Bisphosphonates Act Directly on the Osteoclast to Induce Caspase Cleavage of Mst1 Kinase during Apoptosis
1999; Elsevier BV; Volume: 274; Issue: 49 Linguagem: Inglês
10.1074/jbc.274.49.34967
ISSN1083-351X
AutoresAlfred A. Reszka, Judit Halasy-Nagy, P. Masarachia, Gideon A. Rodan,
Tópico(s)Bone health and osteoporosis research
ResumoBisphosphonates (BPs) include potent inhibitors of bone resorption used to treat osteoporosis and other bone diseases. BPs directly or indirectly induce apoptosis in osteoclasts, the bone resorbing cells, and this may play a role in inhibition of bone resorption. Little is known about downstream mediators of apoptosis in osteoclasts, which are difficult to culture. Using purified osteoclasts, we examined the effects of alendronate, risedronate, pamidronate, etidronate, and clodronate on apoptosis and signaling kinases. All BPs induce caspase-dependent formation of pyknotic nuclei and cleavage of Mammalian Sterile 20-like (Mst) kinase 1 to form the active 34-kDa species associated with apoptosis. Withdrawal of serum and of macrophage colony stimulating factor, necessary for survival of purified osteoclasts, or treatment with staurosporine also induce apoptosis and caspase cleavage of Mst1. Consistent with their inhibition of the mevalonate pathway, apoptosis and cleavage of Mst1 kinase induced by alendronate, risedronate, and lovastatin, but not clodronate, are blocked by geranylgeraniol, a precursor of geranylgeranyl diphosphate. Together these findings suggest that BPs act directly on the osteoclast to induce apoptosis and that caspase cleavage of Mst1 kinase is part of the apoptotic pathway. For alendronate and risedronate, these events seem to be downstream of inhibition of geranylgeranylation. Bisphosphonates (BPs) include potent inhibitors of bone resorption used to treat osteoporosis and other bone diseases. BPs directly or indirectly induce apoptosis in osteoclasts, the bone resorbing cells, and this may play a role in inhibition of bone resorption. Little is known about downstream mediators of apoptosis in osteoclasts, which are difficult to culture. Using purified osteoclasts, we examined the effects of alendronate, risedronate, pamidronate, etidronate, and clodronate on apoptosis and signaling kinases. All BPs induce caspase-dependent formation of pyknotic nuclei and cleavage of Mammalian Sterile 20-like (Mst) kinase 1 to form the active 34-kDa species associated with apoptosis. Withdrawal of serum and of macrophage colony stimulating factor, necessary for survival of purified osteoclasts, or treatment with staurosporine also induce apoptosis and caspase cleavage of Mst1. Consistent with their inhibition of the mevalonate pathway, apoptosis and cleavage of Mst1 kinase induced by alendronate, risedronate, and lovastatin, but not clodronate, are blocked by geranylgeraniol, a precursor of geranylgeranyl diphosphate. Together these findings suggest that BPs act directly on the osteoclast to induce apoptosis and that caspase cleavage of Mst1 kinase is part of the apoptotic pathway. For alendronate and risedronate, these events seem to be downstream of inhibition of geranylgeranylation. bisphosphonate farnesyl diphosphate pamidronate alendronate Mammalian Sterile 20-like risedronate clodronate geranylgeraniol nitrogen-containing bisphosphonate tartrate-resistant acid phosphatase osteoclast phosphate-buffered saline macrophage colony-stimulating factor HEPES buffered solution lovastatin etidronate Bisphosphonates (BPs)1include potent inhibitors of bone resorption used for the treatment of osteoporosis, Paget's disease, bone metastases, and other bone diseases. It is generally accepted that BPs inhibit bone resorption by acting directly or indirectly on osteoclasts, cells of hematopoietic origin. Until recently the molecular mechanism of action of BPs was not known (1Fleisch H. Endocr. Rev. 1998; 19: 80-100Crossref PubMed Scopus (0) Google Scholar, 2Rodan G.A. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 375-388Crossref PubMed Scopus (244) Google Scholar). Recent pharmacological studies suggest that nitrogen-containing BPs (N-BPs), such as alendronate (ALN), risedronate (RIS), ibandronate, and pamidronate (PAM), act on the cholesterol biosynthesis pathway (3Luckman S.P. Hughes D.E. Coxon F.P. Russell R.G.G. Rogers M.J. J. Bone Miner. Res. 1998; 13: 581-589Crossref PubMed Scopus (1076) Google Scholar, 4Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (654) Google Scholar). N-BPs were recently shown to inhibit either isopentenyl diphosphate synthase or the downstream enzyme, farnesyl diphosphate (FPP) synthase, or both (5van Beek E. Pieterman E. Cohen L. Löwik C. Papapoulos S. Biochem. Biophys. Res. Commun. 1999; 255: 491-494Crossref PubMed Scopus (193) Google Scholar), while clodronate (CL2) had little or no effect on these enzymes. Inhibition of either enzyme in this pathway blocks cholesterol biosynthesis, as well as farnesylation and geranylgeranylation. We recently showed that only geranylgeraniol (GGOH) prevented the effects of ALN on bone resorption (pit formation) in vitro (4Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (654) Google Scholar), suggesting that geranylgeranylation is rate-limiting for osteoclast function. CL2 and EHDP may inhibit osteoclasts by forming toxic ATP analogs or by inhibiting protein-tyrosine phosphatases (6Frith J.C. Monkkonen J. Blackburn G.M. Russell R.G.G. Rogers M.J. J. Bone Miner. Res. 1997; 12: 1358-1367Crossref PubMed Scopus (397) Google Scholar, 7Auriola S. Frith J. Rogers M.J. Koivuniemi A. Monkkonen J. J. Chromatogr. 1997; 704: 187-195Crossref Scopus (89) Google Scholar, 8Schmidt A. Rutledge S.J. Endo N. Opas E.E. Tanaka H. Wesolowski G. Leu C.T. Huang Z. Ramachandaran C. Rodan S.B. Rodan G.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3068-3073Crossref PubMed Scopus (197) Google Scholar, 9Opas E.E. Rutledge S.J. Golub E. Stern A. Zimolo Z. Rodan G.A. Schmidt A. Biochem. Pharmacol. 1997; 54: 721-727Crossref PubMed Scopus (36) Google Scholar). The final outcome of BP action appears to be osteoclast apoptosis, which was suggested to be the means by which BPs inhibit bone resorption (10Hughes D.E. Wright K.R. Uy H.L. Sasaki A. Yoneda T. Roodman G.D. Mundy G.R. Boyce B.F. J. Bone Miner. Res. 1995; 10: 1478-1487Crossref PubMed Scopus (931) Google Scholar, 11Selander K.S. Monkkonen J. Karhukorpi E.K. Harkonen P. Hannuniemi R. Vaananen H.K. Mol. Pharmacol. 1996; 50: 1127-1138PubMed Google Scholar). It is not known if the induction of apoptosis is the result of direct action of BPs on the osteoclast, and little is known about the biochemical pathways involved. Coxon et al.(12Coxon F.P. Benford H.L. Russell R.G.G. Rogers M.J. Mol. Pharmacol. 1998; 54: 631-638PubMed Google Scholar) have shown the induction of caspases in BP-treated J774 macrophages undergoing apoptosis. However, macrophages may not fully mimic osteoclast responses to bisphosphonates, since apoptosis in these cells is blocked by addition of either farnesyl diphosphate or geranylgeranyl diphosphate (3Luckman S.P. Hughes D.E. Coxon F.P. Russell R.G.G. Rogers M.J. J. Bone Miner. Res. 1998; 13: 581-589Crossref PubMed Scopus (1076) Google Scholar) and only the latter is implicated as rate-limiting for N-BP effects on the osteoclast (4Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (654) Google Scholar). Recent evidence suggests that Mammalian Sterile 20-like (Mst) kinase 1 is a substrate for caspase 3 in several hematopoietic cells and can induce apoptosis in mesenchymal cell lines (13Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (325) Google Scholar, 14Lee K.K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-19166Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 15Kakeya H. Onose R. Osada H. Cancer. Res. 1998; 58: 4888-4894PubMed Google Scholar). The caspase cleavage site of Mst1 (DEMD), situated between the amino-terminal kinase domain and carboxyl-terminal regulatory and dimerization domains, matches the consensus sequence for caspase 3. After Mst1 cleavage, the kinase domain, separated from its regulatory elements, has high catalytic activity. In this study we investigated the effect of several BPs on apoptosis and activation of signaling kinases in purified osteoclasts. BPs induced osteoclast apoptosis and activation of the 34-kDa Mst1 kinase by up to 13-fold, while reducing the activity of Mst1 (59 kDa) and Mst2 (60 kDa) kinases by up to 50%. Slight 34-kDa Mst2 kinase activity was also observed, and two other responsive kinases of 50 and 36 kDa remain unidentified. Induction of apoptosis by withdrawal of serum and macrophage colony-stimulating factor (M-CSF) elicited kinase responses similar to those produced by BPs, while staurosporine activated both full-length Mst and 34-kDa Mst1 kinase activities without altering the 50-kDa kinase activity. The findings indicate that BPs act directly on osteoclasts to induce caspase cleavage of Mst1, as a signaling intermediate in the induction of apoptosis, and suggest that geranylgeranylation is the upstream target of ALN and RIS in inducing apoptosis. Co-cultures of murine osteoblasts and marrow cells were prepared as described by Wesolowski et al. (16Wesolowski G. Duong L.T. Lakkakorpi P.T. Nagy R.M. Tezuka K. Tanaka H. Rodan G.A. Rodan S.B. Exp. Cell Res. 1995; 219: 679-686Crossref PubMed Scopus (80) Google Scholar) with the following modifications. Bone marrow cells were harvested from 6-week-old male Balb/C mice by flushing the marrow spaces of freshly isolated long bones (tibiae and femora) with α-minimal essential medium (Life Technologies, Inc.) containing penicillin/streptomycin (100 IU and 100 μg/ml, respectively). Bone marrow cells were suspended in Oc medium: α-minimal essential medium supplemented with fetal calf serum (10% v/v; HyClone Laboratories, Logan, UT) and 10 nm 1,25-(OH)2 vitamin D3 (Biomol, Plymouth Meeting, PA). Bone marrow cells were then added to subconfluent monolayers of osteoblastic MB 1.8 cells and cultured for 6–7 days at 37 °C in the presence of 5% CO2. Co-cultures were first treated with type I collagenase (Wako Pure Chemical Industries, Osaka, Japan) at a concentration of 1 mg/ml in phosphate-buffered saline (PBS) for 1 h at 37 °C. Suspended osteoblasts were gently aspirated, leaving a mixture enriched in prefusion Ocs and the remaining MB1.8 osteoblasts. All cells were released with EDTA (0.2 g/liter in PBS) for 20 min at 37 °C and then re-plated in Oc medium and cultured for an additional 3 days. Oc-forming cultures, generated as above in 24 well plates, were treated with type 1 collagenase at a concentration of 1 mg/ml (in PBS) for 1 h at 37 °C. Plates were washed twice with PBS and monitored under the microscope for complete removal of osteoblastic cells. Ocs were then maintained in Oc medium supplemented with M-CSF (R&D Systems, Minneapolis, MN) at 5 ng/ml. Ocs were treated with indicated compounds in the Oc medium for 18 h and stained for tartrate-resistant acid phosphatase (TRAP) and with Hoechst nuclear (no. 33342; Sigma) stain as follows. Cells were fixed with 10% formaldehyde (in PBS) and then rinsed with PBS. Cells were then stained with Fast Red Violet LB (Sigma) dissolved in TRAP buffer (sodium acetate (50 mm), sodium tartrate (30 mm), and Triton X-100 (0.1%), naphthol AS-MX phosphate (100 mg/ml), pH 5.0) for 10 min at 37 °C. After TRAP staining, nuclear staining was performed with 5 μg/ml Hoechst stain in PBS for 10 min at room temperature. Cells were washed with water and stored in the dark at 4 °C. Total osteoclast number (i.e. TRAP (+) multinucleated cells ≥250 μm in diameter) in each well and the number of osteoclasts with pyknotic nuclei were quantitated microscopically using a Nikon Diaphot microscope equipped with a 10× objective and an ultraviolet light source. Oc-forming cultures, generated as above in six-well plates, were treated with type XI collagenase (Sigma) in PBS to remove all osteoblasts, followed by EDTA to remove prefusion Ocs. Ocs (≥95% purity) were then maintained in Oc medium supplemented with M-CSF (5 ng/ml). Oc treatments began within 30 min of purification, except when a time course was initiated. For time courses, Ocs were purified simultaneously and later lysed simultaneously. Under these circumstances, treatments were started at indicated times prior to formation of lysates. After treatments, cells were placed on ice and washed twice with ice-cold β-glycerophosphate-HEPES buffered solution (β-HBS): 50 mm HEPES (pH 7.6), β-glycerophosphate (50 mm), EGTA (1 mm), NaCl (150 mm). Cells were washed again with β-HBS-I: β-HBS supplemented with leupeptin (10 μg/ml), aprotinin (10 μg/ml), microcystin LR (1 μm), Na3VO4 (1 mm), dithiothreitol (1 mm), and phenylmethylsulfonyl fluoride (1 mm). Ocs were then lysed in β-HBS-I containing Triton X-100 (0.2%). Protein concentrations were determined using a Bradford reagent kit (Bio-Rad). For immunoprecipitations we used β-HBS-IP, which was β-HBS-I without phenylmethylsulfonyl fluoride and instead supplemented with a protease inhibitor mixture (Sigma): 4-(2-aminoethyl)-benzenesulfonyl fluoride (48 μg/ml), epoxysuccinyl-l-leucylamido (4-guanido)butane (1 μg/ml), bestatin (2.8 μg/ml), leupeptin (2.1 μg/ml (12.1 μg/ml final)), aprotinin (1 μg/ml (11 μg/ml final), pepstatin A (2.1 μg/ml), and 1,10-phenanthroline (20 μg/ml). Anti-Krs1 (Mst2) amino-terminal and anti-Krs2 (Mst1) amino-terminal antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-Krs1+2 carboxyl-terminal antibody was from Zymed Laboratories Inc. (South San Francisco, CA) and anti-Krs1 carboxyl-terminal goat polyclonal antibody was from Santa Cruz Biotechnology. Protein lysates (50 μg) were diluted in 1 ml of ice-cold β-HBS-IP containing Tween 20 (0.05%) and then combined with primary anti-amino-terminal (3 μg) or anti-carboxyl-terminal (2 μg of each) Mst1 and/or Mst2 antibodies. Rabbit anti-goat secondary antibody (1 μl; ICN Pharmaceuticals, Aurora, OH) was added to increase binding to protein A-conjugated agarose beads (10 μl bead volume; Sigma). Lysate-antibody-bead mixtures were gently mixed overnight at 4 °C. Beads were then washed three times with ice-cold β-HBS-IP containing Tween 20 (0.05%) and the supplemental protease inhibitor mixture described above. Antibodies and antigen were released by suspending the beads in Laemmli sample buffer (Bio-Rad) containing 5% 2-mercaptoethanol and mixed vigorously at 37 °C in a Thermomixer (Brinkmann, Westbury, NY). Kinase assays were performed in the gel using methods based on Kameshita and Fujisawa (17Kameshita I. Fujisawa H. Anal. Biochem. 1989; 183: 139-143Crossref PubMed Scopus (435) Google Scholar) and Gotoh et al. (18Gotoh Y. Nishida E. Yamashita T. Hoshi M. Kawakami M. Sakai H. Eur. J. Biochem. 1990; 193: 661-669Crossref PubMed Scopus (323) Google Scholar). Briefly, 10% SDS-polyacrylamide gels were cast with myelin basic protein (0.2 mg/ml) added prior to polymerization. Lanes were loaded with equivalent amounts of protein lysate (5–10 μg/lane) or the entirety of each immunoprecipitation, and then gels were electrophoresed. Gels were washed twice at room temperature with Buffer A: 50 mm HEPES, pH 7.6; 5 mm 2-mercaptoethanol supplemented with isopropanol (20%), followed by two washes with Buffer A. Proteins were denatured with urea (6 m in Buffer A) at room temperature and renatured at 4 °C with urea at 3m, 1.5 m, and then 0.75 m (in Buffer A), followed by three washes with Buffer A containing Tween-20 (0.05%). Gels were then washed twice with Kinase Buffer (20 mm HEPES, pH 7.6, 5 mm MnCl2, 2 mm dithiothreitol) at 30 °C. Similar, but weaker kinase activities were obtained when 20 mm MgCl2 was used instead of 5 mm MnCl2. Kinase reactions lasted 30 min at 30 °C using Kinase Buffer containing 0.02 mm ATP with ∼1000 cpm/pmol [γ-32P]ATP. Kinase reactions were stopped and unincorporated [γ-32P]ATP was removed with six washes using 5% trichloroacetic acid/1% NaPPi (w/v). Gels were then stained, destained, and dried using standard techniques. Gels were exposed to phosphorimaging screens and scanned and analyzed using a Molecular Dynamics Storm system and associated software (Sunnyvale, CA). N-BPs were shown to disrupt the cytoskeleton, abolish the ruffled border, and induce apoptosis in osteoclasts and macrophages (3Luckman S.P. Hughes D.E. Coxon F.P. Russell R.G.G. Rogers M.J. J. Bone Miner. Res. 1998; 13: 581-589Crossref PubMed Scopus (1076) Google Scholar, 10Hughes D.E. Wright K.R. Uy H.L. Sasaki A. Yoneda T. Roodman G.D. Mundy G.R. Boyce B.F. J. Bone Miner. Res. 1995; 10: 1478-1487Crossref PubMed Scopus (931) Google Scholar, 19Sato M. Grasser W. Endo N. Atkins R. Simmons H. Thompson D.D. Golub E. Rodan G.A. J. Clin. Invest. 1991; 88: 2095-2105Crossref PubMed Scopus (904) Google Scholar). We found that GGOH prevents alendronate inhibition of osteoclastic bone resorption (4Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (654) Google Scholar), suggesting that geranylgeranyl diphosphate, an isoprenylation precursor derived from the mevalonate pathway or from GGOH, is rate-limiting for osteoclast activity. Geranylgeranylated G-proteins regulate cytoskeletal organization, vesicular trafficking, and apoptosis (20Sebti S. Hamilton A.D. Curr. Opin. Oncol. 1997; 9: 557-561Crossref PubMed Scopus (56) Google Scholar, 21Zhang F.L. Casey P.J. Annu. Rev. Biochem. 1996; 65: 241-269Crossref PubMed Scopus (1743) Google Scholar). Focusing on the latter, we examined the effects of GGOH and of the caspase inhibitor Z-VAD-FMK on ALN-, RIS-, CL2-, and EHDP-induced apoptosis in purified Ocs. Ocs were identified by TRAP staining as described under “Experimental Procedures,” and apoptosis in these cells was scored based on the presence of pyknotic nuclei. This method correlated with TUNEL staining in this system (data not shown). Base-line apoptosis in untreated Ocs maintained for 18 h was 9%. Typically, nuclei were deployed in ringlike structures (Fig.1 A). Treatment with ALN (30 μm) for 18 h increased the number of cells with nuclear condensation (apoptosis) by 3-fold (Fig.1, B and E). RIS (30 μm) produced similar effects (Fig. 1 E). Both GGOH (Fig. 1, Cand E) and Z-VAD-FMK (Fig. 1, D and E) prevented ALN- or RIS-induced apoptosis, reducing the number of cells with condensed nuclei to control levels. However, while GGOH prevented both nuclear condensation and disappearance of the ringlike structure in N-BP-treated Ocs, Z-VAD-FMK-treated cells showed normal nuclear morphology without the ringlike arrangement. Instead, these nuclei were clustered in one or more regions of the osteoclast. Apoptosis induced by CL2 and EHDP was also suppressed by Z-VAD-FMK (Fig. 1 E) but was not affected by GGOH. Apoptosis induced by the withdrawal of M-CSF was also prevented by Z-VAD-FMK (Fig. 1 E). Several geranylgeranylated proteins, including members of the Ras family of small GTPases, activate signaling pathways and control apoptosis through their regulation of protein kinases. We assessed kinase activity in purified murine Oc-like cells obtained as described under “Experimental Procedures.” In-gel kinase assays of Oc lysates (Fig. 2) showed that treatment with ALN (panel A), PAM (panel B), RIS (panel C), or CL2 (panel D) for 12–20 h (treatment times are indicated above each lane) increased the activity of 34- and 36-kDa kinases 5–13-fold when compared with untreated controls (0 h) or to osteoclasts treated for 1–2 h, respectively. After 16 h, 50-, 59-, and 60-kDa kinases showed decreases in activity of up to 50% (most notable in Figs. 2 D and 3). ALN and RIS triggered these kinase responses at 10–100 μm. At 30 μm, RIS (Fig. 2 C) elicited a slightly higher activation of the 34- and 36-kDa kinases than ALN (Fig. 2 A). PAM had no effect at 10 or 30 μm, was slightly effective at 60 μm (data not shown), while 100 μm PAM (Fig. 2 B) elicited about the same response as 30 μm ALN (Fig. 2 A) or RIS (Fig. 2 C). However, at 100 μm, PAM-treated cultures also showed the accumulation of debris in the medium, which was not observed with ALN or RIS. Treatment with EHDP (300 μm; Fig. 2 E) for 2–24 h activated the 34-kDa kinase less than 2-fold and there was no effect on kinases migrating at 36, 50, 59, or 60 kDa. In many experiments, EHDP elicited no response at all (data not shown). Tiludronate up to 300 μm did not significantly alter kinase activities (data not shown).Figure 3Alendronate-activated 34-kDa kinase is immunoprecipitated by anti-Mst1 kinase antibodies. Ocs were prepared as described in Fig. 2. Osteoclasts were left untreated (lanes 1, 3, 5,7, and 9) or were treated with 30 μm ALN (lanes 2, 4,6, 8, and 10) for 21 h prior to the preparation of protein lysates. In-gel kinase assays were used to analyze kinase activities in crude lysates (lanes 1 and 2) and in the following immunoprecipitates: anti-Mst1 amino-terminal (lanes 3 and4), anti-Mst2 amino-terminal (lanes 5and 6), anti-Mst1 and -Mst2 carboxyl-terminal (lanes 7 and 8) and rabbit-anti goat IgG control (lanes 9 and 10). Radioactive bands were visualized by phosphorimaging. Molecular mass markers (in kDa) are indicated to the right. The identity of Mst1 (59 kDa), Mst2 (60 kDa), and cleaved Mst1 (34 kDa) are indicated to the left.View Large Image Figure ViewerDownload (PPT) The Oc cultures used in these experiments were 95% pure by cell count, suggesting that the BPs act directly on the osteoclast. Since Ocs in these populations are large and multinucleated, they may contribute ≥99% of the protein and kinase signals in these assays. To examine the potential contribution of kinase signals from the minor population of contaminating osteoblasts, we examined kinase responses to ALN in pure MB1.8 osteoblast cultures (Fig. 2 F). Untreated MB1.8 osteoblasts show a different kinase profile than Oc controls; the 60-kDa kinase in untreated MB1.8 cells appears to migrate as a single band, and the 50-kDa kinase is a more distinct doublet (panel E, lane 1). Additional kinase activity is observed at 43 kDa, possibly corresponding to ERK1, ERK2, or p38 kinases, while no activity was observed at 34 or 36 kDa. After treatment with ALN (30 μm) for 2–24 h, no changes in kinase activity were observed, indicating that the responses to BPs described above occurred in the Ocs. To identify the kinases that respond to BPs in the osteoclast, we used antibodies against kinases with molecular masses in the observed range and assayed immunoprecipitates using in-gel kinase assay. The kinases activated by ALN (Fig. 3) and other BPs (data not shown) were only immunoprecipitated with antibodies against Mst1 or Mst2, also know as kinase responsive to stress (Krs) 2 and 1, respectively. Antibodies directed to the amino terminus of either Mst1 (Fig. 3, lanes 3 (control) and 4(ALN)) or Mst2 (lanes 5 (control) and6 (ALN)) immunoprecipitated both kinases in the 59-kDa (Mst1)/60-kDa (Mst2) doublet in Oc lysates of control and ALN-treated cells. Mst1:Mst1 dimerization has been described and involves a carboxyl-terminal domain that is conserved in Mst2 (22Creasy C.L. Ambrose D.M. Chernoff J. J. Biol. Chem. 1996; 271: 21049-21053Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Consistent with the formation of Mst1:Mst2 heterodimers, either type of antibody precipitated both Mst1 and Mst2. Anti-Mst1 immunoprecipitated Mst1 to a greater extent than Mst2, and vice versa. This is explained by assuming random association, where anti-Mst1 precipitates Mst1:Mst1, Mst1:Mst2, and Mst2:Mst1, but not Mst2:Mst2 (yielding a 2:1 ratio), while anti-Mst2 will give a similar ratio favoring Mst2. Although bands were not separated sufficiently for quantitation, the data are consistent with this ratio. Immunodepletion with a mixture of both anti-Mst1 and Mst2 antibodies removed >95% of all kinase activity migrating at 59/60 kDa, suggesting that this activity doublet was comprised primarily of these kinases (data not shown). Several studies have recently described an apoptosis response, associated with caspase cleavage of the 59-kDa Mst1 and Mst2 kinases to form catalytically active species migrating at 34–36 kDa (13Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (325) Google Scholar, 14Lee K.K. Murakawa M. Takahashi S. Tsubuki S. Kawashima S. Sakamaki K. Yonehara S. J. Biol. Chem. 1998; 273: 19160-19166Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 15Kakeya H. Onose R. Osada H. Cancer. Res. 1998; 58: 4888-4894PubMed Google Scholar). Caspase cleavage of Mst1 occurs at the DEMD sequence (residues 323–326), while Mst2 is cleaved at DELD (residues 319–322). The catalytically active 34-kDa kinase domain is readily immunoprecipitated using antibodies directed only to the Mst amino terminus (13Graves J.D. Gotoh Y. Draves K.E. Ambrose D. Han D.K.M. Wright M. Chernoff J. Clark E.A. Krebs E.G. EMBO J. 1998; 17: 2224-2234Crossref PubMed Scopus (325) Google Scholar). Anti-Mst1 amino-terminal antibody (Fig. 3, lanes 3 (control) and 4 (ALN)) immunoprecipitated the 34-kDa kinase activated by the BPs in the osteoclast, while anti-Mst2 was much less effective (lanes 5 and6). This suggested that the 34-kDa kinase activated by BPs was predominantly the amino-terminal fragment of Mst1. Antibodies directed to the non-catalytic carboxyl-terminal domain of Mst1 and Mst2 immunoprecipitated only the full-length Mst1 and Mst2 isoforms from both untreated (lane 7) and ALN-treated (lane 8) osteoclast lysates. To determine whether the 34-kDa Mst1 kinase was generated as a result of caspase activities, purified Ocs were treated with ALN (Fig.4, lanes 4–6), RIS (lanes 7–9), or CL2 (lanes 10–12) in the absence or presence of the caspase inhibitors: Z-VAD-FMK (lanes 5, 8, and11) or Z-DEVD-FMK (lanes 6,9, and 12). Kinase activity of 34-kDa Mst1 was reduced by either caspase inhibitor, relative to cells treated with ALN, RIS, or CL2, alone (lanes 4, 7, and 10, respectively). These data suggest that BP-induced activation of the 34-kDa species resulted from caspase cleavage of full-length Mst1. In the absence of BP treatment, both caspase inhibitors reduced base-line 34-kDa Mst1 activities (lanes 2 and 3) relative to control (lane 1). The findings indicate that the activity migrating at 36-kDa kinase was also caspase-dependent, although the identity of this kinase remains to be determined. Analyses of p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase using GST-ATF2 as a kinase substrate for in-gel kinase assays and using phosphospecific antibodies to probe immunoblots showed that these kinases were not activated in osteoclasts treated with ALN (data not shown). Recent studies suggest that ALN and other N-BPs act on the Oc by inhibiting protein geranylgeranylation, related to their inhibition of the mevalonate pathway (4Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (654) Google Scholar, 5van Beek E. Pieterman E. Cohen L. Löwik C. Papapoulos S. Biochem. Biophys. Res. Commun. 1999; 255: 491-494Crossref PubMed Scopus (193) Google Scholar). To examine if inhibition of the mevalonate pathway is sufficient to induce caspase cleavage of Mst1 kinase, osteoclast cultures were treated with the hydroxymethylglutaryl-CoA reductase inhibitor, lovastatin (LOV) for 2–24 h. In-gel kinase assays (Fig.5 A) showed that, like N-BPs (Fig. 2), LOV induced 34-kDa Mst1 and 36-kDa kinases and decreased Mst1, Mst2, and 50-kDa kinase activities. The profile of these responses was similar to that of ALN, PAM, and RIS (Fig. 2,A–C, respectively) indicating that inhibition of the mevalonate pathway can induce Mst1 kinase cleavage in these cells. Recent studies showed that induction of apoptosis in J774 macrophages by N-BPs or statins was blocked by the addition of either geranylgeranyl or farnesyl precursors (3Luckman S.P. Hughes D.E. Coxon F.P. Russell R.G.G. Rogers M.J. J. Bone Miner. Res. 1998; 13: 581-589Crossref PubMed Scopus (1076) Google Scholar, 23Luckman S.P. Coxon F.P. Ebetino F.H. Russell R.G.G. Rogers M.J. J. Bone Miner. Res. 1998; 13: 1668-1678Crossref PubMed Scopus (240) Google Scholar). This is consistent with FPP synthase as the target for N-BP action but does not separate between geranylgeranylation and farnesylation as rate-limiting targets in N-BP action on the Oc. To distinguish between these two potential N-BP targets (4Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (654) Google Scholar), we treated purified osteoclasts with ALN (Fig.5 B, lanes 4–6), RIS (lanes 7–9), and LOV (lanes 10–12) in the absence or presence of farnesol (lanes 2,5, 8, and 11) or GGOH (lanes 3, 6, 9, and12). Increase in 34-kDa Mst1 activity with either BP or LOV was blocked by GGOH but not by farnesol, although a partial reduction in 34-kDa kinase activity was seen in the presence of farnesol in some experiments (data not shown and Ref. 4Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (654) Google Schol
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