Woo et al. 10.1073/pnas.0505663103. |
Fig. 5. Effects of reveromycin A (RM-A) on caspase 3 and cytochrome c in mature bone-resorbing osteoclasts (OCs). Purified OCs were cultured for 1 h with or without zVAD-fmk (50 mM) in the presence of receptor activator of NF-kB ligand (RANKL) (100 ng/ml) and then treated for 4 h with or without RM-A (1 mM). Cell lysates were used for Western blotting to detect the proform and active form of caspase 3 (A) and cytosolic cytochrome c (B).
Fig. 6. Effects of concanamycin A (CM-A) on RM-A-induced apoptosis in OCs. Purified OCs were cultured with (b, d, and f) or without (a, c, and e) CM-A (100 nM) on cover glasses in the presence of RANKL (100 ng/ml) for 1 h. After culture, cells were visualized by acridine orange staining (a and b). Purified OCs were cultured with CM-A (100 nM) on cover glasses for 1 h and then were treated with or without RM-A (1 mM) for 6 h. After treatment, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP) (c and d), and nuclei (e and f) were visualized by Hoechst 33258 staining. (Scale bar: 100 mm.)
Fig. 7. Effects of RM-A on caspase 3-like activity in OCs and RAW 264 cells. (A) OCs formed from a RAW 264 cell culture were pretreated for 3 h with or without destruxin B (DB, 3 mM). When OCs are treated with DB, the cells reversibly are changed to nonfunctional osteoclasts that lack actin ring in vitro (1) and then treated for 6 h with or without RM-A (1 mM) in acidic (pH 5.5) or neutral (pH 7.5) medium. (B) RAW 264 cells were seeded and treated for 6 h with RM-A in acidic (pH 5.5) or neutral (pH 7.5) medium. Cell lysates were used for measurement of caspase 3-like enzyme activity.
Fig. 8. Histomorphometric analysis of trabecular bone in proximal tibia from RM-A-treated rats. RM-A (20 mg/kg) or saline was i.v. injected into 4-week-old male Sprague-Dawley rats twice daily for 3 days (four animals per group). Calcein (8 mg/kg) was s.c. injected on day 0 and day 2, respectively, for in vivo fluorescent labeling of mineralization sites. Tibiae were removed on day 3, and their sections were prepared. (Aa and Ab) TRAP staining of OCs in the primary spongiosa. Arrows indicate TRAP (+) OCs. (Ac and Ad) Toluidine blue staining in the primary spongiosa. Arrows indicate osteoblasts along the bone surface. (Ae and Af) Fluorescent micrographs showing double-labeled mineralization in the primary spongiosa. (B) Histomorphometric parameters of bone formation. (Ba) Number of osteoclasts/bone perimeter (No. Oc/B. Pm /100 mm). (Bb) Osteoblast surface/bone surface (Ob. S/BS; %). (Bc) Bone formation rate/bone surface (BFR/BS; mm3/cm2/year). (Bd) Bone volume/total tissue volume (BV/TV; %). (Be) Osteoid surface/bone surface (OS/BS; %). (Bf) Osteoid thickness (O. Th; mm). Values represent means ± SD.
Fig. 9. Effects of RM-A on the stomach and kidney of ovariectomized (OVX) mice. RM-A (4 mg/kg) or saline was administered s.c. twice daily to OVX mice starting 1 day after ovariectomy (five animals per group). After 4 weeks of treatment, mice were killed, and the stomach and kidneys were removed for histological analysis. (A) Histological analysis of H&E-stained sections of stomach and kidney. (B) Body weight. *, P > 0.05 vs. OVX + SA. SA, saline. (C) Uterine weight. Values represent means ± SD.
1. Nakagawa, H., Takami, M., Udagawa, N., Sawae, Y., Suda, K., Sasaki, T., Takahashi, N., Wachi, M., Nagai, K. & Woo, J. T. (2003) Bone 33, 443-455.
Supporting Materials and Methods
MTT Assay. Cells were treated with various concentrations of drugs for time periods in a-MEM adjusted to pH 7.5 or 5.5. After drug treatment, MTT was added, followed by incubation at 37°C for 1 h in a CO2 incubator. Supernatants were carefully removed and dissolved with DMSO. Absorbance at 570 nm was measured using a microplate reader.
Measurement of Total Protein Synthesis.
RAW 264 cells were grown to confluence in 35-mm culture dishes, and then medium was removed. Cells were washed twice with PBS, which was then replaced with 2 ml of methionine-free a-MEM with 20 mCi (1 Ci = 37 GBq) of L-[35S]methionine. Serial dilutions of reveromycin A (RM-A) were simultaneously added. Cells were labeled for 4 h at various intervals after addition of RM-A (1, 3, 10, or 30 mM as final concentrations) at the start of the experiment. After labeling, cells were quickly washed twice with ice-cold PBS and lysed with 500 ml of ice-cold lysis buffer containing 50 mM HEPES (pH 8.0), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 0.5 mM DTT, 0.2% Sarkosyl, 1 mM NaF, 1 mM sodium vanadate, 0.5 mM PMSF, 0.5 mg/ml leupeptin, 1 mg/ml aprotinin, and 1 mg/ml pepstatin. Lysates were centrifuged at 12,000 rpm for 10 min at 4°C, and supernatants were collected. The cellular fraction was added to 10 ml of aqueous counting scintillant (ACS) II sol (Amersham Pharmacia Biotech) for determination of radioactivity. Radioactivity was counted using a liquid scintillation counter.In Vitro
Aminoacyl-tRNA Synthetase Assay. Mature bone-resorbing osteoclasts (OCs) that were formed from bone marrow cell culture were scraped off the dishes, suspended in buffer A (100 mM Tris•HCl, pH 8.0/10 mM MgCl2/1 mM DTT/1 mM EDTA/10 mM NaF/1 mM PMSF/100 units/ml aprotinin), and then disrupted by sonication. After centrifugation, the supernatant containing the extracted proteins was analyzed for aminoacyl-tRNA synthetase activity by aminoacylation of tRNA. Aminoacyl-tRNA synthetase activity was assayed in a reaction buffer containing the cell extract (250 mg of proteins), 20 mM imidazole-HCl (pH 7.5), 75 mM MgCl2, 0.5 mM DTT, 100 units/ml tRNA (Sigma), and 3 mM ATP in a total volume of 100 ml. For the isoleucyl-tRNA synthetase assay, reaction buffer containing 1 mM isoleucine and 10 mCi/ml [3H]isoleucine (PerkinElmer Life and Analytical Sciences) was used. For the valyl-tRNA synthetase assay, reaction buffer containing 1 mM valine and 10 mCi/ml [3H]valine (PerkinElmer Life and Analytical Sciences) was used. Each sample was pretreated with RM-A at specific concentrations for 10 min, and then amino acids, tRNA, and ATP were added and the mixture was incubated for 20 min at 25°C. The reaction was stopped by addition of 450 ml of 1 mg/ml BSA and 500 ml of ice-cold 10% trichloroacetic acid. Labeled tRNA was transferred to a glass microfiber filter (Whatman), washed five times with ice-cold 5% trichloroacetic acid, and counted in a liquid scintillation counter.Bone Resorption Assay in Organ Cultures.
After 24-h preincubation, bones were cultured for 72 h with or without RM-A, in the presence of parathyroid hormone (PTH) in DMEM containing 15% heat-inactivated horse serum (GIBCO, Paisley, U.K.) and 100 units/ml penicillin. At the end of the culture period, bones were extracted with 0.1 M HCl. Radioactivity in media and bone extracts was determined by liquid scintillation spectrophotometry. Resorption was quantified as the percentage (%) of 45Ca released into the medium.Lysate Preparation and Caspase Activity Measurement.
Cells were lysed in lysis buffer (100 mM Tris•HCl, pH 7.5/1 % Triton-X 100/1 mM DTT/0.1 mM PMSF). Cell lysates were centrifuged, and supernatants were collected. Supernatants (30 mg of proteins) were incubated in lysis buffer with 10 mM Ac-DEVD-MCA (Peptide Institute, Osaka), a fluorogenic caspase 3 substrate for 1 h at 37°C. Fluorescence (excitation at 355 nm, emission at 460 nm) was measured with a fluorometer (PerkinElmer Life and Analytical Sciences).Western Blotting for Caspase 3 and Cytochrome c.
Purified OCs (for caspase 3) treated with drugs were washed with PBS and lysed in lysis buffer containing 50 mM Tris•HCl (pH 7.4), 1% Triton X-100, 1 mM DTT, 0.5 mM EDTA, and a protease inhibitor mixture (Complete; Roche Diagnostics). After centrifugation, supernatants were collected. Purified OCs (for cytochrome c) treated with drugs were harvested and resuspended in cell extract buffer (50 mM PIPES/50 mM KCl/5 mM MgCl2/1 mM DTT) with complete protease inhibitors. The suspension was placed on ice for 15 min, and the cells were homogenized with a teflon Dounce homogenizer. Supernatants were centrifuged at 21,000 ´ g for 20 min at 4°C and were collected as cytosolic extracts. Postnuclear lysates (30 mg per lane) for caspase 3 and cytosolic extracts for cytochrome c were separated by SDS/PAGE and analyzed by Western blotting using ECL detection reagents (Amersham Biosciences). Antibodies for caspase 3 (Santa Cruz Biotechnology) and cytochrome c (BD Pharmingen) were used.