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. 2018 Nov;175(21):4183-4192.
doi: 10.1111/bph.14477. Epub 2018 Sep 18.

The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1-bone morphogenic protein 2 longevity axis

Ming Zhao  1 , Seon-Yle Ko  2 , I Ross Garrett  3 , Gregory R Mundy  3 , Gloria E Gutierrez  3 , James R Edwards  4
Affiliations

The polyphenol resveratrol promotes skeletal growth in mice through a sirtuin 1-bone morphogenic protein 2 longevity axis

Ming Zhao et al. Br J Pharmacol. 2018 Nov.

Abstract

Background and purpose: The polyphenol resveratrol (RSV) exists in high quantities in certain foods (e.g. grapes and nuts). However, the capacity of RSV to confer physiological health benefits and a biological mechanism through which this might occur remains unclear.

Experimental approach: Aged, RSV-treated (300 mg·kg-1 ·day-1 ) and genetically modified [endothelial NOS (eNOS-/- )] female mice were assessed using histomorphometric and μCT analysis. Alongside in vivo analysis, molecular siRNA knockdown and pharmacological manipulation of eNOS, BMP2 and sirtuin 1 (SIRT1) and functional cellular assays in an osteoblast cell line panel, explored the mechanism through which RSV might impact overall bone volume.

Key results: RSV promoted osteoblast activity and bone growth in vivo. RSV dose-dependently and simultaneously increased alkaline phosphatase (ALP) and eNOS levels. Similarly, NO-donor treatment increased ALP, runt homology transcription factor 2, BMP2 and stimulated bone formation, whilst eNOS-deficient mice displayed a bone loss phenotype. Moreover, RSV-induced increase in ALP and BMP2 expression was blocked in eNOS-/- osteoblasts and by BMP-inhibitor noggin. The longevity-linked SIRT1 enzyme was positively regulated by RSV and SIRT1 deletion reduced eNOS, BMP2 and ALP. Like eNOS deletion, loss of SIRT1 blocked RSV-induced osteoblast activity; however, SIRT1 levels remained unchanged in eNOS-/- mice, indicating RSV activation of SIRT1 stimulates BMP2 release via eNOS. This signalling axis is supported by decreased SIRT1, eNOS and BMP2 confirmed in old versus young bone.

Conclusions and implications: These findings suggest a new mechanism of action in bone remodelling and the ageing skeleton, where RSV positively impacts bone homeostasis via SIRT1 activation of BMP2.

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Figures

Figure 1
Figure 1
RSV stimulates osteoblasts in vitro and bone formation in vivo. ALP activity of MC3T3‐E1 (A) and 2T3 (B) osteoblast cells treated with RSV (1–10 μM) or vehicle for 2 days (n = 5); normalization to total cell protein. (C) Mineralized matrix formation rates of 2T3 osteoblasts cultured in osteogenic media and RSV (1 μM, n = 5) or vehicle over 2 weeks (von Kossa staining). Normal C57Bl/6 mice (2 month) treated with resveratrol (300 mg·kg−1·day−1) in prepared chow, versus control chow over 10 weeks (n = 10). Bone volume (BV/TV) and trabecular parameters assessed in dissected tibia by μCT scanning (D–G), and cellular distribution by histomorphmetric analysis following H&E or TRAP staining (H–I) (*P < 0.05 vs. vehicle).
Figure 2
Figure 2
eNOS and NO‐donors stimulate bone growth. eNOS mRNA expression in MC3T3 (A) and 2T3 (B) cells treated with RSV (1–100 μM) or vehicle for 24 h (n = 5) determined by real time PCR. Nitrite (NO2 −) levels (C) or total eNOS protein (D) from 2T3 or MC3T3 cells treated with RSV (1–100 μM) or vehicle (n = 5) for 48 h, determined by colorimetric assay. ALP levels from primary osteoblasts treated with NO‐donor (NOC22, 0.1–10 μM) or vehicle for 4 days (n = 5) normalized to total cell protein (E). Calvariae from newborn mice cultured with NO‐donor (NOC22, 0.1–1 μM) or vehicle control for 4 days (n = 5) processed for histology and H&E staining (F). mRNA expression of osteoblast marker genes Runx2, osteocalcin (OCN) and BMP2 determined in 2T3 cells treated with NOC22 (1.0 μM) or vehicle for 24 h (n = 5) by real time PCR (G). BMP2 protein levels assessed in conditioned media from NO‐donor‐treated osteoblasts (NOC22 or SNP, 3–200 μM, 48 h, n = 5), by ELISA (with rhBMP2 as standard) (H). The μCT analysis of dissected tibia from homozygous eNOS knockout (−/−) or wild‐type (+/+) control mice (4 month, n = 10) (I) and trabecular bone volume (J) and BMD (K) analysis (*P < 0.05 vs. vehicle; *P < 0.01 vs. wild‐type control animals).
Figure 3
Figure 3
The eNOS‐SIRT1 axis is necessary for the pro‐osteogenic effects of RSV. Different effects on ALP levels (A) BMP2 gene expression (qPCR, B) and promoter activity (C) in primary osteoblasts from eNOS knockout (−/−) or wild‐type (+/+) control mice (n = 5) treated with RSV (5 μM) or vehicle (24 h). RSV‐induced ALP levels (5 μM) in the presence of the BMP inhibitor noggin (500 ng·mL−1, 48 h, n = 5), normalized to total protein (D). SIRT1 gene expression in 2T3 osteoblasts following RSV (1–100 μM) treatment, quantified by real time PCR (E), along with eNOS (F) and BMP2 (G) mRNA levels transfected with SIRT1 siRNA or scramble control (n = 5) with and without RSV treatment (5 μM). RSV‐induced ALP levels (5 μM) in the presence of SIRT1 (or scrambled control) siRNA (H) (n = 5) (*P < 0.05 vs. vehicle treated WT/scrambled ctrl; # P < 0.001 vs. RSV‐treated WT/scrambled ctrl).
Figure 4
Figure 4
Ageing decreases bone volume and eNOS‐BMP2 expression. μCT analysis of tibia of young (3 month) or aged (12 month) mice (n = 10) (A) determining BV/TV (B) and BMD (C). Gene expression changes (real time PCR) of eNOS (D), BMP2 (E) in ageing long bones (n = 10). Proposed mechanism of RSV action within osteoblasts (F) (*P < 0.05).
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