![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ENDOCRINE AND REPRODUCTIVE
Gene Regulation, Bone and Enabling Biology (J.E.O., R.J.S.G., Y.L.M., D.L.H., R.R.M., X.Y., T.F., R.L.C., Q.Q.Z., S.C., K.T., H.U.B.), Lead Generation Biology (R.E.), and Discovery Chemistry Research (Y.X.), Lilly Research Labs, Eli Lilly and Company, Indianapolis, Indiana; and St. Vincent's Institute of Medical Research (T.J.M.), Fitzroy, Victoria, Australia
Received for publication
July 30, 2003
Accepted
December 31, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
; Yasuda et al., 1998). OPG functions as a decoy receptor and blocks the binding of the receptor activator of NF-
B (RANK) ligand (RANKL, a TNF family member) to its receptor RANK, thereby inhibiting both osteoclast formation and activity. Transgenic mice overexpressing OPG develop osteopetrosis due to the failure of osteoclast formation (Simonet et al., 1997), and targeted deletion of the OPG gene results in severe early-onset osteopenia (Bucay et al., 1998; Mizuno et al., 1998). Evidence that OPG produced locally in bone acts as a paracrine factor to decrease bone resorption comes from studies using bone organ and cell cultures from mice rendered null for the OPG gene (Udagawa et al., 2000). An engineered form of OPG, which has a very prolonged half-life (OPG-Fc), has been used in pharmacological studies in human subjects and experimental animals (Hofbauer and Heufelder, 2000; Bekker et al., 2001; Kostenuik and Shalhoub, 2001). Treatment with OPG-Fc inhibits osteoclastogenesis and the bone resorption response to ovariectomy, parathyroid hormone (PTH)-related protein (PTHrP), 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], interleukin-1
, TNF
, low calcium, tumor-induced humoral hypercalcemia, bone metastasis, periodontal disease, and adjuvant arthritis (Simonet et al., 1997; Kong et al., 1999a; Morony et al., 1999; Capparelli et al., 2000; Min et al., 2000; Teng et al., 2000). Consistent with its important regulatory role in bone resorption, OPG expression by stromal/osteoblastic cells is altered both in vitro and in vivo in response to the many osteotrophic factors, which either enhance or inhibit osteoclast formation (Brandstrom et al., 1998; Horwood et al., 1998; Takai et al., 1998; Vidal et al., 1998a,b; Hofbauer et al., 1999; Nakagawa et al., 1999; Hofbauer and Heufelder, 2000; Onyia et al., 2000; Ma et al., 2001). Agents that decrease OPG production generally have the opposite effect on the production of RANKL leading to the conclusion that osteoclast formation is enhanced by factors that favor an increase in the ratio of RANKL to OPG (Horwood et al., 1998; Ma et al., 2001).
Based on the results of these in vitro and in vivo studies and on the features of genetically manipulated mice, it is apparent that the control of osteoclast formation and activity by OPG might provide a useful approach to the treatment and prevention of clinical conditions characterized by enhanced osteoclastic bone loss (such as osteoporosis, malignant bone disease, rheumatoid arthritis, Paget's disease of bone, hypercalcemia of malignancy, and periodontal disease). Currently OPG-Fc is in early phase evaluation in humans as a potential therapeutic (Bekker et al., 2001), but one limitation for its widespread use is the fact that it is a large protein that is not orally bioavailable, and thus requires parenteral administration. Furthermore, prolonged use of such a modified version of OPG raises the possibility of an immune response. The likelihood that the physiological function of OPG is primarily a paracrine regulator (Udagawa et al., 2000) provides a basis for the development of small molecules that could selectively stimulate endogenous OPG expression to levels sufficient to result in a therapeutic benefit similar to that obtained with parenteral administration of OPG. Such agents could be useful for the treatment of clinical conditions characterized by enhanced bone resorption. In the present study, we developed a high-throughput, cell-based screen to identify compounds that activate the OPG gene promoter and show these to be effective in preventing the increased osteoclast formation and bone resorption associated with either pharmacological treatment or models of disease.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-gal), fused with -galactosidase (-gal) reporter gene of the p-gal-Basic reporter vector (BD Biosciences Clontech, Palo Alto, CA), was performed using standard cloning procedures as previously described (Thirunavukkarasu et al., 2000). All constructs were verified by restriction mapping and DNA sequencing using the dideoxy-chain termination method. Cell Culture, Transfection Assays, and Analyses. The rat osteoblast-like osteogenic sarcoma cell line, UMR 106, and the human osteoblast-like osteosarcoma cell line, SaOS-2 were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (3:1) (Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) plus 2 mM glutamine (Invitrogen). All cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2. Experiments were initiated when cells were approximately 70 to 80% confluent.
For stable transfection experiments, the OPG promoter construct pOPG5.9-gal (-5917 to +1) or SV40 promoter -gal was cotransfected with a second plasmid pRc/CMV (Invitrogen) encoding the neomycin gene into UMR 106 cells using Fugene 6 (Roche Applied Science, Indianapolis, IN) as previously described (Thirunavukkarasu et al., 2000). Details of the selection and characterization of the stable clones used in this study for OPG promoter analysis have been described (Thirunavukkarasu et al., 2001). Stable clones of UMR 106 cells expressing Runx2 (an osteoblast-specific promoter) or 6XOSE (contains six copies of the Runx2 binding site) fused to luciferase reporter gene were prepared as previously described (Ducy and Karsenty, 1995; Krishnan et al., 2003). The stable clones were stimulated with recombinant human TGF-1 (10 ng/ml, R&D Systems, Minneapolis, MN), trichostatin A (1 x 10-4 to 1 x 10-1 µM, Calbiochem, San Diego, CA), sodium butyrate (5 x 10-3 to 5 x 102 µM, Sigma-Aldrich, St. Louis, MO), or compounds (0-30 µM) for 24 h. Cell extracts were assayed for -gal activity using the -Gal reporter gene assay kit (Roche Applied Science) as recommended by the manufacturer. The luciferase reporter gene assay, high sensitivity (Roche Applied Science) was used for analysis of luciferase in the cell extracts. Assays were done in white, opaque Microlite 2+ 96-well plates (Dynex, Chantilly, VA). Luminescence was measured in a MLX microtiter plate luminometer (Dynex) and light integration measured at 2 s (RLU summed).
High-Throughput Screening of Compounds. A stable UMR 106 clone containing the -gal reporter gene under the control of a 5.9-kb human OPG promoter (OPG--gal) was used for screening. Each compound of the library (Research Triangle Park Laboratories, Eli Lilly and Company, Research Triangle Park, NC) was dissolved in DMSO at 1 mg/ml. The UMR 106 cells containing OPG--gal were plated in 96-well plates (Corning, Cambridge, MA) and incubated for 24 h with each compound (final concentration was 10 µM in medium), following serum withdrawal for 12 to16 h. Cell lysates were then assayed for -gal activity as described. All results from each experiment were compared with control wells (DMSO only) and TGF-1-treated positive control. Compounds that showed 
50% of the activity of TGF-1 were studied further.
OPG ELISA Assay. To quantify the amount of OPG secreted into the cell culture medium, SaOS-2 cells (50,000 cells/well in a 96-well plate) were treated with TGF-1 (5 ng/ml), trichostatin A (5 x 10-9 to 5 x 101 µM), sodium butyrate (5 x 10-8 to 5 x 102 µM), or compounds (0-30 µM) for 48 h. OPG secreted into the culture medium was analyzed using a sandwich ELISA, utilizing rabbit polyclonal antiserum directed against recombinant human OPG, as described previously (Onyia et al., 2000; Ma et al., 2001; Yang et al., 2002). A standard curve was generated using recombinant human OPG. Similarly, serum circulating OPG levels were determined by sandwich ELISA with rabbit polyclonal anti-human OPG IgG. Briefly, 96-well plates (IMMUNO 4 flat bottom; Dynatech Labs, Chantilly, VA) were coated with 0.5 µg/well purified rabbit anti-OPG antibody (IgG) (diluted in 100 µl of carbonate/bicarbonate buffer, Sigma-Aldrich), incubated at 4°C overnight, and blocked for 1 h at room temperature with 200 µl/well Blocker casein in PBS (Pierce Endogen, Rockford, IL). The plate was incubated with 100 µl of serum diluted in 1% bovine serum albumin PBS with 0.05% Tween 20 (TPBS) for 1 h and followed by three washes with TPBS. To each well was added 100 µl of diluted biotinylated anti-OPG IgG for 1 h and followed by 100 µl/well streptavidin-HRP conjugate (Zymed Laboratories, South San Francisco, CA) for 30 min with TPBS washing. ELAST ELISA amplification system (PerkinElmer Life Sciences, Boston, MA) was applied before color development according to the manufacturer's protocol. Color was developed with 100 µl/well TMB Microwell Peroxidase Substrate System (KPL, Inc., Gaithersburg, MD) for 15 min, stopped with 100 µl/well 1 N phosphoric acid, and analyzed by reading the absorbance at 450 nm.
Isolation of Poly A+ RNA and Northern Blotting. In UMR 106 cells, the effects of TGF-1 (10 ng/ml) on OPG mRNA expression were evaluated by Northern blot analysis following 24 h of treatment. Time-dependent effects of Cmpds 1, 2, and 3 on OPG and RANKL mRNA expression were evaluated in human SaOS-2 cells treated with 10 µM compound for 2, 24, or 48 h. Cultures (four T150 flasks/group) of cells were grown (as described above) to 80 to 90% confluence and then changed into medium containing 0.1% FBS overnight. The cells were then treated with the indicated concentrations of compound for the specified time periods. Total RNA was extracted from the osteoblast cultures by adding Ultraspec-II RNA (Biotecx Laboratories, Inc., Houston, TX) directly to the culture flasks. The resulting cell lysates were passed several times through a 10-ml pipette before collection. Poly A+ RNA was isolated from total RNA using Oligotex (QIAGEN, Valencia, CA) according to the manufacturer's protocol and quantitated by spectrophotometry. The absorbance at 260 nm was determined and the 260/280-nm absorbance ratio was calculated to ensure the absence of protein contamination. Samples of poly A+ RNA (2 µg) were denatured in 0.04 M MOPS, pH 7.0, 10 mM sodium acetate, 1 mM EDTA, 2.2 M formaldehyde, and 50% formamide at 60°C for 10 min and size fractionated by electrophoresis through 1% agarose gels in 2.1 M formaldehyde and 1x MOPS and transferred to nylon membranes (Brightstar-Plus; Ambion Inc., Austin, TX). The nylon membranes were air-dried, and the RNA samples were cross-linked to the membranes by UV irradiation in a Stratalinker (Stratagene, La Jolla, CA). Migration of 28 S and 18 S ribosomal RNA was determined by ethidium bromide staining. DNA probes were labeled by the random primer method (Invitrogen) using [-32P]dCTP. Prehybridization and hybridization were carried out at 48°C in NorthernMax buffers (Ambion Inc.). After hybridization, membranes were washed for 30 min at room temperature in buffer containing 2x SSC and 0.1% SDS and then 30 min at 48°C in 0.2x SSC and exposed to Biomax MS X-ray film (Eastman Kodak, Rochester, NY) at -70°C. Autoradiograms were quantitated by scanning laser densitometry (2400 Gel Scan XL; Amersham Biosciences Inc., Piscataway, NJ). Labeled bands were quantitated as densitometric units and normalized to that of the GAPDH signals to correct for variations in RNA transfer and gel loading. The data were expressed as -fold change versus untreated control samples.
Generation of Radiolabeled Probes for Northern Analysis. To generate radioactive probes for Northern analysis, the inserts containing OPG or RANKL cDNA were released from the plasmid by restriction digest. Rat GAPDH cDNA probes were cloned using polymerase chain reaction with specific primer pairs as published previously (Onyia et al., 1995, 2000). Twenty-five nanograms of cDNA were labeled by the random primer method (Invitrogen) using [-32P]dCTP (Amersham Biosciences Inc.). Free nucleotides were removed by centrifugation through a Centricon-50 column (Millipore Corporation, Bedford, MA).
Coculture Assay of Bone Marrow Cells and BALC Cells. The effects of recombinant human OPG or compounds on osteoclast differentiation were studied in cocultures of bone marrow cells and BALC cells (a murine calvarial-derived cell line) as previously described (John et al., 1996). Briefly, bone marrow cells from the femurs of male BALB/C mice (aged 8-9 weeks; The Jackson Laboratory, Bar Harbor, ME) were seeded into 24-well cluster dishes (5 x 104 mononuclear cells/cm2) in growth media [RPMI 1640 media (Invitrogen) containing 5% heat-inactivated FBS (Hyclone) and 1% antibiotic/antimycotic solution (Invitrogen)]. BALC cells (1.5 x 104 cells/cm2) were cocultured with the bone marrow cells. The cultures were treated with 1,25(OH)2D3 (10-8 M, BIOMOL Research Laboratories, Plymouth Meeting, PA) in the presence or absence of 0.01 to 30 µM compound or OPG for 6 days with fresh medium and reagents added on day 3. The cultures were fixed with formalin, stained for tartrate-resistant acid phosphatase (TRAP), and the number of TRAP-positive cells with three or more nuclei were quantitated per well as previously described (John et al., 1996).
Calvarial Organ Culture. Calvariae were dissected from mouse pups (aged 3-5 days; Taconic Farms Inc., Germantown, NY), cleaned of soft tissue, and the calvariae were cut in half along the sagittal suture (Fukayama et al., 1988). Half-calvariae were placed in 17 x 100 mm polypropylene round bottom tubes that contained 1 ml of Dulbecco's modified Eagle's medium (Invitrogen) with 15% heat-inactivated horse serum (Hyclone), 2.8 mM L-glutamine (Invitrogen), 10 units/ml sodium heparin (Elkin-Sinn, Cherry Hill, NJ), and penicillin/streptomycin (100 units/ml and 100 µg/ml, respectively; Invitrogen) in the presence or absence of trichostatin A (0.5-50 nM), sodium butyrate (0.5-50 mM), Cmpd 1 (0.1-30 µM), or Cmpd 5 (0.1-30 µM). The tubes were gassed with 50% oxygen, 5% carbon dioxide, and 45% nitrogen, sealed, and incubated for 24 h at 37°C on a Roller Drum (New Brunswick Scientific Co., Inc., Edison, NJ). Following the 24-h preincubation, the media were changed and fresh compound in media or control media alone was added, which did or did not contain 1 nM human PTH 1-38 (Zeneca Inc., Wilmington, DE). After 72 h in culture, the medium was collected and ionized calcium was analyzed using a Ciba-Corning 634 ISE Ca2+/pH analyzer (Chiron Diagnostics Corp., East Walpole, MA).
Parathyroidectomized Rat Model. Weanling, Sprague-Dawley female rats (Taconic Farms, Inc.) weighing 60 to 70 g were used as previously described (Ma et al., 2001). Animals were parathyroidectomized by vendor and delivered to our facility 2 to 3 days postsurgery. They were maintained on a 12h light/dark cycle at 22°C with access to tap water ad libitum. To minimize the gut and kidney effects on serum calcium, rats were fed a calcium-free diet containing 0.02% calcium, and 0.3% phosphorus (TD 99171; Teklad, Madison, WI) during the experimental period. Rats were pretreated with vehicle or Cmpd 5 (0.5, 5, and 50 mg/kg) daily by subcutaneous (s.c.) injection for 3 days with 4 rats in each group. At 2 h after the last dosing, synthetic human PTH 1-38 (1 µg/100 g/6 h; Zeneca Inc.) was given s.c. via Alzet pump (Durect Corp., Palo Alto, CA) for 6 h. Cmpd 5 was first dissolved in 5% DMSO, then 10% castor oil, and finally water was added to the final solution. PTH was prepared in a vehicle of acidified saline containing 2% heat-inactivated rat serum. Blood was collected under isoflurane anesthesia at indicated time points for the measurement of blood ionized calcium (Ciba-Corning 634 Ca2+/pH analyzer) and serum OPG. Bones were collected for histologic analyses as described (Ma et al., 1995). All animal studies were approved by the Eli Lilly and Company Animal Care and Use Committee.
Rat Mammary Carcinoma Tumor Bone Metastasis Model. Cells of the rat mammary carcinoma tumor 13762 (2 x 106 cells/rat in a 200-µl volume) were injected into the left ventricle of 7- to 8-week-old Fischer-344 female rats (Harlan, Indianapolis, IN) (Alvarez et al., 2003). Cmpd 5 (0.1 and 1 mg/kg/day) was administered for 4 weeks by s.c. injection starting at 1 week after cell inoculation. After 3 and 4 weeks of treatment (4 and 5 weeks postinoculation), tumor progression was evaluated using a high resolution Faxitron X-ray system (model MX-20; Faxitron X-ray Corp., Buffalo Grove, IL).
Image Analysis of Osteolytic Lesions. Radiographic images were analyzed for tumor-induced osteolysis. Lesions visible in tibiae, femurs, and fibulae were counted, and the margins were traced to determine lesion number and area using an Image-Pro Plus System (version 3.0.1, Media Cybernetic, Silver Spring, MD).
Rat Adjuvant Arthritis Model. Male, 200 g Lewis rats (Harlan) were injected with lipoidal amine 7.5 mg/kg at the base of the tail as described (Benslay and Bendele, 1991; Bendele, 2001). Rats were treated for 5 days with Cmpd 5 s.c. at 1 and 3 mg/kg/day starting on day 9 (n = 9). On day 15 paw and spleen weight were recorded with a precise balance. Ankle joints were collected for histological analysis.
Quantitation of Osteoclast Number. Bones were fixed in 10% buffered formalin for 2 days, decalcified in Decalcifier I (Surgipath, Richmond, IL), and processed for embedding in paraffin. Longitudinal cut, 5-µm thick, decalcified sections were stained by 0.1% toluidine blue for proximal tibial metaphysis or hemotoxylin-eosin staining (ankle joint). Osteoclast number was measured on the entire marrow region within the cortical shell between 0.67 and 2 mm proximal tibial metaphysis or 1 mm (distal tibial metaphysis) distal to the growth plate metaphyseal junction under 20x magnification. Trabecular bone surface was measured using a digitizing image analyzing system named Osteomeasure (OsteoMetrics, Inc. Atlanta GA). The osteoclast numbers were then normalized to trabecular bone surface (Ma et al., 1995).
Statistical Analysis. Blood ionized calcium, osteoclast number, serum OPG, bone lesion number, total bone lesion area, paw and spleen weight, and trabecular area and osteoclast number in the calcaneum were presented as mean ± S.E.M. Differences in group means were assessed by analysis of variance using Fisher's protected least significant difference for which the significance level was P < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-gal was selected, isolated, and characterized for responsiveness to TGF-1, an agent known to induce OPG transcription (Takai et al., 1998; Thirunavukkarasu et al., 2001). This clone demonstrated a 5-fold increase in OPG promoter activity in response to TGF-1, a pattern reminiscent of its effect on endogenous OPG mRNA and protein expression (Fig. 1A) (Thirunavukkarasu et al., 2001). For screening, OPG--gal-containing cells were treated with individual synthetic organic compounds at a concentration of 10 µM for 24 h, and compounds that induced OPG--gal activity to 50% of the levels seen with TGF-1 were then analyzed further. Compounds that were found to stimulate OPG--gal in a concentration-dependent manner were further evaluated in a second UMR 106 stable cell clone, which contained a SV40 promoter--gal reporter (SV40--gal) and produced -gal constitutively. Only those compounds, which selectively induced OPG--gal and had no detectable activity on SV40--gal (up to 30 µM tested), were further studied. Based on this approach, after screening approximately 387,000 compounds, 3 structurally unrelated compounds Cmpds 1, 2, and 3 that selectively induced the OPG promoter (Table 1) and not the SV40 promoter (Fig. 1B) were chosen for further studies.
|
|
Effects on OPG mRNA and Protein. To confirm that the increase in OPG transcription/promoter was reflected in endogenous mRNA and protein, we evaluated the effects of the compounds on OPG production by rat UMR 106 cells and human SaOS-2 osteoblast-like osteosarcoma cells. An OPG ELISA was used to evaluate secreted OPG in the cell culture media whereas the changes in mRNA were quantitated by Northern blotting of Poly A+ RNA. Cmpds 1, 2, and 3 increased OPG protein levels in both SaOS-2 (Fig. 1C) and UMR 106 cells (data not shown). In SaOS-2 cells, the increase in protein expression was also associated with a time-dependent increase in OPG mRNA expression (Fig. 1D). At the time points of peak OPG mRNA expression, no effects were observed on the levels of mRNA for RANKL, the OPG cognate ligand, or GAPDH (Fig. 1D). Up-regulation of GAPDH expression was observed at time points following peak OPG mRNA expression (Fig. 1D), and the reason for this increase is unclear. The time course for onset of OPG activation (6-24 h) was compound-dependent. Together these results provide evidence that these small molecules induce OPG expression at least in part via activation of transcription.
Selectivity and Structure-Activity Relationships. Of the three compounds, we focused further analysis on Cmpd 1 (a benzamide derivative), which we believed would be a more likely candidate for drug development based on its chemical structure (stability and structural simplicity). Four additional active compounds (Table 2 and Fig. 2A) were identified by analysis in the OPG promoter and secretion assays of 115 compounds that are substructurally related to Cmpd 1. The structural similarities and activity of the five active (Cmpds 1, 4, 5, 6, and 7) and two structurally related inactive compounds were studied (Table 2 and Fig. 2A). All five active compounds stimulated OPG promoter and protein expression but had no effect on the SV40 promoter activity (Table 2, Fig. 2, A-B). In the OPG promoter assay, Cmpds 1, 4, and 5 had similar efficacies and potencies whereas Cmpds 6 and 7 were less potent and efficacious. In the OPG secretion assay all five active compounds had a comparable efficacy but Cmpd 5 was 16- to 100-fold more potent (Table 2). Structure activity analysis demonstrated that activity was retained when the position occupied by the methoxy group in Cmpd 1 was either replaced with a tertbutyl (Cmpd 4), a nitrile (Cmpd 7), or left unoccupied (Cmpd 6). Addition of a 3 pyridylmethyl carbomate group (Cmpd 5) resulted in increased potency. An addition of a pyrimidine ring (compare Cmpds 6 and 8) or the replacement of the position 2 carbon in the pyrimidine ring of Cmpd 4 (compare Cmpds 4 and 9) resulted in loss of activity. To demonstrate further the selectivity of this series for the OPG gene, we compared the effect of Cmpd 5 (the most potent of this series) on the OPG promoter with activity on other promoters, SV40 (a generic constitutive promoter), Runx2 (a bone osteoblast specific promoter), and 6XOSE (which contains 6 copies of the Runx2 binding site, Fig. 2B). Cmpd 5 had no significant effect on transcription of any of the other promoters examined suggesting that the compound activity is specific for OPG and not likely a generalized, nonspecific effect on transcription. These results are consistent with a structure activity relationship and confirm the effect of this compound class on OPG expression.
|
|
The benzamide derivative, Cmpd 5, has been shown to inhibit histone deacetylase (HDAC) activity (Suzuki et al., 1999). Therefore, we evaluated the effects of trichostatin A and sodium butyrate, both of which are broad spectrum HDAC inhibitors, on OPG promoter activity and secretion. Although we were able to confirm the HDAC inhibitory effects of Cmpd 5 (data not shown), neither trichostatin A nor sodium butyrate stimulated OPG promoter activity or protein secretion (Fig. 3, A-B) in contrast to the results with Cmpd 5 (Table 2, Fig. 2A). In the calvarial bone resorption model, Cmpd 5 but not trichostatin A or sodium butyrate, inhibited PTH-induced bone resorption (Fig. 3C). Based on these results, we concluded that the HDAC inhibitory activity of Cmpd 5 is unrelated to its activity on OPG expression and bone resorption.
|
Small Molecule Inducers of OPG Secretion Inhibit Osteoclast Differentiation and Bone Resorption in Vitro. Organ and cell cultures have been used to show that, in the bone microenvironment, osteoblast-derived OPG inhibits osteoclast formation (from hematopoietic precursors) and decreases bone resorption (Udagawa et al., 2000). Using the same strategy, we evaluated the ability of Cmpds 1 and 5 to inhibit osteoclast formation in vitro (in coculture of mouse bone marrow cells and BALC stromal-osteoblasts) and bone resorption in calvariae treated with PTH to stimulate bone resorption. Like exogenous OPG, Cmpds 1 and 5 inhibited osteoclastogenesis (Fig. 4A). Although both compounds inhibited osteoclast formation in a concentration-dependent manner, Cmpd 5 was approximately 20-fold more potent (IC50, 0.022 versus 0.47 µM). Similarly, we also observed a concentration-dependent inhibitory effect of Cmpds 1 and 5 on PTH-induced bone resorption in calvarial organ culture (Fig. 4B) with Cmpd 5 being approximately 20-fold more potent (estimated IC50, 0.93 versus 25.76 µM).
|
Cmpd 5 Inhibits Bone Resorption in Vivo. To demonstrate in vivo efficacy, we evaluated Cmpd 5 for antiresorptive activity in parathyroidectomized (PTX) rats challenged with PTH infusion. Antiresorptive activity was quantitated by inhibition of PTH-induced hypercalcemia. As we have recently shown, PTH-induced resorption in this model is associated with reciprocal regulation of OPG and RANKL (decreased OPG and increased RANKL) (Ma et al., 2001). PTX rats were treated once a day for 3 days with Cmpd 5 and were then challenged with PTH infusion for 6 h at 2 h after injection on the 3rd day (50 h). Blood ionized calcium levels were determined before and after PTH infusion (50, 53, and 56 h). As expected, PTH challenge caused an increase in blood ionized calcium and increased osteoclast number (Fig. 5, A-C and F; data not shown). In this model, exogenous OPG blocks the effects of PTH infusion (data not shown). Cmpd 5 dose-dependently inhibited PTH-induced hypercalcemia and also inhibited basal blood ionized calcium levels to below normal range at the higher doses (15, 35, and 50 mg). The effects on basal blood ionized calcium were observed in the absence of PTH infusion (50 h). In a separate experiment with three doses of Cmpd 5 (low, medium, and high doses, Fig. 5B), we confirmed that the inhibitory effect on PTH-induced hypercalcemia was associated with decreased osteoclast number (Fig. 5, C, G, and H). At the highest dose (50 mg/kg/day), the histologic sections suggested a decrease in cellularity which might indicate toxicity (Fig. 5, F versus H). PTH decreased serum OPG levels in the PTX rats and a trend toward an increase in serum OPG in Cmpd 5-treated rats was observed (Fig. 5D, compared with PTH control); however, the effect did not reach statistical significance.
|
Previous studies have demonstrated that exogenously administered OPG inhibits osteoclastogenesis and bone resorption in experimental models of arthritis and tumor metastasis (Kong et al., 1999a; Capparelli et al., 2000). Therefore, we explored the ability of Cmpd 5 to inhibit tumor-induced osteoclastogenesis, osteolysis, and skeletal tumor burden in a rat model of experimental bone metastasis. Injection of rat mammary carcinoma cell line 13762 into the left ventricle of Fischer-344 rats resulted in the development of radiographically evident lytic bone lesions in the tibiae, fibulae, and femurs. Treatment with Cmpd 5, 0.1 or 1 mg/kg/day for 3 to 4 weeks significantly decreased the number and area of radiographically evident lytic bone lesions (Fig. 6, A-C). Histologic evaluation of the proximal tibiae confirmed that the decrease in lytic lesion number and area was associated with decreased osteoclastic bone resorption (data not shown). No additional clinical toxic syndromes were seen in Cmpd 5-treated rats when compared with vehicle controls during the 4-week experimental period.
|
Last, the efficacy of Cmpd 5 in a lipoidal amine-induced adjuvant arthritic rat model was evaluated (Benslay and Bendele, 1991; Bendele, 2001). Lipoidal amine-induced adjuvant arthritis is characterized by severe inflammation, increased osteoclast number, extensive local bone and cartilage destruction, loss of bone density/volume, and crippling. At the onset of disease (day 9), animals were treated with vehicle or Cmpd 5 (1 or 3 mg/kg) for 5 days. Cmpd 5 partially blocked the severity of inflammation as measured by paw and spleen weight (Fig. 7A). Histologically (when compared with control) lipoidal amine-treated animals showed a decreased trabecular area and increased osteoclast number in the calcaneum (Fig. 7, A and B). Cmpd 5 treatment partially blocked the lipoidal amine-induced decrease in trabecular area and increase in osteoclast number (Fig. 7A).
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Capparelli et al., 2000; Croucher et al., 2001). A considerable body of evidence has established the central importance of osteoclast formation in the processes of formation and maintenance of bone metastases and the effectiveness of inhibitors of bone resorption in prevention and treatment (Mundy, 2002). The results obtained with the breast cancer-bone metastasis model indicate that stimulating OPG production locally in bone might provide an effective therapeutic approach to decreasing and/or preventing osteolysis in metastatic bone disease.
Adjuvant arthritis induced in the rat by lipoidal amine injection results in severe inflammation and bone destruction. The bone protective effect of Cmpd 5 in these experiments is consistent with its promotion of OPG production, but the mechanism for the partial inhibition of inflammation is intriguing and deserves further investigation. Since OPG neutralizes RANKL and given the important role of the RANKL/RANK pathway in immunomodulation, it is tempting to speculate that this effect may be mediated via increased OPG production. This thesis is supported in part by the finding that RANKL-null T cells have a significantly reduced production of both Th1 and Th2 cytokines (Kong et al., 1999b). By contrast, previous studies have shown in experimental models in the rat of adjuvant-induced arthritis (Kong et al., 1999a) and of collagen-induced arthritis (Romas et al., 2002) that OPG-Fc treatment at onset of disease prevents bone and cartilage destruction but not inflammation. However, the exogenous OPG used in these studies (OPG-Fc) is a shortened long-acting peptide (stabilized by the Fc fragment) and might not have all the biological properties of the endogenous full-length protein.
The results of this study provide proof of the concept that small molecular weight compounds that stimulate the OPG gene can be discovered. Additionally, we provide evidence that such molecules inhibit bone resorption in three in vivo models. Cmpd 5, which was most extensively tested, does possess HDAC inhibitory activity. Inhibition of HDAC activity induces histone hyperacetylation and regulates transcription of some genes through alteration in chromatin structure. HDAC inhibitors are being evaluated for treatment of cancer (Johnstone, 2002). Based on our evaluation of trichostatin A and sodium butyrate, we concluded that the effects of Cmpd 5 on OPG promoter activity and protein secretion are probably not due to inhibition of HDAC activity since trichostatin A and sodium butyrate did not mimic the effects of Cmpd 5. Additionally, trichostatin A and sodium butyrate did not inhibit calvarial bone resorption, which suggests that inhibition of HDAC activity alone is not sufficient to block bone resorption.
It is concluded that modulation of OPG in the bone microenvironment can be as effective as exogenously administered OPG-Fc in inhibiting the growth of tumor deposits in bone and the periarticular resorption of adjuvant arthritis. The described small molecules increase OPG transcription, mRNA, and protein, but it is not yet known whether these effects are explained entirely by the enhanced OPG promoter activity. Together, these data strongly support the concept that small molecules that selectively stimulate endogenous OPG expression could have therapeutic benefits similar to those of native OPG in reducing both the formation and the activity of osteoclasts. This could provide a new approach to development of drugs for treatment of diseases of increased bone resorption, such as osteoporosis, Paget's disease, osteolytic bone metastases, hypercalcemia of malignancy, and rheumatoid arthritis.
|
Footnotes |
|---|
ABBREVIATIONS: OPG, osteoprotegerin; TNF, tumor necrosis factor; RANK, receptor activator of nuclear factor B; RANKL, RANK ligand; -gal, -galactosidase; HDAC, histone deacetylase; PTH, parathyroid hormone; TGF-, transforming growth factor beta; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TRAP, tartrate-resistant acid phosphatase; PTX, parathyroidectomized; RLU, relative light units; Cmpd, compound; SV40, simian virus 40; MOPS, 3-(N-morpholino)propanesulfonic acid; SSC, standard saline citrate; 1,25(OH)2D3, 1,25-dihydroxyvitamin D3.
1 Contributed equally to this manuscript. 
Address correspondence to: Dr. Rachelle J. S. Galvin, BLDG 98C/B, DC 0403, Lilly Research Labs, Eli Lilly and Company, Indianapolis, IN 46285. E-mail: RJSGalvin{at}lilly.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Alvarez E, Westmore M, Galvin RJS, Clapp CL, Considine EL, Smith SJ, Keyes K, Iversen PW, Delafuente DM, Sulaimon S, et al. (2003) Properties of bisphosphonates in the 13762 rat mammary carcinoma model of tumor induced bone resorption. Clin Cancer Res 9: 5705-5713.
Bekker PJ, Holloway D, Nakanishi A, Arrighi M, Leese PT, and Dunstan CR (2001) The effect of a single dose of osteoprotegerin in postmenopausal women. J Bone Miner Res 16: 348-360.[CrossRef][Medline]
Bendele AM (2001) Animal models of rheumatoid arthritis. J Musculoskel Neuron Interact 1: 377-385.
Benslay DN and Bendele AM (1991) Development of a rapid screen for detecting and differentiating immunomodulatory vs. anti-inflammatory compounds in rats. Agents Actions 34: 254-256.[CrossRef][Medline]
Brandstrom H, Jonsson KB, Vidal O, Ljunghall S, Ohlsson C, and Ljunggren O (1998) Tumor necrosis factor-alpha and -beta upregulate the levels of osteoprotegerin mRNA in human osteosarcoma MG-63 cells. Biochem Biophys Res Commun 248: 454-457.[CrossRef][Medline]
Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Capparelli C, Scully S, Tan HL, Xu W, Lacey DL, et al. (1998) Osteoprotegerin-deficient mice develop early onset osteoporosis and arterial calcification. Genes Dev 12: 1260-1268.
Capparelli C, Kostenuik PJ, Morony S, Starnes C, Weimann B, Van G, Scully S, Qi M, Lacey DL, and Dunstan CR (2000) Osteoprotegerin prevents and reverses hypercalcemia in a murine model of humoral hypercalcemia of malignancy. Cancer Res 60: 783-787.
Croucher PI, Shipman CM, Lippitt J, Perry M, Asosingh K, Hijzen A, Brabbs A, van Beek E, Holen I, Skerry T, et al. (2001) Osteoprotegerin inhibits the development of osteolytic bone disease in multiple myeloma. Blood 98: 3534-3540.
Ducy P and Karsenty G (1995) Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol Cell Biol 15: 1858-1869.[Abstract]
Fukayama S, Bosma TJ, Goad DL, Voelkel EF, and Tashjian AH (1988) Human parathyroid hormone (PTH)-related protein and human PTH: comparative biological activities on human bone cells and bone resorption. Endocrinology 123: 2841-2848.
Hofbauer LC and Heufelder AE (2000) Clinical review 114: hot topic. The role of receptor activator of nuclear factor-kappa B ligand and osteoprotegerin in the pathogenesis and treatment of metabolic bone diseases. J Clin Endocrinol Metab 85: 2355-2363.
Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Spelsberg TC, and Riggs BL (1999) Estrogen stimulates gene expression and protein production of osteoprotegerin in human osteoblastic cells. Endocrinology 140: 4367-4370.
Horwood NJ, Elliott J, Martin TJ, and Gillespie MT (1998) Osteotropic agents regulate the expression of osteoclast differentiation factor and osteoprotegerin in osteoblastic stromal cells. Endocrinology 139: 4743-4746.
John V, Hock JM, Short LL, Glasebrook AL, and Galvin RJS (1996) A role for CD8(+) T-lymphocytes in osteoclast differentiation in-vitro. Endocrinology 137: 2457-2463.[Abstract]
Johnstone R (2002) Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov 1: 287-299.[CrossRef][Medline]
Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, Capparelli C, Li J, Elliott R, McCabe S, et al. (1999a) Activated T cells regulate bone loss and joint destruction in adjuvant arthritis through osteoprotegerin ligand. Nature (Lond) 402: 304-309.[CrossRef][Medline]
Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C, Morony S, Oliveirados-Santos AJ, Van G, Itie A, et al. (1999b) OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature (Lond) 397: 315-323.[CrossRef][Medline]
Kostenuik PJ and Shalhoub V (2001) Osteoprotegerin: a physiological and pharmacological inhibitor of bone resorption. Curr Pharm Des 7: 613-635.[CrossRef][Medline]
Krishnan V, Moore TL, Ma YFL, Helvering LM, Frolik CA, Valasek KM, Ducy P, and Geiser AG (2003) Parathyroid hormone bone anabolic action requires Cbfa1/Runx2-dependent signaling. Mol Endocrinol 17: 423-435.
Ma YF, Jee WS, Ke HZ, Lin BY, Liang XG, Li M, and Yamamoto N (1995) Human parathyroid hormone-(1-38) restores cancellous bone to the immobilized, osteopenic proximal tibial metaphysis in rats. J Bone Miner Res 10: 496-505.[Medline]
Ma YL, Cain RL, Halladay DL, Yang X, Zeng Q, Miles RR, Chandrasekhar S, Martin TJ, and Onyia JE (2001) Catabolic effects of continuous human PTH (1-38) in vivo is associated with sustained stimulation of RANKL and inhibition of osteoprotegerin and gene-associated bone formation. Endocrinology 142: 4047-4054.
Min H, Morony S, Sarosi I, Dunstan CR, Capparelli C, Scully S, Van G, Kaufman S, Kostenuik PJ, Lacey DL, et al. (2000) Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calcification by blocking a process resembling osteoclastogenesis. J Exp Med 192: 463-474.
Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, Kanno T, Sato Y, Nakagawa N, Yasuda H, Mochizuki S, et al. (1998) Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin. Biochem Biophys Res Commun 247: 610-615.[CrossRef][Medline]
Morony S, Capparelli C, Lee R, Shimamoto G, Boone T, Lacey DL, and Dunstan CR (1999) A chimeric form of osteoprotegerin inhibits hypercalcemia and bone resorption induced by IL-1beta, TNF-alpha, PTH, PTHrP and 1,25(OH)2D3. J Bone Miner Res 14: 1478-1485.[CrossRef][Medline]
Mundy G (2002) Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2: 584-593.[CrossRef][Medline]
Nakagawa N, Yasuda H, Yano K, Mochizuki S, Kobayashi N, Fujimoto H, Shima N, Morinaga T, Chikazu D, Kawaguchi H et al. (1999) Basic fibroblast growth factor induces osteoclast formation by reciprocally regulating the production of osteoclast differentiation factor and osteoclastogenesis inhibitory factor in mouse osteoblastic cells. Biochem Biophys Res Commun 265: 158-163.[CrossRef][Medline]
Onyia JE, Bidwell J, Herring J, Hulman J, and Hock JM (1995) In vivo, human parathyroid hormone fragment (hPTH 1-34) transiently stimulates immediate early response gene expression but not proliferation in trabecular bone cells of young rats. Bone 17: 479-484.[Medline]
Onyia JE, Miles RR, Yang Y, Halladay DL, Hale J, Glasebrook A, McClure D, Seno D, Churgay L, Chandrasekhar S, et al. (2000) In vivo demonstration that parathyroid hormone (hPTH 1-38) inhibits the expression of osteoprotegerin (OPG) in bone with the kinetics of an immediate early gene. J Bone Miner Res 15: 863-871.[CrossRef][Medline]
Romas E, Sims N, Hards D, Lindsay M, Quinn J, Ryan P, Dunstan C, Martin T, and Gillespie M (2002) Osteoprotegerin reduces osteoclast numbers and prevents bone erosions in collagen-induced arthritis. Am J Pathol 161: 1419-1427.
Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, et al. (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89: 309-319.[CrossRef][Medline]
Suzuki T, Ando T, Tsuchiya K, Fukazawa N, Saito A, Mariko Y, Yamashita T, and Nakanishi O (1999) Synthesis and histone deacetylase inhibitory activity of new benzamide derivatives. J Med Chem 42: 3001-3003.[CrossRef][Medline]
Takai H, Kanematsu M, Yano K, Tsuda E, Higashio K, Ikeda K, Watanabe K, and Yamada Y (1998) Transforming growth factor-beta stimulates the production of osteoprotegerin/osteoclastogenesis inhibitory factor by bone marrow stromal cells. J Biol Chem 273: 27091-27096.
Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, Ellen RP, and Penninger JM (2000) Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection. J Clin Investig 106: R59-R67.
Thirunavukkarasu K, Halladay DL, Miles RR, Yang X, Galvin RJS, Chandrasekhar S, Martin TJ, and Onyia JE (2000) The osteoblast-specific transcription factor Cbfa1 contributes to the expression of osteoprotegerin, a potent inhibitor of osteoclast differentiation. J Biol Chem 275: 25163-25172.
Thirunavukkarasu K, Miles RR, Halladay DL, Yang X, Galvin RJ, Chandrasekhar S, Martin TJ, and Onyia JE (2001) Stimulation of osteoprotegerin (OPG) gene expression by transforming growth factor-beta (TGF-beta). Mapping of the OPG promoter region that mediates TGF-beta effects. J Biol Chem 276: 36241-36250.
Udagawa N, Takahashi N, Yasuda H, Mizuno A, Itoh K, Ueno Y, Shinki T, Gillespie MT, Martin TJ, Higashio K, et al. (2000) Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function. Endocrinology 141: 3478-3484.
Vidal NO, Brandstrom H, Jonsson KB, and Ohlsson C (1998a) Osteoprotegerin mRNA is expressed in primary human osteoblast-like cells: down-regulation by glucocorticoids. J Endocrinol 159: 191-195.[Abstract]
Vidal ON, Sjogren K, Eriksson BI, Ljunggren O, and Ohlsson C (1998b) Osteoprotegerin mRNA is increased by interleukin-1 alpha in the human osteosarcoma cell line MG-63 and in human osteoblast-like cells. Biochem Biophys Res Commun 248: 696-700.[CrossRef][Medline]
Yang X, Halladay D, Onyia JE, Martin TJ, and Chandrasekhar S (2002) Protein kinase C is a mediator of the synthesis and secretion of osteoprotegerin in osteoblast-like cells. Biochem Biophys Res Commun 290: 42-46.[CrossRef][Medline]
Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, et al. (1998) Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95: 3597-3602.
This article has been cited by other articles:
|
|
 |
|
  T. J. Martin and K. W. Ng New Agents for the Treatment of Osteoporosis IBMS BoneKEy, November 1, 2007; 4(11): 287 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Martin Current, New and Emerging Anti-Resorptive Drugs; Antibody Blockade Of RANKL Action IBMS BoneKEy, May 1, 2006; 3(5): 42 - 46. [Full Text] [PDF]
|
|
|
|
|
|
T. J. Martin New Aspects of Wnt Signalling Revealed by Mouse and Human Genetics IBMS BoneKEy, October 1, 2005; 2(10): 7 - 11. [Full Text] [PDF]
|
|
|
|
 |
|
 L. C. Hofbauer and M. Schoppet Clinical Implications of the Osteoprotegerin/RANKL/RANK System for Bone and Vascular Diseases JAMA, July 28, 2004; 292(4): 490 - 495. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
0.05, versus PTX-PTH control. *, indicates osteoclasts.
, P