Stimulation Of Wnt/?-catenin Signaling To Improve Bone Development In Ovariectomized Mice By Naringin Via Interacting With AMPK And Akt

Background:Osteoporosis has become an epidemic problem among aging population,especially postmenopausal women.Decreased bone mass and microarchitectural deterioration of bone tissues give rise to high risk of fractures of hip,vertebrae,pelvis and wrist,causing disability and even death.Osteoporosis treatment should be given more attention.Development and maintenance of bones rely on the interaction between two critical cell types,osteoblasts and osteoclasts.Osteoblasts differentiated from common mesenchymal precursors are required in bone formation,which are tightly controlled by several transcription factors and affected by surrounding cells or tissues via cell-cell signaling.Wnt/?-catenin signaling was proposed to be involved in osteoblastogenesis.It was reported that the activation of Wnt/?-catenin signaling was remarkably triggered by osteogenic inducers(BIO),and BIO induced osteogenesis activated Wnt/?-catenin signaling even in the presence of ethanol which Ied to the shift of multipotential mesenchymal stem cells in bone marrow(BMSCs)from osteogenesis towards adipogenesis.Wnt proteins are signaling molecules that affect cell proliferation,differentiation and survival.The canonical Wnt signaling pathway employs the clustering between Wnt proteins,Frizzled(FZD),and low-density lipoprotein receptor-related protein 5/6(LRP5/6)receptors,resulting in phosphorylation,which in turn stabilizes ?-catenin through suppressing glycogen synthase kinase-3(GSK-3)[12,13].Unphosphorylated?-catenins accumulate in the nucleus and bind to lymphoid enhancer factor(LEF)/T-cell factor(TCF)transcription factors while phosphorylated ?-catenins are degraded.The activation of LEF/TCF transcription factors enables the expressions of osteogenic specific genes.In addition,the lack of osteoclastogenesis inhibitory factors and overexpression of soluble osteoclast differentiation factors were documented in severe osteoporosis.Naringin has been proposed as an active component which can promote osteoblastogenesis and abrogate osteoclastogenesis.Importantly,it was reported that naringin exerts oestrogen-like activities and significantly elevates cell proliferation,total protein content and alkaline phosphatase(ALP)activities in rat UMR-106 cells.Additionally,activations of the PI3K,Akt,c-Fos/c-Jun and AP-1 pathways were found to be involved in the stimulatory effects of naringin on bone morphogenetic protein-2(BMP-2)expression in MC3T3-E1 osteoblastic cells.Research objectiveThis study aimed to investigate the influence of naringin on osteoblasts and bone development.To reveal the mechanism of naringin function,we studied the impacts of dorsomorphin blocking AMP-activated protein kinase(AMPK)and Akti-1/2 inhibiting Akt 1 and 2 along with naringin on osteoblast-like cells UMR-106 in vitro and bone development of mice in vivo.Materials and MethodsAnimalsTwenty four weeks old female mice were prepared.Five mice were sham-operated while the rest of the mice underwent ovariectomy(OVX)to induce bone loss at the age of one month.After recovering for 2 weeks,OVX mice were randomly divided into three groups:OVX vehicle(PBS),OVX naringin(5 nM)and OVX naringin with PTH.Control group is sham vehicle.Mice were pair-fed with the control diet,received treatment orally for 6 weeks according to previous study.At the end of treatment,bone specimens from femur,tibia and lumbar spine were collected and stored at-20? until analysis.Naringin extraction and identification10.5 mg naringin Naringin was extracted from 3.0 kg of drynaria fortunei.Naringin and was then analyzed by high performance liquid chromatography(HPLC).Culture of rat osteoblastic UMR-106 cellsUMR-106 cells were cultured iin 96-well,24-well or 6-well plates respectively for different assays.Different concentrations of naringin(0,0.5,1,5,10 and 20 nM)were examed to determine a concentration exerting maximum effects on cell proliferation.For the assessments of the mRNA and protein expressions of ?-catenin,cells were treated by vehicle(PBS)as control,Wnt3a(10 ng/ml),Dkk1(100 ng/ml),naringin(10nM),naringin(10nM)+ Dkk1(100ng/ml)and naringin(10nM)+ FH535(10?M).When indicated,cells were divided into four groups treated by control,naringin(10nM),naringin(10nM)+ dorsomorphin(100nM),and naringin(10nM)+Akti-1/2(200nM),respectively.Isolation of Nuclei and CytosolNuclei and cytosol were isolated using a nuclear extract kit according to the manufacturer's instructions.Briefly,cells were harvested and then were stored in-800? until use.Cell proliferation assayThe cell proliferation rate employed a BrdU incorporation assay.Cells were fixated and then stained with the BrdU antibody by using a BrdU staining kit.O.D.(Optical Density)has measured at 450 nM.Real?time quantitative reverse transcriptase-polymerase chain reaction(RT-PCR)analysisTotal RNA from cells were extracted by RNeasy Mini Kit.Quality and quantity of RNA were measured by NanoDrop 8000 spectrophotometer.1000 ng RNA was used for each reaction to produce cDNA using high capacity cDNA reverse transcription kit following manufac-turer's instructions.The reaction was initiated at 25? for 5 min,annealed at 50? and elongated at 70?.cDNA products were diluted by adding RNase and DNase free water till 250 ?l and frozen at-20? before gene expression assay.Each PCR re-action mixture contained 10 ?l 1 ×PCR master mix,5 ?l diluted cDNA,4 ?l water and 1 ?1 probe.Gene expression was measured with quantitative real-time RT-PCR system.Western blot analysisThe ?-eaten in(?92 kDa)expressed was determined by Western blot.Background color was reduced carefully by washing with TTBS.The results were visualized using ECL kit and observed by GeneGnome mechine.Transient transfection and ER-mediated luciferase activity assayCells were transfected by LipofectamineTM 2000 reagent.Er-mediated dual luciferase plasmids were transfected into the cells.After overnight transfection,the cells were treated with vehicle,naringin(10 nM)or naringin with Dkkl(100 ng/ml)for 24 h.After treatment,the cells were lysed with lysis buffer and luciferase activity was measured using Dual Luciferase.Reporter Assay System and the signal were detected by TD-20/20 Luminometer.Alkaline phosphatase assayALP activity was measured directly on the monolayer of cell cultures on a ninety-six-well microplate after treatments with vehicle,naringin or PTH.The absorbance of color change was measured at 405 nm in a microplate reader.A Bradford protein assay was carried out to normalize ALP expression and as ALP activity was expressed U/l per mg protein.Assessment of bone properties by peripheral quantitative computed tomography(pQCT)Bone mineral density(BMD),cross-sectional area and stress-strain index(SSI)in the distal femur,proximal tibia and lumbar spine region LI were measured using a StraTecXCT2000 machine.Mid-shaft and distal/proximal regions of femur and tibia were scanned.All scans were performed using the protocol designed for studying isolated small bones.Bonm stiffness measuresBone stiffness was measured using a specified 3-point bending test.Load-defonmation curve was plotted simultaneously.Bone stiffness was determined from the load-deformation curve.ResultsNaringin enhanced Wnt/?-catenin signaling pathwayWe first examined the effects of different concentrations of naringin on UMR-106 cell proliferation.10 nM was employed as the optimal dosage in further investigation on the functions of naringin in Wnt/p-catenin signaling pathway.To investigate the influences of naringin on ?-catenin regulation,UMR-106 cells were divided into six groups:control,Wnt3a,DKK1,10nM naringin,Dkk1+naringin and FH535+naringin respectively,and the mRNA level of ?-catenin in each group was measured.The results showed a striking increase in the mRNA level of ?-catenin(p<0.001)by Wnt3a treatment.In contrast,DKK1 significantly reduced mRNA level of ?-catenin(p<0.001),due to its inhibitory role in Wnt signaling pathway.Naringin treatment significantly elevated the mRNA level of p-catenin(p<0.01)compared to the control group.DKK1+naringin and FH535+naringin treatments significantly reduced mRNA levels of ?-catenin compared to control group(DKK1+naringin,p<0.05;FH535+naringins p<0.01).In comparison with naringin treatment,DKK1+naringin and FH535+naringin consistently inhibited the mRNA levels of ?-catenin(DKK1+naringin,p<0.01;FH535+naringin,p<0.001).In western blot analysis,Wnt3a dramatically elevated the protein expression of?-catenin(p<0.001)whereas DKK1 led to a significant decrease in the protein level of ?-catenin(p<0.01).In addition,naringin treatment markedly promoted the protein expression of ?-catenin(p<0.01).It was also observed that DKK1+naringin and FH535+naringin treatments significantly reduced the protein expressions of ?-catenin compared to control group(DKK1+naringin,p<0.05;FH535+naringin,p<0.05)or naringin treatment group(DKK1+naringin,p<0.01;FH535+naringin,p<0.01).Taken together these results were consistent with the results of the mRNA levels of ?-catenin in each group,which clearly demonstrated that naringin treatment enhanced Wnt/?-catenin signaling pathway.Phosphorylation of ?-catenin by naringin via AMPK and Akt signalingWe investigated the protein expressions of phospho-?-catenin in UMR-106 cells treated by control,10 nM naringin,10 nM naringin+100nM dorsomorphin,and 10 nM naringin+200 nM Akti-1/2,using phospho-?-eatenin(Ser552)antibody.Naringin significantly increased Ser552 phosphorylation of ?-catenin(p<0.001)compared to control group.Importantly,the additions of Akti-1/2 or dorsomorphin reduced the Ser552 phosphorylation on ?-catenin(naringin + Akti-1/2,p<0.01;naringin + dorsomorphin,P<0.01).Compared to control,naringin + Akti-1/2 and naringin + dorsomorphin significantly induced Ser552 phosphorylation on ?-catenin(naringin +dorsomorphin,p<0.01;naringin + Akti-1/2,P<0.05).These results indicated that naringin interacted with AMPK and Akt to induce Ser552 phosphorylation of ?-catenin.we transfected UMR-106 cells with a luciferase reporter inserted downstream of Wnt-responsive element(WRE).The LEF/TCF transcriptional activity in UMR-106 cells was significantly improved within 8 hours after naringin treatment(p<0.001),naringin + dorsomorphin treatment(p<0.001)and naringin + Akti-1/2 treatment(p<0.01),compared with control.This result clearly indicates the LEF/TCF transcriptional activity was repressed by additions of Akti-1/2(p<0.01)or dorsomorphin(p<0.001)compared with naringin treatment alone.It would be interesting to point out that 24 hours after naringin treatment,the increase in the transcriptional activity was attenuated to about half of that of 8 hours,reflecting the half-life of naringin-induced activation of the ?-catenin pathway.Naringin stimulated ?-catenin translocationWe found that UMR-106 cells with naringin treatment displayed ?-catenin accumulation in cell nucleus,and this finding was further confirmed by western blot results.Indeed,naringin treatment induced translocation of ?-catenin,accounting for the increased relative expression of ?-catenin(p<0.001)in the nucleus of UMP-106 cells treated by 10 nM naringin compared to its expression in the nucleus of control cells.?-catenin translocation was repressed when Akti-1/2 or dorsomorphin was applied together with naringin,suggesting that naringin could participate in the regulations of AMPK or Akt signaling to regulate the activation of ?-catenin.Naringin facilitated bone developent in miceAfter we measured the effects of naringin,5 mg/Kg/Day naringin treatment was utilized in further experiments of this study.Mice were sham-operated as control or underwent OVX to induce bone loss.After 2 weeks recovery,OVX mice were randomly divided into three groups,OVX vehicle,OVX naringin and OVX parathyroid hormone(PTH).We found the ALP activity was significantly decreased in OVX vehicle mice compared to sham vehicle mice(p<0.05),whereas naringin and PTH treatment significantly recovered ALP activation in OVX mice.Moreover,BrdU was used to detect proliferating cells in sham and OVX mice.OVX led to a significantly reduction of cell proliferation compared to sham mice(p<0.001).OVX mice treated by naringin or PTH showed significantly increased proliferating cells compared to OVX vehicle mice.These results indicate that naringin exerts protective function on bone development.Compared to sham mice,OVX dramatically decreased the total and trabecular BMD in the distal femur,proximal tibia and lumbar spine L-1 regions,indicating OVX can be used as a mimic of osteoporosis.5 mg/Kg/Day treatment of naringin on OVX mice strikingly improved both total and trabecular BMD at all of the regions compared to OVX mice.However,dorsomorphin and Akti-1/2 treatments diminished the effects of naringin on total and trabecular BMD.OVX mice also showed decreased total and trabecular cross-sectional areas at all of the three sites compared to sham mice,whereas 5 mg/Kg/Day naringin significantly reversed these decreases.Dorsomorphin treatment on OVX mice treated by naringin significantly reduced total cross-sectional area at distal femur and proximal tibia,as well as trabecular cross-sectional area at proximal tibia,compared to naringin treatment on OVX mice.Only trabecular cross-sectional area at proximal tibia was significantly decreased in OVX mice treated by naringin and Akti-1/2,compared to that of OVX mice treated by naringin.SSI,referring to bone strength determined by pQCT,was significantly weakened by OVX at the three sites compared to sham mice,while naringin treatment markedly improved SSI in OVX mice.Dorsomorphin had a significant negative influence on SSI at distal femur and proximal tibia,while Akti-1/2 significantly reduced SSI at proximal tibia compared to naringin treatment on OVX mice.These above results demonstrated the positive influence of naringin on bone development and confirmed the involvement of naringin in AMPK and Akt signaling in vivo.Bone stiffness was also measured in distal femur,proximal tibia and lumbar spine L-1 in the sham and OVX mice.OVX diminished bone stiffness at all of the three regions tested.Naringin recovered the bone stiffness loss in OVX mice.The relevance between naringin and AMPK and Akt signaling was further supported by the reductions of bone stiffness by dorsomorphin or Akti-1/2 treatments on OVX mice treated by naringin.ConclusionIn conclusion,our study highlights a new finding on the mechanism of naringin functions in bone development.Actions of naringin involve the recruitments of AMPK and Akt signaling.We confirmed in this study that naringin treatment exerts protective role in bone development,which is consistent with other studies.Our results suggest that naringin facilitates phosphorylation of ?-catenin at Ser552 residues which stabilizes ?-catenin and leads to the translocation of ?-catenin into the nucleus.The functional relationship between naringin and AMPK and Akt should be taken into consideration for the understanding of the mechanical pathway of naringin.Finally our current study supports the role of naringin as a potential therapeutic agent in treating osteoporosis.