Mod Rheumatol (2013) 23:1133–1139 DOI 10.1007/s10165-012-0809-4
ORIGINAL ARTICLE
Role of FK506 binding protein 5 (FKBP5) in osteoclast differentiation Miho Kimura • Tatsuo Nagai • Reiko Matsushita Atsushi Hashimoto • Toshiyuki Miyashita • Shunsei Hirohata
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Received: 24 October 2012 / Accepted: 29 November 2012 / Published online: 21 December 2012 Ó Japan College of Rheumatology 2012
Abstract Objectives We previously disclosed the enhanced expression of FK506 binding protein 5 (FKBP5) messenger RNA (mRNA) in bone marrow (BM) CD34? cells in rheumatoid arthritis (RA), in which systemic osteoporosis takes place. Since BM CD34? cells are precursors of osteoclasts, it is possible that FKBP5 overexpression might lead to osteoporosis by affecting osteoclastogenesis. We therefore explore the influences of FKBP5 in osteoclast differentiation. Methods Stable transfectants of RAW264.7 overexpressing murine FKBP5 gene were established. Osteoclast differentiation was induced by receptor activator of nuclear factor kappa B (NF-jB) ligand and was evaluated by tartrate-resistant acid phosphatase (TRAP) staining and pit formation assay. Results FKBP5 transfectants of RAW264.7 generated higher numbers of TRAP-positive multinucleated cells with increased activity of pit formation on calcium phosphate-coated culture slides than mock transfectants. The enhancement of osteoclast differentiation of FKBP5 transfectants was only partially inhibited by N-acetyl L-cysteine. Finally, glucocorticoid enhanced FKBP5
M. Kimura T. Nagai R. Matsushita A. Hashimoto S. Hirohata (&) Department of Rheumatology and Infectious Diseases, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan e-mail:
[email protected] T. Miyashita Department of Molecular Genetics, Graduate School of Medical Science, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan
mRNA expression as well as osteoclast differentiation of RAW264.7 cells in a dose-dependent manner. Conclusions These results indicate that FKBP5 promotes osteoclast differentiation by a mechanism distinct from NF-jB activation. Moreover, the data suggest that FKBP5 might play a role in bone destruction and development of osteoporosis in RA as well as in glucocorticoid-induced osteoporosis. Keywords Rheumatoid arthritis Osteoporosis Glucocorticoid RANKL NF-jB
Introduction Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by the destruction of bone and cartilage in the joints [1]. It should be noted that the prevalence of systemic osteoporosis, such as vertebral osteoporosis, is increased in RA patients even if they do not receive glucocorticoids [2]. Thus, RA itself is regarded as a risk factor for osteoporosis [3]. Although several factors have been proposed, the precise mechanism for the development of systemic osteoporosis in RA has not been delineated. Bone-resorbing osteoclasts, cells formed by the fusion of mononuclear progenitors of the monocyte/macrophage lineage, play essential roles in regulating bone morphogenesis and remodeling. It is well established that two molecules are required for osteoclastogenesis: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) [4, 5]. RANKL, a member of the tumor necrosis factor (TNF) cytokine superfamily, is critical for differentiation and fusion of precursors into mature osteoclasts [6–10]. Thus, the binding of RANKL to its receptor RANK induced activation of
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TNF receptor-associated factor 6 (TRAF6), which is linked to the nuclear factor jB (NF-jB) and c-Jun N-terminal kinase (JNK) pathways [11–13]. Accumulating evidence suggests that abnormalities in bone marrow (BM) play a role in the pathogenesis of RA [14, 15]. Interestingly, Nakamura et al. [16] recently disclosed that several genes including FK506 binding protein 5 (FKBP5) are upregulated in BM mononuclear cells from RA patients. We have also demonstrated that the expression of mRNA for FKBP5 in BM CD34? cells was higher in RA than that in osteoarthritis (OA) [17]. Recently, it has been reported that FKBP5 is an essential cofactor for the inhibitor of kappaB kinase alpha (IKKa), which phosphorylates the inhibitor of NF-jB alpha (IjBa) [18], resulting in nuclear translocation and activation of NF-jB [19]. Since BM CD34? cells are pluripotent hematopoietic stem cells, overexpression of FKBP5 can result in multiple effects. Previous studies have also revealed that BM CD34? cells differentiate also into osteoclasts in the presence of M-CSF and RANKL [20]. It is therefore possible that overexpression of FKBP5 in BM CD34? cells affects differentiation of osteoclasts, thus accounting for the development of systemic osteoporosis in RA patients. The current studies were undertaken to examine the role of FKBP5 in osteoclast differentiation. For this purpose, we established FKBP5 transfectant clones of mouse macrophage cell line RAW264.7, which has been widely utilized for analysis of RANKL-induced osteoclast formation [21, 22].
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pA-Neo was transfected into RAW264.7 cells using the AmaxaTM 4D-Nucleofector (Lonza, Basel, Switzerland) (FKBP5 RAW). pEGFP-C1 (Clontech, Mountain View, CA, USA) was used as control (mock RAW). G418 (600 lg/mL; Roche, Basel, Switzerland) was added to the cells 48 h after transfection to select stably transfected clones. Western blot analysis Cell lysates of mock RAW and FKBP5 RAW clones were collected in cell lysis buffer [1 % Triton X-100, 10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA)] with protease inhibitor [1 mM phenylmethylsulfonyl fluoride (PMSF), 18 lg/mL aprotinin, 50 lg/mL leupeptin, 1 mM benzamidine, 0.7 lg/mL pepstatin]. Electrophoresis was performed on 10 % sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) gel and transferred onto PVDF plus membrane using Horizblot 2M semi-dry blotting system (Atto, Tokyo, Japan). The membrane was incubated with goat polyclonal antibodies against FKBP51 (F-13; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by incubation with peroxidase-conjugated donkey anti-goat Ig (Santa Cruz). The blots were developed with enhanced chemiluminescence (ECL; GE Healthcare-Amersham, Buckinghamshire, UK) and filmed by LAS-400miniEPUV (Fujifilm, Tokyo, Japan). RT-PCR
Materials and methods Cell culture The murine macrophage cell line RAW264.7, obtained from Dainippon Pharma (Kyoto, Japan), was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Nikken Bio Medical Laboratory, Kyoto, Japan) supplemented with 10 % fetal bovine serum (FBS; SigmaAldrich, St. Louis, MO, USA), 100 unit/mL penicillin G, 100 lg/mL streptomycin, and 0.3 mg/mL L-glutamine as complete medium. Establishment of stable transfectants overexpressing FKBP5
cDNA was prepared from 1 lg of total RNA isolated from purified RAW264.7 cells incubated with various concentrations of prednisolone (Shionogi, Osaka, Japan) for overnight. Real-time quantitative PCR of FKBP5 was performed with a LightCycler 4.1 (Roche Diagnostics, Lewes, UK) and SYBR Premix Ex Taq II (Takara Bio, Shiga, Japan) with the following primers: (forward) GTG TCCATGCATCAAGCCAAAG, and (reverse) AGCCAGC CACGTTCAATCATC (MA063705, Takara Bio). Amplification was performed according to the standard protocol recommended by the manufacturer. FKBP5 PCR results were calibrated to the copy number of glyceraldehyde 3-phosphate dehydrogenase (GAPDH, MA050371; Takara Bio) obtained from the same cDNA samples. Induction of osteoclast differentiation
The open reading frame for murine FKBP5 was polymerase chain reaction (PCR)-amplified from a complementary DNA (cDNA) clone (clone ID: 4237766) obtained from Open Biosystems (Thermo Fisher Scientific, Lafayette, CO) and subcloned into pCAGGS-IRES-eGFP-pA (Unitech, Chiba, Japan). Then, pCAGGS-FKBP5-IRES-eGFP-
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FKBP5 RAW clones or mock RAW were plated at 1 9 104 per well in 8-well chamber plates (Nunc, Roskilde, Denmark) with 200 lL of culture media supplemented with 30 ng/mL RANKL (PeproTech, Rocky Hill, NJ, USA) for 5 days, after which osteoclast differentiation was assessed.
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In some experiments, N-acetyl L-cysteine (NAC; SigmaAldrich) was added to the cultures. TRAP staining To identify osteoclast differentiation, standard TRAP staining procedure was performed according to the manufacturer’s instructions at the termination of the culture (Wako TRAP/ALP staining kit; Wako, Osaka, Japan). The number of TRAP-positive multinucleated cells containing more than 3 nuclei was determined under light microscopy. Measurement of bone resorption activity To estimate bone resorption activity, FKBP5 RAW clones or mock RAW were cultured for 14 days with RANKL (40 ng/mL) and M-CSF (PeproTech) (10 ng/mL) on BD BioCoatTM OsteologicTM multitest slides, which consisted of submicron synthetic calcium phosphate thin films coated onto culture vessels (Becton–Dickinson, Bedford, MA, USA). After the cultures, the cells were removed using 6 % NaOCl and 5.2 % NaCl, and the area of the resorption pits formed in 10 fields of view for mock RAW or for each FKBP5 RAW clone was calculated under light microscopy at 409 magnification using WinROOF software (Mitani, Tokyo, Japan). Statistical analysis Student’s t test or 1-way analysis of variance (ANOVA, Tukey’s multiple-comparison test) were used where appropriate.
Results Osteoclast differentiation of FKBP5-transfected RAW264.7 cells (FKBP5 RAW) To investigate the effect of FKBP5 on osteoclastogenesis, we used murine macrophage cell line RAW264.7, which is known to differentiate into osteoclasts by RANKL. RAW264.7 cells were stably transfected with plasmid containing FKBP5 (FKBP5 RAW) or mock plasmid (mock RAW). The expression of FKBP5 protein in various clones was examined by Western blotting. As can be seen in Fig. 1, different clones of FKBP5 RAW were found to express FKBP5 strongly, and were used in further experiments. FKBP5 RAW and mock RAW were cultured under the stimulation of RANKL for 5 days. As shown in Fig. 2a, b, FKBP5 RAW showed significant increase in the formation of TRAP-positive multinucleated cells compared with mock RAW after 5 days of cultures. To confirm that these
Fig. 1 Protein expression of FKBP5 in RAW264.7 cells transfected with FKBP5 gene. RAW264.7 cells were stably transfected with plasmid containing FKBP5 (FKBP5 RAW) or mock plasmid (mock RAW). The expression of FKBP5 protein in various clones was examined by Western blotting. Three clones that showed high expression of FKBP5 (expressed as FKBP5 RAW clones no. 1, 3, 7) were selected for further experiments, and mock RAW was used as control. Lane 1 mock RAW clone, lanes 2–9 FKBP5 RAW clones
TRAP-positive multinucleated cells possess the characteristics of osteoclast, resorption assay was carried out. FKBP5 RAW or mock RAW were cultured with RANKL and M-CSF on calcium phosphate-coated culture plates for 14 days. As can be seen in Fig. 3a, b, FKBP5 RAW formed significantly larger areas of resorption on the bottom of the plate compared with mock RAW, confirming that TRAPpositive multinucleated cells induced from FKBP5 RAW upregulated bone resorption activity. Activation of NF-jB transcription factor is an essential step for osteoclast differentiation [18, 19, 21–23]. In addition, FKBP5 has been shown to facilitate the phosphorylation of IkBa, resulting in nuclear translocation of NF-jB [19]. To investigate the mechanism of FKBP5mediated enhancement of osteoclast differentiation, we tested the effect of NAC. As shown in Fig. 4, osteoclast formation was markedly inhibited by NAC in mock RAW. However, NAC significantly, but only modestly, suppressed osteoclast differentiation in FKBP5 RAW. The results indicate that the upregulation of osteoclastogenesis in FKBP5 RAW cannot be accounted for by increased activation of NF-jB. Glucocorticoid induced FKBP5 overexpression and enhanced osteoclast differentiation of RAW264.7 cells Previous studies disclosed that glucocorticoid upregulated the expression of FKBP5 in human lymphoblastic cells, prostate cancer cells, and lung cancer cells [24–26]. Consistently, prednisolone upregulated the expression of mRNA for FKBP5 in RAW264.7 cells in a dose–response manner (Fig. 5a). More importantly, prednisolone also enhanced the differentiation of TRAP-positive multinucleated cells from RAW264.7 cells in the presence of RANKL in a dose–response manner (Fig. 5b). The dose– response effect on FKBP5 expression was almost identical to that on the differentiation of TRAP-positive multinucleated cells. The results therefore strongly suggest that glucocorticoid increases osteoclast differentiation from
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1136 Fig. 2 Effect of FKBP5 overexpression on the differentiation of osteoclasts of RAW264.7 cells. a FKBP5 RAW or mock RAW clones (1 9 104/well) were incubated with RANKL (30 ng/mL) for 5 days, and the morphological changes were examined under light microscopy (TRAP staining, original magnification 940). b The numbers of TRAPpositive cells with 3 or more nuclei (arrows) were counted and calculated. Results are shown as mean ± standard deviation (SD) of the mean values of 4 independent experiments. Statistical significance was evaluated by one-way ANOVA with Tukey’s multiple-comparison test
Fig. 3 Effect of FKBP5 overexpression on the differentiation of osteoclasts of RAW264.7 cells. FKBP5 RAW or mock RAW (1 9 104/well) were cultured for 14 days with RANKL (40 ng/mL) and M-CSF (10 ng/mL) on BD BioCoatTM OsteologicTM multitest slides, after which bone resorption activity was examined. a A representative picture of resorption under light microscopy is shown (original magnification 940). b Resorption area was determined and quantitated using WinROOF software. Mean values obtained from 10 random fields from 3 wells of mock RAW (30 fields) and from 10 random fields from each well of 3 different FKBP5 RAW (30 fields) were calculated. Results are shown as mean ± standard error of the mean (SEM) of the mean values of 3 independent experiments. Statistical significance was evaluated by one-tailed Student’s t test
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RANKL-stimulated RAW264.7 cells through upregulation of FKBP5 expression.
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Fig. 4 Effect of N-acetyl L-cysteine (NAC) on the differentiation of osteoclasts of RAW264.7 cells. FKBP5 RAW or mock RAW were cultured with RANKL (30 ng/mL) in the presence or absence of NAC (1 mM) for 5 days followed by TRAP staining. The numbers of TRAP-positive cells with 3 or more nuclei were counted and calculated as mean value of mock RAW clones and that of FKBP5 RAW clones. Results are shown as mean ± SEM of the 3 independent experiments. Statistical significance was evaluated by one-way ANOVA with Tukey’s multiple-comparison test
previously disclosed that the expression of mRNA for FKBP5 in BM CD34? cells was upregulated in RA compared with that in OA [17]. On the other hand, the prevalence of systemic osteoporosis has been found to be higher in RA patients [3]. However, the mechanism for the systemic osteoporosis of RA has not been fully understood. Osteoporosis results from increased osteoclast activity. Since CD34? cells are the progenitors of osteoclasts [20, 27], it was possible that FKBP5 overexpression could lead to increased osteoclast generation. In fact, in the present study, the overexpression of FKBP5 in RAW264.7 cells resulted in increased generation of TRAP-positive multinucleated osteoclasts, expressing higher bone resorption activity. The data therefore strongly suggest that enhanced FKBP5 expression in BM CD34? cells might be involved in the development of systemic osteoporosis in RA as well as in the bone destruction. Recent studies reported that FKBP5 is an essential cofactor for IKKa, which phosphorylates IkBa [18], resulting in proteosome-mediated degradation of IkBa, and nuclear translocation and activation of NF-jB [19]. However, in the present study, an inhibitor of NF-jB (NAC) only modestly suppressed osteoclast differentiation in FKBP5 RAW. Therefore, it is suggested that the enhanced generation of osteoclasts from FKBP5 RAW should involve mechanisms other than NF-jB activation. Notably, it has been reported that RANKL induces the activation of NF-jB and stress-activated protein kinase (SAPK)/JNK through TRAF6 signaling and subsequently the activation of the transcription factor NFATc1 [28–32]. It has been also shown that a series of mitogen-activated protein kinases (MAPKs) are essential for osteoclast differentiation
Fig. 5 Effect of glucocorticoid on osteoclast differentiation and FKBP5 mRNA expression of RAW264.7 cells. a RAW264.7 cells were treated with various concentrations of prednisolone for overnight, and FKBP5 mRNA expression was examined by RTPCR. The ratio of the copy number of FKBP5 mRNA to that of GAPDH mRNA was calculated. Results are shown as mean ± SEM
of 3 independent experiments. b RAW264.7 cells (1 9 104/well) were incubated with RANKL (30 ng/mL) in the presence of various concentrations of prednisolone for 5 days followed by TRAP staining. The number of TRAP-positive multinucleated cells was counted. Results are shown as mean ± SEM of 3 independent experiments. Statistical significance was evaluated by paired-sample t test
Discussion It has been well appreciated that abnormality in BM plays an important role in the pathogenesis of RA [16]. We
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and activation [33–35]. Therefore, NFATc1 and MAPKs could be other candidates for targets of FKBP5. On the other hand, recent studies have disclosed that 2 transcriptional repressors, B cell lymphoma 6 (Bcl6) and B lymphocyte-induced maturation protein-1 (Blimp1), play a critical role in osteoclast differentiation [36]. Thus, overexpression of Bcl6, which suppresses NFATc1, inhibited osteoclastogenesis in vitro. By contrast, Bcl6-deficient mice showed accelerated osteoclast differentiation. Moreover, it has been shown that Bcl6 is a direct target of Blimp1 and that mice lacking Blimp1 in osteoclasts present osteopetrosis caused by impaired osteoclastogenesis due to Bcl6 upregulation [36]. It is therefore possible that FKBP5 could affect the Blimp1–Bcl6–osteoclastic molecule axis. Further studies are required to clarify this point. Previous studies revealed that glucocorticoid induced the expression of FKBP5 in several cells or tissues in vivo and in vitro [24–26]. Consistently, glucocorticoid enhanced the expression of mRNA for FKBP5 in RAW264.7 cells in the present study. More importantly, glucocorticoid also upregulated the generation of osteoclasts from RAW264.7 cells at the same doses that enhanced the expression of FKBP5 mRNA. Since the overexpression of FKBP5 in RAW264.7 cells has also been demonstrated to have direct effects on their differentiation into osteoclasts, these results support the conclusion that glucocorticoid-induced osteoporosis is mediated at least in part by the overexpression of FKBP5. The effects of FKBP5 in human cells need to be explored to examine whether FKBP5 might be a therapeutic target for human osteoporosis. FKBP5 belongs to the immunophilin protein family, which plays an important role in immunoregulation and basic cellular processes through affecting protein folding and trafficking. It has been shown that nuclear translocation of the glucocorticoid receptor was delayed by FKBP5 in mammalian cells [37]. On the other hand, recent studies have demonstrated that apoptosis and cell cycle arrest of osteoblastic MC3T3-E1 cells are mediated by activation of glucocorticoid receptor in vitro [38]. Moreover, it has been also disclosed that selective glucocorticoid receptor modulation by Compound A, a novel glucocorticoid receptor modulator, maintains bone mineral density in mice in vivo [39]. It is thus likely that FKBP5 can inhibit the suppressive effects of glucocorticoid on osteoblast activity. Further studies are therefore required for a complete understanding of the role of FKBP5 in bone metabolism. In summary, the results of the present study demonstrate that FKBP5 has a direct positive effect on the differentiation of osteoclasts through a mechanism distinct from NF-jB activation. Moreover, the data also indicate that glucocorticoid also upregulates the differentiation of osteoclasts through the enhanced expression of FKBP5. Further studies to delineate the precise role of FKBP5 in
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the signal transduction would be important for an understanding of its role in the pathogenesis of RA as well as osteoporosis. Acknowledgments The authors wish to thank Ms. Terumi Mizuno for her excellent technical assistance. This work was supported by a Parents’ Association Grant from Kitasato University School of Medicine and grants from Astellas Pharma Inc., Tokyo, and Eisai Co., Ltd., Tokyo, Japan. Conflict of interest
None.
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