Anti-Osteoclastogenic Activity of Praeruptorin A via Inhibition of p38/Akt-c-Fos-NFATc1 Signaling and PLCγ-Independent Ca2+ Oscillation

et al. (2014) Anti-Osteoclastogenic Activity of Praeruptorin A via Inhibition of p38/Akt-c-Fos-NFATc1 Signaling and PLCc-Independent Ca2+ Oscillation. PLoS ONE 9(2): e88974. doi:10.1371/journal.pone.0088974 Anti-Osteoclastogenic Activity of Praeruptorin A via Inhibition of p38/Akt-c-Fos-NFATc1 Signaling and PLCc- 2+ Independent Ca Oscillation Jeong-Tae Yeon Kwang-Jin Kim Sik-Won Choi Seong-Hee Moon Young Sik Park Byung Jun Ryu Jaemin Oh Min Seuk Kim Munkhsoyol Erkhembaatar Young-Jin Son Seong Hwan Kim Yin Tintut, University of California, Los Angeles, United States of America Background: A decrease of bone mass is a major risk factor for fracture. Several natural products have traditionally been used as herbal medicines to prevent and/or treat bone disorders including osteoporosis. Praeruptorin A is isolated from the dry root extract of Peucedanum praeruptorum Dunn and has several biological activities, but its anti-osteoporotic activity has not been studied yet. Materials and Methods: The effect of praeruptorin A on the differentiation of bone marrow-derived macrophages into osteoclasts was examined by phenotype assay and confirmed by real-time PCR and immunoblotting. The involvement of NFATc1 in the anti-osteoclastogenic action of praeruptorin A was evaluated by its lentiviral ectopic expression. Intracellular Ca2+ levels were also measured. Results: Praeruptorin A inhibited the RANKL-stimulated osteoclast differentiation accompanied by inhibition of p38 and Akt signaling, which could be the reason for praeruptorin A-downregulated expression levels of c-Fos and NFATc1, transcription factors that regulate osteoclast-specific genes, as well as osteoclast fusion-related molecules. The anti-osteoclastogenic effect of praeruptorin A was rescued by overexpression of NFATc1. Praeruptorin A strongly prevented the RANKL-induced Ca2+ oscillation without any changes in the phosphorylation of PLCc. Conclusion: Praeruptorin A could exhibit its anti-osteoclastogenic activity by inhibiting p38/Akt-c-Fos-NFATc1 signaling and PLCc-independent Ca2+ oscillation. - Funding: This work was supported by the Korea Research Institute of Chemical Technology projects grant (SI-1304) and the Inter-ER Cooperation Projects (R0002019) of Korea Institute for Advancement of Technology, which were funded by the Korea Ministry of Knowledge Economy. MSK was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF-2012R1A1A1038381), funded by the Ministry of Education, Science and Technology (MEST). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Bone fracture is a public health problem because it occurs easily in patients with bone-related disorders including osteoporosis. As the elderly population is rapidly increasing, the medical costs of hospitalization caused by fractures have been become a serious social issue [1,2]. Bone homeostasis depends on the balance between osteoblastic bone formation and osteoclastic bone resorption, but an imbalance caused by an increased number of osteoclasts or overactivation can lead to impaired bone structure and low bone mass, which are common characteristics in patients with bone disorders [3,4]. Therefore, a method to pharmaceutically inhibit osteoclast differentiation is one of the therapeutic strategies for preventing and/or treating bone disorders and related fractures [5]. Clinically, osteoclast-targeting bisphosphonates have been widely used to treat patients with osteoporosis and/or prevent osteoporotic fracture. The orally available bisphosphonates principally inhibit the activation of osteoclasts by binding to hydroxyapatite [6], but recently bisphosphonate-related side effects including acute phase response, hypocalcaemia, secondary hyperparathyroidism, upper gastrointestinal tract problems, musculoskeletal pain, rental toxicity and osteonecrosis of the jaw have been reported [7]. Therefore, there have been several basic and clinical efforts to find anti-osteoporotic phytochemicals in order to minimize adverse side effects [810]. Praeruptorin A (Fig. 1A) is isolated from the dry root extract of Peucedanum praeruptorum Dunn, which has been used as an herbal medicine with several pharmacological activities [1114]. Recently, praeruptorin A has been shown to suppress the lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells [15]. Since LPS is a potent stimulator of both inflammation and bone resorption [16,17] and several phytochemicals exhibit dual anti-inflammatory and anti-resorptive activities [18,19], in this study, the anti-osteoclastogenic activity of praeruptorin A was investigated. The functional involvement of praeruptorin A in osteoclast differentiation is not yet clearly understood. Materials and Methods Reagents Praeruptorin A was purchased from Stanford Chemicals (CA). Mouse soluble receptor activator of nuclear factor-kB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF) were purchased from R&D Systems (MN). Penicillin, streptomycin, cell culture medium, and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (NY). The CCK-8 assay kit was from Dojindo Molecular Technologies (ML). Antibodies against c-Fos, NFATc1 and actin were from Santa Cruz Biotechnology (CA). Antibodies against p-p38, p38, p-JNK, JNK, p-ERK, ERK, Akt, p-Akt (Ser473), PLCc and p-PLCc were obtained from Cell Signaling Technology (MA). Osteoclast Differentiation This study was carried out in strict accordance with the recommendations in the Standard Protocol for Animal Study of Korea Research Institute of Chemical Technology (KRICT; Permit No. 2012-7D-02-01). The protocol (ID No. 7D-M1) was approved by the Institutional Animal Care and Use Committee of KRICT (IACUC-KRICT). All efforts were made to minimize suffering. In detail, after cervical dislocation, bone marrow cells were obtained from 5-wk-old male ICR mice (Damool Science, Daejeon, Korea) by flushing femurs and tibias with a-MEM supplemented with antibiotics (100 units/ml penicillin and 100 mg/ml streptomycin). Bone marrow cells were cultured for 1 day on a culture dish in a-MEM supplemented with 10% FBS and M-CSF (10 ng/ml). Non-adherent bone marrow cells were plated on a Petri dish and cultured for 3 days in the presence of M-CSF (30 ng/ml). After non-adherent cells were washed out, adherent cells were used as bone marrow-derived macrophages (BMMs). For osteoclastogenesis, BMMs (16104 cells/well in a 96-well plate or 36105 cells/well in a 6-well plate) were cultured in the presence of M-CSF (30 ng/ml) and RANKL (10 ng/ml) for 4 days. Tartrate-resistant Acid Phosphatase (TRAP) Staining and Activity Assay Cells were fixed with 3.7% formaldehyde for 5 min, permeabilized with 0.1% Triton X-100 for (...truncated)