Corneal and conjunctival epithelial cells are components of the contiguous layer of ocular surface epithelium, but they exhibit distinct morphological characteristics. The expression of corneal epithelial cell specific proteins-may be regulated by exogenous cytodifferentiation factors associated with the underlying basement membrane. This hypothesis will be tested by assessing the ability of the corneal basement membrane to induce the expression of a corneal-specific protein called keratin K12 in ocular surface epithelium of non-corneal origin. These studies will be performed in an in vitro model in which conjunctival epithelium is maintained in culture on corneal extracellular matrix substrates. Conjunctival epithelial cell expression of the corneal-specific keratin K12 will be assayed at the protein level using metabolic labeling, autoradiography and Western immunoblotting to detect de novo keratin biosynthesis in the cells. Oligonucleotide probes complimentary to keratin Kl2 mRNA will be synthesized and characterized using the amino-terminal or carboxy-terminal amino acid sequence of keratin Kl2 as templates. Alternately, a cDNA library will be constructed from corneal epithelial cells and screened immunochemically using a polyclonal antiserum directed against the keratin Kl2 to isolate clones encoding for the Kl2 gene. Labeled molecular probes will be used in Northern blotting and in situ hybridization analyses to confirm the presence of, and determine the cellular localization of, keratin mRNA in cultured conjunctival epithelium. An analysis of the corneal and conjunctival basement membrane composition using selective extraction techniques coupled with biochemical and immunochemical methodologies will be undertaken to identify unique proteins of the corneal basement membrane which may function as cytodifferentiation factors. Information gained from these studies may lead to a greater understanding of the role of the extracellular matrix in establishing and maintaining cell phenotype in ocular epithelial tissues, and of the molecular and cellular mechanisms by which phenotypic cytodifferentiation signals are transduced from matrix to cell. In addition, an understanding of the type of extracellular matrix environment which is necessary to maintain a corneal epithelial cell phenotype may lead to more successful management of corneal trauma and disease using conjunctival epithelial tissue grafting techniques.
