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Endocrinology Vol. 142, No. 12 5371-5378
Copyright © 2001 by The Endocrine Society

PTH-CALCITONIN-VITAMIN D-BONE

Estrogen Reduces the Depth of Resorption Pits by Disturbing the Organic Bone Matrix Degradation Activity of Mature Osteoclasts

Vilhelmiina Parikka, Petri Lehenkari, Mirja-Liisa Sassi, Jussi Halleen, Juha Risteli, Pirkko Härkönen and H. Kalervo Väänänen

Institution of Biomedicine, Department of Anatomy and Medicity Research Laboratory, University of Turku (V.P., J.H., P.H., H.K.V.), FIN-20520 Turku, Finland; Turku Graduate School of Biomedical Sciences (V.P.), Departments of Surgery and Anatomy (P.L.), and Department of Clinical Chemistry (M.-L.S., J.R.), University of Oulu, FIN-90014 Oulun Yliopisto, Finland

Address all correspondence and requests for reprints to: H. Kalervo Väänänen, Department of Anatomy, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. E-mail: kalervo.vaananen{at}utu.fi


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Decreased E2 levels after menopause cause bone loss through increased penetrative resorption. The reversal effect of E2 substitution therapy is well documented in vivo, although the detailed mechanism of action is not fully understood. To study the effects of E2 on bone resorption, we developed a novel in vitro bone resorption assay in which degradation of inorganic and organic matrix could be measured separately. E2 treatment significantly decreased the depth of resorption pits, although the area resorbed was not changed. Electron microscopy further revealed that the resorption pits were filled with nondegraded collagen, suggesting that E2 disturbed the organic matrix degradation. Two major groups of proteinases, matrix metalloproteinases (MMPs) and cysteine proteinases, have been suggested to participate in organic matrix degradation by osteoclasts. We show here that MMP-9 released a cross-linked carboxyl-terminal telopeptide of type I collagen from bone collagen, and cathepsin K released another C-terminal fragment, the C-terminal cross-linked peptide of type I collagen. E2 significantly inhibited the release of the C-terminal cross-linked peptide of type I collagen into the culture medium without affecting the release of cross-linked carboxyl-terminal telopeptide of type I collagen in osteoclast cultures. These results suggest that organic matrix degradation is initiated by MMPs and continued by cysteine proteases; the latter event is regulated by E2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTROGEN DEFICIENCY in both postmenopausal women and ovariectomized animals causes a substantial decrease in bone mass, leading to osteopenia and increased fracture risk. The efficiency of E2 replacement therapy in preventing bone loss has been known for a long time (1). However, the exact cellular and molecular mechanisms behind the effects of E2 on osteoclasts have remained questionable. Several studies support the hypothesis that E2 decreases osteoclast formation by suppressing the production of the proosteoclastogenic cytokines IL-1, IL-6, and TNF{alpha} by stromal cells (2, 3, 4) or T cells (5). Recently, E2 was also demonstrated to suppress receptor activator of nuclear factor-{kappa}B ligand and macrophage colony-stimulating factor-induced differentiation of monocytic precursors into osteoclasts through an ER-dependent mechanism that does not require mediation by stromal cells (6). However, demonstration of direct inhibitory effect of E2 on resorption activity of mature osteoclasts has turned out to be difficult (7, 8, 9, 10), although some contradictory results have also been published (11, 12, 13).

Osteoclasts resorb bone by secreting acid and proteolytic enzymes into an extracellular resorption lacuna. Acid solubilizes the inorganic matrix, thus making the organic matrix available for the proteases. We and others (14, 15) have shown that osteoclasts transcytose the degraded bone material, which allows continuous penetration and generation of deep excavations into mineralized bone matrix. Matrix metalloproteinases (MMPs), particularly MMP-9 (16, 17), and cysteine proteinases, particularly cathepsin K (18, 19), have been suggested to play major roles in degradation of the organic matrix, which is composed mainly of type I collagen.

Two different circulating C-terminal fragments of type I collagen, known as ICTP (cross-linked carboxyl-terminal telopeptide of type I collagen) and CTx (C-terminal cross-linked peptide of type I collagen), have been used as markers of bone degradation in vivo. These markers are known to respond differently to changes in bone resorption activity. Serum and urinary CTx levels are significantly elevated in postmenopausal women compared with premenopausal women and are decreased in response to E2 replacement therapy (20, 21). In contrast, serum ICTP levels are only slightly elevated in postmenopausal women (22) and do not respond to hormone replacement or alendronate therapy to the same extent as CTx (22, 23, 24). Although the circulating ICTP levels do not reflect the state of physiological bone turnover, ICTP is a valuable marker when monitoring tissue degradation during pathological conditions such as rheumatoid arthritis (25) and bone metastases (26). Our hypothesis was that ICTP and CTx fragments might be released from bone collagen due to the action of different proteases that are differentially regulated by E2.

In this article we demonstrate the direct effect of E2 on resorption activity of mature osteoclasts. We found that E2 reduced the depth of the resorption pits without clear effects on the total area resorbed. We also demonstrated that ICTP is released from bone collagen by MMP-9, and CTx is released by cathepsin K, and that E2 inhibited the release of CTx but had no effect on the release of ICTP in in vitro bone resorption assay. We provide evidence suggesting that organic bone matrix degradation is initiated by MMPs and continued by cysteine proteinases, the latter event apparently being regulated by E2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
All cell culture reagents were obtained from Life Technologies, Inc. (Grand Island, NY). The pure anti-E, ICI 182,780, was a gift from Dr. A. E. Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK). 17ß-E2 (Sigma, St. Louis, MO) and ICI 182,780 were dissolved in ethanol as a 10-mM stock solution. E-64 (trans-epoxysuccinyl-L-leucylamido-[4-guanidino]butane) was purchased from Sigma, and batimastat (BB-94) was provided by British Biotech (Oxford, UK). Recombinant human cathepsin K was a gift from Dr. Maxine Gowen (SmithKline Beecham, King of Prussia, PA), and human MMP-9 was a gift from Dr. Karl Tryggvason (Karolinska Institute, Stockholm, Sweden). p-Aminophenylmercuric acetate, tetramethylrhodamine isothiocyanate (TRITC)- and peroxidase-conjugated wheat-germ agglutinin lectins (WGA-lectin), Hoechst 33258, and leukocyte acid phosphatase kit 387-A were purchased from Sigma.

Osteoclast cultures
A mixed rat bone cell population was cultured on bovine bone slices or tetracycline-labeled swine bone slices as described in detail previously (27). Briefly, osteoclasts were mechanically harvested from the long bones of 1-d-old rats and allowed to attach to bone slices for 30 min, after which nonattached cells were washed away. The cell population still contained osteoblasts and other stromal cells in addition to osteoclasts. All cells on bone slices were cultured for 2–3 d in {alpha}MEM buffered with 20 mM HEPES containing 100 IU penicillin, 100 µg streptomycin/ml, and 10% heat-inactivated FCS. Culture medium contained no E-mimic pH indicators (phenol red), and the level of E in FCS was below the level of detection (1 pM) in a specific immunoassay. All individual experiments were performed with osteoclasts isolated from bones of the same animal pool. The animals were maintained in accordance with the guidelines of the Turku University ethical committee for the use and care of experimental animals.

After the culture period, bone slices with cells were fixed in 3% paraformaldehyde in PBS. Cells were stained with tartrate-resistant acid phosphatase to detect osteoclasts and with the DNA-binding fluorochrome Hoechst 33258 to visualize nuclei. Multinucleated TRAP-positive cells were counted as osteoclasts.

Resorption area measurement
After the number of osteoclasts was determined, all cells were removed from bone slices. The total area of resorption pits was determined using WGA-lectin according to Selander and co-workers (28). Briefly, lacunae were stained with peroxidase-conjugated WGA-lectin for 40 min, after which diaminobenzidine solution was added to the bone slices for 10 min. The area resorbed was quantitated using an image analysis system (MCID/M2, Imaging Research, Inc., Brock University, Ontario, Canada) with an Intel 403 E microcomputer linked to an Image 1280 image processor (Matrox Dorral, Québec, Canada). The average size of bone slices was about 5 x 5 mm.

Resorption depth analysis
To visualize the depth of the resorption pits cells were cultured on tetracycline-labeled (14) bone slices. Tetracycline labeling was performed as follows. A growing piglet was daily injected with oxytetracycline at a dose of 20 mg/kg BW for 6 wk. After the pig was killed, thin bone slices were prepared from the cortex of the femurs, and only labeled area from the outer surface of the bone was used for measurements. The pits were stained with TRITC-conjugated WGA-lectin and analyzed using a laser scanning confocal microscope (Aristoplan CLSM, Leica Corp. Lasertechnik, Heidelberg, Germany). The recorded parameters are schematically presented in Fig. 2BGo. Parameter 1 represents the depth of a lacuna from the bone surface to the upper level of the organic matrix, parameter 2 represents the lower level of the organic layer stained with WGA-lectin, and parameter 3 represents the depth of the demineralized lacuna. The depth of the resorption pits was determined using the maximum depth of the pit.



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  		Figure 2. Method for the resorption depth measurement. A FESEM image of an osteoclast resorption lacuna demonstrates that the depth of a resorption pit can be divided into three different measurable parameters (A). Parameters are presented schematically in B: parameter 1, the upper level of WGA-lectin staining that reveals the depth from which both mineral and proteins are removed; parameter 2, the lower level of WGA staining that shows the thickness of the remaining protein layer; and parameter 3, the level of tetracycline labeling that indicates the depth from which the mineral has been removed. A confocal microscope image of a resorption pit on tetracycline-prelabeled bone slice visualized by TRITC-conjugated WGA-lectin in C and with tetracycline fluorescence in D readily shows the described parameters.

 
Biochemical methods
Bone collagen degradation was determined by measuring the amounts of two different types of collagen fragments from the same culture medium. CTx released into the culture medium was measured using a commercial ELISA, CrossLaps for culture (Osteometer Biotech, Herlev, Denmark). RIA for ICTP has been described previously (29). MMP-9 activity was measured from culture medium using a specific MMP-9 activity assay (Amersham Pharmacia Biotech, Little Chalfont, UK).

Field emission scanning electron microscope (FESEM) analysis
Bone slices, after cell removal, were fixed with 5% glutaraldehyde in PBS buffer (pH 7.4) overnight (12 h). Subsequently, samples were dehydrated in ascending ethanol series, then processed for critical point drying (Balzers Union, Balzers, Lichtenstein) and Au/Pg sputtered (E-5100, Polaron Equipment Ltd., East Grinstead, UK). Finally, the samples were examined by FESEM (JSM-6300F, JEOL, Peabody, MA) to study the morphology of resorption lacunae.

Cathepsin K and MMP-9 treatment
Osteoclasts were cultured for 2 d on small bovine bone slices (5 x 5 x 0.1 mm). All cells were removed, and bone slices were incubated with either cathepsin K or MMP-9 enzymes. For cathepsin K treatment, bone slices were incubated with 6 µg recombinant human cathepsin K in 100 mmol sodium acetate, 5 mmol/liter EDTA, and 5 mmol/liter cysteine buffer (pH 5.5) at 37 C for 10 h (30) in the presence and absence of 50 µM E-64. For MMP-9 treatment, bone slices were incubated with 3 µg p-aminophenylmercuric acetate-activated human MMP-9 enzyme in 20 mM Tris-HCl and 1 mM CaCl2 (pH 7.0) at 37 C for 6 h in the presence and absence of 6 µM BB-94.

Statistical analysis
All results are presented as the mean ± SEM. Statistical evaluation was performed using ANOVA and t test. When ANOVA revealed significant differences, further analysis was performed using a t test. Differences between control and treated groups were considered statistically significant at P < 0.05. The results of the t test are shown in the figures as asterisks. A single asterisk indicates a P value between 0.05 and 0.01, two asterisks indicate a P value between 0.01 and 0.001, and three asterisks indicate P < 0.001.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of E2 on osteoclast number and resorption area
We used a well established osteoclast resorption assay to investigate whether E2 has direct effects on bone resorption. Isolated rat osteoclasts were cultured for 48 h on bovine bone slices in the presence of different concentrations of E2. The number of osteoclasts or osteoclast-made resorption pits was not changed by E2, and the total area resorbed was only slightly affected (Fig. 1aGo). A concentration range from 100 fM to 100 nM was used (not all concentrations are shown in the figure). All experiments shown are representative of at least four similar experiments.



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  		Figure 1. The effect of E2 on number of osteoclasts, number of resorption pits, total resorption area, and number of nuclei per osteoclast (a). A mixed rat bone cell population was cultured on bovine bone slices for 48 h in the presence of vehicle or 10 nM or 100 pM E2. Neither the number or size of osteoclasts nor the number or area of resorption pits was affected. The absolute resorption values for control groups presented in this figure were 3,137 ± 342 (no of resorption pits) and 1,998,070 ± 96,059 µm2 (resorption area/bone slice). E2 significantly and dose-dependently reduced the depth of the resorption lacunae (b), and the effect was seen with all depth parameters. Parameters are described in Fig. 2Go. The anti-E, ICI 182,780, abolished the inhibitory effect of 10 nM E2 at 500 nM. The experiment was repeated four times, and each time the depth of 60–75 lacunae was analyzed. Data are the mean ± SEM. The P values from t tests between control and other groups are shown by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
Effect of E2 on resorption depth
For resorption area measurement the pits were stained with peroxidase-conjugated WGA-lectin. Although we were not able to see any effect on the plain area we could detect fainter staining of the pits at the higher E2 concentrations used. This observation led us to study the pits in detail and suggested an effect not determinable by plain area measurement. To evaluate the penetration of osteoclasts into the bone matrix, a new assay was developed (Fig. 2Go). Tetracycline labeling of bones allowed separate detection of mineral (tetracycline) vs. collagen (WGA-lectin stain) removal. The average depth of the resorption pit was significantly and dose-dependently reduced by E2 (Fig. 1bGo). The effect of E2 on the depth of the resorption pits was significantly opposed by pure anti-E ICI 182,780, which almost completely abolished the reducing effect of 10 nM E2 at 500 nM.

These results suggested that E2 could reduce the resorption capacity of mature osteoclasts, especially the penetrative resorption. However, E2 did not influence the number of osteoclasts, indicating that the inhibitory effect of E2 was not due to the changes in cell numbers in this model. We next studied whether this could be due to the changes in the average size of the osteoclasts. To study this, we counted the average number of osteoclast nuclei from bone slices cultured in the presence of E2. One hundred osteoclasts were randomly selected from four bone slices per group. The number of nuclei per cell were 6.56 ± 2.92, 6.5 ± 2.65, and 6.4 ± 2.55 (mean ± SD) in control, 100 pM E2, and 10 nM E2 cultures, respectively (Fig. 1aGo). None of the treatment groups differed significantly from the control.

Effect of E2 on resorption markers
As the depth of resorption pits was significantly reduced by E2 without any effect on the number of osteoclasts, it was obvious that resorption activity of individual cells was altered by E2. The concentration of released collagen fragments, which have been described as markers of bone resorption in vivo, was determined to ensure the role of E2 on the resorption event. The amount of CTx released into culture medium was significantly and dose-dependently reduced by E2; the CTx concentration after treatment with 100 nM E2 was only 18.8% of the control value (Fig. 3aGo). On the contrary, ICTP levels in culture medium after the addition of E2 remained at the control level (108.5% of the control).



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  		Figure 3. The effects of E2 (a), 50 µM cysteine proteinase inhibitor E-64 (b), and 6 µM MMP inhibitor BB-94 (c) on the number of osteoclasts, total resorption area, medium CTx, and ICTP concentrations. Statistical analysis reveals that the medium CTx concentration was significantly and dose-dependently reduced by E2, although the medium ICTP level remained the same. E-64 significantly reduced medium CTx levels, but the resorption area after E-64 treatment was only slightly decreased. Instead, BB-94 significantly reduced both the resorption markers and the area resorbed by osteoclasts. All data are presented as a percentage of the control. Initial values for control groups presented in this figure were 298 ± 12 (number of osteoclasts), 3,610,250 ± 290,417 µm2 (resorption area/bone slice), 3.00 ± 0.52 nM (medium CTx concentration), and 4.05 ± 0.47 µg/liter (ICTP concentration). The medium volume per well was 1 ml. Data are the mean ± SEM. The P values from t tests between control and other groups are shown by asterisks.

 
Effects of proteinase inhibitors on bone resorption
As degradation of organic bone matrix was significantly disturbed in the presence of E2, we concluded that some proteinases could be directly affected by E2. The effects of two proteinase inhibitors, the cysteine proteinase inhibitor E-64 and the MMP inhibitor BB-94 (batimastat), on osteoclast resorption activity were studied. The number of attached osteoclasts in cultures treated with E-64 (50 µM) or BB-94 (6 µM) did not differ from those in control groups (Fig. 3Go, b and c). The resorbed area was only slightly reduced after E-64 treatment (Fig. 3bGo), but BB-94 dramatically reduced the total resorption area (Fig. 3cGo). A reduction in the resorption area in BB-94 culture was parallel to the reduction in the number of resorption pits (data not shown). Like E2, both E-64 and BB-94 strongly inhibited the release of CTx fragments into culture medium, which in the case of BB-94 could be explained by the reduced resorption area. BB-94, but not E2 or E-64, reduced the level of ICTP fragments in the culture medium (Fig. 3Go, a–c).

FESEM analysis of resorption pits
On the basis of the above-mentioned observations, it was obvious that E2 and the cysteine proteinase inhibitor E-64 behaved differently than BB-94, which reduced both the resorption area and the release of both resorption markers. To further investigate the difference between the inhibitors, the morphology of individual resorption pits was studied using FESEM. Although the depth and appearance of pits can widely vary even on the same bone slice, the above-mentioned groups clearly differed from each other. After analyzing a large number of resorption pits, typical lacunae from each group were selected and presented as examples in Fig. 4Go. The addition of E2 or E-64 led to the formation of abnormally shallow resorption pits filled with nondegraded collagen (Fig. 4Go, i and l). The appearance of lacunae on BB-94-treated bone slices resembled that of control lacunae (Fig. 4Go, f and c), but the number of lacunae was significantly reduced compared with the control value (Fig. 4Go, a and d).



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  		Figure 4. FESEM images of a rat osteoclast made resorption lacunae on bovine bone slices. Control bone slices (a–c) and bone slices after BB-94 treatment (d–f), E2 treatment (g–i), or E-64 treatment (j–l) are presented. Enlargements are described in the figure. Both E2 and E-64 treatments led to the formation of resorption pits, which were filled with undegraded collagen, thus appearing remarkably shallow. BB-94 did not affect the morphology of the resorption lacunae, but the number of pits was notably decreased.

 
Degradation of bone collagen by recombinant cathepsin K and MMP-9
The release of the collagen degradation products ICTP and CTx was further studied to understand the role of specific enzymes on bone resorption. We incubated preresorbed bovine bone slices in the presence of cathepsin K or MMP-9 and also in the presence of their inhibitors, E-64 and BB-94, respectively. Spontaneous release of ICTP and CTx was low. Addition of cathepsin K caused a marked release of CTx into the buffer solution (Fig. 5aGo). Instead, cathepsin K in the same experiment reduced ICTP levels even below the control level. E-64 completely abolished the effect of cathepsin K.



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  		Figure 5. The roles of cathepsin K and MMP-9 in releasing the collagen I degradation products ICTP and CTx. Osteoclast-resorbed bone slices were incubated with enzymes after removal of cells, and collagen degradation products were detected with the immunoassays. Cathepsin K treatment caused an increase in the release of CTx fragments from bone collagen and a decrease in the amount of ICTP measured. E-64 completely abolished these events (a). MMP-9 caused increased release of both degradation products, and BB-94 abolished these effects (b). Data are the mean ± SEM. The P values from t tests between control and other groups are shown by asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). #, P < 0.001 when the group is compared with the enzyme (MMP-9 or cathepsin K)-treated group.

 
Incubation of bone slices with recombinant MMP-9 caused a significant increase in the amount of ICTP released into the buffer solution (Fig. 5bGo). CTx levels after MMP-9 treatment were also elevated, suggesting that CTx assay (CrossLaps) also recognizes longer peptides containing the CrossLaps antigen EKAHDGGR. The effect of MMP-9 was completely abolished by BB-94.

MMP-9 activity measurement
To further specify the possible effect of E2 on MMP-9, we measured MMP-9 enzyme activity in control, E2-treated, and BB-94-treated cultures after a 3-d culture period. The addition of E2 did not affect enzyme activity (Fig. 6Go), suggesting that resorption inhibition by E2 was not due to the changes in MMP-9 expression or activity. BB-94 significantly decreased the activity of MMP-9.



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  		Figure 6. MMP-9 enzyme activity was measured from resorption culture medium after a 3-d culture period. Rat bone cells were cultured in the presence of E2 (100 nM) or MMP inhibitor BB-94 (6 µM), after which media were collected, and enzyme activities were measured. Data are the mean ± SEM. ***, P < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
E2 deficiency after ovariectomy is known to result in severe osteopenia (31). Penetrative resorption, observed especially in post- and perimenopausal women, leads to instability of trabecular bone architecture, thus increasing the fracture risk. However, early postmenopausal women respond to E replacement therapy with an inhibition of the development of a pronounced increase in osteoclastic erosion depth that is otherwise seen (32). Although the bone-saving effects of E2 at organ and tissue levels in vivo are quite evident, the cellular and molecular details and possible direct effect of E2 on osteoclasts have remained poorly understood. Osteoclasts have been demonstrated to have ER (11, 33), which indicates that osteoclasts might be capable of directly responding to E treatment. Oursler et al. (34) originally showed that E2 decreases the expression of some lysosomal enzymes in osteoclasts. The resorption lacuna is known to reveal several properties of a late endosome-lysosome compartment (35, 36), and cathepsin K, a member of lysosomal cysteine proteinase family, has been shown to be down-regulated at the mRNA level by E2 (12, 37). However, osteoblasts also contain ER and could mediate the effects of E on osteoclast. Although we demonstrate here that in addition to the osteoclast differentiation, E has an effect on the osteoclast resorption activity, the exact molecular mechanism remains unknown.

In this study we show that E2 had only a modest reducing effect on the resorption area, although it markedly decreased the depth of resorption lacunae in vitro. This finding was confirmed by measuring collagen breakdown products from the culture medium. Together these results suggested that the inhibitory effect of E2 on the mature osteoclast is not related to the early phase of the resorption cycle, but mainly affects those later events allowing deeper penetration of cells into the mineralized matrix. As cysteine proteinases and MMPs have been considered to be the main candidates for enzymes involved in the degradation of organic bone matrix, we further studied the roles of these two enzymes in the collagen degradation event.

The cysteine proteinase cathepsin K has been found to be abundantly and selectively expressed in osteoclasts (19), and the lack or inhibition of cathepsin K is known to reduce the resorption activity (38, 39). Instead, cathepsins B and L, which have previously been proposed to be involved in bone resorption (40, 41), have recently been shown to be expressed only at very low levels in osteoclasts (19). In addition, cathepsin S-, B-, or L-specific inhibitors had no effect on bone resorption (39), suggesting that the main target of the cysteine proteinase inhibitor E-64 in osteoclasts is cathepsin K. In our resorption assay E-64 and E2 behaved similarly, significantly reducing the amount of CTx released into the culture medium, but only slightly inhibiting the resorption area.

Our results demonstrated that cathepsin K treatment released the eight-amino acid-containing CTx fragment known as CrossLaps antigen from bone collagen in an event that was completely inhibited by the cysteine proteinase inhibitor E-64. Our results are in accordance with data from patients with mutation of the cathepsin K gene (42, 43). In these pycnodysostosis patients, urinary CTx levels have been reported to be significantly reduced, although the levels of serum ICTP, a larger fragment derived from the carboxyl-terminus of type I collagen, are elevated (44). This seemingly paradoxical result is reasonable, because it has been shown previously that cathepsin K destroys the immunoreactivity of the ICTP antigen (30). This was confirmed by our present study in which the amount of ICTP released was significantly reduced in the presence of cathepsin K enzyme. This indicates that most of the released ICTP fragments are further cleaved with cathepsin K, which is likely to also happen in vivo and to be prevented in pycnodysostosis patients.

Several MMPs have been suggested to be present in cells of the osteoclast lineage, although only MMP-9 has been shown to be highly and predominantly expressed in osteoclasts (17, 45). We found that the MMP inhibitor BB-94 significantly reduced the total resorption area, although the number of osteoclasts was not changed. In the presence of BB-94, the levels of both collagen degradation markers (CTx and ICTP) were decreased to the same extent as the resorption area. Our results showed that MMP-9 treatment released the ICTP fragment from bone collagen in an event that was completely inhibited by the MMP inhibitor BB-94. However, we also observed a significant increase in the amount of CTx released into the culture medium during MMP-9 treatment. This suggests that the CrossLaps assay, which is based on an eight-amino acid peptide located after the cross-link, is also able to recognize longer peptides that contain the sequence EKAHDGGR.

FESEM analysis revealed that resorption pits in E2-treated cultures were clearly different from controls. The pits were more shallow and filled with a dense collagen network, indicating incomplete collagen degradation. Our morphological findings further suggest that resorption pits after E2 treatment are similar to those in E-64-treated cultures. This observation together with the reduced CTx levels in E2- and E-64-treated groups without clear inhibition of the resorption area suggest that the inhibitory effect of E2 on matrix resorption could be due to the cathepsin inhibition. As cathepsin K is the most abundantly expressed cathepsin in osteoclasts, and its collagenolytic activity is known to be unique among proteinases (46), it is the most likely enzyme to be affected by E2. Our results with BB-94 and E-64 were in agreement with data described previously by Holliday and co-workers (47) proposing a distinct role of MMPs and cysteine proteinases in bone resorption. In our study cysteine proteinase inhibition did not remarkably affect the area resorbed, but degradation of organic matrix was clearly disturbed. Lacunae after BB-94 treatment resembled control lacunae, but the number of pits was dramatically reduced.

A reduction in the number of resorption pits in BB-94-treated cultures was parallel to the reduction in total resorption area, suggesting that the initiation of osteoclast-mediated resorption was disturbed by MMP inhibition. This is in agreement with an earlier study by Sato and co-workers (48), who demonstrated osteoclastic MMPs to be involved in the migration of purified osteoclasts through a collagen layer without further effect on the actual resorption event. On the basis of our present and earlier data we conclude that some MMPs, the enzymes with neutral pH optima, are involved in the initiation of bone resorption, whereas cysteine proteinases, the enzymes with acid pH optima, are essential for competent matrix degradation.

Our results support the idea that E has an inhibitory effect on mature osteoclasts. The major effect of E2 is to decrease the resorption depth via a receptor-mediated mechanism. This is, as far as we know, the first study to show the effect of E2 on collagen degradation in vitro, thus providing an excellent experimental model to study the mechanism of E2 action. Our results could also help to explain the occurrence of penetrative resorption in E2 deficiency in vivo. These results also emphasize the importance of qualitative analysis of the resorption pits in a widely used resorption pit assay.


   Acknowledgments
 
We thank Sari Alatalo and Tiina Laitala-Leinonen for their practical advice concerning some experimental procedures, and Tiina Holappa for her skilful technical assistance.


   Footnotes
 
This work was supported by grants from the Academy of Finland, National Technology Agency, Finland, and Turku Graduate School of Biomedical Sciences (to V.P.).

Abbreviations: CTx, C-Terminal cross-linked peptide of type I collagen; FESEM, field emission scanning electron microscope; ICTP, cross-linked carboxyl-terminal telopeptide of type I collagen; MMP, matrix metalloproteinase; TRITC, tetramethylrhodamine isothiocyanate; WGA-lectin, wheat-germ agglutinin lectins.

Received May 17, 2001.

Accepted for publication August 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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