Mechanism Of FAM20 Family Proteins Regulating The Development Of Articular Cartilage In Mice

Background and ObjectivesFAM 20(Family with Sequence Similarity 20)protein is a new kinase family that phosphorylates secreted protein and proteoglycan,including FAM20A,FAM20B and FAM20C,which play an important role in the development of embryo.Fam20C is an important protein kinase in the process of protein phosphorylation,and its deletion will cause the disorder of osteogenesis and biomineralization.Fam20Bis a xylose kinase that regulates glycosaminoglycan assembly during glycosylation of extracellular matrix proteins,and its deletion will cause the decrease of extracellular matrix proteoglycan,thus affecting cell function.At present,in the research of developmental biology,the influence of Fam20 protein family on biological development has been paid more and more attention,and the research on the relationship between Fam20Band Fam20C and long bone joint development has made some progress,all of which play biological functions indirectly in tissues and cells through phosphorylation of substrate proteins.Fam20C has been proved to be the pathogenic gene of Raine syndrome,which is characterized by fatal sclerosing skeletal dysplasia and non-fatal hypophosphatemia syndrome.Scholars have proved that Fam20C can regulate the formation of enamel and dentin and promote the mineralization of bone.However,the role of Fam20C in the development of articular cartilage has not been studied in detail.At the same time,as a pseudo-kinase that promotes the function of Fam20C,some studies suggest that Fam20A is not an important factor in promoting the function of Fam20C in bone formation,however,whether Fam20A is important in promoting the function of Fam20C in cartilage development has never been mentioned.Therefore,in the first part of this study,the phenotype of articular cartilage was observed through the mouse model of Fam20C mutation at different sites,and the possible role of Fam20C in cartilage development was preliminarily revealed.At the same time,the long bone joint and temporomandibular joint were compared to discuss whether their functions in primary cartilage and secondary cartilage are the same.At last,by observing Wnt1-cre;Fam20a^f/f,Osr2-cre;Fam20a^f/fand Col1-cre;Fam20a^f/f.The articular cartilage phenotype of Fam20a^f/fmice to explore whether Fam20A can promote Fam20C in articular cartilage development.In recent years,the role of Fam20Bin development has gradually become a research hotspot.It has been found that FAM20B can affect the development of various tissues such as the spine,long bones and joints through its proteoglycan assembly.Some scholars have found that Fam20B-dependent proteoglycan can influence the process of endochondral ossification after long bone joint delivery through Wnt and other signaling pathways.However,although the temporomandibular joint of craniofacial region is a synovial joint,it is different from the long bone joints of limbs.Because the temporomandibular joint develops at the late embryonic stage,its cells come from the developed mandibular periosteum and lack of secondary ossification center,condylar cartilage is considered as secondary cartilage,and its development process is quite different from that of primary cartilage.Therefore,the second part of this study passed Wnt1-cre;The Fam20B^f/fmice specifically knock out Fam20Bin the cells derived from the neural crest of road.by observing its phenotype and the changes of chondrocyte proliferation and differentiation,the role of Fam20Bin the development of temporomandibular joint was clarified,and the different biological functions of proteoglycans assembled by Fam20Bin secondary cartilage and primary cartilage were discussed.In the third part of the study,this study observed that the expression pattern Ihh,BMP and other signal pathways are changed in Wnt1-cre;The Fam20B^f/fmice reveals the possible ways in which the proteoglycan assembled by Fam20Baffects the development of condyle and joint disc of temporomandibular joint,and further improves the molecular mechanism of the development of temporomandibular joint.To sum up,this study will be divided into three parts to explore the influence of Fam20 protein family on the development mechanism of articular cartilage.The first part will focus on analyzing the phenotypic changes of long articular cartilage and temporomandibular joint cartilage in mice when Fam20C and Fam20A are conditionally knocked out.The second part focuses on the analysis of phenotypic changes of temporomandibular joint in Wnt1-cre;Fam20B^f/fmice,and the role of proteoglycan assembled by Fam20Bin differentiation and maturation of temporomandibular joint disc and condylar cartilage were discussed.In the third part,by observing the changes of signal factors in Wnt1-cre;Fam20B^f/f mice were studied to explore the mechanism of Fam20Bassembled proteoglycan affecting the differentiation and maturation of temporomandibular joint disc and condylar cartilage.Methods(1)mating Fam20C-D437N mice with Wnt1-cre mice can obtain Wnt1-cre;Fam20C-D437N mice.Wnt1-cre can be obtained by mating Fam20C-G374R mice with Wnt1-cre mice.Fam20C-G374R mice.At first,Fam20a ^f/+male was crossed with Wnt1-cre,Osr2-cre or 2.3Kb Col1a1-cre female to obtain Wnt1-cre;Fam20a ^f/+,Osr2-cre;Fam20a^f/+and 2.3Kb Col1a1-cre;Fam20a ^f/+mice.Then,Wnt1-cre;Fam20a ^f/+,Osr2-cre;Fam20a^f/+and 2.3Kb Col1a1-cre;Fam20a ^f/+mice was obtained by mating Fam20a ^f/+mice with Fam20a^f/f mice to get Wnt1-cre;Fam20a ^f/f,Osr2-cre;Fam20a^f/f and 2.3Kb Col1a1-cre;Fam20a ^f/fmice.The Fam20B^f/fmice were mixed with Wnt1-cre mice.Wnt1-cre can be obtained by mating Fam20B^f/-mice.Embryo of Fam20B^f/fmice.The Fam20B^f/fmice were mated with p Mes-Ihh mice to obtain Fam20B^f/-;p Mes-Ihh mice.In the same way,Fam20B^f/-;p Mes-Ihh and Wnt1-cre;Fam20B^f/+can be obtained by mating in a cage to get Wnt1-cre;Fam20B^f/f mice and Wnt1-cre;p Mes-Ihh mice.(2)X-gal/Lac Z staining was used to observe the expression pattern of Fam20A;(3)Masson staining was used to observe the histological morphology of mice.(4)Alizarin red/arsine blue staining was used to observe the structural changes of bone cartilage in mice.(5)X-ray photographs were used to observe the changes of bone morphology and density.(6)The mineralization degree of tissues was observed by Von-Kossa staining.(7)The content and distribution of acidic mucopolysaccharide in tissues were observed by saffron-solid green staining.(8)IHC method was used to detect the changes and distribution of Col2,Col10,Aggrecan,MMP-13,p-smad1/5/8,p-Erk,osterix,Sox9 and other proteins in temporomandibular joint tissues of mice.(9)Brd U labeling method was used to detect the proliferation of mouse tissue cells.(10)TUNEL method was used to detect apoptosis in mouse tissues.(11)the m RNA expression and distribution of Ihh,PThrp,Gli2 and Pthc1in mouse condyle were detected by in situ hybridization.(12)measurement and counting are analyzed by Image J software;Data drawing(histogram)is carried out by Graph Pad Prism 5 software.The comparison of data between samples was statistically analyzed by independent sample t-test,with p<0.05(*)as the standard with statistical differences,and p<0.01(**)and p<0.001(***)as the highly statistical differences.Results1.the influence of FAM20C and FAM20A on the development of bone and joint after birth(1)Col1-cre;Fam20C^f/fmice affected the differentiation process of epiphysealchondrocytes during the development of long bone joints after birth,and a large number of chondrocytes stayed in the stage of hypertrophic chondrocytes,which inhibited the mineralization of epiphyseal cartilage to form new trabeculae.Col1-cre;Fam20C^f/f-D446N mice and Col1-cre;Fam20C^f/f-D437N mice affected the differentiation process of epiphyseal chondrocytes during the development of long bone joints after birth.A large number of chondrocytes stayed in the stage of hypertrophic chondrocytes,but the mineralization process of secondary ossification center was promoted,and short but dense trabeculae were formed in cancellous bone.Col1-cre;Fam20C^f/f-G374R mice showed abnormal epiphyseal cartilage morphology during the development of long bone joints after birth,and a large number of irregular chondrocytes invaded the osteogenic area.(2)Col1-cre;Fam20C^f/fmice and Col1-cre;Fam20C-D446N The phenotype of mice in condylar cartilage of temporomandibular joint after birth is similar.Compared with wild-type mice,there are more hypertrophic chondrocytes and less ossification under condyle.In Col1-cre;Fam20C-D437N mice,a large number of heterogenous chondrocytes proliferated abnormally under the condylar periosteum in the condylar cartilage of temporomandibular joint after birth.In the temporomandibular joint of Col1-cre;Fam20C-G374R mice after birth,the condyle shape is narrow,and the hypertrophic chondrocytes proliferate excessively and invade the osteogenic area.(3)Fam20A existed in bone marrow and epiphysis of long bone joints of mice after birth,but was not expressed in cartilage.Fam20A is ubiquitous in the mandible of postnatal mice,but it does not appear in molars,incisor nipples and cartilage.In addition,the distribution of Fam20A in long bone and mandible decreased with the development of mice.(4)Knocking out Fam20A in mesenchyme will not cause abnormal development of long bone joint,mandible and temporomandibular joint.2.Knocking out Fam20Bin neural crest mesenchymal cells inhibited the phenotypic analysis of the development of condylar cartilage and articular disc of TMJ.(1)Compared with wild-type mice,In the condylar cartilage of temporomandibular joint of Wnt1-cre;Fam20B^f/fmice,there are fewer hypertrophic chondrocytes,and immature chondrocytes and mature soft cells are densely arranged and disordered,accounting for a relatively high proportion of condylar chondrocytes E15.5 The polysaccharide anion in condylar cartilage mesenchymal decreased,the mineralization ability of hypertrophic chondrocytes weakened,and the ossification of bone collar was abnormal.(2)Compared with wild-type mice,Temporomandibular joint of Wnt1-cre;Fam20B^f/fmice can't form normal joint disc structure,but only loose joint disc structure with thin shape and irregular arrangement of internal cells.At the same time,there are a large number of cell residues in the lower articular cavity,which can not complete the cavitation process of the articular cavity,and the disc-like structure can not be separated from the condylar head at last.(3)Compared with wild-type mice,In Wnt1-cre;Fam20B^f/fmice,the content of proteoglycan in condylar cartilage mesenchyme of temporomandibular joint decreased sharply,and there was no Aggrecan in fibrocartilage layer,immature chondrocyte layer and mature chondrocyte layer at the top of condyle,while matrix metalloproteinase 13 in extracellular matrix of condylar cartilage of temporomandibular joint increased significantly in Wnt1-cre;Fam20B^f/fmice.(4)Compared with wild-type mice,Col2 and Col10 in condylar cartilage of Wnt1-cre;Fam20B^f/fmice decreased significantly,and the differentiation process of condylar cartilage was inhibited(5)Compared with wild-type mice,In Wnt1-cre;Fam20B^f/fmice,the proliferation ability of condylar chondrocytes decreased,and the apoptosis signal at the junction of bone collar and hypertrophic chondrocytes increased.Compared with wild-type mice,the apoptotic signal of inferior articular cavity decreased.3.Knocking out Fam20Bin neural crest mesenchymal cells inhibits the development of condylar cartilage and articular disc of temporomandibular joint(1)The abnormal activation of Ihh signal in condyle of Wnt1-cre;Fam20B^f/fmice increased its expression range.compared with wild-type mice,Ptch1 and Gli2 were found in condylar cartilage of Wnt1-cre;Fam20B^f/fmice,it was obviously up-regulated like Ihh,but in wild-type mice,PThrp signal which should be positively activated by Ihh in fibrochondrocyte layer was in the expression of Wnt1-cre;Fam20B^f/fwas down-regulated obviously.This indicates that up-regulated Ihh factor plays its role in adjacent chondrocytes,but it cannot positively activate PTHr P signaling factor through remote signaling.(2)Over-expression of Ihh in neural crest mesenchymal cells did not appear the similar phenotype of Wnt1-cre;Fam20B^f/fmice,Wnt1-cre;p Mes-Ihh mice showed a large number of hypertrophy chondrocytes,increased condyle length and thickened articular disc.These results suggest that Ihh may play a positive role in the proliferation,differentiation and hypertrophy of chondrocytes in TMJ,which is different from Ihh in the osteogenesis of long bone cartilage.(3)At Wnt1-cre;Fam20B^f/fmouse condylar chondrocytes,BMP classical signaling pathway is abnormally activated in condylar cartilage mucous layer cells and perichondrium,suggesting that the differentiation pattern of condylar chondrocytes may be changed.And the expression of Sox9 in temporomandibular joint disc of Wnt1-cre;Fam20B^f/fmice decreased obviously,suggesting that the articular disc-like tissue formed in Wnt1-cre;Fam20B^f/fmice lacks fibrocartilage,so it cannot form real articular disc tissue.Conclusion(1)Gene mutations at different sites of Fam20C can cause different abnormal manifestations of chondrocytes,and Fam20c plays different functions in primary cartilage and secondary cartilage.Fam20A does not play a key role in the development of articular cartilage.(2)In Wnt1-cre;Fam20B^f/fmice,the loss of proteoglycan function delayed the differentiation of condylar chondrocytes.The articular disc can't be formed due to the lack of collagen fibers,and the apoptosis of inferior articular cavity is inhibited,which leads to the joint disc can't be separated.(3)In the process of condylar differentiation of temporomandibular joint,BMP and IHH signaling pathway act together to regulate the differentiation of condylar chondrocytes.However,the deficiency of proteoglycan inhibited Ihh remote regulation of PThrp,which resulted in the failure to form a complete joint disc tissue and affected the cavitation of the subarticular cavity.