SMOC2 Is Involved In The Composition Of The Extracellular Matrix Of The Cartilage

[Background]Articular cartilage is a highly specialized tissue covering the end of bones which enables a stable and smooth movement of the joint surfaces and,equally important,the dissipation of mechanical load under physiological conditions.The extracellular matrix(ECM)of the cartilage mainly consists of hyaline cartilage,a high collagen Ⅱcontent and rich proteoglycan matrix,such as aggrecan,laminins,fibronectin and so on.SMOC2(SPARC related modular calcium binding 2)is located on human chromosome 6q27,and encodes a secreted calcium-binding protein from the BM-40/SPARC/osteonectin family of secreted matricellular proteins.Many members of the SPARC protein family have been associated with diseases of the skeletal system.SMOC1,one of the SPARC family and highly homologous to SMOC2,has been associated with osteoporosis,shortening of the tibia and femur,abnormal limb development by disturbing the BMP signaling pathway.SMOC2 has been involved in the process of skeletal development.Mutations of SMOC2 gene can lead to abnormal tooth development and abnormalities of skull,and rheumatoid arthritis.Overexpressing SMOC2 in osteoprogenitor MC3T3-E1 cells inhibits osteogenic differentiation and extracellular matrix mineralization.Moreover,SMOC2 is involved in the fibrosis progression of lung,liver and kidney.The knockout of the SMOC2 gene can alleviate the progression of fibrosis and reduce the inflammatory response by inactivating NF-κB pathway.[objective]In view of the above background,while the important role of SMOC2 in skeletal development confirmed,we will aim to investigate the interaction between SMOC2 and other components the extracellular matrix of the cartilage,to analyze the relationship between SMOC2 and BMP pathway.[Materials and Methods]1.The knee joints from 21-day wild-type mouse were obtained.The extracted tissue proteins were used for Western Blot to detect the expression of SMOC2 protein in various tissues.The paraffin sections of knee joints were used for immunohistochemical staining to detect the expression of SMOC2 in the knee joints.Paraffin sections of knee joints were performed with immunofluorescence staining to determine whether SMOC2 colocalized with other cartilage extracellular matrix proteins.2.HEK293T cells were stably transfected with the human SMOC2-MYC lentivirus.Then the expression vectors of other cartilage extracellular matrix proteins(such as BGN,FMOD,COL9A1,etc.)were transfected with PEI into the stable overexpressing SMOC2-WT-MYC cells.The PLA assay and CoIP assay were utilized to analyze whether SMOC2 bound with these proteins.3.Lentiviral vectors containing only the EC domain or the FTST domain(without the EC domain)of SMOC2 were constructed and were stably transfected into HEK293T cells respectively.After being cotransfected with other ECM genes expressing vector,the domain of the SMOC2 binding with other cartilage ECM proteins was be determined by using The PLA assay and CoIP assay.4.Western Blot was carried out using total proteins extracted from the stable overexpressing SMOC2-WT-MYC cells in order to analyze the phosphorylation of SMAD1/5/9.Activated BMP receptors expressing vector(CaALK2,CaALK3 or CaALK6)was transfected into the above cell lines to analyze the phosphorylation of SMAD1/5/9.After HEK293T cells were stimulated with exogenous rhSMOC2,the total protein was extracted by RIPA buffer and the phosphorylation of SMAD 1/5/9 was analyzed by Western Blot.By this way,the effect and the mechanism of exogenous rhSMOC2 on BMP pathway was determined.[Rresults]1.By using western Blot and immunohistochemical staining,we found that SMOC2 was widely expressed in mouse tissues and highly expressed in the proliferative zone of epiphyseal growth plate of knee joints and articular cartilages.By immunofluorescence staining,we found that SMOC2 and BGN or FMOD co-localized in and articular cartilages.2.In the HEK293T cells co-overexpressed SMOC2 and other ECM gene,the interaction between SMOC2 and BGN,FMOD,COL9A1 or COMP respectively was identified by the PLA assay,and was further confirmed by CoIP assay.The binding between SMOC2 and HSPG2 was demonstrated by using PLA assay in the stable expression of SMOC2 C28/I2 cells.3.CoIP assay of HEK293T cell lines only highly expressed EC domain or FTST domain and one of BGN,FMOD or COL9A1 found that SMOC2 binds to BGN or FMOD through FTST domain and SMOC2 binds to COL9A1 through EC domain.4.Overexpression of SMOC2 in HEK293T cells blocked the phosphorylation of SMAD1/5/9.It suggested that SMOC2 was an tagonizes of BMP signaling pathway.Cotransfecting with activated BMP receptors(CaALK2,CaALK3 or CaALK6)caused phosphorylation of Smad 1/5/9 and inhibition of BMP signaling pathway by SMOC2 was rescued.Meanwhile,Serum-starved HEK293T cells were incubated with rhSMOC2,combined with the addition of BMP2 or not.The results of Western Blot showed that low concentrations of rhSMOC2 resulted SMAD1/5/9 phosphorylation,while high concentrations of rhSMOC2 blocked SMAD 1/5/9 phosphorylation.[Conclusions]1.SMOC2 is involved in the composition of the extracellular matrix of the cartilage by interacting with other ECM components.SMOC2 binds to MATN3,COMP or HSPG2.SMOC2 binds to BGN or FMOD through the FTST domain and binds to COL9A1 through the EC domain.2.In HEK293T cells,overexpression of SMOC2 blocked the phosphorylation of SMAD1/5/9 suggested that BMP pathway was inhibited.In the presence of a constitutively active BMP receptor,SMOC2 could not antagonizes BMP activity.It indicated that SMOC2 might inhibit BMP pathway by competing to bind with BMP receptors,not act downstream the receptor.3.Low concentration of exogenous rhSMOC2 enhanced BMP2 pathway by competitive binding of BMP2 to other ECM proteins,thereby activating BMP pathway.High concentration of rhSMOC2 would directly bind to BMPRIB,thereby inhibiting the BMP pathway.