Role Of Rictor In Senile Osteoporosis And Its Regulatory Mechanisms

1 BackgroundOsteoporosis(OP)is an asymptomatic bone disease characterized by low bone mineral density and deterioration of microarchitecture of the skeleton,which leads to an increased fracture risk.At present,nearly 100 million people were diagnosed with osteoporosis in our country,ranking the highest in the world.As osteoporosis-related fractures are the main cause of bone pain,fractures and disability or death in the elderly,osteoporosis is becoming a significant health care issue of the society.In postmenopausal women,estrogen deficiency cause rapid bone loss,i.e.postmenopausal osteoporosis(PMOP).In additon,both male and female both show a relatively slow and age-related bone loss,i.e.senile osteoporosis.Age-related bone loss is more obvious over 60 years of age.Postmenopausal osteoporosis and age-related bone loss are both primary osteoporosis,accounting for 85-90%of all cases.So far,most of the studies focus on PMOP,while little is known about senile osteoporosis.The mechanism of senile osteoporosis is still unclear.As the aging of population,the trend of age-related bone loss has increased year by year,especially in densely populated and aging Southern China.Epidemiological investigations showed that 20%-30%aged men died from complications of fracture every year in Guangzhou Province.At present the key scientific problems to be solved is gaining insight into the mechanism of age-related bone loss and senile osteoporosis and looking for effective prevention against these two dieases.Senile osteoporosis is charactered by bone mass loss,slowed bone transformation,fat accumulation in bone marrow and inverse proportion of the adipocytes in bone marrow to the cancellous bone mass.It been reported that along with the age increase,the differentiation of the bone marrow stromal cells(BMSC)to adipocytes increased,and the differentiation of BMSC to osteoblast differentiation decreased.The lifespan of osteoblast shortened and apoptosis increased,leading to insufficient bone formation.The expression of cytokines inducing osteoclast differentiation,including RANKL,TNF-a and IL-1,remained the same or increased.The differentiation of osteoclasts increased and lifespan lengthened,leading to high-leveled bone resorption.The unbalance between bone formation and bone resorption cause bone loss.Most of the studies related to senile osteoporosis focus on molecules which control the differentiation of BMSC to adipocytes in the elderly.However,the adhesion of osteoblast precursors on bone surface and the function/apoptosis of osteoblast also play important roles in osteoblast number and bone formation.Further studies in these molecular mechanism will shed light on the pathogenesis of age-related bone loss and senile osteoporosis.Senile osteoporosis is a natural process of degeneration and aging,associated with aging,lack of estrogen,genetic,endocrine,nutritional and physical factors.Aging is a determining factor of age-related bone loss and senile osteoporosis.Lack of estrogen once considered the common and main reason of postmenopausal osteoporosis and senile osteoporosis.Recently,aging and reactive oxygen species(ROS)are regarded as the direct reasons of bone loss and osteoporosis.Estrogen increase ROS and oxidative stress,which then cause osteoporosis.No matter men or women,the bone loss happens as early as 30 years old,much earlier than the age of dramatical decreasing of sex hormone.In addition,animal and cell experiments showed that aging and aging related oxidative stress are the basic cause of bone loss.ROS regulates the proliferation,differentiation and survival of osteoblast and osteoclast.Increased ROS is main reason of the onset of senile osteoporosis.On the one hand,ROS promote osteoclast differentiation and improve the ability of bone resorption.On the other hand,ROS inhibit BMSC differentiation to the osteoblast,destroy cell membrane,increase the permeability of osteoblast and promote cell apoptosis.In additon,ROS can promote bone matrix degradation.The molecular mechanism of ROS on the osteoblast has not yet been clearly elucidated.ROS targeting microRNA and regulating its downstreamers has not been reported.Mammals rapamycin target protein(mTOR)is a relatively conservative serine/threonine protein kinase.mTORC1 is activated by nutrients(amino acids),growth factors(insulin,IGF,etc.)and cellular energy status(AMP/ATP ratio),and regulates several growth related process including protein synthesis,ribosome biogenesis,lipid synthesis,nutrient import,and autophagy.There are two kinds of mTOR complexes:mTORCl and mTORC2.mTORCl is sensitive to raprmycin,composed by mTOR,Raptor,mLST8 and PRAS40.mTORC2,containing mTOR,mLST8,Rictor,PRR5 and mSinl,is not sensitive to rapamycin.mTORC2 regulates the actin cytoskeleton,cell survival and other processes via phosphorylation of AGC kinase family members including Akt,SGK1 and PKC.Rictor is a key protein of mTORC2 and recent studies have shown that Rictor plays an important role in regulating cytoskeleton and osteogenic differentiation.However,the role of cytoskeleton in senile osteoporosis had not been reported.Relatively few studies focus on mTOR in bone metabolism.Studies show that mTORCl play an important role in the differentiation of osteoblast proliferation.Rapamycin can inhibit or promote osteoblast differentiation in different cells.There are two studies using animal models.There are two studies using animal models.Application of Rapamycin on immature mice decreased bone mineral density(BMD),and inhibited bone loss caused by ovariectomised(OVX).These results suggested that bone development in mice need mTORCl activity,but mTORCl activation may also be involved in bone loss caused by the lack of estrogen.Therefore,the regulation of mTORCl on bone metabolism is very complex,and may be different in different cells,the different stages of development and age or different disease states.mTORC2 regulates the cytoskeleton and cell motility,and plays roles in cell survival through phosphorylation of Akt(S473).Roles and mechanisms of rictor/mTORC2 in senile osteoporosis is also not reported.In this study,we proposed that increasing activity of mTOR2 may be one of the pathogenesis of senile osteoporosis,and research on that has not been reported.This research will provide a new mechanism for pathogenesis of age-related and senile osteoporosis,and provide new strategies and targets for the treatment of osteoporosis.2 ObjectiveThere are five points of innovation in this tudy as follows:Firstly,Rictor is decreased in primary osteoporosis,its expression decline with age.Furthermore,the attentchment ability of osteoblast and osteogenesis have declined in osteoporosis group.Secondly,Rictor-KO mice accelerate the occurrence of osteoporosis.The apoptosis of osteoblast increased in Rictor-KO mice.While the osteoblast differentiation and adhesion ability were inhibited by Rictor.Thirdly,the role of Rictor in senile osteoporosis and its regulatory machanisms have been studied,miR-218 targeting Rictor and the down-regulate its expression.And the downstream functional protein of Rictor was inhibited.Rictor were verified in osteoblast,together with their interaction.Last but not least,ROS active miR-218 in senile osteoporosis mice.And the expression of Rictor can be downregulated by activated miR-218.Which lead to increasing apoptotic osteoblast,decreasing the differentiation of osteoblast and attachment ability of osteoblast.3 Materials and Methods1 The effect of aging on osteoblast differentiation,adhesion and function.A total of 12 C57/B6 mices were obtained from the Experimental Animal Research Center of Southern Medical University.The animals were divided into two groups 16 and 3 months old.Femur was fixated by 4%paraformaldehyde(LPL)and scanned by ?CT to analyze the distal femur bone parameters.Decalcified bone was embedded with parpffin wax and sectioned.H&E staining,Trap staining and immunohistochemical staining were performed.Adipocytes,osteoblast and osteoclast have been counted and analyzed.Long bone of 3 and 16 months old mice were scaned by electron scanning microscope(ESM)to observe the surface of cancellous of distal femur,and cell adhesion,differentiation,proliferation andapopotsis wre detected.2 expression of Rictor and its downstream functional proteins in osteoporosis.The skull or femur from 3 and 16 months old mice were lysed for proteomic analysis to find out the disparitied protein between two groups.And Western blot were performed to verify the protein and its downstream protein we picked.Other organs from 3 and 16 months mice were also lysed,for western blot and compared with bone lysate.3 osteoblastic specific knockout of Rictor impairs bone formation and accelerates aged-related osteoporosisWe propagate osteoblast-specific knockout Rictor mice using cre-loxp system.Gene type was identified by PCR.Bone tissue of 2 and 6 months old KO/WT mice were detected by western blot and immunohistochemical to verify the knockout effect.Long bones were dissected and scanned by micro-CT for bone mass analysis.These long bone was fixated by 4%LPL.Decalcified bone was embedded with parpaffin wax and sectioned.H&E staining,Trap staining and immunohistochemical staining were performed.Adipocytes,osteoblast and osteoclast have been counted and analysised.And TUNEL staining was performed to detect the apoptotic cell.The surface of cancellous of distal femur was analysed by electron scanning microscope(ESM).4 Rictor is essential for osteoblast differentiation,adhesion and survivalPrimary calvarial osteoblast were isolated from Rictor-KO(OSX+,Rictorfl/fl)and WT(OSX-,Rictorfl/fl)Osteoblastic induction cultured for 7 days followed by ALP staining.Proliferation test was performed by CCK8,western blot,adhesion test.The expression of Paxillin was detected by western bolt and immunofluorescence localization to test F-actin and Paxillin.5 Rictor is a target of miR-218 in osteoblastBone total RNA was extracted from 3 and 16 months mice.mRNA of Rictor have been tested was measured by QPCR.MG63 cell were transfected miR-218 inhibitor or mimics to detected Rictor expression.Luciferase assay were performed in MG63 or MC3T3-E1 cell transfected with miR-218 plasmid to detecte interaction between Rictor and miR-218.miR-218 mutation were constructed plasmid for further verify.6 miR-218 represses osteoblast adhesion and survival by targeting Rictor during agingMG63 or MC3T3-E1 cell were transfected with miR-218 inhibitor or mimics,and cell adhesion were detecdted.Cells were also collected and lyzed for western blot of expression of Rictor and its downstream function proteins,Paxillin and apoptin.7 Reactive oxygen species stimulates miR-218-mediated rictor downregulaiton in aged mice osteoblast.A total of 20 C57/B6 mices were obtained from the Experimental Animal Research Center of Southern Medical University.The animals were randomly divided into two groups.For each group,n=10.Experimental group treated with NAC(2mg/ml)in drinking water,while control group drinking water without NAC for 3 months.Long bone from experimental group and control group was fixated by 4%LPL.Decalcified bone was embedded with parpffin wax and sectioned.H&E staining,Trap staining and immunohistochemical staining were performed.Adipocytes,osteoblast and osteoclast have been counted and analysed.And TUNEL staining were performed to detect the apoptotic cell.4 Results1 aging affects differentiation adhesion and function.micro-CT scanning indicated that in 16 months mice Th.N and BMD were relatively low compared with 3 months mices.16M group shows severe bone loss.Trap staining showed that osteoclasts decreased.Osteoclast wasn't contribute to osteoporosis.Primary calvarial osteogenesis of 16M group was restrained and proliferation rate was slower than 3M group.The expression of cleaved-PARP increased in 16M group.SEM indicated that Th.N became less and thinner in 16M groups.Cells adhesed to the bone surface obviously reduced,and bone matrix destroyed,leaving with large number of cell prits.2 expression of Rictor and its downstream functional pretein in osteoporosisProteomic analyses of bone shows that the expression of Rictor was decreased obviously in 16M group compared with 3M group.Western blot resuot in other organs were consistent with this result.The expression of downstream proteins of Rictor P-Akt(S473),Paxillin,P-Paxillin(T118)of Rictor were decline.But the expression of cleaved-PARP rised.3 Deletion of rictor in osteoblast impairs bone formation and accelerates aged-related osteoporosisOsteoblastic specific knockout of Rictor animal model was constructed using cre-loxp system.OSX-cre,Rictorfl/fl(Rictor-KO)and Rictorfl/fl(WT)mice were obtained.The micro-CT scanning indicated that Th.N and BMD are relatively lower in Rictor-KO group compared with WT group in 6 months old.PCNA staining indicated that the number of proliferation cells relatively reduced,And apoptin cleaved-PAR increased in 6 months old group.SEM indicated that Th.N became less and thinner in Rictor-KO groups,and cells adhered to the bone surface obviously reduced,leaving with a large number of cell prits.4 Rictor is essential for osteoblast adhesion,function and survivalIn vitro experiments indicated that the differentiation of primary BMSC isolated from Rictor-KO group was inhibited and cell attachment was declined,compared with WT group,.And the expression downstream proteins of Rictor P-Akt(S473),Paxillin,P-Paxillin(T118)of Rictor were declined.But the expression of cleaved-PARP rised in Rictor-KO group.5 Rictor is a target of miR-218 in osteoblastQPCR analysis showed no obvious changes of Rictor mRNA,but significant increase in protein level in 16M group.In MG63 or MC3T3-E1 cells transfected with miR-218 inhibitor,the expression of Rictor,and cell attachment were increased.In MG63 or MC3T3-E1 cells transfected with miR-218 mimics,the expression of Rictor and cess attachment ability was reduced.While the expression of cleaved-PARP increased.Luciferase assay indicated that Rictor was interacted with miR-218 and this interaction diaappeared in the miR-218 mutation group.6 miR-218 represses osteoblast adhesion and survival by targeting Rictor during agingQPCR analysis showed the expression of miR-218 increased significantly in 9 months old C57/B6 mice.The expression of Rictor and the downstream proteins p-Akt(S473),Paxillin,P-Paxillin(T118)were decreased in MG63 or MC3T3-E1 cell transfected with miR-218 mimics.While the expression of cleaved-PARPwas increased.Conversely,the expression of Rictor and the downstream proteins p-Akt(S473),Paxillin,P-Paxillin(T118)were decreased in MG63 or MC3T3-Elwhen transfected miR-218 inhibitor.7 Reactive oxygen species stimulates miR-218-mediated rictor downregulaiton in aged mice osteoblastThe expression of miR-218 was significantly up-regulated in MG63 or MC3T3-E1treated with H2O2 for 24h.Compared with control group,NAC treated group showed higher BMD and osteoblast.The BMD of experimental group was higher than control group.And the number of osteoblast cell increased treated with NAC,miR-218 declined obvious and expressiong of Rictor raised relatively in NAC group.5 ConclusionsWe discussed that Rictor/mTORC2 signaling pathways played an important role in senile osteoporosis,and studied its regulating mechanism of upstream and downsteam by using genetic knockout mice and cell model.(1)Reduction of osteoblast number own to decreased of osteoblastic differentiation ability,decreasing cell proliferation and increasing apoptosis.This phenotype is one of the cytology mechanism of age-related osteoporosis.(2)Rictor is decreased in primary osteoporosis,its expression decline with age.Rictor dwon-regulated the osteoblastic differentiation and attachment..(3)Osteoblast specific knockout of Rictor showed accelerated aged-related osteoporosis by dwon-regulating the osteoblastic differentiation and attachment,decreasing cell proliferation and increasing apoptosis(4)miR-218 targeting Rictor and the downstream of Rictor were verified in osteoblast,together with their interaction.miR-218 dwon-regulated the osteoblastic differentiation and attachment by targeting Rictor.(5)ROS increase the expression of miR-218.And Rictor was targeted by miR-218,downregulated the expression of Rictor and the osteoblastic differentiation and attachment,decreasing cell proliferation and increasing apoptosis.NAC can release the senile osteoporosisTherefore,Our study not only verify that the down-regulation of Rictor/mTORC2 signaling is one of the pathology mechanisn of senile osteoporosis,but also build the connection of ROS,miR-218,Rictor/mTORC2 and senile osteoporosis.Our parper illustrates a new mechanism lead to senile osteoporosis.ROS increase the expression of miR-218.And Rictor was targeted by miR-218,down-regulated the expression of Rictor and the osteoblastic differentiation and attachment,decreasing cell proliferation and increasing apoptosis.This study provide new view for the regulation of bone metabolism,and a new target for prevention and healing of senile osteoporosis.