The Role Of BMPR1A Signaling Pathway In Controlling Osteroblasts Quantity And Terminal Differentiation

Backgrounds:Bone morphogenetic proteins(BMPs)were discovered and named by Marshall Urist,who initially identified the ability of an unknown factor in bone to induce ectopic bones in muscle.Afterwards BMPs were confirmed to be involved in many organs’ formation and development,and played an important role in many kinds of cells.Binding of BMPs to their receptor complexes may trigger Smad or non-Smad signaling cascades.In recent years,researches about BMPs signaling pathway in different animal models,different cells and different timing of manipulation shows that it could led to the change of bone mass,but the certain mechanism of these change was unknown and was quite controversial.Moreover the majority of these researches focused on the change of bone mass,seldom attention was paid to bone qualitys.BMP type I receptors,type IA(BMPR1A or ALK3)as well as other BMPs and BMPRII were members of the transforming growth factor-b(TGF-b)superfamily.BMPR1 A was a powerful receptor of BMP2 and BMP4,and it could directly impact the function of BMP2 and BMP4 in the signaling pathway.BMP2 was a bone inducing factor which could induce mesenchymal stem cells hyperplasia,differentiation into osteoblasts and chondrocytes.BMP2 also participated in osteoclasts formation,differentiation,maturity and activation directly or indirectly;BMP4 was a key factor of ossification which could lead to heterotopic ossification early lesions as well as stimulate COL1,ALP and OCN expression and induce mesenchymal stem cells differentiate into chondrocytes and facilitate chondrocytes maturity.Osteroblasts were differentiated form mesenchymal stem cells and they mainly participated in bone formation and bone restoration procedure.Vibrant osteroblasts were located near the surface of bone formation area.Mature osteroblasts compounded and secreted extracellular matrix proteins,including all kinds of collagens and non-collagenous proteins,formed osteoid and initiated bone formation.In adults,bone formation was under strict control to maintain the bone homeostasis.Osteroblasts may undergo several processes as proliferation,maturity of extracellular matrix,extracellular matrix mineralization and osteroblasts apoptosis,and many factors could regulate these processes to have a influence in bone formation.In consideration of the importance of the number and activity of osteroblasts in bone homeostasis and BMP signaling pathway playing key role in bone formation and bone resorption,our first aim was to observe the effect of conditional knockout of BMPR1 A in osteroblasts on osteroblasts numbers and activity,as well as the change on bone mass.Secondly,we aimed to observe the influence of BMP Signaling Pathway in osteroblasts terminal differentiation,the change in bone mineralization and collagen synthesis,and the possible mechanisms.Methods:Part I: the effect of Deletion of BMPR1 A in osteoblasts on controlling bone mass1.Set up osteoblasts BMPR1 A knockout mice model and observed for their body size.The body weight and length of tibia were measured;2.Histological sections of mice stainning with H&E,X-Ray and Micro-CT were applied to observe bone structure and bone mass.3.TRAP staining was used to assay osteoclast activities,Brd U staining was used to observe osteoblasts proliferation,TUNEL was used assay to evaluate cell apoptosis.Part II: the role of Deletion of BMPR1 A in osteoblasts in osteoblasts terminal differentiation1.Von Kossa staining,Masson staining and Sirius red staining were performed on skeleton sections with half decalcification to analysis bone mineralization and the content of collagen.2.Immunohistochemisty were used to analysis osteoblastogenesis and mineralization level.3.The concentrations of calcium and phosphate in serum were measured.4.Scanning electron microscope was used to obsereve bone mass in processed lower limbs and teeth of mice.5.Cell culture system was applied to evaluate osteocytes mineralization with Alizarin red staining to analysis cell cycle with Flow Cytometer,to analysis osteoblastogenesis and osteoclastogenesis with reverse transcription PCR(RT-PCR).Results:1.Disruption of BMPR1 A in osteoblasts increased bone mass which was mainly affacted by enhanced osteogenic function and subdued osteoclastic function(1)Compared with 3.2Cre and DMP1 Cre could confirm that 3.2Cre specificity express in osteoblasts.(2)The BMPR1 A knockout mice(c KO mice)showed no obvious difference in development,but they showed decreased body weight and shorten lower limbs.(3)X-Ray,Micro-CT and H&E staining results showed cancellous bone volume significantly increased in c KO mice.(4)Osteoblasts were more active in c KO mice.Expression of osteoblast marker Run X2 and Osterix was increased,as well as the proliferation of osteoblasts,and the apoptosis rate of osteoblast was decreased.(5)Osteoclasts activity was decreased in c KO mice.There was less osteoclasts in c KO mice and the ratio of RANKL/OPG was reduced.2.The terminal differentiation of osteoblast was obstructed in c KO mice(1)The mineralization level of osteoblasts was decreased in c KO mice both in vivo and in vitro.(2)Collagen content of c KO mice was reduced and more type III collagen was detected,and also the arrangement of those collagen was disorganized compared with the controls.(3)Immunohistochemistry staining showed the factor of mineralization inhibition OPN and FGF23 expression and the concentration of serum calcium increased.(4)Cell cycle analysis showed more osteoblasts was restraining at G2/M.Conclusions:1.BMPR1 A knockout in osteoblasts could increase the number of osteoblasts,as well as increase bone mass.The BMP singling pathway played an important role in maintaining bone volme mainly by controlling the number of osteroblasts.2.BMPR1 A knockout in osteoblasts impairs the terminal differentiation in osteoblasts,decresed mineralization level,reduced collagen synthesis,resulted in low bone quality.