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H. M. Girao, S. Catarino, P. Pereira; Regulation of Intercellular Communication in Retinal Endothelial Cells Is Mediated by Upp-Dependent Stabilization of Connexin 43 at Plasma Membrane. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5886.
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Intercellular communication through gap junctions (GJIC) is important in maintaining the integrity of the blood-retinal barrier. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. The purpose of this study is to investigate the molecular mechanisms involved in regulation of connexin 43 (Cx43) stability at plasma membrane in retinal endothelial cells.
To investigate the interaction of Cx43 with ZO-1 and Nedd4, Cx43 was selectively immunoprecipitated (IP), followed by western blot (WB) and probing with antibodies against ZO-1 or Nedd4. To investigate the role of the proteasome in Cx43-ZO-1 interaction, cells were incubated either in the presence or absence of proteasome inhibitors, and then Cx43 was IP, followed by WB and probing with antibodies against ZO-1. Alternatively, cells were fixed and simultaneously stained with antibodies against Cx43 and ZO-1, and subsequently imaged by confocal microscopy. To evaluate the role of the ubiquitin ligase Nedd4 on the interaction between Cx43 and Eps15 and further internalization, cells were transfected with a dominant negative form of Eps15, and Cx43 distribution subsequently investigated by confocal microscopy. To demonstrate that interaction of Cx43 with Eps15 depends on prior interaction of Nedd4 with Cx43, cells were transfected with a mutated form of Cx43 that does not interact with Nedd4.
Data obtained in this study indicates that the proteasome regulates interaction between Cx43 and ZO-1 most likely by degrading an ancillary (and still unknown) protein that binds to the C-terminus of Cx43 preventing interaction with ZO-1. When interaction between Cx43 and ZO-1 is disrupted, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane, leading to deregulation of intercellular communication. Moreover, ovexpression of a dominant negative form of Eps15 results in Cx43 accumulation at the plasma membrane. However, when interaction between Cx43 and Nedd4 is prevented, this accumulation is no longer observed.
Data suggests a new pathway whereby alterations in proteasome activity and Cx43 ubiquitination may impact on GJIC, contributing to endothelial cell dysfunction associated with the breakdown of the blood-retinal barrier, as observed in diabetic retinopathy.
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