Anat Cell Biol.  2010 Dec;43(4):310-316. 10.5115/acb.2010.43.4.310.

Amorphigenin inhibits Osteoclast differentiation by suppressing c-Fos and nuclear factor of activated T cells

• Kim BG
• Kwak HB
• Choi EY
• Kim HS
• Kim MH
• Kim SH
• Choi MK
• Chun CH
• Oh J
• Kim JJ

• KMID: 1455345
• DOI: http://doi.org/10.5115/acb.2010.43.4.310

Abstract

Among the several rotenoids, amorphigenin is isolated from the leaves of Amopha Fruticosa and it is known that has anti-proliferative effects and anti-cnacer effects in many cell types. The main aim of this study was to investigate the effects of amorphigenin on osteoclast differentiation in vitro and on LPS treated inflammatory bone loss model in vivo. We show here that amorphigenin inhibited RANKL-induced osteoclast differentiation from bone marrow macrophages in a dose dependent manner without cellular toxicity. Anti-osteoclastogenic properties of amorphigenin were based on a down-regulation of c-fos and NFATc1. Amorphigenin markedly inhibited RANKL-induced p38 and NF-kappaB pathways, but other pathways were not affected. Micro-CT analysis of the femurs showed that amorphigenin protected the LPS-induced bone loss. We concluded that amorphigenin can prevent inflammation-induced bone loss. Thus we expect that amorphigenin could be a treatment option for bone erosion caused by inflammation.

Keyword

Osteoporosis; Osteoclast; NFATc1

MeSH Terms

Bone Marrow
Down-Regulation
Femur
Inflammation
Macrophages
NF-kappa B
Osteoclasts
Osteoporosis
Rotenone
T-Lymphocytes
NF-kappa B
Rotenone

Figure

• 

  Fig. 1 Amorphigenin inhibits osteoclast differentiation in BMMs. (A) BMMs were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) in the presence of amorphigenin for 4 days. Then the cells were fixed in 3.7% formalin, permeabilized in 0.1% Triton X-100, and stained for TRAP. Arrows indicate the multinuclear osteoclasts. Arrowheads indicate the mononuclear osteoclasts (B) TRAP-positive cells were counted as osteoclasts. Askerisks indicate statistical difference from the control.

• 

  Fig. 2 Effect of amorphigenin on cytotoxicity. BMMs were seded onto 96-well plate. Then the cells were cultured for 3 days with the indicated concentration of amorphigenin in the presence of M-CSF (30 ng/ml). After 3 days, XTT reagents were added to each well. The optical density was read at 450 nm in an ELISA plate reader after 4 hours of incubation. Cell viability was determined relative to the control.

• 

  Fig. 3 Effects of amorphigenin in RANKL signaling. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. Cell lysates were performed by Wesern blotting with the indicated antibodies.

• 

  Fig. 4 Amorphigenin suppresses RANKL-induced gene expression. BMMs were pretreated with or without amorphigenin and then stimulated with RANKL (100 ng/ml) for the indicated times. Total RNA was isolated from the treated cells. The mRNA expression of c-fos, NFATc1, TRAP, OSCAR, and GAPDH was analyzed by RT-PCR.

• 

  Fig. 5 Amorphigenin inhibits RANKL-mediated c-Fos and NFATc1 expression. BMMs were pretreated with or without amorphigenin and stimulated with RANKL (100 ng/ml) for the indicated times. The cells were lysed in lysis buffer. c-Fos, NFATc1, or actin proteins were detected by Western blotting with an anti-c-fos, and anti-NFATc1, or anti-actin-antibody, respectively.

• 

  Fig. 6 Suppression of LPS-induced bone loss by amorphigenin. Mice were injected intraperitonealy with amorphigenin (10 µg/g of body weight) on days -1, 1, 3, 5, and 7 days. LPS was injected intraperitoneally on days 0 and 4. The mice were killed 8 days after the first injection of LPS, and femurs were collected. (A) The femurs were imaged with a micro-CT machine. (B) Bone volume/tissue volume (BV/TV) was analyzed using the histomorphometric results. Asterisks indicate statistical difference between the indicated groups.

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