Identification of a Novel Phosphonocarboxylate Inhibitor of Rab Geranylgeranyl Transferase That Specifically Prevents Rab Prenylation in Osteoclasts and Macrophages
2001; Elsevier BV; Volume: 276; Issue: 51 Linguagem: Inglês
10.1074/jbc.m106473200
ISSN1083-351X
AutoresFraser P. Coxon, Miep Helfrich, Banafshé Larijani, M Muzylak, James E. Dunford, Deborah Marshall, Alastair McKinnon, Stephen A. Nesbitt, Michael A. Horton, Miguel C. Seabra, Frank H. Ebetino, Michael J. Rogers,
Tópico(s)Click Chemistry and Applications
ResumoNitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass ∼22–26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22–26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [3H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [14C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts. Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass ∼22–26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22–26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [3H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [14C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts. farnesyl diphosphate bisphosphonate protein:farnesyl transferase protein:farnesyl transferase inhibitor all-trans geranylgeraniol geranylgeranyl diphosphate protein:geranylgeranyl transferase protein:geranylgeranyl transferase inhibitor Rab escort protein (2-(3-pyridinyl)-1-hydroxyethylidene-1,1-bisphosphonate) scanning electron microscopy tetramethylrhodamine isothiocyanate phosphate-buffered saline 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Protein prenylation is a post-translational modification involving the transfer of an isoprenoid lipid moiety from farnesyl diphosphate (FPP)1 or geranylgeranyl diphosphate (GGPP) to a conserved C-terminal cysteine residue contained within characteristic prenylation motifs of target proteins, mostly small GTP-binding proteins (small GTPases). Prenylation (either farnesylation or geranylgeranylation) is essential for the function of the modified proteins, because it is required both for membrane association and for specific protein-protein interactions (1Seabra M.C. Cell Signal. 1998; 10: 167-172Crossref PubMed Scopus (224) Google Scholar). FPP and GGPP, which are sequentially produced by condensation reactions involving FPP synthase and GGPP synthase, are products of the mevalonate pathway, which is also responsible for the biosynthesis of cholesterol. The process of prenylation is carried out by one of three protein:prenyl transferase enzymes, the specificity being determined by the prenylation motif in the protein substrate. Proteins with a cysteine residue four positions from the C terminus (CAAX motif) are modified by either protein:farnesyl transferase (FTase), which farnesylates proteins such as Ras and lamins, or protein:geranylgeranyl transferase I (GGTase I), which geranylgeranylates small GTPase proteins of the Rho family (e.g. Rho, Rac, and Cdc42; molecular mass, ∼21 kDa) and others such as Rap. A distinct protein:geranylgeranyl transferase (Rab GGTase, also known as GGTase II) geranylgeranylates small GTPases of the Rab family (molecular mass, 22–26 kDa) on two C-terminal cysteine residues contained in motifs such as CCXX,X CX C, or XX CC (2Glomset J.A. Farnsworth C.C. Annu. Rev. Cell Biol. 1994; 10: 181-205Crossref PubMed Scopus (278) Google Scholar). This modification also requires the participation of an additional protein, Rab escort protein (REP), which binds unprenylated Rab and presents it to Rab GGTase (3Anant J.S. Desnoyers L. Machius M. Demeler B. Hansen J.C. Westover K.D. Deisenhofer J. Seabra M.C. Biochemistry. 1998; 37: 12559-12568Crossref PubMed Scopus (65) Google Scholar). Several effective and specific inhibitors of both FTase and GGTase I have been developed, such as the peptidomimetic inhibitors FTI-277 and GGTI-298 (4Lerner E.C. Qian Y. Blaskovich M.A. Fossum R.D. Vogt A. Sun J. Cox A.D. Der C.J. Hamilton A.D. Sebti S.M. J. Biol. Chem. 1995; 270: 26802-26806Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 5McGuire T.F. Qian Y. Vogt A. Hamilton A.D. Sebti S.M. J. Biol. Chem. 1996; 271: 27402-27407Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). However, a specific inhibitor of Rab GGTase has not yet been identified. Although metabolites of the monoterpene limonene are able to inhibit one or more protein:prenyl transferase, none of these specifically inhibits Rab GGTase. For example, perillyl alcohol inhibits Rab GGTase, but also GGTase I in cell-free lysates and intact 3T3 cells (6Ren Z. Elson C.E. Gould M.N. Biochem. Pharmacol. 1997; 54: 113-120Crossref PubMed Scopus (67) Google Scholar), whereas other monoterpenes, such as perillic acid, inhibit FTase and GGTase I only (7Hardcastle I.R. Rowlands M.G. Barber A.M. Grimshaw R.M. Mohan M.K. Nutley B.P. Jarman M. Biochem. Pharmacol. 1999; 57: 801-809Crossref PubMed Scopus (71) Google Scholar). Recently, some bisphosphonate drugs have been shown to act by preventing protein prenylation (8Luckman 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 (1073) Google Scholar). Bisphosphonates (BPs) are synthetic inhibitors of bone resorption, a property that has led to their use in the treatment of bone diseases characterized by excessive resorption, such as post-menopausal osteoporosis and tumor-associated bone disease (9Russell R.G.G. Rogers M.J. Bone. 1999; 25: 97-106Crossref PubMed Scopus (768) Google Scholar). BPs that contain a nitrogen in the structure of one of their two side chains (10Rogers M.J. Gordon S. Benford H.L. Coxon F.P. Luckman S.P. Monkkonen J. Frith J.C. Cancer. 2000; 88: 2961-2978Crossref PubMed Google Scholar), such as risedronate (RIS), inhibit the function of bone-resorbing osteoclasts by preventing protein prenylation in these cells because of inhibition of FPP synthase (11Dunford J.E. Thompson K. Coxon F.P. Luckman S.P. Hahn F.M. Poulter C.D. Ebetino F.H. Rogers M.J. J. Pharmacol. Exp. Ther. 2001; 296: 235-242PubMed Google Scholar, 12van Beek E. Pieterman E. Cohen L. Lowik C. Papapoulos S. Biochem. Biophys. Res. Commun. 1999; 264: 108-111Crossref PubMed Scopus (469) Google Scholar, 13Bergstrom J.D. Bostedor R.G. Masarachia P.J. Reszka A.A. Rodan G. Arch. Biochem. Biophys. 2000; 373: 231-241Crossref PubMed Scopus (373) Google Scholar). This results in the depletion of FPP and GGPP required for prenylation of small GTPase proteins that are essential for osteoclast function. Loss of geranylgeranylated proteins appears to be the cause of the anti-resorptive effects of these BPs on osteoclasts, because loss of osteoclast function can be overcome by the addition of geranylgeraniol, which bypasses inhibition of FPP synthase and replenishes the cells with a substrate for protein geranylgeranylation (14Fisher J.E. Rogers M.J. Halasy J.M. Luckman S.P. Hughes D.E. Masarachia P.J. Wesolowski G. Russell R.G.G. Rodan G.A. Reszka A.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 133-138Crossref PubMed Scopus (653) Google Scholar, 15van Beek E. Lowik C. van der Pluijm G. Papapoulos S. J. Bone Miner. Res. 1999; 14: 722-729Crossref PubMed Scopus (247) Google Scholar). In addition, we have suggested that bisphosphonates disrupt osteoclast function as a result of loss of geranylgeranylation of small GTPases by GGTase I, such as Rho, Rac, and Cdc42, because an inhibitor of GGTase I (GGTI-298) closely mimics the effects of BPs (16Coxon F.P. Helfrich M.H. Van't Hof R. Sebti S. Ralston S.H. Hamilton A. Rogers M.J. J. Bone Miner. Res. 2000; 15: 1467-1476Crossref PubMed Scopus (352) Google Scholar). Because of the lack of a specific inhibitor of Rab GGTase, the effect of loss of prenylation of Rab small GTPases in osteoclasts could not be assessed. There is evidence that some bisphosphonates and their analogues may be able to inhibit other enzymes of the mevalonate pathway. Some nitrogen-containing BPs can also inhibit squalene synthase (17Amin D. Cornell S.A. Gustafson S.K. Needle S.J. Ullrich J.W. Bilder G.E. Perrone M.H. J. Lipid Res. 1992; 33: 1657-1663Abstract Full Text PDF PubMed Google Scholar, 18Ciosek Jr., C.P. Magnin D.R. Harrity T.W. Logan J.V.H. Dickson Jr., J.K. Gordon E.M. Hamilton K.A. Jolibois K.G. Kunselman L.K. Lawerence R.M. Mookhtiar K.A. Rich L.C. Slusarchyk D.A. Sulsky R.B. Biller S.A. J. Biol. Chem. 1993; 268: 24832-24837Abstract Full Text PDF PubMed Google Scholar), which is involved in the synthesis of cholesterol and also uses FPP as a substrate. Furthermore, a recent study has demonstrated that certain bisphosphonate analogues of FPP are able to inhibit FTase (19Holstein S.A. Cermak D.M. Wiemer D.F. Lewis K. Hohl R.J. Bioorg. Med. Chem. 1998; 6: 687-694Crossref PubMed Scopus (97) Google Scholar). NE10790 (see Fig. 1 A) is an analogue of the nitrogen-containing BP RIS, in which one of the phosphonate groups is replaced with a carboxylate group (20Ebetino F.H. Bayless A.V. Ambugey J.I Ibbotsen K.J. Dansereau S. Ebrahiimpour A. Phosphorus, Sulfur, and Silicon. 1996; 109–110: 217-220Crossref Scopus (4) Google Scholar). NE10790 retains the ability to inhibit bone resorption in vivo, although its anti-resorptive potency in rodents is markedly reduced compared with RIS (20Ebetino F.H. Bayless A.V. Ambugey J.I Ibbotsen K.J. Dansereau S. Ebrahiimpour A. Phosphorus, Sulfur, and Silicon. 1996; 109–110: 217-220Crossref Scopus (4) Google Scholar). At least part of this loss of potency is due to the fact that NE10790 has reduced affinity for bone, because the loss of one of the phosphonate groups allows binding of only one calcium ion (21van Beek E.R. Lowik C.W. Ebetino F.H. Papapoulos S.E. Bone. 1998; 23: 437-442Crossref PubMed Scopus (107) Google Scholar). However, it remains unclear whether this compound is also less effective at affecting osteoclast function at the cellular level or indeed whether it inhibits bone resorption by the same molecular mechanism as nitrogen-containing BPs (that is, by inhibition of FPP synthase). NE10485 (see Fig. 1 A) is an analogue of NE10790 in which the nitrogen of the heterocyclic group is methylated and the hydroxyl group attached to the central carbon is replaced with hydrogen. The anti-resorptive potency of this compound has not been characterized. In this study we demonstrate that NE10790 is a poor inhibitor of FPP synthase and does not inhibit protein prenylation indiscriminately. Rather, this compound selectively prevents the geranylgeranylation of Rab small GTPases in several cell types in vitro, including osteoclasts, as a result of specific inhibition of Rab GGTase. NE10790, NE10485, and RIS (see Fig. 1 A) were provided by Procter and Gamble Pharmaceuticals (Cincinnati, OH). The drugs were dissolved in PBS, and the pH was adjusted to 7.4 with 1n NaOH and then filter-sterilized by using a 0.2-μm filter. Mevastatin was purchased from Sigma and converted from the lactone as described by Luckman et al. (8Luckman 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 (1073) Google Scholar). [3H]GGOH was from American Radiochemicals Ltd. (St Louis, MO). [14C]Mevalonic acid lactone, Enhance reagent, [1-3H] trans-FPP, and [1-3H]trans-GGPP were purchased from PerkinElmer Life Sciences. Solvent was removed from [3H]GGOH and [14C]mevalonic acid lactone by evaporating in nitrogen. Recombinant human K-Ras, Rho, Rab1a, Rab escort protein (REP1) and recombinant human FTase, GGTase I, and Rab GGTase were purified as described previously (22Seabra M.C. James G.L. Methods Mol. Biol. 1998; 84: 251-260PubMed Google Scholar). All other reagents were from Sigma unless stated otherwise. The number of viable J774 macrophage cells was determined by MTT assay as previously described (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 (238) Google Scholar). J774 cells were seeded at a density of 104cells/well into 96-well plates and then treated with RIS, NE10790, or NE10485 the following day in replicates of six wells. 48 h later, the reduction of MTT reagent was measured. Mature osteoclasts were isolated from 2-day-old New Zealand White rabbits as previously described (16Coxon F.P. Helfrich M.H. Van't Hof R. Sebti S. Ralston S.H. Hamilton A. Rogers M.J. J. Bone Miner. Res. 2000; 15: 1467-1476Crossref PubMed Scopus (352) Google Scholar). Briefly, the long bones from each rabbit were removed and minced in α-minimum essential medium (Life Technologies, Inc.). After allowing the bone pieces to settle, the supernatant was transferred to a fresh tube, and fetal calf serum was added to a final concentration of 10%. The cells were seeded into 6-well or 96-well plates (Costar, Cambridge, MA). The following day, contaminating adherent cells in 6-well plates were removed by treatment with 0.001% Pronase, 0.002% EDTA in PBS. The remaining adherent cells (>95% tartrate-resistant acid phosphatase-positive osteoclasts) were rinsed twice in PBS and then cultured in fresh α-minimum essential medium containing 10% fetal calf serum plus treatments under investigation. Detection of prenylated proteins in J774 macrophages and purified rabbit osteoclasts was carried out as described previously (16Coxon F.P. Helfrich M.H. Van't Hof R. Sebti S. Ralston S.H. Hamilton A. Rogers M.J. J. Bone Miner. Res. 2000; 15: 1467-1476Crossref PubMed Scopus (352) Google Scholar, 24Coxon F.P. Benford H.L. Russell R.G.G. Rogers M.J. Mol. Pharmacol. 1998; 54: 631-638PubMed Google Scholar). Briefly, the cells were depleted of mevalonate by incubation with 5 μm mevastatin for 4 h and then transferred into fresh medium containing 5 μm mevastatin and either 7.5 μCi/ml [14C]mevalonic acid lactone or 30 μCi/ml [3H]GGOH, plus RIS, NE10790, NE10485, FTI-277, or GGTI-298. After 18 h the cells were lysed in RIPA buffer (1% (v/v) Nonidet P-40, 0.1% (w/v) sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate in PBS, plus 1:100 (v/v) Sigma protease inhibitor mixture), and then 50 μg of cell lysate from each treatment were electrophoresed on 12% polyacrylamide-SDS gels under reducing conditions. After electrophoresis, the gels were fixed in 10% (v/v) acetic acid, 40% (v/v) methanol, 50% (v/v) distilled water, and then14C-labeled gels were dried, and labeled proteins were visualized on a Bio-Rad Personal FX Imager after exposure to a Kodak phosphorimaging screen. 3H-Labeled gels were incubated in Enhance for 30 min prior to drying. 3H-Labeled proteins were then visualized by exposing the gel to preflashed Hyperfilm-MP (Amersham Pharmacia Biotech) for 6 days at −70 °C. J774 cells were seeded at 2 × 106 cells/well in a 6-well plate and then metabolically labeled the following day with [14C]mevalonic acid as described above. Whole cell lysates were prepared in 1 ml of RIPA buffer and assayed for protein content, and then equal amounts of protein (∼500 μg) were incubated with 2 μg of rabbit polyclonal Rab6 antibody or 1 μg/ml goat polyclonal Rap1A antibody (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for 1 h. 20 μl of protein G-Sepharose were then added, and incubation was continued overnight. For immunoprecipitation of Ras, lysates were incubated at 4 °C overnight with 20 μl of Ha-Ras antibody conjugated to agarose beads (Oncogene Science, Manhasset, NY). The beads were pelleted by centrifugation at 2,500 rpm for 5 min in a microcentrifuge and washed four times in RIPA buffer. 20 μl of 2× Laemmli sample buffer were added to each Sepharose pellet and boiled for 5 min, and the entire sample was electrophoresed on 12% polyacrylamide-SDS gels. Radiolabeled bands were then visualized as described above. Purified rabbit osteoclasts and J774 macrophages were treated for 24 h and then lysed in RIPA buffer. After determining the protein content, 20 μg (J774 lysate) or 60 μg (osteoclast lysate) from each sample were electrophoresed under reducing conditions on 12% polyacrylamide-SDS gels. The proteins were transferred on to polyvinyl difluoride membrane by semi-dry transfer and blocked overnight with 5% (w/v) skimmed milk in TBS with Tween 20. Membranes were then hybridized with 0.5 μg/ml rabbit polyclonal Rab6 antibody or 1 μg/ml goat polyclonal Rap1A antibody, followed by 1 μg/ml anti-rabbit or anti-goat IgG-peroxidase conjugate. Blots were visualized after incubation with SuperSignal (Pierce), using a Bio-Rad FluorS Max imager. FPP synthase was assayed as described previously (11Dunford J.E. Thompson K. Coxon F.P. Luckman S.P. Hahn F.M. Poulter C.D. Ebetino F.H. Rogers M.J. J. Pharmacol. Exp. Ther. 2001; 296: 235-242PubMed Google Scholar). Briefly, 40 μl of assay buffer (50 mm Tris, pH 7.7, 10 mm NaF, 2 mmMgCl2, 1 mg/ml bovine serum albumin, 0.5 mmdithiothreitol) containing 2 nmol [1-14C]isopentenyl diphosphate (4 μCi/mmol) and 2 nmol GPP were prewarmed to 37 °C. The assay was initiated by the addition of 1 μl of recombinant human FPP synthase (with an activity of 8 pmol FPP/min) diluted to 10 μl with assay buffer. The assay was allowed to proceed for 30 min and was terminated by the addition of 200 μl of saturated NaCl. The samples were then extracted with 1 ml of water-saturated butan-1-ol, and the amount of radioactivity in the upper phase was determined by mixing 0.5 ml of the butyl alcohol with 4 ml of general purpose scintillant. This was then counted using a Packard Tricarb 1900CA scintillation counter. To determine the effects of NE10790, RIS, and NE10485 on FPP synthase activity, the compounds were diluted to 5× final concentration in assay buffer and were preincubated with the enzyme preparation for 10 min prior to initiation of the reaction. The activity of recombinant human farnesyl transferase was determined by assessing the amount of [3H]farnesyl transferred from [3H]FPP to recombinant K-Ras (22Seabra M.C. James G.L. Methods Mol. Biol. 1998; 84: 251-260PubMed Google Scholar). The final concentrations of reagents in a standard reaction mix were 50 mm Tris-Cl, pH 7.2, 150 mm KCl, 10 mm ZnCl2, 3 mm MgCl2, 0.2% (v/v) octyl-d-glucopyranoside (NOGA detergent), 1 mm dithiothreitol, 0.9 μm FPP, 0.3 μm [3H]FPP (15–30 Ci/mmol), 40 nm FTase, and 15 μm K-Ras. NE10790, NE10485, and RIS were aliquoted into siliconized microcentrifuge tubes (Fisher) at 4 °C and added to the reaction mix. The reactions were initiated by transferring the tubes to 37 °C and incubated for 15 min, and then reactions were stopped by the addition of 400 μl of ethanol:HCl (9:1 v/v). The protein was allowed to precipitate at room temperature for 30 min and was then filtered on to glass microfiber filters (GF/C, Whatman). The tubes were washed three times with 1 ml of ethanol, and then the amount of radioactivity on each filter quantified by liquid scintillation counting following the addition of 5 ml of scintillation mixture. The assays were in duplicate and were repeated three times independently to verify reproducibility. The activity of recombinant human GGTase I and Rab GGTase were determined by assessing the amount of [3H]geranylgeranyl transferred from [3H]GGPP to Rho A and Rab1a, respectively (22Seabra M.C. James G.L. Methods Mol. Biol. 1998; 84: 251-260PubMed Google Scholar). The final concentrations in the GGTase I reaction mix were 50 mmTris, pH 7.2, 150 mm KCl, 5 mmMgCl2, 0.3 mm Nonidet P-40, 1 mmdithiothreitol, 5 μm GGPP, 0.5 μm[3H]GGPP (15–30 Ci/mmol), 58 nmGGTase I, and 10 μm RhoA. Final concentrations in the Rab GGTase reaction mix were 25 mm Tris, pH 7.2, 5 mm MgCl2, 1 mm dithiothreitol, 3.6 μm GGPP, 0.4 μm [3H]GGPP, 0.04 μm Rab GGTase, 10 μm Rab1a, and 2.5 μm REP1. The assay was carried out as outlined above for the FTase assay, including a negative control (lacking REP1) for the Rab GGTase assay. The assays were in duplicate and were repeated three times independently to verify reproducibility. Osteoclast number, F-actin "rings," and resorptive activity of mature rabbit osteoclasts in vitro were assessed as described previously (16Coxon F.P. Helfrich M.H. Van't Hof R. Sebti S. Ralston S.H. Hamilton A. Rogers M.J. J. Bone Miner. Res. 2000; 15: 1467-1476Crossref PubMed Scopus (352) Google Scholar). Briefly, rabbit osteoclasts were allowed to adhere for 2 h on 5-mm-diameter elephant tusk dentine discs in 96-well plates and then cultured with fresh α-minimum essential medium in the presence or absence of RIS, NE10790, or NE10485. 48 h later, intracellular F-actin was visualized by staining with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin, and the number of actin rings, defined as a distinct and complete ring of podosomes, per disc was then counted (actin rings are an indication of osteoclast polarization, and their presence correlates highly with active resorption). The discs were then stained for tartrate-resistant acid phosphatase by incubating with naphthol-ASBI-phosphate, hexazotized pararosanilin, and 50 mm tartrate in acetate buffer, pH 5.5, at 37 °C for 30 min (25van't Hof R.J. Tuinenburg-Bol R.A. Nijweide P.J. Int. J. Exp. Pathol. 1995; 76: 205-214PubMed Google Scholar). To detect the total number of osteoclasts in the cultures, the number of multinucleated (>2 nuclei/cell), tartrate-resistant acid phosphatase-positive cells/disc was then counted. To assess osteoclastic resorption, the discs were immersed in 20% (w/v) sodium hypochlorite to remove all cells, and then resorption pits in the mineral surface were visualized by reflected light microscopy. The area of the pits/disc was then examined using a Zeiss Axiolab reflective light microscope and quantified using software developed in-house based on Aphelion (ADCIS, France) ActiveX components. Scanning electron microscopy (SEM) was carried out on osteoclasts that had been seeded on to dentine discs and treated as described above. After 48 h, the cells were fixed in 2.5% (v/v) glutaraldehyde and 2.5 mm MgCl2 in 0.89m phosphate buffer, pH 7.2, for 20 h at room temperature. Thereafter they were washed in 0.1 m phosphate buffer, pH 7.2, post-fixed in osmium tetroxide for 1.5 h, then washed in distilled water, and dehydrated through a graded series of ethanol solutions. The samples were then critical point dried from CO2, glued onto aluminum stubs with colloidal silver adhesive, and sputter coated with 20 nm platinum and examined in a Jeol JSM-35CF scanning electron microscope operating at 10 kV. To quantify the results, osteoclasts throughout the whole dentine disc were identified and scored according to their morphology. "Normal" osteoclasts were defined as those that were well spread, with numerous microvilli on the basolateral surface (the membrane not apposed to the bone surface). "Retracted" osteoclasts were defined as those in which the peripheral membrane adjacent to the dentine surface was completely retracted or showed evidence of retraction fibers. "Domed" osteoclasts were defined as those in which one or more regions of the basolateral membrane were both raised and devoid of microvilli. For morphological studies, rabbit osteoclasts were cultured as above for 48 h on dentine slices without reagents or with 10–100 μm RIS or 500–1000 μmNE10790. The cultures were fixed for 10 min in a 1:1 (v:v) mixture of minimum essential medium and fixation buffer (3.5% (w/v) paraformaldehyde, 2% (w/v) sucrose in PBS). The cells were then permeablized in Triton buffer (20 mm Hepes, 300 mm sucrose, 50 mm NaCl, 3 mmMgCl2, 0.5% (v/v) Triton X-100, 0.5% (w/v) sodium azide, in PBS at pH 7.0) at 4 °C and then immunostained for paxillin using a mouse monoclonal antibody (10 μg/ml) and secondary fluorescein isothiocyanate-conjugated anti-mouse Ig polyclonal antibodies (1:40 dilution) (Dako, Denmark). Resorbing osteoclasts were identified by their characteristic F-actin ring structure (described above) after staining with TRITC-phalloidin conjugate (Molecular Probes) at 5 units/ml. The dentine surface was examined by simultaneous confocal reflection microscopy where resorbed areas could be identified by their reduced reflection. Scanning laser confocal microscopy was performed on a Leica TCS NT system (Heidelberg, Germany). Fluorescent images were collected in sequential 1-μm steps through the osteoclasts for fluorescein isothiocyanate and TRITC fluorochromes and reflection at 488-, 568-, and 647-nm emission wavelengths, respectively, and displayed in the xy and zx planes. For electron microscopic analysis, rabbit osteoclasts were treated with or without 1 mm NE10790 for 48 h on dentine slices. The cells were fixed in 2.5% (v/v) glutaraldehyde in phosphate buffer (0.1 m, pH 7.4) for a minimum of 24 h and then demineralized in the same fixative with 2.5% (w/v) EDTA for ∼48 h. The samples were then washed in buffer, postfixed in osmium tetroxide, dehydrated in ethanol, and embedded in Epon. Ultrathin sections were stained using uranyl acetate and lead citrate and then examined using a Philips EM201 microscope. RIS dramatically and dose-dependently reduced the number of viable J774 cells with an IC50 of approximately 30 μm (Fig.1 B). NE10790 also reduced viable J774 cell number but was ∼40 times less potent than RIS (IC50, ∼1.2 mm). By contrast, NE10485 had no effect on cell viability at concentrations up to 3 mm. RIS potently inhibited recombinant human FPP synthase in vitro with an IC50 value of 10 nm ± 10 nm(n = 4; Fig. 1 C). By contrast, neither NE10790 nor NE10485 affected FPP synthase at concentrations up to 100 μm, although at higher concentrations up to 1 mm, both NE10790 and NE10485 partially inhibited (30–50%) FPP synthase activity in vitro. The effect of NE10790, RIS, and NE10485 on protein prenylation was investigated by examining the incorporation of [14C] mevalonic acid into prenylated proteins in J774 cells in vitro. Whereas 100 μm RIS inhibited the incorporation of [14C]mevalonic acid into all prenylated small GTPases (21–26 kDa) and higher molecular mass proteins (∼60 kDa, most likely farnesylated lamin B and prelamin A), 1.5 mm NE10790 inhibited incorporation of [14C]mevalonic acid into bands of prenylated small GTPases of higher molecular mass only (22–26 kDa proteins; most likely Rab GTPases based on molecular mass) (Fig.2 A). The effect of NE10790 was dose-dependent, with complete inhibition of incorporation of [14C]mevalonic acid into 22–26-kDa proteins at a concentration of 1 mm (Fig. 2 B). The IC50 for inhibition of Rab prenylation in J774 cells was calculated from densitometric analysis of the 22–26-kDa bands and was determined to be 560 ± 120 μm (n = 3). The intense radiolabel at the dye front, which probably represents isoprenoids such as GGPP (8Luckman 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 (1073) Google Scholar), was unaffected by treatment with NE10790 but was completely absent in lysates of RIS-treated cells (Fig. 2 A). 1.5 mm NE10485 had no effect on either protein prenylation or the abundance of radiolabeled isoprenoids at the dye front (Fig. 2 A). The effect of NE10790 on incorporation of [14C]mevalonic acid into prenylated proteins in J774 cells was compared with the effects of peptidomimetic inhibitors of FTase and GGTase I. An FTase inhibitor, FTI-277 (10 μm), prevented incorporation of [14C]mevalonic acid into bands of around 60 kDa (farnesylated lamins), whereas 15 μm GGTI-298, an inhibitor of GGTase I, reduced incorporation of [14C]mevalonic acid into a broad band with a molecular mass of 21 kDa, most likely consisting of geranylgera
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