mice in the lumbar spine (Fig. 1AGo, column 2 vs. column 1) and femur (Fig. 2AGo, column 2 vs. column 1) whereas the BMD was substantially increased by pretreatment with OPG (Figs. 1AGo and 2AGo, column 3 vs. column 1). In PBS-pretreated OVX mice, PTH treatment also markedly increased the BMD of the lumbar spine (Fig. 1AGo, column 5 vs. column 4, 25% increase) and the femur (Fig. 2AGo, column 5 vs. column 4, 21% increase), compared with the control group (PBS treated). In alendronate-pretreated animals, when PTH was administered alone, the BMD increase was 68 and 61% less in the lumbar spine and the femur, respectively, than that observed in the absence of pretreatment with an anticatabolic (Figs. 1AGo, column 8 vs. column 5, P < 0.05, and 2AGo, column 8 vs. column 5, P < 0.05), whereas a combination of PTH plus alendronate was able to significantly increase the BMD by 27% (Fig. 1AGo, column 9 vs. column 6, P < 0.001) and 17% (Fig. 2AGo, column 9 vs. column 6, P < 0.001), i.e. to almost the same extent as in the group treated with PTH but without prior anticatabolic therapy. In contrast, the effect on BMD achieved by adding PTH after OPG pretreatment was 15% greater in the lumbar spine (P < 0.05) and 7% greater in the femur (P < 0.05) than the BMD achieved with PTH treatment preceded by PBS (Figs. 1AGo and 2AGo, column 12 vs. column 5). However, the combination of PTH plus OPG after OPG pretreatment failed to further increase the BMD (Figs. 1AGo and 2AGo, column 13 vs. column 12). The greater increases in BMD occurring with PTH alone after OPG pretreatment, compared with PTH alone after alendronate pretreatment (Fig. 1AGo, column 8 vs. column 12, 12%, P < 0.05; and Fig. 2AGo, column 12 vs. column 8, 5%, P < 0.05), reflected the greater increases in BMD produced by OPG relative to alendronate before initiating the PTH treatment (Figs. 1AGo and 2AGo, column 3, compared with column 2). Similarly, the greater increases in BMD occurring with concomitant treatment of PTH plus OPG relative to concomitant treatment of PTH plus alendronate may have been due to the greater increases in OPG relative to alendronate produced at the end of the 60 d of treatment with OPG relative to alendronate (Fig. 1Go, A and B, column 11, compared with column 7, P < 0.01 in each case).


Figure 1
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  		FIG. 1. A, BMD of the lumbar spine after 30 d of pretreatment with PBS, alendronate (ALN), or OPG (pretreatment period, d 0–30) or after an additional 30 d of treatment with the single or combined therapies noted (treatment period, d 30–60). B, Net increase in lumbar spine calculated as BMD at d 60 for single or combined therapies minus BMD at d 30 for the pretreatment agent administered for that group, i.e. PBS, ALN, or OPG. In B, column 7 shows the sum of the net increases in BMD elicited by ALN plus PTH (individually depicted in columns 4 and 5), and column 12 represents the sum of the net increases in BMD elicited by OPG plus PTH (individually depicted in columns 9 and 10). Data are shown as the mean ± SEM (n = 7). *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with vehicle (PBS) in each group; {wedge}, P < 0.05, compared with PTH in PBS pretreated group; #, P < 0.05, compared with OPG+PTH.

 

Figure 2
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  		FIG. 2. A, BMD of the whole femur after 30 d of pretreatment with PBS, alendronate (ALN), or OPG (pretreatment period, d 0–30) or after an additional 30 d of treatment with the single or combined therapies noted (treatment period, d 30–60). B, Net increase in femur calculated as BMD at d 60 for single or combined therapies minus BMD at d 30 for the pretreatment agent administered for that group, i.e. PBS, ALN, or OPG. In B, column 7 shows the sum of the net increases in BMD elicited by ALN plus PTH (individually depicted in columns 4 and 5), and column 12 represents the sum of the net increases in BMD elicited by OPG plus PTH (individually depicted in columns 9 and 10). Data are shown as the mean ± SEM (n = 7). *, P < 0.05; **, P < 0.01; and ***, P < 0.001, compared with vehicle (PBS) in each group; {wedge}, P < 0.05, compared with PTH in PBS pretreated group; #, P < 0.05, compared with OPG+PTH.

 
Therefore, to determine whether pretreating with anticatabolic agents was having an enhancing or inhibiting effect on PTH, we compared the observed net effects on BMD produced by the treatments by subtracting BMD on d 30 (the final day of pretreatment) from BMD on d 60 (the final day of treatment) (Figs. 1BGo and 2BGo). In addition, we determined the predicted net effects of the combination therapy with alendronate by adding the net effects, after alendronate pretreatment, of PTH alone with the net effect of alendronate alone (Figs. 1BGo and 2BGo, column 7), and we determined the predicted net effects of the combination therapy with OPG by adding the net effect, after OPG pretreatment, of PTH alone with the net effect of OPG alone (Figs. 1BGo and 2BGo, column 12). The results indicated that in both the lumbar spine and femur, the increases in BMD in response to PTH treatment after pretreatment with alendronate was diminished relative to BMD increases in response to PTH after pretreatment with PBS (Figs. 1BGo and 2BGo, column 5 vs. column 2); however, the reduction was not significant after pretreatment with OPG (Figs. 1BGo and 2BGo, column 10 vs. column 2). In contrast, continued treatment with alendronate did not further diminish the blunted response to PTH, whereas continued treatment with OPG did (Figs. 1BGo and 2BGo, columns 6 and 11). Furthermore, in both the lumbar spine and femur, concomitant treatment of PTH with alendronate produced BMD increases that were at least as great as or greater than the predicted increase (Figs. 1BGo and 2BGo, column 6 vs. column 7), whereas with OPG the observed increase in BMD was lower than the predicted increase (Figs. 1BGo and 2BGo, column 11 vs. column 12). Of great importance, however, was that in all cases PTH treatment after pretreatment with either anticatabolic agent provided enhanced benefit relative to continued treatment with either anticatabolic agent alone (Figs. 1BGo and 2BGo, column 5 vs. columns 4 and 10 vs. column 9).

The femur samples obtained from all groups were also analyzed ex vivo by microCT analysis. The results demonstrated an increase in bone density in all treated groups, compared with vehicle (Fig. 3AGo). Furthermore, the increase in trabecular bone of the lumbar spine and femur of animals pretreated with OPG alone was greater than with alendronate-pretreated animals. These results closely resembled those of von Kossa staining of femur sections (Fig. 3BGo). MicroCT analysis of BV/TV (Fig. 3CGo) showed similar findings to the results obtained from densitometry (Figs. 1Go and 2Go).


Figure 3
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  		FIG. 3. MicroCT and histological analyses of bone specimens after pretreatment with PBS, alendronate (ALN), or OPG followed by treatment with the single or combined agents noted. A, MicroCT analysis of the femur. B, Representative sections of Von Kossa staining of the femur showing mineralized bone. C, BV/TV in femur. Data are shown as mean ± SEM (n = 4). *, P < 0.05, **, P < 0.01, and ***, P < 0.001, compared with PBS-pretreated, PTH-treated animals.

 
Effect of single and combination therapies on osteoclasts after pretreatment with anticatabolic agents
OPG pretreatment for a month dramatically decreased the number of osteoclasts (data not shown), and this was sustained during the treatment period with PBS and OPG (Fig. 4Go, A and B). Treatment with PTH alone or OPG plus PTH for 30 d failed to further increase the TRAP-positive cells (Fig. 4Go, A and B). Alendronate pretreatment also decreased the number of osteoclasts but to a lesser extent (data not shown), and this was sustained during the subsequent treatment phase with PBS. The reductions were slightly more marked with continued alendronate administration during the treatment phase (Fig. 4Go, A and B). PTH alone significantly increased the number of TRAP-positive osteoclasts in alendronate-pretreated animals. Alendronate in combination with PTH reduced the increase in resorbing surface seen with PTH treatment alone (Fig. 4BGo).


Figure 4
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  		FIG. 4. Effect of single and combination therapies on the number and activity of osteoclasts after pretreatment with PBS, alendronate (ALN), or OPG. A, Representative sections of osteoclasts, demonstrating TRAP staining. B, Quantitation of the number of TRAP-positive osteoclasts per bone perimeter. C, Serum levels of TRAP-5b. Data are the mean ± SEM (n = 10). *, P < 0.05, **, P < 0.01, and ***, P < 0.001, compared with the PBS-treated group in PBS-pretreated animals.

 
The circulating levels of the bone resorption marker TRAP-5b (Fig. 4Go C) correlated well with the number of osteoclasts and resorbing surface perimeter observed by histology and histomorphometry (Fig. 4Go, A and B, respectively).

Effect of single and combination therapies on osteoblasts after pretreatment with anticatabolic agents
PTH treatment increased the osteoblast perimeter over the bone perimeter (Ob.Pm/B.Pm) by 110% in PBS-pretreated mice (Fig. 5Go, A and B). Alendronate and OPG pretreatment reduced Ob.Pm/B.Pm by 35% (P < 0.05) and 48% (P < 0.05), respectively, from the PBS pretreatment control, and these reductions were sustained after PBS treatment and after treatment with either alendronate or OPG, respectively (Fig. 5BGo). Nevertheless, PTH alone increased the Ob.Pm/B.Pm in both alendronate- and OPG-pretreated groups (by 97 and 36%, respectively, Fig. 5Go, A and B). Similar respective increases were observed when PTH was used in combination with either anticatabolic agent. These increases were less than the increases of osteoblast perimeter in the animals treated with PTH after PBS pretreatment (Fig. 5Go, A and B).


Figure 5
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  		FIG. 5. Effects of single and combination therapies on the number and activity of osteoblasts after pretreatment with PBS, alendronate (ALN), or OPG. A, Representative sections of osteoblasts, denoted by immunohistochemical staining for the PTH receptor. B, Quantitation of osteoblast perimeter per bone perimeter. C, Serum levels of osteocalcin. Data are shown as mean ± SEM (n = 7). *, P < 0.05, and ***, P < 0.001, compared with the PBS-treated group in PBS-pretreated animals.

 
Compared with PBS pretreatment, both alendronate and OPG pretreatment reduced circulating levels of the bone formation marker, osteocalcin, but the reductions were greater with OPG pretreatment (25% reduction for alendronate and 76% for OPG, i.e. column 4 relative to column 1 and column 8 relative to column 1). These decreases were sustained after PBS treatment and after treatment with either alendronate or OPG, respectively (Fig. 5CGo). PTH treatment increased the osteocalcin levels in the PBS pretreated group by 37%, (P < 0.05) but was less effective in the groups pretreated with the anticatabolics, particularly alendronate (Fig. 5CGo).

Effects of single and combination therapies on expression of skeletal genes after pretreatment with anticatabolic agents
PTH treatment, after pretreatment with PBS, markedly increased both osteocalcin and RANKL gene expression (Fig. 6Go, A and B). PTH-induced increases in osteocalcin and RANKL were, however, reduced by continued treatment with alendronate after alendronate pretreatment (Fig. 6Go, A and B, column 6 relative to column 2) and continued treatment with OPG after OPG pretreatment (Fig. 6Go, A and B, column 10 relative to column 2).


Figure 6
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  		FIG. 6. Gene expression of osteocalcin (A) and RANKL (B) in skeletal tissue of OVX mice pretreated and then treated as indicated. Osteocalcin, RANKL, and GAPDH were determined by real-time PCR as described in Materials and Methods, and the ratios of osteocalcin relative to GAPDH and RANKL relative to GAPDH are depicted for each treatment. Data are shown as mean ± SEM (n = 3). *, P < 0.05, compared with the PBS-treated in each group.

 
Effect of single and combination therapies on bone formation in vivo after pretreatment with anticatabolic agents
PTH injection after PBS pretreatment markedly increased the total calcein labeled mineralizing surfaces and also the MAR (Fig. 6Go), whereas PTH alone or in combination with alendronate produced more modest increases in these parameters in the alendronate-pretreated group. OPG pretreatment almost completely eliminated the mineralizing surfaces and blocked the MAR (data not shown), and this was sustained during the 30-d treatment period with either PBS or OPG (Fig. 6Go, B and C). PTH treatment alone or in combination with OPG was able to slightly increase the mineralizing surface and MAR. These results are consistent with the levels of bone formation markers (Fig. 5Go) in these animals.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that concomitant inhibition of bone resorption by administration of anticatabolic agents with stimulation of bone formation by exogenously administered PTH has a beneficial effect on various indices, reflecting the skeletal anabolic activity of PTH. In that study, OPG and alendronate, two mechanistically distinct anticatabolics, prolonged the beneficial effect of PTH and improved bone volume, mechanical strength, and appendicular and axial BMD in aged oophorectomized mice (17). However, there is substantial evidence from animal models and human studies indicating that bisphosphonates, when given in combination with PTH, will inhibit the anabolic effects of PTH(8, 9, 10, 11). In most of those studies, PTH(1–34) treatment was introduced after anticatabolics were already in use. To explore whether prior administration of an anticatabolic would produce different results from a cotreatment protocol in our animal model and simulate the results achieved in humans, we explored the effect of anticatabolic pretreatment on various indices of the anabolic response to exogenous PTH in oophorectomized mice. We also assessed whether pretreatment with two anticatabolics that use different molecular mechanisms, i.e. a bisphosphonate and an inhibitor of RANKL, would similarly interfere with the bone-forming actions of PTH when administered alone or concomitantly with the anticatabolic. One of the limitations of our study was clearly the use of single doses of each anticatabolic agent; however, the combinatorial design of this large in vivo study clearly precluded the use of multiple doses.

Our studies show that in association with the more potent effect of OPG on inhibiting bone resorption, OPG pretreatment produced a more profound increase in bone accrual than alendronate. Consequently, in this 30-d pretreatment period, bone formation must have exceeded resorption to a greater extent than in the alendronate-treated animals. This bone accrual was sustained during the second, 30-d phase of the study, even when only PBS was used for treatment (Figs. 1–3GoGoGo), demonstrating the longevity of the effects of both agents. Furthermore, no further major change in bone accrual occurred, even when each anticatabolic agent was continued during the second phase of the study (Figs. 1–3GoGoGo). Consequently, any modest additional reduction of resorption occurring during the continued treatment with either anticatabolic agent (Fig. 4Go) was associated with a reduction in bone formation (Fig. 7Go).


Figure 7
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  		FIG. 7. In vivo bone formation induced by single and combination therapies after pretreatment with PBS, alendronate (ALN), or OPG. Mineralizing surfaces (A) and MARs (B) from lumbar spine cross-sections from PBS-pretreated, ALN-pretreated, or OPG-pretreated animals. Data are shown as mean ± SEM (n = 4). *, P < 0.05, **, P < 0.01, and ***, P < 0.001, compared with the PBS-treated group in PBS-pretreated animals; {wedge}, P < 0.05, compared with the PBS-treated group in OPG-pretreated animals.

 
OPG produced almost total inhibition of MAR and mineralizing surface, and these indices were only minimally increased by PTH (Fig. 7Go). However, if one calculates the level of an index of bone formation induced by PTH after OPG pretreatment, e.g. the level of Ob. Pm/B.Pm (from Fig. 5Go), relative to the level of an index of resorption occurring with PTH after OPG pretreatment, e.g. by number of osteoclasts per bone perimeter (from Fig. 4Go), a ratio of 3.5 is obtained. If one calculates the level of the same index of bone formation induced by PTH after alendronate pretreatment relative to the level of the same index of bone resorption, a ratio of 0.16 is obtained. Consequently the PTH anabolic action relative to its catabolic action is reduced more with alendronate pretreatment than with OPG. Similar results are obtained using the bone turnover markers osteocalcin (from Fig. 5Go) and TRAP-5b (from Fig. 4Go) as indices of formation and resorption, respectively. This appeared to be reflected in the BMD results obtained for PTH treatment after pretreatment with OPG or alendronate. Consequently, determining the levels of the MAR or the mineralizing surface per se do not appear to reflect the net bone anabolic response to PTH as reliably as assessing the level of suppression of bone formation relative to the level of suppression of bone resorption, even when both are markedly suppressed, as after OPG pretreatment.

Pretreatment with each anticatabolic agent, however, reduced the anabolic efficacy of PTH(1–34) (Figs. 1–3GoGoGo), although the mechanisms used by each agent may have been both similar and discrete. Both anticatabolics inhibit bone resorption due to inhibition of the activity of mature multinucleated osteoclasts. Consequently, both may have reduced the osteoblast pool on which PTH acts by inhibiting the release of either osteoblast-stimulating factors from these mature osteoclasts or osteoblast growth factors from resorbed bone matrix (22). This is therefore consistent with the important regulatory role that resorbing osteoclasts can exert on osteoblast pools, even in the presence of PTH.

In view of the fact that direct effects of alendronate on inhibiting cells of the osteoblast lineage has been reported, at least in vitro (17, 23, 24), it is possible that direct interference with the PTH target pool of osteoblasts may have occurred. However, when PTH was added with alendronate, a slight increase in the anabolic effect of PTH was observed (Figs. 1–3GoGoGo). This may have reflected the slight further decrease in bone resorption that was observed by continued use of alendronate (Fig. 4Go) and that may have accentuated the anabolic activity of PTH as was previously observed by us in cotreatment studies of anticatabolics with PTH in treatment-naïve animals (17). On the other hand, use of PTH in association with OPG in the second phase of the current studies tended to slightly reduce rather than enhance the effectiveness of PTH. This may reflect the unique activity of this inhibitor of the RANKL pathway to also inhibit the full spectrum of the osteoclastic lineage (in contrast to bisphophonates). Thus, continued use of OPG in this model may have inhibited the release of osteoblast-stimulating factors from such osteoclast progenitors, which may have had a delayed impact in partially compromising the efficacy of PTH. Overall, therefore, our results indicate that pretreatment with both anticatabolics is able to blunt the anabolic response to PTH, although the mechanisms that they exhibit to produce this effect is likely different.

It is possible, but unclear at present, whether the blunting of the PTH effect would be reduced with anticatabolic agents that are more short lasting, irrespective of their precise site of action as antiresorptives; that is, it is possible that a more transient inhibition could be beneficial in increasing the osteoblast pool, whereas sustained inhibition of resorption could be detrimental. Newer agents, such as inhibitors of cathepsin K, a cysteine protease that is involved in osteoclastic cleavage of collagen (25), and inhibitors of {alpha}Vß3 integrin (the vitronectin receptor) through which osteoclasts bind to bone extracellular matrix proteins (26) are being evaluated as potential agents of value in osteoporosis treatment. It will therefore be of interest to determine the capacity of such agents to interact with PTH.

Notwithstanding our observations on the reduction of PTH action on bone by the two anticatabolic agents we examined, BMD and bone volume were increased when PTH was added after pretreatment with either anticatabolic treatment. Similar results have been found in the lumbar spine in human studies using PTH(1–34) and alendronate (13). Consequently, although the maximum anabolic effect of PTH(1–34) may be achieved by omitting prior treatment with an anticatabolic, our results indicate that increased benefit can still accrue by using an anabolic agent after using either class of anticatabolic.


   Acknowledgments
 
The authors acknowledge the excellent facilities of the Centre for Bone and Periodontal Research at McGill University, where imaging and histology were performed. We thank Dr. Paul Kostenuik (Amgen) for providing important materials and assistance with the study and his very thoughtful review of the manuscript and Stephen Adamu for kindly performing the biomarker assays.


   Footnotes
 
First Published Online March 22, 2007

Abbreviations: BMD, Bone mineral density; BV/TV, bone volume per tissue volume; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MAR, mineral apposition rate; microCT, microcomputed tomography; Ob.Pm/B.Pm, osteoblast perimeter over the bone perimeter; OPG, osteoprotegerin; OVX, ovariectomized; RANKL, receptor activator of nuclear factor-{kappa}B ligand; TRAP, tartrate-resistant acid phosphatase.

This work was supported by the Canadian Institutes of Health Research (CIHR) (to D.G.). R.S. is a Bone Scholar of the CIHR Skeletal Health Training Program.

Author Disclosures: R.S. and Q.X. have nothing to declare. D.G. consults intermittently for Amgen, Lilly, and Merck.

Received November 6, 2006.

Accepted for publication March 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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