2013 Volume 36 Issue 5 Pages 796-801
6,4′-Dihydroxy-7-methoxyflavanone (DMF) is a flavonoid isolated from Heartwood Dalbergia odorifera. It has been known that DMF has antioxidant, anti-inflammatory and neuroprotective effects. DMF, however, the efficacy of bone related diseases has not been reported. In this study, we determined DMF’s efficacy on osteoclasts differentiation and function using in vitro bone marrow macrophage osteoclast differentiation culture system. DMF inhibited receptor activators of nuclear factor kappa-B ligand (RANKL) induced osteoclastogenesis dose dependently. In addition, DMF decreased osteoclast function through disruption of actin ring formation and consequently suppression of the pit-forming activity of mature osteoclasts. Mechanistically, DMF inhibited RANKL-induced expression of nuclear factor of activatied T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and c-Fos via inhibition of mitogen activated protein kinases (MAPKs) pathway. Collectively, the inhibition of osteoclasts differentiation and function by DMF suggests that DMF can be a potential therapeutic molecule for osteoclastogenic bone diseases such osteoporosis, rheumatoid arthritis and periodontal diseases.
Bone composed two different lineage cells like osteoblast-osteocytes and osteoclasts. Osteoclasts were multinucleated cells, resorb bone, maintained calcium homeostasis and helping normal bone remodeling. Enhanced resorptive activity by imbalanced bone remodeling causes various bone diseases such as osteoporosis, paget’s disease, hip fracture, rheumatoid and periodontal disease.1) Differentiation and maturation of osteoclasts is controlled by many factors, including macrophage colony stimulating factor (M-CSF) and receptor activators of nuclear factor κB ligand (RANKL).2,3)
RANKL is members of the tumor necrosis factor (TNF) superfamily.4) RANKL interacts RANK trigger activation of tumor necrosis factor receptor-associated factor-6 (TRAF6) which subsequently induces mitogen-activated protein (MAP) kinases and transcription factors included nuclear factor-kappa B (NF-κB), activator protein 1 (AP-1), nuclear factor of activatied T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1).5,6) These transcription factors play an essential role in the regulation of genes involved in osteoclast differentiation and bone resorptive activity.7,8)
Current treatement of osteoporosis was based on to increase bone formation (estrogen, PTH) and inhibits bone resorption (estrogen, calcitonin, calcium, bisphosphonates).9–13) Estrogen-replacement therapy (ERT) was used to be popular treatment and prevention of postmenoposal osteoporosis. However, risk of breast cancer, stroke, and heart attack increases by excessive ERT.14) Bisphosphonates are the most widely used drug to cure osteoporosis. But, it was poorly absorbed from the gastrointestinal tract and show side effects like osteonecrosis of jaw and atypical fracture.15) Furthermore, PTH cannot be given orally, expensive, limited indication and concern about osteosarcoma has led to recommendation of a 2 years maxium treatment course.16) Thus, new agents for management of osteoporosis are needed. Natural source products have been used successfully for identification and development of therapeutic agents.17,18)
6,4′-Dihydroxy-7-methoxyflavanone (DMF), flavonoid, was isolated from the heartwood of Dalbergia (D.) odorifera as previous study.19) Previous phytochemical studies of D. odorifera have reported the isolation of flavonoid, quinines and phenolic constituents.20–22) Flavonoids and related plant-derived phenolic compounds are well known to have a wide range of biological activities such as anti-inflammation, anti-cancer, anti-osteoporosis.23–27) However, the anti-osteoporotic effects of DMF have not been studied yet. In this study, we studied the effect of DMF on osteoclastogenesis in vitro.
DMF was isolated from D. odorifera as the same method described previously.19) The structure is shown in Fig. 1. Recombinant murine macrophage colony-stimulating factor (M-CSF) and recombinant soluble receptor activators of nuclear factor kappa-B ligand (sRANKL) were purchased from PeproTech EC Ltd. (London, U.K.). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Amresco Inc. (OH, U.S.A.). Naphtol AS-MX phosphate, fast red LB salt, ρ-nitrophenylphosphate, 4,6-diamidino-2-phenylindole (DAPI) and Actin antibody were purchased Sigma-Aldrich Chemicals (St. Louis, MO, U.S.A.). Alexa Fluor 488-conjugated phalloidin was purchased from Molecular Probes Inc. (Eugene, OR, U.S.A.) and primary antibodies for phospho-c-Jun N-terminal kinase (JNK), JNK, phospho-extracellular signal-regulated kinase (ERK), ERK, phospho-p38, p38 and rabbit polyclonal antibodies from Cell Signaling Technology Inc. (Beverly, MA, U.S.A.). An anti-c-Fos mouse antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.) and anti-NFATc1 mouse antibody was obtained from BD Biosciences (San Jose, CA, U.S.A.). The enhanced chemiluminescence (ECL) Western blotting detection system was from Amersham (GE Healthcare Life Science, Tokyo, Japan). The Osteo Assay Plate was purchased from Corning (Corning, New York, U.S.A.).
Isolation of bone marrow derived macrophages (BMMs) from male ICR mice was performed as described previously.28) Male ICR mice (4–6 weeks of age) were purchased from Orient Bio (Gyeonggido, Korea). The animal study was performed in accordance with the guidelines of the Animal Experiment Committee of Keimyung University (KM 2011–40R).
The BMCs were cultured in α-minimal essential medium (α-MEM) supplied with 10% fetal bovine serum and 100 U/mL of penicillin, and 100 µg/mL streptomycin with the addition of M-CSF (100 ng/mL) in 60 mm dishes. After 4 d culture, non-attached cells were used as bone marrow macrophages (BMMs) as osteoclast precursors after removal of non-adherent cells. BMMs were replaced 96 well plates (5×104 cells/well) presence M-CSF (100 ng/mL) for 3 d, treated with RANKL (100 ng/mL) and cultured another 3 d until the differentiated into multinucleated mature osteoclasts (OCLs).
Cell Viability AssayCell viability was determined by the MTT assay. BMMs were plated in 96 well plated at 5×104 cells/well in 180 µL. DMF was added various concentrations and were grown for 3 d. After 20 µL of MTT reagent (2.5 mg/mL) was added to each well and incubated for 4 h, the supernatants were discarded, cells were dissolved in dimethylsulfoxide (DMSO). The absorption was measured at 550 nm using a microplate reader (Molecular Device, CA, U.S.A.).
Tartrate Resistant Acid Phosphatase (TRAP) Staining and Measurement of TRAP ActivityThe cells were fixed with 10% formaldehyde for 10 min and ethanol–acetone for 1 min, and then stained with TRAP solution. TRAP positive multinucleated cells containing more than 5 nuclei were counted and captured under a microscope. In order to measure TRAP activity, cells were fixed and then 10 mm citrate buffer (pH 4.6) containing 10 mm sodium tartarate and 3.7 mm ρ-nitrophenylphosphate was added to the cell-containing 96 well plates. After incubated 30 min, the reaction mixtures were transferred into new plates containing an equal volume of 0.1 n NaOH. The absorption was measured at 405 nm. TRAP activity was calculated as percent of control.
Actin Ring StainingMature osteoclasts (mOCs) possessing actin ring structures were formed from BMMs cells. Cells were fixed 10% formaldehyde 10 min and stained with Alexa Flour 488-conjugated phalloidin in the dark and then washed with cold phosphate-buffered saline (PBS).
DAPI StainingFor DAPI staining, cells were fixed as following actin ring staining methods and added 1 µg/mL DAPI in PBS for 1 h. Cells showing fragmented chromatin were considered apoptotic. The distribution of DAPI staining in mOCs was visualized under a fluorescence microscope Olympus IX71 (Olympus, Tokyo, Japan).
Bone Resorption Pit Formation AssayFor bone resorption pit formation, BMMs were seeded at 5×104 cells/well onto calcium phosphate (Ca-P) nanocrystal-coated plates and incubatied with M-CSF and RANKL for 7 d. After incubation of cells in bleach solution for 5 min at room temperature, cells were washed twice with distilled water and dried 3 to 5 h. Resorption pits were observed using a microscope (Olympus, Tokyo, Japan). The resoprtion area was calculated using IMT isolution FL 9.1 (Vancouver, BC, Canada) software.
Western Blot AnalysisBMMs were washed with PBS, lysed in a Ripa buffer containing protease and phosphatase inhibitor cocktail (Thermo, U.S.A.) and centrifugation at 13000 rpm for 15 min. Protein concentration was determined Bradford methods using manufacturer’s kit (Bio-Rad, U.S.A.). Equal amount of each lysate was separated in sodium dodecyl sulfate (SDS)-polyacrylamide gel. After the electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membrane was blocked with 5% skim milk in TBS buffer, incubated primary antibodies (NFATc1, c-Fos, phospho-JNK, JNK, phospho-ERK, ERK, phospho-p38, p38, β-actin) at 4°C overnight, washed, incubated secondary antibody, horseradish peroxidase conjugated anti-rabbit Immunoglobulin G (IgG) and anti-mouse IgG, and detected with ECL-plus (GE Healthcare Life Science). The immunoreactive bands were analyzed by LAS4000 (GE Healthcare Life Science).
Statistical AnalysisAll experiments were replicated at least 3 times. The means and standard deviations (S.D.) calculated by Sigma Plot software 10.1. Student’s t-test were used to assess the statistical significance of differences. Differences were considered significant as at p<0.05.
As shown in Fig. 1, 6,4′-dihydroxy-7-methoxyflavanone (DMF) is one of flavonoids with methylations on hydroxyl groups (methoxy bonds) and two hydroxyl groups.19) The effect of DMF on BMM cells viability was examined by MTT assay (Fig. 2A). BMM cells were cultured 3 d in various concentrations of DMF (0–30 µm). DMF did not affect cell viability at concentration of < 30 µm. To determine the effects of DMF on osteoclast differentiation, BMMs were differentiated into osteoclasts after treatment with M-CSF and RANKL. As shown in Fig. 2B, DMF inhibited TRAP activity at 3 to 30 µm significantly. This inhibition effect of DMF (10–30 µm) on osteoclastogenesis was reconfirmed by inhibition of the formation of multinuclear OCLs in dose dependent manner (Fig. 2C). Baicalein was used positive control and showed a significantly inhibit osteoclast differentiation at concentration 10–20 µm. These results indicate that DMF significantly inhibits osteoclastogenesis.
(A) BMMs were cultured with or without DMF for 72 h 96 well plates. Cell viability were determined by MTT assays and (B) TRAP activity was measeured. Mouse BMMs were cultured with M-CSF (100 ng/mL) and RANKL (100 ng/mL) and with or without DMF. (C) After cultured for 3 d, the cells were fixed and stained for TRAP. Results were presented as means±S.D. (n>3). * p>0.05, ** p>0.01. Baicalein (10, 20 µm) was used positive control.
Actin ring formation is essential for bone resorption in mature osteoclasts. The actin ring was stained with Allexa-488 Flour conjugated phalloidin. The mature osteoclasts treated with DMF for 2 d were changed their morphology with disruption of actin ring formation (Fig. 3A). To determine whether DMF induces apoptosis, nuclear condensation and fragmentation were assayed by DAPI staining. DMF did not induce nuclear condensation or fragmentation of mature osteoclasts at this condition (Fig. 3B). Baicalein was used positive control. It also induced distruption of the actin ring structure and induced apoptosis at 20 µm. In addition, these results suggest that DMF can disrupt actin ring formation of mature osteoclasts.
(A) Actin ring formation, (B) Nuclear staining by using DAPI staining. BMM were cultured with M-CSF (100 ng/mL) and RANKL (100 ng/mL) for 5 d. mOC were treated with DMF in presence of M-CSF and RANKL. After cultured for 48 h, the cells were fixed with praformaldehyde. After fixation incubated with Alexa Fluor 488-conjugated phalloidin (A) or stained with DAPI (B). Baicalein (20 µm) was used positive control.
To test the effect of DMF on osteoclast function, we measured bone resorption activity of osteoclasts. M-CSF and RANKL-induced mature osteoclasts were treated with various concentration of DMF. Resorption pits formation on Ca-P coated plate was dose dependently inhibited by DMF (Figs. 4A, 4B). These results indicate that DMF has inhibitory function on mature osteoclasts. In addition, baicalein also inhibited the bone resorption activity.
BMMs were cultured for 5 d with the indicated concentration of DMF in the presence of M-CSF (100 ng/mL) and RANKL (100 ng/mL). (A) A photograoh of the bone-resorption activity of OCLs. The data are representative of three independent experiments. (B) The resorption area was calculated using software. The results shown are representative of three independent experiments. Results were presented as means±S.D. (n>3). * p>0.05, ** p>0.01. Baicalein (20 µm) was used positive control.
Many studies have reported that the activation of MAPKs suppressed osteoclast differentiation.29) To define the molecular mechanism of DMF on the inhibition of osteoclast differentiation, we investigated the effects of DMF on RANKL induced phosphorylation of MAPKs that has been known to play important roles during osteoclastogenesis. The phosphorylation of MAPKs induced by RANKL in BMM cells, reached the maximum levels at 15 min, and decreased thereafter.30) At a concentration of 30 µm, DMF strongly inhibited RANKL-induced phosphorylation of JNK without decrease of the phosphorylation of ERK and p38 MAPK (Fig. 5). It also suppressed c-Fos and NFATc1 expression.
BMM cells were preincubated in the presence of DMF for 1 h and then treated with RANKL (100 ng/mL) for 15 min. Cell lysates were collected and separated by 10% SDS-PAGE. The levels of phosphorylated and non-phosphorylated p38 MAPK, ERK and JNK were determined by Western blotting (A). RANKL (100 ng/mL) treated 24 h, the level of NFATc1 and c-Fos were determined by Western blot (B). The results shown are representative of three independent experiments.
DMF, a class of flavonoid, was isolated from the heartwood of D. odorifera T. Chen (Leguminosae).19) The heartwood of the D. odorifera T. Chen (Leguminosae) is used to treat ischemia, swelling and rheumatic pain in China and Korea. Previous studies showed that DMF displays anti-oxidative and anti-inflammatory heme oxygenase-1 (HO-1) inducer in mouse hippocampal HT22 cells and BV2 microglia cells.20,21,31,32) However, other biological effects of DMF have not been reported.
The oriental medicine has been widely used in the prevention and the treatment of fracture and joint diseases. Recently, several studies have suggested that natural source of small molecules prevent osteoporosis by modulate osteoclastogenesis. For example, saurolactam, sauchinone isolated from Saururus chinensis33,34) inhibited bone destruction and osteoclast formation caused by lipopolysaccharide in an animal model. Baicalein isolated from Scutellaria baicalensis35) inhibited osteoclast differentiation and induced apoptosis of mature osteoclasts to inhibit bone resorption. Furthermore, honokiol, a neolignan, isolated from Magnolia obovata36) also inhibited osteoclast differentiation and function in vitro. In this study, we further demonstrated that DMF inhibited osteoclast differentiation and modulated their function.
Osteoclasts are multi-nucleated cells that are differentiated from hematopoietic cells of the monocyte/macrophage family. The differentiation of this cell is mainly regulated by M-CSF, RANKL, or osteoprotegerin. Coincident treatment of mouse BMM cells with M-CSF and RANKL induced osteoclast differentiation. DMF did not affect the proliferation. However, DMF inhibited osteoclast differentiation induced by RANKL from BMM cells.
In osteoclast, RANKL binding to RANK prompt the activation of MAPKs signaling pathways.37) Also, MAPKs are important for the induction of c-Fos and NFATc1 during osteoclast differentiation.29) JNK has been reported to play a role in osteoclast differentiation and bone resorptive activity. In our results, DMF inhibited the JNK pathway in BMM cells. Since NFATc1 is a critical transcription factor in RANKL-induced osteoclastogenesis, the down regulation of NFATc1 by DMF effectively suppressed osteocast differentiation.
The formation and maintenance of actin rings on the bone surface are essential in order to conduct their major function of bone resorption in mature osteclasts, so the formation of actin rings can be said to be a bone resorption factor.38,39) DMF also inhibited osteoclast bone resorption activity due to the disruption of actin ring formation. Besides, DMF directly induced disruption of the actin rings and nuclear fragmentation in mature osteoclasts and resulted in the inhibition of bone resorption. These findings suggest that DMF suppressed bone resorption through both its inhibitory effect on osteoclast differentiation and function.
In conclusion, we have demonstrated that DMF suppresses osteoclast differentiation by inhibiting RANKL induced MAPK signaling pathways and attenuates bone resorption by disrupting the actin rings in mature osteoclasts. DMF could be useful for the treatment of bone diseases associated with excessive bone resorption. In the present study, we demonstrated for first time that DMF has inhibition of osteoclast differentiation and anti-resorptive activity. Therefore, it could be good candidate to develop a therapeutic drug for osteoporosis treatment.
The present research has been conducted by the Settlement Research Grant of Keimyung University in 2011.
