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Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-κB and MAPK signaling pathways
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways Qiang Xu a, b, 1, Guiping Chen a, b, 1, Xuqiang Liu a, b, Min Dai a, b, *, Bin Zhang a, b, ** a
Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Artificial Joints Engineering and Technology Research Center of Jiangxi Province, Nanchang, Jiangxi province, 330006, China b Multidisciplinary Therapy Center of Musculoskeletal Tumor, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi province, 330006, China
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Article history: Received 20 November 2018 Accepted 29 November 2018 Available online xxx
The receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL)-RANK regulatory axis is a major regulator of osteoclast differentiation and activation. Icariin, a flavonol glycoside isolated from the Epimedium herb, has been reported to prevents bone loss in ovariectomized mice and inhibits wear particle-induced osteolysis. However, the molecular mechanism through which icariin inhibits RANKLinduced osteoclastogenesis has not been fully understood. Therefore, we aimed to investigate the effects of icariin on RANKL-induced osteoclastogenesis and to elucidate the mechanism underlying this effect. Our results showed that RANKL-induced osteoclastogenesis was inhibited by icariin in bone marrow macrophages (BMMs) and RAW264.7 cells, and that this effect was due to suppression of NF-kB and mitogen-activated protein kinase (MAPK) activation. In addition, icariin inhibited F-actin ring formation and attenuated the bone resorption ability of mature osteoclasts. Collectively, our results indicate that icariin may be a promising potential candidate for the treatment of osteolytic diseases such as osteoporosis. Moreover, our findings lay the foundation for understanding and intervening in osteoclastrelated diseases at the molecular level. © 2018 Elsevier Inc. All rights reserved.
Keywords: Icariin NF-kB RANKL MAPK Osteoclastogenesis Osteoporosis
1. Introduction Osteoporosis is an highly prevalent disease that imposes a serious economic burden on society. It is characterized by impaired bone mass and bone strength, destruction of bone microstructure, and increased bone fragility [1]. The increase in the estimated number of individuals with osteoporosis or osteopenia (a precursor to osteoporosis) is expected to continue, especially with the increase in the aging population [2]. Several studies have shown that osteoporosis is caused by excessive osteoclast activity that
* Corresponding author. Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Artificial Joints Engineering and Technology Research Center of Jiangxi Province, Nanchang, Jiangxi province, 330006, China. ** Corresponding author. Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Artificial Joints Engineering and Technology Research Center of Jiangxi Province, Nanchang, Jiangxi province, 330006, China. E-mail addresses: [email protected] (Q. Xu), [email protected] (G. Chen), [email protected] (X. Liu), [email protected] (M. Dai), [email protected] (B. Zhang). 1 Qiang Xu, Guiping Chen contributed equally to this work and should be considered co-first authors.
contributes to pathological bone resorption [3e5]. Increased bone resorption indicates that osteoclasts play a key role in the development of osteoporosis, and therefore, they are considered a viable therapeutic target for osteoporosis treatment [6]. Osteoclasts are multinucleated cells that are generated through differentiation of hematopoietic progenitors of the monocyte/ macrophage lineage [7]. Receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL) is the most important cytokine in the process of osteoclast differentiation. RANKL binds to its receptor RANK, which is expressed on the surface of osteoclast precursor cells, thereby inducing their differentiation and maturation into osteoclasts [7,8]. Thus, inhibition of osteoclast formation via modulation of the RANKL-induced signaling pathways is a viable therapeutic strategy for the treatment of osteoclast-associated diseases such as osteoporosis. Currently, the main treatments for osteoporosis include a humanized monoclonal antibody of RANKL, bisphosphonates, selective estrogen receptor modulators, and calcitonin. However, these treatments often produce adverse reactions, including osteonecrosis of the jaw, gastrointestinal discomfort, breast cancer, hypercalcemia, and cardiovascular diseases [9e11]. Thus, it is a clinical and social imperative to explore
https://doi.org/10.1016/j.bbrc.2018.11.201 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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novel agents for the treatment of osteoporosis in view of the current therapeutic drugs options. The deficiencies in current therapeutic options have led to an increasing interest in bioactive natural compounds that can be used as potential alternatives for osteoporosis treatment. Icariin is the most abundant flavonoid constituent in Herba Epimedii, and has been shown to be effective in bone fracture healing, joint disease, and improvement of gonadal function [12,13]. Studies have shown that icariin can play a dual role in restoring bone remodeling by stimulating bone formation while simultaneously inhibiting bone resorption [14]. Wang et al. reported that the icariin-mediated increase in bone formation is a result of multiple signal transduction pathways, including up-regulation of BMP, NO, MAPK, and Wnt pathways and down-regulation of the extracellular signalregulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways [15]. In addition, icariin increases bone mineral density in postmenopausal women and in rat models of ovariectomy-induced bone loss by inhibiting osteoclast formation [15]. Moreover, icariin has a preventive and therapeutic effect against wear particleinduced osteolysis and aseptic loosening [16]. Recently, icariin was shown to play an important role in negatively regulating inflammatory responses in vitro and in vivo [16]. Several studies showed that icariin also inhibits the expression of tumor necrosis factor (TNF)-a and interleukin(IL)-1b [17,18]. However, the molecular mechanism by which icariin interacts with RANKL-induced osteoclastogenesis has not been reported in vitro. Therefore, the purpose of this study was to investigate the effects of icariin on RANKL-induced osteoclast formation, and to elaborate the potential mechanisms underlying this effect. 2. Materials and methods 2.1. Cell viability assay In order to evaluate the cytotoxic effect of icariin, we used the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's instructions, as reported previously [19]. Briefly, bone marrow macrophages (BMMs) were added to 96-well plates at 8 103 cells/well in triplicate and cultured for 24 h in alpha modification of Eagle's medium (a-MEM, Gaithersburg, MD, USA) containing 30 ng/mL macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA), 10% fetal bovine serum (FBS; Gibco-BRL, Sydney, Australia), and 1% penicillin/streptomycin. BMMs were cultured with the following concentrations of icariin for 48 h: 0, 107, 106, 105, 104, 103, 102, and 101 M. Next, 10 mL of CCK-8 substrate was added to each well, and the plate was incubated at 37 C under 5% CO2 for 2 h. The absorbance of each well was measured at 450 nm with an ELX800 microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The cell viability relative to the control group was calculated using the following formula: (experimental group optimal density (OD) e blank OD)/(control group OD e blank OD). Similarly, the toxic effects of icariin were also tested on the RAW264.7 cell line. 2.2. Osteoclast differentiation assay BMMs were seeded in a 96-well plate at a density of 8 103 cells/well in a-MEM with 30 ng/mL M-CSF, 50 ng/mL RANKL (PeproTech, Rocky Hill, NJ, USA), and different concentrations of icariin (0, 107, 106, and 105 M). In addition, RAW264.7 cells cultured at 2 103 cells/well in a 96-well plate were treated with icariin at various concentrations (0, 107, 106, and 105 M) in the presence of 50 ng/mL RANKL. Both were supplemented with fresh medium every 2 days until mature osteoclasts were observed. Next, the cells were fixed with 4% paraformaldehyde and stained for
tartrate-resistant acid phosphatase (TRAP; Sigma-Aldrich, St. Louis, MO, USA) activity. The number of mature osteoclasts (TRAP-positive cells with 3 nuclei) was counted, and their spread area was measured. 2.3. F-actin ring evaluation with immunofluorescence confocal microscopy Osteoclasts derived from RAW264.7 cells were induced by RANKL, cultured on glass coverslips, and treated with different icariin concentrations (0, 107, and 105 M) until mature osteoclasts were observed in the control wells. Cells were fixed for 20 min at room temperature with 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100, and washed three times with phosphate-buffered saline (PBS). F-actin was stained with tetramethylrhodamine isothiocyanate-coupled phalloidin(Invitrogen, SanDiego, CA, United States), nuclei were stained with 40 ,6-diamidino-2-phenylindole(DAPI), and then the coverslips were mounted on microscope slides with ProLong Diamond Antifade Mounting medium (Invitrogen). Images were obtained using a LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using Zeiss ZEN software. 2.4. Bone resorption pit assay Bovine bone slices were placed in a 96-well plate, and then RAW264.7 cells were added at 2 103 cells/well in Dulbecco's Modified Eagle's Medium(DMEM) containing 50 ng/mL RANKL. Cells were treated with 0, 107, and 105 M icariin for 48 h, at which point mature osteoclasts were observed. The cells adhering to the bone slices were removed with a brush. Resorption pits were visualized using a scanning electron microscope (SEM, FEI Quanta 250), and the proportion area of resorbed bone slices was computed using Image J software (National Institutes of Health, Bethesda, MD, USA). 2.5. Western blot RAW264.7 cells were lysed by adding RIPA lysis buffer (Applygen Technologies Inc, Beijing, China) containing protease inhibitor (Sigma, Rockford, USA) and phosphatase inhibitor (Sigma, Rockford, USA) in concentrations specified by the respective manufacturers. The lysate was centrifuged at 12,000 g for 10 min, and the supernatant was collected. Protein concentration was determined using the bicinchoninic acid assay. The samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The PVDF membranes were blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and then incubated overnight at 4 C with specific antibodies against nuclear factor of activated T cells c1 (NFATc1), c-fos, TRAP, cathepsin K, IkBa, p-ERK1/2, ERK 1/2, p-JNK1/2, JNK 1/2, p-p38, p38, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cambridge, MA, USA). The membranes were washed in TBS-T, and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the membranes were washed with TBS-T, and developed using the enhanced chemiluminescence Western Blot Detection Reagent kit (GE Healthcare, Parramatta, Australia). Odyssey image scanning software v3.0 (LiCOR Biosciences, Lincoln, NE, USA) was used to obtain images of the membranes, and the protein bands were subjected to densitometric analysis with Image J software.
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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2.6. Statistical analysis All data were obtained from three or more independent experiments. The data from the cell experiments are expressed as the means ± standard deviations. Data were analyzed using unpaired Student's t-test with SPSS 23.0 software (SPSS Inc., Chicago, USA). Pvalues < 0.05 were considered statistically significant. 3. Results 3.1. Icariin inhibits RANKL-induced osteoclast formation without any cytotoxicity To examine the effect of icariin on RANKL-induced osteoclastogenesis, BMMs were stimulated with M-CSF and RANKL in the presence of different concentrations of icariin (0, 107, 106, and 105 M). Interestingly, the BMMs treated with icariin showed a significant concentration-dependent decrease in mature osteoclast formation (Fig. 1AeC). Additionally, to determine if the inhibitory effects of icariin were due to cytotoxicity, we used the CCK-8 assay to measure the effect of icariin on BMMs proliferation and survival. Our data indicated that the cell viability was distinct affected by treatment with icariin at concentrations of 103, 10-2 and 101 M (Fig. 1D). The inhibitory effect of icariin on osteoclastogenesis was further confirmed in RAW264.7 cells (Fig. 1EeH). 3.2. Icariin inhibits osteoclast function in vitro The formation of highly polarized F-actin rings and bone resorption are essential for osteoclast function. Therefore, we first explored the impact of icariin on F-actin ring formation in RANKLinduced osteoclasts that were derived from RAW264.7 cells. F-actin ring formation was inhibited in a concentration-dependent manner in cells treated with different concentrations of icariin (0, 107, 105 M; Fig. 2A and B). Next, to further test the effect of icariin on osteoclast-mediated bone resorption, we seeded equal numbers of RAW264.7 cell-derived pre-osteoclasts (4e5 days RANKL
Fig. 2. Icariin inhibited F-actin ring formation and osteoclast-mediated bone resorption. (A) RAW264.7 cells were treated with different concentrations of icariin in the presence of RANKL, and incubated until mature osteoclasts were observed. F-actin rings and cell nuclei were stained with tetramethylrhodamine isothiocyanate-coupled phalloidin and DAPI, respectively, and observed under a confocal microscope. (B) Quantification of F-actin rings. (C) RAW264.7 cells were stimulated with RANKL, and different concentrations of icariin until mature osteoclasts were formed. Scanning electron microscope images of bone resorption pits. (D) Percentage of resorption area. * P < 0.05; **P < 0.01, versus the control.
stimulation) onto bovine bone slices, and treated these slices with different concentrations of icariin (0,107, 105 M) for 48 h. We observed a significant decrease in bone resorption pits in mature osteoclasts treated with icariin compared to that in untreated controls (Fig. 2C and D). 3.3. Icariin inhibits the expression of osteoclastogenesis-related markers RANKL works synergistically with NF-kB to activate signaling molecules such as NFATc1, c-fos, and activator protein 1(AP-1), which are considered master controllers and are critical factors for osteoclast differentiation [20e22]. Among the osteoclastassociated genes induced by NF-kB and NFATc1, TRAP and cathepsin K play a significant role in bone resorption [23,24]. RANKL-induced expression of NFATc1, c-fos, TRAP, and cathepsin K was conspicuously inhibited by icariin in a dose-dependent manner (Fig. 3A and B).
Fig. 1. Icariin inhibited RANKL-induced osteoclast formation in a concentrationdependent manner without any cytotoxicity. (A) Bone marrow macrophages (BMMs) were treated with various concentrations of icariin followed by M-CSF(30 ng/mL) and RANKL (50 ng/mL) stimulation for 5 days. Cells were then fixed with 4% paraformaldehyde and subjected to tartrate-resistant acid phosphatase (TRAP) staining. (B, C) The number and spread area of TRAP-positive multinuclear cells was measured. (D) BMMs were plated in 96-well plates, and stimulated with M-CSF (30 ng/mL) and different concentrations of icariin for 48 h. Cell viability was measured using the CCK-8 assay. (E) RAW264.7 cells were treated with various concentrations of icariin followed by RANKL (50 ng/mL) stimulation for five days. Cells were then fixed with 4% paraformaldehyde and subjected to TRAP staining. (F, G) The number and spread area of TRAP-positive multinuclear cells was measured. (H) RAW264.7 cells were plated in 96well plates with different concentrations of icariin for 48 h. Cell viability was measured using the CCK-8 assay. All experiments were carried out at least three times, and statistical significance was determined using an unpaired Student s t-test. *P < 0.05; ** P < 0.01, versus the control.
3.4. Icariin inhibits the RANKL-induced activation of NF-kB and MAPK during osteoclastogenesis To examine potential mechanisms through which icariin modulates signaling pathways, we performed western blotting to measure the effect of icariin on the expression of various signaling proteins. To further explore the effects of icariin on the NF-kB signaling pathways involved in RANKL-induced osteoclast formation, we assessed the impact of icariin on RANKL-mediated NF-kBIkBa expression in RAW264.7 cells. It was expected that RANKL treatment would significantly decrease the level of IkBa. However, IkBa degradation was significantly impaired in the presence of
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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icariin (Fig. 4A and B). Additionally, we further explored the effect of icariin on MAPK activation in RAW264.7 cells. The three major MAPK cascades (P38, ERK, and JNK) were all suppressed, and further quantitative analysis showed a significant decrease in phosphorylated MAPK after treatment with icariin compared to that in the control group (Fig. 4A and B). Collectively, icariin inhibits osteoclast formation by regulating NF-kB and MAPK signaling pathways. 4. Discussion
Fig. 3. Icariin inhibits the expression of osteoclastogenesis-related markers. (A) RAW264.7 cells were cultured with or without the indicated concentrations of icariin in the presence of RANKL(50 ng/mL) for 96 h. Western blot was performed to measure the expression of NFATc1, c-fos,TRAP, and cathepsin K. (B) The levels of NFATc1, c-fos, TRAP, and cathepsin K were quantified using Image Lab software. All experiments were performed at least three times, and statistical significance was determined using an unpaired Student s t-test. *P < 0.05; **P < 0.01, versus the control.
Fig. 4. Icariin inhibits RANKL-induced activation of NF-kB and MAPK signaling pathways. (A) RAW264.7 cells were pre-incubated for 30 min with indicated concentrations of icariin, and then stimulated by the addition of RANKL (50 ng/mL) for 1 h. Western blot was used to quantitatively determine the protein expression of IkBa, phosphorylated ERK(p-ERK), ERK, p-JNK, JNK, p-p38, p38, and GAPDH. (B) Densitometric analysis for protein quantification was performed using Image Lab software, and the following values were calculated: IkBa/GAPDH, p-ERK/ERK, p-JNK/JNK, p-p38/p38. * P < 0.05; **P < 0.01, versus the RANKL-treated control group.
Bone remodeling is mediated by two basic physiological processes, osteoblastogenesis and osteoclastogenesis, which comprise highly coordinated, dynamic, and constant homeostasis of bone formation and bone resorption [25e27]. Long-term excessive osteoclast formation disrupts this balance, resulting in skeletal diseases such as osteoporosis [27]. Therefore, we urgently need feasible therapeutic strategies to treat osteoporosis by inhibiting osteoclast formation. In this study, we demonstrated that icariin had a beneficial effect on the suppression of osteoclast formation and resorption activity. Downstream regulatory factors such as c-fos [28] and NFATc1 [21] play key roles in osteoclastogenesis. Among them, NFATc1 is considered a critical factor for the regulation of many osteoclastspecific genes such as TRAP and cathepsin K [29,30]. In the present study, we found that RANKL-induced expression of NFATc1, cfos, and cathepsin K proteins were downregulated after treatment with icariin. This finding explains our observation that icariin inhibits RANKL-induced expression of osteoclast transcription factors c-Fos and NFATc1, as well as the suppression of downstream osteoclast-specific markers. The binding of RANKL to its receptor, RANK, leads to the recruitment of adaptor molecules such as TNF receptor-associated factor 6 (TRAF6) [31], followed by the activation of signaling pathways, including the MAPK and NF-kB pathways [32,33]. In the classical NF-kB pathway, the activated IkB kinase complex induces the phosphorylation and degradation of IkBa, which leads to the release and phosphorylation of NF-kB component proteins, followed by nuclear translocation of NF-kB and binding to a DNA target site to activate the expression of genes involved in osteoclastogenesis [34]. Studies have confirmed that knocking out the p65 gene results in osteopetrosis due to the lack of osteoclast formation, proving the important role of NF-kB signaling in osteoclast differentiation [35]. In the present study, we demonstrated that icariin significantly inhibits NF-kB activation in RANKL-induced RAW264.7 cells by degradation of IkBa. Thus, these data suggest that icariin inhibits the activation of the NF-kB signaling pathway, resulting in the inhibition of osteoclast formation. Furthermore, the MAPK signaling pathway plays an essential in inducing and activating osteoclast formation and function, mainly via three major signaling cascades: ERK-MAPK, JNK-MAPK, and p38-MAPK [13]. In the current study, we found that pre-treatment with icariin inhibited the RANKL-induced phosphorylation of all three MAPKs in RAW264.7 cells, which suggested that the inhibitory effect of icariin on osteoclast differentiation may be mediated via the regulation of MAPK phosphorylation in mature osteoclasts. However, the interaction between ERK-MAPK, JNK-MAPK, and p 38-MAPK pathways and the precise mechanisms via which these pathways are involved in osteoclast formation and function remain unclear and should be investigated in future studies. In conclusion, our findings demonstrate that icariin inhibits RANKL-induced osteoclast formation and function in vitro. Moreover, these inhibitory effects of icariin occur through the suppression of NF-kB and MAPK activation. Taken together, these findings
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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indicate that icariin represents a promising agent for the treatment of osteoclast-related diseases such as osteoporosis.
[14]
Authors’ contribution MD and BZ designed the study. QX and Gp C performed the experiments. Xq L analyzed the data. QX wrote the paper. BZ, and MD reviewed and edited the manuscript. All authors read and approved the manuscript.
[15]
Competing interests
[16]
The authors declare that they have no competing interests.
[17]
Abbreviations
[18]
BMM, bone marrow macrophage; MAPK, mitogen-activated protein kinase; M-CSF, macrophage colony-stimulating factor; NAFTc1, nuclear factor of activated T cells c1; NF-kB, nuclear factorkB; PVDF, polyvinylidene difluoride; RANKL, receptor activator of nuclear factor-kB ligand; TNF, tumor necrosis factor; TRAF6, TNF receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase.
[19]
[20] [21]
[22]
Acknowledgements [23]
All research costs were supplied by the following two project grants:National Natural Science Foundation for Youths (Grant No.81601912). National Natural Science Foundation (Grant No.81660365). References
[24] [25] [26] [27]
[1] T.D. Rachner, S. Khosla, L.C. Hofbauer, Osteoporosis: now and the future, Lancet 377 (2011) 1276e1287. [2] I.M. Alexander, Pharmacotherapeutic management of osteoprosis and osteopenia, Nurs. Pract. 34 (2009) 30e40, quiz 41. [3] G.A. Rodan, T.J. Martin, Therapeutic approaches to bone diseases, Science 289 (2000) 1508e1514. [4] G. Holt, C. Murnaghan, J. Reilly, et al., The biology of aseptic osteolysis, Clin. Orthop. Relat. Res. 460 (2007) 240e252. [5] Y.Y. Kong, W.J. Boyle, J.M. Penninger, Osteoprotegerin ligand: a regulator of immune responses and bone physiology, Immunol. Today 21 (2000) 495e502. [6] T.C. Phan, J. Xu, M.H. Zheng, Interaction between osteoblast and osteoclast: impact in bone disease, Histol. Histopathol. 19 (2004) 1325e1344. [7] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation, Nature 423 (2003) 337e342. [8] T. Nakashima, M. Hayashi, H. Takayanagi, New insights into osteoclastogenic signaling mechanisms, Trends Endocrinol. Metabol. 23 (2012) 582e590. [9] H.G. Bone, R. Chapurlat, M.L. Brandi, et al., The effect of three or six years of denosumab exposure in women with postmenopausal osteoporosis: results from the FREEDOM extension, J. Clin. Endocrinol. Metab. 98 (2013) 4483e4492. [10] H. Fleisch, Bisphosphonates in osteoporosis, Eur. Spine J. 12 (2003) S142eS146. [11] E. Barrett-Connor, L. Mosca, P. Collins, et al., Effects of raloxifene on cardiovascular events and breast cancer in postmenopausal women, N. Engl. J. Med. 355 (2006) 125, 17. [12] X. Zhang, T. Liu, Y. Huang, et al., Icariin: does it have an osteoinductive potential for bone tissue engineering? Phytother Res. 28 (2014) 498e509. [13] N.D. Zhang, T. Han, B.K. Huang, et al., Traditional Chinese medicine formulas
[28]
[29]
[30]
[31]
[32]
[33]
[34] [35]
5
for the treatment of osteoporosis: implication for antiosteoporotic drug discovery, J. Ethnopharmacol. 189 (2016) 61e80. (a) L.G. Ming, K.M. Chen, C.J. Xian, Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling, J. Cell. Physiol. 228 (2013) 513e521; (b) Z. Wang, D. Wang, D. Yang, et al., The effect of icariin on bone metabolism and its potential clinical application, Osteoporos. Int. 29 (2018) 535e544; (c) H. Nian, M.H. Ma, S.S. Nian, et al., Antiosteoporotic activity of icariin in ovariectomized rats, Phytomedicine 16 (2009) 320e326. G. Zhang, L. Qin, Y. Shi, Epimedium-derived phytoestrogen flavonoids exert beneficial effect on preventing bone loss in late postmenopausal women: a 24-month randomized, double-blind and placebo-controlled trial, J. Bone Miner. Res. 22 (2007) 1072e1079. J. Cui, M. Zhu, S. Zhu, et al., Inhibitory effect of icariin on Ti-induced inflammatory osteoclastogenesis, J. Surg. Res. 192 (2014) 447e453. M.H. Liu, J.S. Sun, S.W. Tsai, et al., Icariin protects murine chondrocytes from lipopolysaccharide-induced inflammatory responses and extracellular matrix degradation, Nutr. Res. 30 (2010) 57e65. C.Q. Xu, B.J. Liu, J.F. Wu, et al., Icariin attenuates LPS-induced acute inflammatory responses: involvement of PI3K/Akt and NF-kappaB signaling pathway, Eur. J. Pharmacol. 642 (2010) 146e153. X. Liu, J.F. Chin, X. Qu, et al., The beneficial effect of praeruptorin C on osteoporotic bone in ovariectomized mice via suppression of osteoclast formation and bone resorption, Front. Pharmacol. 8 (2017) 627. H. Takayanagi, The role of NFAT in osteoclast formation, Ann. N. Y. Acad. Sci. 1116 (2007) 227e237. H. Takayanagi, S. Kim, T. Koga, et al., Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts, Dev. Cell 3 (2002) 889e901. T. Yamashita, Z. Yao, F. Li, et al., NF-kappaB p50 and p52 regulate receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor-induced osteoclast precursor differentiation by activating c-Fos and NFATc1, J. Biol. Chem. 282 (2007) 18245e18253. K. Harada, H. Itoh, Y. Kawazoe, et al., Polyphosphate-mediated inhibition of tartrate-resistant acid phosphatase and suppression of bone resorption of osteoclasts, Plos One 8 (2013) e78612. Z. Li, M. Kienetz, M.M. Cherney, et al., The crystal and molecular structures of a cathepsin K: chondroitin sulfate complex, J. Mol. Biol. 383 (2008) 78e91. E. Seeman, Bone modeling and remodeling, Crit. Rev. Eukaryot. Gene Expr. 19 (2009) 219e233. V.D. Myneni, E. Mezey, Regulation of bone remodeling by vitamin K2, Oral Dis. 23 (2017) 1021e1028. H.J. Kim, H.J. Yoon, S.Y. Kim, et al., A medium-chain fatty acid, capric acid, inhibits RANKL-induced osteoclast differentiation via the suppression of NFkappaB signaling and blocks cytoskeletal organization and survival in mature osteoclasts, Mol. Cell. 37 (2014) 598e604. P. Monje, J. Hernandez-Losa, R.J. Lyons, et al., Regulation of the transcriptional activity of c-Fos by ERK. A novel role for the prolyl isomerase PIN1, J. Biol. Chem. 280 (2005) 35081e35084. M. Matsumoto, M. Kogawa, S. Wada, et al., Essential role of p38 mitogenactivated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1, J. Biol. Chem. 279 (2004) 45969e45979. Y. Kim, K. Sato, M. Asagiri, et al., Contribution of nuclear factor of activated T cells c1 to the transcriptional control of immunoreceptor osteoclastassociated receptor but not triggering receptor expressed by myeloid cells-2 during osteoclastogenesis, J. Biol. Chem. 280 (2005) 32905e32913. B.G. Darnay, J. Ni, P.A. Moore, et al., Activation of NF-kappaB by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappaBinducing kinase. Identification of a novel TRAF6 interaction motif, J. Biol. Chem. 274 (1999) 7724e7731. G. Takaesu, J. Ninomiya-Tsuji, S. Kishida, et al., Interleukin-1 (IL-1) receptorassociated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway, Mol. Cell Biol. 21 (2001) 2475e2484. G. Pearson, F. Robinson, G.T. Beers, et al., Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions, Endocr. Rev. 22 (2001) 153e183. Y. Abu-Amer, NF-kB signaling and bone resorption, Osteoporos. Int. 24 (2013) 2377e2386. B.F. Boyce, L. Xing, Functions of RANKL/RANK/OPG in bone modeling and remodeling, Arch. Biochem. Biophys. 473 (2008) 139e146.
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways Qiang Xu a, b, 1, Guiping Chen a, b, 1, Xuqiang Liu a, b, Min Dai a, b, *, Bin Zhang a, b, ** a
Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Artificial Joints Engineering and Technology Research Center of Jiangxi Province, Nanchang, Jiangxi province, 330006, China b Multidisciplinary Therapy Center of Musculoskeletal Tumor, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi province, 330006, China
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a b s t r a c t
Article history: Received 20 November 2018 Accepted 29 November 2018 Available online xxx
The receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL)-RANK regulatory axis is a major regulator of osteoclast differentiation and activation. Icariin, a flavonol glycoside isolated from the Epimedium herb, has been reported to prevents bone loss in ovariectomized mice and inhibits wear particle-induced osteolysis. However, the molecular mechanism through which icariin inhibits RANKLinduced osteoclastogenesis has not been fully understood. Therefore, we aimed to investigate the effects of icariin on RANKL-induced osteoclastogenesis and to elucidate the mechanism underlying this effect. Our results showed that RANKL-induced osteoclastogenesis was inhibited by icariin in bone marrow macrophages (BMMs) and RAW264.7 cells, and that this effect was due to suppression of NF-kB and mitogen-activated protein kinase (MAPK) activation. In addition, icariin inhibited F-actin ring formation and attenuated the bone resorption ability of mature osteoclasts. Collectively, our results indicate that icariin may be a promising potential candidate for the treatment of osteolytic diseases such as osteoporosis. Moreover, our findings lay the foundation for understanding and intervening in osteoclastrelated diseases at the molecular level. © 2018 Elsevier Inc. All rights reserved.
Keywords: Icariin NF-kB RANKL MAPK Osteoclastogenesis Osteoporosis
1. Introduction Osteoporosis is an highly prevalent disease that imposes a serious economic burden on society. It is characterized by impaired bone mass and bone strength, destruction of bone microstructure, and increased bone fragility [1]. The increase in the estimated number of individuals with osteoporosis or osteopenia (a precursor to osteoporosis) is expected to continue, especially with the increase in the aging population [2]. Several studies have shown that osteoporosis is caused by excessive osteoclast activity that
* Corresponding author. Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Artificial Joints Engineering and Technology Research Center of Jiangxi Province, Nanchang, Jiangxi province, 330006, China. ** Corresponding author. Department of Orthopedics, The First Affiliated Hospital of Nanchang University, Artificial Joints Engineering and Technology Research Center of Jiangxi Province, Nanchang, Jiangxi province, 330006, China. E-mail addresses: [email protected] (Q. Xu), [email protected] (G. Chen), [email protected] (X. Liu), [email protected] (M. Dai), [email protected] (B. Zhang). 1 Qiang Xu, Guiping Chen contributed equally to this work and should be considered co-first authors.
contributes to pathological bone resorption [3e5]. Increased bone resorption indicates that osteoclasts play a key role in the development of osteoporosis, and therefore, they are considered a viable therapeutic target for osteoporosis treatment [6]. Osteoclasts are multinucleated cells that are generated through differentiation of hematopoietic progenitors of the monocyte/ macrophage lineage [7]. Receptor activator of nuclear factor-kB (NF-kB) ligand (RANKL) is the most important cytokine in the process of osteoclast differentiation. RANKL binds to its receptor RANK, which is expressed on the surface of osteoclast precursor cells, thereby inducing their differentiation and maturation into osteoclasts [7,8]. Thus, inhibition of osteoclast formation via modulation of the RANKL-induced signaling pathways is a viable therapeutic strategy for the treatment of osteoclast-associated diseases such as osteoporosis. Currently, the main treatments for osteoporosis include a humanized monoclonal antibody of RANKL, bisphosphonates, selective estrogen receptor modulators, and calcitonin. However, these treatments often produce adverse reactions, including osteonecrosis of the jaw, gastrointestinal discomfort, breast cancer, hypercalcemia, and cardiovascular diseases [9e11]. Thus, it is a clinical and social imperative to explore
https://doi.org/10.1016/j.bbrc.2018.11.201 0006-291X/© 2018 Elsevier Inc. All rights reserved.
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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novel agents for the treatment of osteoporosis in view of the current therapeutic drugs options. The deficiencies in current therapeutic options have led to an increasing interest in bioactive natural compounds that can be used as potential alternatives for osteoporosis treatment. Icariin is the most abundant flavonoid constituent in Herba Epimedii, and has been shown to be effective in bone fracture healing, joint disease, and improvement of gonadal function [12,13]. Studies have shown that icariin can play a dual role in restoring bone remodeling by stimulating bone formation while simultaneously inhibiting bone resorption [14]. Wang et al. reported that the icariin-mediated increase in bone formation is a result of multiple signal transduction pathways, including up-regulation of BMP, NO, MAPK, and Wnt pathways and down-regulation of the extracellular signalregulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways [15]. In addition, icariin increases bone mineral density in postmenopausal women and in rat models of ovariectomy-induced bone loss by inhibiting osteoclast formation [15]. Moreover, icariin has a preventive and therapeutic effect against wear particleinduced osteolysis and aseptic loosening [16]. Recently, icariin was shown to play an important role in negatively regulating inflammatory responses in vitro and in vivo [16]. Several studies showed that icariin also inhibits the expression of tumor necrosis factor (TNF)-a and interleukin(IL)-1b [17,18]. However, the molecular mechanism by which icariin interacts with RANKL-induced osteoclastogenesis has not been reported in vitro. Therefore, the purpose of this study was to investigate the effects of icariin on RANKL-induced osteoclast formation, and to elaborate the potential mechanisms underlying this effect. 2. Materials and methods 2.1. Cell viability assay In order to evaluate the cytotoxic effect of icariin, we used the Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Inc., Kumamoto, Japan) according to the manufacturer's instructions, as reported previously [19]. Briefly, bone marrow macrophages (BMMs) were added to 96-well plates at 8 103 cells/well in triplicate and cultured for 24 h in alpha modification of Eagle's medium (a-MEM, Gaithersburg, MD, USA) containing 30 ng/mL macrophage colony-stimulating factor (M-CSF; PeproTech, Rocky Hill, NJ, USA), 10% fetal bovine serum (FBS; Gibco-BRL, Sydney, Australia), and 1% penicillin/streptomycin. BMMs were cultured with the following concentrations of icariin for 48 h: 0, 107, 106, 105, 104, 103, 102, and 101 M. Next, 10 mL of CCK-8 substrate was added to each well, and the plate was incubated at 37 C under 5% CO2 for 2 h. The absorbance of each well was measured at 450 nm with an ELX800 microplate reader (Bio-Tek Instruments Inc., Winooski, VT, USA). The cell viability relative to the control group was calculated using the following formula: (experimental group optimal density (OD) e blank OD)/(control group OD e blank OD). Similarly, the toxic effects of icariin were also tested on the RAW264.7 cell line. 2.2. Osteoclast differentiation assay BMMs were seeded in a 96-well plate at a density of 8 103 cells/well in a-MEM with 30 ng/mL M-CSF, 50 ng/mL RANKL (PeproTech, Rocky Hill, NJ, USA), and different concentrations of icariin (0, 107, 106, and 105 M). In addition, RAW264.7 cells cultured at 2 103 cells/well in a 96-well plate were treated with icariin at various concentrations (0, 107, 106, and 105 M) in the presence of 50 ng/mL RANKL. Both were supplemented with fresh medium every 2 days until mature osteoclasts were observed. Next, the cells were fixed with 4% paraformaldehyde and stained for
tartrate-resistant acid phosphatase (TRAP; Sigma-Aldrich, St. Louis, MO, USA) activity. The number of mature osteoclasts (TRAP-positive cells with 3 nuclei) was counted, and their spread area was measured. 2.3. F-actin ring evaluation with immunofluorescence confocal microscopy Osteoclasts derived from RAW264.7 cells were induced by RANKL, cultured on glass coverslips, and treated with different icariin concentrations (0, 107, and 105 M) until mature osteoclasts were observed in the control wells. Cells were fixed for 20 min at room temperature with 4% paraformaldehyde, permeabilized for 5 min with 0.1% Triton X-100, and washed three times with phosphate-buffered saline (PBS). F-actin was stained with tetramethylrhodamine isothiocyanate-coupled phalloidin(Invitrogen, SanDiego, CA, United States), nuclei were stained with 40 ,6-diamidino-2-phenylindole(DAPI), and then the coverslips were mounted on microscope slides with ProLong Diamond Antifade Mounting medium (Invitrogen). Images were obtained using a LSM5 confocal microscope (Carl Zeiss, Oberkochen, Germany) and analyzed using Zeiss ZEN software. 2.4. Bone resorption pit assay Bovine bone slices were placed in a 96-well plate, and then RAW264.7 cells were added at 2 103 cells/well in Dulbecco's Modified Eagle's Medium(DMEM) containing 50 ng/mL RANKL. Cells were treated with 0, 107, and 105 M icariin for 48 h, at which point mature osteoclasts were observed. The cells adhering to the bone slices were removed with a brush. Resorption pits were visualized using a scanning electron microscope (SEM, FEI Quanta 250), and the proportion area of resorbed bone slices was computed using Image J software (National Institutes of Health, Bethesda, MD, USA). 2.5. Western blot RAW264.7 cells were lysed by adding RIPA lysis buffer (Applygen Technologies Inc, Beijing, China) containing protease inhibitor (Sigma, Rockford, USA) and phosphatase inhibitor (Sigma, Rockford, USA) in concentrations specified by the respective manufacturers. The lysate was centrifuged at 12,000 g for 10 min, and the supernatant was collected. Protein concentration was determined using the bicinchoninic acid assay. The samples were then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The PVDF membranes were blocked with 5% skim milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TBS-T), and then incubated overnight at 4 C with specific antibodies against nuclear factor of activated T cells c1 (NFATc1), c-fos, TRAP, cathepsin K, IkBa, p-ERK1/2, ERK 1/2, p-JNK1/2, JNK 1/2, p-p38, p38, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Cambridge, MA, USA). The membranes were washed in TBS-T, and then incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the membranes were washed with TBS-T, and developed using the enhanced chemiluminescence Western Blot Detection Reagent kit (GE Healthcare, Parramatta, Australia). Odyssey image scanning software v3.0 (LiCOR Biosciences, Lincoln, NE, USA) was used to obtain images of the membranes, and the protein bands were subjected to densitometric analysis with Image J software.
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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2.6. Statistical analysis All data were obtained from three or more independent experiments. The data from the cell experiments are expressed as the means ± standard deviations. Data were analyzed using unpaired Student's t-test with SPSS 23.0 software (SPSS Inc., Chicago, USA). Pvalues < 0.05 were considered statistically significant. 3. Results 3.1. Icariin inhibits RANKL-induced osteoclast formation without any cytotoxicity To examine the effect of icariin on RANKL-induced osteoclastogenesis, BMMs were stimulated with M-CSF and RANKL in the presence of different concentrations of icariin (0, 107, 106, and 105 M). Interestingly, the BMMs treated with icariin showed a significant concentration-dependent decrease in mature osteoclast formation (Fig. 1AeC). Additionally, to determine if the inhibitory effects of icariin were due to cytotoxicity, we used the CCK-8 assay to measure the effect of icariin on BMMs proliferation and survival. Our data indicated that the cell viability was distinct affected by treatment with icariin at concentrations of 103, 10-2 and 101 M (Fig. 1D). The inhibitory effect of icariin on osteoclastogenesis was further confirmed in RAW264.7 cells (Fig. 1EeH). 3.2. Icariin inhibits osteoclast function in vitro The formation of highly polarized F-actin rings and bone resorption are essential for osteoclast function. Therefore, we first explored the impact of icariin on F-actin ring formation in RANKLinduced osteoclasts that were derived from RAW264.7 cells. F-actin ring formation was inhibited in a concentration-dependent manner in cells treated with different concentrations of icariin (0, 107, 105 M; Fig. 2A and B). Next, to further test the effect of icariin on osteoclast-mediated bone resorption, we seeded equal numbers of RAW264.7 cell-derived pre-osteoclasts (4e5 days RANKL
Fig. 2. Icariin inhibited F-actin ring formation and osteoclast-mediated bone resorption. (A) RAW264.7 cells were treated with different concentrations of icariin in the presence of RANKL, and incubated until mature osteoclasts were observed. F-actin rings and cell nuclei were stained with tetramethylrhodamine isothiocyanate-coupled phalloidin and DAPI, respectively, and observed under a confocal microscope. (B) Quantification of F-actin rings. (C) RAW264.7 cells were stimulated with RANKL, and different concentrations of icariin until mature osteoclasts were formed. Scanning electron microscope images of bone resorption pits. (D) Percentage of resorption area. * P < 0.05; **P < 0.01, versus the control.
stimulation) onto bovine bone slices, and treated these slices with different concentrations of icariin (0,107, 105 M) for 48 h. We observed a significant decrease in bone resorption pits in mature osteoclasts treated with icariin compared to that in untreated controls (Fig. 2C and D). 3.3. Icariin inhibits the expression of osteoclastogenesis-related markers RANKL works synergistically with NF-kB to activate signaling molecules such as NFATc1, c-fos, and activator protein 1(AP-1), which are considered master controllers and are critical factors for osteoclast differentiation [20e22]. Among the osteoclastassociated genes induced by NF-kB and NFATc1, TRAP and cathepsin K play a significant role in bone resorption [23,24]. RANKL-induced expression of NFATc1, c-fos, TRAP, and cathepsin K was conspicuously inhibited by icariin in a dose-dependent manner (Fig. 3A and B).
Fig. 1. Icariin inhibited RANKL-induced osteoclast formation in a concentrationdependent manner without any cytotoxicity. (A) Bone marrow macrophages (BMMs) were treated with various concentrations of icariin followed by M-CSF(30 ng/mL) and RANKL (50 ng/mL) stimulation for 5 days. Cells were then fixed with 4% paraformaldehyde and subjected to tartrate-resistant acid phosphatase (TRAP) staining. (B, C) The number and spread area of TRAP-positive multinuclear cells was measured. (D) BMMs were plated in 96-well plates, and stimulated with M-CSF (30 ng/mL) and different concentrations of icariin for 48 h. Cell viability was measured using the CCK-8 assay. (E) RAW264.7 cells were treated with various concentrations of icariin followed by RANKL (50 ng/mL) stimulation for five days. Cells were then fixed with 4% paraformaldehyde and subjected to TRAP staining. (F, G) The number and spread area of TRAP-positive multinuclear cells was measured. (H) RAW264.7 cells were plated in 96well plates with different concentrations of icariin for 48 h. Cell viability was measured using the CCK-8 assay. All experiments were carried out at least three times, and statistical significance was determined using an unpaired Student s t-test. *P < 0.05; ** P < 0.01, versus the control.
3.4. Icariin inhibits the RANKL-induced activation of NF-kB and MAPK during osteoclastogenesis To examine potential mechanisms through which icariin modulates signaling pathways, we performed western blotting to measure the effect of icariin on the expression of various signaling proteins. To further explore the effects of icariin on the NF-kB signaling pathways involved in RANKL-induced osteoclast formation, we assessed the impact of icariin on RANKL-mediated NF-kBIkBa expression in RAW264.7 cells. It was expected that RANKL treatment would significantly decrease the level of IkBa. However, IkBa degradation was significantly impaired in the presence of
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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icariin (Fig. 4A and B). Additionally, we further explored the effect of icariin on MAPK activation in RAW264.7 cells. The three major MAPK cascades (P38, ERK, and JNK) were all suppressed, and further quantitative analysis showed a significant decrease in phosphorylated MAPK after treatment with icariin compared to that in the control group (Fig. 4A and B). Collectively, icariin inhibits osteoclast formation by regulating NF-kB and MAPK signaling pathways. 4. Discussion
Fig. 3. Icariin inhibits the expression of osteoclastogenesis-related markers. (A) RAW264.7 cells were cultured with or without the indicated concentrations of icariin in the presence of RANKL(50 ng/mL) for 96 h. Western blot was performed to measure the expression of NFATc1, c-fos,TRAP, and cathepsin K. (B) The levels of NFATc1, c-fos, TRAP, and cathepsin K were quantified using Image Lab software. All experiments were performed at least three times, and statistical significance was determined using an unpaired Student s t-test. *P < 0.05; **P < 0.01, versus the control.
Fig. 4. Icariin inhibits RANKL-induced activation of NF-kB and MAPK signaling pathways. (A) RAW264.7 cells were pre-incubated for 30 min with indicated concentrations of icariin, and then stimulated by the addition of RANKL (50 ng/mL) for 1 h. Western blot was used to quantitatively determine the protein expression of IkBa, phosphorylated ERK(p-ERK), ERK, p-JNK, JNK, p-p38, p38, and GAPDH. (B) Densitometric analysis for protein quantification was performed using Image Lab software, and the following values were calculated: IkBa/GAPDH, p-ERK/ERK, p-JNK/JNK, p-p38/p38. * P < 0.05; **P < 0.01, versus the RANKL-treated control group.
Bone remodeling is mediated by two basic physiological processes, osteoblastogenesis and osteoclastogenesis, which comprise highly coordinated, dynamic, and constant homeostasis of bone formation and bone resorption [25e27]. Long-term excessive osteoclast formation disrupts this balance, resulting in skeletal diseases such as osteoporosis [27]. Therefore, we urgently need feasible therapeutic strategies to treat osteoporosis by inhibiting osteoclast formation. In this study, we demonstrated that icariin had a beneficial effect on the suppression of osteoclast formation and resorption activity. Downstream regulatory factors such as c-fos [28] and NFATc1 [21] play key roles in osteoclastogenesis. Among them, NFATc1 is considered a critical factor for the regulation of many osteoclastspecific genes such as TRAP and cathepsin K [29,30]. In the present study, we found that RANKL-induced expression of NFATc1, cfos, and cathepsin K proteins were downregulated after treatment with icariin. This finding explains our observation that icariin inhibits RANKL-induced expression of osteoclast transcription factors c-Fos and NFATc1, as well as the suppression of downstream osteoclast-specific markers. The binding of RANKL to its receptor, RANK, leads to the recruitment of adaptor molecules such as TNF receptor-associated factor 6 (TRAF6) [31], followed by the activation of signaling pathways, including the MAPK and NF-kB pathways [32,33]. In the classical NF-kB pathway, the activated IkB kinase complex induces the phosphorylation and degradation of IkBa, which leads to the release and phosphorylation of NF-kB component proteins, followed by nuclear translocation of NF-kB and binding to a DNA target site to activate the expression of genes involved in osteoclastogenesis [34]. Studies have confirmed that knocking out the p65 gene results in osteopetrosis due to the lack of osteoclast formation, proving the important role of NF-kB signaling in osteoclast differentiation [35]. In the present study, we demonstrated that icariin significantly inhibits NF-kB activation in RANKL-induced RAW264.7 cells by degradation of IkBa. Thus, these data suggest that icariin inhibits the activation of the NF-kB signaling pathway, resulting in the inhibition of osteoclast formation. Furthermore, the MAPK signaling pathway plays an essential in inducing and activating osteoclast formation and function, mainly via three major signaling cascades: ERK-MAPK, JNK-MAPK, and p38-MAPK [13]. In the current study, we found that pre-treatment with icariin inhibited the RANKL-induced phosphorylation of all three MAPKs in RAW264.7 cells, which suggested that the inhibitory effect of icariin on osteoclast differentiation may be mediated via the regulation of MAPK phosphorylation in mature osteoclasts. However, the interaction between ERK-MAPK, JNK-MAPK, and p 38-MAPK pathways and the precise mechanisms via which these pathways are involved in osteoclast formation and function remain unclear and should be investigated in future studies. In conclusion, our findings demonstrate that icariin inhibits RANKL-induced osteoclast formation and function in vitro. Moreover, these inhibitory effects of icariin occur through the suppression of NF-kB and MAPK activation. Taken together, these findings
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201
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indicate that icariin represents a promising agent for the treatment of osteoclast-related diseases such as osteoporosis.
[14]
Authors’ contribution MD and BZ designed the study. QX and Gp C performed the experiments. Xq L analyzed the data. QX wrote the paper. BZ, and MD reviewed and edited the manuscript. All authors read and approved the manuscript.
[15]
Competing interests
[16]
The authors declare that they have no competing interests.
[17]
Abbreviations
[18]
BMM, bone marrow macrophage; MAPK, mitogen-activated protein kinase; M-CSF, macrophage colony-stimulating factor; NAFTc1, nuclear factor of activated T cells c1; NF-kB, nuclear factorkB; PVDF, polyvinylidene difluoride; RANKL, receptor activator of nuclear factor-kB ligand; TNF, tumor necrosis factor; TRAF6, TNF receptor-associated factor 6; TRAP, tartrate-resistant acid phosphatase.
[19]
[20] [21]
[22]
Acknowledgements [23]
All research costs were supplied by the following two project grants:National Natural Science Foundation for Youths (Grant No.81601912). National Natural Science Foundation (Grant No.81660365). References
[24] [25] [26] [27]
[1] T.D. Rachner, S. Khosla, L.C. Hofbauer, Osteoporosis: now and the future, Lancet 377 (2011) 1276e1287. [2] I.M. Alexander, Pharmacotherapeutic management of osteoprosis and osteopenia, Nurs. Pract. 34 (2009) 30e40, quiz 41. [3] G.A. Rodan, T.J. Martin, Therapeutic approaches to bone diseases, Science 289 (2000) 1508e1514. [4] G. Holt, C. Murnaghan, J. Reilly, et al., The biology of aseptic osteolysis, Clin. Orthop. Relat. Res. 460 (2007) 240e252. [5] Y.Y. Kong, W.J. Boyle, J.M. Penninger, Osteoprotegerin ligand: a regulator of immune responses and bone physiology, Immunol. Today 21 (2000) 495e502. [6] T.C. Phan, J. Xu, M.H. Zheng, Interaction between osteoblast and osteoclast: impact in bone disease, Histol. Histopathol. 19 (2004) 1325e1344. [7] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation, Nature 423 (2003) 337e342. [8] T. Nakashima, M. Hayashi, H. Takayanagi, New insights into osteoclastogenic signaling mechanisms, Trends Endocrinol. Metabol. 23 (2012) 582e590. [9] H.G. Bone, R. Chapurlat, M.L. Brandi, et al., The effect of three or six years of denosumab exposure in women with postmenopausal osteoporosis: results from the FREEDOM extension, J. Clin. Endocrinol. Metab. 98 (2013) 4483e4492. [10] H. Fleisch, Bisphosphonates in osteoporosis, Eur. Spine J. 12 (2003) S142eS146. [11] E. Barrett-Connor, L. Mosca, P. Collins, et al., Effects of raloxifene on cardiovascular events and breast cancer in postmenopausal women, N. Engl. J. Med. 355 (2006) 125, 17. [12] X. Zhang, T. Liu, Y. Huang, et al., Icariin: does it have an osteoinductive potential for bone tissue engineering? Phytother Res. 28 (2014) 498e509. [13] N.D. Zhang, T. Han, B.K. Huang, et al., Traditional Chinese medicine formulas
[28]
[29]
[30]
[31]
[32]
[33]
[34] [35]
5
for the treatment of osteoporosis: implication for antiosteoporotic drug discovery, J. Ethnopharmacol. 189 (2016) 61e80. (a) L.G. Ming, K.M. Chen, C.J. Xian, Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling, J. Cell. Physiol. 228 (2013) 513e521; (b) Z. Wang, D. Wang, D. Yang, et al., The effect of icariin on bone metabolism and its potential clinical application, Osteoporos. Int. 29 (2018) 535e544; (c) H. Nian, M.H. Ma, S.S. Nian, et al., Antiosteoporotic activity of icariin in ovariectomized rats, Phytomedicine 16 (2009) 320e326. G. Zhang, L. Qin, Y. Shi, Epimedium-derived phytoestrogen flavonoids exert beneficial effect on preventing bone loss in late postmenopausal women: a 24-month randomized, double-blind and placebo-controlled trial, J. Bone Miner. Res. 22 (2007) 1072e1079. J. Cui, M. Zhu, S. Zhu, et al., Inhibitory effect of icariin on Ti-induced inflammatory osteoclastogenesis, J. Surg. Res. 192 (2014) 447e453. M.H. Liu, J.S. Sun, S.W. Tsai, et al., Icariin protects murine chondrocytes from lipopolysaccharide-induced inflammatory responses and extracellular matrix degradation, Nutr. Res. 30 (2010) 57e65. C.Q. Xu, B.J. Liu, J.F. Wu, et al., Icariin attenuates LPS-induced acute inflammatory responses: involvement of PI3K/Akt and NF-kappaB signaling pathway, Eur. J. Pharmacol. 642 (2010) 146e153. X. Liu, J.F. Chin, X. Qu, et al., The beneficial effect of praeruptorin C on osteoporotic bone in ovariectomized mice via suppression of osteoclast formation and bone resorption, Front. Pharmacol. 8 (2017) 627. H. Takayanagi, The role of NFAT in osteoclast formation, Ann. N. Y. Acad. Sci. 1116 (2007) 227e237. H. Takayanagi, S. Kim, T. Koga, et al., Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts, Dev. Cell 3 (2002) 889e901. T. Yamashita, Z. Yao, F. Li, et al., NF-kappaB p50 and p52 regulate receptor activator of NF-kappaB ligand (RANKL) and tumor necrosis factor-induced osteoclast precursor differentiation by activating c-Fos and NFATc1, J. Biol. Chem. 282 (2007) 18245e18253. K. Harada, H. Itoh, Y. Kawazoe, et al., Polyphosphate-mediated inhibition of tartrate-resistant acid phosphatase and suppression of bone resorption of osteoclasts, Plos One 8 (2013) e78612. Z. Li, M. Kienetz, M.M. Cherney, et al., The crystal and molecular structures of a cathepsin K: chondroitin sulfate complex, J. Mol. Biol. 383 (2008) 78e91. E. Seeman, Bone modeling and remodeling, Crit. Rev. Eukaryot. Gene Expr. 19 (2009) 219e233. V.D. Myneni, E. Mezey, Regulation of bone remodeling by vitamin K2, Oral Dis. 23 (2017) 1021e1028. H.J. Kim, H.J. Yoon, S.Y. Kim, et al., A medium-chain fatty acid, capric acid, inhibits RANKL-induced osteoclast differentiation via the suppression of NFkappaB signaling and blocks cytoskeletal organization and survival in mature osteoclasts, Mol. Cell. 37 (2014) 598e604. P. Monje, J. Hernandez-Losa, R.J. Lyons, et al., Regulation of the transcriptional activity of c-Fos by ERK. A novel role for the prolyl isomerase PIN1, J. Biol. Chem. 280 (2005) 35081e35084. M. Matsumoto, M. Kogawa, S. Wada, et al., Essential role of p38 mitogenactivated protein kinase in cathepsin K gene expression during osteoclastogenesis through association of NFATc1 and PU.1, J. Biol. Chem. 279 (2004) 45969e45979. Y. Kim, K. Sato, M. Asagiri, et al., Contribution of nuclear factor of activated T cells c1 to the transcriptional control of immunoreceptor osteoclastassociated receptor but not triggering receptor expressed by myeloid cells-2 during osteoclastogenesis, J. Biol. Chem. 280 (2005) 32905e32913. B.G. Darnay, J. Ni, P.A. Moore, et al., Activation of NF-kappaB by RANK requires tumor necrosis factor receptor-associated factor (TRAF) 6 and NF-kappaBinducing kinase. Identification of a novel TRAF6 interaction motif, J. Biol. Chem. 274 (1999) 7724e7731. G. Takaesu, J. Ninomiya-Tsuji, S. Kishida, et al., Interleukin-1 (IL-1) receptorassociated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway, Mol. Cell Biol. 21 (2001) 2475e2484. G. Pearson, F. Robinson, G.T. Beers, et al., Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions, Endocr. Rev. 22 (2001) 153e183. Y. Abu-Amer, NF-kB signaling and bone resorption, Osteoporos. Int. 24 (2013) 2377e2386. B.F. Boyce, L. Xing, Functions of RANKL/RANK/OPG in bone modeling and remodeling, Arch. Biochem. Biophys. 473 (2008) 139e146.
Please cite this article as: Q. Xu et al., Icariin inhibits RANKL-induced osteoclastogenesis via modulation of the NF-kB and MAPK signaling pathways, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.11.201