Inhibitory Effect of Chrysanthemum zawadskii Herbich var. latilobum Kitamura Extract on RANKL-Induced Osteoclast Differentiation

Abstract

Chrysanthemum zawadskii Herbich var. latilobum Kitamura, known as "Gujulcho" in Korea, has been used in traditional medicine to treat various inflammatory diseases, including rheumatoid arthritis. However, these effects have not been tested on osteoclasts, the bone resorbing cells that regulate bone metabolism. Here, we investigated the effects of C. zawadskii Herbich var. latilobum Kitamura ethanol extract (CZE) on osteoclast differentiation induced by treatment with the receptor activator of NF- κ B ligand (RANKL). CZE inhibited osteoclast differentiation and formation in a dose-dependent manner. The inhibitory effect of CZE on osteoclastogenesis was due to the suppression of ERK activation and the ablation of RANKL-stimulated Ca(2+)-oscillation via the inactivation of PLC γ 2, followed by the inhibition of CREB activation. These inhibitory effects of CZE resulted in a significant repression of c-Fos expression and a subsequent reduction of NFATc1, a key transcription factor for osteoclast differentiation, fusion, and activation in vitro and in vivo. These results indicate that CZE negatively regulates osteoclast differentiation and may be a therapeutic candidate for the treatment of various bone diseases, such as postmenopausal osteoporosis, rheumatoid arthritis, and periodontitis.

Figure 1

Effects of Chrysanthemum zawadskii Herbich var. latilobum Kitamura extract (CZE) on cell viability. (a) BMMs were cultured with indicated concentrations of CZE for 1 day. (b) BMMs were cultured with 25 μg/mL CZE or without (control) for 4 days. Cell viability was measured as described in the materials and methods. Data are the mean of three independent experiments (± SD).

Figure 2

Effects of CZE on osteoclast differentiation. BMMs were cultured with various concentrations of CZE under RANKL and M-CSF treatment for 4 days. (a) Osteoclasts were stained for TRAP. (b) TRAP⁺ multinuclear cells (MNCs) with more than 3 nuclei were counted as mature osteoclasts. (c) Total TRAP activity from TRAP⁺-mono-, di- and multinuclear cells was measured as described in the materials and methods. Data are expressed as the mean ± SD and are representative of at least three independent experiments. *P < 0.05, **P < 0.01, and ^† P < 0.001 versus control (0 μg/mL CZE). Scale bar = 200 μm.

Figure 3

Effects of CZE on the expression of osteoclast differentiation marker genes. BMMs were cultured with RANKL and M-CSF treatment in the presence or absence of CZE (25 μg/mL) for 4 days. The expression of osteoclast differentiation marker genes was measured by real-time PCR. The expression of mRNA levels was normalized with GAPDH and described as fold change of mRNA level. Data are expressed as the mean ± SD and are representative of at least three independent experiments. *P < 0.05, and ^† P < 0.01 versus control (0 μg/mL CZE).

Figure 4

Effects of CZE on RANKL-induced intracellular signaling and the expression of transcription factors in osteoclasts. BMMs were treated with M-CSF and RANKL in the presence or absence of CZE (25 μg/mL) for the indicated time. Lysate (30 μg) was subjected to SDS-PAGE and analyzed by immunoblotting. (a) MAPK (ERK, JNK, and p38) activation was measured by using their respective antibodies. (b)-(c) The expression of c-Fos and NFATc1 was detected by anti-c-Fos and NFATc1 antibody, respectively. Fold change normalized by actin is presented in the right panel. Data are representatively obtained from three independent experiments and are expressed as the mean ± SD. *P < 0.05, and ^† P < 0.01 versus control (0 μg/mL CZE).

Figure 5

Effect of CZE in RANKL-stimulated costimulatory signals in osteoclasts. BMMs were cultured with M-CSF and RANKL in the presence or absence of CZE (25 μg/mL) for 1 day. (a)-(b) RANKL-induced Ca²⁺-oscillation was measured with the Ca²⁺ indicator, Fura-2AM. (c) BMMs were cultured with M-CSF and RANKL in the presence of CZE for 1 day. RANKL-induced Ca²⁺-oscillation by acute treatment of CZE was measured. (d)-(e) BMMs were cultured with M-CSF and RANKL in presence or absence of CZE (25 μg/mL) for the indicated time. Lysate (30 μg) was subjected to SDS-PAGE, and the activation of PLCγ2 (d) and CREB (e) was analyzed by immunoblotting. All data are representative of at least three independent experiments.

Figure 6

Schematic diagram of the effect of CZE on RANKL-induced osteoclastogenesis. RANKL/RANK interaction may lead to the activation of MAPKs followed by c-Fos expression and alternatively activation of PLCγ2 inducing calcium signaling, which is critical for NFATc1 activation, followed by CREB activation and induction of c-Fos and NFATc1. CZE inhibited both RANKL-induced ERK and PLCγ activation signaling pathways.