Full Length ArticleResveratrol inhibits adipocyte differentiation and cellular senescence of human bone marrow stromal stem cells
Graphical abstract
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.
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