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Geraniin suppresses RANKL-induced osteoclastogenesis in vitro and ameliorates wear particle-induced osteolysis in mouse model Fei Xiao, Zanjing Zhai, Chuan Jiang, Xuqiang Liu, Haowei Li, Xinhua Qu, Zhengxiao Ouyang, Qiming Fan, Tingting Tang, An Qin, Dongyun Gu www.elsevier.com/locate/yexcr

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S0014-4827(14)00274-2 http://dx.doi.org/10.1016/j.yexcr.2014.07.005 YEXCR9672

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Experimental Cell Research

Received date: 21 May 2014 Revised date: 28 June 2014 Accepted date: 1 July 2014 Cite this article as: Fei Xiao, Zanjing Zhai, Chuan Jiang, Xuqiang Liu, Haowei Li, Xinhua Qu, Zhengxiao Ouyang, Qiming Fan, Tingting Tang, An Qin, Dongyun Gu, Geraniin suppresses RANKL-induced osteoclastogenesis in vitro and ameliorates wear particle-induced osteolysis in mouse model, Experimental Cell Research, http://dx.doi.org/10.1016/j.yexcr.2014.07.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Geraniin suppresses RANKL-induced osteoclastogenesis in vitro and ameliorates wear particle-induced osteolysis in mouse model

Fei Xiao a, 1, Zanjing Zhai a, 1, Chuan Jiang a, Xuqiang Liu a, Haowei Li a, Xinhua Qu a, Zhengxiao Ouyang a, b

a

, Qiming Fan a, Tingting Tang a, An Qin a, * and Dongyun Gu a, *

Department of Orthopedics, Shanghai Key Laboratory of Orthopedic Implant, Shanghai Ninth People’s

Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China

b

Department of Orthopaedics, Hunan Provincial Tumor Hospital and Tumor Hospital of Xiangya School

of Medicine, Central South University, Changsha, Hunan 410013, The People's Republic of China

*Corresponding author. Address: Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, China.

Tel.: +86 021 23271159; fax: +86 0 21 63139920

E-mail address: [email protected] (A. Qin); [email protected] (D.Y. Gu). 1

These authors contributed equally to this work.ABSTRACT

Wear particle-induced osteolysis and subsequent aseptic loosening remains the most common complication that limits the longevity of prostheses. Wear particle-induced osteoclastogenesis is known to be responsible for extensive bone erosion that leads to prosthesis failure. Thus, inhibition of osteoclastic bone resorption may serve as a therapeutic strategy for the treatment of wear particle induced osteolysis. In this study, we demonstrated for the first time that geraniin, an active natural compound derived from Geranium thunbergii, ameliorated 1   

particle-induced osteolysis in a Ti particle-induced mouse calvaria model in vivo. We also investigated the mechanism by which geraniin exerts inhibitory effects on osteoclasts. Geraniin inhibited RANKL-induced osteoclastogenesis in a dose-dependent manner, evidenced by reduced osteoclast formation and suppressed osteoclast specific gene expression. Specially, geraniin inhibited actin ring formation and bone resorption in vitro. Further molecular investigation demonstrated geraniin impaired osteoclast differentiation via the inhibition of the RANKL-induced NF-κB and ERK signaling pathways, as well as suppressed the expression of key osteoclast transcriptional factors NFATc1 and c-Fos. Collectively, our data suggested that geraniin exerts inhibitory effects on osteoclast differentiation in vitro and suppresses Ti particle-induced osteolysis in vivo. Geraniin is therefore a potential natural compound for the treatment of wear particle induced osteolysis in prostheses failure.

Keywords: Geraniin; Osteoclastogenesis; Osteolysis; ERK; NF-κB

2   

Introduction Arthroplasty is one of the most successful procedures for treating terminal stage degenerative and inflammatory arthritis [1, 2]. Although complications after joint arthroplasty are relatively rare, aseptic prosthetic loosening remains the most common long-term complication limiting the longevity of prostheses [3, 4]. Wear particles released from prosthetic implants are widely recognized as the key trigger causing the development of peri-prosthetic osteolysis [1, 2, 5]. Thus, elimination of the production of wear particles by improving material quality and/or reducing wear particle-induced biological effects by means of particular compounds are key strategies to overcome aseptic loosening.

Active bone resorbing osteoclasts are critical for the development of osteolysis [6, 7]. Wear debris stimulates the secretion of cytokines, including tumor necrosis factor-α, interleukin-6, interleukin-1,  prostaglandin E2, matrix metalloproteinase, and other proinflammatory cytokines [1, 8-10]. These cytokines can subsequently induce local inflammation and thus initiate the recruitment of osteoclast precursors to the bone–implant interface. Consequently, functional osteoclasts formed and started resorbing bone around the prosthesis, leading to peri-prosthetic osteolysis [6]. Therefore, identification of compounds that can effectively inhibit wear particle-induced activation of osteoclasts is crucial for the prevention of osteolysis [11].

Current molecular findings have demonstrated that the differentiation of osteoclasts is mainly depending on two essential cytokines, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B (RANK) ligand (RANKL) [12, 13]. M-CSF is crucial for providing proliferation and survival signals to osteoclast precursor cells and upregulating RANK expression that is prerequisite for osteoclast differentiation [12]. The binding of RANKL and RANK induces trimerization of RANK and subsequently activates tumor necrosis factor receptor-associated factor 6 (TRAF6), thereby resulting in a cascade of intracellular events, such

3   

as the activation of NF-κB signaling pathway, the mitogen-activated protein kinases (MAPK) signaling pathways and the nuclear factor of activated T -cells1 (NFATc1) signaling pathway, which are essential for osteoclast formation [12-14]. Given the importance of NF-κB and MAPK signalling pathway in osteoclast formation, screening active compounds that can targete modulation of signaling pathways involved in osteoclast differentiation is an effective strategy for the treatment of osteoclast-related osteolysis diseases.

Geraniin (Fig. 3A), the main polyphenolic component of Geranuim thunbergii, is an important Chinese herbal medicine that has been used widely as an antidiarrheic drug. Several studies have demonstrated that geraniin possesses a wide range of pharmacological effects, including radioprotective [15], antiviral [16], antioxidant [17], antitumor [18], and antihypertensive [19] activities. Interestingly, previous study has revealed that geraniin inhibited NF-κB activity in RAW 264.7 cells [20]. In agreement with these findings, our RAW 264.7 cells stably transfected with an NF-κB-luciferase reporter construct further confirmed the inhibitory effect. Hereby, we hypothesized that geraniin may represent a novel treatment for osteoclast-related diseases, such as particle-induced peri-implant osteolysis. Therefore, the aim of the present study was to clarify the effects of geraniin on wear-debris-induced osteolysis, as well as to unveil the mechanisms by which geraniin affects osteoclasts. We report that geraniin has the ability to prevent wear particle-induced osteolysis.

2. Materials and methods

2.1 Titanium particles

Pure titanium particles (1–3-μm diameter) were obtained from Johnson Mathey Chemicals (Ward Hill, MA, USA) and prepared by continuous washing in 100% ethanol for 48 h, to remove adherent endotoxins [21]. Particles were then resuspended in sterile phosphate-buffered saline (PBS) solution at a concentration of 300

4   

mg/mL and stored at 4°C until required for use. The endotoxin level of particle suspension was determined by a Limulus assay (Biowhittaker, Walkersville, MD), according to the manufacturer’s instructions.

2.2 Media and reagents

Geraniin, purchased from Meilun Biotech (Dalian, China). Alpha-MEM, fetal bovine serum (FBS), and penicillin were purchased from Gibco BRL (Gaithersburg, MD, USA). Soluble mouse recombinant M-CSF and RANKL were purchased from R&D Systems (Minneapolis, MN, USA). Tartrate-resistant acid phosphatase (TRAP) staining solution was obtained from Sigma-Aldrich (St Louis, MO, USA). A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technology (Kumamoto, Japan). Primary antibodies targeting β-actin, IκBα, phospho-AKT, AKT, phospho-ERK, ERK, phospho-JNK, JNK, phospho-p38, p38, and NFATc1 were purchased from Cell Signaling Technology (Danvers, MA, USA).

2.3 Cell viability assay

The anti-proliferative effect of geraniin on bone marrow macrophages (BMMs) was assessed with a CCK-8 kit according to the manufacturer’s instructions. BMMs were plated in 96-well plates at a density of 1 × 104 cells/well, in triplicate, and cultured in 100 μL complete α-MEM medium for 24 h, to which different doses of geraniin (0, 1.25, 2.5, 5, 10, 20, or 40 μM) had been added. Subsequently, 10 μL CCK-8 reagent was added to each well. After 2 h incubation, the optical density (OD) at 450 nm (630 nm as reference) was read using an ELX800 absorbance microplate reader (Bio-Tek, Winooski, VT, USA). Experiments were repeated independently three times.

2.4 In vitro osteoclastogenesis assay

5   

In vitro osteoclastogenesis assays were performed to examine the effects of geraniin on osteoclast differentiation. BMMs were prepared as previously described [21-24]. Briefly, cells extracted from the femur and tibiae of a 6-week-old C57/BL6 mouse were allowed to proliferate in complete cell culture medium containing 30 ng/mL M-CSF, in a T-75cm² flask. Cells were washed during medium change in order to deplete residual stromal cells. At 90% confluence, cells were washed with PBS three times and then trypsinized for 30 min to harvest BMMs adhering to the bottom of the flasks. BMMs were then plated in 96-well plates at a density of 8 × 103 cells/well, in triplicate, and incubated in a humidified incubator containing 5% CO2, at 37°C for 24 h. Cells were then treated with various concentrations of geraniin (0, 1.25, or 5 µM) plus M-CSF (30 ng/mL) and RANKL (50 ng/mL). After 5 days, cells were fixed and stained for TRAP activity. TRAP+ cells containing more than five nuclei were counted as osteoclasts. Experiments were repeated independently at least three times.

2.5 Resorption pit assay and Actin ring-formation assay

A bone resorption assay was performed as previously described [23, 24]. BMMs were seeded onto bovine bone slices (2.4 × 104 cells/cm2). After 48 h, cells were treated with 50 ng/mL RANKL, 30 ng/mL M-CSF, and 0, 1.25, or 5 μM geraniin, until mature osteoclasts formed. Cells that had adhered to bone slices were then removed by mechanical agitation and sonication. Resorption pits were visualized under a scanning electron microscope (FEI Quanta 250), and the bone resorption area was quantified using Image J software (National Institutes of Health, Bethesda, MD, USA). Experiments were repeated independently at least three times.

Actin ring formation in osteoclasts cultured on bone was analyzed using confocal microscopy. Osteoclasts were fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100 in PBS for 5 min, and then incubated with rhodamine-conjugated phalloidin for 15 min. Actin ring distribution was visualized using a LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany). 6   

2.6 Quantitative polymerase chain reaction (PCR) analysis

For real-time PCR, 10 × 104 BMMs were seeded in each well of a 24-well plate and cultured in complete medium containing α−MEM, 10% FBS, 100 U/mL penicillin, M-CSF (30 ng/mL), and RANKL (50 ng/mL). Cells were then treated with or without geraniin (5 μM) for the indicated times. Total RNA was prepared using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions, and cDNA was synthesized from 1 μg of total RNA using reverse transcriptase (TaKaRa Biotechnology, Otsu, Japan). Real-time PCR was performed using a SYBR Premix Ex Tag kit (TaKaRa Biotechnology) and an ABI 7500 Sequencing Detection System (Applied Biosystems, Foster City, CA, USA). PCR was performed under the following conditions: 40 cycles each involving 5 s of denaturation at 95°C and 34 s of amplification at 60°C. All PCRs were performed in triplicate and levels were normalized to those of the housekeeping gene Actb. The following primer

sets

were

used

as

previously

described

[23]:

mouse

β-actin:

forward,

5 ′

-TCTGCTGGAAGGTGGACAGT-3 ′ and reverse, 5 ′-CCTCTATGCCAACACAGTGC-3′; mouse NFATc1: forward, 5′-CCGTTGCTTCCAGAAAATAACA-3′ and reverse, 5′-TGTGGGATGTGAACTCGGAA-3′; mouse TRAP:

forward,

5

′

-CTGGAGTGCACGATGCCAGCGACA-3

′

and

reverse,

5

′

-TCCGTGCTCGGCGATGGACCAGA-3′; mouse cathepsin K: forward, 5′-CTTCCAATACGTGCAGCAGA-3′ and

reverse,

5

′

-TCTTCAGGGCTTTCTCGTTC-3

′

;

mouse

CTR:

forward,

5

′

-TGCAGACAACTCTTGGTTGG-3′ and reverse, 5′-TCGGTTTCTTCTCCTCTGGA-3′; mouse c-Fos: forward, 5'-CCAGTCAAGAGCATCAGCAA-3', reverse, 5'-AAGTAGTGCAGCC CGGAGTA-3'; and mouse V-ATPase d2: forward, 5′-AAGCCTTTGTTTGACGCTGT-3′ reverse, 5′-TTCGATGCCTCTGTGAGATG-3′.

2.7 Luciferase reporter gene activity assay

7   

The effects of geraniin on RANKL-induced NF-κB and NFATc1 activation were measured using RAW264.7 cells that had been stably transfected with an NF-κB- or NFATc1-luciferase reporter construct, as previously described [22, 25]. Briefly, cells were seeded into 48-well plates and cultured for 24 h, and then pre-treated with or without the indicated concentrations of geraniin for 1 h, followed by addition of RANKL (50 ng/mL) for 8 h, when investigating NF-κB activation, and for 24 h when investigating NFATc1 activation. Luciferase activity was measured using the Promega Luciferase Assay System (Promega, Madison, WI, USA) and normalized to that of the vehicle control. Experiments were repeated independently at least three times.

2.8 Confocal microscopy for NF-κB localization

RAW264.7 cells were seeded at a density of 1×104 cells in 6-well plates containing sterile cover slips. After incubated with complete α-MEM medium at 37°C for 24 h, the cells were treated with geraniin for 4 h, followed by stimulation with RANKL (50 ng/mL) for 20 min. After incubation, cells were washed twice with 1× PBS, fixed in 4% paraformaldehyde for 20 min and then washed with 1×PBS three times before permeabilized by 0.1%Triton-X 100 for 30 min at room temperature. After blocking with 3% BSA-PBS for 1 h at room temperature, cells were incubated with anti-p65 subunit antibody diluted 1:100 in PBS at 4°C overnight. Nuclear stained with 0.1μg/mLDAPI (Sigma-Aldrich) in PBS at 37°C for 10 minutes in the dark. After washed three times with PBS, the nuclear translocation of p65 was imaged using a NIKON A1Si spectral detector confocal system (Nikon, Tokyo, Japan).

2.9 Western blotting analysis

Western blotting analysis was carried out as previously described [26]. RAW264.7 cells were seeded in 6-well plates at a density of 6 × 105 cells/well. When the cells were confluent, they were pre-treated with or without geraniin for 4 h. Cells were then stimulated with 50 ng/mL RANKL for 0, 5, 10, 20, or 30 min. Cells were lysed 8   

using radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA), and the protein concentration was determined using a BCA protein assay (Thermo Fisher Scientific). Lysate proteins (30 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were then blocked with 5% skimmed milk in TBS–Tween (TBS: 0.05 M Tris, 0.15 M NaCl pH 7.5; 0.2% Tween-20) for 1 h, and incubated with primary antibodies diluted in 1% (w/v) skimmed milk powder in TBS–Tween overnight at 4°C. Membranes were incubated with the appropriate secondary antibodies conjugated with IRDye 800CW (molecular weight: 1162 Da). Immunoblots were visualized by Odyssey V3.0 image scanning (Li-Cor Biosciences, Lincoln, NE, USA). Experiments were repeated independently at least three times.

2.10 Calvarial osteolysis model

Animal studies were conducted in accordance with the principles and procedures approved by the Animal Care Committee of Shanghai Jiao Tong University, China. The in vivo mouse calvaria experiments were performed as described previously [11, 21]. Briefly, 20 healthy 8-week-old male C57BL/6 mice were assigned randomly into four groups: sham PBS control (sham), Ti particles with PBS (vehicle), and Ti particles with low (1 mg•kg-1) and high (4 mg•kg-1) concentrations of geraniin (low and high geraniin groups, respectively). The concentration of geraniin was consided to be safety according the previous research, which affirmed that geraniin was non-toxic when used at a dose of 250 mg•kg-1 intraperitoneally in 6-8 weeks old male C57BL/6 mice[27].  The mice were anesthetized, and the cranial periosteum was separated from the calvarium by sharp dissection. Then, 30 mg of Ti particles were embedded under the periosteum at the middle suture of the calvaria. No adverse effects or fatalities were noted. Two days postimplantation, geraniin or PBS were injected intraperitoneally, every other day for 14 days, before mice were sacrificed in a CO2 chamber and further analyzed.

9   

2.11 μCT scanning

After mice were sacrificed, calvaria were removed and fixed in 4% formaldehyde in PBS (pH 7.4) for 24 h; they were analyzed using high-resolution micro-computed tomography (µCT; Skyscan 1072; Skyscan, Aartselaar, Belgium). In order to reduce metal artifacts, the wear particles were removed before μCT scanning. Calvaria were scanned completely using 9-µm thick slices. After reconstruction, a square region of interest (ROI) around the midline suture was chosen for further qualitative and quantitative analysis: bone mineral density (BMD), bone volume to tissue volume ratio (BV/TV), the number of porosity, and percentage of total porosity of each sample were determined.

2.12 Histological and histomorphometric analysis

After μCT scanning, the calvaria samples were decalcified in 10% EDTA for ca. 1 month, and then embedded in paraffin. Histological sections were prepared for hematoxylin and eosin as well as TRAP staining. The specimens were then examined and photographed using a high quality microscope. The number of TRAP+ multinucleated osteoclasts, the percentage of osteoclast surface per bone surface (OcS/BS, %) and the bone area were assessed for each sample.

2.13 Statistical analysis

Values are presented as the mean ± SD of results obtained from three or more experiments. Statistical analyses were performed using one-way ANOVA followed by the Student–Newman–Keul test. P-values of less than 0.05 were considered significant.

3. Results

10   

3.1 Therapeutic effects of geraniin on Ti particle-induced osteolysis in vivo

In order to investigate the therapeutic effect of geraniin on osteolysis, the in vivo effect of geraniin on wear particles induced osteolysis is determined. Therefore, As clearly seen by three-dimensional reconstructions of μCT images, severe osteolysis was induced by Ti particles, as evidenced by extensively eroded surface of the calvaria in the vehicle group, as compared to that of the negative control (sham group; Fig. 1A). In contrast, the extent of wear particle-induced bone destruction was significantly reduced by treatment with geraniin in a dose-dependent manner, where bone loss in mice treated in the high geraniin group was markedly less than that in the low geraniin group (Fig. 1A). This result was further confirmed by quantitative analysis of bone mass parameters. Geraniin treatment ameliorated particle-induced osteolysis, significantly increased BV/TV (Fig. 1B), and markedly reduced the number of resorptive pits and the percentage of total porosity of the calvaria (Fig. 1B).

Furthermore, histological assessment and histomorphometric analysis confirmed the amelioration of particle-induced bone erosion by geraniin. The presence of Ti particles induced intensive inflammatory infiltration of lymphocytes and macrophages into the injection site. Multiple osteoclasts lined along the eroded bone surface, as revealed by TRAP staining (Fig. 2A). In contrast, treatment with geraniin markedly diminished these Ti particle-mediated effects, which was illustrated by reduced number of TRAP+ osteoclasts in both low and high geraniin groups (Fig. 2A). Moreover, histomorphometric analysis of OcS/BS, the number of osteoclasts, and the bone area confirmed that geraniin prevented Ti particle-induced osteolysis (Fig. 2B).

3.2 Geraniin inhibits early-stage osteoclastogenesis without cytotoxic effects

To unveil the mechanisms on how geraniin prevented wear particle induced osteolysis, the effect of geraniin on osteoclastogenesis was investigated in vitro. BMMs were treated with M-CSF and RANKL in the absence or 11   

presence of different concentrations of geraniin for 5 days. Numerous TRAP+ multinucleated osteoclasts formed in the control group (Fig. 3C). In contrast, osteoclast formation was inhibited by geraniin treatment in a dose-dependent manner. Osteoclast formation was reduced to approximately 10% of control levels by treatment with 5 μM geraniin (Figs. 3C, 3D). No cytotoxic effect of geraniin on BMMs was detected for doses at 0–5 μΜ (Fig. 3B). To examine at which stage geraniin inhibited osteoclastogenesis, 5 μM geraniin was added to culture medium at 0, 2, or 4 days during osteoclast differentiation. Geraniin treatment at earlier stages (days 0 and 2) significantly suppressed osteoclastogenesis (Fig. 3E, 3F). However, exposure of osteoclastic precursor cells to geraniin at later stage (day 4) did not affect osteoclast formation. Thus, geraniin inhibited early-stage osteoclast differentiation at non-cytotoxic doses.

3.3 Geraniin impairs osteoclastic bone resorption and actin ring formation

Since geraniin inhibited osteoclastogenesis, we then examined whether geraniin could impair osteoclastic bone-resorptive function in vitro. Thus, BMMs were cultured on bone slices without or with geraniin. In the control group, mature osteoclasts extensively resorbed bone matrix. On the contrary, the treatment of osteoclasts with geraniin at 1.25 µM effectively inhibited osteoclast-mediated bone resorption by approximately 50% (Fig. 4A, 4B). There is almost no resorptive pit when osteoclasts are treated with geraniin at higher concentrations (5 μΜ).

A well-polarized F-actin ring is a prerequisite for efficient bone resorption [28]. Thus, we futher assessed the effect of geraniin on F-actin ring formation. Clear actin ring structures were observed in the untreated control group (Fig. 4C). However, the actin ring structure was significantly disrupted when BMMs were incubated with 1.25 or 5 μM geraniin (Fig. 4D), suggesting that the inhibitory effects of geraniin on bone resorption is correlated with F-actin ring disruption. 12   

3.4 Geraniin suppresses RANKL-induced osteoclast-specific gene expression

The expression of osteoclast-specific genes in response to RANKL is up-regulated during osteoclast differentiation [12]. Therefore, the osteoclastic marker gene expression profile was examined to further confirm the inhibitory effect of geraniin on osteoclast formation. As shown in Fig. 5A, the expression of all osteoclast-specific genes, including Trap, Ctr, Ctsk, Nfatc1, Fos, and Atp6v0d2 (V-ATPase d2), was gradually induced under RANKL stimulation. However, the induction of these genes was suppressed markedly by the presence of geraniin in a time-dependent manner. In addition, geraniin dose-dependently suppressed osteoclastic gene expression at 1.25 and 5 μΜ (Fig. 5B). Taken together, these data further confirmed that geraniin suppressed osteoclast formation and inhibited the expression of osteoclast-specific genes.

3.5 Geraniin inhibits RANKL-induced NF-κB and ERK activation

It has been reported that RANKL activates a variety of signaling pathways, including those of p38, JNK, ERK, and NF-κB, which are critical for osteoclastogenesis [1, 12, 14]. To further explore the underlying mechanisms through which geraniin mediated osteoclast formation, RANKL-induced signaling pathways were investigated. The activation of NF-κB signaling pathway plays a crucial role in osteoclastogenesis [12]. IκBa, an inhibitory subunit of NF-κB, was degraded upon 5 min of RANKL stimulation in the control group (Fig. 6A, 6B). But this degradation was notably inhibited by geraniin. Figure 6D shows that, most p65 were located in the nucleus in the control group. However, the nuclear translocation of p65 was substantially suppressed when incubation occurred with geraniin, as demonstrated by the retention in the cytoplasm of the p65 proteins. This finding was further confirmed by luciferase reporter analysis. NF-κB transcriptional activity was evidently activated by RANKL (Fig. 6E). However, this activation NF-κB was partially suppressed by geraniin in a dose-dependent manner. 13   

Besides the NF-κB signaling pathway, activation of the mitogen-activated protein kinases (MAPK) plays a critical role in osteoclastogenesis [29]. To investigate the effects of geraniin on MAPKs, we examined RANKL-induced phosphorylation of p38, JNK, and ERK by western blotting. Geraniin attenuated ERK phosphorylation, without affecting that of JNK or p38 (Fig. 6A, 6C).

Furthermore, NFATc1 and c-Fos are crucial transcription factors during osteoclast differentiation [30]. We therefore assessed the expression of NFATc1 and c-Fos after geraniin treatment. Consistent with the gene expression profile results, our western blot analysis confirmed the suppression of NFATc1 and c-Fos protein expression after geraniin treatment (Fig. 6F, 6G). Furthermore, luciferase reporter assays demonstrated that RANKL induced NFATc1 transcriptional activation, but this was inhibited dose-dependently by geraniin (Fig. 6H).

Collectively, these results suggested that geraniin participates in the regulation of the RANKL-activated NF-κB and ERK signaling pathways, and inhibits key transcription factors such as c-Fos and NFATc1, which contribute to the inhibition of osteoclast formation in vitro and prevention of wear particle induced osteolysis in vivo.

4. Discussion

Arthroplasty is generally used to treat the most severe joint diseases. However, peri-prosthetic osteolysis and subsequent aseptic loosening caused by wear particles currently remain the most important problems in arthroplasty surgery [1, 2, 5]. It is generally accepted that continuous generation of prosthetic wear particles from the articulating interface  stimulate macrophages, fibroblasts, foreign body giant cells, and T lymphocytes to produce a series of proinflammatory cytokines, which would  excessive induce macrophages differentiating into mature and functional osteoclasts that result in local osteolysis [1, 31, 32]. Osteolysis resulting from bone loss exacerbates prosthesis loosening, while the increasing loosening further aggravates mechanical wear and 14   

induces more severe osteolysis [1, 2, 31, 33]. Since the last century, a variety of methods have been explored to reduce the wear particles, such as optimizing implant design, improving the material properties and developing surgical techniques. Unfortunately, the wear particle generation is inevitable due to the relative motion between the prosthesis components and the continuous degradation of the material. Therefore, reducing bone loss by inhibition of periprosthetic osteoclast function seems as an alternative effective approach to prevent wear particles induced osteolysis and prosthesis loosening. Here, we confirmed that geraniin attenuated Ti particle-induced osteolysis in a murine model. Our findings suggest that geraniin is a candidate compound for the treatment of pathological bone loss, such as seen in peri-prosthetic osteolysis.   

Ti particle-induced mouse calvarial osteolysis was established as the model to explore the potential protective effect(s) of geraniin during pathological bone destruction. Previous studies revealed that wear debris-stimuli significantly upregulated osteoclast formation and resorption [8-10]. In the present study, we found that severe osteolysis was induced by Ti particles. However, the extensive bone erosion was significantly suppressed by geraniin treatment (Fig. 1). As mentioned above, aseptic local inflammation associated with wear debris initiating the recruitment of osteoclast precursors to the bone–implant interface, leading to periprosthetic osteolysis [1]. Since the TRAP+ osteoclast number was significantly decreased by geraniin administration (Fig. 2), the amelioration of bone destruction by geraniin was thought to be mainly due to the suppression of osteoclastogenesis. However, bone homeostasis depends on functional balance between bone-resorbing osteoclasts and bone-forming osteoblasts, further investigations will be needed to understand the signalling mechanisms mediating the effects of AP on osteoblasts.

During osteoclast differentiation, RANKL binding to RANK induces the activation of several vital signaling pathways, such as the NF-κB pathway, the MAPK pathways, and the NFATc1 pathway [12, 14, 34]. A

15   

preliminary step in NF-κB activation involves phosphorylation and degradation of IκBα, which allows nuclear translocation of NF-κB proteins (such as p65) and its binding to DNA target sites to trigger the expression of osteoclastogenesis-related genes [35]. Since NF-κB plays a vital role in the osteoclastogenesis, targeting of NF-κB may potentially reduce periprosthetic osteolysis and thereby improving the longevity of joint replacements [36]. In our study, we found that the RANKL-induced activation of NF-κB was suppressed by geraniin (Fig. 6E), as demonstrated by the inhibition of IκBα degradation (Fig. 6A) and p65 translocation (Fig. 6D). This result was consistent with a previous study that revealed that geraniin inhibited NF-κB activity in RAW 264.7 cells [20].  The inhibition of NF-κB activity by geraniin is further supported by our finding that geraniin suppressed early stage, but not late stage, osteoclast formation (Fig. 3E). Interestingly, phosphorylation of ERK, but not of p38 or JNK, was inhibited in geraniin-treated cells, suggesting that geraniin may disrupt osteoclast formation via multiple targets (Fig. 6A). Further investigations are necessary to unveil the direct binding target site of geraniin in osteoclast precursors.

The downstream signaling events of NF-κB activation during osteoclastogenesis involve the activation of c-Fos followed by NFATc1 [37]. Within 1 h after RANKL stimulation, NF-κB proteins were recruited to the NFATc1 promoter, inducing NFATc1 autoamplification during osteoclastogenesis [38]. Additionally, the inhibition of NF-κB as well as down-regulation of c-Fos could decrease NFATc1, which subsequently inhibits osteoclastogenesis [37]. In this study, we showed that inhibition of NF-κB activation and ERK phosphorylation by geraniin markedly downregulated RANKL-induced NFATc1 and c-Fos expression (Fig. 6F), resulting in impaired osteoclast formation. This was further supported by the downregulation of NFATc1-regulated osteoclast marker genes, such as TRAP, CTR, and CTSK by geraniin (Fig. 5).

16   

In conclusion, the present study demonstrated the inhibitory effects of geraniin on osteoclastogenesis and osteoclast function in vitro and prevented wear particle induced osteolysis in vivo. Moreover, the mechanisms of these inhibitory effects of geraniin are mediated via the suppression of the NF-κB and ERK signaling pathways. The results suggest that geraniin may be a therapeutic candidate for the treatment of osteolysis induced by prosthesis-derived wear particles.

Disclosures

The authors have no conflicts of interest.

Acknowledgements This study was supported by a National Nature Science Foundation of China (Grant No. 31170901), a scientific research grant from the National Natural Science Foundation for the Youth of China (Grant No. 81201364), a scientific research grant for youth of Shanghai (Grant No. ZZjdyx 2097) and Doctoral Innovation Foundation from Shanghai Jiaotong University School of Medicine (BXJ201330).

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Figure captions

19   

Fig. 1. Histomorphometric analysis of calvaria in mice treated with geraniin (Gr) for 14 days after surgery. (A) Representative three-dimensional reconstructed μCT images from each group. (B) Bone mineral density (BMD), bone volume to tissue volume ratio (BV/TV), number of pits, and the percentage of total porosity of each sample was measured (*P < 0.05; ** P < 0.01).

Fig. 2. Histological staining of calvaria sections. (A) Representative hematoxylin and eosin (H&E) staining and TRAP staining. (B) Percentage osteoclast surface per bone surface (OcS/BS, %) and bone area at the suture site and adjacent osteolysis region in H&E-stained calvaria sections; TRAP+ cell number in TRAP-stained sections （** P < 0.01）.

Fig. 3. Geraniin (Gr) inhibits RANKL-induced osteoclastogenesis in a dose-dependent manner without cytotoxicity. (A) The structure of Gr. (B) Bone marrow-derived macrophages (BMMs) were treated with 30 ng/mL M-CSF, 50 ng/mL RANKL, and various concentrations of geraniin, as indicated, for 48 h or 96 h. Cell viability was then measured using a CCK-8 assay. (C) BMMs were treated with different concentrations of Gr followed by 30 ng/mL M-CSF and 50 ng/mL RANKL for 5 days. Cells were fixed with 4% paraformaldehyde and subjected to tartrate-resistant acid phosphatase (TRAP) staining. (D) The number and area of TRAP+ cells was counted, TRAP activity was measured by assessing optical density at 405 nm. (E) BMMs were cultured and M-CSF (30 ng/mL), RANKL (50 ng/mL), and Gr (5 μM) were added on day 0, 2, or 4. At the end of 5 days, cells were stained for TRAP expression. (F) TRAP+ multinucleated osteoclasts were counted. Gr-untreated, RANKL-exposed cells served as a control. All experiments were carried out at least three times, independently (** P < 0.01).

Fig.4. Geraniin (Gr) impairs osteoclast-mediated bone resorption and actin ring formation. (A) Bone marrow-derived macrophages (BMMs) were stimulated with M-CSF (30 ng/mL), RANKL (50 ng/mL), and the 20   

indicated concentrations of Gr, until mature osteoclasts formed. SEM images of bone resorption pits are shown. (B) Resorption pit areas were measured using Image J and are presented graphically. (C) BMMs were incubated and then stimulated with M-CSF (30 ng/mL), RANKL (50 ng/mL), and the indicated concentrations of Gr, until mature osteoclasts formed. Then, cells were fixed and stained for F-actin. Actin ring distribution was visualized under confocal microscopy. (D) The number of osteoclasts with an intact actin ring was counted. All experiments were carried out at least three times (** P < 0.01).

Fig. 5. Geraniin (Gr) suppresses RANKL-induced gene expression. Bone marrow-derived macrophages (BMMs) were cultured in the presence of M-CSF (30 ng/mL), RANKL (50 ng/mL), and 5 μΜ Gr (A) for 0, 1, 3, or 5 days, or with M-CSF (30 ng/mL), RANKL (50 ng/mL), and the indicated concentrations of Gr for 5 days (B). RANKL-induced osteoclast-specific gene expressions were analyzed by real-time PCR. mRNA expression levels of the targeted genes were normalized relative to the expression of ACTB (β-actin). All experiments were performed at least three times (*P < 0.05; ** P < 0.01).

Fig. 6. Geraniin (Gr) inhibited RANKL-induced NF-κB and ERK signaling pathways. (A): RAW264.7 cells were pretreated with or without Gr for 4 h prior to RANKL stimulation (50 ng/mL) for the indicated time periods. Cells were lysed for western blotting with the indicated antibodies. (B) and (C): Gray levels of IκBa and phosphorylation of ERK were analyzed and normalized to that of GAPDH using image J software, and are presented graphically. (D): RAW264.7 cells were plated at a density of 1×104 cells in 6-well plates and treated with Gr for 4 h, followed by stimulation with RANKL (50 ng/mL) for 20 min. The localization of p65 was visualized by immunofluorescence analysis. (E) and (H): RAW264.7 cells that had been stably transfected with a NF-κB or NFATc1 luciferase reporter construct were pretreated with the indicated concentrations of Gr for 1 h, and then incubated in the absence or presence of RANKL for 8 h (NF-κB reporter) or 24 h (NFATc1 reporter), 21   

prior to determination of luciferase activity. (F) BMMs were cultured with 30 ng/mL M-CSF and 50 ng/mL RANKL, with or without 5μM Gr, for 0, 1, or 3 days. Cell lysates were then analyzed by western blotting with antibodies against NFATc1, c-Fos, and GAPDH. All experiments were performed at least three times (*P < 0.05; ** P < 0.01).

22   

Highlights: 1. Geraniin suppresses osteoclasts formation and function in vitro. 2. Geraniin impairs RANKL-induced nuclear factor-κB and ERK signaling pathway. 3. Geraniin suppresses osteolysis in vivo. 4. Geraniin may be used for treating osteoclast related diseases.

23