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L. K. Bossmann, D. Banerjee, D. Madgwick, V. Akoyev, D. J. Takemoto; Overexpression of Cx46 Causes Proteasome-Dependent Degradation of Cx43 in Lens. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4604.
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© ARVO (1962-2015); The Authors (2016-present)
Vertebrate lens primarily contains three connexin isoforms; connexin 43(Cx43), connexin 46(Cx46) and connexin 50(Cx50). The outer layer of epithelial cells in lens mainly contain Cx43 and Cx50 whereas the inner layer of differentiating fiber cells and mature fiber cells degrade Cx43 and preferentially contain Cx46 and Cx50. The purpose of this study is to investigate if overexpression of Cx46 mediates the degradation of Cx43 by the proteasomal pathway in lens.
The rat full length Cx46 cDNA was cloned into PEGFP-N3 vector and rabbit lens NN1003A epithelial cells were stably or transiently transfected with the construct. The level of Cx43 protein was determined by western blot and the level of Cx43 mRNA was determined by reverse transcriptase (RT)-PCR in transfected cells. The cells overexpressing Cx46 protein were treated with protease inhibitor cocktail, 100uM of ALLN (N-Acetyl-Leu-Leu-Nle-CHO) + 10uM of clasto-Lactacystin β-Lactone to assess effects on Cx43 degradation. The increased ubiquitination of Cx43 in NN1003A cells overexpressing Cx46-GFP protein was investigated by immunoprecipitation studies where Cx43 was pulled down, from whole cell lysate, by anti-Cx43 antibody followed by immunoblot with anti-ubiquitin antibody.
Overexpression of Cx46 protein caused a decrease in endogenous Cx43 protein level both in stably or transiently transfected rabbit lens NN1003A epithelial cells. In transiently transfected cells the level of Cx43 protein was reduced at 36 h and 48 h after Cx46-GFP construct transfection. Cx43 mRNA level was not altered indicating reduction in Cx43 protein level is not due to inhibition of transcription. Treatment with protease inhibitor ALLN showed no change in Cx43 protein level even in NN1003A cells overexpressing Cx46 protein. Immunoprecipitation study showed increase in ubiquitin conjugation of Cx43 in cells that overexpress Cx46 as compared to control.
Our study demonstrates that overexpression of Cx46, initiates proteasome-dependent degradation of another gap junction protein, Cx43, in rabbit lens NN1003A epithelial cells.
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