The Mechanism Of AMPK/mTOR/p70S6K Signaling Pathway-mediated Autophagy In Osteoprotegerin-induced Inhibition Of Osteoclastogenesis

Bone is an important tissues and organs of animal,and has the function of supporting the body and movement,and also involves in the regulation of the complex environment.Bone formation and bone resorption are coupled processes during bone remodeling in normal bone skeleton system.However,bone resorption exceeds the rate of bone formation causing osteoporosis,exhibiting loss of bone mass induced by augments of the number and activity of osteoclasts,which is responsible for bone resorption.There are many complicated factors to regulate the differentiation and function of osteoclasts.At present,the widely accepted critical central axis for controlling osteoclast differentiation is "osteoprotegerin(OPG)/receptor activator of nuclear factor kappa-B ligand(RANKL)/receptor activator of nuclear factor kappa-B(RANK)".RANKL binds to the membrane receptor RANK to promote the formation and function of osteoclasts.In addition,OPG as a decoy receptor for RANKL can block the RANKL binding to RANK,inhibiting the differentiation of function of osteoclasts indirectly.Adenosine monophosphate(AMP)-activated protein kinase(AMPK)/mammalian target of rapamycin(mTOR)/ribosomal protein S6 kinase beta 1(S6K1,also known as p70S6K)signaling pathway-mediated autophagy also involves osteoclast differentiation,which by increasing the expression of autophagy marker proteins and the number of autophagosomes can provide the support for the differentiation and survival of osteoclasts.The current study aims to explore the mechanism of AMPK/mTOR/p70S6K signaling pathway-mediated autophagy during OPG-induced inhibition of osteoclasts differentiation,which inducing by bone marrow monocytes/macrophages(BMMs)and RAW264.7 cells in the presence of RANKL and macrophage colony-stimulating factor(M-CSF).1.The effects of AICAR activates autophagy on osteoclast differentiationTo explore the effects of 5-Aminoimidazole-4-carboxamide ribonucleotide(AICAR)on autophagy during osteoclast differentiation.In current part of the experiment,BMMs and RAW264.7 cells were induced to differentiate into osteoclasts for 3.5 days in the presence of RANKL(60 ng/mL)and M-CSF(30 ng/mL).Add different concentration of AICAR(0,0.05,0.5,and 5 mM)to differentiated osteoclast for 3 h,and to detect the formation of tartrate-resistant acid phosphatase(TRAP)positive osteoclast using the TRAP staining kit,and the expression of cathepsin K(CTSK),c-Fos,nuclear factor of activated T-cells cytoplasmic 1(NFATc 1),Beclinl,microtubule-associated protein 1A/1B-light chain 3(LC3),p62(SQSTM1/sequestomel),p-mTOR/mTOR,and AMPK signaling pathway-related proteins using western blot.EGFP-pmCherry-LC3 plasmid was transfected,and LC3 puncta was recorded using laser confocal microscopy.The results showed the formation of TRAP positive osteoclast was inhibited and the expression of CTSK,c-Fos,NFATc 1,p62,p-mTOR/mTOR,Ras homologue in the brain(Rheb),and p-p70S6K/p70S6K were decreased significantly(P<0.05 or P<0.01)after AICAR treatments,as well as increased LC3 puncta and the expression of Beclinl,LC3 ?,p-AMPK?/AMPK?,and tuberous sclerosis complex 2(TSC2)were increased significantly(P<0.05 or P<0.01).These data demonstrated that activation of AMPK attenuated osteoclast differentiation by enhancing autophagy and regulating downstream signaling pathway.2.OPG inhibits osteoclast differentiation by AMPK signaling pathwayTo explore the role of OPG inhibits osteoclast differentiation through AMPK signaling pathway.In current part of the experiment,BMMs and RAW264.7 cells were added the OPG(40 ng/mL)for treatment of different time(0,1.5,3,and 6 h),or were added the OPG of different concentration(0,20,40,and 80 ng/mL)for 3 h.The formation of TRAP positive osteoclast was detected using the TRAP staining kit,and the expression of CTSK,c-Fos,NFATc1,Beclin1,LC3 ?)p62,autophagy-related genes(Atgs),mTORC1,and AMPK signaling pathway-related proteins were measured using western blot.Reactive oxygen species(ROS)level was detected by flow cytometry.Autophagosomes and autolysosomes were observed by transmission electron microcopy.LC3 puncta was recorded using laser confocal microscopy.The results showed that the expression of Beclinl and LC3 ? were increased highly(P<0.01)treatment with OPG(40 ng/mL)for 3h,but the expression of p62 was decreased(P<0.01).The formation of TRAP positive osteoclast was inhibited treatment with different concentration of OPG.The expression of CTSK,c-Fos,NFATc1,p62,p-mTOR/mTOR,regulatory-associated protein of mTOR(Raptor),Rheb,and p-p70S6K/p70S6K were decreased significantly(P<0.05 or P<0.01)by different concentration of OPG,but increased the number of LC3 puncta and autophagosomes and upregulated significantly(P<0.05 or P<0.01)the expression of Beclinl,LC3 ?,p-AMPK?/AMPK?,and tuberous sclerosis complex 2(TSC2).There are no effects on ROS level and G protein beta subunit-like(G?L)expression.These data demonstrated that OPG inhibits osteoclast differentiation by enhancing autophagy and AMPK signaling pathway.3.Autophagy regulates OPG-induced inhibition of osteoclasts differentiationTo explore the influence of autophagy on OPG-induced inhibition of osteoclast differentiation.In current part of the experiment,BMMs and RAW264.7 cells were pretreated with 10 ?mol/L chloroquine(CQ)and 5 ?mol/L rapamycin(Rap)for 0.5 h and 1 h respectively,and then add OPG(40 ng/mL)treatment for 3 h.The formation of TRAP positive osteoclast was detected using the TRAP staining kit,the expression of c-Fos,NFATc1,Beclinl,p62,and AMPK signaling pathway-related proteins were measured using western blot.LC3 puncta was recorded using laser confocal microscopy.The results showed the number of TRAP positive osteoclast was reduced significantly(P<0.05 or P<0.01)by treating with chloroquine(CQ)and rapamycin(Rap),increasing the number of LC3 puncta.Compare with chloroquine(CQ)group,the expression of c-Fos was decreased significantly(P<0.01)by treating with chloroquine(CQ)+ OPG group,upregulated significantly(P<0.05 or P<0.01)the expression of p62,Rheb,and p-p70S6K/p70S6K,but has no effect on p-AMPK?/AMPKa and TSC2 expression.Compare with rapamycin(Rap)group,the expression of c-Fos,p62,Rheb,and p-p70S6K/p70S6K were decreased significantly(P<0.05 or P<0.01)by treating with rapamycin(Rap)+ OPG group,upregulated significantly(P<0.05 or P<0.01)the expression of Beclinl,p-AMPK?/AMPK?,and TSC2.These data demonstrated that autophagy regulates OPG-induced inhibition of osteoclast differentiation by AMPK/mTOR/p70S6K signaling pathway.4.The effects of 3-MA on OPG-induced inhibition of osteoclast differentiationTo explore the effects of 3-methyladenine(3-MA)on OPG-induced inhibition of osteoclasts differentiation.In current part of the experiment,BMMs and RAW264.7 cells were pretreated with 5 ?mol/L 3-MA for 0.5 h,and then add OPG(40 ng/mL)treatment for 3 h The formation of TRAP positive osteoclast was detected using the TRAP staining kit,the expression of CTSK,c-Fos,NFATc1,Beclinl,LC3 ?,p62,Atgs,mTORC1,and AMPK signaling pathway-related proteins were measured using western blot.EGFP-pmCherry-LC3 plasmid was transfected,and LC3 puncta was recorded using laser confocal microscopy.Autophagosomes and autolysosomes were observed by transmission electron microcopy.The results showed the number of TRAP positive osteoclast was increased by treating with 3-MA+OPG group compare with OPG group,but downregulated significantly(P<0.05 or P<0.01)the CTSK,c-Fos,NFATc1,Beclinl,Atg5,Atg12,p-AMP??/AMPK? and TSC2 expression and the autophagosomes and autolysosomes number,and increased significantly(P<0.05 or P<0.01)the LC3 ?,Atg7,mTORC1,Rheb,and p-p70S6K/p70S6K expression and LC3 puncta.These data demonstrated that 3-MA inhibits autophagy during OPG-induced inhibition of osteoclast differentiation by AMPK/mTOR/p70S6K signaling pathway5.The effects of BAF on OPG-induced inhibition of osteoclast differentiationTo explore the effects of bafilomycin A1(BAF)on OPG-induced inhibition of osteoclasts differentiation.In current part of the experiment,BMMs and RAW264.7 cells were incubated with 0.1?mol/L BAF for 0.5 h in advance,and then add OPG(40 ng/mL)treatment for 3 h.The formation of TRAP positive osteoclast was detected using the TRAP staining kit,the expression of CTSK,c-Fos,NFATc1,Beclinl,LC3 ?,p62,Atgs,mTORC1,and AMPK signaling pathway-related proteins were measured using western blot.EGFP-pmCherry-LC3 plasmid was transfected,and LC3 puncta was recorded using laser confocal microscopy.The results showed the expression of CTSK,c-Fos,NFATc 1,LC3 II,p62,p-mTOR/mTOR,Raptor,Rheb,and p-p70S6K/p70S6K were increased significantly(P<0.05 or P<0.01)and LC3 puncta was increased by treating with BAF+OPG group compared with OPG group,dowmegulated significantly(P<0.01)the expression of Beclinl,Atg5,Atg7,and Atg12,but has no effect on G?L,p-AMPK?/AMPK?,and TSC2 expression.These data demonstrated that BAF negatively regulate autophagy by the phosphorylation of mTOR and p70S6K during OPG-induced inhibition of osteoclast differentiation6.The effects of AMPK-mediated autophagy on OPG-induced inhibition of osteoclast differentiationTo explore the effects of AMPK-mediated autophagy on OPG-induced inhibition of osteoclasts differentiation.In current part of the experiment,BMMs and RAW264.7 cells were incubated with different concentration Compound C(Com C)(0,1.25,2.5,5,and 10?mol/L)or AMPK ?1/2 siRNA(20 nmol/L),and then add OPG(40 ng/mL)treatment for 3 h.Morphological dynamic change was monitored using RTCA systems.The formation of TRAP positive osteoclast was detected using the TRAP staining kit,the expression of CTSK,c-Fos,NFATc1,Beclin1,LC3 ?,p62,Atgs,mTORC1,and AMPK signaling pathway-related proteins were measured using western blot.EGFP-pmCherry-LC3 plasmid was transfected,and LC3 puncta was recorded using laser confocal microscopy.The results showed the cell index was reduced by treating with 10 ?mol/L Com C,the number of TRAP positive osteoclast was increased significantly(P<0.01)by treating with 1.25 ?mol/L Com C group,Com C(1.25?mol/L)+ OPG group,and siAMPK+OPG group.The expression of CTSK,c-Fos,LC3 ?,p62,p-mTOR/TOR,Raptor,Rheb,and p-p70S6K/p70S6K and LC3 puncta were increased significantly(P<0.05 or P<0.01),downregulated significantly(P<0.05 or P<0.01)the expression of NFATc1,Beclinl,Atg5,Atg7,Atg12,p-AMPK?/AMPK?,and TSC2.These data demonstrated that AMPK signaling pathway involves OPG-induced inhibition of osteoclast differentiation by autophagy.In summary,activated AMPKa can induce autophagy resulting in the regulation negatively of osteoclast differentiation.AMPK phosphorylation and autophagy were activated and augment of autophagosomes number during OPG-induced inhibition of osteoclast differentiation.Moreover,inhibition of autophagy or AMPK? phosphorylation can attenuate the suppression of osteoclasts by OPG.