Injuries and disorders of the cornea are common causes of impaired vision. For the better management and treatment of corneal healing problems, it is important to understand the mechanisms of normal development and regeneration of corneal tissues. A corneal epithelial protein has been identified (HEBM1), that appears to be involved in corneal epithelial differentiation during fetal development, in maintaining normal adult cornea, and in would healing. The previous study indicates that HEBM1 is a novel serine/threonine protein kinase (PK). The proposed studies will test the hypotheses that this novel PK is involved in a signal transduction pathway that a) mediates limbal to corneal epithelial phenotypic transition in normal adult, and b) vice versa during would healing. HEBM1 will be characterized by determining its substrate and inhibitor specificity, the mechanisms involved in regulation of its synthesis and enzymatic activity, and its role in regulating phenotypic features of corneal epithelial cells. To study its regulatory role, the temporal relationship between changes in its expression and activation, and limbal to corneal epithelial phenotypic transition, will be evaluated in vitro using rabbit and human limbal explant cultures. Another approach will be to express HEBM1 in different cell types (e.g. corneal fibroblasts or lens epithelial cells) that normally do not express HEBM1 (by transfecting them with cDNA encoding for HEBM1) and to then examine the morphological and phenotypic changes in these transfected cells that do express HEBM1. Cytoskeletal structures are now well-recognized as dynamic structures that are involved in various cellular activities which are important for embryonic development and would healing. Two immunologically related actin filament (microfilament)-associated proteins that are recognized by the same monoclonal antibody (AFAP50 and 180) and a vimentin (intermediate filament)-associated protein (IFAP130) have been identified and are found to be expressed by activated fibroblasts in response to wounding. The following hypotheses about the functions of these proteins (based on preliminary studies) will be tested: a) AFAP50 is the chain elongation factor, EF1a; b) AFAP180 is an actin binding protein; c) IFAP130 is a novel protein that binds both actin and vimentin; d) increased expressions of AFAP50/180 and IFAP130 are associated with the migratory activity of corneal fibroblasts. The experiments will include direct biochemical analyses of the interactions of these proteins with actin and/or vimentin filaments (using purified proteins), characterization of their structure-function relationship by analyzing their cDNA and deduced amino acid sequences, and evaluation of the role of these proteins in migratory activity during wound healing (i.e., changes in the expression and intracellular distribution of these proteins and their associated cytoskeletal networks). These studies of the molecular mechanisms that regulate important cellular activities during differentiation and regeneration of corneal tissues will yield insights that may lead to better diagnosis and treatment of corneal development and healing problems.
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