Systematic Analysis And Charactrazation Of TGFβ Receptors In Osteogenic Differentiation Induced By Bmp6

BACKGROUND: Bone morphogenetic protein(BMPs) family ofcytokines comprises over20different ligands that belong to thetransforming growth factorβ(TGFβ) superfamily,except for BMP-1.BMPsare multi-functional growth factors determining embryonic mesodermdorsal–ventral (DV) axis development, maintenance and regeneration oftissues and organs,playing an important role in proliferation, differentiation,maintain phenotype and apoptosis of adult cells. The most biologic activityof BMPs is to induce stem cells to osteoblastic differentiation, which areutilized extensively in bone tissue engineering. Among those, BMP6is asignificant osendochondrale effect factor.As a secreted protein, the initialstep of signal transduction is to bind of the ligand to distinct receptors,forming the ligand–receptor oligomerization complex and subsequentlyphosphorylate transcriptional factors to regulate downstream targets.TGFβreceptors are transmembrane serine/threonine kinase receptors, composedof type I and II subtypes. The receptors contain an extracellular ligandbinding domain,an transmembrane domain and an intracellular kinase domain,so they are bridges to carry on in BMP6signal pathway. To date,thefunction mechanisms and binding receptors of BMP6maintain no overalland system analysis.OBJECTIVE: To characterize of TGFβ receptors in osteogenicdifferentiation induced by BMP6,and provide experiment evidence ofreceptors to the further gene function.METHODS:1mesenchymal stem cell C3H10, mouse myoblastic cellC2C12and mouse osteoblastic cell MC3T3were co-stimulated withdn-receptor adenovirus and BMP6conditional medium respectively,luciferase reportor assay, ALP staining and ALP activity quantity weredetected to initial screen the dn-receptors which have stronger affinity withBMP6to inhibit osteogenesis.2the initial screening dn-rceptors were further identified,mineralization was detected by Alizarin Red staining in C3H10, C2C12and MC3T3cells,special osteogenesis marker OPN and OC expressionwere detected by immunohistochemistry(IHC),Smad6and Smad7expression were observed by real time PCR, intranuclear transferation ofSmad1/5/8was observed by laser scanning confocal microscope(LSCM).The C2C12cells treated with dn-rceptors and BMP6adenovirus wasinjected into nude mice subcutaneously to observe ectopic osteogeneticmass size and calcification degree by histologic staining.3.sh-BMPRⅡ,sh-ActRⅡ,sh-ActR Ⅱ B and negative control(HK) were constructed, identified and selected most of the sequence ofinterference..4. Endogenous expression level of TGFβ receptor in C3H10, C2C12and MC3T3cell line were detected by real time PCR.C3H10cell wastreated with si-ALK2,si-ALK3,sh-BMPRⅡ,sh-ActR Ⅱ and HK,ALPstaining,ALP activity quantity,ALP mRNA expression were detected toanalysis the inhibition of osteogenetic function after interferingendogenous receptor expression.RESULTS:1C3H10, C2C12and MC3T3cells co-stimulated with11distinct dn-receptors and BMP6respectively,showed luciferase activity byluciferase reportor assay induced by BMP6was inhibited, ALP activity byALP staining and quantity was decreased, when dn-ALK2,dn-ALK3,dn-ALK6,dn-BMPRⅡ,dn-ActRⅡ or dn-ActRⅡB treated.2ALP activity was decreased in a dose-dependent manner infectedwith dn-ALK2,dn-ALK3,dn-BMPRⅡ,dn-ActR Ⅱ. Moreover,dn-ALK2,dn-ALK3, dn-BMPRⅡ, dn-ActRⅡ and dn-ActRⅡB could reducemineralization by Alizarin Red staining,decrease expression of OPN andOC by IHC, inhibit expression of Smad6mRNA and Smad7mRNA byreal time PCR,decrease intranuclear transferation of Smad1/5/8by LSCM.In vivo, subcutaneous osteogenetic mass size was smaller than the RFPcontrol, HE staining,alcian blue staining and Mason trichrome stainingshowed that neo-osteogenetic cells decrease and calcification degree reduce.3sh-BMPRⅡ,sh-ActRⅡ,sh-ActR Ⅱ B and negative control(HK)were constructed of three different successfully, identified right by enzymeelectrophoresis, and selected which was the strongest interferencesequence by real time PCR to the next.4. Endogenous expression level of ALK6and ActR ⅡB in C3H10,C2C12and MC3T3cell line was little detected by real time PCR.C3H10cell was treated with si-ALK2,si-ALK3,sh-BMPRⅡ,sh-ActR Ⅱand HKrespectively,ALP staining and ALP activity quantity showed RNAidecrease osteogenetic function induced by BMP6,ALP mRNA expressiondecreased by RT-PCR.CONCLUSIONS: BMP6is the important acting factors inducingosteogenic differentiation of stem cells,which can bind to ALK2,ALK3,BMPRⅡ and ActRII receptors in the membrane of C3H10, C2C12andMC3T3cell line, activation of Smad transcription factors to play aregulatory function of bone. ALK6and ActRⅡ B havestrong affinity withBMP6,but they may be not physiological receptors during osteogenicdifferentiation induced by BMP6.