β-Boswellic Acid Inhibits RANKL-Induced Osteoclast Differentiation and Function by Attenuating NF-κB and Btk-PLCγ2 Signaling Pathways

Abstract

Osteoporosis is a systemic metabolic bone disorder that is caused by an imbalance in the functions of osteoclasts and osteoblasts and is characterized by excessive bone resorption by osteoclasts. Targeting osteoclast differentiation and bone resorption is considered a good fundamental solution for overcoming bone diseases. β-boswellic acid (βBA) is a natural compound found in Boswellia serrata, which is an active ingredient with anti-inflammatory, anti-rheumatic, and anti-cancer effects. Here, we explored the anti-resorptive effect of βBA on osteoclastogenesis. βBA significantly inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclasts induced by receptor activator of nuclear factor-B ligand (RANKL) and suppressed bone resorption without any cytotoxicity. Interestingly, βBA significantly inhibited the phosphorylation of IκB, Btk, and PLCγ2 and the degradation of IκB. Additionally, βBA strongly inhibited the mRNA and protein expression of c-Fos and NFATc1 induced by RANKL and subsequently attenuated the expression of osteoclast marker genes, such as OC-STAMP, DC-STAMP, β3-integrin, MMP9, ATP6v0d2, and CtsK. These results suggest that βBA is a potential therapeutic candidate for the treatment of excessive osteoclast-induced bone diseases such as osteoporosis.

Keywords: bone resorption; osteoclast; osteoporosis; β-boswellic acid.

Figure 1

β-boswellic acid (βBA) inhibits RANKL-induced osteoclast differentiation without cytotoxicity. (A) Mouse bone marrow-derived macrophages (BMMs) were cultured for 3 days at the indicated concentration of βBA in the presence of M-CSF (30 ng/mL). Cell viability was analyzed by XTT assay. (B) BMMs were cultured for 4 days in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL) with the control (DMSO) or βBA at the indicated concentration. After culturing, cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with TRAP staining solution. (C) TRAP-positive multinucleated cells (TRAP⁺ MNCs; N > 3) were counted as osteoclasts. Scale bar, 100 μm. Data represent means ± SD (n = 3). ** p < 0.01, *** p < 0.001 as compared with DMSO control.

Figure 2

β-boswellic acid (βBA) suppresses the bone-resorbing activity of mature osteoclasts. Mature osteoclasts were prepared with co-cultured osteoblasts and bone marrow cells on a collagen matrix plate. Mature osteoclasts were cultured in a 48-well plate for 24 h, in a hydroxyapatite-coated plate (HA) for 24 h, or in dentin slices for 48 h with or without βBA (30 μM). The cells attached to 48-well plates were stained with a TRAP solution (top), and the cells on HA (middle) and dentin slices (bottom) were removed with 10% bleach solution. The dentin slices were counterstained with hematoxylin solution, and the resorbed surface was photographed under a light microscope. The number of TRAP-positive multinucleated cells (TRAP⁺ MNCs; N > 3) was counted, and the relative pit areas in the HA and dentin slices were quantified using Image-Pro Plus (Ver 4.5) software. Scale bar, 100 μm. Data represent means ± SD (n = 3). *** p < 0.001 vs. as compared with DMSO control.

Figure 3

β-boswellic acid (βBA) downregulates RANKL-induced phosphorylation of IκB, Btk, and PLCγ2 and IκB degradation. Mouse bone marrow-derived macrophages (BMMs) were starved with serum-free α-MEM media for 3 h. BMMs were pre-treated with DMSO control or βBA (30 μM) for 1 h and then stimulated with RANKL (100 ng/mL) at the indicated times. (A) The cell lysates were analyzed by Western blotting with antibodies. β-actin was used as internal control. (B) Quantification of relative ratio of band intensity was performed using Image J software. Data represent means ± SD (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control at 0 min; ^# p < 0.05, ^## p < 0.01, ^### p < 0. as compared with DMSO control at the indicated time.

Figure 4

β-boswellic acid (βBA) inhibits RANKL-induced c-Fos and NFATc1 expression. Mouse bone marrow-derived macrophages (BMMs) were incubated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of βBA (30 μg/mL) for the indicated time. DMSO was used as control. (A) Total RNA was isolated from cells using TRIzol reagent, and mRNA expression levels of c-Fos and NFATc1 were analyzed by real-time RT-PCR. Data represent means ± SD (n = 3). ** p < 0.01, *** p < 0.001 vs. control at 0 h; ^# p < 0.05, ^### p < 0.001 as compared with DMSO control at the indicated time. (B) Protein expression levels of c-Fos and NFATc1 were evaluated by immunoblotting. β-actin was used as the internal control.

Figure 5

β-boswellic acid (βBA) inhibits RANKL-induced mRNA expression of OC-STAMP, DC-STAMP, β3-integrin, MMP9, ATP6v0d2, and CtsK. Mouse bone marrow-derived macrophages (BMMs) were incubated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of βBA (30 μM) for the indicated time. Total RNA was isolated from cells using TRIzol reagent, and the mRNA expression of OC-STAMP, DC-STAMP, β3-integrin, MMP9, ATP6v0d2, and CtsK was analyzed by real-time RT-PCR. Data represent means ± SD (n = 4). *** p < 0.001 vs. control at 0 h; ^### p < 0.001 as compared with DMSO control at the indicated time.