Continued integrity of the oral integument requires that the wounded gingiva re-epithelialize. In this process, cells at the margin of a wound are induced by a new environment to attach, spread, migrate, and eventually invade through the wound extracellular matrix (ECM). Substantial data from this and other laboratories indicate that the major group of ECM receptors, the integrins, are critical in mediating interactions of cells with the ECM. This proposal seeks to investigate the hypothesis that after injury one subset of integrin receptors promotes the initial out-migration of activated keratinocytes on the wound matrix. Migration then proceeds until the cells contact the opposing wound margin, at which time movement is restricted, and a second set of integrin-mediated signals restricts movement and induces formation of new hemidesmosomes and basement membrane. The process of re- epithelization is also likely to be modulated by wound cytokines, such as TGF-beta, and requires keratinocytes to elaborate specific proteases, including plasminogen activators and metalloproteinases. The proposed studies will use model systems of freshly dissociated and cultured human gingival keratinocytes (HGK), explants of human gingival epithelium, and tissue biopsies of human gingiva to analyze three stages of wound healing: activation, migration and reformation of stable attachment such as hemidesmosomes. The activation of integrin receptors immediately after HGK dissociation will be assessed using functional and biochemical assays to characterize HGK interaction with ligands in the wound bed. Changes in integrin synthesis, assembly, and expression over time will also be followed by pulse-chase labeling and flow cytometry of freshly dissociated and cultured basal HGK. The ligand specificity of specific HGK integrins will be analyzed by cell attachment, affinity chromatography, and receptor-binding assays. The possibility that certain integrins, particularly alpha3beta1 and alpha6beta4, are important in promoting and then halting migration will be examined. Also to be studied are the possibility that cell migration is regulated by HGK deposition of an ECM carpet containing epiligrin/kalinin; the expression of specific proteases during and after HGK activation and the possible modulating effect of the wound cytokine TGF-beta on integrin profiles, migratory response, ECM deposition, and protease expression. These studies will increase understanding of the dynamic processes of activation, migration and reorganization of stable epithelial attachments that occur in gingival wound healing. They are highly relevant to the migration of junctional epithelium in the progression of periodontitis, as well as healing of the junctional epithelium after periodontal surgical therapy.
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