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Endocrinology Vol. 144, No. 9 4106-4116
Copyright © 2003 by The Endocrine Society

Collagenase Cleavage of Type I Collagen Is Essential for Both Basal and Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor-Induced Osteoclast Activation and Has Differential Effects on Discrete Bone Compartments

R. Chiusaroli, A. Maier, M. C. Knight, M. Byrne, L. M. Calvi, R. Baron, S. M. Krane and E. Schipani

Cell Biology and Orthopedics, Yale University (R.C., R.B.), New Haven, Connecticut 06520; and Endocrine Unit (A.M., M.C.K., L.M.C., E.S.) and Arthritis Unit (M.B., S.M.K.), Massachusetts General Hospital-Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Dr. E. Schipani, Endocrine Unit, Massachusetts General Hospital-Harvard Medical School, Boston, Massachusetts 02114. E-mail: schipani{at}helix.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Expression of a constitutively active PTH/PTHrP receptor in cells of osteoblast lineage in vivo (CL2+) causes increases in trabecular bone volume and trabecular bone formation and, conversely, a decrease in the periosteal mineral apposition rate. Collagenase-3 (matrix metalloprotease-13) is a downstream target of PTH action. To investigate the relevance of collagenase cleavage of type I collagen for the CL2+ bone phenotype, we bred CL2+ animals with mice carrying a mutated col1{alpha}1 gene that encodes a protein resistant to digestion by collagenase-3 and other collagenases (rr). Adult tibias and parietal bones from 4-wk-old double-mutant animals (CL2+/rr) and from control littermates were analyzed. Trabecular bone volume was higher in CL2+/rr than in CL2+ mice. This increase occurred despite a modest reduction in bone formation rate, which was, however, still significantly higher that in wild-type littermates, and therefore must reflect decreased bone resorption in rr mice. Osteoclast number was increased in CL2+/rr animals compared with either wild-type or CL2+ mice, suggesting that collagenase-dependent collagen cleavage affected osteoclast function rather than osteoclast number and/or differentiation. Interestingly, the periosteal mineral apposition rate was similar in CL2+/rr and CL2+ animals and was significantly lower than that in wild-type animals. Our study provides evidence that collagenase activity is important for both basal and PTH/PTHrP receptor-dependent osteoclast activation. Furthermore, it indicates that a mild impairment of osteoclast activity is still compatible with increased osteoblast function. Lastly, it supports the hypothesis that collagenases can be a downstream effector of PTH/PTHrP receptor action in trabecular bone, but not in periosteum.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN BONE remodeling, the activities of osteoblasts, the bone-forming cells, and osteoclasts, multinucleated giant cells of hemopoietic origin, whose physiological role is to resorb bone, must be carefully balanced to maintain skeletal and calcium homeostasis (1, 2). The importance of understanding the factors controlling this process is highlighted by metabolic bone disorders such as osteoporosis, in which the imbalance of bone formation and resorption leads to net bone loss.

PTH is a major regulator of mineral ion metabolism and bone turnover, mainly through activation of the PTH/PTHrP receptor, a seven-transmembrane, G protein-coupled receptor (3). Functional PTH/PTHrP receptors are present on cells of the osteoblast lineage rather than on osteoclasts (4). PTH acts on these mesenchymal cells, which, through direct cell-cell contact mediated by cell-bound ligands such as receptor activator of nuclear factor-{kappa}B ligand and production of soluble ligands, modulates osteoclast recruitment and activation (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). The chronic hypersecretion of PTH, as seen in patients with primary hyperparathyroidism, is responsible for an increase in both bone resorption and bone formation. In most cases this results in preservation of cancellous bone and decreased thickness of cortical bone (16, 17, 18). In vivo administration of amino-terminal fragments of PTH has been shown to have either anabolic or catabolic effects on bone, the net result depending on dose, method of administration (continuous vs. intermittent), and bone compartment (trabecular vs. cortical) (19, 20, 21, 22, 23, 24). To date, the mechanisms underlying this dual effect are incompletely understood. As PTH analogs are among the few anabolic agents currently proposed for treatment of osteoporosis (25), elucidation of their mechanisms of action has great relevance for therapeutic intervention.

Collagenase, in particular, collagenase-3 [matrix metalloprotease-13 (MMP-13)], is a known downstream target of PTH action (26, 27). Collagenase-3 is a MMP normally expressed in cells of the osteoblast lineage and in hypertrophic chondrocytes during embryonic development and adult skeletal remodeling (26, 27, 28). It has been suggested that collagenases could act as a coupling factor for activation of osteoclasts (29, 30). In this regard, it has been shown that collagenase cleavage of the extracellular matrix modifies cell-matrix interactions and cell motility and function by increasing the availability of integrin-binding sites at the extracellular membrane surface (31, 32, 33, 34). Mice that produce a mutant, collagenase-resistant type I collagen have been generated (35). The mutation was targeted to Col1a1 in the region that encodes the single site in the helical domain of the {alpha}1(I) chains where collagenase cleavage occurs. Mutant animals that express the targeted mutation on both alleles (rr) of Col1a1 produce type I collagen molecules that cannot be cleaved by collagenase-3 and other collagenases. These mice display an increase in periosteal bone formation (28) and are resistant to PTH-induced bone resorption (35).

We have recently developed transgenic mice (CL2+ mice) expressing constitutively active PTH/PTHrP receptors in cells of the osteoblast lineage under the control of the mouse {alpha}1(I) collagen gene promoter (36). The CL2+ mice have a vivid postnatal phenotype, characterized by increased trabecular bone volume and trabecular mineral apposition rate, decreased thickness of cortical bone and periosteal mineral apposition rate, and increased number of differentiated osteoclasts in both trabecular and cortical bone. In the present study we generated double-mutant mice CL2+/rr, and we investigated their adult bone phenotype. The goal of the study was 2-fold: 1) to investigate in vivo the role of collagenase-mediated collagen cleavage in generating the CL2+ transgenic phenotype, and 2) to establish whether collagenase could be involved in the differential effects that activation of the PTH/PTHrP receptor has on cortical and trabecular bone remodeling, respectively.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of CL2+/rr mice
Generation of CL2+ and rr mice (Col1a1tml Jae) has been previously reported (35, 36). To generate CL2+/rr double-mutant animals, FVB male mice hemizygous for a mutant human PTH/PTHrP receptor cDNA driven by a 2.3-kb fragment of the collagen 1{alpha}1 promoter (CL2+) were bred with C57 females carrying the homozygous Col1a1 mutation (rr); appropriate matings were then carried out. Genotyping of the mutant animals was performed as previously described (35, 36). Appropriate institutional animal care and use committee approval was obtained for this study.

Preparation of collagens and digestion with collagenases
Collagens were extracted from mouse tails and digested with recombinant mouse collagenase-3 as previously described (37).

Histological analysis
For histological analysis, transgenic mice and sex-matched, wild-type littermates were killed by cervical dislocation 4 wk after birth. Tissues removed from transgenic and wild-type mice were fixed and stored as previously described (38). In selected cases, hindlimbs and/or skulls were decalcified (38), and paraffin blocks were prepared by standard histological procedures. For selected samples, tartrate-resistant acid phosphatase (TRAP) staining was performed using an acid phosphatase detection kit (Sigma-Aldrich Corp., St. Louis, MO).

Histomorphometry
For histomorphometric analysis, transgenic mice and wild-type littermates were killed by cervical dislocation at 4 wk of age after being previously injected with calcein (30 mg/kg) 7 and 2 d before sacrifice. Bone specimens were fixed in 4% formalin and then embedded in methylmethacrylate as previously described (36). Five-micrometer sections were cut and stained with Toluidine Blue or with the von Kossa method for calcified tissues, or with Alcian Blue to visualize cartilage remnants in trabecular bone [1% Alcian Blue (Sigma-Aldrich Corp.) in 0.2 M HCl, stain for 20 min at 37 C]. Ten-micrometer sections from the same samples were cut and placed on slides without staining, and coverslips were applied for dynamic measurements. Histomorphometric analysis was carried out with an Osteomeasure system (Osteometrics Inc., Atlanta, GA), using standard procedures (39) in a blind fashion. Tibial sections were measured in the proximal metaphysis beginning 340 µm below the chondro-osseous junction. Tibial cortical thickness and periosteal mineral appositional rates were measured beginning 680 µm from the chondro-osseous junction on the anterofibular side. Coronal sections of the parietal bone were measured immediately frontal to the interparietal bone, excluding 300 µm on either side of the sagittal suture. A minimum of six animals per group were examined. Statistical analysis was performed using ANOVA, with P < 0.05 accepted as significant; error bars represent ±SD.

In situ hybridization
Tissues were fixed in 3.7% formaldehyde/PBS overnight at 4 C, processed, embedded in paraffin, and cut. In situ hybridization was performed as described previously (40) using complementary 35S-labeled riboprobes.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of CL2+/rr double-mutant mice
CL2+ transgenic male mice on the FVB/N background were mated with heterozygous female r mutant animals on the C57BL/6/SV129J background. After appropriate matings, double-mutant Cl2+/rr mice carrying both a constitutively active PTH/PTHrP receptor in cells of the osteoblast lineage and a mutant collagen resistant to collagenase activity were generated. Cl2+/rr mice were viable, but significantly smaller than either Cl2+ or rr littermates. Resistance of the mutant collagen to collagenase activity in the mixed FVB/N-C57BL/6-SV129J backgrounds was confirmed by enzymatic digestion with recombinant mouse collagenase-3 of collagen extracted from tails of Cl2+, rr, Cl2+/rr, and wild-type littermates as well as r+ littermates as previously described (35) (Fig. 1Go). Note that collagen extracted from the mice heterozygous for the collagenase resistance mutation (r+) was partially cleaved, but less than that in wild-type animals (++), and there was no cleavage of the rr collagen.



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  		FIG. 1. Collagen digestion by collagenase. Collagen was extracted from mouse tails and digested with recombinant mouse MMP-13 (r-mouse MMP-13); the digested collagen products were run on a polyacrylamide gel and stained with Coomassie Blue. Note that heterozygosity for the r mutation causes partial resistance and homozygosity produces complete resistance to collagen cleavage by mouse MMP-13.

 
Osteopetrotic phenotype of collagenase-resistant (rr) mice
X-Ray analysis performed on tibiae showed a mild, but clear, increase in radiodensity in the metaphyseal area of tibiae isolated from rr mice compared with wild-type specimens (Fig. 2AGo). The von Kossa staining confirmed the increase in trabeculation of the metaphyseal regions of the long bones from rr animals (Fig. 2BGo), which extends well beyond the area normally occupied by the secondary spongiosa. The trabeculae in the mutant specimens were mineralized as shown by von Kossa staining (Fig. 2BGo) and histomorphometry (data not shown). These trabeculae, similar to those in the primary spongiosa, all contained unresorbed cartilage remnants, as demonstrated by Alcian Blue staining (Fig. 3Go).



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  		FIG. 2. A, X-Ray analysis performed on tibias from 4-wk-old wild-type, rr, CL2+, and CL2+/rr mice (left to right). A mild, but clear, increase in radiodensity in the metaphyseal area of rr tibias is shown. An increase in radiodensity is also observed in the bone marrow area of tibiae from CL2+ mice, whereas cortical bone appeared less dense. In the double-mutant CL2+/rr mice, a dramatic increase in radiodensity is evident all along the bone marrow cavity, whereas the cortical bone again appeared less dense. The long bones from CL2+ mice are also shorter and misshapen, and this phenotype is more severe in the double-mutant CL2+/rr mice. B, Von Kossa staining performed on tibias from 4-wk-old wild-type, rr, CL2+, and CL2+/rr mice (left to right). A moderate increase in trabeculation of the metaphyseal regions of rr long bones, which extends well beyond the area normally occupied by the secondary spongiosa, is clearly evident. A more profound increase in trabeculation was detected in CL2+/rr mice compared with either rr or CL2+ double-mutant mice. Furthermore, in the Cl2+/rr mice, the ectopic trabeculae extended well into the diaphysis and all along the bone shaft to completely obliterate the bone marrow cavity (data not shown).

 


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  		FIG. 3. Alcian Blue staining of tibias from 4-wk-old wild-type and rr mice shows the unresorbed cartilage core in all trabeculae of rr bones.

 
Histomorphometric analysis confirmed a mild, but significant, increase in trabecular number and volume and a sharp decrease in the trabecular separation (Fig. 4AGoGo). No fibrotic appearance of bone marrow was observed (data not shown). To investigate whether the increased trabecular bone volume seen in the mutant rr mice was secondary solely to decreased bone resorption or, in addition, to augmented osteoblast activity, dynamic histomorphometry was performed. Four-week-old rr animals showed a 30% decrease in trabecular bone formation rate (BFR) compared with wild-type littermates (Fig. 4BGoGo). Trabecular mineral apposition rate (MAR), an index of osteoblast function, was also decreased by 30% in rr mice compared with normal littermates (Fig. 4BGoGo). Therefore, rr mice displayed an increase in trabecular bone volume despite an overall decrease in osteoblast activity in trabecular bone. Interestingly, both osteoclast number and surface were increased in the long bones of rr mutant mice (Fig. 4BGoGo). This finding is consistent with an impairment of osteoclast function in rr mutant animals.



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  		FIG. 4. Trabecular bone histomorphometry performed on tibias from wild-type (white), rr (orange), CL2+ (red), and CL2+/rr (dark red) mice. A, Structural parameters; B, dynamic and cellular parameters. a, P < 0.05 vs. wild-type; b, P < 0.05 vs. CL2+.

 


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  		FIG. 4A. Continued

 
Cortical bone thickness was similar in both 4-wk-old rr mice and their wild-type littermates, at least at 4 wk of age (Fig. 6AGo). The increase in periosteal MAR observed in the long bones of the rr mice did not reach statistical significance (Fig. 6AGo). Conversely, in agreement with our previous studies, both outer and inner periosteal MARs were significantly increased in calvarial bones from rr animals compared with their wild-type littermates (Fig. 6BGo). Calvarial thickness was also increased in rr mice compared with that in wild-type mice. At this age, no difference in the number of empty osteocyte lacunae was observed in long bones and calvariae in rr mice compared with controls or transgenics (data not shown). In a previous study (28) the number of empty lacunae in bones from the collagenase-resistant mice increased with increasing age, and older animals have not yet been examined. Lastly, the number of osteoclasts present in the calvarial endosteum was not statistically different between these two groups (Fig. 6BGo).



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  		FIG. 6. Cortical bone histomorphometry performed on tibias (A) and calvariae (B) from wild-type (white), rr (orange), CL2+ (red), and CL2+/rr (dark red) mice. a, P < 0.05 vs. wild-type.

 
Effect of resistance to collagenase activity on the phenotype of CL2+ mice in both trabecular and cortical bone
Consistent with our previous study (36), the tibiae isolated from CL2+ transgenic mice showed a clear increase in radiodensity compared with wild-type specimens (Fig. 2AGo). Furthermore, the mutant bones appeared to be shorter and misshapen (Fig. 2AGo). The overall phenotype was even more severe in the CL2+/rr double-mutant animals (Fig. 2AGo).

The von Kossa staining revealed a profound increase in metaphyseal trabeculation in CL2+/rr mice compared with either rr or CL2+ animals (Fig. 2BGo). The bony trabeculae extended well into the diaphysis and virtually obliterated the bone marrow cavity of tibiae isolated from CL2+/rr animals (Fig. 2BGo). Consistent with this finding, in situ hybridization analysis with osteocalcin cRNA revealed the presence of numerous cells expressing osteocalcin throughout the diaphysis of long bones isolated from CL2+/rr animals (Fig. 5AGo). Histomorphometric analysis confirmed the histological findings. In particular, trabecular number and volume appeared to be further augmented, and trabecular separation further decreased in tibiae of CL2+/rr mice compared with CL2+ transgenic animals, whereas trabecular thickness was similar in both wild-type and mutant specimens (Fig. 4AGoGo). In agreement with our previous data (36), the trabecular BFR and MAR were higher in CL2+ mice than in wild-type littermates. Interestingly, both parameters, i.e. trabecular BFR and MAR, were significantly lower in CL2+/rr animals than in CL2+ mutants, although significantly higher than in controls (Fig. 4BGoGo). Osteoclast number and surface were sharply increased in CL2+/rr animals compared with either wild-type or CL2+ mice (Fig. 4BGoGo). The histomorphometric data were confirmed by in situ hybridization analysis with the antisense riboprobe for TRAP, a specific marker of differentiated osteoclasts (Fig. 5BGo). Consistent with our analysis of the rr mice (see above), these findings suggest that inhibition of collagenase-dependent collagen cleavage somewhat diminished osteoblast activity in trabecular bone and partially impaired osteoclast activity with a final net increase in trabecular bone volume.



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  		FIG. 5. In situ hybridization with 35S-labeled osteocalcin (A) and TRAP (B) cRNAs in serial sections of decalcified proximal tibia of 4-wk-old wild-type and CL2+/rr littermates; dark-field views are shown.

 
Cortical bone thickness was diminished in CL2+ mice, as previously reported (36), due at least in part to a sharp decrease in the periosteal MAR. Interestingly, this phenotype was not corrected to any degree in the double CL2+/rr mice (Fig. 6Go, A and B). We then measured both inner and outer periosteal MAR in calvarial bones from 4-wk-old wild-type, CL2+, and CL2+/rr mice, respectively. As expected, we observed a clear decrease in periosteal MAR in CL2+ calvaria, which still persisted in the CL2+/rr mutants (Fig. 6BGo). The number of osteoclasts was found profoundly increased in the calvariae from CL2+ mice compared with wild-type mice, consistent with the previous study. Again, no statistical difference could be found between CL2+ and CL2+/rr mice in this respect (Fig. 6BGo).

Taken together, these data indicate that collagenase-dependent collagen cleavage has a profound effect on the trabecular bone phenotype of CL2+ mice by decreasing osteoclast and, to a lesser extent, osteoblast activity, with the net result of a further increase in trabecular bone volume. In contrast, few, if any, changes were observed in the cortical bone of CL2+/rr animals compared with CL2+ mutants.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
As described in our previous study (36), constitutively active PTH/PTHrP receptors in cells of the osteoblast lineage stimulate a vivid anabolic response in trabecular bone. The increase in trabecular bone volume in CL2+ transgenic mice was related to an increase in both osteoblast number and activity. The endosteal MAR was clearly augmented in CL2+ transgenic animals, although in the periosteum of the same animals, no increase in bone formation was detected by dynamic histomorphometry. Therefore, activation of the PTH/PTHrP receptor has different effects on the cells of periosteal, trabecular, and endosteal compartments, respectively. The mechanisms underlying this different responsiveness are, however, unknown.

Numerous in vitro and in vivo observations reveal that PTH is important in the regulation of collagenase-3 expression (26, 27, 31, 41, 42, 43). Mice that are resistant to collagenase action (rr) display changes in both bone resorption and formation (28, 35). We therefore undertook mating of CL2+ transgenic mice with rr mice to explore the possible role of collagenase activity in mediating, at least in part, the anabolic and/or catabolic effects of the PTH/PTHrP receptor in the various bone compartments.

In this study we found an inhibition of osteoclastic bone resorption in the trabecular bone of rr mice. Trabecular bone formation was not quantified in our earlier studies of rr mice. In contrast to other osteopetrotic models, rr mice do not display abnormalities in tooth eruption. They, nevertheless, exhibit an osteopetrotic phenotype characterized by trabeculae with bone-encased cartilage found in and beyond the area normally occupied by the secondary spongiosa. Furthermore, in both rr and CL2+/rr mice, osteoclast number and surface are increased. Similar findings have been reported for other osteopetrotic models, in which osteoclast attachment and/or function, rather than proliferation or differentiation, are impaired (44, 45). Taken together, our data indicate that collagen cleavage by collagenase is critical for proper osteoclast function, not only after PTH treatment (35), but also at a basal, steady state level. To the best of our knowledge this is the first time that an osteopetrotic phenotype, albeit mild, has been shown to result from an impairment of neutral collagenase activity.

Collagenase digestion could be required for proper activity of cathepsin K. Alternatively, proteolytic cleavage on type I collagen can result in unwinding of the cleaved ends to reveal cryptic binding sites for the {alpha}vß3 integrin (33), which is known to be the major integrin in osteoclasts (45, 46). Therefore, impairment of collagenase cleavage could affect osteoclast activity by interfering with the generation of new sites for binding to cell surface integrins (47). Furthermore, the effects of the collagen mutation on osteoclastic bone resorption could be mediated indirectly through effects on osteoblasts.

Bone resorption is dramatically increased in CL2+ mice. In the double-mutant CL2+/rr animals this increase is partially impaired, causing a further increase in trabecular bone volume. The finding shows that PTH/PTHrP receptor-induced bone resorption requires proper collagen cleavage. Therefore, PTH/PTHrP receptor may modulate bone resorption at three levels: 1) the RANK/RANKL system by which it controls osteoclast differentiation and survival; 2) stimulating production of collagenase by osteoblasts and partial collagenase cleavage of collagen revealing cryptic binding sites for {alpha}vß3 that affect osteoclast function, and 3) collagenase cleavage, as an integral part in the resorption process (29, 31, 33).

It is shown in Fig. 2AGo that the CL2+ tibia is slightly shorter than the wild-type tibia, whereas that in the rr mouse is the same length. The double-mutant tibia, however, is clearly much shorter and wider than all of the others. Many osteopetrotic mice are short-limbed and runted, with club-shaped long bones. Whereas in rr mice the defect is not severe enough to result in short, runted mice with club-shaped bones, this does occur in CL2+/rr animals.

BFR and MAR are modestly impaired in both rr and CL2+/rr mice, respectively. The mechanisms that lead to this impairment have not yet been defined. Suppression of bone resorption is associated with reduced bone formation in many animal models where bone resorption is reduced due to genetic defects or pharmacological treatment. It is possible that growth factors released during bone resorption have an important impact on the osteoblast phenotype of the rr mutant mice. Exposure to cryptic integrin binding sites in the helical structure of collagen that are made available by collagenase cleavage may be essential for osteoblast activity as well (29, 48). Similar signals have indeed been shown to be essential for the viability and activity of other cell types (33, 49).

In any case we emphasize that in CL2+/rr animals, BFR and MAR, despite being modestly decreased compared with findings in CL2+ mice, are still significantly higher than those in wild-type littermates. In clinical trials, human PTH-(1–34), given by daily sc injections, induces a marked increase in trabecular bone mass, but does not alter cortical bone (25). The lack of an anabolic effect on cortical bone could be related to an increase in bone resorption not adequately compensated by an equivalent increase in bone formation. Therefore, the coadministration of an inhibitor of resorption could be beneficial, provided that the anabolic effect of PTH in the trabecular bone compartment could still be obtained. Conversely, it is possible that the PTH effect on bone formation may require increases in osteoclast recruitment and activity to prepare the bone surface for the subsequent deposition of new matrix (30), and/or allow the release from the matrix itself of indispensable growth factors (2, 50, 51, 52). The published reports on this issue, using different species and different drug regimens, lead to variable conclusions. It is thus uncertain whether resorption is necessary for PTH-dependent bone formation. Although some osteopetrotic mouse mutants indicate that in many pathological settings the arrest of bone resorption does not necessarily determine the arrest of bone formation (53), there is evidence supporting the hypothesis that osteoclasts can regulate osteoblast/stromal cell functions, especially in conditions of increased bone turnover. Our findings in CL2+/rr mice clearly indicate that a mild impairment of osteoclast activity is still compatible with increased osteoblast function.

In general, the remodeling process is rapid in trabecular bone and slower in cortical bone, in particular at the periosteal surface, and the periosteum in most locations is naturally devoid of osteoclast activity. Whether these differences are due to the fact that osteoprogenitors in the periosteum are intrinsically different from osteoprogenitors in the marrow cavity, or whether it is the different local microenvironments that modulate the process of osteoblast proliferation and differentiation is indeed an open question. In our model, osteoblasts in the periosteum display a different response to collagenase resistance compared with cells in the trabecular and endosteal compartments. Periosteal osteoblasts have a somewhat increased activity in rr mice, the opposite of what occurs in trabecular osteoblasts. This increase could not, however, rescue the dramatic reduction of the periosteal MAR and cortical thickness observed in mice expressing constitutively active PTH/PTHrP receptors. In this regard, the increases in cortical thickness and MAR observed in rr mice were completely reversed by the constitutively activated PTH/PTHrP receptor, further evidence for an overall different behavior of osteoblasts in trabecular vs. periosteal/cortical bone.

In summary, our data show the following. Osteoclast function is partially impaired in rr as well as CL2+/rr mutant mice. Trabecular osteoblast activity is lower in CL2+/rr than in CL2+ mice, although still very high compared with that in wild-type mice. The rr mutation does not rescue the reduced periosteal osteoblast function observed in Cl2+ mice. Our study provides clear evidence that collagenase-dependent cleavage of type I collagen is associated with osteoclast activation and/or function both under basal conditions and upon activation of the PTH/PTHrP receptor. Further, it supports of the concept that collagenase is a downstream effector of PTH/PTHrP receptor action in trabecular/endosteal bone compartments, but not in the periosteum.


   Acknowledgments
 
We thank Dr. H. M. Kronenberg for critical review of the manuscript and helpful discussion.


   Footnotes
 
This work was supported by NIH Grants AR-44815 (to S.K.), AR-44855 (to E.S. and S.K.), and AR-45354 (to R.B.).

Abbreviations: BFR, Bone formation rate; MAR, mineral apposition rate; MMP, matrix metalloprotease; TRAP, tartrate-resistant acid phosphatase.

Received February 25, 2003.

Accepted for publication May 30, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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