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Inhibitory effect of minocycline on osteoclastogenesis in mouse bone marrow cells
archives of oral biology 56 (2011) 924–931
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Inhibitory effect of minocycline on osteoclastogenesis in mouse bone marrow cells Tsuneyasu Nagasawa, Michitsugu Arai, Akifumi Togari * Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
article info
abstract
Article history:
Objective: To study the effects of minocycline hydrochloride (MINO) on the formation of
Accepted 2 February 2011
tartrate-resistant acid phosphatase (TRAP) staining-positive multinucleated osteoclast-like cells in mouse bone marrow cells (BMCs) treated with 1a,25(OH)2D3 or soluble receptor
Keywords:
activator of nuclear factor-kB ligand (s-RANKL).
Minocycline
Materials and methods: Mouse BMCs were cultured in alpha-modified minimum essential
Tetracycline
medium containing foetal calf serum (10%) and tetracyclines (2.5, 5 and 10 mM), such as
Osteoclast
MINO, tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OXT) or doxycycline
Osteoclastogenesis
(DOXY) in the presence of 1a,25(OH)2D3 (10 nM) or s-RANKL (20 ng/ml) for 7 days, and the
Bone marrow cells
number of TRAP staining-positive osteoclast-like cells was counted. In RNA isolated from
RANKL
BMCs treated with 1a,25(OH)2D3 or s-RANKL in the presence or absence of MINO, the expressions of osteoclast differentiation relating to mRNA were analysed by reverse transcription-polymerase chain reaction. Cell viability was examined in mouse BMCs and rabbit osteoclasts treated with MINO (0.25–20 mM and 2–50 mM, respectively) for 24 h. Results: MINO, TC, OXT or DOXY inhibited 1a,25(OH)2D3-induced osteoclast-like cell formation in mouse BMCs dose dependently. MINO suppressed 1a,25(OH)2D3-induced up-regulation of mRNA expressions of TRAP, cathepsin K, carbonic anhydrase II, and calcitonin receptor, but not RANKL. MINO inhibited s-RANKL-induced osteoclast-like cell formation and up-regulation of mRNA expressions for nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation; however, MINO had no effects on the viability of mouse BMCs and rabbit osteoclasts. Conclusion: MINO inhibits RANKL-induced osteoclastogenesis via down-regulation of NFATc1 mRNA expression in osteoclast precursor cells. # 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Tetracyclines are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl transfer RNA to the ribosomal acceptor site, and are broad-spectrum antimicrobial agents, exhibiting activity against a wide range of aerobic and anaerobic Gram-positive and Gram-negative bacteria.1,2 In dentistry, tetracyclines have long been used extensively in the management of periodontal diseases
characterised by the formation of periodontal pockets, loss of periodontal support and pathological alveolar bone resorption.3,4 Minocycline hydrochloride (MINO), a semi-synthetic derivative of tetracycline, is one of the most active antibiotics against periodontal pathogens, and has been used to suppress connective tissue breakdown during periodontal disease.5–10 Apart from antimicrobial activity, tetracyclines also exhibit additional pharmacological properties in the management of periodontal diseases.11 Inhibitory effects of tetracyclines on bone resorption have been demonstrated in both in vitro and
* Corresponding author. Tel.: +81 52 757 6742; fax: +81 52 751 2588. E-mail address: [email protected] (A. Togari). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.02.002
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in vivo studies. In vitro, parathyroid hormone (PTH)-induced bone resorption in organ culture was inhibited by not only antibiotic tetracyclines but also a chemically modified tetracycline (CMT) without antibiotic activity.11–13 Bettany et al.14,15 demonstrated that doxycycline (DOXY) and CMT induced apoptosis in 24-h cultures of two monocyte/macrophage cell lines (U937 and RAW264 cells) and mature rabbit osteoclasts, suggesting that tetracyclines act in the apoptosis of osteoclast precursors and mature osteoclasts. In vivo, oral administration of MINO or CMT prevents trabecular bone loss in ovariectomised (OVX) rats and streptozotocin-induced diabetic rats, a model of postmenopausal osteoporosis and diabetic osteoporosis, respectively.16–18 Furthermore, both DOXY and non-antimicrobial CMT inhibited periodontal bone resorption induced by Escherichia coli endotoxin in rats.19 This evidence indicated that tetracyclines inhibit pathologically accelerated bone resorption independent of antimicrobial activity. Osteoclasts are bone-resorbing multinucleate large cells derived from the haematopoietic monocyte–macrophage lineage, having characteristics of mRNA expressions, such as tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), carbonic anhydrase II (CAII), and cathepsin K.20,21 1a,25(OH)2D3, PTH, and PGE2 increase the receptor activator of nuclear factor-kB (RANK) ligand (RANKL) production in osteoblasts/stromal cells to induce the formation of osteoclast-like cells from osteoclast precursors.20,21 The precise molecular mechanisms underlying osteoclast differentiation have been clarified in recent studies by Takayanagi’s group.22–26 They showed that the binding of RANKL to RANK in osteoclast precursor cells specifically induced the nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation via the tumour necrosis factor receptor-associated factor 6 (TRAF6) and c-Fos signalling pathways, and that RANKL evoked Ca2+ oscillations that led to calmodulin–calcineurin-mediated activation of NFATc1. They also demonstrated that the selective Ca2+ chelator BAPTA-AM suppressed RANKL-induced NFATc1 expression and osteoclastogenesis in bone marrow-derived monocyte/macrophage precursor cells. Chemically, tetracyclines are known as chelating agents of Ca2+ independent of their antibiotic activity.27,28 It was demonstrated that pretreatment of osteoclasts with either MINO or DOXY significantly attenuated the peak cytosolic Ca2+ response to extracellular Ca2+.27 In the present study, we hypothesise that tetracyclines including MINO probably have an inhibitory effect on osteoclastogenesis via osteoclast differential signalling pathway in osteoclast precursor cells by their Ca2+ chelating action. To clarify the hypothesis, the effects of MINO, TC, OXT or DOXY on the formation of TRAP staining positive osteoclastlike cells were examined in mouse BMCs treated with 1a,25(OH)2D3 or s-RANKL, a simple and convenient culture system for experimental osteoclastogenesis.29 Then, the effects of MINO on the mRNA expression of osteoclast phenotypes, transcriptional factors involved in osteoclast differentiation and dendritic cell-specific transmembrane protein (DC-STAMP), an essential molecule for cell–cell fusion,30–32 were also examined. Here we demonstrated that MINO inhibited s-RANKL-induced osteoclastogenesis via
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down-regulation of NFATc1 mRNA expression in mouse BMC culture.
2.
Materials and methods
2.1.
Materials
Six-week-old ddY male mice and 10-day-old Japan white male rabbits were purchased from Japan SLC Inc. (Hamamatsu, Japan). Animals were treated in accordance with the Guidelines for Animal Experiments at the School of Dentistry, Aichi-Gakuin University. Minocycline hydrochloride (MINO), tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OXT), doxycycline hydrochloride hemiethanolate hemihydrate (DOXY) and foetal calf serum (FCS) were purchased from Sigma Co. (St. Louis, MO, USA). Recombinant mouse soluble RANKL (s-RANKL) was purchased from R&D (London, UK), 1a,25(OH)2D3 from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA, USA) and alphamodified minimum essential medium (a-MEM) from Invitrogen Co. (Carlsbad, CA, USA). The CellTiter-GloTM Luminescent Cell Viability Assay, a commercial kit, was purchased from Promega Co. (Madison, WI, USA). All other chemicals were of reagent grade.
2.2. The formation of tartrate-resistant acid phosphatasepositive multinucleate osteoclast-like cells in mouse BMC culture Mouse BMCs were obtained from tibiae as previously described29 and cultured in plastic dishes in a-MEM containing FCS (10%), penicillin (100 IU/ml) and streptomycin (100 mg/ml) in humidified air with CO2 (5%) at 37 8C. To examine osteoclast formation, we cultured BMCs (1 106 cells) in a-MEM (1 ml) containing FCS (10%) and tetracyclines (2.5, 5 and 10 mM) in 24well plates for 7 days in the presence of osteoclastogenesisinducing drugs, such as 1a,25(OH)2D3 (10 nM) and s-RANKL (20 ng/ml). 1a,25(OH)2D3 and s-RANKL were used to examine if tetracyclines have some effects on osteoclastogenesis via osteoblastic function, such as RANKL production and via RANKL–RANK interaction-induced osteoclast differentiation signalling in precursor cells, respectively. To avoid aspirating all of the non-adherent BMCs, including osteoclast precursors, we replaced one-half (0.5 ml) of the supernatant of the culture medium with fresh medium every 2 days, and added drugs at the beginning of cultures and at each medium change. After 7 days in culture, adherent cells were fixed with a mixture of 10% formalin in phosphate-buffered saline and ethanol– acetone (50:50, v/v), and stained for TRAP by incubating the cells in sodium acetate buffer (0.1 M, pH 5) containing naphthol AS-MX phosphate and red violet LB salt in the presence of sodium tartrate (10 mM), as described previously.33 Cells containing 3 or more nuclei were designated as osteoclast-like cells. Moreover, to examine the bone-resorbing capability of osteoclast-like cells, we cultured BMCs in 24-well plates in which a dentine slice had been placed at the bottom. After 7 days in culture, the cells were removed from the dentine slices, and resorption pits on the slices were stained with haematoxylin.
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Table 1 – RT-PCR primers. Primers (source) TRAP (S70805) Cathepsin K (NM_00780) CAII (K00811) CTR (U18542) RANK (NM_009399) RANKL (AB008426) OPG (AB013898) M-CSF (X05010) c-FOS (V00727) PU.1 (M_32370) MITF (NM_008601) NFATc1 (NM_198429) DC-STAMP (AY517483) GAPDH (NM_002046)
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Sequences (50 –30 )
Product length (bp)
TTGAGGACGTGTTCTCTGACC TGGGCTGCTGACTGGCAAAGT GGCCAACTCAAGAAGAAAACT ATAGCCCACCACCAACACTGC TTGGCTGTTTTGGGCTATTTT CGCTTTGATCTTTCTATTCTT AAGATGAGGCAAACCCACGAG CCCGAGGAGCACTAACTACGC AACCTCCAGTCAGCAAGAAGT GTCACAGCCCTCAGAATCCAC GCCATTTGCACACCTCACCAT AGAATTGCCCGACCAGTTTTT CCTTGCCCTGACCACTCTTAT ACATCTATTCCACACTTTTGC AATCCCCAACGAGTCAGCAAC GGCCCCCAACAGTCAGCAAGA AGAAGGGGCAAAGTAGAGCAG TGAGAAGGGGCAGGGTGAAGG CTTCCCTTATCAAACCTTGTC AGGTGAGCTTCTTCTTGACTT AGACCTGACATGTACGACAAC TATGCAGGGCTACTGATAAAG CAACGCCCTGACCACCGATAG GGCTGCCTTCCGTCTCATAGT CTAAGGAGAAGAAAACCCTTG CAGCATAGAAGACAACAATCC ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGTA
249 402 354 319 373 316 407 319 361 398 397 392 332 452
Sources are GenBank accession numbers.
2.3.
Isolation of mature osteoclasts from rabbit long bones
Purified mature osteoclasts were prepared from rabbit tibia and femora as reported previously.34 Briefly, long bones from 10-dayold rabbits were minced with scissors and agitated with a vortex mixer. An aliquot of unfractionated bone cells was seeded onto 0.24% collagen gel (Nitta Gelatin, Tokyo) coated on 10-cm tissue culture dishes and incubated. Two hours later, non-adherent cells were washed off and the remaining osteoclasts were then removed from the gels with 0.1% collagenase solution (Wako Pure Chemical Co., Osaka). Isolated cells fulfilled the criteria of osteoclasts; TRAP staining-positive multinuclear cells with bone-resorbing activity and actin ring formation.
2.4.
Analysis of mRNA expression by RT-PCR
For the experiment conducted to examine mRNA expression, BMCs were cultured for 5 days in 6-cm-diameter plastic dishes in a-MEM (5 ml) containing minocycline (10 mM) in the presence of 1a,25(OH)2D3 (10 nM) or s-RANKL (20 ng/ml). For the experiment conducted to examine mRNA expression, mouse BMCs were cultured in 6-cm-diameter plastic dishes in a-MEM containing FCS (10%), penicillin (100 IU/ml), and streptomycin (100 mg/ml) in humidified air with CO2 (5%) at 37 8C. One half of the culture medium was replaced with fresh medium every 2 days. RNA was extracted from the cells by the guanidinium-thiocyanate method.35 The reverse transcription-polymerase chain reaction (RT-PCR) was performed by standard methods. Briefly, cDNA was first synthesised using random primers and Moloney murine leukaemia virus reverse
transcriptase (Gibco-BRL; Life Technologies, Grand Island, NY, USA), followed by PCR amplification using synthetic gene primers specific to mouse genes. The oligonucleotides used as primers for PCR are shown in Table 1. Each PCR amplification mixture (25 ml) contained 2.5 mg cDNA. PCR amplification was performed using the GeneAmp PCR System (Perkin Elmer/ Cetus, Norwalk, CT, USA) under the following conditions: denaturation at 95 8C for 15 s, annealing at 55 8C for 30 s, and elongation at 72 8C for 30 s. PCR products were electrophoresed on a 2% Nusive GTG agarose gel (FMC Bioproducts, Rockland, ME, USA), stained with ethidium bromide, and detected with a fluorescence imager analyser (FluorImager 575; Amersham Pharmacia Biotech, Sunnyvale, CA, USA).
2.5. Cell viabilities of BMCs and mature osteoclasts treated with minocycline Cell viabilities of BMCs were estimated using a commercial assay kit, CellTiter-GloTM Luminescent Cell Viability Assay, which is based on quantification of the ATP level as previously described.36 Briefly, mouse BMCs (103 cells) were cultured in 96-well plates for 24 h in a-MEM (100 ml) containing minocycline (0.25–20 mM), and then the ATP level as an index of the number of viable cells was measured by determining the luminescent signal. Cell viabilities in mature osteoclasts were estimated by counting the number of cells under the light microscope. Purified mature osteoclasts were seeded in 24-well plastic dishes in a-MEM (1 ml). One hour after seeding, one group (8 wells) of cells was fixed with formalin and stained for TRAP,
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Fig. 1 – Effect of minocycline on osteoclast-like cell formation induced by 1a,25(OH)2D3 in mouse BMCs. BMCs were treated with 1a,25(OH)2D3 (10 nM), or minocycline (2.5–10 mM) for 7 days. The cells were fixed and stained for TRAP activity, and then the number of TRAP-positive multinucleate osteoclast-like cells (TRAP+ MNCs) was counted. BMCs were also cultured on dentine slices under the same culture conditions. After a 7-day culture period, the dentine slices were stained with haematoxylin. Photomicrographs of TRAP+ MNCs (A) and resorption pits (B) of non-treated control culture (a), and cultures treated with 1a,25(OH)2D3 (b), or 1a,25(OH)2D3 + minocycline ((c–e) 2.5 mM, 5 mM, and 10 mM minocycline, respectively). Bar = 0.1 mm. (C) Number of TRAP+ MNCs. Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from 1a,25(OH)2D3 alone according to Tukey’s multiple comparison test.
and the number of adherent TRAP-positive osteoclasts was counted (0 h). Other osteoclasts were further cultured for 24 h in a-MEM without MINO (control) or with MINO (2–50 mM), and the numbers of adherent TRAP-positive osteoclasts were counted in the same manner.
2.6.
Statistical analysis
3.2. Effect of MINO on the mRNA expressions of osteoclast phenotypes and osteoclastogenesis-related proteins in mouse BMC culture treated with 1a,25(OH)2D3 As shown in Fig. 3, mRNA expressions of osteoclast phenotypes, such as TRAP, cathepsin K, CAII and CTR were upregulated in mouse BMCs treated with 1a,25(OH)2D3 (10 nM) alone but not 1a,25(OH)2D3 and MINO (10 mM). The expression
All data are presented as the means standard error of the mean (S.E.M.). Statistical analysis was carried out by one-way analysis of variance (Tukey’s Multiplan Comparison Tests) using Prism version 4 (GraphPad Software Inc., San Diego, CA), and p < 0.05 was considered significant.
3.
Results
3.1. Effect of tetracyclines on the formation of TRAPpositive osteoclast-like cells in mouse BMC culture treated with 1a,25(OH)2D3 As shown in Fig. 1, TRAP-positive osteoclast-like cells and resorbing pits were clearly observed in cultures treated with 1a,25(OH)2D3 (10 nM) alone. In contrast, the formation of TRAP-positive cells and resorbing pits was decreased dose dependently by the addition of MINO. No TRAP-positive cells or resorbing pits were detected in mouse BMCs treated with 1a,25(OH)2D3 and MINO (10 mM), the same as the non-treated control. As shown in Fig. 2, 1a,25(OH)2D3-induced osteoclastogenesis was dose-dependently inhibited by treatment with TC, DOXY or OXY.
Fig. 2 – Effects of tetracyclines on osteoclast-like cell formation induced by 1a,25(OH)2D3 in mouse BMCs. BMCs were treated with 1a,25(OH)2D3 (10 nM) alone, or with tetracycline, doxycycline or oxytetracycline (2.5–10 mM) for 7 days. Cells were fixed and stained for TRAP activity; and then the number of TRAP-positive multinucleate osteoclast-like cells (TRAP+ MNCs) was counted. Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from each 1a,25(OH)2D3 alone according to Tukey’s multiple comparison test.
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Fig. 3 – Effect of minocycline on the mRNA expression of TRAP, cathepsin K, CAII, CTR, RANKL, OPG and M-CSF in mouse BMC culture treated with 1a,25(OH)2D3. Total RNA for RT-PCR analysis was isolated from mouse BMCs cultured for 5 days in a-MEM with no additional agents (lane 1), 10 nM 1a,25(OH)2D3 (lane 2), or 1a,25(OH)2D3 and 10 mM MINO (lane 3). DNA size markers (fX 174/Hae III digest) are shown in the left lanes (S). Numbers in parentheses indicate cycles for PCR amplification. Results using primers specific to GAPDH are shown for comparison.
of RANKL mRNA was up-regulated in mouse BMCs treated with 1a,25(OH)2D3 in the presence or absence of MINO. In contrast, OPG and M-CSF mRNAs were constitutively expressed in BMCs, and these mRNA expressions were not influenced by 1a,25(OH)2D3 with or without MINO.
3.3. Effect of MINO on the formation of TRAP-positive osteoclast-like cells in mouse bone marrow cell cultures treated with s-RANKL As shown in Fig. 4, MINO (2.5–10 mM) suppressed s-RANKLinduced TRAP-positive osteoclast-like cell formation dose dependently.
3.4. Effect of MINO on the expressions of mRNA for NFATc1, DC-STAMP and multiple transcription factors in mouse BMC culture treated with s-RANKL As shown in Fig. 5, expressions of mRNA for NFATc1 and DCSTAMP were up-regulated by s-RANKL (20 ng/ml) alone but not by s-RANKL and MINO (10 mM). In contrast, multiple transcription factors, such as cFOS, PU.1 and MITF mRNA, were constitutively expressed in BMCs, and these mRNA expressions were not influenced by RANKL with or without MINO.
3.5. Effect of MINO on the survival of mouse BMCs and rabbit osteoclasts As shown in Fig. 6, treatment with MINO at any concentration (1.25–20 mM) did not influence the viability of BMCs (Fig. 6A), and the number of rabbit osteoclasts was significantly decreased in all groups treated with MINO (2–50 mM) or without MINO (control) by about 50% of the initial number (Fig. 6B); however, no significant differences in cell number were observed amongst all groups.
Fig. 4 – Effect of minocycline on osteoclast-like cell formation induced by s-RANKL in mouse BMCs. BMCs were treated with s-RANKL (20 ng/ml) alone or with minocycline (2.5–10 mM) for 7 days. Cells were fixed and stained for TRAP activity, and then the number of TRAPpositive multinucleate osteoclast-like cells (TRAP+ MNCs) was counted. (A) Photomicrographs of TRAP+ MNCs of cultures treated with s-RANKL (a), s-RANKL + minocycline ((b–d) 2.5 mM, 5 mM, and 10 mM minocycline, respectively). Bar = 0.1 mm. (B) Number of TRAP+ MNCs. Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from s-RANKL alone according to Tukey’s multiple comparison test.
4.
Discussion
In the initial experiments of the present study, MINO, TC, OXY and DOCY inhibited 1a,25(OH)2D3-induced osteoclastogenesis in a dose dependent manner (Figs. 1 and 2) suggesting that tetracyclines commonly have an inhibitory effect on osteoclastogenesis. Thereafter, we used MINO to elucidate the mechanism underlying inhibition of osteoclast differentiation by tetracyclines. RT-PCR analysis (Fig. 3) showed that mRNA expressions of RANKL and osteoclast phenotypes were upregulated in BMCs treated with 1a,25(OH)2D3, and the addition of MINO (10 mM) decreased mRNA expressions of osteoclast phenotypes except RANKL to the non-treated control levels with no changes in OPG and M-CSF mRNA expression. MINO also inhibited s-RANKL-induced osteoclast-like cell formation dose-dependently (Fig. 4). Most of these observations suggested that MINO inhibited osteoclastic differentiation via downstream of RANKL–RANK interaction in osteoclast precursor cells.37 As shown in Fig. 5, NFATc1 and DC-STAMP mRNA expressions in BMCs up-regulated by s-RANKL were reduced by the addition of MINO without any changes in mRNA expressions of c-FOS, PU.1 and MITF, suggesting that MINO might inhibit NFATc1 and DC-STAMP mRNA expressions via an independent pathway from those mediated signalling pathway in osteoclast precursor cells.38
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Fig. 5 – Effect of minocycline on the expressions of mRNA for NFATc1, DC-STAMP, c-FOS, PU.1 and MITF in mouse BMCs culture treated with s-RANKL. Total RNA for RT-PCR analysis was isolated from mouse BMCs cultured for 5 days in a-MEM with no additional agents (lane 1), 20 ng/ml s-RANKL (lane 2), or s-RANKL and 10 mM minocycline (lane 3). DNA size markers (fX 174/Hae III digest) are shown in the left lane (S). Numbers in parentheses indicate cycles for PCR amplification. Results using primers specific to GAPDH are shown for comparison.
Our results are well consistent with the study of Takayanagi’s group22–26 already demonstrated that RANKL-induced Ca2+ oscillations result in an activation of NFATc1 and this effect was inhibited by a Ca2+ chelator BAPTA-AM. They also
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demonstrated that calcineurin inhibitors, cyclosporine A and FK506, suppressed RANKL-induced osteoclastogenesis.23 Judging from these previous observations and present results, it is possible that Ca2+ chelator tetracyclines block the calcium– calmodulin–calcineurin signalling pathway to inhibit NFATc1 mRNA expression and osteoclast-like cell formation in mouse BMCs treated with s-RANKL. Previously, Bettany et al.14,15 demonstrated that tetracycline derivatives induced apoptosis in osteoclastic cell lines and rabbit osteoclasts. However, treatment with MINO did not show any influence on the viability of BMCs and isolated rabbit osteoclasts (Fig. 6). Differences between the results of our study and Bettany’s studies14,15 may have been caused by differences in the experimental conditions, such as cells and drug concentrations. In Bettany’s studies14,15 DOXY and CMT at a concentration of 25–50 mg/ml (about 50–100 mM) induced the apoptosis of monocyte and macrophage cell lines, whereas in the present study MINO at 20 mM did not induce the apoptosis of mouse BMCs; however, in rabbit osteoclasts, almost the same range of drug concentrations was used in Bettany’s studies (20–40 mM DOXY) and the present study (20– 50 mM MINO), suggesting a difference in the cell toxicity of DOXY and MINO. Further studies are needed to elucidate differences in their cell toxicity. A therapeutically useful property of tetracyclines for the treatment of periodontal disease was found previously. Golub et al.4,5 demonstrated that tetracyclines inhibited the activity of mammalian collagenase regardless of antimicrobial efficacy, and Ramamurthy et al.19 showed that tetracyclines with or without antimicrobial activity inhibited matrix metalloproteinase (MMP)-mediated periodontal bone resorption in rats. These investigations indicate that tetracyclines can inhibit the breakdown of connective tissues mediated by excessive collagenolytic activity. In the present study, we demonstrated that MINO inhibited s-RANKL induced osteoclast-like cell formation in mouse BMCs via the down-regulation of NFATc1
Fig. 6 – Effect of minocycline on the survival of mouse BMCs and rabbit osteoclasts. (A) Mouse BMCs (103 cells) were seeded in 96-well plates and cultured for 24 h in a-MEM without minocycline (control) or with minocycline (1.25–20 mM). Cell viabilities were estimated by quantification of the ATP concentration using a commercial assay kit, the CellTiter-GloTM Luminescent Cell Viability. Values are given as a percentage of the control, and are the mean W S.E.M. (n = 8). (B) Rabbit osteoclasts isolated from the femur and tibia were seeded in 24-well plates in a-MEM. After seeding, osteoclasts adhering to the plate bottom (8 wells) were fixed with formalin, stained for TRAP, and counted as the initial number of osteoclasts (0 h). Other osteoclasts were further cultured for 24 h in a-MEM without minocycline (control) or with minocycline (2– 50 mM), and the number of surviving adherent TRAP-positive osteoclasts was counted (24 h). Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from 0 h and +++p < 0.001, statistically different from 24 h control according to Tukey’s multiple comparison test.
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mRNA expression. This effect of MINO may also contribute to the management of periodontal diseases with pathological alveolar bone resorption.
5.
Conclusion
MINO inhibited RANKL-induced osteoclastogenesis via the down-regulation of NFATc1 mRNA expressions in osteoclast precursor cells, suggesting the usefulness of tetracyclines against pathologically accelerated bone resorption. Funding: None. Competing interests: None declared. Ethical approval: Not required.
Acknowledgment This study was partly supported by a grant-in-aid from the Strategic Research AGU-Platform Formation (2008-2012).
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12. Gomes BC, Golub LM, Ramamurthy NS. Tetracyclines inhibit parathyroid hormone-induced bone resorption in organ culture. Experientia 1984;40(11):1273–5. 13. Rifkin B, Gomes BC, Ramamurthy NS, McNamara TF, Golub LM. Effect of a chemically-modified tetracycline (CMT) on bone resorption in organ culture. J Cell Biol 1987;105(abstract):216. 14. Bettany JT, Wolowacz RG. Tetracycline derivatives induce apoptosis selectively in cultured monocytes and macrophages but not in mesenchymal cells. Adv Dent Res 1998;12(2):136–43. 15. Bettany JT, Peet NM, Wolowacz RG, Skerry TM, Grabowski PS. Tetracyclines induce apoptosis in osteoclasts. Bone 2000;27(1):75–80. 16. Williams S, Wakisaka A, Zeng QQ, Barnes J, Seyedin S, Martin G, et al. Effect of minocycline on osteoporosis. Adv Dent Res 1998;12(2):71–5. 17. Sasaki T, Ramamurthy NS, Golub LM. Long-term therapy with a new chemically modified tetracycline (CMT-8) inhibits bone loss in femurs of ovariectomized rats. Adv Dent Res 1998;12(2):76–81. 18. Bain S, Ramamurthy NS, Impeduglia T, Scolman S, Golub LM, Rubin C. Tetracycline prevents cancellous bone loss and maintains near-normal rates of bone formation in streptozotocin diabetic rats. Bone 1997;21(2):147–53. 19. Ramamurthy NS, Rifkin BR, Greenwald RA, Xu J, Liu Y, Turner G, et al. Inhibition of matrix metalloproteinasemediated periodontal bone loss in rats: a comparison of 6 chemically modified tetracyclines. J Periodontol 2002;73(7):726–34. 20. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423(6937):337–42. 21. Takahashi N, Udagawa N, Suda T. A new member of tumor necrosis factor ligand family, ODF/OPGL/TRANCE/RANKL, regulates osteoclast differentiation and function. Biochem Biophys Res Commun 1999;256:449–55. 22. Asagiri M, Takayanagi H. The molecular understanding of osteoclast differentiation. Bone 2007;40:251–64. 23. Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 2002;3(6):889–901. 24. Koga T, Inui M, Inoue K, Kim S, Suematsu A, Kobayashi E, et al. Costimulatory signals mediated by the ITAM motif cooperate with RANKL for bone homeostasis. Nature 2004;428(6984):758–63. 25. Asagiri M, Sato K, Usami T, Ochi S, Nishina H, Yoshida H, et al. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J Exp Med 2005;202(9):1261–9. 26. Negishi-Koga T, Takayanagi H. Ca2+–NFATc1 signaling is an essential axis of osteoclast differentiation. Immunol Rev 2009;231(1):241–56. 27. Donahue HJ, Iijima K, Goligorsky MS, Rubin CT, Rifkin BR. Regulation of cytoplasmic calcium concentration in tetracycline-treated osteoclasts. J Bone Miner Res 1992;7(11):1313–8. 28. Grenier D, Huot MP, Mayrand D. Iron-chelating activity of tetracyclines and its impact on the susceptibility of Actinobacillus actinomycetemcomitans to these antibiotics. Antimicrob Agents Chemother 2000;44(3):763–6. 29. Takahashi N, Yamana H, Yoshiki S, Roodman GD, Mundy GR, Jones SJ, et al. Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow cultures. Endocrinology 1988;122(4):1373–82. 30. Hartgers FC, Vissers JL, Looman MW, van Zoelen C, Huffine C, Figdor CG, et al. DC-STAMP, a novel multimembranespanning molecule preferentially expressed by dendritic cells. Eur J Immunol 2000;30(12):3585–90.
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31. Kukita T, Wada N, Kukita A, Kakimoto T, Sandra F, Toh K, et al. RANKL-induced DC-STAMP is essential for osteoclastogenesis. J Exp Med 2004;200(7):941–6. 32. Yagi M, Miyamoto T, Sawatani Y, Iwamoto K, Hosogane N, Fujita N, et al. DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells. J Exp Med 2005;202(3):345–51. 33. Minkin C. Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 1982;34(3):285–90. 34. Tezuka K, Sato T, Kamioka H, Nijweide PJ, Tanaka K, Matsuo T, et al. Identification of osteopontin in isolated rabbit osteoclasts. Biochem Biophys Res Commun 1992;186(2):911–7. 35. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–
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chloroform extraction. Anal Biochem 1987;162(1): 156–9. 36. Wesierska-Gadek J, Gueorguieva M, Ranftler C, ZerzaSchnitzhofer G. A new multiplex assay allowing simultaneous detection of the inhibition of cell proliferation and induction of cell death. J Cell Biochem 2005;96(1):1–7. 37. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999;20(3):345–57. 38. Sato M, Morii E, Takebayashi-Suzuki K, Yasui N, Ochi T, Kitamura Y, et al. Microphthalmia-associated transcription factor interacts with PU.1 and c-Fos: determination of their subcellular localization. Biochem Biophys Res Commun 1999;254(2):384–7.
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Inhibitory effect of minocycline on osteoclastogenesis in mouse bone marrow cells Tsuneyasu Nagasawa, Michitsugu Arai, Akifumi Togari * Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan
article info
abstract
Article history:
Objective: To study the effects of minocycline hydrochloride (MINO) on the formation of
Accepted 2 February 2011
tartrate-resistant acid phosphatase (TRAP) staining-positive multinucleated osteoclast-like cells in mouse bone marrow cells (BMCs) treated with 1a,25(OH)2D3 or soluble receptor
Keywords:
activator of nuclear factor-kB ligand (s-RANKL).
Minocycline
Materials and methods: Mouse BMCs were cultured in alpha-modified minimum essential
Tetracycline
medium containing foetal calf serum (10%) and tetracyclines (2.5, 5 and 10 mM), such as
Osteoclast
MINO, tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OXT) or doxycycline
Osteoclastogenesis
(DOXY) in the presence of 1a,25(OH)2D3 (10 nM) or s-RANKL (20 ng/ml) for 7 days, and the
Bone marrow cells
number of TRAP staining-positive osteoclast-like cells was counted. In RNA isolated from
RANKL
BMCs treated with 1a,25(OH)2D3 or s-RANKL in the presence or absence of MINO, the expressions of osteoclast differentiation relating to mRNA were analysed by reverse transcription-polymerase chain reaction. Cell viability was examined in mouse BMCs and rabbit osteoclasts treated with MINO (0.25–20 mM and 2–50 mM, respectively) for 24 h. Results: MINO, TC, OXT or DOXY inhibited 1a,25(OH)2D3-induced osteoclast-like cell formation in mouse BMCs dose dependently. MINO suppressed 1a,25(OH)2D3-induced up-regulation of mRNA expressions of TRAP, cathepsin K, carbonic anhydrase II, and calcitonin receptor, but not RANKL. MINO inhibited s-RANKL-induced osteoclast-like cell formation and up-regulation of mRNA expressions for nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation; however, MINO had no effects on the viability of mouse BMCs and rabbit osteoclasts. Conclusion: MINO inhibits RANKL-induced osteoclastogenesis via down-regulation of NFATc1 mRNA expression in osteoclast precursor cells. # 2011 Elsevier Ltd. All rights reserved.
1.
Introduction
Tetracyclines are a family of antibiotics that inhibit protein synthesis by preventing the attachment of aminoacyl transfer RNA to the ribosomal acceptor site, and are broad-spectrum antimicrobial agents, exhibiting activity against a wide range of aerobic and anaerobic Gram-positive and Gram-negative bacteria.1,2 In dentistry, tetracyclines have long been used extensively in the management of periodontal diseases
characterised by the formation of periodontal pockets, loss of periodontal support and pathological alveolar bone resorption.3,4 Minocycline hydrochloride (MINO), a semi-synthetic derivative of tetracycline, is one of the most active antibiotics against periodontal pathogens, and has been used to suppress connective tissue breakdown during periodontal disease.5–10 Apart from antimicrobial activity, tetracyclines also exhibit additional pharmacological properties in the management of periodontal diseases.11 Inhibitory effects of tetracyclines on bone resorption have been demonstrated in both in vitro and
* Corresponding author. Tel.: +81 52 757 6742; fax: +81 52 751 2588. E-mail address: [email protected] (A. Togari). 0003–9969/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2011.02.002
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in vivo studies. In vitro, parathyroid hormone (PTH)-induced bone resorption in organ culture was inhibited by not only antibiotic tetracyclines but also a chemically modified tetracycline (CMT) without antibiotic activity.11–13 Bettany et al.14,15 demonstrated that doxycycline (DOXY) and CMT induced apoptosis in 24-h cultures of two monocyte/macrophage cell lines (U937 and RAW264 cells) and mature rabbit osteoclasts, suggesting that tetracyclines act in the apoptosis of osteoclast precursors and mature osteoclasts. In vivo, oral administration of MINO or CMT prevents trabecular bone loss in ovariectomised (OVX) rats and streptozotocin-induced diabetic rats, a model of postmenopausal osteoporosis and diabetic osteoporosis, respectively.16–18 Furthermore, both DOXY and non-antimicrobial CMT inhibited periodontal bone resorption induced by Escherichia coli endotoxin in rats.19 This evidence indicated that tetracyclines inhibit pathologically accelerated bone resorption independent of antimicrobial activity. Osteoclasts are bone-resorbing multinucleate large cells derived from the haematopoietic monocyte–macrophage lineage, having characteristics of mRNA expressions, such as tartrate-resistant acid phosphatase (TRAP), calcitonin receptor (CTR), carbonic anhydrase II (CAII), and cathepsin K.20,21 1a,25(OH)2D3, PTH, and PGE2 increase the receptor activator of nuclear factor-kB (RANK) ligand (RANKL) production in osteoblasts/stromal cells to induce the formation of osteoclast-like cells from osteoclast precursors.20,21 The precise molecular mechanisms underlying osteoclast differentiation have been clarified in recent studies by Takayanagi’s group.22–26 They showed that the binding of RANKL to RANK in osteoclast precursor cells specifically induced the nuclear factor of activated T-cells c1 (NFATc1), a key regulator of osteoclast differentiation via the tumour necrosis factor receptor-associated factor 6 (TRAF6) and c-Fos signalling pathways, and that RANKL evoked Ca2+ oscillations that led to calmodulin–calcineurin-mediated activation of NFATc1. They also demonstrated that the selective Ca2+ chelator BAPTA-AM suppressed RANKL-induced NFATc1 expression and osteoclastogenesis in bone marrow-derived monocyte/macrophage precursor cells. Chemically, tetracyclines are known as chelating agents of Ca2+ independent of their antibiotic activity.27,28 It was demonstrated that pretreatment of osteoclasts with either MINO or DOXY significantly attenuated the peak cytosolic Ca2+ response to extracellular Ca2+.27 In the present study, we hypothesise that tetracyclines including MINO probably have an inhibitory effect on osteoclastogenesis via osteoclast differential signalling pathway in osteoclast precursor cells by their Ca2+ chelating action. To clarify the hypothesis, the effects of MINO, TC, OXT or DOXY on the formation of TRAP staining positive osteoclastlike cells were examined in mouse BMCs treated with 1a,25(OH)2D3 or s-RANKL, a simple and convenient culture system for experimental osteoclastogenesis.29 Then, the effects of MINO on the mRNA expression of osteoclast phenotypes, transcriptional factors involved in osteoclast differentiation and dendritic cell-specific transmembrane protein (DC-STAMP), an essential molecule for cell–cell fusion,30–32 were also examined. Here we demonstrated that MINO inhibited s-RANKL-induced osteoclastogenesis via
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down-regulation of NFATc1 mRNA expression in mouse BMC culture.
2.
Materials and methods
2.1.
Materials
Six-week-old ddY male mice and 10-day-old Japan white male rabbits were purchased from Japan SLC Inc. (Hamamatsu, Japan). Animals were treated in accordance with the Guidelines for Animal Experiments at the School of Dentistry, Aichi-Gakuin University. Minocycline hydrochloride (MINO), tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OXT), doxycycline hydrochloride hemiethanolate hemihydrate (DOXY) and foetal calf serum (FCS) were purchased from Sigma Co. (St. Louis, MO, USA). Recombinant mouse soluble RANKL (s-RANKL) was purchased from R&D (London, UK), 1a,25(OH)2D3 from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA, USA) and alphamodified minimum essential medium (a-MEM) from Invitrogen Co. (Carlsbad, CA, USA). The CellTiter-GloTM Luminescent Cell Viability Assay, a commercial kit, was purchased from Promega Co. (Madison, WI, USA). All other chemicals were of reagent grade.
2.2. The formation of tartrate-resistant acid phosphatasepositive multinucleate osteoclast-like cells in mouse BMC culture Mouse BMCs were obtained from tibiae as previously described29 and cultured in plastic dishes in a-MEM containing FCS (10%), penicillin (100 IU/ml) and streptomycin (100 mg/ml) in humidified air with CO2 (5%) at 37 8C. To examine osteoclast formation, we cultured BMCs (1 106 cells) in a-MEM (1 ml) containing FCS (10%) and tetracyclines (2.5, 5 and 10 mM) in 24well plates for 7 days in the presence of osteoclastogenesisinducing drugs, such as 1a,25(OH)2D3 (10 nM) and s-RANKL (20 ng/ml). 1a,25(OH)2D3 and s-RANKL were used to examine if tetracyclines have some effects on osteoclastogenesis via osteoblastic function, such as RANKL production and via RANKL–RANK interaction-induced osteoclast differentiation signalling in precursor cells, respectively. To avoid aspirating all of the non-adherent BMCs, including osteoclast precursors, we replaced one-half (0.5 ml) of the supernatant of the culture medium with fresh medium every 2 days, and added drugs at the beginning of cultures and at each medium change. After 7 days in culture, adherent cells were fixed with a mixture of 10% formalin in phosphate-buffered saline and ethanol– acetone (50:50, v/v), and stained for TRAP by incubating the cells in sodium acetate buffer (0.1 M, pH 5) containing naphthol AS-MX phosphate and red violet LB salt in the presence of sodium tartrate (10 mM), as described previously.33 Cells containing 3 or more nuclei were designated as osteoclast-like cells. Moreover, to examine the bone-resorbing capability of osteoclast-like cells, we cultured BMCs in 24-well plates in which a dentine slice had been placed at the bottom. After 7 days in culture, the cells were removed from the dentine slices, and resorption pits on the slices were stained with haematoxylin.
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Table 1 – RT-PCR primers. Primers (source) TRAP (S70805) Cathepsin K (NM_00780) CAII (K00811) CTR (U18542) RANK (NM_009399) RANKL (AB008426) OPG (AB013898) M-CSF (X05010) c-FOS (V00727) PU.1 (M_32370) MITF (NM_008601) NFATc1 (NM_198429) DC-STAMP (AY517483) GAPDH (NM_002046)
Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense Sense Antisense
Sequences (50 –30 )
Product length (bp)
TTGAGGACGTGTTCTCTGACC TGGGCTGCTGACTGGCAAAGT GGCCAACTCAAGAAGAAAACT ATAGCCCACCACCAACACTGC TTGGCTGTTTTGGGCTATTTT CGCTTTGATCTTTCTATTCTT AAGATGAGGCAAACCCACGAG CCCGAGGAGCACTAACTACGC AACCTCCAGTCAGCAAGAAGT GTCACAGCCCTCAGAATCCAC GCCATTTGCACACCTCACCAT AGAATTGCCCGACCAGTTTTT CCTTGCCCTGACCACTCTTAT ACATCTATTCCACACTTTTGC AATCCCCAACGAGTCAGCAAC GGCCCCCAACAGTCAGCAAGA AGAAGGGGCAAAGTAGAGCAG TGAGAAGGGGCAGGGTGAAGG CTTCCCTTATCAAACCTTGTC AGGTGAGCTTCTTCTTGACTT AGACCTGACATGTACGACAAC TATGCAGGGCTACTGATAAAG CAACGCCCTGACCACCGATAG GGCTGCCTTCCGTCTCATAGT CTAAGGAGAAGAAAACCCTTG CAGCATAGAAGACAACAATCC ACCACAGTCCATGCCATCAC TCCACCACCCTGTTGCTGTA
249 402 354 319 373 316 407 319 361 398 397 392 332 452
Sources are GenBank accession numbers.
2.3.
Isolation of mature osteoclasts from rabbit long bones
Purified mature osteoclasts were prepared from rabbit tibia and femora as reported previously.34 Briefly, long bones from 10-dayold rabbits were minced with scissors and agitated with a vortex mixer. An aliquot of unfractionated bone cells was seeded onto 0.24% collagen gel (Nitta Gelatin, Tokyo) coated on 10-cm tissue culture dishes and incubated. Two hours later, non-adherent cells were washed off and the remaining osteoclasts were then removed from the gels with 0.1% collagenase solution (Wako Pure Chemical Co., Osaka). Isolated cells fulfilled the criteria of osteoclasts; TRAP staining-positive multinuclear cells with bone-resorbing activity and actin ring formation.
2.4.
Analysis of mRNA expression by RT-PCR
For the experiment conducted to examine mRNA expression, BMCs were cultured for 5 days in 6-cm-diameter plastic dishes in a-MEM (5 ml) containing minocycline (10 mM) in the presence of 1a,25(OH)2D3 (10 nM) or s-RANKL (20 ng/ml). For the experiment conducted to examine mRNA expression, mouse BMCs were cultured in 6-cm-diameter plastic dishes in a-MEM containing FCS (10%), penicillin (100 IU/ml), and streptomycin (100 mg/ml) in humidified air with CO2 (5%) at 37 8C. One half of the culture medium was replaced with fresh medium every 2 days. RNA was extracted from the cells by the guanidinium-thiocyanate method.35 The reverse transcription-polymerase chain reaction (RT-PCR) was performed by standard methods. Briefly, cDNA was first synthesised using random primers and Moloney murine leukaemia virus reverse
transcriptase (Gibco-BRL; Life Technologies, Grand Island, NY, USA), followed by PCR amplification using synthetic gene primers specific to mouse genes. The oligonucleotides used as primers for PCR are shown in Table 1. Each PCR amplification mixture (25 ml) contained 2.5 mg cDNA. PCR amplification was performed using the GeneAmp PCR System (Perkin Elmer/ Cetus, Norwalk, CT, USA) under the following conditions: denaturation at 95 8C for 15 s, annealing at 55 8C for 30 s, and elongation at 72 8C for 30 s. PCR products were electrophoresed on a 2% Nusive GTG agarose gel (FMC Bioproducts, Rockland, ME, USA), stained with ethidium bromide, and detected with a fluorescence imager analyser (FluorImager 575; Amersham Pharmacia Biotech, Sunnyvale, CA, USA).
2.5. Cell viabilities of BMCs and mature osteoclasts treated with minocycline Cell viabilities of BMCs were estimated using a commercial assay kit, CellTiter-GloTM Luminescent Cell Viability Assay, which is based on quantification of the ATP level as previously described.36 Briefly, mouse BMCs (103 cells) were cultured in 96-well plates for 24 h in a-MEM (100 ml) containing minocycline (0.25–20 mM), and then the ATP level as an index of the number of viable cells was measured by determining the luminescent signal. Cell viabilities in mature osteoclasts were estimated by counting the number of cells under the light microscope. Purified mature osteoclasts were seeded in 24-well plastic dishes in a-MEM (1 ml). One hour after seeding, one group (8 wells) of cells was fixed with formalin and stained for TRAP,
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Fig. 1 – Effect of minocycline on osteoclast-like cell formation induced by 1a,25(OH)2D3 in mouse BMCs. BMCs were treated with 1a,25(OH)2D3 (10 nM), or minocycline (2.5–10 mM) for 7 days. The cells were fixed and stained for TRAP activity, and then the number of TRAP-positive multinucleate osteoclast-like cells (TRAP+ MNCs) was counted. BMCs were also cultured on dentine slices under the same culture conditions. After a 7-day culture period, the dentine slices were stained with haematoxylin. Photomicrographs of TRAP+ MNCs (A) and resorption pits (B) of non-treated control culture (a), and cultures treated with 1a,25(OH)2D3 (b), or 1a,25(OH)2D3 + minocycline ((c–e) 2.5 mM, 5 mM, and 10 mM minocycline, respectively). Bar = 0.1 mm. (C) Number of TRAP+ MNCs. Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from 1a,25(OH)2D3 alone according to Tukey’s multiple comparison test.
and the number of adherent TRAP-positive osteoclasts was counted (0 h). Other osteoclasts were further cultured for 24 h in a-MEM without MINO (control) or with MINO (2–50 mM), and the numbers of adherent TRAP-positive osteoclasts were counted in the same manner.
2.6.
Statistical analysis
3.2. Effect of MINO on the mRNA expressions of osteoclast phenotypes and osteoclastogenesis-related proteins in mouse BMC culture treated with 1a,25(OH)2D3 As shown in Fig. 3, mRNA expressions of osteoclast phenotypes, such as TRAP, cathepsin K, CAII and CTR were upregulated in mouse BMCs treated with 1a,25(OH)2D3 (10 nM) alone but not 1a,25(OH)2D3 and MINO (10 mM). The expression
All data are presented as the means standard error of the mean (S.E.M.). Statistical analysis was carried out by one-way analysis of variance (Tukey’s Multiplan Comparison Tests) using Prism version 4 (GraphPad Software Inc., San Diego, CA), and p < 0.05 was considered significant.
3.
Results
3.1. Effect of tetracyclines on the formation of TRAPpositive osteoclast-like cells in mouse BMC culture treated with 1a,25(OH)2D3 As shown in Fig. 1, TRAP-positive osteoclast-like cells and resorbing pits were clearly observed in cultures treated with 1a,25(OH)2D3 (10 nM) alone. In contrast, the formation of TRAP-positive cells and resorbing pits was decreased dose dependently by the addition of MINO. No TRAP-positive cells or resorbing pits were detected in mouse BMCs treated with 1a,25(OH)2D3 and MINO (10 mM), the same as the non-treated control. As shown in Fig. 2, 1a,25(OH)2D3-induced osteoclastogenesis was dose-dependently inhibited by treatment with TC, DOXY or OXY.
Fig. 2 – Effects of tetracyclines on osteoclast-like cell formation induced by 1a,25(OH)2D3 in mouse BMCs. BMCs were treated with 1a,25(OH)2D3 (10 nM) alone, or with tetracycline, doxycycline or oxytetracycline (2.5–10 mM) for 7 days. Cells were fixed and stained for TRAP activity; and then the number of TRAP-positive multinucleate osteoclast-like cells (TRAP+ MNCs) was counted. Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from each 1a,25(OH)2D3 alone according to Tukey’s multiple comparison test.
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Fig. 3 – Effect of minocycline on the mRNA expression of TRAP, cathepsin K, CAII, CTR, RANKL, OPG and M-CSF in mouse BMC culture treated with 1a,25(OH)2D3. Total RNA for RT-PCR analysis was isolated from mouse BMCs cultured for 5 days in a-MEM with no additional agents (lane 1), 10 nM 1a,25(OH)2D3 (lane 2), or 1a,25(OH)2D3 and 10 mM MINO (lane 3). DNA size markers (fX 174/Hae III digest) are shown in the left lanes (S). Numbers in parentheses indicate cycles for PCR amplification. Results using primers specific to GAPDH are shown for comparison.
of RANKL mRNA was up-regulated in mouse BMCs treated with 1a,25(OH)2D3 in the presence or absence of MINO. In contrast, OPG and M-CSF mRNAs were constitutively expressed in BMCs, and these mRNA expressions were not influenced by 1a,25(OH)2D3 with or without MINO.
3.3. Effect of MINO on the formation of TRAP-positive osteoclast-like cells in mouse bone marrow cell cultures treated with s-RANKL As shown in Fig. 4, MINO (2.5–10 mM) suppressed s-RANKLinduced TRAP-positive osteoclast-like cell formation dose dependently.
3.4. Effect of MINO on the expressions of mRNA for NFATc1, DC-STAMP and multiple transcription factors in mouse BMC culture treated with s-RANKL As shown in Fig. 5, expressions of mRNA for NFATc1 and DCSTAMP were up-regulated by s-RANKL (20 ng/ml) alone but not by s-RANKL and MINO (10 mM). In contrast, multiple transcription factors, such as cFOS, PU.1 and MITF mRNA, were constitutively expressed in BMCs, and these mRNA expressions were not influenced by RANKL with or without MINO.
3.5. Effect of MINO on the survival of mouse BMCs and rabbit osteoclasts As shown in Fig. 6, treatment with MINO at any concentration (1.25–20 mM) did not influence the viability of BMCs (Fig. 6A), and the number of rabbit osteoclasts was significantly decreased in all groups treated with MINO (2–50 mM) or without MINO (control) by about 50% of the initial number (Fig. 6B); however, no significant differences in cell number were observed amongst all groups.
Fig. 4 – Effect of minocycline on osteoclast-like cell formation induced by s-RANKL in mouse BMCs. BMCs were treated with s-RANKL (20 ng/ml) alone or with minocycline (2.5–10 mM) for 7 days. Cells were fixed and stained for TRAP activity, and then the number of TRAPpositive multinucleate osteoclast-like cells (TRAP+ MNCs) was counted. (A) Photomicrographs of TRAP+ MNCs of cultures treated with s-RANKL (a), s-RANKL + minocycline ((b–d) 2.5 mM, 5 mM, and 10 mM minocycline, respectively). Bar = 0.1 mm. (B) Number of TRAP+ MNCs. Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from s-RANKL alone according to Tukey’s multiple comparison test.
4.
Discussion
In the initial experiments of the present study, MINO, TC, OXY and DOCY inhibited 1a,25(OH)2D3-induced osteoclastogenesis in a dose dependent manner (Figs. 1 and 2) suggesting that tetracyclines commonly have an inhibitory effect on osteoclastogenesis. Thereafter, we used MINO to elucidate the mechanism underlying inhibition of osteoclast differentiation by tetracyclines. RT-PCR analysis (Fig. 3) showed that mRNA expressions of RANKL and osteoclast phenotypes were upregulated in BMCs treated with 1a,25(OH)2D3, and the addition of MINO (10 mM) decreased mRNA expressions of osteoclast phenotypes except RANKL to the non-treated control levels with no changes in OPG and M-CSF mRNA expression. MINO also inhibited s-RANKL-induced osteoclast-like cell formation dose-dependently (Fig. 4). Most of these observations suggested that MINO inhibited osteoclastic differentiation via downstream of RANKL–RANK interaction in osteoclast precursor cells.37 As shown in Fig. 5, NFATc1 and DC-STAMP mRNA expressions in BMCs up-regulated by s-RANKL were reduced by the addition of MINO without any changes in mRNA expressions of c-FOS, PU.1 and MITF, suggesting that MINO might inhibit NFATc1 and DC-STAMP mRNA expressions via an independent pathway from those mediated signalling pathway in osteoclast precursor cells.38
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Fig. 5 – Effect of minocycline on the expressions of mRNA for NFATc1, DC-STAMP, c-FOS, PU.1 and MITF in mouse BMCs culture treated with s-RANKL. Total RNA for RT-PCR analysis was isolated from mouse BMCs cultured for 5 days in a-MEM with no additional agents (lane 1), 20 ng/ml s-RANKL (lane 2), or s-RANKL and 10 mM minocycline (lane 3). DNA size markers (fX 174/Hae III digest) are shown in the left lane (S). Numbers in parentheses indicate cycles for PCR amplification. Results using primers specific to GAPDH are shown for comparison.
Our results are well consistent with the study of Takayanagi’s group22–26 already demonstrated that RANKL-induced Ca2+ oscillations result in an activation of NFATc1 and this effect was inhibited by a Ca2+ chelator BAPTA-AM. They also
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demonstrated that calcineurin inhibitors, cyclosporine A and FK506, suppressed RANKL-induced osteoclastogenesis.23 Judging from these previous observations and present results, it is possible that Ca2+ chelator tetracyclines block the calcium– calmodulin–calcineurin signalling pathway to inhibit NFATc1 mRNA expression and osteoclast-like cell formation in mouse BMCs treated with s-RANKL. Previously, Bettany et al.14,15 demonstrated that tetracycline derivatives induced apoptosis in osteoclastic cell lines and rabbit osteoclasts. However, treatment with MINO did not show any influence on the viability of BMCs and isolated rabbit osteoclasts (Fig. 6). Differences between the results of our study and Bettany’s studies14,15 may have been caused by differences in the experimental conditions, such as cells and drug concentrations. In Bettany’s studies14,15 DOXY and CMT at a concentration of 25–50 mg/ml (about 50–100 mM) induced the apoptosis of monocyte and macrophage cell lines, whereas in the present study MINO at 20 mM did not induce the apoptosis of mouse BMCs; however, in rabbit osteoclasts, almost the same range of drug concentrations was used in Bettany’s studies (20–40 mM DOXY) and the present study (20– 50 mM MINO), suggesting a difference in the cell toxicity of DOXY and MINO. Further studies are needed to elucidate differences in their cell toxicity. A therapeutically useful property of tetracyclines for the treatment of periodontal disease was found previously. Golub et al.4,5 demonstrated that tetracyclines inhibited the activity of mammalian collagenase regardless of antimicrobial efficacy, and Ramamurthy et al.19 showed that tetracyclines with or without antimicrobial activity inhibited matrix metalloproteinase (MMP)-mediated periodontal bone resorption in rats. These investigations indicate that tetracyclines can inhibit the breakdown of connective tissues mediated by excessive collagenolytic activity. In the present study, we demonstrated that MINO inhibited s-RANKL induced osteoclast-like cell formation in mouse BMCs via the down-regulation of NFATc1
Fig. 6 – Effect of minocycline on the survival of mouse BMCs and rabbit osteoclasts. (A) Mouse BMCs (103 cells) were seeded in 96-well plates and cultured for 24 h in a-MEM without minocycline (control) or with minocycline (1.25–20 mM). Cell viabilities were estimated by quantification of the ATP concentration using a commercial assay kit, the CellTiter-GloTM Luminescent Cell Viability. Values are given as a percentage of the control, and are the mean W S.E.M. (n = 8). (B) Rabbit osteoclasts isolated from the femur and tibia were seeded in 24-well plates in a-MEM. After seeding, osteoclasts adhering to the plate bottom (8 wells) were fixed with formalin, stained for TRAP, and counted as the initial number of osteoclasts (0 h). Other osteoclasts were further cultured for 24 h in a-MEM without minocycline (control) or with minocycline (2– 50 mM), and the number of surviving adherent TRAP-positive osteoclasts was counted (24 h). Values are the mean W S.E.M. (n = 8). ***p < 0.001, statistically different from 0 h and +++p < 0.001, statistically different from 24 h control according to Tukey’s multiple comparison test.
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mRNA expression. This effect of MINO may also contribute to the management of periodontal diseases with pathological alveolar bone resorption.
5.
Conclusion
MINO inhibited RANKL-induced osteoclastogenesis via the down-regulation of NFATc1 mRNA expressions in osteoclast precursor cells, suggesting the usefulness of tetracyclines against pathologically accelerated bone resorption. Funding: None. Competing interests: None declared. Ethical approval: Not required.
Acknowledgment This study was partly supported by a grant-in-aid from the Strategic Research AGU-Platform Formation (2008-2012).
references
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