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The Sirt1 Activators SRT2183 and SRT3025 Inhibit RANKL-Induced Osteoclastogenesis in Bone Marrow-Derived Macrophages and Down-Regulate Sirt3 in Sirt1 Null Cells.

Gurt I, Artsi H, Cohen-Kfir E, Hamdani G, Ben-Shalom G, Feinstein B, El-Haj M, Dresner-Pollak R - PLoS ONE (2015)

Bottom Line: SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target.However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions.In sirt1 osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology and Metabolism Service, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT
Increased osteoclast-mediated bone resorption is characteristic of osteoporosis, malignant bone disease and inflammatory arthritis. Targeted deletion of Sirtuin1 (Sirt1), a key player in aging and metabolism, in osteoclasts results in increased osteoclast-mediated bone resorption in vivo, making it a potential novel therapeutic target to block bone resorption. Sirt1 activating compounds (STACs) were generated and were investigated in animal disease models and in humans however their mechanism of action was a source of controversy. We studied the effect of SRT2183 and SRT3025 on osteoclastogenesis in bone-marrow derived macrophages (BMMs) in vitro, and discovered that these STACs inhibit RANKL-induced osteoclast differentiation, fusion and resorptive capacity without affecting osteoclast survival. SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target. However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions. In sirt1 osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated. Our findings suggest that SRT2183 and SRT3025 activate Sirt1 and inhibit RANKL-induced osteoclastogenesis in vitro however under conditions of Sirt1 deficiency can affect Sirt3. As aging is associated with reduced Sirt1 level and activity, the influence of STACs on Sirt3 needs to be investigated in vivo in animal and human disease models of aging and osteoporosis.

No MeSH data available.


Related in: MedlinePlus

SRT2183 activates AMPK and deacetylates RelA/p65 K310 in RANKL-induced BMMs.(A) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; AMPKα- AMP-activated protein kinase alpha. (B) The effect of SRT2183 on ACC phosphorylation. Western blot analysis of pACC and ACC in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; ACC-Acetyl CoA Carboxylase. (C) The effect of SRT2183 on Sirt1 protein level in RANKL-stimulated osteoclasts. Western blot analysis of Sirt1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (D) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (E) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test; *P<0.05 versus vehicle-treated BMMs.
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pone.0134391.g003: SRT2183 activates AMPK and deacetylates RelA/p65 K310 in RANKL-induced BMMs.(A) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; AMPKα- AMP-activated protein kinase alpha. (B) The effect of SRT2183 on ACC phosphorylation. Western blot analysis of pACC and ACC in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; ACC-Acetyl CoA Carboxylase. (C) The effect of SRT2183 on Sirt1 protein level in RANKL-stimulated osteoclasts. Western blot analysis of Sirt1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (D) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (E) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test; *P<0.05 versus vehicle-treated BMMs.

Mentions: Previous work has shown that Sirt1 is closely coupled to AMP-activated protein kinase (AMPK) activity in a mutually enforcing mechanism [25]. Moreover, AMPK regulates osteoclast differentiation and function, and AMPKα1 deficiency in mice causes enhanced osteoclast differentiation and fusion [26]. We therefore investigated AMPK activation and discovered increased phosphorylation of AMPKα and its target acetyl CoA carboxylase (ACC) in SRT2183-treated cells, indicating AMPK stimulation (Fig 3A and 3B). Of note, increased Sirt1 level in SRT2183-treated cells was also observed (Fig 3C), and can result from AMPK activation, as AMPK was shown to positively regulate Sirt1 level [27].


The Sirt1 Activators SRT2183 and SRT3025 Inhibit RANKL-Induced Osteoclastogenesis in Bone Marrow-Derived Macrophages and Down-Regulate Sirt3 in Sirt1 Null Cells.

Gurt I, Artsi H, Cohen-Kfir E, Hamdani G, Ben-Shalom G, Feinstein B, El-Haj M, Dresner-Pollak R - PLoS ONE (2015)

SRT2183 activates AMPK and deacetylates RelA/p65 K310 in RANKL-induced BMMs.(A) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; AMPKα- AMP-activated protein kinase alpha. (B) The effect of SRT2183 on ACC phosphorylation. Western blot analysis of pACC and ACC in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; ACC-Acetyl CoA Carboxylase. (C) The effect of SRT2183 on Sirt1 protein level in RANKL-stimulated osteoclasts. Western blot analysis of Sirt1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (D) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (E) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test; *P<0.05 versus vehicle-treated BMMs.
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pone.0134391.g003: SRT2183 activates AMPK and deacetylates RelA/p65 K310 in RANKL-induced BMMs.(A) The effect of SRT2183 on AMPKα phosphorylation (Thr172). Western blot analysis of pAMPKα and AMPKα in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; AMPKα- AMP-activated protein kinase alpha. (B) The effect of SRT2183 on ACC phosphorylation. Western blot analysis of pACC and ACC in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. p- phosphorylated; ACC-Acetyl CoA Carboxylase. (C) The effect of SRT2183 on Sirt1 protein level in RANKL-stimulated osteoclasts. Western blot analysis of Sirt1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. (D) The effect of SRT2183 on IκBα protein level. Western blot analysis of IκBα and GAPDH in SRT2183- and vehicle-treated BMMs 24 hours post RANKL stimulation. (E) The effect of SRT2183 on p65 acetylation (Lys310). Western blot analysis of p65K310 ac and p65 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test; *P<0.05 versus vehicle-treated BMMs.
Mentions: Previous work has shown that Sirt1 is closely coupled to AMP-activated protein kinase (AMPK) activity in a mutually enforcing mechanism [25]. Moreover, AMPK regulates osteoclast differentiation and function, and AMPKα1 deficiency in mice causes enhanced osteoclast differentiation and fusion [26]. We therefore investigated AMPK activation and discovered increased phosphorylation of AMPKα and its target acetyl CoA carboxylase (ACC) in SRT2183-treated cells, indicating AMPK stimulation (Fig 3A and 3B). Of note, increased Sirt1 level in SRT2183-treated cells was also observed (Fig 3C), and can result from AMPK activation, as AMPK was shown to positively regulate Sirt1 level [27].

Bottom Line: SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target.However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions.In sirt1 osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated.

View Article: PubMed Central - PubMed

Affiliation: Endocrinology and Metabolism Service, Department of Medicine, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.

ABSTRACT
Increased osteoclast-mediated bone resorption is characteristic of osteoporosis, malignant bone disease and inflammatory arthritis. Targeted deletion of Sirtuin1 (Sirt1), a key player in aging and metabolism, in osteoclasts results in increased osteoclast-mediated bone resorption in vivo, making it a potential novel therapeutic target to block bone resorption. Sirt1 activating compounds (STACs) were generated and were investigated in animal disease models and in humans however their mechanism of action was a source of controversy. We studied the effect of SRT2183 and SRT3025 on osteoclastogenesis in bone-marrow derived macrophages (BMMs) in vitro, and discovered that these STACs inhibit RANKL-induced osteoclast differentiation, fusion and resorptive capacity without affecting osteoclast survival. SRT2183 and SRT3025 activated AMPK, increased Sirt1 expression and decreased RelA/p65 lysine310 acetylation, critical for NF-κB activation, and an established Sirt1 target. However, inhibition of osteoclastogenesis by these STACs was also observed in BMMs derived from sirt1 knock out (sirt1-/-) mice lacking the Sirt1 protein, in which neither AMPK nor RelA/p65 lysine 310 acetylation was affected, confirming that these effects require Sirt1, but suggesting that Sirt1 is not essential for inhibition of osteoclastogenesis by these STACs under these conditions. In sirt1 osteoclasts treated with SRT2183 or SRT3025 Sirt3 was found to be down-regulated. Our findings suggest that SRT2183 and SRT3025 activate Sirt1 and inhibit RANKL-induced osteoclastogenesis in vitro however under conditions of Sirt1 deficiency can affect Sirt3. As aging is associated with reduced Sirt1 level and activity, the influence of STACs on Sirt3 needs to be investigated in vivo in animal and human disease models of aging and osteoporosis.

No MeSH data available.


Related in: MedlinePlus