Role Of Core Binding Factor Beta In Bone And Cartilage Development

Core Binding Factor alpha1(Cbfa1), also called Runx2, plays a centralrole in osteoblastogenesis and skeleton development. Mutations of Runx2lead to completely lose of ossification, and skeletal development isobviously inhibited in Runx2heterozygotes, the skeletal developmentdefects of which is similar like human disease Cleidocranial Dysplasia(CCD), Runx2mutations have been detected in some CCD patients.Core binding factor beta (Cbfb) forms heterodimer with Cbfa, whichcan stabilize Cbfa’s interaction with DNA and enhance thetranscriptional activity. Cbfb knock out mosue die at midgestationbecause of failure of fetal liver hematopoiesis. Cbfb–/–Tg and CbfbGFP/GFPmouse, which rescued fetal liver hematopoiesis, can survive before birth,and show skeletal development defects, which is similar to Runx2mutatemouse. Some CCD patients, those who show normal Runx2, showdecreased Cbfb expression, this indicates that Cbfb is candidate gene for CCD.All of these transgenic mouse die at birth because of respiratorydisturbance caused by serious ossification defects, which make itimpossible to detect Cbfb role in postnatal bone development, also thedetailed mechanism, and skeletal defects of mature Cbfb knock outmouse. To study the mechanism of Cbfb in postnatal skeletaldevelopment and skeletal defects of mature Cbfb mutate mouse canprovide new approach to dignose and cure CCD.Objective: To knock out Cbfb in mesenchymal cells and chondrocytesrespectively with Cre/LoxP conditional knock out system, avoiding thedeath of transgenic mouse after birth, and build a new CCD mouse model;to study Cbfb function in postnatal skeletal development and the skeletalphenoltype of mature Cbfb conditional knock out mouse, to furtherinvestigate Cbfb role in CCD.Method: Generate mesenchymal and chondrocyte specific Cbfbconditional knock out mouse model by crossing Cbfb-floxed mouse withthe mosue which can express Cre recombinase driven by Prx1andCol2a1promoter respectively. Study skeletal development defects ofnewborn and mature Cbfb mutant mouse, investigate mechanism of Cbfbin regulating differentiation and maturation of osteoblast and chondrocyte.Culture mesenchymal specific Cbfb conditional knock out osteoblast in vitro, detect differentiation and maturation of osteoblast.Result: Cbfbf/fPrx1-Cre mouse can survive after birth and growmature, exhibiting CCD-like skeletal defects, ossification of calvarial isdelayed, clavicle is very small, shortening of limbs become more obviousas the mouse grow mature. Endochondral ossification is delayed inCbfbf/fPrx1-Cre mouse, proliferation and maturation of chondrocyte isinhibited. Cbfbf/fPrx1-Cre mouse mouse show lower expression of Ihh,Cyclin D1, ColX, and higher expression of PTHrP-R in chondrocyte.Osteoblast function is weakened with lower expression of osteopontinand osteocalcin, primary spongiosa formation is retarded, which is verysevere in mature mouse. In vitro osteoblastogenesis is impaired in Cbfbf/fPrx1-Cre mesenchymal cells, which also show lower osteoblast function.Cbfbf/fCol2a1-Cre mouse also can survive after birth, endochondralossification is inhibited, proliferation and hypertrophy is retarded,intramembranous ossification is normal.Conclusion: We build a new CCD mosue model by mesenchymal andchondrocyte specific Cbfb conditional knock out, which can survive afterbirth, Cbfb play important role in osteoblastogenesis and regulatingosteoblast function, and Cbfb can regulate chondrocyte proliferation andmaturation by regulating expression of Ihh, Cyclin D1and PTHrP-R.Mutation of Cbfb is an important reason of CCD.