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Inhibition of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ)
Immunology Letters 142 (2012) 34–40
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Inhibition of receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ) Erdenezaya Odkhuu, Naoki Koide, Abedul Haque, Bilegtsaikhan Tsolmongyn, Yoshikazu Naiki, Shoji Hashimoto, Takayuki Komatsu, Tomoaki Yoshida, Takashi Yokochi ∗ Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Article history: Received 16 September 2011 Received in revised form 9 November 2011 Accepted 5 December 2011 Available online 13 December 2011 Keywords: Pyrroloquinoline quinine Receptor activator of nuclear factor-B ligand Osteoclast Nuclear factor of activated T cells c-Fos Type I interferon receptor
a b s t r a c t The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-B ligand (RANKL)induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKLstimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-B and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN--mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN- signaling due to augmented IFNAR expression. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Osteoclasts are bone-resorbing multinuclear cells derived from hematopoietic stem cells [1]. The interaction between receptor activator of nuclear factor (NF)-B (RANK) and RANK ligand (RANKL) is essential for osteoclast differentiation and activation [2,3]. The binding of RANKL and RANK on osteoclast progenitor cells triggers the activation of tumor necrosis factor receptor-associated factor 6 (TRAF6) [4] and subsequently the activation of NF-B and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and stress-activated
Abbreviations: AP1, activated protein 1; CREB, cAMP responsive element binding protein; ERK, extracellular signal-regulated kinase; IFNAR, type I interferon receptor; IFN, interferon; iNOS, inducible type of nitric oxide synthase; JAK1, janus kinase 1; MAPK, mitogen-activated protein kinase; NFATc1, nuclear factor of activated T cells 1; NF-B, nuclear factor-B; PQQ, pyrroloquinoline quinine; RANKL, receptor activator of nuclear factor-B ligand; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; SD, standard deviation; STAT1, signal transducer and activator of transcription 1; TRAF6, tumor necrosis factor receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase. ∗ Corresponding author. Tel.: +81 561 62 3311x2269; fax: +81 561 63 9187. E-mail address: [email protected] (T. Yokochi). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.12.001
protein kinase/c-Jun N-terminal kinase (SAPK/JNK) [5,6]. Nuclear factor of activated T cells (NFATc1) is a downstream transcription factor in the RANKL/RANK signal pathway and plays a crucial role on the osteoclastogenesis [3,7]. NFATc1 as a key molecule of osteoclastogenesis induces a series of osteoclast-specific genes, including cathepsin K, tartrate-resistant acid phosphatase (TRAP), calcitonin receptor and osteoclast-associated receptor [7,8]. c-Fos is also an essential transcription factor for osteoclastogenesis [9] and positively regulates osteoclastogenesis via NFATc1 activation. On the other hand, c-Fos induces interferon (IFN)- [10] as a strong inhibitor of osteoclastogenesis and negatively regulates osteoclastogenesis through type I interferon receptor (IFNAR) [11]. Pyrroloquinoline quinine (PQQ) possesses a variety of functions ranging from classical vitamin to anti- and pro-oxidant [12,13]. PQQ is widely dispersed in animals [13–15]. Dietary in vivo experiments reveal that PQQ deficiency exhibits growth impairment, compromised immune responsiveness and abnormal reproductive performance [16,17]. PQQ might be involved in bone metabolism via nitric oxide biosynthesis [12]. However, there is no report on the role of PQQ on RANKL-induced osteoclast formation. Recently, PQQ has been reported to regulate several intracellular signaling pathways, including Ras-related ERK1/2 activation [18], CREBdependent mitochondriogenesis [19], and JAK/STAT activation [13].
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It is of interest to clarify the effect of PQQ on RANKL-induced osteoclast formation, especially RANKL-mediated intracellular signal pathway. In this study we aimed to clarify if and how PQQ affected RANKL-induced osteoclast formation in RAW 264.7 murine macrophage-like cells or mouse peritoneal macrophages. Here, we report a putative inhibitory mechanism of RANKL-induced osteoclast formation by PQQ.
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stained for TRAP activity at day 6 or 8, respectively. Osteoclast like cells were counted as previously described [21,22]. 2.5. TRAP staining
2. Materials and methods
A TRAP staining kit was obtained from Primary Cell (Sapporo, Japan). TRAP staining was carried out according to the manufacturer’s instruction as described elsewhere [23]. The images were taken with a digital camera attached to the microscope.
2.1. Materials
2.6. Immunoblotting
Recombinant murine soluble RANKL and PQQ disodium salt was purchased from PeproTech EC (Princeton, NJ, USA) and Wako (Osaka, Japan), respectively. A series of antibodies to p65 NF-B, p38, SAPK/JNK, ERK1/2, STAT1 and their phosphorylated forms, and c-Jun, c-Fos and rabbit IgG were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies to TRAF6 and IFNAR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and an antibody to NFATc1 and JAK1 were from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mouse IgG antibody was obtained from Pierce (Rockford, IL, USA).
Immunoblotting was performed as described previously [24]. Briefly, cells were cultured with or without PQQ in the presence of RANKL (100 ng/ml) for indicated time and then lysed in a lysis buffer. Cytoplasmic and nuclear fractions of proteins were isolated by a nuclear extract kit (Active Motif, Carlsbad, CA, USA). Protein concentrations were measured using bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL, USA) and cell lysates were diluted using 2× sample buffer and boiled for 5 min. Equal amounts of protein (0.02 mg) were analyzed by polyacrylamide gel electrophoresis under reducing conditions and transferred to membranes by electroblotting. After blocking with 5% skim milk in phosphate-buffered saline, membranes were treated with appropriately diluted antibodies. Resulting immune complexes were reacted with 1:2000 or 1:500 dilutions of horseradish peroxidaseconjugated goat anti-rabbit and anti-mouse antibody, respectively. Finally, labeled antigen bands were detected with a chemiluminescence reagent, supersignal west dura (Pierce) and analyzed using an AE6955 light capture system with a CS analyser (Atto, Tokyo, Japan). For reprobing, the membranes were stripped with the restore western blot striping buffer (Thermo Scientific, Rockford, IL, USA) for 15 min and treated with corresponding antibodies. Prestained protein markers from BioDynamics Laboratory (Tokyo, Japan) were used to estimate molecular mass.
2.2. Cell culture The murine macrophage cell line, RAW 264.7, was obtained from Riken Cell Bank (Tsukuba, Japan) and maintained in ␣-MEM medium containing 5% heat inactivated fetal calf serum (GibcoInvitrogen, Carlsbad, CA, USA), antibiotics, antimycotics and non essential amino acid (Invitrogen, Carlsbad, CA, USA) at 37 ◦ C under 5% CO2 . Peritoneal cells were collected from BALB/c mice (Japan SLC, Hamamatsu, Japan) 3 days after an intraperitoneal injection of 1 ml sterile 10% thioglycollate (Remel, Kansas City, MO, USA). Thioglycollate-elicited peritoneal cells were obtained by washing out the peritoneal cavity with DMEM medium (Gibco-BRL, Gaithersburg, MD). The cells were suspended in DMEM medium containing 10% fetal calf serum and incubated for 3 h. After removal of nonadherent cells, adherent cells as peritoneal macrophages were further cultured for 24 h and used for osteoclast formation. The animal experiments were carried out following the Guide for Care and Use of Laboratory Animals, Aichi Medical University. 2.3. Cell viability Cell viability was assessed by a MTT assay and trypan blue dye exclusion test, respectively. RAW 264.7 cells were treated with various concentrations of PQQ in a 96-well plate for 24 h. Cell viability was determined by MTT activity using 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Dojindo, Kumamoto, Japan) according to the instruction. The optical density at 570 nm was determined with a microplate reader. Trypan blue dye exclusion tested 6 day after osteoclastogenesis as previously described [20]. All measurements were corrected for the interference of PQQ at this wavelength.
2.7. Laser flow cytometry RAW 264.7 cells were seeded at 1 × 106 cells/ml in a 35-mm culture dish for 12 h and then treated with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24 h. The cells were incubated with a FITC conjugated antibody to F4/80 (Biolegend, San Diego, CA, USA) or an isotype-matched irrelevant antibody for 30 min. The cell surface expression of F4/80 was analyzed by a fluorescence activated cells sorter (BD FACS Calibur, San Jose, CA, USA) and the fluorescence intensity is shown in a log scale. 2.8. Statistical analysis Statistical analysis was performed using Student’s t-test and values of P < 0.05 were considered significant. All experiments were performed independently at least three times. Data represent the mean value of triplicates ± SD. 3. Results
2.4. Osteoclast formation 3.1. The effect of PQQ on RANKL-induced osteoclast formation RAW 264.7 cells were cultured in 96-well dishes at a density of 5000 cells per well and were allowed to adhere overnight. The cells were cultured with 0.25 ml fresh media containing RANKL at 100 ng/ml. Peritoneal macrophage were treated with RANKL at 100 ng/ml and macrophage-colony stimulating factor (M-CSF) at 50 ng/ml (R and D systems, Minneapolis, MN, USA) for 8 days. The culture medium including reagents was replaced every 3 days after the cultivation. RAW 264.7 cells and peritoneal macrophages were
Since RAW 264.7 macrophage-like cells differentiate into osteoclast-like cells as described elsewhere [25], the effect of PQQ on RANKL-induced osteoclast formation was examined using the cells. RAW 264.7 cells were incubated with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 6 days. A number of TRAP-positive and multinuclear cells identified as osteoclasts appeared in the culture with the addition of RANKL.
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On the other hand, PQQ at 10 M significantly inhibited RANKLinduced osteoclast formation (Fig. 1A). PQQ at 0.1 M did not prevent the appearance of TRAP-positive cells but markedly inhibited that of multinuclear cells. The inhibitory action of PQQ against RANKL-induced osteoclast formation was also examined by mouse peritoneal macrophages (Fig. 1B). Peritoneal macrophages were incubated with various concentrations of PQQ in the presence RANKL (100 ng/ml) and M-CSF (50 ng/ml). PQQ inhibited the osteoclast formation in peritoneal macrophages as well as RAW 264.7 cells. The cytotoxicity of PQQ against RAW 264.7 cells and mouse peritoneal macrophages were shown with a MTT assay in Fig. 1C. No cytotoxicity of PQQ was also confirmed by a trypan blue dye exclusion test (data not shown). 3.2. The effect of PQQ on the expression of the F4/80 macrophage maturation marker on RANKL-treated RAW 264.7 cells The expression of a macrophage maturation marker F4/80 is reported to be downregulated during RANKL-induced osteoclastic differentiation [26]. Therefore, the effect of PQQ on the F4/80 surface expression on RANKL-treated cells was examined. RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24 h. The cells were stained with FITC-conjugated anti-F4/80 antibody and the fluorescence intensity indicating the F4/80 expression was determined by FACS analysis. PQQ prevented the reduction in the F4/80 expression although RANKL significantly reduced the fluorescence intensity (Fig. 2). 3.3. Characterization of an inhibitory action of PQQ on RANKL-induced osteoclast formation The dose-dependent effect of PQQ on the number of osteoclasts appearing was examined (Fig. 3A). RAW 264.7 cells were incubated with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 6 days and the number of osteoclasts appearing was determined. PQQ at 0.1 M significantly reduced the number of osteoclasts appearing at day 6. The osteoclast number was markedly inhibited in the presence of PQQ at 10 M. In addition, PQQ was confirmed to exhibit no cytotoxicity against RAW 264.7 cells with a MTT assay and trypan blue dye exclusion test. Next, PQQ was added into the cultures at various stages of the culture period to clarify the crucial stage for inhibition of RANKL-induced osteoclast formation. The culture period was subdivided into three stages (days 0–2, days 2–4, and days 4–6) as described elsewhere [27]. The addition of PQQ significantly inhibited RANKL-induced osteoclast formation at any stage of the culture period (Fig. 3B). 3.4. The effect of PQQ on RANKL-induced NFATc1 and c-Fos expression NFATc1 is known as a key transcription factor triggering RANKLinduced osteoclast formation [8,28]. Therefore, the effect of PQQ on RANKL-induced NFATc1 activation was examined (Fig. 4A). RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 24 h. RANKL definitely induced NFATc1 expression whereas PQQ significantly inhibited it. The inhibition was roughly dependent on the concentrations of PQQ. Since RANKL leads to the NFATc1 expression via the activation of AP1 consisting of c-Fos and c-Jun [6,29,30], the effect of PQQ on the RANKL-induced c-Fos expression was also examined (Fig. 4A). PQQ inhibited the expression of c-Fos as well as NFATc1. Further, the time course of RANKL-induced NFATc1 and c-Fos expression was followed in the presence or absence of PQQ (Fig. 4B). PQQ inhibited RANKL-induced NFATc1 and c-Fos expression 24, 48 and 72 h after the RANKL treatment.
Fig. 1. Effect of PQQ on RANKL-induced osteoclast formation. (A) RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6 days. (B) Peritoneal macrophages were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 8 days. TRAP staining was carried out to identify osteoclasts. Scale bar indicates 100 M. Values are shown as Mean ± SD. *P < 0.05 vs RANKL + M-CSF. (C) The cytotoxicity of PQQ against RAW 264.7 cells and peritoneal macrophages was determined by a MTT assay. RAW 264.7 cells and peritoneal cells were treated with RANKL and RANKL + M-CSF, respectively.
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Fig. 2. Effect of PQQ on the expression of F4/80 macrophage maturation marker. RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24 h. The cells were stained with an anti-F4/80 antibody conjugated with FITC. The fluorescent intensity is expressed on a log scale. A typical result of three independent experiments is shown.
3.5. The effect of PQQ on RANKL-induced NF-B and MAPKs activation RANKL activates TRAF6 via RANK [4] and subsequently triggers the activation of NF-B and a series of MAPKs [31]. The effect of PQQ on RANKL-induced NF-B and MAPKs activation was examined (Fig. 5). RAW 264.7 cells were cultured with or without PQQ in the presence of RANKL (100 ng/ml) for 30 min as an early stage (Fig. 5A). PQQ (10 M) alone as well as RANKL induced the phosphorylation of p38, ERK1/2, JNK, IKK and p65 NF-B. Combined treatment with PQQ and RANKL also resulted in their phosphorylation. There was no significant difference in the phosphorylation of those signaling molecules among the experimental groups. Further, RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6, 12 or 18 h as a late
Fig. 4. Effect of PQQ on RANKL-induced NFATc1 and c-Fos expression. (A) RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 24 h. (B) RAW 264.7 cells were cultured with PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24, 48 or 72 h. Immunoblotting bands were quantified by densitometry and normalized against -actin. A typical result of three independent experiments is shown.
stage (Fig. 5B). PQQ did not affect RANKL-induced NF-B and MAPKs phosphorylation at 6, 12 and 18 h, suggesting that PQQ did not affect RANKL-induced NF-B and MAPKs activation. RANKL activates NFB and MAPKs via TRAF6 [4]. Therefore, the effect of PQQ on the TRAF6 expression was examined (Fig. 5A and B). PQQ did not affect the TRAF6 expression 30 min, 6 h, 12 h or 18 h after the treatment, excluding an impaired TRAF6 expression. 3.6. The effect of PQQ on the IFN-ˇ signaling in RANKL-stimulated cells
Fig. 3. Inhibitory actions of PQQ on RANKL-induced osteoclast formation. RAW 264.7 cells were cultured with various concentrations of PQQ for 24 h (A) or with PQQ (10 M) for various exposure time periods of RANKL (100 ng/ml) (B). Values are shown as Mean ± SD. **P < 0.01, *P < 0.05 vs RANKL alone.
IFN- is produced in response to RANKL and inhibits osteoclastogenesis via down-regulation of c-Fos protein level [10,11]. In fact, this study demonstrated reduced expression of c-Fos (Fig. 4). Therefore, a possibility was raised that PQQ might inhibit the osteoclast formation through affecting RANKL-induced IFN- production. Therefore, the effect of PQQ on RANKL-induced IFN- production was examined (Fig. 6A). RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 8 h and the IFN- production was determined with an enzyme-linked immunosorbent assay. RANKL induced the IFN production whereas PQQ did not significantly affect it. Next, we examined the effect of PQQ on the expression of IFNAR (Fig. 6B). PQQ significantly augmented the expression of IFNAR at 6, 12, 24 and 48 h when RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml). Moreover, the effect of PQQ on the expression of downstream signaling molecules of IFNAR, such as JAK1 and STAT1, was examined (Fig. 6C). PQQ significantly enhanced phosphorylation of STAT1 (pSTAT) as well
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Fig. 5. Effect of PQQ on RANKL-induced NF-B and MAPKs activation. (A) RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 30 min. (B) RAW 264.7 cells were cultured with PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6, 12 or 18 h. A typical result of three independent experiments is shown.
as augmented expression of JAK1 and STAT1 in the presence of RANKL at 24 h. Furthermore, the expression of an inducible type of nitric oxide synthase (iNOS), which is IFN- inducible protein, was markedly enhanced by PQQ. The effect of PQQ on nuclear translocation of pSTAT1 was also examined in order to confirm the activation of JAK/STAT signaling. PQQ significantly augmented the nuclear translocation of pSTAT1 at 24 h (Fig. 6D).
4. Discussion In the present study we demonstrate that PQQ inhibits RANKLinduced osteoclast formation via impaired activation of NFATc1, which is a key transcription factor regulating RANKL-induced osteoclast differentiation [8,28]. The RANKL-induced NFATc1 expression is mediated by the activation of AP1 consisting of c-Fos
Fig. 6. Effect of PQQ on the IFN- signaling in RANKL-stimulated cells. (A) RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 8 h and the level of IFN- in the culture supernatant was determined with an enzyme-linked immunosorbent assay. Values are shown as Mean ± SD. (B) RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6, 12, 24 or 48 h. (C and D) The total protein (C) or cytoplasmic and nuclear fraction (D) were isolated 24 h after stimulation of RANKL (100 ng/ml) with or without PQQ (10 M) and the expression of a series of signaling molecules were analyzed with immunoblotting. Bands were quantified by densitometry and normalized against -actin or CREB. A typical result of three independent experiments is shown.
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and c-Jun [6,29,30]. The present study demonstrates the reduced c-Fos expression in PQQ-treated cells. Therefore, the reduced c-Fos expression is suggested to cause the impaired activation of NFATc1, followed by the inhibition of RANKL-induced osteoclast formation. The c-Fos expression is regulated by IFN- as a negative feedback mechanism [10,11]. IFN- is produced in response to RANKL and triggers the JAK/STAT signaling via IFNAR [32]. Subsequently, IFN-inducible genes activated by JAK/STAT signaling inhibit the c-Fos expression [10]. Therefore, the augmented IFN- signaling causes reduced c-Fos expression and further impaired NFATc1 activation. Surprisingly, the present study demonstrates that PQQ augments the expression of IFNAR but not the production of IFN-, suggesting that PQQ enhances the activation of JAK1 and STAT1 via augmented IFNAR expression. The augmented JAK1/STAT1 signaling may reduce the c-Fos expression, leading to impaired NFATc1 expression. In fact, PQQ repletion is reported to upregulate the JAK/STAT pathways [33]. Further, the number of osteoclasts notably increases in the bone of the IFN-−/− [10] or IFNAR−/− [32] mice, compared to the wild-type mice. Therefore, PQQ is possible to enhance the IFN--dependent JAK/STAT pathway via augmented IFNAR expression. However, it is still unknown how PQQ augments the expression of IFNAR. In addition, RANKL-induced osteoclast formation is negatively regulated via several other mechanisms. Osteoprotegerin as the decoy receptor of RANKL is known to inhibit the osteoclastogenesis [34]. IFN-␥ inhibits it via impaired TRAF6 activation [35]. RANKL-induced NFATc1 activation is dependent on both the TRAF6-NF-B and the MAPK-AP1 (c-Fos) [3,11]. The binding of RANKL to RANK induces the trimerization of RANK and TRAF6 [3], which leads to the activation of NF-B and a series of MAPKs [31]. In this study, PQQ does not affect the activation of TRAF6, NF-B and MAPKs at the early and late stage after RANKL stimulation. However, PQQ has been recently reported to influence the MAPK pathway. MAPKKK12, an activator of the JNK/SAPK, and p38␣ MAPK are upregulated by PQQ repletion and deprivation [33], respectively. The discrepancy might be due to the difference in the experimental system between the in vitro culture system and the in vivo system. A macrophage maturation marker, F4/80, is not expressed on osteoclast [36]. Moreover, the expression of a macrophage maturation marker F4/80 is reported to be downregulated during RANKL-induced osteoclast differentiation [26]. In fact, RANKL stimulation reduces the F4/80 expression on RAW 264.7 cells whereas PQQ prevents the reduction in the surface expression of F4/80 macrophage maturation marker. It is consistent with the idea that PQQ inhibits the RANKL-induced osteoclast differentiation in RAW 264.7 cells and keeps the cells as macrophages. No alteration in the surface expression of F4/80 also supports the inhibition of osteoclast differentiation by PQQ. For animals and humans, there has been constant exposure to PQQ [14]. In animals, PQQ is reported to participate in a range of biological functions with apparent survival benefits (e.g., improved neonatal growth [16] and reproductive performance [17]). There are also benefits from PQQ supplementation related to cognitive, immune, and antioxidant functions, as well as protection from cardiac and neurological ischemic events [13,37]. Although PQQ is not currently viewed as a vitamin, PQQ could be defined as vital to life [13]. We have for the first time demonstrated the regulatory action of PQQ on RANKL-induced osteoclastogenesis. In conclusion, PQQ inhibits RANKL-induced osteoclastogenesis through the inactivation of NFATc1 in RAW 264.7 macrophage-like cells. PQQ augments the IFNAR expression and subsequently the IFN- signaling. The enhanced IFN- signaling causes the impaired NFATc1 activation via reduced c-Fos expression. PQQ present ubiquitously in mammal tissues may control osteoclastic resorption through regulating osteoclastogenesis.
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Conflict of interest statement The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and a grant of Strategic Research Foundation Grantaided Project for Private Universities from Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT), 2011–2015 (S1101027). We are grateful to K. Takahashi and A. Morikawa for the technical assistance.
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[24] Koide N, Naiki Y, Morikawa A, Tumurkhuu G, Dagvadorj J, Noman AS, et al. Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7. Microbiol Immunol 2009;53:295–300. [25] Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci USA 1999;96:3540–5. [26] Lean JM, Matsuo K, Fox SW, Fuller K, Gibson FM, Draycott G, et al. Osteoclast lineage commitment of bone marrow precursors through expression of membrane-bound TRANCE. Bone 2000;27:29–40. [27] Sakai S, Takaishi H, Matsuzaki K, Kaneko H, Furukawa M, Miyauchi Y, et al. 1Alpha, 25-dihydroxy vitamin D3 inhibits osteoclastogenesis through IFN-betadependent NFATc1 suppression. J Bone Miner Metab 2009;27:643–52. [28] 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;3:889–901. [29] Ikeda F, Nishimura R, Matsubara T, Tanaka S, Inoue J, Reddy SV, et al. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J Clin Invest 2004;114:475–84. [30] Wagner EF, Eferl R. Fos/AP-1 proteins in bone and the immune system. Immunol Rev 2005;208:126–40. [31] Matsumoto M, Sudo T, Saito T, Osada H, Tsujimoto M. Involvement of p38 mitogen-activated protein kinase signaling pathway in osteoclastogenesis
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Inhibition of receptor activator of nuclear factor-B ligand (RANKL)-induced osteoclast formation by pyrroloquinoline quinine (PQQ) Erdenezaya Odkhuu, Naoki Koide, Abedul Haque, Bilegtsaikhan Tsolmongyn, Yoshikazu Naiki, Shoji Hashimoto, Takayuki Komatsu, Tomoaki Yoshida, Takashi Yokochi ∗ Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
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Article history: Received 16 September 2011 Received in revised form 9 November 2011 Accepted 5 December 2011 Available online 13 December 2011 Keywords: Pyrroloquinoline quinine Receptor activator of nuclear factor-B ligand Osteoclast Nuclear factor of activated T cells c-Fos Type I interferon receptor
a b s t r a c t The effect of pyrroloquinoline quinine (PQQ) on receptor activator of nuclear factor-B ligand (RANKL)induced osteoclast formation was examined using RAW 264.7 macrophage-like cells. RANKL led to the formation of osteoclasts identified as tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in the culture of RAW 264.7 cells. However, PQQ inhibited the appearance of osteoclasts and prevented the decrease of F4/80 macrophage maturation marker on RANKL-stimulated cells, suggesting a preventive action of PQQ on RANKL-induced osteoclast differentiation. PQQ inhibited the activation of nuclear factor of activated T cells (NFATc1), a key transcription factor of osteoclastogenesis, in RANKLstimulated cells. On the other hand, PQQ did not inhibit the signaling pathway from RANK/RANKL binding to NFATc1 activation, including NF-B and mitogen-activated protein kinases (MAPKs). PQQ augmented the expression of type I interferon receptor (IFNAR) and enhanced the IFN--mediated janus kinase (JAK1) and signal transducer and activator of transcription (STAT1) expression. Moreover, PQQ reduced the expression level of c-Fos leading to the activation of NFATc1. Taken together, PQQ was suggested to prevent RANKL-induced osteoclast formation via the inactivation of NFATc1 by reduced c-Fos expression. The reduced c-Fos expression might be mediated by the enhanced IFN- signaling due to augmented IFNAR expression. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Osteoclasts are bone-resorbing multinuclear cells derived from hematopoietic stem cells [1]. The interaction between receptor activator of nuclear factor (NF)-B (RANK) and RANK ligand (RANKL) is essential for osteoclast differentiation and activation [2,3]. The binding of RANKL and RANK on osteoclast progenitor cells triggers the activation of tumor necrosis factor receptor-associated factor 6 (TRAF6) [4] and subsequently the activation of NF-B and mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and stress-activated
Abbreviations: AP1, activated protein 1; CREB, cAMP responsive element binding protein; ERK, extracellular signal-regulated kinase; IFNAR, type I interferon receptor; IFN, interferon; iNOS, inducible type of nitric oxide synthase; JAK1, janus kinase 1; MAPK, mitogen-activated protein kinase; NFATc1, nuclear factor of activated T cells 1; NF-B, nuclear factor-B; PQQ, pyrroloquinoline quinine; RANKL, receptor activator of nuclear factor-B ligand; SAPK/JNK, stress-activated protein kinase/c-Jun N-terminal kinase; SD, standard deviation; STAT1, signal transducer and activator of transcription 1; TRAF6, tumor necrosis factor receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase. ∗ Corresponding author. Tel.: +81 561 62 3311x2269; fax: +81 561 63 9187. E-mail address: [email protected] (T. Yokochi). 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.12.001
protein kinase/c-Jun N-terminal kinase (SAPK/JNK) [5,6]. Nuclear factor of activated T cells (NFATc1) is a downstream transcription factor in the RANKL/RANK signal pathway and plays a crucial role on the osteoclastogenesis [3,7]. NFATc1 as a key molecule of osteoclastogenesis induces a series of osteoclast-specific genes, including cathepsin K, tartrate-resistant acid phosphatase (TRAP), calcitonin receptor and osteoclast-associated receptor [7,8]. c-Fos is also an essential transcription factor for osteoclastogenesis [9] and positively regulates osteoclastogenesis via NFATc1 activation. On the other hand, c-Fos induces interferon (IFN)- [10] as a strong inhibitor of osteoclastogenesis and negatively regulates osteoclastogenesis through type I interferon receptor (IFNAR) [11]. Pyrroloquinoline quinine (PQQ) possesses a variety of functions ranging from classical vitamin to anti- and pro-oxidant [12,13]. PQQ is widely dispersed in animals [13–15]. Dietary in vivo experiments reveal that PQQ deficiency exhibits growth impairment, compromised immune responsiveness and abnormal reproductive performance [16,17]. PQQ might be involved in bone metabolism via nitric oxide biosynthesis [12]. However, there is no report on the role of PQQ on RANKL-induced osteoclast formation. Recently, PQQ has been reported to regulate several intracellular signaling pathways, including Ras-related ERK1/2 activation [18], CREBdependent mitochondriogenesis [19], and JAK/STAT activation [13].
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It is of interest to clarify the effect of PQQ on RANKL-induced osteoclast formation, especially RANKL-mediated intracellular signal pathway. In this study we aimed to clarify if and how PQQ affected RANKL-induced osteoclast formation in RAW 264.7 murine macrophage-like cells or mouse peritoneal macrophages. Here, we report a putative inhibitory mechanism of RANKL-induced osteoclast formation by PQQ.
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stained for TRAP activity at day 6 or 8, respectively. Osteoclast like cells were counted as previously described [21,22]. 2.5. TRAP staining
2. Materials and methods
A TRAP staining kit was obtained from Primary Cell (Sapporo, Japan). TRAP staining was carried out according to the manufacturer’s instruction as described elsewhere [23]. The images were taken with a digital camera attached to the microscope.
2.1. Materials
2.6. Immunoblotting
Recombinant murine soluble RANKL and PQQ disodium salt was purchased from PeproTech EC (Princeton, NJ, USA) and Wako (Osaka, Japan), respectively. A series of antibodies to p65 NF-B, p38, SAPK/JNK, ERK1/2, STAT1 and their phosphorylated forms, and c-Jun, c-Fos and rabbit IgG were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies to TRAF6 and IFNAR were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and an antibody to NFATc1 and JAK1 were from BD Biosciences (Franklin Lakes, NJ, USA). Anti-mouse IgG antibody was obtained from Pierce (Rockford, IL, USA).
Immunoblotting was performed as described previously [24]. Briefly, cells were cultured with or without PQQ in the presence of RANKL (100 ng/ml) for indicated time and then lysed in a lysis buffer. Cytoplasmic and nuclear fractions of proteins were isolated by a nuclear extract kit (Active Motif, Carlsbad, CA, USA). Protein concentrations were measured using bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL, USA) and cell lysates were diluted using 2× sample buffer and boiled for 5 min. Equal amounts of protein (0.02 mg) were analyzed by polyacrylamide gel electrophoresis under reducing conditions and transferred to membranes by electroblotting. After blocking with 5% skim milk in phosphate-buffered saline, membranes were treated with appropriately diluted antibodies. Resulting immune complexes were reacted with 1:2000 or 1:500 dilutions of horseradish peroxidaseconjugated goat anti-rabbit and anti-mouse antibody, respectively. Finally, labeled antigen bands were detected with a chemiluminescence reagent, supersignal west dura (Pierce) and analyzed using an AE6955 light capture system with a CS analyser (Atto, Tokyo, Japan). For reprobing, the membranes were stripped with the restore western blot striping buffer (Thermo Scientific, Rockford, IL, USA) for 15 min and treated with corresponding antibodies. Prestained protein markers from BioDynamics Laboratory (Tokyo, Japan) were used to estimate molecular mass.
2.2. Cell culture The murine macrophage cell line, RAW 264.7, was obtained from Riken Cell Bank (Tsukuba, Japan) and maintained in ␣-MEM medium containing 5% heat inactivated fetal calf serum (GibcoInvitrogen, Carlsbad, CA, USA), antibiotics, antimycotics and non essential amino acid (Invitrogen, Carlsbad, CA, USA) at 37 ◦ C under 5% CO2 . Peritoneal cells were collected from BALB/c mice (Japan SLC, Hamamatsu, Japan) 3 days after an intraperitoneal injection of 1 ml sterile 10% thioglycollate (Remel, Kansas City, MO, USA). Thioglycollate-elicited peritoneal cells were obtained by washing out the peritoneal cavity with DMEM medium (Gibco-BRL, Gaithersburg, MD). The cells were suspended in DMEM medium containing 10% fetal calf serum and incubated for 3 h. After removal of nonadherent cells, adherent cells as peritoneal macrophages were further cultured for 24 h and used for osteoclast formation. The animal experiments were carried out following the Guide for Care and Use of Laboratory Animals, Aichi Medical University. 2.3. Cell viability Cell viability was assessed by a MTT assay and trypan blue dye exclusion test, respectively. RAW 264.7 cells were treated with various concentrations of PQQ in a 96-well plate for 24 h. Cell viability was determined by MTT activity using 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Dojindo, Kumamoto, Japan) according to the instruction. The optical density at 570 nm was determined with a microplate reader. Trypan blue dye exclusion tested 6 day after osteoclastogenesis as previously described [20]. All measurements were corrected for the interference of PQQ at this wavelength.
2.7. Laser flow cytometry RAW 264.7 cells were seeded at 1 × 106 cells/ml in a 35-mm culture dish for 12 h and then treated with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24 h. The cells were incubated with a FITC conjugated antibody to F4/80 (Biolegend, San Diego, CA, USA) or an isotype-matched irrelevant antibody for 30 min. The cell surface expression of F4/80 was analyzed by a fluorescence activated cells sorter (BD FACS Calibur, San Jose, CA, USA) and the fluorescence intensity is shown in a log scale. 2.8. Statistical analysis Statistical analysis was performed using Student’s t-test and values of P < 0.05 were considered significant. All experiments were performed independently at least three times. Data represent the mean value of triplicates ± SD. 3. Results
2.4. Osteoclast formation 3.1. The effect of PQQ on RANKL-induced osteoclast formation RAW 264.7 cells were cultured in 96-well dishes at a density of 5000 cells per well and were allowed to adhere overnight. The cells were cultured with 0.25 ml fresh media containing RANKL at 100 ng/ml. Peritoneal macrophage were treated with RANKL at 100 ng/ml and macrophage-colony stimulating factor (M-CSF) at 50 ng/ml (R and D systems, Minneapolis, MN, USA) for 8 days. The culture medium including reagents was replaced every 3 days after the cultivation. RAW 264.7 cells and peritoneal macrophages were
Since RAW 264.7 macrophage-like cells differentiate into osteoclast-like cells as described elsewhere [25], the effect of PQQ on RANKL-induced osteoclast formation was examined using the cells. RAW 264.7 cells were incubated with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 6 days. A number of TRAP-positive and multinuclear cells identified as osteoclasts appeared in the culture with the addition of RANKL.
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On the other hand, PQQ at 10 M significantly inhibited RANKLinduced osteoclast formation (Fig. 1A). PQQ at 0.1 M did not prevent the appearance of TRAP-positive cells but markedly inhibited that of multinuclear cells. The inhibitory action of PQQ against RANKL-induced osteoclast formation was also examined by mouse peritoneal macrophages (Fig. 1B). Peritoneal macrophages were incubated with various concentrations of PQQ in the presence RANKL (100 ng/ml) and M-CSF (50 ng/ml). PQQ inhibited the osteoclast formation in peritoneal macrophages as well as RAW 264.7 cells. The cytotoxicity of PQQ against RAW 264.7 cells and mouse peritoneal macrophages were shown with a MTT assay in Fig. 1C. No cytotoxicity of PQQ was also confirmed by a trypan blue dye exclusion test (data not shown). 3.2. The effect of PQQ on the expression of the F4/80 macrophage maturation marker on RANKL-treated RAW 264.7 cells The expression of a macrophage maturation marker F4/80 is reported to be downregulated during RANKL-induced osteoclastic differentiation [26]. Therefore, the effect of PQQ on the F4/80 surface expression on RANKL-treated cells was examined. RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24 h. The cells were stained with FITC-conjugated anti-F4/80 antibody and the fluorescence intensity indicating the F4/80 expression was determined by FACS analysis. PQQ prevented the reduction in the F4/80 expression although RANKL significantly reduced the fluorescence intensity (Fig. 2). 3.3. Characterization of an inhibitory action of PQQ on RANKL-induced osteoclast formation The dose-dependent effect of PQQ on the number of osteoclasts appearing was examined (Fig. 3A). RAW 264.7 cells were incubated with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 6 days and the number of osteoclasts appearing was determined. PQQ at 0.1 M significantly reduced the number of osteoclasts appearing at day 6. The osteoclast number was markedly inhibited in the presence of PQQ at 10 M. In addition, PQQ was confirmed to exhibit no cytotoxicity against RAW 264.7 cells with a MTT assay and trypan blue dye exclusion test. Next, PQQ was added into the cultures at various stages of the culture period to clarify the crucial stage for inhibition of RANKL-induced osteoclast formation. The culture period was subdivided into three stages (days 0–2, days 2–4, and days 4–6) as described elsewhere [27]. The addition of PQQ significantly inhibited RANKL-induced osteoclast formation at any stage of the culture period (Fig. 3B). 3.4. The effect of PQQ on RANKL-induced NFATc1 and c-Fos expression NFATc1 is known as a key transcription factor triggering RANKLinduced osteoclast formation [8,28]. Therefore, the effect of PQQ on RANKL-induced NFATc1 activation was examined (Fig. 4A). RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 24 h. RANKL definitely induced NFATc1 expression whereas PQQ significantly inhibited it. The inhibition was roughly dependent on the concentrations of PQQ. Since RANKL leads to the NFATc1 expression via the activation of AP1 consisting of c-Fos and c-Jun [6,29,30], the effect of PQQ on the RANKL-induced c-Fos expression was also examined (Fig. 4A). PQQ inhibited the expression of c-Fos as well as NFATc1. Further, the time course of RANKL-induced NFATc1 and c-Fos expression was followed in the presence or absence of PQQ (Fig. 4B). PQQ inhibited RANKL-induced NFATc1 and c-Fos expression 24, 48 and 72 h after the RANKL treatment.
Fig. 1. Effect of PQQ on RANKL-induced osteoclast formation. (A) RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6 days. (B) Peritoneal macrophages were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) and M-CSF (50 ng/ml) for 8 days. TRAP staining was carried out to identify osteoclasts. Scale bar indicates 100 M. Values are shown as Mean ± SD. *P < 0.05 vs RANKL + M-CSF. (C) The cytotoxicity of PQQ against RAW 264.7 cells and peritoneal macrophages was determined by a MTT assay. RAW 264.7 cells and peritoneal cells were treated with RANKL and RANKL + M-CSF, respectively.
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Fig. 2. Effect of PQQ on the expression of F4/80 macrophage maturation marker. RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24 h. The cells were stained with an anti-F4/80 antibody conjugated with FITC. The fluorescent intensity is expressed on a log scale. A typical result of three independent experiments is shown.
3.5. The effect of PQQ on RANKL-induced NF-B and MAPKs activation RANKL activates TRAF6 via RANK [4] and subsequently triggers the activation of NF-B and a series of MAPKs [31]. The effect of PQQ on RANKL-induced NF-B and MAPKs activation was examined (Fig. 5). RAW 264.7 cells were cultured with or without PQQ in the presence of RANKL (100 ng/ml) for 30 min as an early stage (Fig. 5A). PQQ (10 M) alone as well as RANKL induced the phosphorylation of p38, ERK1/2, JNK, IKK and p65 NF-B. Combined treatment with PQQ and RANKL also resulted in their phosphorylation. There was no significant difference in the phosphorylation of those signaling molecules among the experimental groups. Further, RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6, 12 or 18 h as a late
Fig. 4. Effect of PQQ on RANKL-induced NFATc1 and c-Fos expression. (A) RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 24 h. (B) RAW 264.7 cells were cultured with PQQ (10 M) in the presence of RANKL (100 ng/ml) for 24, 48 or 72 h. Immunoblotting bands were quantified by densitometry and normalized against -actin. A typical result of three independent experiments is shown.
stage (Fig. 5B). PQQ did not affect RANKL-induced NF-B and MAPKs phosphorylation at 6, 12 and 18 h, suggesting that PQQ did not affect RANKL-induced NF-B and MAPKs activation. RANKL activates NFB and MAPKs via TRAF6 [4]. Therefore, the effect of PQQ on the TRAF6 expression was examined (Fig. 5A and B). PQQ did not affect the TRAF6 expression 30 min, 6 h, 12 h or 18 h after the treatment, excluding an impaired TRAF6 expression. 3.6. The effect of PQQ on the IFN-ˇ signaling in RANKL-stimulated cells
Fig. 3. Inhibitory actions of PQQ on RANKL-induced osteoclast formation. RAW 264.7 cells were cultured with various concentrations of PQQ for 24 h (A) or with PQQ (10 M) for various exposure time periods of RANKL (100 ng/ml) (B). Values are shown as Mean ± SD. **P < 0.01, *P < 0.05 vs RANKL alone.
IFN- is produced in response to RANKL and inhibits osteoclastogenesis via down-regulation of c-Fos protein level [10,11]. In fact, this study demonstrated reduced expression of c-Fos (Fig. 4). Therefore, a possibility was raised that PQQ might inhibit the osteoclast formation through affecting RANKL-induced IFN- production. Therefore, the effect of PQQ on RANKL-induced IFN- production was examined (Fig. 6A). RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 8 h and the IFN- production was determined with an enzyme-linked immunosorbent assay. RANKL induced the IFN production whereas PQQ did not significantly affect it. Next, we examined the effect of PQQ on the expression of IFNAR (Fig. 6B). PQQ significantly augmented the expression of IFNAR at 6, 12, 24 and 48 h when RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml). Moreover, the effect of PQQ on the expression of downstream signaling molecules of IFNAR, such as JAK1 and STAT1, was examined (Fig. 6C). PQQ significantly enhanced phosphorylation of STAT1 (pSTAT) as well
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Fig. 5. Effect of PQQ on RANKL-induced NF-B and MAPKs activation. (A) RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 30 min. (B) RAW 264.7 cells were cultured with PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6, 12 or 18 h. A typical result of three independent experiments is shown.
as augmented expression of JAK1 and STAT1 in the presence of RANKL at 24 h. Furthermore, the expression of an inducible type of nitric oxide synthase (iNOS), which is IFN- inducible protein, was markedly enhanced by PQQ. The effect of PQQ on nuclear translocation of pSTAT1 was also examined in order to confirm the activation of JAK/STAT signaling. PQQ significantly augmented the nuclear translocation of pSTAT1 at 24 h (Fig. 6D).
4. Discussion In the present study we demonstrate that PQQ inhibits RANKLinduced osteoclast formation via impaired activation of NFATc1, which is a key transcription factor regulating RANKL-induced osteoclast differentiation [8,28]. The RANKL-induced NFATc1 expression is mediated by the activation of AP1 consisting of c-Fos
Fig. 6. Effect of PQQ on the IFN- signaling in RANKL-stimulated cells. (A) RAW 264.7 cells were cultured with various concentrations of PQQ in the presence of RANKL (100 ng/ml) for 8 h and the level of IFN- in the culture supernatant was determined with an enzyme-linked immunosorbent assay. Values are shown as Mean ± SD. (B) RAW 264.7 cells were cultured with or without PQQ (10 M) in the presence of RANKL (100 ng/ml) for 6, 12, 24 or 48 h. (C and D) The total protein (C) or cytoplasmic and nuclear fraction (D) were isolated 24 h after stimulation of RANKL (100 ng/ml) with or without PQQ (10 M) and the expression of a series of signaling molecules were analyzed with immunoblotting. Bands were quantified by densitometry and normalized against -actin or CREB. A typical result of three independent experiments is shown.
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and c-Jun [6,29,30]. The present study demonstrates the reduced c-Fos expression in PQQ-treated cells. Therefore, the reduced c-Fos expression is suggested to cause the impaired activation of NFATc1, followed by the inhibition of RANKL-induced osteoclast formation. The c-Fos expression is regulated by IFN- as a negative feedback mechanism [10,11]. IFN- is produced in response to RANKL and triggers the JAK/STAT signaling via IFNAR [32]. Subsequently, IFN-inducible genes activated by JAK/STAT signaling inhibit the c-Fos expression [10]. Therefore, the augmented IFN- signaling causes reduced c-Fos expression and further impaired NFATc1 activation. Surprisingly, the present study demonstrates that PQQ augments the expression of IFNAR but not the production of IFN-, suggesting that PQQ enhances the activation of JAK1 and STAT1 via augmented IFNAR expression. The augmented JAK1/STAT1 signaling may reduce the c-Fos expression, leading to impaired NFATc1 expression. In fact, PQQ repletion is reported to upregulate the JAK/STAT pathways [33]. Further, the number of osteoclasts notably increases in the bone of the IFN-−/− [10] or IFNAR−/− [32] mice, compared to the wild-type mice. Therefore, PQQ is possible to enhance the IFN--dependent JAK/STAT pathway via augmented IFNAR expression. However, it is still unknown how PQQ augments the expression of IFNAR. In addition, RANKL-induced osteoclast formation is negatively regulated via several other mechanisms. Osteoprotegerin as the decoy receptor of RANKL is known to inhibit the osteoclastogenesis [34]. IFN-␥ inhibits it via impaired TRAF6 activation [35]. RANKL-induced NFATc1 activation is dependent on both the TRAF6-NF-B and the MAPK-AP1 (c-Fos) [3,11]. The binding of RANKL to RANK induces the trimerization of RANK and TRAF6 [3], which leads to the activation of NF-B and a series of MAPKs [31]. In this study, PQQ does not affect the activation of TRAF6, NF-B and MAPKs at the early and late stage after RANKL stimulation. However, PQQ has been recently reported to influence the MAPK pathway. MAPKKK12, an activator of the JNK/SAPK, and p38␣ MAPK are upregulated by PQQ repletion and deprivation [33], respectively. The discrepancy might be due to the difference in the experimental system between the in vitro culture system and the in vivo system. A macrophage maturation marker, F4/80, is not expressed on osteoclast [36]. Moreover, the expression of a macrophage maturation marker F4/80 is reported to be downregulated during RANKL-induced osteoclast differentiation [26]. In fact, RANKL stimulation reduces the F4/80 expression on RAW 264.7 cells whereas PQQ prevents the reduction in the surface expression of F4/80 macrophage maturation marker. It is consistent with the idea that PQQ inhibits the RANKL-induced osteoclast differentiation in RAW 264.7 cells and keeps the cells as macrophages. No alteration in the surface expression of F4/80 also supports the inhibition of osteoclast differentiation by PQQ. For animals and humans, there has been constant exposure to PQQ [14]. In animals, PQQ is reported to participate in a range of biological functions with apparent survival benefits (e.g., improved neonatal growth [16] and reproductive performance [17]). There are also benefits from PQQ supplementation related to cognitive, immune, and antioxidant functions, as well as protection from cardiac and neurological ischemic events [13,37]. Although PQQ is not currently viewed as a vitamin, PQQ could be defined as vital to life [13]. We have for the first time demonstrated the regulatory action of PQQ on RANKL-induced osteoclastogenesis. In conclusion, PQQ inhibits RANKL-induced osteoclastogenesis through the inactivation of NFATc1 in RAW 264.7 macrophage-like cells. PQQ augments the IFNAR expression and subsequently the IFN- signaling. The enhanced IFN- signaling causes the impaired NFATc1 activation via reduced c-Fos expression. PQQ present ubiquitously in mammal tissues may control osteoclastic resorption through regulating osteoclastogenesis.
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Conflict of interest statement The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.
Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and a grant of Strategic Research Foundation Grantaided Project for Private Universities from Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT), 2011–2015 (S1101027). We are grateful to K. Takahashi and A. Morikawa for the technical assistance.
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