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Keywords:

  • zoledronic acid;
  • osteoblast-like cells;
  • RANKL;
  • osteoprotegerin;
  • TNF-α converting enzyme

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Bisphosphonates are used to prevent osteoclast-mediated bone loss. Zoledronic acid inhibits osteoclast maturation indirectly by increasing OPG protein secretion and decreasing transmembrane RANKL expression in human osteoblasts. The decreased transmembrane RANKL expression seems to be related to the upregulation of the RANKL sheddase, TACE.

Introduction: Bisphosphonates (BPs) exhibit high affinity for hydroxyapatite mineral in bone and are used extensively to treat malignancy-associated bone disease and postmenopausal bone loss by inhibiting osteoclast (OC)-mediated bone resorption.

Materials and Methods: We examined the effect of the most potent nitrogen-containing BP available, zoledronic acid (ZOL), on the expression of RANKL and osteoprotegerin (OPG), critical factors in the regulation of OC formation and activation, in primary osteoblast (OB)-like cells derived from human bone, using flow cytometry, ELISA, semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), in situ immunofluorescence staining, and Western blotting.

Results: Our studies show that ZOL, while not significantly affecting RANKL or OPG gene expression, markedly increased OPG protein secretion and reduced transmembrane RANKL protein expression in OB-like cells. The reduction in transmembrane RANKL expression was preceded by a marked increase in the expression of the metalloprotease-disintegrin, TNF-α converting enzyme (TACE). In addition, the decreased transmembrane expression of RANKL could be partially reversed by a TACE inhibitor, TAPI-2.

Conclusions: Our studies indicate that ZOL, in addition to its direct effects on mature OCs, may inhibit the recruitment and differentiation of OCs by cleavage of transmembrane RANKL in OB-like cells by upregulating the sheddase, TACE.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

Bisphosphonates (BPs) are stable pyrophosphate analogs that have been successfully used to treat postmenopausal and glucocorticoid-induced osteoporosis, as well as Paget's disease of bone, metastatic and osteolytic bone disease, and hypercalcemia of malignancy.(1) There is good evidence that the inhibition of bone resorption in patients receiving BPs is caused by the inhibition of osteoclast (OC) precursor proliferation, differentiation,(2,3) and the induction of apoptosis in mature OCs.(4–7)

The development of OCs is controlled by osteoclastogenic factors synthesized by osteoblasts (OBs), including RANKL and osteoprotegerin (OPG). RANKL (also known as TRANCE/OPGL/ODF) plays a pivotal role in osteoclastogenesis by providing an essential signal to OC progenitors through the membrane-anchored receptor RANK.(8) Signal transduction through RANK(8–11) leads to OC differentiation and functional activation.(12,13) This signaling pathway can be disrupted by a naturally occurring decoy receptor for RANKL, termed osteoprotegerin (OPG), which blocks the interaction between RANKL and RANK.(12,14) Bone marrow OB/stromal cells secrete both RANKL and OPG,(9,13,15) and OC formation is determined principally by the relative ratio of RANKL to OPG in the bone marrow (BM) microenvironment.

In addition to their direct effect on OC, BPs can modulate the expression of a number of OB-derived osteoclastogenic factors.(16–20) In fact, recent studies show that BPs can decrease RANKL mRNA expression in a rat osteoblast cell line(21) and increase OPG mRNA and protein expression in human OBs.(22) However, to date, no study has reported the effect of BPs on the RANKL protein expression in human OB-like cells. In this study, we show that the treatment of primary human OB-like cells with the potent nitrogen-containing BP, zoledronic acid (ZOL), resulted in a downregulation of membrane-associated RANKL protein expression. This marked decrease in protein expression was not associated with a significant change in RANKL gene expression, raising the possibility that ZOL may regulate transmembrane RANKL expression post-translationally. In support of this concept, we show that this decrease in transmembrane RANKL expression was preceded by a marked increase in the expression of the metalloprotease-disintegrin TNF-α converting enzyme (TACE), a known RANKL sheddase.(23,24) Furthermore, the decreased membrane expression of RANKL could be partially reversed by a TACE inhibitor, TAPI-2. Therefore, our studies suggest that ZOL, in addition to its direct effects on mature bone-resorbing OCs, may affect the recruitment and differentiation of OC by decreasing the level of membrane-associated RANKL expression in human OB-like cells.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

OB-like cell cultures

Human OB-like cells were cultured from trabecular bone explants of bone biopsy or bone marrow aspirates. The specimens were obtained from osteoarthritis patients, during routine knee and hip replacements, from the Department of Orthopaedic Surgery and Trauma at the Royal Adelaide Hospital. Alternately, bone marrow was obtained from young healthy donors as part of an institutional ethics-approved Normal Bone Marrow Donor Program. Bone explants were cultured in T-75 tissue-culture flasks in α-MEM (JRH Biosciences) supplemented with 10% fetal calf serum (FCS; CSL Limited, Victoria, Australia), 2 mM glutamine (JRH Biosciences), 50 IU/ml penicillin, 50 μg/ml streptomycin sulfate (CSL Biosciences), and 100 μM L-ascorbate-2-phosphate (ASC-2P; WAKO). Medium was changed twice weekly for 5–6 weeks, and cultures were incubated at 37°C in the presence of 5% CO2 until confluent. Single-cell suspensions were obtained from confluent primary OB-like cell cultures by enzymatic digestion. The cells were washed twice in PBS and digested in a 2-ml mixture of collagenase (1 mg/ml, Worthington Biochemical Corp.) and dispase (1 mg/ml; Boehringer Mannheim) for 45 minutes at 37°C. The detached cells were transferred to a 50-ml Falcon tube, following one wash with 10 ml PBS. The remaining adherent cells were treated with Trypsin-EDTA (0.05% trypsin, 0.53 mM EDTA-4 Na; GIBCO Invitrogen Corp.) for 5 minutes at 37°C. After washing with Hank's balanced salt solution (HBSS) supplemented with 5% FCS (HHF), cells were mixed and pelleted by centrifugation at 274g for 5 minutes and used as described below.

Reverse transcriptase-polymerase chain reaction amplification of DNA

Total cellular RNA was extracted from OB-like cells by lysing in 1 ml of TRIzol II (Invitrogen Life Technologies) per 5–10 × 106 cells as recommended by the manufacturer. First strand cDNA was synthesized from 1 μg of total RNA using Superscript II (Invitrogen Life Technologies). cDNA was used as a template for polymerase chain reaction (PCR) amplification to generate products corresponding to mRNA encoding the various gene products. Each PCR reaction contained 1 μl of cDNA, 1 U of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Norwalk, CT, USA), 100 ng of the forward and reverse primers, 0.2 mM dNTPs (Biotech), 1.5 mM MgCl2, 2 μl of 10× PCR buffer, and diethylpyrocarbonate (DEPC) H2O to a final volume of 20 μl. DNA was amplified under the following typical cycling conditions: denaturation at 94°C for 1 minute, annealing (60°C) for 1 minute, extension at 72°C for 1 minute (22 and 30 cycles for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and TACE primer pairs, respectively), and a final 10-minute extension performed at 72°C, such that all products were assayed in the exponential phase of the amplification curve. PCR amplification products were separated by electrophoresis on a 2% wt/vol agarose gel and visualized by SYBR Gold (Quantum Scientific) staining at 570 nm. The relative amounts of PCR products were determined by quantitating the intensity of the bands using a Fluroimager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA, USA). Amplified products are represented as a ratio of the specific PCR product/GAPDH PCR product. A negative control was performed with all PCR reactions, which had no cDNA added to the PCR reaction mixture. Primers used were as follows: GAPDH (417 bp): forward 5′-CACTGACACGTTGGCAGTGG-3′ and reverse 5′-CATGGAGAAGGCTGGGGCTC-3′; TACE (483 bp): forward 5′-AAGAGTGTGATCCTGGCATC-3′ and reverse 5′-CATCCTGTACTCGTTTCTCAC-3′.

Flow cytometric analysis

Cultures of OB-like cells were cultured in the presence or absence of 5 μM ZOL (hydrated disodium salt, molecular weight 401.6; Novartis Pharma) and/or 100 μM TAPI-2 (Peptides International, Louisville, KY, USA), a hydroxamate-based inhibitor of matrix metalloproteinases (MMP) and TACE. At days 3 and 5, OB-like cells were detached with 2 mM EDTA and subsequently incubated in blocking buffer (HBSS supplemented with 20 mM HEPES, 1% normal human serum, 1% bovine serum albumin [BSA] and 5% FCS) at a concentration of 5 × 106 cells/ml on ice for 30 minutes. For each condition, aliquots of 2 × 105 cells were incubated with 100 μl of 10 μg/ml RANKL mAb (anti-human TRANCE mAb; R&D Systems) on ice for 1 h. An isotype-matched negative control antibody, IgG2b (1A6.11), was used under identical conditions. The cells were washed twice in HHF and resuspended in 50 μl HHF containing 1:50 dilution of fluorescein isothiocyanate (FITC)-conjugated IgG (Southern Biotechnology Associates Inc., Birmingham, AL, USA). After incubation with secondary antibody for a further 45 minutes at 4°C, cells were washed and fixed in 250 μl FACS fix (1% vol/vol of formalin, 2% wt/vol of D-glucose, and 0.02% wt/vol of sodium azide in PBS). Flow cytometry was performed using an Epics-XL-MCL analyser (Beckman Coulter). Typically, for each sample, 10,000 events were stored as list mode data for further analysis using WinMDI software (Windows Multiple Document Interface Flow Cytometry Application, 1993–1998 Joseph Trotter).

Intracellular antigen detection

Enzymatically detached single-cell suspensions of OB-like cells were incubated in blocking buffer for 30 minutes on ice and subsequently washed twice in PBS. After fixing in 1% (wt/vol) paraformaldehyde (BDH Chemical Ltd.) in PBS for 20 minutes at room temperature, cells were washed twice in wash medium (HHF containing 0.1% saponin [wt/vol, Sigma]) and cells resuspended with 100 μl of purified rabbit anti-RANKL (Santa Cruz Biotechnologies) at a final concentration of 20 μg/ml (diluted in wash medium). The isotype-matched negative control antibody, rabbit anti-IgG (α-MAP, kindly provided by Dr GJ Atkins, Department of Orthopaedics and Trauma, University of Adelaide) was used under identical conditions. After a 60-minute incubation at 4°C, the cells were washed twice in wash medium and resuspended in 100 μl of a 1:200 dilution of sheep anti-rabbit IgG biotin (Rockland Inc., Gilbertsville, PA, USA). After incubation at 4°C for 45 minutes, cells were washed twice, resuspended in a 1:50 dilution of streptavidin-FITC (Caltag Laboratories), and incubated at 4°C for 30 minutes, washed, fixed, and analyzed as described above.

OPG measurement by ELISA

The amount of OPG liberated into the culture medium by the OB-like cells after ZOL treatment was measured using an ELISA. Each well of a 96-well plate (MaxiSorp; Nalge Nunc International, Rochester, NY, USA) was coated overnight with 100 μl of 2 μg/ml anti-human OPG mAb (MAB 805; R&D Systems) diluted in PBS. Plates were washed four times with 100 μl Wash Buffer (PBS containing 0.05% [vol/vol] Tween 20; PBS-T), and “blocked” by the addition of 250 μl of Blocking Buffer (PBS containing 1% BSA). After a 2-h incubation at room temperature, plates were washed as above, and 100 μl of the rhOPG standard (human OPG-Fc, 805-OS; R&D Systems) or a 1/20 dilution of each test sample was added in triplicate wells. The plates were washed as above, and 100 μl of 100 ng/ml biotinylated α-huOPG monoclonal antibody (R&D Systems), diluted in “detection antibody diluent” (Tris buffered saline containing 0.1% BSA), was added to each well and incubated for 2 h at room temperature. The plates were washed four times, and 100 μl of 1:2000 streptavidin-conjugated horse radish peroxidase (Genzyme, Cambridge, MA, USA) in Blocking Buffer was added to each well, incubated at room temperature for 20 minutes, and washed as above, and 100 μl of TMB substrate reagent (Sigma Chemical Co., St Louis, MO, USA) was added and incubated in the dark for 20 minutes at room temperature. The reaction was stopped with 0.5 M sulfuric acid, and the absorbance at 450 nm was measured using a BIO-RAD Model 3550 microplate reader (BioRad).

In situ immunofluorescence staining

To examine the intracellular localization of TACE before and after ZOL treatment, human OB-like cells were plated in 8-well chamber slides at a concentration of 1 × 104 cells/well. Cells were cultured without or with a range of ZOL concentrations for 3 and 5 days. Cells were washed three times with 200 μl PBS and blocked in Blocking Buffer for 30 minutes on ice. After washing with PBS twice, cells were fixed in 1% (wt/vol) paraformaldehyde in PBS for 20 minutes at room temperature and permeabilized by washing cells with wash medium twice. The fixed cells were probed with 10 μl of fluorescein-conjugated mouse monoclonal anti-human TACE or an equivalent amount of negative control IgG1-FITC (Immunotech, Marseille, France) for 1 h on ice. To amplify the signal, 100 μl of a 1:50 dilution of goat anti-mouse IgG-biotin (Southern Biotechnology Associates Inc.) was incubated with the cells for 45 minutes, washed as above, and subsequently resolved by the addition of 100 μl of a 1:50 dilution of streptavidin-FITC. The cells were washed, fixed with 200 μl of FACS fix slides mounted with anti-fade solution (1.8% wt/vol of DABCO, 2 mM Tris pH 7.5 in glycerol), and examined using an Olympus BH2-RFCA fluorescence microscope (Olympus, Tokyo, Japan).

Western blot analysis of TACE expression

Human OB-like cells were plated into T75 flasks at a concentration of 8 × 105 cells/flask and treated with ZOL for 3 and 5 days. Cells were washed with ice-cold PBS twice and lysed in 1 ml of lysis buffer (1% [vol/vol] NP-40 and 1× protease inhibitor cocktail tablets [Roche] in TSE [50 mM Tris-HCl, 150 mM NaCl, and 1 mM EDTA]) for 30 minutes on ice. Lysates were centrifuged to remove detergent-insoluble material, and the final protein concentration was determined using a BCA protein assay, as recommended by the manufacturer (Pierce, Rockford, IL, USA). Approximately 20 μg of protein was diluted in an equal volume of 2× reducing buffer (125 mM Tris-HCl, pH 6.8, 20% glycerol, 4% SDS, 10% β-mercaptoethanol, and 0.0025% bromophenol blue). Samples were boiled for 5 minutes, separated on 10% SDS-PAGE, transferred to PVDF membranes (Amersham Pharmacia Biotech), and blocked with 5% (wt/vol) powdered milk in PBS-T for 1 h at room temperature. Membranes were probed with the anti-TACE (Sigma) or control α-MAP polyclonal antisera for 1 h at room temperature. The membranes were subsequently washed three times with PBS-T and incubated with an anti-rabbit IgG alkaline phosphatase (1:10,000; Amersham Life Sciences) for 1 h. The protein bands were visualized by adding ECF substrate (diethanolamine; Amersham Pharmacia Biotech) and scanned using a Typhoon fluorimager (Molecular Dynamics) at a wavelength of 488 nm. To ensure equal loading of samples, the membranes were sequentially probed with an antibody to goat anti-β actin (1:500; Santa Cruz Biotechnologies) and rabbit anti-goat IgG alkaline phosphatase (1:5000, Pierce Biotechnology) and subsequently resolved as described as above.

Statistical analysis

Results are expressed as the mean ± SEM. Student's t-test and one-way ANOVA were used to determine the statistical significance of differences between the means of experiments for normal distribution data. The Wilcoxon test was used for nonparametric data. A probability value ≤0.05 was considered to be statistically significant. Analyses were applied to experiments carried out at least three times.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

ZOL decreases RANKL protein expression in OB-like cells

We have previously found that ZOL, at concentrations ranging from 10−8 to 10−5 M, mediates differential effects on OB cell proliferation, viability, and gene expression (Pan B, To LB, Fendlay DM, Farrugia AN, Green J, Gronthos S, Lynch K, Evdokiou A, Atkins GJ, Zannettino ACW, unpublished data, 2003). Based on this work, we chose a concentration of 5 μM ZOL for the studies described below. Although substantially higher than the doses of ZOL used in studies by Viereck et al.,(22) work by Chen et al.,(25) suggest that this concentration reflects the concentration that OBs in the bone microenvironment would be exposed to in areas of active resorption. Using indirect immunofluorescence and flow cytometry, we examined the effect of ZOL treatment on both cell surface and intracellular RANKL protein expression in human OB-like cells. As seen in Fig. 1, culture of OB-like cells with ZOL for 3 days resulted in a significant decrease in transmembrane RANKL expression (p = 0.007, t-test) as evidenced by the decrease in the mean fluorescence intensity from 16.21 ± 0.21 (Fig. 1A) to 4.87 ± 1.13 (Fig. 1B). In contrast, the decrease in intracellular RANKL expression was not significant (p = 0.16, t-test), with a decrease in the mean fluorescence intensity from 10.98 ± 2.29 (Fig. 1C) to 9.10 ± 1.06 (Fig. 1D). The ZOL-mediated decrease of both transmembrane and intracellular RANKL expression was less obvious at day 5 (data not shown) than that observed at day 3 and may be caused by the consumption of ZOL over this period of culture.

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Figure FIG. 1.. ZOL-mediated downregulation of RANKL expression is predominantly at the level of cell surface expression. The expression of both membrane-associated and intracellular RANKL protein was measured using indirect immunofluorescence and flow cytometry. Fluorescence histograms showing the expression of RANKL protein (A and B) at the cell surface and (C and D) within the cytoplasm of OB-like cells are presented. Culture of OB-like cells in 5 μM ZOL for 3 days resulted in a significant decrease (p = 0.007, t-test) in transmembrane RANKL expression (B; MnX = 4.87 ± 1.13) compared with untreated cells (A; MnX = 16.21 ± 0.21). In contrast, the decrease in intracellular RANKL expression was not significant (p = 0.16, t-test), with a decrease in the mean fluorescence intensity from (C) 10.98 ± 2.29 to (D) 9.10 ± 1.06. The data shown are representative of results obtained with cells from three donors.

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ZOL increases OPG protein expression in OB-like cells

Consistent with the studies of Viereck et al.,(22) we found that ZOL exposure caused an increase in OPG protein expression in primary human OB-like cells. After the addition of 5 μM ZOL, conditioned media were collected at different time-points, and the level of secreted OPG was measured by ELISA. Our previous studies (Pan B, To LB, Fendlay DM, Farrugia AN, Green J, Gronthos S, Lynch K, Evdokiou A, Atkins GJ, Zannettino ACW, unpublished data, 2003) showed that at a concentration of 5 μM, ZOL induced both cytostasis and cell death in a proportion of the human OB-like cell cultures examined. To account for this reduction in cell number, the level of OPG in conditioned medium was normalized to viable cell number. As shown in Fig. 2, the level of secreted OPG in the conditioned media increased in a time-dependent manner. Compared with untreated controls, the level of OPG was significantly increased in ZOL-treated cultures at 72 h (89.4 ± 4.2 versus 48.1 ± 1.2 ng per 5 × 104 cell, p = 0.00076, ANOVA) and 120 h (210.3 ± 7.5 versus 54.8 ± 8.1 ng per 5 × 104 cells; p = 0.00015, ANOVA).

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Figure FIG. 2.. ZOL increased OPG protein expression. The level of OPG protein in the conditioned media harvested from human OB-like cells, at the indicated time-points, was measured by ELISA. Compared with untreated controls, a significant increase of OPG level was observed in ZOL-treated cultures at 72 h (89.4 ± 4.2 vs. 48.1 ± 1.2 ng per 5 × 104 cells; p = 0.00076, ANOVA) and 120 h (210.3 ± 7.5 vs. 54.8 ± 8.1 ng per 5 × 104 cells; p = 0.00015, ANOVA). Three independent experiments were performed, and similar results were observed with cells from three donors.

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ZOL increases the expression of TACE

Although found to significantly decrease transmembrane RANKL protein expression and increase OPG protein secretion, ZOL did not alter the expression of either RANKL or OPG mRNA in OB-like cells (data not shown), suggesting that ZOL may post-translationally regulate transmembrane RANKL expression. Whereas Schlondorff et al.(24) have reported the existence of at least two additional proteases capable of pervanadate-activated RANKL shedding, other studies have shown that TACE plays an essential role in phorbol ester-stimulated RANKL shedding.(23,26) These studies prompted us to examine whether ZOL might regulate the expression of RANKL protein on the OB-like cell surface by modulating the expression of TACE. As shown in Fig. 3, ZOL significantly increased TACE expression at the mRNA level (p < 0.005, Wilcoxon) in human OB-like cells as early as 2 h after its addition. We next examined the expression of TACE protein after the addition of ZOL. Compared with untreated OB-like cells, which expressed low levels of immunofluorescence-detectable TACE protein (Fig. 4C), ZOL treatment resulted in a dramatic upregulation of TACE expression (Fig. 4D). The majority of TACE in ZOL-treated cells was localized to the perinuclear compartment, with some diffuse staining consistent with endoplasmic reticulum localization (Fig. 4D). Western blot analysis of cell lysates confirmed that ZOL upregulated TACE expression in human OB-like cells, at both days 3 and 5, with maximum effect at day 3 (Fig. 5). An antibody to the C-terminal portion of TACE detected four bands when proteins were separated under reducing conditions (Fig. 5A). Two major bands of approximately 106 and 88 kDa, respectively, have been described by others(27) and are thought to represent immature full-length TACE (106 kDa) and a smaller, activated form that lacks the prodomain (88 kDa). The ratio of the two major TACE bands was determined by densitometry and plotted relative to β-actin. As seen in Fig. 5B, 5 μM ZOL increased the expression of TACE protein almost 2-fold in human OB-like cells.

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Figure FIG. 3.. ZOL increased gene expression of TACE. (A) The expression of TACE mRNA in human OB-like cells was examined using semiquantitative RT-PCR. (B) ZOL at a concentration of 5 μM significantly upregulated gene expression of TACE (p < 0.005, Wilcoxon) as early as 2 h after its addition and increased TACE mRNA expression almost 2-fold at 120 h. Three independent experiments were performed, and similar results were observed in cells obtained from three donors.

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Figure FIG. 4.. ZOL increased TACE protein expression in human OB-like cells. To localize TACE in human OB-like cells, in situ immunofluorescence staining was performed. While low levels of TACE protein were detectable in (C) untreated cells, culture of OB-like cells in ZOL resulted in upregulation of TACE expression, (D) localized to the perinuclear compartment and endoplasmic reticulum. An isotype-matched negative control antibody was used to stain OB-like cells under the identical conditions (B) with or (A) without ZOL treatment. Three independent experiments were performed, and the data shown are representative of similar results obtained from two donors. Original magnification, ×40.

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Figure FIG. 5.. (A) Western blot analysis of TACE expression in human OB-like cells. ZOL increased TACE expression, as shown by the increased intensity of a 106-kDa full-length TACE protein and 88-kDa active forms of TACE lacking the prodomain. β-actin was used as an internal control. (B) The density of TACE was quantitated relative to β-actin and plotted as a histogram. Three independent experiments were performed, and similar results were observed in cells obtained from two donors.

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TAPI-2 partially reverses the shedding of membrane RANKL mediated by ZOL

To obtain additional evidence that increased TACE expression was responsible for the reduced expression of transmembrane RANKL in OB-like cells, the TACE inhibitor, TAPI-2, was used. Human OB-like cells were cultured in the presence of 5 μM ZOL and 100 μM TAPI-2 for 3 days before staining with an antibody to RANKL. Consistent with results presented in Fig. 1, when OB-like cells were cultured in ZOL alone, a significant decrease in the percentage of cells expressing membrane RANKL was noted (47.3% in untreated control cells versus 8.6% in cultures treated with 5 μM ZOL, in a representative example shown in Figs. 6A versus 6B). However, when the cells were treated with both 5 μM ZOL and 100 μM TAPI-2, this decrease was partially restored, with 35.5% of the cells now positive for membrane RANKL expression (Fig. 6C). When the data were expressed as the percentage of inhibition of transmembrane RANKL expression (Fig. 6D), they showed that ZOL caused a 57.4 ± 12.4% inhibition of RANKL expression, whereas TAPI-2 was able to reverse this inhibition to 16.1 ± 8.0% (p = 0.02, t-test) of control cells.

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Figure FIG. 6.. TAPI-2 partially reversed the shedding of transmembrane RANKL mediated by ZOL. Using flow cytometric analysis, a marked decrease in the expression of transmembrane RANKL in OB-like cells was observed in cultures treated with (B) ZOL compared with (A) untreated cells. However, when cells were treated with both ZOL (5 μM) and TAPI-2 (100 μM), the percentage of cells expressing membrane RANKL was partially restored (B: 8.6% to C: 35.5%). When the data were expressed as the percentage of inhibition of transmembrane RANKL expression (D), ZOL caused a 57.4 ± 12.4% inhibition of RANKL expression, whereas TAPI-2 was able to reverse this inhibition to 16.1 ± 8.0% (p = 0.02, t-test) of control cells. The data shown in A-C are representative of similar results obtained with cells from three donors.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

BPs have been used for the past three decades for the treatment of OC-mediated bone resorption seen in a variety of disease states.(1) The nitrogen-containing BPs, like ZOL, function by inhibiting enzymes of the mevalonate pathway,(6,28–30) leading to the inhibition of OC activity and induction of OC apoptosis.(5,7,31) BPs are also thought to mediate their effects, in part, by regulating the production of critical OB-derived osteoclastogenic factors, including interleukin-6 (IL-6)(19) and RANKL.(21)

Although difficult to determine, a number of studies have attempted to measure the local concentration of BP to which OB-like cells would be exposed to in the bone microenvironment. Sato et al.(32) estimated that the concentration of alendronate at a resorptive site could be in the order of 800 μM, whereas Usui et al.(33) calculated that the incadronate concentration around bone tumors was approximately 10–30 μM. Although in vivo studies with both neonatal and adult laboratory rodents indicate preferential accumulation of labeled BP at the osteoclastic surface,(32,34) labeling was also consistently observed at osteoblastic surfaces. With regard to ZOL, recent studies by Chen et al.(25) showed that peak plasma levels of 1 μM were obtained after intravenous administration of a 4-mg dose. Given the high affinity of BPs for bone mineral, it is highly likely that OBs in the bone microenvironment would be exposed to ZOL concentrations several-fold higher than the peak plasma level. Based on these findings and our previous work (Pan B, To LB, Fendlay DM, Farrugia AN, Green J, Gronthos S, Lynch K, Evdokiou A, Atkins GJ, Zannettino ACW, unpublished data, 2003), 5 μM ZOL was used in the studies reported herein. Although having little effect on RANKL and OPG mRNA expression, treatment of primary human OBs with ZOL led to a significant decrease in the expression of transmembrane RANKL and a significant increase in the level of secreted OPG protein. Whereas Mackie et al.(21) similarly demonstrated that the nitrogen-containing BP pamidronate downregulated RANKL mRNA in the rat osteosarcoma cell line UMR 106–01 at a concentration of 10 μM, they did not detect any alteration in OPG gene expression. In contrast, Viereck et al.(22) demonstrated that ZOL at a concentration of 0.01 μM not only increased the mRNA level of OPG but also increased OPG protein level as measured by ELISA. Despite the fact that clear similarities exist between our study and those of others, valid comparisons are difficult to make because the target cells and concentration and/or BPs used differ between studies. Nonetheless, the change in the RANKL-to-OPG ratio observed in our study supports the idea that ZOL also mediates its effects on OCs through its effects on OBs.

The decrease in transmembrane RANKL expression, unaccompanied by a commensurate loss in total RANKL protein, suggested that ZOL may mediate its effects post-translationally. Like TNF-α, RANKL is a type II membrane-anchored polypeptide, which is shed from the plasma membrane by TACE or related metalloprotease(s).(23,24) The metalloprotease/disintegrin family of proteins (also referred to as ADAMs [a disintegrin and metalloprotease]) and MDC (metalloprotease, disintegrin, cysteine-rich proteins) have been implicated in a number of cellular processes. TACE (ADAM17) is required for the release of transforming growth factor (TGF)-α, p75 TNF receptor (p75 TNFR), L-selectin, and β-amyloid precursor protein from the cell transmembrane.(35,36) Our data revealed that the loss of RANKL protein from the transmembrane was preceded by an elevation of TACE gene expression as early as 2 h after ZOL treatment. Both in situ immunofluorescence staining and Western blot analyses indicated that ZOL augmented the expression of TACE protein. Although some mature form of TACE was detected on the cell surface by flow cytometry (data not shown), consistent with the findings of Schlondorff et al.,(37) the majority of TACE was detected in the perinuclear compartment after ZOL treatment. This raised the possibility that TACE may function intracellularly to diminish the function of RANKL by decreasing its availability at the membrane surface. The partial reversion of the ZOL-mediated downregulation of membrane RANKL expression in OB-like cells by the TACE inhibitor, TAPI-2, provides further support for this notion. Because TAPI-2 could not totally reverse the effect of ZOL, additional, as yet uncharacterized metalloproteinases, may also be involved in the cleavage of transmembrane RANKL.

In summary, in addition to direct effects on cells of the osteoclast lineage, this study shows that ZOL may inhibit bone resorption by reducing transmembrane RANKL expression and increasing OPG secretion in OB-like cells. This would have a net effect of reducing the ratio of RANKL/OPG, which would lead to a decreased capacity of OB-like cells to support OC formation. Our study supports the concept that the decrease in transmembrane RANKL expression after exposure to ZOL may be caused, in part, by an elevation in TACE protein expression. TACE, which is predominantly localized to the perinuclear compartment, may be critical in cleaving transmembrane RANKL protein and thereby reducing its function in supporting localized de novo OC formation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES

This work was supported by grants from the National Health and Medical Research Council of Australia. The authors gratefully acknowledge Drs Stan Gronthos, Andreas Evdokiou, and Gerald Atkins for helpful discussions.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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