Abstract
BackgroundExcessive bone resorption by osteoclasts causes pathological bone destruction, seen in various bone diseases. There is accumulating evidence that certain herbal extracts have beneficial effects on bone metabolism. The fruits of Alpinia oxyphylla has been traditionally used for the treatment of diarrhea and enuresis. In this study, we investigated the effects of water extract of the fruits of Alpinia oxyphylla (WEAO) on osteoclast differentiation and osteoclast-mediated bone destruction.MethodsFor osteoclast differentiation assay, mouse bone marrow-derived macrophages (BMMs) were cultured in the presence of RANKL and M-CSF. RANKL signaling pathways and gene expression of transcription factors regulating osteoclast differentiation were investigated by real-time PCR and Western blotting. A constitutively active form of NFATc1 was retrovirally transduced into BMMs. Bone resorbing activity of mature osteoclast was examined on a plate coated with an inorganic crystalline calcium phosphate. The in vivo effect against bone destruction was assessed in a murine model of RANKL-induced osteoporosis by micro-computed tomography and bone metabolism marker analyses.ResultsWEAO dose-dependently inhibited RANKL-induced osteoclast differentiation from BMMs by targeting the early stages of osteoclast differentiation. WEAO inhibited RANKL-induced expression of NFATc1, the master regulator of osteoclast differentiation. Overexpression of a constitutively active form of NFATc1 blunted the inhibitory effect of WEAO on osteoclast differentiation, suggesting that NFATc1 is a critical target of the inhibitory action of WEAO. WEAO inhibited RANKL-induced expression of c-Fos, an upstream activator of NFATc1, by suppressing the classical NF-κB signaling pathway. WEAO also inhibited RANKL-induced down-regulation of Id2 and MafB, negative regulators of NFATc1. WEAO does not directly affect bone resorbing activity of mature osteoclasts. In accordance with the in vitro results, WEAO attenuated RANKL-induced bone destruction in mice by inhibiting osteoclast differentiation.ConclusionsThis study demonstrates that WEAO exhibits a protective effect against bone loss by inhibiting RANKL-induced osteoclast differentiation. These findings suggest that WEAO might be useful for the prevention and treatment of bone diseases associated with excessive bone resorption.
Highlights
Excessive bone resorption by osteoclasts causes pathological bone destruction, seen in various bone diseases
WEAO inhibits RANKL-induced osteoclast differentiation To determine whether WEAO affects osteoclast differentiation, we examined the effect of WEAO on RANKLinduced osteoclast differentiation from its precursor cells, bone marrow-derived macrophages (BMMs)
When WEAO was added to the BMM cultures simultaneously with RANKL, osteoclast differentiation was inhibited by WEAO in a dose-dependent manner (Figure 1A and B)
Summary
Excessive bone resorption by osteoclasts causes pathological bone destruction, seen in various bone diseases. We investigated the effects of water extract of the fruits of Alpinia oxyphylla (WEAO) on osteoclast differentiation and osteoclast-mediated bone destruction. Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) are critical cytokines required for osteoclast differentiation [3,4,5]. M-CSF stimulates the expression of RANK, the receptor for RANKL, in osteoclast precursors [5]. Accumulating evidence suggests that nuclear factor of activated T cells cytoplasmic 1 (NFATc1) integrates RANKL signaling for terminal differentiation of osteoclasts [1]. RANKL stimulates the expression and activation of NFATc1 in osteoclast precursors by regulating other transcription factors. It was reported that RANKL suppresses the expression of transcriptional repressors such as inhibitors of differentiation/DNA binding (Ids), v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (MafB), and interferon regulatory factor 8 that inhibit NFATc1 expression and osteoclast differentiation [13]
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