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D. K. Madgwick, V. Akoyev, D. J. Takemoto; Cx46 Overexpression Initiates the Degradation of Cx43 in the Lens. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4074.
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The overexpression of Cx46 in N/N1003A lens epithelial cells may facilitate a decrease in Cx43 protein levels during the terminal differentiation of lens epithelial cells into fiber cells. The purpose of this study is to elucidate whether overexpression of Cx46 inhibits Cx43 synthesis or enhances its proteasomal degradation.
90% confluent N/N1003A rabbit lens epithelial cells were stably transfected with Gfp-Cx46 or Gfp-Cx50 plasmids and selected with G418-DMEM media. Cx46-overexpressing cells were treated with two proteasome inhibitors (ALLN, 100Î¼M; clasto-Lactacystin Î²-Lactone, 10Î¼M) for 4 hours. Cx43 and Cx50 protein levels were then determined in the N/N-Cx46 overexpressing cells by Western Blot using anti-N-terminal Cx43 antibodies (Fred Hutchinson Cancer Research Center, P1E11) or anti-Cx50 antibodies (Zymed). Total RNA was isolated from transiently transfected (Gfp or Gfp-Cx46 plasmid) or control N/N1003A cells at 24, 48, and 72 hour time periods after transfection and characterized by RT-PCR using Cx43-specific primers. To chronicle morphological changes in cell-culture appearance in both Gfp-Cx46 and Gfp-Cx50 transfected cells, phase-contrast pictures were taken at various time intervals.
Overexpression of Gfp-Cx46 (Cx46OE) caused a decrease in endogenous Cx43 protein levels as determined by Western Blot while endogenous Cx50 levels were not altered. Treatment of lens epithelial cells overexpressing Gfp-Cx46 with proteasome inhibitors restored Cx43 protein levels to normal. RT-PCR confirmed that Cx43 synthesis levels were normal in the Gfp-Cx46 containing cells. N/N-Cx46OE cells exhibited morphological changes that were not seen in N/N-Cx50OE cells.
Overexpression of Cx46 in lens epithelial cells initiates post-transcriptional degradation of endogenous Cx43 via a proteasomal pathway.
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