Abstract
BackgroundConnexin43 (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. The association of Cx43 with cholesterol enriched lipid raft domains was recently demonstrated. The objective of this study is to assess if products of cholesterol oxidation (oxysterols) affect gap junction intercellular communication (GJIC).ResultsPrimary cultures of lens epithelial cells (LEC) were incubated with 7-ketocholesterol (7-Keto), 25-hydroxycholesterol (25-OH) or cholesterol and the subcellular distribution of Cx43 was evaluated by immunofluorescence confocal microscopy. The levels of Cx43 present in gap junction plaques were assessed by its insolubility in Triton X-100 and quantified by western blotting. The stability of Cx43 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 Cx43 at the plasma membrane and an increase in intercellular communication through gap junction. However, incubation with cholesterol or 25-OH did not lead to significant alterations on subcellular distribution of Cx43 nor in intercellular communication. Data further suggests that increased intercellular communication results from increased stability of Cx43 at the plasma membrane, presumably forming functional gap-junctions, as suggested by decreased solubility of Cx43 in 1% Triton X-100. The increased stability of Cx43 at the plasma membrane seems to be specific and not related to disruption of endocytic pathway, as demonstrated by dextran uptake.ConclusionsResults 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 Cx43 assembled in gap junction plaques.
Highlights
Connexin43 (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions
The gap junctions allow the passage of small molecules between metabolically active epithelial cells, which produces most of the ATP used by the lens, and the fully differentiated fiber cells that present low metabolic activity
In this study we evaluated the effect of the products of cholesterol oxidation on subcellular distribution of Cx43 and gap junction intercellular communication (GJIC) in lens epithelial cells (LEC)
Summary
Connexin (Cx43) is an integral membrane protein that forms intercellular channels called gap junctions. Intercellular communication in the eye lens relies on an extensive network of gap junctions essential for the maintenance of lens transparency. Each connexon is composed of six subunits, the connexins These channels allow passage of small molecules, with a molecular mass below 1 kDa, such as small metabolites, ions, and second messengers [1]. Cell Communication and Signaling 2004, 2 http://www.biosignaling.com/content/2/1/2 junctions is well illustrated in the eye lens where inner fiber cells fully depend on a complex network of gap junctions for nutrition and signalling [2,3]. The gap junctions allow the passage of small molecules between metabolically active epithelial cells, which produces most of the ATP used by the lens, and the fully differentiated fiber cells that present low metabolic activity. Three connexin genes are expressed in the vertebrate lens; α1 (Cx43) connexin that is expressed mostly in epithelial cells [5]; α3 (Cx46) and α8 (Cx50) connexins which are expressed in fiber cells [6,7]
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