Preliminary Study On The Role And Mechanism Of Smoc2 Gene In Bone Development Of Mice

BackgroundSMOC2(SPARC-related modular calcium-binding protein 2)is a secretory calcium-binding extracellular matrix protein,which belongs to the BM-40 protein family,also known as secreted protein acidic and rich in cysteines(SPARC).The SMOC2 gene is located at 6q27,with 232kb of genomic DNA in size,consisting of 13 exons and encoding a 446 amino acids protein.SMOC2 protein was first identified in the extracellular matrix of articular cartilage.SMOC2 plays an important role in the development of bones and teeth It has been found that mutation of SMOC2 gene cause autosomal recessive dental dysplasia with major microdontia,oligodontia.Craniofacial skeletal and dental dysplasia were manifested in smoc2 knockout zebrafish.SMOC2 was expressed not only in mouse costal cartilage,but also in rat proximal tibial growth plate chondrocytes.SMOC2 is significantly expressed in the anterior and posterior limbs and parotid arch of embryonic mice.Smoc2 deficiency could ameliorate kidney,lung and liver fibrosis through inhibiting TGF-?/NF-kB pathway.Smoc2 knock-out mice showed reduced tooth size,altered enamel prism patterning,and spontaneous age-induced periodontal bone and root lossMultiple Epiphyseal Dysplasia(MED)is inherited skeletal dysplasia which affects the development of long bone epiphysis.It is a rare disease with incidence of 1/10 000 to 1/20 000.The onset of the disease begins in early childhood with joint pain and stiffness,mild to moderate short stature,and early onset osteoarthritis.Radiological examination showed femoral epiphyseal dysplasia in childhood,knee joint dysplasia,short fingers,scoliosis and irregular endplate in the spine.Adults also have flattened femoral condyle,depression of the superficial trochlear groove and lateral tibial plateau,and decreased sphericity of the humerus and femoral head.The predominant pathological feature of MED is the delayed and irregular ossification of numerous epiphyses.,which eventually lead to the destruction of articular cartilage and the early onset of osteoarthritis.So far,8 genes responsible for MED were identified.Studies have shown that MED is usually caused by abnormal osteogenesis in the epiphyseal growth plate,such as decreased proliferation or abnormal differentiation,abnormal apoptosis of chondrocytes in the hypertrophic area or delayed calcification.Our group found that the SMOC2 p.L359R mutation is a pathogenic mutation of an autosomal dominant MED family characterized by early-onset osteoarthritis and short stature.p.L359R mutation knock-in mice showed small body in size coinciding with the affected.Experimental results in vitro suggested that mutant SMOC2 protein decreased the ability of binding to HSPG and COL9 matrix proteins,and increasing dissociative mutant SMOC2 antagonized the BMP-SMAD1/5/9 pathway by competitive binding to BMPR1 receptors with BMP2.In view of the fact that the mouse Smoc2 gene is located on chromosome 17,the total length of the genome is about 125.3 kb,there are 13 exons,which are highly homologous to human SMOC2 gene(84.19%homology),and its coding protein is consistent with human SMOC2 protein structure.Therefore,the construction of Smoc2 gene knockout mouse model is one of the important strategies to study the function of the gene.Objectives1.To construct Smoc2 gene knockout mice;2.To observe the abnormal phenotype of Smoc2 knockout mice;3.To explore the role of Smoc2 gene underlying the bone developmentMethods1.Smoc2 gene knockout mice was constructed by using CRISPR/Cas9 technology.The knockout efficiency was tested,and the fertility of mice was counted.2.The body length and weight of mice were measured from P8 and the growth curve of mice was drawn.The body length of P30 mice was furtherly measured by using X-ray.The whole skeleton of 6-month-old mice was stained with Alcian blue and Alizarin red,and the body length was measured with Vernier caliper.The lengths of skull,sternum,scapula,humerus,radius,femur and tibia were measured respectively.The morphology of knee joint,distal femur and proximal tibia was detected by micro-CT,and the changes of bone mineral density and bone mass were analyzed.Paraffin sections of kidney,lung and heart of mice were prepared,and their morphology was observed by hematoxylin-eosin staining.3.Double staining of skeleton with Alcian blue and alizarin red was performed on P0 and E18.5 mice.4.After the paraffin sections of mouse sternum were prepared,the development of sternum was observed by hematoxylin-eosin staining and Safranine/fast green staining.5.After preparing the paraffin sections of mouse tibia,the morphological development of epiphyseal growth plate was observed by hematoxylin-eosin staining and Safranine/fast green staining.The expression of markers related to proliferation,hypertrophy and apoptosis of chondrocytes were detected by immunostaining,real-time quantitative PCR and Western-BlotResultsThe Smoc2 knockout mice were constructed by CRISPR/Cas9 technology,and the knockout efficiency was tested at the RNA level and protein level by reverse transcription PCR and Western Blot.The results showed that the Smoc2 knockout mice were successfully constructed.The fertility of the Smoc2 knockout mice conformed to Mendelian inheritance.The growth curve observed that the Smoc2 knockout mice were shorter and lighter than wild-type mice.Compared with wild-type mice,the average length of P30 knockout mice was reduced by 8.68%,and the average body weight was reduced by 12.37%.In 6-month-old mice,the average length of the sternum was reduced by 10.54%,the average length of the skull was reduced by 6.70%,the average length of the scapula was reduced by 6.54%,the average length of the humerus was reduced by 10.67%,and the average length of the tibia was reduced by 5.68%.It shows that this change occurs during embryonic development and will continue to adulthood.At the same time,we firstly observed the osteoporosis in the knee joint of 6-month-old mice,including bone loss and reduction of trabecular boneThe whole skeleton from P0 and E18.5 mice were staining with Alcian blue and alizarin red,and results showed that the osteogenesis of spinal vertebrae was smaller and the osteogenic differentiation of sternum,phalanges and phalanges was delayed.To analyze the growth plate of proximal tibial epiphysis form P0 mice by using H&E staining,the reduced proliferative zones,disorganized proliferative chondrocytes,and expanded hypertrophic zones in mutant mouse tibial growth platesit were detected.These suggest that chondrocytes differentiate into hypertrophic state in advance,which affected the growth of the growth plate.To determine the mechanism of the reduction of proliferative zones and the expansion of hypertrophic zones in mutant mouse tibial growth plates,we analyzed the proliferation,differentiation and apopotosis of chondrocytes in growth plate from PO mice.The low proliferating cell nuclear antigen(PCNA),a marker of proliferation was observed.The results of immunohistochemical staining of the growth plate showed the ignificantly increased expression of CollagenX,Aggrecan and RUNX2 proteins,and significantly expanded expression region of IHH protein in the growth plate of Smoc2 knockout mice were.The results of real-time quantitative PCR showed that the mRNA transcription of CollagenX,Aggrecan,Runx2 and Ihh were significantly up-regulated in growth plate of knockout mice.Western blot results showed that the expression of CollagenX protein,Aggrecan and IHH protein increased.CollagenX and RUNX2 are markers of hypertrophic chondrocytes and IHH is a marker of prehypertrophic chondrocytes.The high expression level of these markers can indicate the differentiation level of chondrocytes.VEGF is a marker of chondrocyte apoptosis in the growth plate.The results of immunohistochemistry,real-time quantitative PCR and Western blot showed that Vegf expression decreased,which suggested that the differentiation and apoptosis of chondrocytes are abnormal in the growth plate of proximal tibia.PTHrP-IHH negative feedback regulatory loop plays an important role in chondrocyte differentiation.PTHrP can negatively regulate chondrocyte maturation by increasing the activity of SOX9,a transcription factor that inhibits chondrocyte hypertrophy.In our study,we found that the results of immunohistochemical staining showed that the contents of SOX9 and PTHrP increased,the results of real-time quantitative PCR showed that Pthrp transcription increased significantly,and the results of Western blot showed that the expression of SOX9 protein increased significantly.It is suspected that the increase of Ihh activates the negative feedback regulation of Pthrp to reduce Ihh.ALP is a marker of osteoblast maturation and a marker of bone metabolism.The results of immunohistochemistry,real-time quantitative PCR and Westernblot showed that the content of ALP decreased.The decreased ALP indicates that osteoblast maturation may be impaired,or fewer osteoblasts,which is consistent with the delayed osteogenic differentiation observed in Alcian blue-alizarin red double staining.These findings confirm that Smoc2 gene plays an important role in bone growth and developmentInnovation and significanceIn this study,Smoc2 knockout mice were successfully constructed,and the role of Smoc2 gene in growth and development was observed wholy and partly.We found firstly that smoc2 gene knockout resulted in low weight,shortened body length,delayed bone osteogenesis and abnormal growth plate cartilage differentiation.We found firstly that Smoc2 gene knockout result in osteoporosis in mice.