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Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

Li J, Zeng L, Xie J, Yue Z, Deng H, Ma X, Zheng C, Wu X, Luo J, Liu M - Sci Rep (2015)

Bottom Line: In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems.At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis.As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Fengxian District Central Hospital and East China Normal University Joint Center for Translational Medicine, Shanghai Key laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

No MeSH data available.


Related in: MedlinePlus

XN blocks RANKL-induced Ca2+/NFATc1 signaling pathway.(A) The effect of XN on RANKL-induced Ca2+ oscillation. The M-CSF incubated BMMs were pretreated with or without XN (5 uM) for 24 hours, and then incubated with or without RANKL (30 ng/ml) for another 60 hours. The Ca2+ oscillation was analyzed as described in Materials and Methods. (B) The effect of XN on RANKL-induced activity of NFAT. RAW264.7 cells were cotransfected with NFAT-luciferase reporter gene and Renilla gene. After 36 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured as described in Materials and Methods. Results are expressed as fold activity over the activity of the control. (C,D) The effect of XN on NFATc1 binding to Cathepsin K (Ctsk) promoter region by Chromatin immunoprecipitation (ChIP) assays. BMMs incubated with RANKL (30 ng/ml) were treated with or without XN for indicated time (C). Or BMMs incubated with RANKL (30 ng/ml) were treated with indicated concentration of XN for 48 hours (D). The Chromatin DNA were immunoprecipitated with control IgG or the anti-NFATc1 antibody and subjected to quantitative real-time PCR with primers specific for NFATc1-binding sites. (E) NFATc1 prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with NFATc1 or vector control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were fixed and stained for TRAP activity (left). Original magnification, ×40. The numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). Column, means of three experiments conducted in triplicate; bar, SD. ***P < 0.001.
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f5: XN blocks RANKL-induced Ca2+/NFATc1 signaling pathway.(A) The effect of XN on RANKL-induced Ca2+ oscillation. The M-CSF incubated BMMs were pretreated with or without XN (5 uM) for 24 hours, and then incubated with or without RANKL (30 ng/ml) for another 60 hours. The Ca2+ oscillation was analyzed as described in Materials and Methods. (B) The effect of XN on RANKL-induced activity of NFAT. RAW264.7 cells were cotransfected with NFAT-luciferase reporter gene and Renilla gene. After 36 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured as described in Materials and Methods. Results are expressed as fold activity over the activity of the control. (C,D) The effect of XN on NFATc1 binding to Cathepsin K (Ctsk) promoter region by Chromatin immunoprecipitation (ChIP) assays. BMMs incubated with RANKL (30 ng/ml) were treated with or without XN for indicated time (C). Or BMMs incubated with RANKL (30 ng/ml) were treated with indicated concentration of XN for 48 hours (D). The Chromatin DNA were immunoprecipitated with control IgG or the anti-NFATc1 antibody and subjected to quantitative real-time PCR with primers specific for NFATc1-binding sites. (E) NFATc1 prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with NFATc1 or vector control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were fixed and stained for TRAP activity (left). Original magnification, ×40. The numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). Column, means of three experiments conducted in triplicate; bar, SD. ***P < 0.001.

Mentions: RANKL-induced Ca2+/NFATc1 signaling activation is among the very early molecular events in osteoclastogenesis6. Our observations indicated that XN inhibited RANKL-induced osteoclast differentiation at an early stage. Therefore, we employed three separate approaches to investigate whether XN inhibits RANKL-induced Ca2+/NFATc1 signaling pathway. First, using calcium imaging assay, we examined whether XN suppressed RANKL-induced Ca2+ oscillations. Our results showed that XN at 5 μM completely diminished the amplitude and frequency of Ca2+ oscillations induced by RANKL (Fig. 5A). Second, using NFAT-luciferase reporter gene assay, we found that the activity of NFAT, which is the downstream transcription factor of Ca2+ oscillation and the master regulator of osteoclastogenesis, was dose-dependently suppressed by XN in response to RANKL stimulation (Fig. 5B). Finally, using ChIP assay, XN concentration- and time-dependently suppressed the recruitment of NFATc1 on Ctsk promoter. To further confirm that XN inhibited osteoclast differentiation by suppressing RANKL-induced NFATc1 activity, we assessed whether overexpression of NFATc1 could rescue the XN-induced suppression of osteoclast differentiation. Our results showed that XN inhibited osteoclast formation in control RAW264.7 cells, but had little inhibitory effect on osteoclastogenesis in NFATc1-overexpressed cells (Fig. 5E), suggesting that NFATc1 prevented XN induced inhibition of osteoclastogenesis. All of the results demonstrate that XN suppresses osteoclast formation by inhibiting Ca2+/NFATc1 signaling pathway.


Inhibition of Osteoclastogenesis and Bone Resorption in vitro and in vivo by a prenylflavonoid xanthohumol from hops.

Li J, Zeng L, Xie J, Yue Z, Deng H, Ma X, Zheng C, Wu X, Luo J, Liu M - Sci Rep (2015)

XN blocks RANKL-induced Ca2+/NFATc1 signaling pathway.(A) The effect of XN on RANKL-induced Ca2+ oscillation. The M-CSF incubated BMMs were pretreated with or without XN (5 uM) for 24 hours, and then incubated with or without RANKL (30 ng/ml) for another 60 hours. The Ca2+ oscillation was analyzed as described in Materials and Methods. (B) The effect of XN on RANKL-induced activity of NFAT. RAW264.7 cells were cotransfected with NFAT-luciferase reporter gene and Renilla gene. After 36 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured as described in Materials and Methods. Results are expressed as fold activity over the activity of the control. (C,D) The effect of XN on NFATc1 binding to Cathepsin K (Ctsk) promoter region by Chromatin immunoprecipitation (ChIP) assays. BMMs incubated with RANKL (30 ng/ml) were treated with or without XN for indicated time (C). Or BMMs incubated with RANKL (30 ng/ml) were treated with indicated concentration of XN for 48 hours (D). The Chromatin DNA were immunoprecipitated with control IgG or the anti-NFATc1 antibody and subjected to quantitative real-time PCR with primers specific for NFATc1-binding sites. (E) NFATc1 prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with NFATc1 or vector control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were fixed and stained for TRAP activity (left). Original magnification, ×40. The numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). Column, means of three experiments conducted in triplicate; bar, SD. ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f5: XN blocks RANKL-induced Ca2+/NFATc1 signaling pathway.(A) The effect of XN on RANKL-induced Ca2+ oscillation. The M-CSF incubated BMMs were pretreated with or without XN (5 uM) for 24 hours, and then incubated with or without RANKL (30 ng/ml) for another 60 hours. The Ca2+ oscillation was analyzed as described in Materials and Methods. (B) The effect of XN on RANKL-induced activity of NFAT. RAW264.7 cells were cotransfected with NFAT-luciferase reporter gene and Renilla gene. After 36 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured as described in Materials and Methods. Results are expressed as fold activity over the activity of the control. (C,D) The effect of XN on NFATc1 binding to Cathepsin K (Ctsk) promoter region by Chromatin immunoprecipitation (ChIP) assays. BMMs incubated with RANKL (30 ng/ml) were treated with or without XN for indicated time (C). Or BMMs incubated with RANKL (30 ng/ml) were treated with indicated concentration of XN for 48 hours (D). The Chromatin DNA were immunoprecipitated with control IgG or the anti-NFATc1 antibody and subjected to quantitative real-time PCR with primers specific for NFATc1-binding sites. (E) NFATc1 prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with NFATc1 or vector control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were fixed and stained for TRAP activity (left). Original magnification, ×40. The numbers of TRAP positive multinucleated (>3 nuclei) osteoclasts were counted (right). Column, means of three experiments conducted in triplicate; bar, SD. ***P < 0.001.
Mentions: RANKL-induced Ca2+/NFATc1 signaling activation is among the very early molecular events in osteoclastogenesis6. Our observations indicated that XN inhibited RANKL-induced osteoclast differentiation at an early stage. Therefore, we employed three separate approaches to investigate whether XN inhibits RANKL-induced Ca2+/NFATc1 signaling pathway. First, using calcium imaging assay, we examined whether XN suppressed RANKL-induced Ca2+ oscillations. Our results showed that XN at 5 μM completely diminished the amplitude and frequency of Ca2+ oscillations induced by RANKL (Fig. 5A). Second, using NFAT-luciferase reporter gene assay, we found that the activity of NFAT, which is the downstream transcription factor of Ca2+ oscillation and the master regulator of osteoclastogenesis, was dose-dependently suppressed by XN in response to RANKL stimulation (Fig. 5B). Finally, using ChIP assay, XN concentration- and time-dependently suppressed the recruitment of NFATc1 on Ctsk promoter. To further confirm that XN inhibited osteoclast differentiation by suppressing RANKL-induced NFATc1 activity, we assessed whether overexpression of NFATc1 could rescue the XN-induced suppression of osteoclast differentiation. Our results showed that XN inhibited osteoclast formation in control RAW264.7 cells, but had little inhibitory effect on osteoclastogenesis in NFATc1-overexpressed cells (Fig. 5E), suggesting that NFATc1 prevented XN induced inhibition of osteoclastogenesis. All of the results demonstrate that XN suppresses osteoclast formation by inhibiting Ca2+/NFATc1 signaling pathway.

Bottom Line: In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems.At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis.As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr.

View Article: PubMed Central - PubMed

Affiliation: Shanghai Fengxian District Central Hospital and East China Normal University Joint Center for Translational Medicine, Shanghai Key laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.

ABSTRACT
Excessive RANKL signaling leads to superfluous osteoclast formation and bone resorption, is widespread in the pathologic bone loss and destruction. Therefore, targeting RANKL or its signaling pathway has been a promising and successful strategy for this osteoclast-related diseases. In this study, we examined the effects of xanthohumol (XN), an abundant prenylflavonoid from hops plant, on osteoclastogenesis, osteoclast resorption, and RANKL-induced signaling pathway using both in vitro and in vivo assay systems. In mouse and human, XN inhibited osteoclast differentiation and osteoclast formation at the early stage. Furthermore, XN inhibited osteoclast actin-ring formation and bone resorption in a dose-dependent manner. In ovariectomized-induced bone loss mouse model and RANKL-injection-induced bone resorption model, we found that administration of XN markedly inhibited bone loss and resorption by suppressing osteoclast activity. At the molecular level, XN disrupted the association of RANK and TRAF6, resulted in the inhibition of NF-κB and Ca(2+)/NFATc1 signaling pathway during osteoclastogenesis. As a results, XN suppressed the expression of osteoclastogenesis-related marker genes, including CtsK, Nfatc1, Trap, Ctr. Therefore, our data demonstrated that XN inhibits osteoclastogenesis and bone resorption through RANK/TRAF6 signaling pathways. XN could be a promising drug candidate in the treatment of osteoclast-related diseases such as postmenopausal osteoporosis.

No MeSH data available.


Related in: MedlinePlus