Effects Of Src Family Kinases On Gap Junction Cx43 In Formation Of Cortical Cataract In Vitro

BackgroundOur previous studies demonstrated that inhibition of SFK activity prevented development of cataract in vitro, but the mechanism is not clear. In a wide variety of cancer cells, Src tyrosine kinase is involved in phosphorylation of Cx43, and then suppresses the communication of gap junctions. However, the lens is an avascular organ with the most abundant and ubiquitously expressed connexin as its critical channels providing pathways for metabolic and electrical coupling between cells. Whether the changes of Cx43 participate in the process of cortical cataract, whether this changes mediated by SFK is still unknown.AimTo investigate the effects of inhibition of Src family kinases (SFKs) activation on connexin43 of gap junction in formation of cataract in vitro.MethodsChicken embryonic day 10 (E10) lenses were cultured in medium M199 with 10% fetal bovine serum in addition with or without PP1, the specific inhibitor of SFKs. They were observed and photographed at every other day in 10-day culture period. The degree of opacification was quantified by using image-analysis software. Transmission electron microscope was used to observe the ultrastructural changes of the lens cells. The histomorphological changes were detected by immunofluorence staining with WGA for cell membranes, and DAPI for cell nuclei. To investigate the expression and distribution of Cx43 protein, we used immunofluorescence staining on cryosections of cultured lenses. For functional changes of Cx43 gap junction of lens epithelium, we employed Lucifer Yellow dye uptake on scraped anterior capsules of cultured lenses. The expression of Cx43 was detected by Western blot and RT-PCR analysis.ResultsThe cultured lenses developed peripheral cortical opacification gradually in 10-day culture period. At culture day 4 and 10, the opacification areas were 26% and 49% in control lenses, whereas in PP1 treated lenses, the lens opacity was prevented, and the opacification areas were decreased to 20% and 14%, respectively(p<0.05). There were no obvious changes on histomorphology and expression of Cx43 protein in both groups at day one. At day 5 and 10, in control lenses, the lens epithelium was detached from the lens fibers, and the dead cells were found in or beneath the lens epithelium. Cx43 was detected at the interface between lens epithelial cells. But in PP1 treated lenses, the lens epithelium was attached with the lens fibers, and the lens epithelial cells were combined tightly with each other. The dead cells decreased dramatically, the structure of lens epithelium maintained almost normal. The expression of Cx43 was detected at the interfaces between lens epithelium and lens fibers, and anterior capsules. Lucifer Yellow dye uptake analysis shown that the dye transfer distances in control lenses were 50.93±13.19,66.67±7.29,99.52±12.67μm, meanwhile in PP1 treated lenses, the distances were 89.54±9.60,69.18±5.49,130.63±10.52μm at day 1, 5 and 10, respectively. The differences at day 1 and 10 were significant (p<0.01). By Western blot assay, the expression of p-Src was significantly low after treatment with PP1, and the expression of Cx43 in PP1 treated lenses was quite lower than that in the control lenses. With RT-PCR, we obtained the similar tendency with Western Blot assay.ConclusionsInhibition of Src activation by PP1 enhances the communication of Cx43 gap junctions in lens epithelial cells, maintains the normal structure of lens epithelium in cultured lenses and blocks formation of cataract.