Corneal and conjunctival-specific extracellular matrix domains located along the ocular surface basement membrane may function in the extrinsic modulation of ocular surface epithelial cell phenotype during embryogenesis, in the normal and diseased adult, and following wounding. Changes in conjunctival epithelial cell phenotype accompanying ocular disorders secondary to dry eye syndromes, and the ability of conjunctival epithelium to resurface corneal wounds with deficiency of limbal stem cells, may be related to external microenvironment influences. Three hypotheses will be tested to determine the role of external factors on conjunctival epithelial cell phenotype in vitro, characterized by the expression of the differentiation-type keratin K4 in assembled intermediate filament networks. The hypothesis that intracellular signaling pathways of conjunctival epithelial cells initiated by interaction with laminin beta2 chain are mediated by beta1 integrin signal transduction complexes will be tested by integrin clustering and ligation studies and phosphotyrosine analysis, utilizing the techniques of immunofluorescence microscopy and Western blot analysis. The hypothesis that conjunctival epithelial cell interaction with extracellular matrix lacking laminin beta2 chain results in hyperphosphorylation of keratin K4 and disassembly of intermediate filament networks will be tested by beta1 integrin affinity modulation using function activating antibodies, antisense oligonucleotides, and inhibitors of signal transduction mediators. The hypothesis that laminin beta2 chain regulates the proliferation and stratification of conjunctival epithelial cells via alpha6 integrin signaling pathways will be tested by analyzing protein tyrosine kinase activity and association of integrin with signal transduction adapter molecules using coimmunoprecipitation and Western blot analysis. Completion of these studies may provide a greater understanding of the role of external environment in the establishment and maintenance of the conjunctival epithelial cell phenotype. This information may be applied to the more successful management of human ocular trauma and disease.
