Mechanistic Study Of Osteoblastic Differentiation Stimulated By Resveratrol

Osteoblasts,bone-forming cells,are derived from mesenchymal stem cells (MSCs) at sites of membranous bone formation,in periosteum of endochondral bones, and in bone marrow stroma.During adult life,bone marrow mesenchymal stem cells are the source of osteoprogenitors.The osteoblastic differentiation program from MSCs is controlled by hormonal and local factors,including those involved in the canonical Wnt/β-catenin signaling.Wnts are a family of secreted proteins that regulate the development and growth of many organs and tissues,including bone.They function through binding to Frizzled(Fz) and low-density lipoprotein receptor-related protein-5 or 6(LRP-5/6) receptors and activating different signaling pathways,including the canonicalβ-catenin pathway.In the absence of stimulation by Wnts,β-catenin protein is constitutively phosphorylated,by CKl and GSK-3β,at both serine and threonine residues in the N-terminal region,which results in ubiquitination and subsequent proteosomal degradation.In the presence of Wnt signaling,unphosphorylatedβ-catenin accumulates in the cytoplasm and subsequently translocates into the nucleus, in which it interacts with T-cell factor(TCF)/lymphoid enhancer factor(LEF) family transcription factors to regulate Wnt target gene expression.The functional importance of the canonical Wnt signaling cascade in bone formation was illustrated by the anomalies in bone formation caused by mutations in genes involved in Wnt signaling.For example,loss-of-fuction mutations in human LRP5 were found to cause the osteoporosis-pseudoglioma syndrome,an autosomal recessive disorder characterized by extremely low bone mineral density and skeletal fragility. Gain-of-function mutations in the same gene,on the other hand,resulted in high bone mass(HBM).Consistent with the observations in humans,bone formation and osteoblast proliferation were decreased in Lrp5-deficient mice,and transgenic mice that expressed the LRP5 G171V mutation in osteoblasts exhibited enhanced osteoblastic activity,reduced osteoblast apoptosis,and a high bone mass phenotype.Resveratrol,found in wine and in numerous plant species including grapes, peanuts,and some Asian medicinal herbs,has been shown to modulate multiple metabolic pathways.Resveratrol has been shown to reduce LDL oxidation,to inhibit platelet aggregation and to block synthesis of proatherogenic eicosanoids.Resveratrol was also shown to affect many cellular responses,such as inhibiting endothelial cell activation and cell proliferation.Recent studies indicate that resveratrol functions as a mimic of calorie restriction,and can activate Sir2,a key component of yeast longevity and the founding member of the sirtuin family of deacetylastes.Resveratrol was also shown to stimulate the differentiation of MSCs into osteoblasts.It increased alkaline phosphatase(ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells in a dose-dependent manner.Resveratrol markedly inhibited adipogenesis and promoted osteoblast differentiation from MSCs. Resveratrol probably inhibits adipocyte formation via the activation of SIRTl,which represses PPAR-γ,2,a fat regulator.However,little is known about the signaling pathway underlying resveratrol-induced osteoblast differentiation.Since Wnt/β-catenin signaling is pivotal in the regulation of osteogenesis,we hypothesized that resveratrol might promote osteoblastic differentiation by modulating Wnt/β-catenin signaling.In this study we investigated the mechanism by which resveratrol induces osteoblastic differentiation from pluripotent mesenchymal cells. PART ONE Resveratrol induces osteoblastic differentiation of ST2 cellsTo confirm the role of resveratrol in promoting osteoblast differentiation from MSCs,we incubated the murine bone marrow stromal cell line ST2 in the OBM (Osteoblast Medium) to induce their differentiation toward osteoblastic lineage.1.Since ALP is well recognized as a biochemical marker for osteoblastic activity, we examined the change of ALP activity of ST2 cells in response to resveratrol.We found that exposure of ST2 cells to 5μM to 50μM resveratrol resulted in a significant increase in ALP activity,and the effects were dose-dependent.Higher concentration of resveratrol,however,was cytotoxic to the cells.2.Expression of osteoblast markers(OCN,Runx2) were studied with quantitative real-time PCR.Resveratrol(10μM) was found to increase the expression of both OCN and Runx2 in ST2 cells.PART TWO Resveratrol up-regulates Wnt signaling pathway independent of Lrp5Wnt/β-catenin signaling plays an important role in regulation of bone formation. To uncover the molecular mechanisms for resveratrol-mediated promotion of osteoblast differentiation,we focused on the Wnt signaling pathway.1.Resveratrol stabilizesβ-catenin in ST2 cells.As an important component of the Wnt signaling pathway,β-catenin serves as a coactivator of TCF/LEF family of DNA binding proteins.The amount ofβ-catenin and its subcellular distribution were examined.Resveratrol treatment resulted in an increase in both totalβ-catenin and nuclearβ-catenin in a dose-dependent way, suggesting that resveratrol induced nuclear accumulation ofβ-catenin in ST2 cells.2.Resveratrol augments the transcriptional activity ofβ-catenin target genes.In the nucleus,β-catenin interacts with TCF/LEF transcription factors and up-regulates Wnt target genes.The luciferase reporters,which have either wild type (TOPFLASH) or mutated(FOPFLASH) binding sites for theβ-catenin-TCF complex, were used to examineβ-catenin-TCF-dependent gene expression.Resveratrol treatment increased TOPFLASH activation by 1.5 to 2.5-fold at concentration as low as 1μM.It had a maximum effect at 10μM.These results demonstrated that resveratrol was able to transactivate genes targeted by TCF transcription factor.The promoter of Runx2 gene contains a single putative TCF site,It was addressed that canonical Wnt/β-catenin directly stimulating Runx2 gene expression via this TCF binding site.The Ruxn2 luciferase reporter containing the TCF element or the mutated were constructed.Transient transfection studies show that resveratrol increased activity of Ruxn2 reporter,while,mutation of the TCF-binding site resulted in loss of activation driven by resveratrol.These results confirm that the resveratrol up-regulate target genes of Wnt/β-catenin signaling pathway3.Activativation of Wnt signaling by resveratrol does not require Lrp5.Lrp5 serves as a Wnt co-receptor in the canonical signaling pathway and acts upstream ofβ-catenin.To study whether resveratrol augments the nuclear accumulation ofβ-catenin via Lrp5,we assessed the effects of resveratrol on the cells in which Lrp5 is knocked down via RNA interference.a.ST2 cells were stably transfected with Lrp5-siRNA(pS-Lrp5) and control vector(pSN).Treatment of ST2 cells with pS-Lrp5 led to a significant reduction in the Lrp5 mRNA as demonstrated by RT-PCR.The expression of markers associated with osteoblast differentiation was significantly down-regulated in pS-Lrp5 cells compared with control pSN cellsb.Resveratrol treatment increased ALP activity in both pS-Lrp5 and pSN cells, indicating that resveratrol-induced increase of ALP activity does not require LrpS.c.β-catenin was significantly increased in both pS-Lrp5 and control cells after incubation in resveratrol for 24h.Resveratrol treatment significantly increased TOPFLASH luciferase activity in both pS-Lrp5 and pSN cellsTaken together,our data indicate that resveratrol up-regulates Wnt signaling pathway downstream of Lrp5. PART THREE Resveratrol induced phosphorylation of GSK-3βis mediated by ERKGSK-3βdown-regulates the Wnt signaling pathway by phosphorylatingβ-catenin and promoting its degradation.The kinase activity of GSK-3βcan be inhibited by its phosphorylation.In this part,we determined whether and how the phophorylation of GSK-3βcontributes to the increased activity ofβ-catenin.1.The level of Ser9 phosphorylation of GSK-3βwas significantly increased under 10μM resveratrol treatment.This result suggests that the increase ofβ-catenin in response to resveratrol is mediated by down-regulation of the kinase activity of GSK-3β.2.Preivous studies showed that the GSK-3βcan be phosphorylated by PI3-K/Akt,MAPK or the ERK pathway.We first evaluated the role of ERK in the GSK-3βphosphorylation by measuring total Erk and the phosphorylated form of Erk. Erk was significantly activated in resveratro-treated cells.Importantly,when ERK pathway was inhibited by PD98059,the phosphorylation of GSK-3βwas decreasedPART FOUR Resveratrol may promote osteoblastic differentiation by augmenting Sirtl and PPAR-γ,Activation of Sirtl has recently been shown to decrease adipocyte development from preadipocytes through inhibition of PPAR-γ2.The effect of resveratrol on the expression of Sirtl was evaluated in this part.1.Resveratrol treatment enhances the expression of SIRT1 at protein level Recent studies indicate that resveratrol functions as a mimic of calorie restriction,and can activate Sir2.We measured SIRT1 levels in mouse ST2 cells and human U2OS treated with resveratrol.Resveratrol induced SIRT1 at protein level in the p53-functional cells.2.Induction of p53 and Erk phosphorylation by resveratrol Resveratrol was also found to induce p53 phosphorylation at serine 15.The phophorylation of p53 was inhibited by ERK inhibitor PD98059.These observations suggest that both Wnt signaling pathway,Erk and p53 play roles in the ostoblastic differentiation in response to resveratrol.3.Resveratrol decreased PPAR-γmRNA level in vitro/vivo To further confirm the enhancing effect of resveratrol on osteoblast differentiation,the influence of resveratrol on PPAR-γexpression was investigated in vitro and in vivo.ST2 cells were cultured for 2 weeks in DMEM supplemented with osteoblast medium containing increasing amount(0,1,5,10,50μM) of Resveratrol.Total cellular RNA was isolated,and gene expression was quantified by using semi-quantitative RT-PCR.Significant reduced level of PPAR-γ,mRNA was observed in treated with ResveratrolAdult rats were treated with low,medium and high dosage of resveratrol or vehicle-treated(4,40 and 100mg//kg of body weight/day,respectively) once a day for consecutive 30 days.The result showed that in consistency with the results of in vitro experiments,resveratrol treatment markedly decreased PPAR-γ,mRNA level in fats of these rats.