Limits...
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 inhibits RANKL-induced osteoclastogenesis and pit formation in sirt1-/- BMMs.(A) Sirt1 expression in WT- and in Sirt1-/--derived osteoclasts. PCR amplification of exons 1–9 of the sirt1 gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from Sirt1Δ/Δ (Sirt1-/-) mice. (B) The effect of SRT2183 on osteoclast differentiation in Sirt1-/--derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in Sirt1-/--derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) 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. (E) 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. (F) 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. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to Polr2a (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's t-test paired (C) or one-sample Student's t-test (H-I); ***P<0.001, ****P<0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4520518&req=5

pone.0134391.g004: SRT2183 inhibits RANKL-induced osteoclastogenesis and pit formation in sirt1-/- BMMs.(A) Sirt1 expression in WT- and in Sirt1-/--derived osteoclasts. PCR amplification of exons 1–9 of the sirt1 gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from Sirt1Δ/Δ (Sirt1-/-) mice. (B) The effect of SRT2183 on osteoclast differentiation in Sirt1-/--derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in Sirt1-/--derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) 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. (E) 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. (F) 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. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to Polr2a (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's t-test paired (C) or one-sample Student's t-test (H-I); ***P<0.001, ****P<0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.

Mentions: To understand the role of Sirt1, the influence of SRT2183 on osteoclastogenesis was evaluated in bone marrow cells derived from sirt1-/- mice. Sirt1Δ/Δ (Sirt1-/-) mice lacking Sirt1 protein were generated from inbred 129/Sv Sirt1+/Δ mice, whereas their littermates WT served as the controls. These KO mice are lacking sirt1 exons 5–7 resulting in no sirt1 protein production (Fig 4A) and have a birth rate lower than 3% [20]. Strikingly, SRT2183 abolished the generation of large multi-nucleated osteoclasts and their resorptive capacity in sirt1-/- BMMs similar to the effect observed in WT-derived osteoclasts, indicating that Sirt1 is not essential for inhibition of osteoclast generation and function under these conditions (Fig 4B and 4C). As expected, RelA/p65 K310 acetylation was not changed in SRT2183-treated sirt1-/- osteoclasts (Fig 4D), as this is a direct Sirt1 target. Furthermore, AMPKα phosphorylation was not affected by SRT2183 administration in sirt1-/--derived osteoclasts (Fig 4E), suggesting that Sirt1 is upstream of and is required for AMPK activation by SRT2183 under these conditions. Consistently IκBα level was unchanged (Fig 4F).


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 inhibits RANKL-induced osteoclastogenesis and pit formation in sirt1-/- BMMs.(A) Sirt1 expression in WT- and in Sirt1-/--derived osteoclasts. PCR amplification of exons 1–9 of the sirt1 gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from Sirt1Δ/Δ (Sirt1-/-) mice. (B) The effect of SRT2183 on osteoclast differentiation in Sirt1-/--derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in Sirt1-/--derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) 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. (E) 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. (F) 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. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to Polr2a (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's t-test paired (C) or one-sample Student's t-test (H-I); ***P<0.001, ****P<0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520518&req=5

pone.0134391.g004: SRT2183 inhibits RANKL-induced osteoclastogenesis and pit formation in sirt1-/- BMMs.(A) Sirt1 expression in WT- and in Sirt1-/--derived osteoclasts. PCR amplification of exons 1–9 of the sirt1 gene (left panel) and Western blot analysis with Sirt1 antibody (right panel) demonstrates complete loss of Sirt1 protein in osteoclasts obtained from Sirt1Δ/Δ (Sirt1-/-) mice. (B) The effect of SRT2183 on osteoclast differentiation in Sirt1-/--derived BMMs. BMMs were inducted to osteoclastogenesis with RANKL in the presence or absence of SRT2183. TRAP staining performed 4 days post induction. (C) The effect of SRT2183 on pit formation in Sirt1-/--derived BMMs stimulated with RANKL. An eroded area (left panel) and pit formation assay (right) are shown. (D) 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. (E) 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. (F) 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. (G-H) The effect of SRT2183 on Sirt3 protein (G) and gene expression (H). Western blot analysis of Sirt3 and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation (G). Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to Polr2a (H). (I) The effect of SRT2183 on superoxide dismutase 2 (Sod2) Lys68 acetylation. Western blot analysis of acetylated (ac) Sod2K68 and Sod2 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. Data are Mean ± SEM (n = 3 independent experiments), analyzed by paired Student's t-test paired (C) or one-sample Student's t-test (H-I); ***P<0.001, ****P<0.0001, versus vehicle-treated BMMs. Magnification X40; scale bar 1mm.
Mentions: To understand the role of Sirt1, the influence of SRT2183 on osteoclastogenesis was evaluated in bone marrow cells derived from sirt1-/- mice. Sirt1Δ/Δ (Sirt1-/-) mice lacking Sirt1 protein were generated from inbred 129/Sv Sirt1+/Δ mice, whereas their littermates WT served as the controls. These KO mice are lacking sirt1 exons 5–7 resulting in no sirt1 protein production (Fig 4A) and have a birth rate lower than 3% [20]. Strikingly, SRT2183 abolished the generation of large multi-nucleated osteoclasts and their resorptive capacity in sirt1-/- BMMs similar to the effect observed in WT-derived osteoclasts, indicating that Sirt1 is not essential for inhibition of osteoclast generation and function under these conditions (Fig 4B and 4C). As expected, RelA/p65 K310 acetylation was not changed in SRT2183-treated sirt1-/- osteoclasts (Fig 4D), as this is a direct Sirt1 target. Furthermore, AMPKα phosphorylation was not affected by SRT2183 administration in sirt1-/--derived osteoclasts (Fig 4E), suggesting that Sirt1 is upstream of and is required for AMPK activation by SRT2183 under these conditions. Consistently IκBα level was unchanged (Fig 4F).

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