Category Archives: Sirtuins

SIRT1 Represses Sclerostin in Mice and may be Anabolic for Bone

Abstract

Sirt1 is a regulator of bone mass and a repressor of Sost encoding for sclerostin, a bone formation inhibitor.

Sirt1, the mammalian ortholog of the yeast Sir2 (silent information regulator 2), was shown to play an important role in metabolism and in age-associated diseases, but its role in skeletal homeostasis and osteoporosis has yet not been studied. Using 129/Sv mice with a germline mutation in the Sirt1 gene, we demonstrate that Sirt1 haplo-insufficient (Sirt1(+/-)) female mice exhibit a significant reduction in bone mass characterized by decreased bone formation and increased marrow adipogenesis. Importantly, we identify Sost, encoding for sclerostin, a critical inhibitor of bone formation, as a novel target of Sirt1. Using chromatin immunoprecipitation analysis, we reveal that Sirt1 directly and negatively regulates Sost gene expression by deacetylating histone 3 at lysine 9 at the Sost promoter. Sost down-regulation by small interfering RNA and the administration of a sclerostin-neutralizing antibody restore gene expression of osteocalcin and bone sialoprotein as well as mineralized nodule formation in Sirt1(+/-) marrow-derived mesenchymal stem cells induced to osteogenesis. These findings reveal a novel role for Sirt1 in bone as a regulator of bone mass and a repressor of sclerostin, and have potential implications suggesting that Sirt1 is a target for promoting bone formation as an anabolic approach for treatment of osteoporosis.

Cohen-Kfir E, Artsi H, Levin A, Abramowitz E…
Endocrinology Dec 2011
PMID: 21952235

Resveratrol and SIRT1 Reduces Sclerostin Expression In-Vitro

Abstract

Low sirtuin 1 levels in human osteoarthritis subchondral osteoblasts lead to abnormal sclerostin expression which decreases Wnt/β-catenin activity.

Wnt/β-catenin (cWnt) signaling plays a key role in osteogenesis by promoting the differentiation and mineralization of osteoblasts, activities altered in human osteoarthritic subchondral osteoblast (OA Ob). Sclerostin (SOST) has been shown to alter cWnt signaling. Sirtuin 1 (SIRT1) acts as a novel bone regulator and represses SOST levels in Ob. However the role of SIRT1 and SOST in OA Ob remains unknown. Herein, we explored the role played by SIRT1 and SOST on the abnormal mineralization and cWnt signaling in OA Ob.
Primary human normal and OA Ob were prepared from tibial plateaus. SOST levels were evaluated by immunohistochemistry, the expression and production of genes by qRT-PCR and WB analysis. Their inhibitions were performed using siRNA. cWnt signaling was measured by the TOPflash TCF/lef luciferase reporter assay. Mineralization was determined by alizarin red staining.
SOST levels were significantly increased in OA Ob compared to normal and were linked with elevated TGF-β1 levels in these cells. SIRT1 expression was significantly reduced in OA Ob compared to normal yet not modified by TGF-β1. Specific inhibition of SIRT1 increased TGF-β1 and SOST expressions in OA Ob, while stimulating SIRT1 activity with β-Nicotinamide mononucleotide reduced the expression of TGF-β1 and SOST, and increased mineralization in OA Ob. Resveratrol also reduced SOST expression in OA Ob. Reduced cWnt signaling, β-catenin levels, and mineralization in OA Ob were all corrected via reducing SOST expression.
These data indicate that high level of SOST is responsible, in part, for the reduced cWnt and mineralization of human OA Ob, which in turn is linked with abnormal SIRT1 levels in these pathological cells.

Abed É, Couchourel D, Delalandre A, Duval N…
Bone Feb 2014
PMID: 24184155

Resveratrol Enhances Osteogenesis via Runx2 and SIRT1 In Vitro

Abstract

Resveratrol mediated modulation of Sirt-1/Runx2 promotes osteogenic differentiation of mesenchymal stem cells: potential role of Runx2 deacetylation.

Osteogenic repair in response to bone injury is characterized by activation and differentiation of mesenchymal stem cells (MSCs) to osteoblasts. This study determined whether activation of Sirt-1 (a NAD(+)-dependent histone deacetylase) by the phytoestrogen resveratrol affects osteogenic differentiation.
Monolayer and high-density cultures of MSCs and pre-osteoblastic cells were treated with an osteogenic induction medium with/without the Sirt-1 inhibitor nicotinamide or/and resveratrol in a concentration dependent manner.
MSCs and pre-osteoblastic cells differentiated to osteoblasts when exposed to osteogenic-induction medium. The osteogenic response was blocked by nicotinamide, resulting in adipogenic differentiation and expression of the adipose transcription regulator PPAR-γ (peroxisome proliferator-activated receptor). However, in nicotinamide-treated cultures, pre-treatment with resveratrol significantly enhanced osteogenesis by increasing expression of Runx2 (bone specific transcription factor) and decreasing expression of PPAR-γ. Activation of Sirt-1 by resveratrol in MSCs increased its binding to PPAR-γ and repressed PPAR-γ activity by involving its cofactor NCoR (nuclear receptor co-repressor). The modulatory effects of resveratrol on nicotinamide-induced expression of PPAR-γ and its cofactor NCoR were found to be mediated, at least in part, by Sirt-1/Runx2 association and deacetylation of Runx2. Finally, knockdown of Sirt-1 by using antisense oligonucleotides downregulated the expression of Sirt-1 protein and abolished the inhibitory effects of resveratrol, namely nicotinamide-induced Sirt-1 suppression and Runx2 acetylation, suggesting that the acetylated content of Runx2 is related to downregulated Sirt-1 expression.
These data support a critical role for Runx2 acetylation/deacetylation during osteogenic differentiation in MSCs in vitro.

Shakibaei M, Shayan P, Busch F, Aldinger C…
PLoS ONE 2012
PMID: 22539994 | Free Full Text


From the introduction:

Resveratrol is a polyphenolic phytoestrogen (trans-3,5, 4′-trihydroxystilbene) found in the skin of red grapes, red vines, various other fruits, peanuts and root extracts of Polygonum cuspidatum [8]. Resveratrol acts as a mixed agonist/antagonist for the estrogen receptors alpha and beta [9]. Through binding to the estrogen receptor, resveratrol is thought to exert beneficial effects on the cardiovascular system and may reverse osteoporosis by a direct stimulatory effect on bone formation in osteoblastic cells [10]. Many of the biological effects of resveratrol have already been demonstrated in the literature; these include cardiovascular protection [11], anticancer activity [12] and stimulation of proliferation and osteoblastic differentiation in human and mouse MSCs [13], [14]. However, its effects on bone are less studied and are particularly relevant to this investigation.

From the discussion:

Resveratrol’s enhancement of osteogenesis was, at least in part regulated by Runx2 with additional contributions by Sirt-1. Resveratrol increases alkaline phosphatase activity in osteoblastic cells [10] an effect that is blocked by tamoxifen, an estrogen antagonist, suggesting that some of resveratrol’s stimulatory actions may be mediated through the estrogen receptor. Gehm et al. have reported that resveratrol acts as a phytoestrogen (i.e. activating the estrogen receptor) and decreases osteoporosis [43]. Moreover, resveratrol is one of the most potent Sirt-1 activators; through binding to a special binding site it induces a conformational change in Sirt-1, lowering the Km for both the acetylated substrate and NAD, thus resulting in increased enzymatic activity [18]. Sirt-1 facilitates the differentiation of MSCs to osteoblasts by directly regulating factors such as Runx2 and by modulation of nuclear receptor co-repressor NCoR and PPAR-γ.

SIRT6 Boosts Bone Formation

Abstract

SIRT6 regulates osteogenic differentiation of rat bone marrow mesenchymal stem cells partially via suppressing the nuclear factor-kappa B signaling pathway.

Sirtuin 6 (SIRT6) is a NAD-dependent deacetylase involved in lifespan regulation. To evaluate the effect of SIRT6 on osteogenesis, rat bone marrow mesenchymal stem cells (rBMSCs) with enhanced or reduced SIRT6 function were developed. We observed that SIRT6 knockdown significantly reduced the mRNA levels of several key osteogenic markers in vitro, including alkaline phosphatase (ALP), Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN), while overexpression of SIRT6 enhanced their expression. Additionally, SIRT6 knockdown activated nuclear factor-kappa B (NF-κB) transcriptional activity and upregulated the expression of acetyl-NF-κB p65 (Lys310). The decreased osteogenic differentiation ability of rBMSCs could be partially rescued by the addition of NF-κB inhibitor BAY 11-7082. Furthermore, SIRT6 overexpression in rBMSCs combined with the use of collagen/chitosan/HA scaffold (CHHS) could significantly boost new bone formation in rat cranial critical-sized defects, as determined by microcomputed tomography and histological examination. These data confirm that SIRT6 is mainly located in the nuclei of rBMSCs and plays an essential role in their normal osteogenic differentiation, partly by suppressing NF-κB signaling.

Sun H, Wu Y, Fu D, Liu Y…
Stem Cells Feb 2014
PMID: 24510807