Study Of The Role Of Metalloproteinase ADAMTS18 In SMG Development And Fibrosis Of Mice

There are three major salivary glands in human including submandibular gland(SMG),parotid gland and sublingual gland.Submandibular gland products and secretes about 65%of saliva.Submandibular gland hypofunciton results in hyposalivation,xerostomia,dental caries and peridentitis which influence a person's quality of life.Therefore,identification of key cellular and molecular mechanisms involved in SMG organogenesis and pathology is important for the development of novel strategies to prevent this disease.ADAMTS(A disintegrin and metalloproteinase with thrombospondin motifs)enzymes are a family of 19 secreted,multi-domain matrix-associated zinc metalloendopeptidases.ADAMTSs have diverse roles in pathological and physiological progression including organ development,morphogenesis,tumorigenesis and angiogenesis by cleaving a variety of extracellular matrix(ECM).ADAMTS18 is a kind of orphan metalloproteinase,which function and substrate remains unclear.Recent studies showed that Adamts18 is a gene with branch tip-enriched expression shared by several organs undergoing branch tip directed morphogenesis,including the lung,kidney,and salivary gland.We previously developed an Adamts18 knockout(Adamts18^-/-)C57BL/6/129Sv mouse strain and showed that Adamts18 is a morphogen for early lung bronchial branching by modulating the production of extracellular microfibrils.The mouse submandibular gland has been used as a model system to study branching morphogenesis.During the process of phenotyping of Adamts18^-/-mice,we observed that more than half of Adamts18^-/-mice developed palpable unilateral or bilateral SMG scleroma at 4 mo.of age.These novel findings prompted further studies on the role of Adamts18 in SMG pathophysiology.Using previously constructed Adamts18 knockout mice,we studied the effects of ADAMTS18 on submandibular gland pathophysiology.The research methods are as followed.1)Determine Adamts18 m RNA spatiotemporal expression in SMG tissues by quantitative real time PCR(q RT-PCR)and in situ hybridization.2)Determine branching morphogenesis by SMG organ culture.3)Compare the differences of expression of proteins involved in branching morphogenesis by q RT-PCR and immunofluorescence(IF).4)Compare the architecture and function of SMG by immunohistochemistry(IHC),western blotting(WB),ELISA and AB-PAS staining.5)WB,ELISA and IHC were used to determine fibrogenic cytokines and inflammatory infiltration.The results showed:1)Adamts18 m RNA was enriched in the epithelium of branching tips at prebud stage(E11.5-E13.5)and pseudoglandular stage(E13.5-E15.5),after E15.5,its expression was decreased but retained by adult SMG tissues.2)After the in vitro culture of embryonic SMG,Adamts18 deficiency lead to reduced number of branching.HE staining showed that the cleft formation and the number of branching of Adamts18^-/-SMG were both reduced at pseudoglandular stage.3)Major molecules involved in SMG epithelium branching including Fn1,Lama1,Lama5,Lamb1,Cdh1did not change significantly in Adamts18^-/-SMG tissues at pseudoglandular stage,while the transcription level of Fbn1 decreased in Adamts18^-/-SMG at E13.5.4)Fibronectin,Laminin and E-cadherin were accumulated at the cleft site in wildtype SMG at E13.5,while Adamts18^-/-SMG had barely cleft formation.5)The expression and distribution of ductal cells marker cytokeratin7(KRT7)and epithelial cells marker aquaporin5(AQP5)did not change significantly in in Adamts18^-/-SMG tissues at 2-wk-old that determined by IF,WB and q RT-PCR.ELISA and AB-PAS staining showed that the secretory function of GCT cells and acinar cells had not significant differences.6)IHC results showed that 8-wk-old,Adamts18^-/-mice began to manifest the first signs of pathologic changes of mild fibrosis and CD11b+cell infiltration in SMG tissues.At 8-mo-old,Adamts18^-/-mice developed unilateral or bilateral SMG scleroma,periductal fibrosis,acinar atrophy and irregular duct ectasis.7)IHC and WB results showed increased Fibronectin and Laminin deposition and dense infiltration of Ig G-positive plasma cells in SMG of 8-mo-old Adamts18^-/-mice.A significant infiltration of CD4+T lymphocytes and CD11b+monocytes and macrophages was also detected in the SMG scleroma of Adamts18^-/-mice.The levels of TGF-?1,IL-6,and IL-33 were significantly increased in Adamts18^-/-SMGs.8)IHC and WB results showed more activated myofibroblasts in SMG of 8-mo-old Adamts18^-/-mice.Together,ADAMTS18 is secreted by SMG epithelial cells,highly expressed at early developmental stages,and gradually decreased after birth but retained by adult SMG tissues.ADAMTS18 modulates cleft formation of epithelium hence regulates branching morphogenesis.Adamts18 deficiency results in remodeling of ECM,induces autoimmune reactions and inflammatory responses,activities myofibroblast leading to fibrogenesis of SMG.This study indicates that ADAMTS18 regulates the early branching morphogenesis of embryonic SMG and plays a role in protecting from spontaneous SMG fibrogenesis via modulating local inflammation,autoimmune reaction,and myofibroblast activation in adult mice.