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R.J. McCully, V. Akoyev, M. Barnett, D. Lin, L. Takemoto, D.J. Takemoto; Connexin 46 Overexpression in Lens Epithelial Cells Causes Fiber Cell Differentiation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1987.
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Overexpression of Cx46 may down regulate the expression of Cx43 and may up regulate the expression of crystallins and/or aquaporin O in cells in culture. This could cause terminal differentiation of the epithelial cell line, implying that Cx46 proteins are involved in the differentiation process. The purpose of this study is to determine the effects of overexpression of Cx46 in a lens epithelial cell line, expressing endogenous Cx43 and Cx50, on growth and differentiation properties.
Cx46 was transfected into 90% confluent N/N 1003A lens epithelial cells, and stably transfected cells were selected in G418–containing DMEM growth media. Cell localization of each connexin protein was determined +/– phorbal–12 myristate 13 acetate (TPA), using immunofluorescence and confocal microscopy. We also determined whether overexpression of Cx46 resulted in the down regulation of Cx50 or Cx43 by utilizing Western blots and PCR. Through these same methods, we were able to determine if the overexpression of Cx46 caused the up regulation of gamma crystallin or aquaporin–0 synthesis, both of which are markers of terminal differentiation in these cells.
N/N 1003A cells overexpressing Cx46 were found to express levels of the protein, gamma crystallin, a protein involved in the differentiation of lens epithelial cells to fiber cells. A shape change in the cells, from a typical N/N lens epithelial cell to the shape of a lens fiber cell was noted. It appears that overexpressing Cx46 in the N/N 1003A cell line will cause crystallin proteins to be expressed and differentiation to occur.
Cx46 may have functions in addition to gap junction activity. Results suggest that Cx46 overexpression may cause lens epithelial cells to differentiate into fiber cells.
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