Ocular transparency and growth of the human lens are dependent on the proper differentiation of lens epithelium into fiber cells. We have for the first time developed an in vitro cell culture system from the fetal human lens epithelial cells which not only shows unabated synthesis of epithelial cell specific polypeptide, alphaB-crystallin(alphaB) but also a progressive expression of fiber cell specific protein BetaBp-crystallin (BetaBp) indicating maintenance of the commitment within these cells to differentiate in vitro. Very importantly, we have shown extra-lenticular presence of alphaB which has suggested a possible regulatory rather than a structural role for this protein. The present study will investigate the regulation of the synthesis of alphaB in the human lens epithelial cells to understand its role in differentiation and de-differentiation, and/or in the functional maintenance of the lens epithelium in normal and diseased states. We will also investigate the molecular mechanistics of the activation of BetaBp gene, upon transition of these cells from a primary to a secondary culture which presents a very relevant paradigm for the study of the activation of a fiber cell specific gene. This investigation will entail isolation and physical characterization by sequencing of human alphaB and BetaBp crystallin genes; delineation of the cis regulatory elements in the two genes within 5-10kb on either side of each gene and identification and characterization of cell-type or differentiation-specific trans acting protein factors involved in the maintenance of the synthesis of BetaB and activation of the synthesis of BetaBp. Because de-differentiation and loss of the organizational competence of the epithelial cells is known to be compromised in pathologies such as anterior and posterior subcapsular opacities, this study will attempt to investigate the role of alphaB e.g. in maintenance of the epithelial phenotype and thereby in the differentiation of epithelium into fiber cells, a developmental process so very innate to a non cataractous lens.
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