7-Ketocholesterol modulates intercellular communication through gap-junction in bovine lens epithelial cells

Abstract: Background: Connexin43 (C×43) is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eyelens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of C×43 with cholesterol enriched lipid raft domain was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols) affect gap junction intercellular communication (GJIC). Results: Primary cultures of lens epithelial cells (LEC) were incubated with 7-ketocholesterol (7-Keto). 25-hydroxycholesterol (25-OH) or cholesterol and the subcellular distribution of C×43 was evaluated by immunofluorescence confocal microscopy. The levels of C×43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of C×43 at the plasma membrane following incubation with oxysterols was evaluated by biotinylation of cell surface proteins. Gap junction intercellular communication was evaluated by transfer of the dye Lucifer yellow. The results obtained showed that 7-keto induces an accumulation of C×43 at the plasma membrane and an increase in intercellular communication through gap junction. However, in cubation with cholesterol or 25- OH did not lead to significant alterations on subcellular distribution of C×43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of C×43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of C×43 in 1% Triton X-100. The increased stability of C×43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake. Conclusions: Results demonstrate, for the first time, that 7-keto induces an increase in gap junction intercellular communication, that is most likely due to an increased stability of protein at the plasma membrane and to increased abundance of C×43 assembled in gap junction plaques.