The corneal epithelium has a variety of cell:cell and cell:substrate contacts which maintain much of its integrity even as it migrates in response to injury. Among the cell:substrate junctions, the hemidesmosomes (HDs) are responsible for most of the adhesion of the epithelium to its substrate. HDs contain the alpha6beta4 integrin as one of their components and other integrin heterodimers are present in cell:cell boundaries of the corneal epithelium.
Aim 1 of this proposal is to determine whether the expression and structure of beta1, beta4, alpha3, alpha5 and alpha6 integrin mRNAs are altered during migration by a) amplifying rat integrin cDNAs from a rat corneal epithelial cell library, b) determining if the expression of rat integrin mRNAs is altered during migration, c) determining the relative abundance of the cytoplasmic integrin mRNA alternative splicing variants and whether there are changes during migration, and d) correlating the data on integrin mRNA expression with data on integrin protein synthesis.
Aim 2 is to determine if the delay in corneal epithelial migration induced by addition of extracts from rat polymorphonuclear leukocytes (PMNs) to debridement wounded corneal organ cultures involves alterations in the amounts or the localization of integrins by a) using immunoblotting, immunoprecipitation, and mRNA quantitation to discover if the PMN extract added to debridement wounds affects protein synthesis in the epithelium by determining the level of integrins, vinculin, alpha-actin, alpha- enolase, and ICAM-1 and b) determining if the addition of purified inflammatory cytokines to corneal organ cultures with epithelial debridement wounds results in a delay in epithelial migration by a mechanism similar to PMN extract.
Aim 3 is to determine whether the disassembly of hemidesmosomes and migration of the corneal epithelium involves phosphorylation and/or proteolytic cleavage of the beta4 subunit by a) determining whether the HD alpha6 and beta4 subunits are phosphorylated on tyrosine in the unwounded cornea and if their phosphorylation state is altered during migration, b) developing polyclonal antisera with specificity against either the entire extracellular domain or the entire cytoplasmic portion of the beta4 molecule, and c) using the antisera to determine if the beta4 molecule undergoes cleavage during epithelial cell migration.
Aim 4 is to determine if integrins at regions of cell:cell interaction are functionally important in maintaining cell:cell contacts in the corneal epithelium by a) establishing cell culture conditions for bovine corneal epithelial cells, b) determining the state of assembly of desmosomes, adherins junctions, and alphav- and the beta1-containing cell:cell junctions in cells cultured in low calcium and after shifting cells to high calcium medium, c) determining the effect of adhesion blocking integrin peptides and antibodies on the ability of cultured corneal epithelial cells to maintain cell:cell contact in low and in high calcium media, and d) determining whether addition of PMN extract to cultured cells disrupts cell:cell contacts and the cell:cell localization of alphav and the beta1 integrins in low and high calcium media. These experiments will help accomplish our goal of obtaining a better understanding, at the molecular level, of the role of integrins in the normal cornea and in epithelial cell migration during wound healing.
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