Role And Regulatory Mechanisms Of MTORC1 In Osteoclastogenesis

Background and aimsHealthy skeletal bone continually remodels itself to adapt to the influences of growth and changes in mechanical loads to maintain mineral homoeostasis and to regulate the bone marrow environment.Bone remodeling involves the removal of old or damaged bone by osteoclasts and the subsequent replacement of new bone formed by osteoblasts.Osteoblasts are derived from mesenchymal stem cells(MSCs)through a multistep differentiation pathway.Osteoclasts are multinucleated cells derived from the monocyte-macrophage lineage and are primarily responsible for resorption of mineralized matrices.An imbalance of bone homeostasis causes various skeletal disorders,such as osteoporosis,rheumatoid arthritis and osteopetrosis.Thus,an improved understanding of the molecular pathways involved in osteoclast differentiation and function will reveal the physiology and pathology of the skeletal system and provide new therapeutic strategies for the treatment of bone remodeling diseases.Osteoclasts develop from monocyte/macrophage lineage precursors under the strict regulation of osteoblast-lineage cells and MSCs,which express two cytokines essential for osteoclast differentiation,macrophage-colony-stimulating factor(M-CSF),and receptor activator of nuclear factor ?B ligand(RANKL).Cell-to-cell contact allows RANKL to bind to its physiologic receptor RANK,which is expressed on the surface of osteoclast-lineage cells.After RANKL-induced RANK stimulation,several key regulatory transcription factors and enzymes are induced to promote the differentiation,proliferation,multinucleation,activation,and survival of osteoclasts.Osteoprotegerin(OPG),a soluble decoy receptor RANKL produced by MSCs and osteoblasts,inhibits osteoclastogenesis by blocking the RANKL-RANK interaction.The discovery of the roles of RANK/RANKL and OPG in osteoclastogenesis represents a major breakthrough in basic biology as RANKL is absolutely essential for osteoclast formation throughout life and this discovery is being translated into clinical practice.Although much has been learned about the differentiation pathways activated by RANKL in osteoclast precursors,comparatively less is known about the molecular mechanisms by which various stimuli control RANKL expression in cells that support osteoclast differentiation.In addition to MSCs and osteoblast-lineage cells,RANKL is abundantly expressed by T and B lymphocytes,mammary epithelial cells,vascular endothelial cells,synovial fibroblasts,cells within periodontal tissue,hypertrophic chondrocytes and some cancer cells.RANKL is inducibly expressed in response to bone resorption-stimulating factors such as parathyroid hormone and interleukin(IL)-11 and is upregulated under conditions of estrogen deficiency,obesity,aging and inflammation.Elucidation of the signals that regulate RANKL/OPG expression under physiological and pathological conditions and their contribution to osteoclastogenesis could further our understanding of bone resorption disorders and may allow us to develop new therapies to inhibit bone loss.The mechanistic target of rapamycin(mTOR)is a conserved protein kinase that forms two distinct functional complexes,termed mTOR complex 1(mTORC1)and mTORC2 17.mTORCl uniquely contains raptor and is the sensitive target of rapamycin that controls protein synthesis through phosphorylation of the translational regulators 4E-BP1 and S6 kinase 1(S6K1).In response to diverse signals including nutrients,growth factors,energy and stresses,mTORC1 is activated by two families of ras-related small GTPases,Rheb and Rags.GTP-bound(active)Rheb is suppressed by tuberous sclerosis complex 1/2(TSC1/2),a functional complex which has GTPase-activating protein(GAP)activity toward Rheb.Loss of TSC1/2 causes cells and tissues to display constitutive mTORC1 activation.mTORC2 is less understood and appears only to sense growth factors to control cell survival and motility.mTORC1 has evolved to be a central signaling hub for coupling cell growth and metabolism to environmental inputs in eukaryotes,and dysregulation of mTORCl signaling is important in the pathology of cancer,diabetes,obesity and aging.However,evidence on the role of mTORC1 in bone remodeling and bone loss-related diseases is limited and elusive,and the underlying mechanisms are not known.Rapamycin has been shown to inhibit or stimulate osteoblastogenesis,and osteoclastogenesis.Whether mTORC1 regulates RANKL/OPG and its contribution to osteoclast formation and bone resorption remain unclear.Here we report that mTORC1 stimulates RANKL,but suppresses OPG expression via negative regulation of P-catenin in a variety of RANKL-expressing cells and in mouse genetic models.Importantly,through this pathway,mTORC1 stimulates osteoclast formation and bone resorption during skeletal bone development and ovariectomy(OVX)-induced bone loss.Thus,our findings establish a link between mTORC1 activation and both the OPG/RANKL/RANK axis and bone remodeling,and regulation of ?-catenin by mTORC1 represents a novel crosstalk between these two signaling pathways critical for development and metabolism.Methods1.Using cre-loxp system,we have successfully created the mice with collecting duct-specific deletion of Tscl,raptor,which were termed CDTsc1KO mice.2.Bone samples were assessed by microCT,to analysis bone mineral density(BMD),calculate osteoclasts number,assess the effects of mTORC1 hyperactivation or inactivation on bone loss.3.Histological and morphological analysis(HE and IHC)to examine the effects of mTORC1 hyperactivation or inactivation on the RANKL expression and morphological appearance of bone.4.Western blot deteted RANKL/OPG expression and ?-catenin expression under mTORCl hyperactivation or inactivation in vitro and in vivo.5?Oral administration of rapamycin to test whether OVX-induced osteoporosis could be prevented by the mTORCl specific inhibitor-rapamycin.6?OP9,MC3T3-E1,MG63,MLO,Y4 cell lines,primary rat BMSC and mouse osteoblasts,B lymphocytes were treated with rapamycin in vitro,detected RANKL/OPG expression level and p-Akt(S473),p-Akt(T308)phospharylated levels.7?MEFTSC2+/+and MEFTSC2-/-cell lines were cultured in viteo,detected RANKL/OPG expression level.8?Sepecific Tscl-siRNA interfered BMSC,detected RANKL/OPG expression levels.9?DN-Akt Ads transfected BMSC in vitro,and treated with rapamycin,then detected RANKL/OPG(3-catenin expression levels,to explore mTORC1 activity in regulation of RANKL/OPG ratio variation.10??-catenin plasmid and specific Tscl-siRNA co-transfected BMSC,detected RANKL/OPG,?-catenin expression levels.11?Specific ?-catenin-siRNA transfected BMSC in vitro,and treated with rapamycin;or treat primary osteoblasts which loss raptor expression with rapamycin,detected protein levels of RANKL/OPG.12?As mTORC1 is a key factor to sense nutritional signals,we remove serum and glucose to stimulate mTORCl,then detected ?-catenin?RANKL/OPG?p-Akt expression levels.Results1?mTORC1 activation correlates with enhanced RANKL expression,osteoclast formation and cancellous bone loss in OVX miceWe established the ovariectomy(OVX)mouse model and administered the mTORC1-specific inhibitor,rapamycin,to determine the association between mTORC1 activity and both RANKL expression and osteoclastogenesis in vivo.Microcomputer tomography(micro-CT)analysis confirmed significant cancellous bone loss in OVX adult female mice,characterized by reduced bone indices including bone mineral density(BMD)(p<0.05),trabecular bone volume per tissue volume(BV/TV)(p<0.05),trabecular thickness(Tb.Th)(p=0.903),and trabecular number(Tb.n)(p<0.05),with concomitant augmentation of trabecular spacing(Tb.Sp)(p<0.05).As previously reported,OVX-induced bone loss was mainly from enhanced osteoclast formation and bone resorption,but not reduced osteoblast formation,as the number of tartrate-resistant acid phosphatase(TRAP)multinucleated cells in the trabecular bone surface of OVX mice significantly increased,while osteoblast number per bone perimeter remained unchanged(P<0.01)compared to the Sham controls.Consistent with elevated osteoclastogenesis in OVX mice,RANKL levels increased,while OPG levels decreased in the bone marrow of OVX mice compared with the Sham controls.Interestingly,phosphorylation levels of S6(S235/236)(downstream effector of mTORCl and S6K1)also increased in the bone marrow and bone marrow stromal cells(BMSC)of these estrogen-deprived animals,indicating that mTORC1 activity is enhanced by OVX and that mTORC1 activation correlates with enhanced RANKL production,osteoclast formation and cancellous bone loss in OVX mice.We further examined the effects of mTORC1 inhibition on bone in OVX mice.Although BMD,BV/TV and BMC were elevated without statistical significance,the trabecular number increased,while trabecular spacing reduced significantly(P<0.05)in OVX mice after 2 months of rapamycin(p.o.,2 mg/kg/d)treatment compared to vehicle-treated OVX mice.Rapamycin treatment did not cause significant changes in body and uterus weight or bone histomorphological indices in the Sham control mice.Similar results were obtained for rapamycin-treated OVX rats in a previous study.These findings suggest that mTORCl inhibition may protect animals from OVX-induced cancellous bone loss.Importantly,the number of osteoclasts in the trabecular bone surface and the protein level of RANKL in the bone marrow of OVX mice were significantly reduced by inhibition of mTORC1.These results indicate the potential role of mTORC1 in the regulation of RANKL/OPG expression,osteoclast formation and bone resorption.2?mTORC1 regulates RANKL/OPG mRNA and protein levels in a variety of RANKL-expressing cellsTo further address the role of mTORC1 in RANKL/OPG production,mTORC1 was activated or inhibited using a chemical inhibitor or through genetic manipulation in a variety of RANKL-expressing cells.As expected,treatment with rapamycin caused a marked decrease in RANKL mRNA levels and an increase in OPG mRNA levels in primary BMSCs,calvarial osteoblasts,mouse osteoblastic MC3T3-E1 and mouse BSMC OP9 cell lines.Accordingly,the protein level of RANKL was downregulated,while OPG expression was upregulated by rapamycin in these cells.Furthermore,knockdown of TSC2,an upstream negative regulator of mTORCl,led to mTORC1 activation and the stimulation of RANKL expression both at the mRNA and protein level in OP9 cells.In TSC2 depleted mouse embryonic fibroblasts(TSC2-/-MEFs),RANKL mRNA level was increased more than times compared to wild-type MEFs(TSC2+/+MEFs),due to the high level of S6(S235/236)phosphorylation and mTORC1 activity in TSC2-/-cells.In addition,calvarial osteoblasts were isolated from Tscl or Raptor flox/-and flox/flox mice and treated with an adeno-cre virus ex vivo to knock down Tscl or Raptor,respectively,and their production of RANKL/OPG capacity was assessed.Deletion of TSC1 significantly enhanced the expression of RANKL and reduced the production of OPG,while deletion of Raptor inhibited mTORC1 and RANKL expression in these osteoblasts.Similarly,RANKL expression was markedly increased in Tscl-deficient B lymphocytes.Interestingly,the regulation of RANKL by mTORC1 was also observed in the osteocyte cell line,MLO-Y4,the branched cell embedded in the matrix of bone tissue which has recently been shown to be a major source of RANKL in supporting osteoclast formation during adult bone remodeling.Collectively,these findings identify the dominant role of mTORC1 in regulation of RANKL/OPG expression in a variety of RANKL-expressing cells.3?mTORCl downregulates ?-catenin via inhibition of Akt and destabilization of P-catenin mRNATo understand the molecular basis of mTORC1 in the regulation of RANKL/OPG,we analyzed gene expression in rapamycin-treated OP9 cells using global mRNA profiles.We found that ?-catenin,a key transcript factor which is critical for osteoblast differentiation and has been shown to inhibit transcription of RANKL,but promote OPG transcription,exhibited a marked increase in mRNA expression in rapamycin-treated OP9 cells.The rapamycin-induced upregulation of P-catenin mRNA and protein levels was further confirmed in primary BMSCs,OP9,MLO-Y4 and MC3T3-E1 cells.Accordingly,transfection of TSC2-specific siRNA led to activation of mTORC1 and reduction of ?-catenin mRNA and protein expression in OP9 cells.Furthermore,TSC2-/-MEFs exhibited a much lower level of?-catenin than Tsc2+/+MEFs.Similarly,deletion of Tsc1 by transfection of osteoblasts isolated from Tsc1 flox/-and flox/flox mice with an adeno-cre virus inhibited the expression of ?-catenin,while deletion of Raptor increased the expression of ?-catenin in these osteoblasts.The protein level of P-catenin in the bone marrow of Tsc1CAG-Cre-ERT mice was significantly reduced compared with Tsc1 flox mice.The reduction of ?-catenin was also observed in B lymphocytes ofTsc1 CD19-Cre mice in which mTORC1 was constitutive activated in B lymphocytes.These evidences indicated that mTORC1 negatively regulated mRNA and protein expression of ?-catenin.It has been shown that ?-catenin is encoded by a labile transcript whose half-life is prolonged by phosphatidylinositol 3-kinase(PI3-K)-Akt signaling.Akt phosphorylates and inhibits the mRNA decay-promoting factor,KSRP,leading to ?-catenin accumulation38.To elucidate the mechanism through which mTORC1 regulates(?-catenin expression,we first examined the effect of mTORC1 on?-catenin mRNA stability.The results showed that rapamycin stabilized ?-catenin mRNA and increased mRNA steady-state levels in OP9 cells.We next investigated the potential role of Akt in the regulation of ?-catenin by mTORC1 and found that mTORCl inhibition resulted in activation of PI3-K/Akt signaling,as manifested by the enhanced phosphorylation of Akt(T308),Akt(S473)and its downstream substrate GSK3-?(S9)in rapamycin-treated cells.In contrast,PI3-K/Akt signaling was inactivated in Tsc2-deficient cells.These responses could stem from negative feedback regulation of PI3-K/Akt signaling or phosphorylation of rictor and suppression of mTORC2 by mTORC1/S6K1 or other unknown mechanisms.Importantly,transfection of the dominant-negative(DN)mutant of Akt prevented the rapamycin-induced accumulation of ?-catenin.Thus,our findings indicate that mTORC1 negatively regulates ?-catenin expression via inhibition of Akt and destabilization of ?-catenin mRNA.4?mTORCl controls RANKL/OPG expression through ?-cateninWe further tested whether mTORCl controls RANKL/OPG expression through(3-catenin.As expected,overexpression of P-catenin suppressed RANKL,but stimulated OPG expression,knockdown of ?-catenin induced RANKL,but reduced OPG expression.Importantly,knockdown of P-catenin prevented the enhanced OPG and decreased RANKL expression by rapamycin treatment.In Raptor-depleted calvarial osteoblasts,downregulation of ?-catenin also reversed enhanced RANKL and reduced OPG expression compared with wild-type osteoblasts.Similarly,transfection of Akt(DN)re-induced RANKL expression in rapamycin-treated OP9 cells.Furthermore,knockdown of KSRP increased ?-catenin and OPG,but reduced RANKL protein levels.Luciferase assay using RANKL and OPG reporter genes containing putative ?-catenin binding sites demonstrated that mTORC1 inhibition repressed RANKL and stimulated OPG transcription activity,while mTORC1 activation enhanced RANKL and inhibited OPG transcription activity.These results further demonstrate that RANKL/OPG lies downstream of Akt and p-catenin to control osteoclast formation.5?mTORC1 regulates RANKL/OPG expression to promote osteoclast formation in vivoWe next assessed the function of mTORCl-regulated expression of RANKL/OPG in osteoclast formation and bone resorption in vivo.RANKL is abundantly expressed by various cell components in bone including BMSCs,osteoblast-lineage cells,T and B lymphocytes,vascular endothelial cells and chondrocytes,and TSC1 deletion in whole body causes fetal lethality.To investigate the actions of mTORC1 activation in adult bone,we generated tamoxifen-inducible Tscl-whole body deletion mice by crossing Tscl-flox mice with mice expressing Cre-ERT(CAG-Cre-ERT)recombinase driven by the chicken ?-actin promoter,thereby obtaining Tscl-CAG-Cre-ERT mice.Adult mice(2-months old)rapidly lost body weight and died 3-4 weeks after an intraperitoneal injection of 1 mg of tamoxifen,likely due to severe intestinal dysfunction.We also detected a marked increase in bone marrow P-S6(S235/236)using Western blotting,confirming that we had successfully established a system for the conditional activation of mTORC1.Interestingly,the production of RANKL was enhanced,while OPG was attenuated in the bone marrow and bone section of Tsc1CAG-Cre-ERT mice 10 days after tamoxifen administration.Consistent with the activation of mTORCl and the enhanced RANKL/OPG ratio,the number of osteoclasts increased significantly in trabecular bone of Tsc1-deficient mice.It is suggested that mTORCl activation enhanced the RANKL/OPG ratio to promote osteoclast formation.However,this model could not rule out the direct action of mTORC1 on osteoclasts and their precursor macrophages.Chondrocytes are the major source of RANKL in supporting osteoclastogenesis at the base of the growth plate and newly formed bone within the growing skeleton.To further identify the contribution of mTORC 1-regulated RANKL/OPG to osteoclastogenesis in vivo,we disrupted the Tsc1 or Raptor gene specifically in the chondrocyte lineage by crossing Tscl or Raptor flox mice with an osteo-chondroprogenitor cell-specific Cre transgenic mouse line(Col2a1-Cre),thereby obtaining TsclCol2al-Cre or RaptorCol2al-Cre mice.Efficient deletion of Tscl or Raptor was observed at the sites of endochondral bone formation,resulting in the activation or inhibition of mTORC1 at these sites.As expected,expression of RANKL was increased,while OPG was decreased in chondrocytes of TsclCol2al-Cre mice.In contrast,expression of RANKL was attenuated,while OPG was enhanced in chondrocytes of RaptorCol2al-Cre mice.Most importantly,a significant increase in osteoclast number beneath the growth plate was observed in Tsc1Col2?1-Cre mice,indicating that mTORC1-upregulated RANKL production in chondrocytes was sufficient to promote osteoclast formation in vivo.In contrast,Raptor-deficiency in chondrocytes attenuated osteoclastogenesis beneath the growth plate.Previous studies demonstrated that T and B lymphocyte expression of RANKL may play an important role in osteoclast formation and elevated bone resorption under pathological conditions.Consistent with the increased RANKL production inTscl-depleted B lymphocytes,osteoclast number was enhanced in trabecular bone of B lymphocyte-specific Tsc1 deletion mice.(CAG-Tscl vs WT,p=0.000;col-Tscl vs WT,p=0.000;col-raptor vs WT,p=0.001;CD19-Tsc1 vs WT,p=0.000)This increased osteoclast number was also found in osteoblast or osteo-chondroprogenitor cell-specific Pten deletion mice,in which mTORC1 activities were enhanced in osteoblasts and chondrocytes.These findings indicate that mTORCl is a crucial regulator of RANKL/OPG expression and osteoclast formation in vivo.6?The schematic model depicting the role of mTORC1 in regulation of RANKL/OPG expression and osteoclast formation through ?-cateninShown are mechanisms that involve the mTORCl-dependent regulation of?-cantenin,RANKL/OPG expression and osteoclastogenesis in RANKL-expressing cells(BMSC,osteoblast,osteocyte or lymphocyte).Activation of Akt inhibits KRSP-dependent ?-catenin mRNA degradation and promotes ?-catenin protein expression,thereby by triggering the nuclear tanslocation of(3-catenin that suppresses RANKL transcription and promotes OPG transcription,leading to the decrease of RANKL/OPG ratio and reduction of osteoclastogenesis.mTORC1 activation causes negative feedback inhibition of Akt and KRSP-dependent degradation of P-catenin mRNA and downregulation of ?-catenin protein expression,thereby enhancing RANKL production that promotes osteoclast differentiation and activation.ConclusionIn summary,using cell-specific mTORC1 activation or inhibition cellular and mouse genetic models,we established the role of mTORC1 in the promotion of RANKL expression and osteoclast formation via negative regulation of P-catenin.This finding not only provides mechanistic insights into how mTORC1 regulates osteoclast formation and bone remodeling,but represents a crosstalk between mTORC1 and ?-catenin,two critical signaling pathways for development and metabolism,and provides a potential link between enhanced mTORC1 activity and both elevated RANKL expression and bone loss in pathological conditions such as aging,obesity and inflammation.Identification and elucidation of key modulators of RANKL expression should aid in the development of therapeutic strategies for the treatment of skeletal diseases.