Protein Kinase A Activation Alleviates Cataract Formation Via Increased Gap Junction Intercellular Communication

Objective:Cataract is the main cause of blindness worldwide.How to effectively and economically prevent and control it has always been a worldwide problem,which has plagued ophthalmologists all over the world.Although surgical intervention is a very effective treatment at present,there is no therapeutic drug available for the prevention and treatment of this disease,and the cost of surgical intervention is high,which is limited for underdeveloped regions and developing countries due to medical conditions and other reasons.Our previous studies showed that protein kinase A（PKA）increases gap junction communication of cells in the lens,which is an important mechanism for maintaining lens transparency.Therefore,we sought a new approach for potential cataract prevention and delay,and observed whether PKA activators could reduce the damage caused by hydrogen peroxide（H₂O₂）or ultraviolet B（UVB）in the mouse lens by enhancing gap connexin channels/hemichannels.The induced active oxygen can further reduce or delay the occurrence and development of cataract.Method:Animal experiments:Four genotype mice were used:wild type（WT）,connexin 50 knockout mice（Connexin,Cx,50 knockout,KO）,connexin 46 knockout mice（Cx46KO）and Cx46 and Cx50 double knockout mice（double,d KO）.Cataract formation in the lens was induced by H₂O₂ or UVB after isolation and culture of mouse lens in vitro.Then the lenses were divided into control group and experimental group for experiment.The difference in cataract between the two groups was observed by comparing the degree of lens opacity,and the level of reactive oxygen species（ROS）in the lens of the two groups was detected by the carboxy H2DCFDA probe.Intra-lens PKA levels were detected using a commercial PKA colorimetric activity kit.The levels of antioxidant proteins in the lens were detected by frozen section of lens tissue,immunofluorescent staining and Western blot.Cell experiments:chicken lens primary cells and embryonic fibroblast cells（chick embryonic fibroblast cells,CEF）are naturally ideal cells lacking gap junction proteins,and the recombinant RCAS of Cx46,Cx50 and Cx46+Cx50 mutants were used in the experimental design（A）Viruses were obtained by point mutation,and then high-titer recombinant RCAS（A）virus was used to infect chicken lens primary cells and CEFs to express exogenous Cx.Use the Annexin V/PI kit double staining method to detect cell apoptosis/necrosis,and use lucifer yellow（LY）/rhodamine dextran（rhodamine dextran,RD）to perform dye transfer experiments to detect gap junctions（GJs）function,dye uptake assay to detect gap junction protein hemichannel Cx Hemichannel function.Results:Connexin（Cx）deletion resulted in a marked increase in cataract formation induced by H₂O₂ and UVB.We further found that PKA activators reduced H₂O₂-or UVB-induced cataracts in WT,Cx50KO,and Cx46KO,but not d KO mice.We therefore investigated PKA levels in mouse lenses and showed that PKA activators increased PKA levels in WT,Cx50KO,Cx46 KO and Cx46/Cx50 d KO lenses in the presence or absence of H₂O₂ and UVB.Cellular experiments were performed to demonstrate that PKA activators increased Cx46 homologous as well as Cx46 and Cx50 heterologous gap connexin channel and hemichannel activity.It was further confirmed that PKA activators reduced reactive oxygen species（ROS）levels in the lenses of WT,Cx46,and Cx50 KO mice,but not in Cx46/Cx50 d KO lenses.PKA activators can enhance glutathione（GSH）transport and protect lens fiber cells from H₂O₂-induced lens fiber cell death by enhancing hemichannel（HCs）function.Reduced expression of anti-oxidative stress genes found in connexin knockout lenses.In addition,loss of connexin（Cx）expression in the lens increased lens opacity induced by H₂O₂ and UVB,which may be related to the loss of connexin expression in the lens leading to decreased expression of anti-oxidative stress genes.Conclusion:1.PKA increases the function of gap junction/hemichannel and reduces the level of reactive oxygen species in the lens,and protects lens fiber cells from oxidative stress through the increase of glutathione transport mediated by connexin hemichannel.2.PKA activation attenuates cataract induced by oxidative stress by increasing gap junction intercellular communication in lens fiber cells.3.The level of antioxidant enzymes in Cx50KO was lower than that in WT and Cx46KO,and the responses of antioxidant proteases in mouse lens fiber cells to H₂O₂ and UVB were different.Therefore,our study could help identify a cost-effective way to prevent and delay the onset and progression of cataracts.