Anti-osteoclastogenic activity of matairesinol via suppression of p38/ERK-NFATc1 signaling axis

Choi et al. BMC Complementary and Alternative Medicine 2014, 14:35 http://www.biomedcentral.com/1472-6882/14/35 RESEARCH ARTICLE Open Access Anti-osteoclastogenic activity of matairesinol via suppression of p38/ERK-NFATc1 signaling axis Sik-Won Choi1†, Kie-In Park2†, Jeong-Tae Yeon3, Byung Jun Ryu1, Kwang-Jin Kim3 and Seong Hwan Kim1* Abstract Background: Matairesinol is a plant lignan present in a wide variety of foodstuffs such as seeds, vegetables and fruits. It has various biological functions including anti-angiogenic, anti-cancer and anti-fungal activities, but its anti-osteoporotic activity, if any, is unknown. Methods: For osteoclast differentiation, primary mouse bone marrow-derived macrophage cells (BMMs) were cultured for 4 days in the presence of RANKL and M-CSF with the vehicle (DMSO) or matairesinol. Cell cytotoxicity was examined by CCK-8 assay. Gene expression of NFATc1, TRAP, OSCAR, v-ATPasev0d2 were observed in the presence or absence of matairesinol (10 μM) for the indicated times. For evaluating the involvement of NFATc1 in the anti-osteoclastogenic action of matairesinol, BMMs were infected with pMX-IRES-GFP or pMX-IRES-CA-NFATc1GFP for 8 h with polybrene, and then infected BMMs were cultured with M-CSF and RANKL for 4 days in the presence or absence of matairesinol (10 μM). MAPK signaling activation was examined by immunoblotting. For measuring the resorptive activity of mature osteoclasts, osteoclasts and osteoblasts were co-cultured on BioCoat Osteologic MultiTest slides, and treated with matairesinol for 24 h. Result: Here we show that matairesinol dose-dependently inhibited the RANKL-induced differentiation of BMMs into osteoclasts by downregulating RANKL-induced expression and activity of NFATc1. Ectopic overexpression of NFATc1 blunted the anti-osteoclastogenic effect of matairesinol implicating NFATc1 in the action of matairesinol. Additionally, matairesinol blocked the RANKL-induced activation of p38 and ERK in BMMs, but had no effect on bone resorption activity in mature osteoclasts. Conclusion: Taken together, our results suggest that the anti-osteoporotic activity of matairesinol could arise from its anti-osteoclastogenic potential via p38/ERK-NFATc1 signaling, but not by way of anti-resorptive action. Keywords: Osteoclast differentiation, Matairesinol, MAP kinases, NFATc1 Background Bone is a highly dynamic tissue continuously remodeled by osteoclasts and osteoblasts, which are responsible for bone resorption and bone formation, respectively [1]. The delicate balance between osteoclast-mediated bone destruction and osteoblast-mediated bone formation is important for maintaining bone mineral density. Multinucleated osteoclasts are formed and functionalized by the fusion of macrophage precursor cells. Specifically, excessive bone resorption by overactivated osteoclasts * Correspondence: † Equal contributors 1 Laboratory of Translational Therapeutics, Pharmacology Research Center, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, P.O. Box 107, Yuseong-gu, Daejeon 305-600, Korea Full list of author information is available at the end of the article is involved in several lytic bone diseases, such as osteoporosis, periodontal disease and rheumatoid arthritis [2,3]. Osteoporosis is a metabolic disease characterized by decreased bone mass and an increased risk of skeletal fracture and is widely recognized as a major public health problem in an aging society [4]. Several antiresorptive agents such as bisphosphonates, calcitonin and estrogen have been developed to treat osteoporosis, but each one has side effects including induction of breast cancer, osteonecrosis and vaginal bleeding [5,6]. Thus, a much safer therapeutic strategy for preventing and/or treating lytic bone diseases including osteoporosis is required. Natural product-derived small molecules have been used as therapeutic agents for preventing and curing a © 2014 Choi et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Choi et al. BMC Complementary and Alternative Medicine 2014, 14:35 http://www.biomedcentral.com/1472-6882/14/35 number of diseases [7,8]. Among them, lignans are phytochemicals elaborated from two phenylpropanoid units in plants and are present in a wide variety of plant foodstuffs including seeds, vegetables, and fruits [9,10]. Matairesinol (Figure 1A), a dibenzylbutyrolactone lignan, has been reported to possess anti-oxidative, estrogenic, or anti-estrogenic activities and reduce the risk of hormone-dependent cancer [11]. However, the detailed anti-osteoporotic activity and mechanism of matairesinol has not been explored. Therefore, we examined the in vitro effect of matairesinol on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and the bone resorptive activity of mature osteoclasts. Methods Reagents and antibodies Penicillin, streptomycin, cell culture medium, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies. Mouse soluble macrophage-colony stimulating factor (M-CSF) and RANKL were purchased from R&D Systems. The CCK-8 assay kit was purchased from Dojindo Molecular Technologies. Antibodies against nuclear factor of activated T cells (NFAT)c1, c-Fos, and actin were purchased from Santa Cruz Biotechnology Page 2 of 8 and antibodies against MAP kinases from Cell Signaling Technology. Matairesinol was purchased from SigmaAldrich and dissolved in DMSO (dimethylsulfoxide; Sigma-Aldrich). Preparation of osteoclast precursor cells All experiments were carried out as described in a previous study, with modifications [12]. All animal procedures were performed according to the guide for the Institutional Animal Care and Use Committee of the Korea Research Institute of Chemical Technology (Protocol ID No. 7D-M1). Five-week-old male ICR (Damul Science Co. Deajeon, Korea) were maintained in a room illuminated daily from 07:00 to 19:00 (12:12 h light/dark cycle), with controlled temperature (23 ± 1°C) and ventilation (10–12 times per hour), and humidity was maintained at 55 ± 5% with free access to a standard animal diet and tap water. Bone marrow cells were obtained from five-week-old male ICR mice by flushing femurs and tibias with α-MEM-containing antibiotics (100 units/ml penicillin, 100 μg/ml streptomycin). Bone marrow cells were cultured on culture dishes for 1 day in α-MEM containing 10% FBS and M-CSF (10 ng/ml). Non-adherent bone marrow cells were plated on Petri dishes and cultured for 3 days in the presence of M-CSF Figure 1 Matairesinol inhibits RANKL-mediated osteoclast differentiation. (A) Structure of matairesinol. (B) BMMs were cultured for 4 days in the presence of RANKL (10 ng/ml) and M-C (...truncated)