The integrin alpha/v/beta6, a receptor for the extracellular matrix proteins fibronectin and tenascin, is highly expressed in both airway and alveolar epithelium during organogenesis and in response to injury or inflammation. Inactivation of the beta6 gene in mice leads to infiltration of the skin and airways with inflammatory cells, suggesting a role for this receptor in down-modulating epithelial inflammation. In vitro, alpha/v/beta6 augments the ability of epithelial cells to proliferate and modulates the expression of several epithelial-derived cytokines. The central goal of this proposal is to identify the signaling pathway or pathways by which alpha/v/beta6 induces these complex changes in cell behavior. This goal is approached through 4 specific aims: 1) Native immunoprecipitation, western blotting, in vitro kinase assays, and in vitro binding assays with cell lysates from stable transfectants expressing intact or mutant alpha/v/beta6 will be used to determine the relationships between activation of known components of integrin-associated signaling complexes and subsequent alpha/v/beta6- induced changes in cell behavior. 2) Preliminary results suggest that alpha/v/beta6 inhibits activation of NF-kappaB, a family of transcription factors involved in regulation of cytokine gene expression. These results will be confirmed, and their relevance to transcription and cytokine gene expression will be assessed in cell lines stably transfected with intact or mutant beta6. 3) To identify the precise structural requirements for beta6-mediated cellular responses, proliferation, cytokine gene expression, and activation of specific signaling intermediates will be assessed in stable cell lines expressing a variety of mutant versions of beta6 containing deletions or point mutations within the C-terminal 11 amino acids. 4) Native immunoprecipitation, focal adhesion plaque isolation, affinity chromatography and the yeast two-hybrid system will be used to identify the proteins responsible for signaling specificity through alpha/v/beta6. By utilizing a variety of mutant versions of beta6 that do or do not support specific functions, it should be possible to rapidly assess the likelihood that each of the proteins thus identified is functionally significant. Together, these studies should provide new information about the mechanisms by which changes in the extracellular matrix can modulate the growth and differentiation of airway epithelial cells and modulate airway inflammation. Since epithelial remodeling and inflammation are important features of a number of diseases of the lungs and airways, this information could be important for understanding the pathophysiology of these diseases and for designing improvements in their treatment.
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