The Mechanism Of Osteoblastic MTORCl In Regulation Of Bone Formation And Intraosseous Angiogenesis

1.BackgroundThe process of bone formation can be divided into four phases:(1)migration of embryonic cells to the site of bone formation;(2)epithelial-mesenchymal interactions;(3)formation of condensations;(4)proliferation and differentiation of chondroblasts and osteoblasts.Bone formation is executed in two ways:(1)endochondral ossification:bone formation originated from cartilage.This process is very complex and comprises a plurality of steps.Postnatal bone molding and fracture repair are both endochondral ossification process.(2)intramembranous ossification:bone formation without involvement of cartilage.During intramembranous ossification,mesenchymal progenitors condense and directly differentiate into osteoblasts.Bone formation occurs since the seventh weeks of embryo development.Intramembranous osteogenesis occurs in the frontal,parietal,temporal bone,collarbone and other flat bones and irregular bones.In sites of future bone,mesenchymal progenitors differentiation to original film of connective tissue,followed by differentiation into preosteoblasts,osteoblasts generate bone tissue.After completion of bone growth,bone maintains specific structure serving for its function.There is a close link between bone structure and function.The configuration and internal micro-structure of bone lay foundation for the function of bone.Bone shape and size are generally not the same.Bone tissue is wrapped in a fibrous sheath(ie periosteum),which is rich in nerve and blood supply.Calcified bone is dynamic rather than static.Modeling or remodeling of microstructure of bone depensd on continuous activity of cells residing in bone.After the formation of bone structure,bone turnover keeps on to maintain a relatively stable state of bone,without changing the morphology and size of bone.This cyclic metabolic process occurs in bone mass is called bone remodeling.Bone remodeling involves cell proliferation,differentiation,apoptosis and transformation.Remodeling is a cyclical metabolism of bone,which showed only an increase or decrease in bone mass without morphogenetic changes of bone.From the micro level,the basic unit of bone remodeling is bone remodeling units(BRU).The elementary process of bone remodeling is:resorption-bone formation-bone still-resorption,so the cycle to cycle.During growth,development,maturation and aging process of human body,shape,bone density and internal structure of bone tissue keep in constantly self-adjustment and self-renewal.Maintenance of distribution,mass,number and microstructure of bone relies on modeling and remodeling,during which cells and blood vessels within bone play essential role.Bone resorption overcomes formation with age,resulting in bone loss.In some pathological processes,such as advanced multiple myeloma,only bone resorption occurred without accompanied by bone remodeling.Multinucleated osteoclasts play main role in the process of bone resorption,number of which directly determines the activity of bone resorption.Bone cells accounted for about 95%of total cells of bone tissue,located within the lacuna formed by coating bone matrix.Although most research on calcium transport in bone tissue focused on osteoblasts and osteoclasts,we can infer that bone cells play potential role in maintaining calcium transport process,which accounted for 95%of the total cells of bone.When refers to osteoblasts,they are cells capable of promoting formation,which not only secrete large amounts of bone collagen and other bone matrix,but also secrete cytokines and some important enzymes,such as matrix metalloproteinases(MMPs),alkaline phosphatase(ALP),osteocalcin(OCN),osteoprotegerin(OPG),RANKL to initiate the process of bone formation.Osteoblasts are couple with osteoclast through these factors and regulate osteoclast formation,ripening and activation.Blood vessels bring oxygen,nutrition and all kinds of regulatory factors and carry off metabolites of bone,such as carbon dioxide,acids,etc.Blood flow in bone accounts for 10%of cardiac blood flow,which provides great convenience for cell renewal,reconstruction and repair of bone.Imbalance of bone homeostasis leads to bone structure and functional disorders.With an aging population,osteoporosis and associated fractures have become a major public health threat.Current treatment of osteoporosis mainly relies on antiresorptive drugs targeting osteoclasts,long-term use of which results in inhibition of bone turnover,accumulation of cracks of micro damage of old bone and increase of bone fragility.Therefore,drugs for promotion of osteoblastic function are in realistic demand,and the strengthening of basic research into cell and bone metabolism is good for the osteoporosis prevention and development of drugs.There are three ways to promote bone formation by targeting osteoblasts:(1)promoting differentiation of mesenchymal stem cells into osteoblast lineage cells;(2)promoting differentiation of preosteoblasts into mature osteoblast;(3)extend the survival time of osteoblast.Therefore,the regulation of osteoblast differentiation is of great significance for promoting bone formation.Osteoblast differentiation process is usually divided into three stages:(1)mesenchymal progenitors,(2)preosteoblasts,(3)mature osteoblasts.Osteoblast differentiation depends on a number of specific transcription factors,such as SOX9,RUNX2,OSX,ATF4.These transcription factors act in different phases of osteoblastic differentiation and determine the developmental stage of osteoblast lineage cells.In addition,differentiation of osteoblasts also depends on a variety of signaling pathways,which regulate osteoblast differentiation by controlling the expression and activity of transcription factors.Among these signaling pathways,mTORC1 is the central signaling that integrates various signals inside and outside to regulate cell growth and metabolism.The important role of mTORC1 signaling in bone development and metabolism has been recognized by the scientific community.However,results from numerous related researches are inconsistent and even contradictory,and the corresponding mechanisms have not been elucidated.Angiogenesis is another important target for treatment of bone structure and function related disease.As we all know,blood supply is a crucial for healing of fracture bone,and reduction of blood vessels in bone directly related to the incidence of osteoporosis.Bone marrow is a highly heterogeneous and vascularized tissue.The diverse cell types populating the bone marrow communicate extensively with each other,and the cell-to-cell cross-talk is vital for correct bone development and homeostasis.The cross-talk between bone-forming osteoblasts and vessel-forming endothelial cells(ECs)is progressively gaining strong support in the scientific community.In particular,osteoblasts secrete angiogenic factors,such as vascular endothelial growth factor(VEGF)and erythropoietin,to mediate the cross-talk between osteoblasts and ECs.However,molecules that couple osteoblasts and ECs to modulate bone remodeling and angiogenesis have not been fully defined,and the signaling pathways that control the production of these molecules in osteoblasts under physiological and pathological conditions are unclear.2.ObjectiveInjury or age-related degenerative diseases may seriously undermine the structure and function of the bone.Bone structure and function-related diseases(such as bone fractures,osteoporosis,etc.)not only seriously affect the quality of life of patients,but also brought a heavy financial burden to the country,society and families,thus exploring new therapies to repair bone structure is of great significance.As is well known,promotion of bone formation by targeting osteoblasts accelerates repair of bone structure and function related diseases;in addition,angiogenesis in bone is another important therapeutic target for bone structure and function related diseases,because blood supply is a crucial factor affecting the rate of fracture healing,and it has been discovered recently that the incidence of osteoporosis is also directly related to the reduction in the number of blood vessels within the bone.mTORC1 signaling pathway exerts wide range of biological effects,but evidence support for its function in osteoblasts and bone homeostasis is limited.This study investigated the role of osteoblastic mTORC1 on maintenance of bone structure and function in two aspects:(1)formation of bone;(2)angiogenesis in the bone.To this end,we use osteoblast-specific knockout mice,osteoblasts and vascular endothelial cells to investigate the role of osteoblastic mTORC1 in the regulation of bone formation and angiogenesis in bone and the mechanisms responsible,so as to add new content for the regulation of bone formation and angiogenesis and provide new theoretical support for the clinical treatment of bone structure and functional disorders in future.3.Materials and Methods3.1 The role of osteoblastic mTORC1 on bone formationTo get a preliminary perspective for the demand of or mTORCl in osteoblast proliferation and differentiation,we extract protein of osteoblast cell line MC3T3-E1 and primary osteoblast cells during their proliferation,differentiation and mineralization,and detect the activity of mTORC1 by western blot.On this basis,we used rapamycin(mTORC1 inhibitor)-treated and specific Tscl knockout(mTORC1 activation)mice and cell models to determine the effect of mTORC1 activation and inactivation on osteoblast proliferation,differentiation and mineralization.We first determine the overall effect of mTORC1 activation and inactivation on osteoblast proliferation,differentiation and mineralization by measurement of body weight of mice,X-ray,micro-CT analysis.Then morphological alteration of osteoblasts were observed by scanning electron microscopy.We produced frozen sections,paraffin sections and plastic sections,and use HE staining to observe the general structure of mice bone.Mice bone were also sectioned without being decalcified and specific staining of bone(Goldner's-Masson trichrome staining)was performed to show special structure(such as osteoid formation and change of bone mineralization,etc.)of bone.Specific staining of cells in bone,such as ALP staining,TRAP staining and immunohistochemical staining of osteoblast-specific markers were performed to determine the number of osteoblasts expression of osteoblastic marker in order to observe changes of osteoblasts in cellular and molecular levels.To test the results in vivo,primary osteoblasts obtained from newborn mouse were cultured and amplified in vitro.Cells were incorporated with Brdu and immunofluorescence staining was performed to detect proliferation of osteoblasts.To preclude the effect of cell proliferation on the differentiation of osteoblasts,we induced osteoblasts with contact inhibition to osteoblast differentiation and intervened mTORCl activity of them at the same time,protein extracted at different time points were detected by western blot to determine alteration in expression of osteoblast marker.Mineralization of osteoblasts was detected by alizarin red staining.Detailed study were also performed to investigate the mechanism through which mTORCl regulate bone formation by osteoblasts.Western blot analysis cells and bone tissues revealed that mTORC1 positively regulate Notch signaling in osteoblasts.Results from co-precipitation(CoIP)and in vitro kinase assays showed that mTORCI promotes phosphorylation of STAT3 at Ser727;Nuclear protein of osteoblasts bound specifically to a double-strand probe containing a consensus STAT3-specific binding sequence in the promoter region of ANp63,and anti-pSTAT3(S727)antibody significantly reduced the binding of STAT3 to p63 promoter.Accordingly,nuclear protein from osteoblasts with higher mTORCl activity showed an increased binding of pSTAT3(S727)to the probe.Using RNA interference method,we found that mTORC1 promotes the expression of Notch ligand Jagged1 and even entire Notch pathway activation through elevation of P63.By using of RNA interference and the Notch inhibitor(DAPT),we found that it is Notch pathway that mediated the regulation of osteoblast differentiation by mTORC1,and increased Runx2 is responsible for the inhibition of osteoblast differentiation by Notch.3.2 The role of osteoblastic mTORC1 on angiogenesis in boneTo investigate the regulation of angiogenesis in bone by osteoblastic mTORC1,we established two mouse models,in which Tscl(mTORC1 is activated)or Raptor(mTORC1 is inactivated)gene were specifically deleted in osteoblast.Through observing the appearance and color of bone,we got a first insight into the blood perfusion of bone in mice.Then long bones of mice were sectioned and immunostained by CD31 to detect the number of blood vessels in bone.To test the results in vitro,we cultured HUVECs in supernatant obtained from cultured primary osteoblasts.Cells were incorporated with Brdu and then underwent immunofluorescence assay to determine the rate of proliferation.Wound healing assay was performed to detect the migration rate of HUVECs,and tube formation assay were executed to detect the direct role of osteoblasts supermatant on angiogenesis of HUVECs in vitro.To explore the mechanism responsible for the regulation of angiogenesis in bone by osteoblastic mTORC1,we detect VEGF expression and secretion by osteoblasts,and detect the VEGF signaling transduction in HUVECs that cultured in supernatant obtained from osteoblasts,results from which revealed that mTORC1 positively regulates expression and secretion of VEGF by osteoblasts.Howerve,the regulation of VEGF by mTORC1 did not explain the angiogenesis phenotype of mice.To characterize the real mechanism responsible,we developed a genome-wide expression profiling microarray to screen angiogenesis/angiostatic factors regulated by mTORC1,from which we found Cxcl9 as a potential explanation for the angiogenesis phenotypes.To determine whether Cxcl9 is responsible for the phenotypes in mice,we performed numerous experiments in vitro and in vivo.Results from in situ hybridization and western blot assay revealed specific expression of Cxcl9 in osteoblasts,and mTORC1 promote Cxcl9 expression at the transcriptional level.Mice received treatment exogenous Cxcl9 or Cxcl9 antibody exhibited normalized angiogenesis in bone,from which we inferred that it is Cxcl9 that mediate the regulation of angiogenesis in bone by mTORC1.Then we explored the mechanism responsible for the regulation of Cxcl9 expression by mTORC1.As STAT1 is reported to regulate Cxcl9 expression,osteoblasts were immunofluorescence-stained with STAT1,results from which revealed that mTORC1 promotes translocation of STAT1 into nuclear.Results from EMSA showed specific binding of STAT1 to Cxcl9 promoter and mTORC1 promoted this binding.Finally,siRNA was used to reduce the expression of STAT1 in osteoblasts,results from which ascertained that it is STAT1 that mediate the regulation of Cxcl9 expression by mTORC1.In order to detect the role of Cxcl9 in osteoporosis after menopause,we constructed the ovariectomized mouse model.Micro-CT was performed to detect bone parameters of mice bone to assure that mouse model is established successfully.Then immunostaining of blood vessels in bone was performed to detect the blood vessel number in the mouse model.Concentration of Cxcl9 in bone marrow and sera were detected by ELISA assay to delineate the correlation between Cxcl9 concentration and blood vessel numbers in bone.Finally,mice were administered with Cxcl9 antibody to test whether Cxcl9 is responsible for the reduced blood vessels and bone mass in the mice model.4.Results4.1 The role of osteoblastic mTORC1 on bone formationDuring proliferation of osteoblasts,mTORC1 activity was positively correlated with growth rate of osteoblast;while in the process of osteoblast differentiation,mTORC1 activity was negatively correlated with differentiation degree of osteoblasts.Rapamycin(mTORC1 inhibitor)inhibit the proliferation of osteoblasts in vitro and in vivo.Intraperitoneal injection of rapamycin resulted in lower bone mass,decreased number of osteoblasts but elevated expression Osx and Ocn in single osteoblast in mice.Specific mTORC1 activation in osteoblast produced immature woven bone in mice.These mice exhibited accelerated proliferation of osteoblasts but inhibited osteoblast differentiation.Osteoblasts number was increased,but expression of Osx and Ocn were both reduced in single osteoblasts in these mice.Impairment of minerlization of bone matrix resulted accumulation of osteoid in bone of these mice.Mechanical study revealed that mTORC1 promotes phosphorylation of STAT3 at Ser727,which then promotes p63 transcription and further elevated jagged1 expression and activity of Notch signaling pathway.Activation of Notch inhibited osteoblast differentiation through downregulation of Runx2 in osteoblasts.4.2 The role of osteoblastic mTORC1 on angiogenesis in boneAngiogenesis in bone was reduced in mice with mTORC1 being activated in osteoblasts.Supernatants from osteoblasts with activated mTORC1 repressed proliferation,migration and tube formation of HUVECs.In contrary,mice with mTORC1 inacitivation in osteoblasts exhibited elevated angiogenesis in bone.Osteoblast with activated mTORC1 expressed and secreted more VEGF,supernatant of which,however,present angiostatic effect on HUVECs.While osteoblasts with impaired mTORC1 expressed and secreted less VEGF,supernatant of them promoted angiogenesis of HUVECs in vitro.Results from Genome-wide expression profiling microarray revealed significant elevated Cxcl9 mRNA levels in osteoblasts with mTORC1 activation.HUVECs maintained in ATscl CM exhibited impaired proliferation and migration.When the cells were grown in the same medium supplemented with anti-Cxcl9,proliferationand migration rates of the cells were significantly elevated to levels higher than cells treated with the control medium.Mechanical study revealed that mTORC1 elevated STAT1 mRNA and protein significantly.Immunostaining of cellular STAT1 showed more concentrated location of STAT1 in the nuclear in osteoblasts with activated mTORC1 and more STAT1 located in plasma of osteoblasts with inactivated mTORC1.Nuclear extract of osteoblasts with activated mTORC1 exhibited more binding of STAT1 to Cxcl9 promoter,while osteoblasts with inactivated mTORC1 showed less binding.Blood vessel number was significantly reduced in mice with osteoporosis after menopause.Concentrations of Cxcl9 in bone marrow and sera were elevated in OVX mice.Administration of Cxcl9 antibody promoted angiogenesis in bone and even bone formation in these mice.5.Conclusions5.1 The role of osteoblastic mTORC1 on bone formation(1)mTORC1 is activated during osteoblast proliferation but is suppressed during their differentiation.(2)mTORC1 inactivation inhibits proliferation and promote differentiation of osteoblasts;on the contrary.mTORC1 activation induces proliferation and inhibit differentiation of osteoblasts.(3)mTORC1 activates Notch signaling pathway via STAT3/P63,and Notch impairs osteoblast differentiation through inhibition of Runx2.(4)In the process of bone formation,demand for mTORC1 activity is phase dependent in osteoblast.Therefore,the stage specific regulation osteoblastic mTORC1 may be beneficial for bone formation.5.2 The role of osteoblastic mTORC1 on angiogenesis in bone(1)Osteoblasts with mTORC1 activation inhibit angiogenesis in bone;osteoblasts with mTORC1 inactivation promote angiogenesis in bone.(2)mTORC1 positively regulates expression and secretion of VEGF in osteoblasts.(3)Osteoblasts express and secrete Cxcl9,which inhibits angiogenesis by blocking VEGF signal transduction in endothelial cells.(4)mTORC1 modulates expression and secretion of Cxcl9 via regulation of expression and nuclear translocation of STAT1.(5)Cxcl9 content is increased in mice with postmenopausal osteoporosis,which is related to the reduced blood vessels and bone mass in mice.Reduction of Cxcl9 cause elevated angiogenesis in bone and increased bone mass in mice.mTORC1/Cxcl9 is expected to be a new target for treatment of post-menopausal osteoporosis.