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Stauntonia hexaphylla (Lardizabalaceae) leaf methanol extract inhibits osteoclastogenesis and bone resorption activity via proteasome-mediated degradation of c-Fos protein and suppression of NFATc1 expression
© Cheon et al. 2015
Received: 2 June 2015
Accepted: 3 August 2015
Published: 14 August 2015
Natural plants, including common vegetables and fruits, have been recognized as essential sources for drug discovery and the development of new, safe, and economical medicaments. Stauntonia hexaphylla (Lardizabalaceae) is widely distributed in Korea, Japan, and China, and is a popular herbal supplement in Korean and Chinese folk medicine owing to its analgesic, sedative, and diuretic properties. However, the exact pharmacological effects of S. hexaphylla extract, particularly its effect on osteoclastogenesis, are not known.
Osteoclast differentiation and function were identified with tartrate-resistant acid phosphatase (TRAP) staining and bone resorption assay, and the underling mechanisms were determined by real-time RT-PCR and western blot analysis.
S. hexaphylla was found to inhibit early-stage receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation in bone marrow macrophages (BMMs) without cytotoxicity and bone-resorbing activity in mature osteoclasts in a dose-dependent manner. This S. hexaphylla-mediated blockade of osteoclastogenesis involved abrogation of the NF-κB, ERK, and c-Src-Btk-PLCγ2 calcium signal pathways. Interestingly, we found that S. hexaphylla down-regulated RANKL-associated c-Fos protein induction by suppressing its translation. Furthermore, ectopic overexpression of c-Fos and NFATc1 rescued the inhibition of osteoclast differentiation by S. hexaphylla. Furthermore, S. hexaphylla inhibited the c-Fos- and NFATc1-regulated expression of genes required for osteoclastogenesis, such as TRAP, OSCAR, β3-integrin, ATP6v0d2, and CtsK.
These findings suggest that S. hexaphylla might be useful for the development of new anti-osteoporosis agents.
Osteoporosis, characterized by abnormally low bone mineral density and micro-architectural degeneration of bone tissue, is a progressive systemic skeletal disease with various causes, including menopause; advanced age; low calcium and vitamin D intake; and lack of exercise due to long-term bed rest, especially in the elderly population [1–3]. Current anti-osteoporosis drugs such as estrogen, raloxifene, bisphosphonates, and calcitonin have been widely used, and their bone protective mechanisms are based on the inhibition osteoclastic bone resorption. However, these drugs have numerous side effects, causing many patients to discontinue their use [4–7]. Hence, to prevent or reverse osteolytic bone diseases, it is necessary to develop novel agents that have fewer undesirable side effects.
Osteoclasts are specialized, multinucleated, and bone-resorbing cells that form part of the mononuclear phagocyte system . They are formed by the fusion of circulating mononuclear precursor cells of hematopoietic origin. Osteoclast differentiation is critically regulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL) [1, 8, 9]. M-CSF stimulates the expression of RANK, the receptor for RANKL, and supports the survival and proliferation of osteoclast precursors . RANKL stimulates differentiation of precursors into osteoclasts by inducing the expression of osteoclast-specific genes, including tartrate-resistant acid phosphatase (TRAP), osteoclast-associated receptor (OSCAR), and cathepsin K (CtsK) [1, 8, 10]. The RANK/RANKL pathway is considered to be an attractive therapeutic target for bone destructive diseases. Although a more detailed mechanism is still expected to be unveiled, the major signaling events have been elucidated: RANK/RANKL binding activates downstream early signaling pathways, including mitogen-activated protein kinase (MAPK) pathways and NF-κB, by recruitment of TNF-receptor associated factor 6 (TRAF6), which regulates transcription factors involved in osteoclast differentiation [11, 12]. Subsequently, RANKL induces the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), key transcription factors for osteoclastogenesis [13–15]. NFATc1 is a master regulator of terminal osteoclastogenesis, and autoamplification of NFATc1 depends on activator protein (AP)-1 complex containing c-Fos and NF-κB . In addition, the RANKL/RANK signaling pathway activates Ca2+ signaling through the activation of phospholipase Cγ (PLCγ) [16, 17]. PLCγ regulates protein kinase C (PKC) activation, intracellular Ca2+ levels, and NFATc1 expression in hematopoietic systems [16, 18]. During osteoclastogenesis, increased Ca2+ levels induce NFATc1 dephosphorylation and NFATc1 translocation into the nucleus. Calcineurin inhibitors, such as FK506 and cyclosporine A, as well as the Ca2+ chelator BAPTA-AM, potently suppress RANKL-induced osteoclastogenesis through inhibition of NFATc1 nuclear translocation .
Recently, many plants and their extracts have been recognized as useful sources for the prevention and treatment of bone-related disorders. Stauntonia hexaphylla (Lardizabalaceae) is widely distributed in thickets in lowlands and foothills in warmer regions of Korea, Japan, and China. S. hexaphylla has been used in Chinese folk medicine as analgesics, sedatives, and diuretics . As part of a search for bioactive compounds, methanol extract from the aerial part of S. hexaphylla was found to exhibit significant cytotoxic activity and anti-cancer effects on HCT116 human colon cancer cells . However, the exactly pharmacological effect of S. hexaphylla remains unknown; in particular, the effect of S. hexaphylla on osteoclast differentiation and pathological bone destruction has not yet been well defined. In this study, we have investigated the effects of S. hexaphylla on osteoclast differentiation and function, and its possible mechanism of action.
Reagents and antibodies
Methanol extract from the S. hexaphylla leaf was purchased from the Korean Plant Extract Bank (Daejeon, Korea). Recombinant soluble human M-CSF and RANKL were obtained from PeproTech EC Ltd. (London, UK). Monoclonal β-actin antibody was obtained from Sigma (St. Louis, MO, USA). Anti-Akt, anti-phospho-Akt, anti-p38, anti-phospho-p38, anti-JNK, anti-phospho-JNK, anti-phospho-IκB, and anti-phospho-PLCγ2 were purchased from Cell signaling Technology Inc. (Beverly, MA, USA). Anti-c-Fos, anti-NFATc1, anti-IκB, and anti-PLCγ2 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fetal bovine serum (FBS), α-minimum essential medium (α-MEM), and penicillin/streptomycin were purchased from Gibco BRL (Grand Island, NY, USA). All other chemicals were of analytical grade or complied with the standards required for cell culture experiments. Cyclohexamide (CHX), MG132, and Ac-Leu-Leu-nle-h (ALLN) were obtained from Calbiochem (San Diego, CA, USA).
Mice and in vitro osteoclastogenesis assay
BMMs isolation was performed in accordance with the guidelines for animal experimentation of the Institutional Animal Care and Use Committee of Wonkwang University (WKU15-48, committee member: Sung Yeon Kim, Jungkee Kwon, Hong Geun Oh, Hong-Seob So, Okjin Kim, Chun-Soo Ko). Five-week-old male ICR mice were purchased from Damul Science (Daejeon, Korea). The mice were housed in cages in a controlled temperature (22–24 °C) and humidity (55–60 %) room with a 12 h light/dark cycle. Bone marrow cells (BMCs) were obtained from 5-week-old male ICR mice by flushing the tibias and femurs with α-MEM supplemented with 10 % FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). To obtained bone marrow macrophages (BMMs) for osteoclast differentiation of primary BMCs, the cells were cultured in α-MEM supplemented with 10 % FBS and M-CSF (10 ng/mL) for 1 day on culture dishes. Non-adherent cells were transferred to 10 cm petri dishes and further cultured in the presence of M-CSF (30 ng/mL) for 3 days. Adherent cells were used as BMMs or as osteoclast precursors, after the non-adherent cells were removed. To generate osteoclasts from the BMMs culture system, cells were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of S. hexaphylla on 48-well plates, placed in an incubator set to 37 and 5 % CO2, and allowed to incubate for 3 days. The cells were fixed in 3.7 % formalin for 10 min, permeabilized with 0.1 % Triton X-100, and then stained with TRAP solution. TRAP-positive multinucleated cells (MNCs) with more than three nuclei were counted as osteoclasts.
PCR primers and quantitative real-time RT-PCR
The oligonucleotides used as primers
Forward primer (5'-3')
Reverse primer (5'-3')
Cell proliferation was measured using the XTT assay kit (Sigma). The BMMs (1 × 104/well) were seeded in 96-well plates with various concentrations of S. hexaphylla and cultured for 3 days in the presence of M-CSF (30 ng/mL). For the last 4 h of culture, 50 μL of XTT solution was added, and the plate was read at 450 nm with an ELISA reader (Molecular Devices, CA, USA).
Western blot analysis
After the specified treatment, cells were washed twice with cold-PBS and lysed with lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1 % Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1 % deoxycholate, and protease inhibitors. The protein concentration was determined using a Bio-Rad DC protein assay kit (Bio-Rad Laboratories., Hercules, CA, USA). The same amount of protein (15–30 μg) was separated using 10 % SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF, Millipore, Bedford, MA, USA) membrane for 90 min at 25 V on an XCell II blot module (Invitrogen, Carlsbad, CA, USA). The PVDF membranes were placed in 5 % nonfat milk in tris-buffered saline with 0.1 % Tween 20 (TBST) for 1 h, washed, and incubated with the primary antibodies for 2 h at room temperature. The membranes were washed in TBST, and incubated for 1 h with various HRP-conjugated donkey anti-rabbit, sheep anti-mouse IgG secondary antibodies. The immunoreactive signals were detected using the Western Chemiluminescent HRP substrate kit (Millipore, Billerica, USA).
Bone resorption assay
Primary osteoblast cells (1 × 106 cells) and BMCs (1 × 107 cells) were cultured on collagen-gel-coated culture dishes for 7 days in the presence of 10−8 M 1,25-dihydroxyvitamin D3 (Sigma) and 10−6 M prostaglandin E2 (PGE2) (Sigma). The co-cultured osteoclasts were detached by 0.1 % collagenase treatment at 37 °C for 10 min and then placed on dentine slices or hydroxyapatite coated plates (Corning, NY, USA) with or without S. hexaphylla. After 24 h, the cells were removed, and the total resorption pits were observed under a microscope and then quantified using Image-Pro Plus version 4.0 (Media Cybernetics, Silver Spring, MD, USA).
Retrovirus preparation and infection
Plat-E retroviral packaging cells were transfected by pMX-IRES-EGFP, pMX-c-Fos-IRES-EGFP, and pMX-CA-NFATc1-IRES-EGFP using X-tremeGENE 9 (Roche, Nutley, NJ, USA) according to the manufacturer’s protocol. The culture supernatant of the retrovirus-producing cells was collected for retroviral infection on BMMs that were cultured with BMCs in M-CSF (30 ng/mL) for 2 days. The BMMs were incubated with the retrovirus soup produced by Plat-E cells together with polybrene (10 μg/mL) and M-CSF (10 ng/mL) for 6 h. Infected BMMs were further cultured in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with or without S. hexaphylla for 3 days. Osteoclast formation was detected by TRAP staining.
The BMMs were incubated with c-Fos retro-virus soup produced by Plat-E cells together with polybrene (10 μg/mL) and M-CSF (10 ng/mL) for 6 h. Infected BMMs were pretreated with or without S. hexaphylla in the presence of M-CSF for 24 h, and then stimulated with RANKL. After 20 h, 2 μg/mL CHX, 5 μM MG132, and 20 μM ALLN were added to the cultures for 4 h before harvest.
Experiments were conducted at least three times, and the data are expressed as the mean ± standard deviation (SD). All statistical analyses were conducted using the Statistical Package for the Social Sciences Software (SPSS; Korean version 14.0). Most of the statistical differences were analyzed using one-way ANOVA followed by Tukey’s post hoc test. P-values of less than 0.05 were considered statistically significant.
S. hexaphylla suppresses RANKL-induced osteoclast differentiation in BMMs and bone resorption by co-cultured mature osteoclast
S. hexaphylla suppresses RANKL-induced ERK, NF-κB, and Ca2+ signaling pathways
S. hexaphylla downregulates RANKL-induced expression of NFATc1 and c-Fos stability
S. hexaphylla inhibits RANKL-induced mRNA expression of OSCAR, TRAP, ATP6v0d2, β3-integrin, and CtsK
There is a growing interest in the efficacy of natural plant products for the prevention and treatment of bone-related disorders such as osteoporosis and rheumatoid arthritis. In this study, we have shown that S. hexaphylla effectively inhibits RANKL-induced osteoclast differentiation at the early stages without any cytotoxicity or bone-resorbing activity by mature osteoclasts (Fig. 1).
RANK/RANKL interaction forms a fundamental cytokine system that is capable of influencing all aspects of osteoclast differentiation, function, and (indirectly) the complete bone regulatory system [10, 12]. The connection between RANK and RANKL induces recruitment and activation of TRAF6, which leads to multiple downstream cascades. The signals are composed of six known signal pathways: ERK, p38, JNK, NF-κB, Src, and Akt. We found that by suppressing RANKL-mediated ERK and NF-κB signaling, S. hexaphylla inhibited osteoclast differentiation (Fig. 2a). In previous studies, compared to WT mice, ERK1 deficiency reduced the number of osteoclast progenitors and the formation of TRAP-positive cells on the trabecular surface . In addition, NF-κB is an important signal mediator of inflammatory and immune reactions, and is a major transcription factor for RANKL-activated osteoclastogenesis . IκB is bound to NF-κB, which prevents it from translocating to the nucleus, and phosphorylation by IKK separates the two proteins. Subsequent ubiquitination and proteasomal degradation of IκB allows NF-κB to translocate into the nucleus and stimulate the transcription of the target gene . NF-κB p50/p52 double knockout mice exhibit osteoclastogenesis defects and severe osteopetrosis [25, 26]. S. hexaphylla also induces osteoclastogenesis through the c-Src-Btk-PLCγ2 signaling pathway (Fig. 2b). c-Src deficiency produces an osteopetrotic skeletal phenotype and affects the bone-resorbing activity of mature osteoclasts [27, 28]. PLCγ activates Ca2+ signaling and NFATc1 in immune cells. PLCγ2 deficiency in mouse cells lowers the expression of NFATc1 RANKL-stimulation relative to wild mice .
Under normal conditions, the RANKL-RANK axis appears to be essential for osteoclastogenesis, and costimulatory immunoreceptors lead to robust induction of c-Fos and NFATc1, which are the necessary and sufficient transcription factors for osteoclast differentiation. The crucial role of c-Fos in osteoclastogenesis was demonstrated by in vivo experiments using genetically modified mice. c-Fos-deficient mice exhibit a severe osteoporotic phenotype due to the failure of osteoclast differentiation . NFATc1-deficient embryonic stem cells are unable to differentiate into osteoclasts in response to RANKL, and the forced expression of NFATc1 leads to the formation of osteoclasts from BMMs in the absence of RANKL . In our study, S. hexaphylla suppressed RANKL-induced NFATc1 mRNA and protein expression. By contrast, c-Fos protein, but not mRNA, expression was diminished (Fig. 3a and b). This suggests that S. hexaphylla-mediated inhibition of NFATc1 expression is not involved in c-Fos transcriptional activity, and instead results from the inhibition of c-Fos translational activity. Moreover, the protein stability assay showed that the reduction in c-Fos protein contributed to the suppression of c-Fos translation via proteasome-dependent degradation (Fig. 3c). Furthermore, the forced expression of c-Fos or CA-NFATc1 rescued the S. hexaphylla-induced inhibition of osteoclast differentiation, which suggests that down-regulation of c-Fos is responsible for the inhibitory effects of S. hexaphylla (Fig. 3d-f).
NFATc1 has a crucial role in the regulation of osteoclastogenic marker genes during RANKL-mediated osteoclast differentiation, and it gradually induces the expression of osteoclast-specific genes, including OSCAR, TRAP, ATP6v0d2, β3-integrin, and CtsK [1, 8, 9]. The present data suggest that S. hexaphylla suppressed the induction of OSCAR, TRAP, ATP6v0d2, β3-integrin, and CtsK (Fig. 4). Several proteolytic enzymes, including OSCAR, TRAP, ATP6v0d2, and CtsK, have been shown to play important roles in degrading the organic bone matrix, and of these proteolytic enzymes have the highest levels in osteoclasts. Matrix-degrading enzymes are also known to be collagen-degrading enzymes, and can directly degrade collagen in hard tissues that have been demineralized. In addition, ανβ3-integrin is known to play a role in the regulation of cell migration and the maintenance of the sealing zone required for effective osteoclastic bone resorption. This indicates that S. hexaphylla extract inhibited bone resorption by disrupting the induction of cytoskeletal organization, leading to the formation of a ruffled border and the adhesion of osteoclasts to the bone matrix.
In conclusion, this is the first study to report that S. hexaphylla efficiently suppresses RANKL-induced osteoclast differentiation and bone-resorbing activity in vitro. S. hexaphylla inhibits osteoclastogenesis via proteasome-mediated degradation of c-Fos protein and suppression of NFATc1 induction. These results suggest that S. hexaphylla has potential as a natural herbal therapy for diseases associated with bone loss.
This study was supported by a grant from Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2059831).
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