Tenuigenin inhibits RANKL-induced osteoclastogenesis by down-regulating NF-κB activation and suppresses bone loss in vivo

Biochemical and Biophysical Research Communications 466 (2015) 615e621

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Tenuigenin inhibits RANKL-induced osteoclastogenesis by down-regulating NF-kB activation and suppresses bone loss in vivo Shuo Yang a, b, Xianan Li b, Liang Cheng a, Hongwei Wu b, Can Zhang a, Kanghua Li a, * a

Department of Orthopedic Surgery, The Xiangya Hospital of Central South University, Changsha, Hunan 410008, PR China Department of Orthopedics, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, Changsha, Hunan 410012, PR China

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 September 2015 Accepted 17 September 2015 Available online 21 September 2015

Tenuigenin, a major active component of polygala tenuifolia root, has been used to treat patients with insomnia, dementia, and neurosis. In this study, we aimed to investigate the effects of tenuigenin on osteoclastogenesis and clarify the possible mechanism. We showed that tenuigenin inhibited receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclast differentiation and bone resorption without cytotoxicity, which was further demonstrated by reduced osteoclast specific gene expression such as TRAP, c-Src, ATP6v0d2, etc. Moreover, the inhibitory effect of tenuigenin was associated with impaired NF-kB activity owing to delayed degradation/regeneration of IkBa and inhibition of p65 nuclear translocation. Consistent with the in vitro results, micro-ct scanning and analysis data showed that tenuigenin suppressed RANKL-induced bone loss in an animal model. Taken together, our data demonstrate that tenuigenin inhibit osteoclast formation and bone resorption both in vitro and in vivo, and comprise a potential therapeutic alternative for osteoclast-related disorders such as osteoporosis and cancer-induced bone destruction. © 2015 Elsevier Inc. All rights reserved.

Keywords: Tenuigenin Osteoclasts Osteolysis RANKL NF-kB

1. Introduction Bone is a rigid yet dynamic organ in a continuous state of remodeling, whose metabolic balance relies on osteoclast and osteoblast. Particularly, osteoclast is the only bone-resorbing cell derived from precursors of the monocyte-macrophage lineage [1]. Over activation of osteoclasts is related with lots of osteolytic bone disorders, including osteoporosis, cancer-induced osteolysis, aseptic loosening after prosthetic replacement, etc [1]. RANKL (receptor activator of NF-kB ligand), a member of the tumor necrosis factor (TNF) receptoreligand family, is the most crucial factor for osteoclasts differentiation both in vivo and in vitro [2,3]. RANKL ablation results in significant retard in osteoclastogenesis and severe osteopetrosis [4]. In the process of osteoclast differentiation, RANKL binds to its receptor RANK, results in TNF receptor associated factor (TRAF) adapter proteins recruitment and NF-kB signaling activation, leading to specific genes expression that have DNA-binding sites unique for NF-kB. NF-kB signaling is essential for both osteoclast differentiation and survival [5e7]. The

* Corresponding author. E-mail address: [email protected] (K. Li). http://dx.doi.org/10.1016/j.bbrc.2015.09.093 0006-291X/© 2015 Elsevier Inc. All rights reserved.

phosphorylation of Inhibitor kBs (IkB) is the prerequisite to NF-kB cascade activation, which is regulated by the IkB kinase (IKK) complex consists of IKK alpha (IKKa), IKK beta (IKKb), and IKK gamma/NEMO [8]. In conditional knockout mouse, previous study showed that IKKa is indispensable for osteoclast differentiation in vitro, but not in vivo. On the other hand, IKKb is required for RANKL-induced osteoclastogenesis both in vitro and in vivo [9]. Especially, IKKb has been shown to be a critical mediator of osteoclast survival and is required for inflammation-induced bone destruction in vivo [9]. In addition, IKK downstream targets p50/52 and p65 are also important for osteoclastogenesis, p65 knockout results in inhibition of osteoclast formation and severe osteopetrosis [10,11]. Tenuigenin (TNG) is a major active component of the Chinese herb Polygala tenuifolia root, also known as ‘Yuan Zhi’ in Chinese Materia Medica. TNG has been extensively applied in traditional Chinese medicine and used to treat patients with insomnia, neurosis, and dementia [12]. Presently, TNG has been reported to have various biological and pharmacological activities such as anti-inflammation, anti-dementia, anti-oxidation, and etc [13,14]. However, the effect of TNG on osteoclastogenesis and RANKL-

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induced NF-kB signaling pathways has not been investigated. In the present study, we investigated the effects of TNG on in vitro osteoclast formation and NF-kB signaling. Our results demonstrate that TNG is a potent inhibitor of osteoclast formation and NF-kB activity and thus might serve as a new therapeutic agent against bone lytic diseases involving increased osteoclastogenesis. 2. Materials and methods 2.1. Media and reagents Tenuigenin (TNG), purity >98%, was obtained from the Guidechem (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Invitrogen-Gibco (Grand Island, NY, USA). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Japan). Soluble mouse recombinant macrophage colonystimulating factor (M-CSF) and RANKL were purchased from R&D system (USA). Tartrate-resistant acid phosphatase (TRAP) staining solution was from SigmaeAldrich (St Louis, MO, USA). IkBa and p65 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). 2.2. Cells and culture system Bone marrowederived macrophages (BMMs) were obtained accordingly [15,16]. Generally, bone marrow cells were extracted from the femur or tibia of a 4-week-old C57/BL6 mouse and incubated in culture medium with M-CSF. Several days later, harvested the adhered cells as BMMs. The complete cell culture medium was DMEM with 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL). The cells were maintained in a humidified atmosphere of 5% CO2 at 37
C. 2.3. Cell viability assay The effect of TNG on BMMs viability of was detected using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Accordingly, cells were pre-treated with indicated concentrations of TNG for 48 h, and with indicated time in 8 mg/ml TNG, followed by addition of 10 mL CCK-8 solution to each well. After 4 h incubation, absorbance was measured at 450 nm using a microplate reader.

Valencia, CA) according to the manufacturer's instructions, and cDNA was synthesized from 3 mg of total RNA using reverse transcriptase (Superscript II Preamplification System; Invitrogen). Realtime PCR was performed by CFX96TM real-time system using SYBR FAST KAPA iCycler qPCR kit. The detector was programmed with the following PCR conditions: 40 cycles of 5 s denaturation at 95
C and 34 s amplification at 60
C. All reactions were run in triplicate and were normalized to the housekeeping gene b-actin. Primers for osteoclastogenic genes used in this study were as follows: mouse bactin: forward, 50 -TTTGATGTCACGCACGATTTCC-30 and reverse, 50 TGTGATGGTGGGAATGGGTCAG-30 ; mouse TRAP: forward, 50 -CTGG AGTGCACGATGCCAGCGACA-30 and reverse, 50 -TCCGTCTCGGCGAT GGACCAGA-30 ; mouse c-Src: forward, 50 -CCAGGCTGAGGAGTGGTACT-30 and reverse, 50 -CAGCTTGCGGATCTTGTAGT-30 ; mouse vATPase d2: forward, 50 -AAGCCTTTGTTTGACGCTGT-30 and reverse, 50 -TTCGATGCCTCTGTGAGATG-30 . 2.7. Western blot analysis Cells were cultured with TNG for indicated time. After that, cells were lysed with RIPA buffer plus PMSF. Collected cell lysates and centrifuged at 12,000 rpm for 5e15 min. Supernatants were collected as samples. Protein (30 mg) was separated on 10% SDSePAGE and transferred to PVDF membranes. The membranes were blocked with 5% BSA in TBST containing 0.05% Tween-20 and probed successively with mouse anti eIkBa and GAPDH overnight at 4
C. Horseradish peroxidase-conjugated rabbit anti-mouse IgG antibodies were used as secondary antibodies for 1 h at room temperature. The signals were detected by exposure in an Odyssey infrared imaging system (LI-COR). 2.8. Luciferase reporter gene activity assay The effect of TNG on RANKL-induced NF-kB activation was conducted using RAW264.7 cells stably transfected with an NF-kBdriven luciferase reporter gene construct (3 kB-Luc-SV40) [17]. To investigate the effect of TNG on NF-kB activity, RAW264.7 cells stably transfected with an NF-kB-driven luciferase reporter gene were pretreated with various doses of TNG for 1 h followed by 100 ng/mL RANKL for 8 h. Luciferase activities were measured using a Promega Luciferase Assay System (Promega, Madison, WI, USA), and normalized to that of the vehicle control. 2.9. Confocal microscopy for NF-kB localization

2.4. In vitro osteoclast differentiation To examine osteoclast formation, BMMs were treated with reagents in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml, PeproTech, USA) in 96 well culture plates (Corning, MA, USA). Cells were fixed and stained for tartrate resistant acid phosphatase (TRAP), a marker enzyme of osteoclasts. 2.5. Bone resorption assay For bone resorption assay, BMMs at 6  103 cells/well were seeded on bone slices in 96-well plates and stimulated with M-CSF (30 ng/mL) and RANKL (100 ng/mL). Four days later, pre-osteoclasts were treated with TNG (8 mg/ml) for another four days. Bone slices were then fixed and subjected to TRAP staining. Pit areas and number were quantified and analyzed. The experiments were repeated at least three times. 2.6. Real-time polymerase chain reaction analysis Total RNA was prepared using RNeasy Mini kit (QIAGEN,

Cells were plated on cover slips and starved for 24 h before treatment. Then cells were treated with TNG for 4 h, followed by stimulation with RANKL (100 ng/ml) for another 30 min. After that, the cells were permeated by 0.1%Triton-X 100 and blocked in 3% BSA for 1 h at room temperature. Antibody of the NF-kB subunit p65 (1:100) was incubated for 12 h at 4
C. For nuclear staining, DAPI solution (SigmaeAldrich) was added and incubated for 5e10 min shielded from light. The nuclear translocation of p65 was imaged using confocal system (Nikon, Tokyo, Japan). 2.10. In vivo experiments 8-week-old C57BL/6 mice were obtained from Hunan Academy of Chinese Medicine. More than 6 mice were examined for each group of experiments. TNG (3 mg/kg) or DMSO was injected intraperitoneally 24 h before the first RANKL injection, and the mice were subsequently injected with TNG (3 mg/kg), RANKL (0.5 mg/kg, R&D) or phosphate buffered saline (PBS) at 24 h intervals for 3 days. A high-resolution micro-CT scanner (Skyscan 1176; Skyscan;

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Fig. 1. TNG inhibits RANKL-induced osteoclast formation and survival in vitro. (A) Structure of TNG. (B) BMMs was treated with various concentrations of TNG for 48 h, and cell viability was measured by using the CCK8 assay. (C) BMMs were treated with 8 mg/ml TNG for the indicated times, and cell viability was measured by using the CCK8 assay. (D) BMMs were cultured with RANKL (100 ng/ml) and M-CSF (30 ng/ml) in the presence of TNG (8 mg/ml) for 6 days. (E) TRAPþ multinucleated cells (MNCs) shown in D were counted. (F) TNG was added during the indicated culture days in the presence of RANKL (100 ng/ml) and M-CSF (30 ng/ml). (G) Mature osteoclasts from BMMs were treated with 8 mg/ml TNG for 24 h (H) TRAPþ MNCs shown in G were counted. Data are expressed as means ± SD from at least three independent experiments, #p < 0.05 versus controls.

Fig. 2. TNG suppresses osteoclastic bone resorption in vitro. (A) BMMs were placed on dentin slices and cultured in the presence of TNG (8 mg/ml) with RANKL (100 ng/ml) and MCSF (30 ng/ml) for 6 days. (B) And (C) The resorbed pit number and pits area were counted. Data are expressed as means ± SD from at least three independent experiments, # p < 0.05 versus controls.

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Aartselaar, Belgium) was used for quantitative analyses of osteolysis in mouse femoral condyle at a resolution of 9 mm, 50 kV, 500 mA. After reconstruction, a square region of interest (ROI) around the femoral condyle was chosen for further quantitative analysis, with the bone volume against tissue volume (BV/TV), bone mineral density (BMD), bone surface against bone volume (BS/BV) and trabecular thickness (Tb.th).

ml) had no cytotoxic effects on cells, as compared to the control cells that received no treatment (Fig. 1B). Furthermore, 8 mg/ml TNG was chosen for subsequent cytotoxic analysis for more days. As shown in Fig. 1C, during the whole culture period, TNG had no inhibitory effect on BMM proliferation. 3.2. TNG inhibits RANKL-osteoclast differentiation, bone resorption and survival in vitro

2.11. Statistical analysis Values are presented as the mean ± S.D. values from three or more experiments. Data were analyzed by Student's t-test for comparisons between two mean values. A value of P < 0.05 was considered significant. 3. Results 3.1. Effects of TNG on cell viability BMMs were treated with various concentrations of TNG for 48 h, and cell viability was assessed using a CCK8 assay. TNG (up to 32 mg/

RANKL is essential in the formation of mature osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF) [18]. Thus, we investigated the effects of TNG on RANKL-induced osteoclast differentiation from BMMs. BMMs were co-cultured with MCSF and RANKL, 5 days later numerous TRAP-positive multinucleated osteoclasts were generated. On the contrary, treatment with TNG significantly suppressed osteoclast formation (Fig. 1D and E). We next examined at which stage TNG blocked osteoclast formation. In RANKL-induced osteoclastogenesis TNG was added on different days (D0e5), and TRAP staining was performed on day 6. TNG effectively inhibited osteoclast formation when added on the first culture days (D0e3) as well as the last culture days (D3e5),

Fig. 3. TNG inhibits RANKL-induced NF-kB activity and p65 nuclear translocation. (A) TNG suppresses RANKL-induced NF-kB-dependent transcription. RAW264.7 cells stably transfected with the NF-kB-luc-SV40 reporter gene were pretreated with various doses of TNG for 1 h followed by 100 ng/mL RANKL for 8 h. Luciferase activities were measured. (B) Representative Western blot image showing TNG delays IkBa degradation. Cell extracts were prepared from BMM cells that were pretreated with or without TNG for 4 h followed by stimulation with RANKL (100 ng/mL) for 0, 10, and 60 min. Proteins extracted were subject to Western blot analysis using primary antibodies for IkBa or GAPDH. (C) Average ratio of IkBa relative to GAPDH. Western blot signal intensities were quantified by image J software. IkBa/GAPDH ratios were normalized to 0 min (D) RAW264.7 cells were plated and treated with TNG for 4 h, followed by stimulation with RANKL (100 ng/mL) for 30 min. The localization of p65 was visualized using immunofluorescence analysis. (E) The percentage of cells showing nuclear translocation was calculated. (F) TNG suppresses RANKL-induced osteoclast specific gene expression. BMMs were cultured with M-CSF (30 ng/mL), RANKL (100 ng/mL) and TNG (8 mg/ml) for 6 days. RANKL-induced gene expression was detected by real-time PCR assay. Expression levels were normalized. Data are expressed as means ± SD from at least three independent experiments, #p < 0.05 versus controls.

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suggests TNG affects both early and late osteoclastogenesis (Fig. 1F). We also investigated the effect of TNG on osteoclasts survival. When mature osteoclasts were incubated with RANKL in the absence or presence of TNG for 24 h, TNG impaired the survival of mature osteoclasts even in the presence of RANKL (Fig. 1G and H). Based on the inhibitory effect of TNG on osteoclast formation and survival, we next examined whether osteoclastic activity was also impaired using an in vitro resorption pit assay on dentin slice. As can be seen in Fig. 2, many resorption pits were generated in RANKL-treated cells. In contrast, TNG strongly inhibited formation of resorption pits by the RANKL-treated cells (Fig. 2). Above all, these results suggest that TNG exerts inhibitory effects on osteoclast formation and survival, which leads to reduced bone resorption activity. 3.3. TNG suppresses RANKL-induced activation of NF-kB In the process of osteoclast differentiation, RANKL binds to its receptor RANK, results in TNF receptor associated factor (TRAF) adapter proteins recruitment and NF-kB signaling activation, leading to specific genes expression that have DNA-binding sites unique for NF-kB. NF-kB is the most important and essential signaling for both osteoclast differentiation and survival [5e7]. To investigate the effects of TNG on NF-kB signaling we employed

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RAW264.7 cells stably transfected with a NF-kB-driven luciferase reporter gene construct (3 kB-Luc-SV40). As shown in Fig. 3A, RANKL-induced a 10-fold increase in NF-kB-mediated luciferase gene expression in relative to negative control. By comparison, TNG significantly inhibited RANKL-induced transcriptional NF-kB activity in a dose dependent manner (Fig. 3A). As aforementioned, during RANKL mediated NF-kB signaling, the inducible phosphorylation and degradation of IkB is required for NF-kB activation. We next examined the effect of TNG on RANKL-induced IkBa degradation. As shown in Fig. 3B, BMMs treated with RANKL alone showed maximal degradation of IkBa after 10 min, and resynthesis of IkBa at 60 min. In comparison, TNG (8 mg/ml) delayed the RANKL-induced degradation/resynthesis of IkBa, most evident after 60 min (Fig. 3B and C). Finally, to further identify the effect of TNG on NF-kB activity, we also evaluated the effect of TNG on RANKL-mediated p65 nuclear translocation. As shown in Fig. 3D, pre-treatment with TNG significantly decreases the nuclear translocation of p65 upon RANKL stimulation, as compared to vehicle pre-treated control cells (Fig. 3D and E). In addition, mature osteoclast specific maker genes such as TRAP, c-Src and ATP6v0d2 were notably down regulated by TNG in the presence of RANKL, further verified the inhibitory effect of TNG on osteoclast formation and function in molecular level (Fig. 3F). Together, these results demonstrate that the anti-osteoclastogenic effect of TNG is due to

Fig. 4. TNG prevents RANKL-induced bone loss in vivo. (A) The femurs were scanned with a high-resolution micro-CT. (B) The calculation of the microstructural indices was performed with the micro-CT data as described in the methods section, including bone mineral density (BMD), bone volume per tissue volume (BV/TV), bone surface/volume ratio (BS/BV), and trabecular thickness (Tb.Th.). Data are expressed as means ± SD from at least three independent experiments, #p < 0.05 versus controls.

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suppression in NF-kB signaling. 3.4. TNG prevents RANKL-induced bone destruction in vivo

Conflict of interest All authors state that they have no conflicts of interest.

To investigate the effect of TNG on RANKL-induced osteoporotic bone loss in vivo, TNG was intraperitoneally injected into the mice 24 h before the first RANKL injection, and the mice (n ¼ 6) subsequently received simultaneous injections of both TNG and RANKL every 24 h for 3 days. Micro-CT scanning was performed accordingly [19]. It was revealed that TNG significantly restored BMD in RANKL-induced osteoporotic mice (Fig. 4). In addition, 2D micro-CT image and quantitative analysis also showed that RANKL-induced bone destruction was significantly reduced by TNG (Fig. 4).

Acknowledgments

4. Discussion

References

Excessive RANKL leads to enhanced osteoclast formation and bone resorption [18]. Reasonably, down regulation of RANKL expression or inhibition of its downstream signals could be a promising strategy to the treatment of bone destruction [20]. In this study, we for the first time demonstrate the inhibitory effects of TNG on RANKL-induced NF-kB activation and osteoclastogenesis. Furthermore, our data verifies that TNG delays IkBa degradation and suppresses the nuclear translocation of p65 upon RANKL stimulation, suggesting that the inhibitory effect of TNG on osteoclastogengesis is due to suppressed NF-kB activation. RANKL is an essential factor in osteoclast formation [18]. Once RANKL combines with its receptor RANK, it recruits adapter TRAF6 and further leads to the activation of several signaling cascades, among those NF-kB is the most important and indispensable one [18,21]. The crucial role of NF-kB signaling in osteoclast formation and survival has been demonstrated thoroughly, such as NF-kB knockout mice displayed severe osteopetrosis and failed osteoclastogenesis [10,22]. In our study, we demonstrated that TNG delayed RANKL-induced degradation/resynthesis of IkBa and significantly inhibited RANKL-induced transcriptional NF-kB activity, thereby suppressing the nuclear translocation and activation of the p65 subunit of NF-kB. This suggested TNG could impair RANKL-induced NF-kB signaling in a concentration-dependent manner, in turn inhibiting efficient transcription of genes involved in osteoclastogenesis. Recently, great efforts have been taken in searching for active compounds that specifically and selectively block NF-kB activation. Such as aspirin, salicylates, sulindac, and etc., these non-steroidal anti-inflammatory drugs have inhibitory effects on NF-kB activation [23e25]. Thalidomide and related agents, cyclopentenone prostaglandins, which are known as immunomodulatory drugs, also have showed the capability of inhibiting NF-kB signaling [26,27]. Meanwhile, significant progress has been made in developing novel NF-kB specific inhibitors [28]. These above mentioned research has provided an authentic evidence for the use of NF-kB inhibitors as treatment therapies of various diseases including inflammation, autoimmune disorders and cancer [29]. In the present study, our data further support and extend the notion that TNG, a selective inhibitor to NF-kB, might also serve as promising compound for the treatment and alleviation of osteoclast-related diseases. In summary, this study demonstrates that TNG efficiently prevents RANKL-induced osteoclastogenesis in vitro as well as osteoclastic bone destruction in vivo. The therapeutic effect of TNG is associated with down-regulation of NF-kB activity, leading to the lowered expression osteoclastic specific genes. These findings indicate that TNG may be useful for the prevention or treatment of osteolytic disorders such as osteoporosis, tumor bone metastasis, and inflammation-elicited bone loss.

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The authors want thank Department of Orthopedic Surgery, The Xiangya Hospital of Central South University for its great support. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.09.093.

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