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gerPrüßmeyer, JessicaLudwig, AndreasLimitierte Proteolyse endothelialer und epithelialer Oberflächenmoleküle durch die Metalloprotease ADAM17info:eu-repo/classification/ddc/570LeukozytEndothelzelleEpithelBiowissenschaften, BiologieMetalloproteaseADAMmetalloproteinasesendothelial cellsepithelial cellsSeveral transmembrane molecules on the surface of endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases ADAM10 and ADAM17. This proteolytic cleavage is called Shedding and results in the release of soluble molecules from the cell surface. In this thesis, junctional adhesion molecule-A (JAM-A) and members of the proteoglycan family (syndecan-1 and syndecan-4) were identified as novel substrates for these proteases. The transmembrane protein JAM-A is a member of the Ig superfamily and is expressed at endothelial and epithelial junctions, and on leukocytes. JAM-A participates in the organization of tight junctions and has been implicated in adhesion and transendothelial migration of neutrophils and mononuclear cells. Syndecans are cell surface proteoglycans that bind and present various proinflammatory mediators. Syndecan-1 and -4 are expressed on lung epithelial cells and a soluble form is released into the bronchoalveolar fluid during inflammation. The results presented here show that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble JAM-A and proteoglycan ectodomains. This release was enhanced by treatment with the phorbolester PMA or the proinflammatory cytokines TNF-alpha and IFN-gamma. The enhanced release was not caused by increased gene induction, but was rather associated with a reduction of the protein present on the cell surface by enhanced Shedding. Both constitutive and induced release required the presence of ADAM17. This was shown by specific inhibitors that allowed discrimination between ADAM10 and ADAM17, by lentiviral silencing experiments and by treatment with the recombinant catalytic domain of ADAM17. In cultured epithelial cell lines, enhanced Shedding of JAM-A or proteoglycans was not associated with increased mRNA expression or enhanced redistribution of ADAMs on the cell surface, but rather with increased activity of cellular ADAM17. This was demonstrated by means of a synthetic substrate assay. Since in vitro experiments showed that treatment with TNF-alpha and IFN-gamma enhanced proteolytic Shedding of the analysed cell surface molecules, this was also tested in an in vivo model. To this end, mice were treated systemically with TNF-alpha/IFN-gamma leading to increased presence of soluble JAM-A in the serum. Both cytokines also stimulated JAM-A release from excised murine aortas which was associated with enhanced ADAM17 activity in the tissue and could be suppressed by treatment with an ADAM17 inhibitor. Soluble syndecan-1 and -4 were released into the bronchoalveolar fluid of mice. Moreover, treatment with TNF-alpha and IFN-gamma increased ADAM17 activity and syndecan release in murine lungs. Further in vitro studies demonstrated that cleavage by ADAM17 generates cellular fragments residing in the cell membrane, undergoing further proteolytic degradation, potentially by gamma-secretase. Functionally, soluble JAM-A blocked migration of cultured endothelial cells, reduced transendothelial migration of isolated neutrophils in vitro and decreased neutrophil infiltration in a murine air pouch model in a LFA-1- and JAM-A-dependent mechanism. These data suggest multiple roles of ADAM17-mediated Shedding. Shedding of JAM-A may be required to facilitate transmigration of neutrophils within the endothelial junctions. On the cell surface Shedding of syndecans may limit the presentation of chemokines. Moreover, soluble syndecan and JAM-A may act as antagonists by interacting with soluble chemokines or integrins on neutrophils and thereby downregulate transmigration. The upregulation of ADAM17 mediated Shedding activity by proinflammatory cytokines further indicates that this protease is an important modulator of endothelial and epithelial surface molecules and leukocyte recruitment during inflammation.Aachen : Publikationsserver der RWTH Aachen University 188 S. : Ill., graph. Darst. (2010). = Aachen, Techn. Hochsch., Diss., 2010info:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/publishedVersionPublikationsserver der RWTH Aachen University2010info:eu-repo/semantics/openAccessDEhttps://publications.rwth-aachen.de/record/51799https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-114051%22StudentsStudent Financial Aid ProvidersTeachersResearchersinfo:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-32584