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 NFATc1 activation in bone marrow-derived macrophages (BMMs).(A) The effect of SRT2183 on NFATc1 protein level. Western blot analysis of NFATc1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. NFATc1- nuclear factor of activated T-cell cytoplasmic 1. (B) The effect of SRT2183 on DC-STAMP protein level. Western blot analysis of DC-STAMP and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. DC-STAMP- dendritic cell-specific transmembrane protein. (C) The effect of SRT2183 on mRNA expression of osteoclast markers and fusion-related genes. SRT2183 or vehicle were co-administrated with RANKL. Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to GAPDH. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test, *P<0.05; **P<0.01; ***P<0.001 compared to vehicle-treated BMMs.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134391.g002: SRT2183 inhibits RANKL-induced NFATc1 activation in bone marrow-derived macrophages (BMMs).(A) The effect of SRT2183 on NFATc1 protein level. Western blot analysis of NFATc1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. NFATc1- nuclear factor of activated T-cell cytoplasmic 1. (B) The effect of SRT2183 on DC-STAMP protein level. Western blot analysis of DC-STAMP and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. DC-STAMP- dendritic cell-specific transmembrane protein. (C) The effect of SRT2183 on mRNA expression of osteoclast markers and fusion-related genes. SRT2183 or vehicle were co-administrated with RANKL. Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to GAPDH. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test, *P<0.05; **P<0.01; ***P<0.001 compared to vehicle-treated BMMs.

Mentions: Time course experiments revealed that SRT2183 treatment at the proliferation phase did not affect osteoclast generation, however osteoclast differentiation and maturation were markedly reduced when SRT2183 was administered at the differentiation stages (Fig 1E a-d). The proliferation of osteoclast precursors was not altered by SRT2183 treatment (Fig 1F). To understand if SRT2183 affects cell survival, viability and apoptosis studies were conducted at the proliferation and differentiation phases. The administration of SRT2183 at the proliferation and differentiation phases did not decrease cell viability or increased apoptosis (Fig 1G and 1H). Accordingly, total protein was unchanged in SRT2183 versus vehicle-treated cells (S1 Fig). These results suggest that SRT2183 inhibits osteoclast differentiation and function but not precursors’ proliferation or cell survival. Consistently, nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), a master transcription factor in osteoclast differentiation [23] as well as its downstream target, dendritic cell-specific transmembrane protein (DC-STAMP), necessary for osteoclast multi-nucleation [24] were reduced in SRT2183-treated cells (Fig 2A and 2B). Similarly, mRNA expression of osteoclast markers and specifically key osteoclast fusion-related genes, Tm7sf4 (encoding for DC-STAMP) and the gene encoding for osteoclast stimulatory trans-membrane protein (OC-STAMP) were significantly decreased in SRT2183-treated osteoclasts (Fig 2C).


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 NFATc1 activation in bone marrow-derived macrophages (BMMs).(A) The effect of SRT2183 on NFATc1 protein level. Western blot analysis of NFATc1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. NFATc1- nuclear factor of activated T-cell cytoplasmic 1. (B) The effect of SRT2183 on DC-STAMP protein level. Western blot analysis of DC-STAMP and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. DC-STAMP- dendritic cell-specific transmembrane protein. (C) The effect of SRT2183 on mRNA expression of osteoclast markers and fusion-related genes. SRT2183 or vehicle were co-administrated with RANKL. Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to GAPDH. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test, *P<0.05; **P<0.01; ***P<0.001 compared to vehicle-treated BMMs.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0134391.g002: SRT2183 inhibits RANKL-induced NFATc1 activation in bone marrow-derived macrophages (BMMs).(A) The effect of SRT2183 on NFATc1 protein level. Western blot analysis of NFATc1 and HSP90 in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. NFATc1- nuclear factor of activated T-cell cytoplasmic 1. (B) The effect of SRT2183 on DC-STAMP protein level. Western blot analysis of DC-STAMP and GAPDH in SRT2183- and vehicle-treated osteoclasts 4 days post RANKL stimulation. DC-STAMP- dendritic cell-specific transmembrane protein. (C) The effect of SRT2183 on mRNA expression of osteoclast markers and fusion-related genes. SRT2183 or vehicle were co-administrated with RANKL. Gene expression analysis by quantitative Real-Time PCR 4 days post RANKL stimulation is shown. Results are relative to GAPDH. Data are Mean ±SEM (n = 3 independent experiments), analyzed by one-sample Student's t-test, *P<0.05; **P<0.01; ***P<0.001 compared to vehicle-treated BMMs.
Mentions: Time course experiments revealed that SRT2183 treatment at the proliferation phase did not affect osteoclast generation, however osteoclast differentiation and maturation were markedly reduced when SRT2183 was administered at the differentiation stages (Fig 1E a-d). The proliferation of osteoclast precursors was not altered by SRT2183 treatment (Fig 1F). To understand if SRT2183 affects cell survival, viability and apoptosis studies were conducted at the proliferation and differentiation phases. The administration of SRT2183 at the proliferation and differentiation phases did not decrease cell viability or increased apoptosis (Fig 1G and 1H). Accordingly, total protein was unchanged in SRT2183 versus vehicle-treated cells (S1 Fig). These results suggest that SRT2183 inhibits osteoclast differentiation and function but not precursors’ proliferation or cell survival. Consistently, nuclear factor of activated T-cell cytoplasmic 1 (NFATc1), a master transcription factor in osteoclast differentiation [23] as well as its downstream target, dendritic cell-specific transmembrane protein (DC-STAMP), necessary for osteoclast multi-nucleation [24] were reduced in SRT2183-treated cells (Fig 2A and 2B). Similarly, mRNA expression of osteoclast markers and specifically key osteoclast fusion-related genes, Tm7sf4 (encoding for DC-STAMP) and the gene encoding for osteoclast stimulatory trans-membrane protein (OC-STAMP) were significantly decreased in SRT2183-treated osteoclasts (Fig 2C).

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