Elsevier

Bone

Volume 133, April 2020, 115252
Bone

Full Length Article
Resveratrol inhibits adipocyte differentiation and cellular senescence of human bone marrow stromal stem cells

https://doi.org/10.1016/j.bone.2020.115252Get rights and content

Highlights

  • Resveratrol inhibits of adipocytic differentiation of hBMSCs, It might regulate bone marrow adiposity.

  • Resveratrol up-regulates osteogenic differentiation of hBMSCs, it may contribute to protection against bone loss.

  • Resveratrol targets senescence, oxidative stress and up regulates endogenous protective anti-oxidant pathway in hBMSCs.

Abstract

Bone marrow adipose tissue (BMAT) is a unique adipose depot originating from bone marrow stromal stem cells (BMSCs) and regulates bone homeostasis and energy metabolism. An increased BMAT volume is observed in several conditions e.g. obesity, type 2 diabetes, osteoporosis and is known to be associated with bone fragility and increased risk for fracture. Therapeutic approaches to decrease the accumulation of BMAT are clinically relevant. In a screening experiment of natural compounds, we identified Resveratrol (RSV), a plant-derived antioxidant mediating biological effects via sirtuin- related mechanisms, to exert significant effects of BMAT formation. Thus, we examined in details the effects RSV on adipocytic and osteoblastic differentiation of tolermerized human BMSCs (hBMSC-TERT). RSV (1.0 μM) enhanced osteoblastic differentiation and inhibited adipocytic differentiation of hBMSC-TERT when compared with control and Sirtinol (Sirtuin inhibitor). Global gene expression profiling and western blot analysis revealed activation of a number of signaling pathways including focal adhesion kinase (FAK). Pharmacological inhibition of FAK using (PF-573228) and AKT inhibitor (LY-294002) (5μM), diminished RSV-induced osteoblast differentiation. In addition, RSV reduced the levels of senescence-associated secretory phenotype (SASP), gene markers associated with senescence (P53, P16, and P21), intracellular ROS levels and increased gene expression of enzymes protecting cells from oxidative damage (HMOX1 and SOD3). In vitro treatment of primary hBMSCs from aged patients characterized with high adipocytic and low osteoblastic differentiation ability with RSV, significantly enhanced osteoblast and decreased adipocyte formation when compared to hBMSCs from young donors. RSV targets hBMSCs and inhibits adipogenic differentiation and senescence-associated phenotype and thus a potential agent for treating conditions of increased BMAT formation.

Introduction

Human bone marrow stromal stem cells (hBMSCs) (also known as marrow mesenchymal or skeletal stem cells) are adult multipotent stem cells of non-hematopoietic origin that possess self-renewal ability and potential to differentiate into multiple mesodermal lineage cells, such as osteoblasts, adipocytes, and chondrocytes [1,2]. Several diseases e.g. obesity, type 2 diabetes and osteoporosis that exhibit increased risk for bone fractures, are characterized by increased bone marrow fat accumulation [3]. For example, magnetic resonance imaging (MRI) in aged osteoporotic men and females has revealed increased marrow fat and reduced bone density [[4], [5], [6]]. A clinical study in children with diabetes type 1 has shown increased bone marrow adipose tissue (BMAT) volume that was inversely correlated with deficits in trabecular bone microarchitecture, bone mass density and bone formation [7]. Similar observations have been reported in young and postmenopausal women with and without type 2 diabetes (T2D) [[8], [9], [10]] and osteoporotic diabetic men [11].

The cellular mechanisms underlying increased BMAT is thought to be caused by enhanced differentiation of BMSCs to adipocytes (AD) and not osteoblastic cells (OB) [12,13]. Several studies have corroborated this notion. Recent studies in mice models of HFD-induced-obesity revealed enhanced adipocyte differentiation of murine BMSCs that was associated with increased in vivo BMAT volume and decreased in trabecular and cortical bone mass [14,15]. Similarly, in a clinical study performed in our group, we observed that hBMSCs obtained from obese persons, exhibited enhanced adipocyte differentiation. Interestingly, in this study enhanced adipocyte differentiation was associated with the existence of a hypermetabolic state characterized by increased oxidative phosphorylation, generation of reactive oxygen species (ROS) and the presence of senescent bone marrow microenvironment that may explain bone fragility observed in obesity [3]. Thus, identifying relevant therapeutic approaches to target BMSCs and to prevent accumulation of BMAT are needed [16,17].

Identifying and testing novel molecules isolated from natural products, for disease prevention and treatment, is a traditional approach in medicine as more than two thirds of common drugs are derived from natural sources [18]. Resveratrol (RS, trans-3,5,40-hydroxystilbene) (RSV) is a natural phenolic compound present in grapes, cranberries and peanuts and is a Sirtuin 1 activator (SIRT1) [[19], [20], [21]]. Sirtuins are considered a class III NAD+ -dependent histone deacetylases (HDAC) that are involved in epigenetic regulation and a number of cellular processes as cell cycle regulation, DNA repair, metabolism, inflammation and cellular senescence/aging [22]. SIRT1 expression levels in mesenchymal stem cells is reduced during aging [23,24] and it induces the deacetylation of SOX2 in the nucleus promoting the activity of SOX2 target genes. SOX2 is transcriptional factor involved in the self renewal and multipotency of BMSCs and other stem cells [25]. In addition, RSV-SIRT1 is upregulated during chondrogenic differentiation of BMSCs via inhibition/deacetylation of NF-kB, inhibition of inflammatory signaling and activation of SOX9 [26].

RSV has anti-oxidant, anti-inflammatory, and estrogenic activity thus relevant for prevention of human diseases including cardiovascular disease, and cancer [[27], [28], [29]]. Several previous studies have reported that RSV exerts significant biological effects on stem cells. Treatment of cardiac stem cells (CSCs) with RSV prior to transplantation, improved cardiac performance in a mouse model of acute myocardial infarction [30]. RSV inhibited the teratoma formation by induced pluripotent stem (iPS) cells transplanted in mice in vivo and enhanced osteogenesis via up-regulation of osteopontin [31]. RSV treatment of myeloma cells cultured from bone marrow aspirates from myeloma patients, inhibited their negative effects on bone formation [32,33]. Simic et al reported a role for SIRT1 in regulating BMSCs differentiation. In aged SIRT1-deficient mice reduction in subcutaneous fat, cortical bone thickness and trabecular volume were observed. These effects may be mediated through β-catenin deacetylation leading to transcriptional activation of genes necessary for mesenchymal stromal stem cells (MSCs) differentiation [34]. RSV induced cell proliferation and osteogenic differentiation of hBMSCs via activation of ERK-dependent MAPK pathway that are linked to RUNX2 activation [35]. Employing human embryonic mesenchymal progenitors, RSV down regulated adipocyte differentiation and upregulated the expression of osteogenic genes RUNX2 and Osteocalcin (OC) via activation of SIRT1/FOXO3A [36]. In senescent murine BMSCs, RSV enhanced osteogenic differentiation via up regulation of core component of mitochondrial contact site mitofilin [37]. Osteogenic differentiation of MSCs from patients with periodontitis was rescued by RSV via inhibiting the inflammatory microenvironment caused by TNFα [38]. Cigarette smoke extract (CSE) affected the primary cilia distribution and osteogenic differentiation of human BMSCs via downregulating hedgehog signaling that was reversed by RSV giving pharmacological potentials for treatment of observed delayed fracture healing in smokers [39].

Picard et al reported in 3T3-L1 preadipocyte cell line, that pharmacological activation of SIRT1 inhibit expression of PPARγ leading to reduction of fat storage in white adipose tissue [40]. Starvation of animals caused activation of SIRT1 that interacted with PPARγ DNA-binding sites and down regulated target genes involved in fat storage [41]. Disruption of SIRT1 in MSCs led to bone loss [42], but SIRT1 over-expression did not increase bone mass yet reduced susceptibility of male mice to cancer, DNA-damage and age-related bone loss [43].

We have previously identified RSV as a molecule with significant regulatory effects on cultured hBMSCs, during a library screen of natural compounds. In the current study, we examined in details the effects of RSV on OB and AD differentiation of hBMSCs and examined the underlying molecular mechanism. We have also tested the ability of RSV to rescue the age-related enhanced AD differentiation of cultured hBMSCs.

Section snippets

Compounds

Resveratrol and Sirtinol were obtained from Selleckchem, Inc. (Selleckchem, Inc., Houston, TX, USA). FAK inhibitor (PF-573228) was purchase from Sigma (Sigma-Aldrich Inc., St. Louis, MO, USA) & AKT inhibitor (LY-294002) from Millipore (EMD Millipore Corporation, Canada). Compounds were dissolved in dimethyl sulfoxide (DMSO) and used at a concentration of 1.0 μM (Supplementary Fig. S1 A&B) and as previously described [44]. Control cells were treated with DMSO as a vehicle.

Cell culture

We used a model for

Effect of resveratrol on osteogenic and adipogenic differentiation of hBMSCs

Initially, we assessed the effect of RSV (1.0 μM) and Sirtinol (1.0 μM) on the osteoblastic differentiation of hBMSC-TERT. Cells were continuously exposed to Sirtinol or Resveratrol in the presence of osteoblastic differentiation media. On day 10, higher ALP activity was observed in RSV-treated compared to Sirtinol- and vehicle-treated control cells (Fig. 1A, left panel). Similar effects were observed in primary hBMSCs (Fig. 1A, middle panel) and primary human adipose tissue-derived MSCs

Discussion

Increased BMAT formation has been associated with decreased bone formation and increased bone fragility in a number of diseases e.g. obesity, diabetes [51], and age-related osteoporosis [52]. One putative cellular mechanism is a re-direction of hMSCs to adipocytes and not osteoblastic cells [53].Thus, identifying possible small molecules that target hMSCs differentiation and revert this altered lineage allocation, has a potential clinical use [54]. In the current study, we have demonstrated

Conclusion

Our study demonstrates that RSV inhibits adipogenic differentiation of hBMSCs through FAK/Akt and MAPK pathways, reduce senescence associated phenotype and oxidative stress. These changes were associated with increased osteoblast differentiation of hBMSCs. Our results suggest a possible role for RSV in enhancing osteoblast commitment of in vitro cultured hBMSCs prior to their use in clinical transplantation protocols. In addition, RSV treatment is a potentially beneficial strategy for

Abbreviations

    RSV

    resveratrol

    hBMSC-TERT

    tolemerized-human bone marrow stromal stem cells

    MAT

    marrow adipose tissue

    BMAT

    bone marrow adipose tissue

    (hATMSCs)

    human primary adipose tissue derived

    FAK

    focal adhesion kinase

    ERK

    extracellular signal regulated kinase

    DMSO

    dimethyl sulfoxide

    hTERT

    human telomerase reverse transcriptase gene

    DMEM

    Dulbecco's modified Eagle's medium

    AIM

    adipogenic induction medium

    PBS

    phosphate-buffered saline

    TC

    tissue culture

    ALP

    alkaline phosphatase

    ALZR

    Alizarin red

    TBHP

    tert-butyl hydroperoxide (H2O2)

    ROS

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and material

Data are available upon request.

Funding

This work was supported by the Deanship of Scientific Research at King Saud University Research Group No. RG-1438-033.

Authors' contributions

D.A. involved in conception, design, performed experiments and manuscript writing; C.L, J.M.K, M.O., M.M., M.A., Y.A., R.A., N.A., performed experiments; N.M.A, M.A., A.A., M.K., were involved in conception and design; N.M.A&M.A obtained funding, conceived the study and finalized manuscript.

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

We would like to thank the Deanship of Scientific Research, King Saud University (Research Group No. RG-1438-033 for funding this work.

References (78)

  • D.S. Yoon

    Different effects of resveratrol on early and late passage mesenchymal stem cells through beta-catenin regulation

    Biochem. Biophys. Res. Commun.

    (2015)
  • B.M. Abdallah

    Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation

    Biochem. Biophys. Res. Commun.

    (2005)
  • K.J. Livak et al.

    Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method

    Methods

    (2001)
  • S. Bhattacharya et al.

    Hepatoprotective properties of kombucha tea against TBHP-induced oxidative stress via suppression of mitochondria dependent apoptosis

    Pathophysiology

    (2011)
  • H.S. Camp et al.

    Adipogenesis and fat-cell function in obesity and diabetes

    Trends Mol. Med.

    (2002)
  • M. Shakibaei et al.

    Resveratrol-mediated SIRT-1 interactions with p300 modulate receptor activator of NF-kappaB ligand (RANKL) activation of NF-kappaB signaling and inhibit osteoclastogenesis in bone-derived cells

    J. Biol. Chem.

    (2011)
  • T. Schilling

    Effects of phytoestrogens and other plant-derived compounds on mesenchymal stem cells, bone maintenance and regeneration

    J. Steroid Biochem. Mol. Biol.

    (2014)
  • M.J. Biggs

    The use of nanoscale topography to modulate the dynamics of adhesion formation in primary osteoblasts and ERK/MAPK signalling in STRO-1+ enriched skeletal stem cells

    Biomaterials

    (2009)
  • S. Shirjang

    Toll-like receptors as a key regulator of mesenchymal stem cell function: an up-to-date review

    Cell. Immunol.

    (2017)
  • V.C. Khanh

    Aging impairs beige adipocyte differentiation of mesenchymal stem cells via the reduced expression of Sirtuin 1

    Biochem. Biophys. Res. Commun.

    (2018)
  • A. Aldahmash

    Human stromal (mesenchymal) stem cells: basic biology and current clinical use for tissue regeneration

    Ann Saudi Med

    (2012)
  • M.F. Pittenger

    Multilineage potential of adult human mesenchymal stem cells

    Science

    (1999)
  • M. Tencerova

    Obesity-associated hypermetabolism and accelerated senescence of bone marrow stromal stem cells suggest a potential mechanism for bone fragility

    Cell Rep.

    (2019)
  • J.F. Griffith

    Vertebral bone mineral density, marrow perfusion, and fat content in healthy men and men with osteoporosis: dynamic contrast-enhanced MR imaging and MR spectroscopy

    Radiology

    (2005)
  • D.K. Yeung et al.

    Osteoporosis is associated with increased marrow fat content and decreased marrow fat unsaturation: a proton MR spectroscopy study

    JMRI

    (2005)
  • J.F. Griffith et al.

    Vertebral marrow fat content and diffusion and perfusion indexes in women with varying bone density: MR evaluation

    Radiology

    (2006)
  • S.C. Chen

    Skeletal fragility and its clinical determinants in children with type 1 diabetes

    J. Clin. Endocrinol. Metab.

    (2019)
  • T. Baum

    Does vertebral bone marrow fat content correlate with abdominal adipose tissue, lumbar spine bone mineral density, and blood biomarkers in women with type 2 diabetes mellitus?

    J. Magn. Reson. Imaging

    (2012)
  • J.M. Patsch

    Bone marrow fat composition as a novel imaging biomarker in postmenopausal women with prevalent fragility fractures

    J. Bone Miner. Res.

    (2013)
  • M. Tencerova

    High-fat diet-induced obesity promotes expansion of bone marrow adipose tissue and impairs skeletal stem cell functions in mice

    J. Bone Miner. Res.

    (2018)
  • A.G. Veldhuis-Vlug et al.

    Clinical implications of bone marrow adiposity

    J. Intern. Med.

    (2018)
  • S. Bornstein

    Metformin Affects Cortical Bone Mass and Marrow Adiposity in Diet-Induced Obesity in Male Mice

    Endocrinology

    (2017)
  • S. Bornstein

    Metformin affects cortical bone mass and marrow adiposity in diet-induced obesity in male mice

    Endocrinology

    (2017)
  • D.J. Newman et al.

    Natural products as sources of new drugs over the 30 years from 1981 to 2010

    J. Nat. Prod.

    (2012)
  • M. Gertz

    A molecular mechanism for direct sirtuin activation by resveratrol

    PLoS One

    (2012)
  • D.S. Mohar et al.

    The sirtuin system: the holy grail of resveratrol?

    J Clin Exp Cardiolog

    (2012)
  • P.R. Élie Abed et al.

    Sirtuins as markers of bone disease: a focus on osteoarthritis and osteoporosis

  • X. Chen

    Intercellular interplay between Sirt1 signalling and cell metabolism in immune cell biology

    Immunology

    (2015)
  • H. Yuan et al.

    The emerging and diverse roles of sirtuins in cancer: a clinical perspective

    Onco Targets Ther

    (2013)
  • Cited by (0)

    View full text