PIAS1 Regulates P53 Sumoylation To Control Apoptosis Of Lens Epithelial Cells Through The P53-Bax Signal Pathway

Protein sumoylation is an important post-translational modification which is implicated in control of DNA synthesis and repair,gene transcription and protein synthesis,and thus actively participates in regulation of many cellular physiological activities including cell division,autophagy,senescence and apoptosis.Cataract is the leading cause for blindness of the whole world.Our previous studies revealed that apoptosis of lens epithelial cells induced by various stress factors acts as a common cellular basis for non-genetic cataractogenesis.Therefore,elucidation of the mechanism for stress-induced lens epithelial cells apoptosis is very important in our understanding of the pathogenic mechanism of cataract formation and development of effective therapeutic strategies.Moreover,our recent studies showed PIAS1 can promote oxidative stress-induced apoptosis of lens epithelial cells.However,how PIAS1 promotes oxidative stress-induced apoptosis of lens epithelial cells remains to be determined.Therefore,this thesis mainly explored the molecular mechanism by which PIAS1 regulates oxidative stress-induced apoptosis of lens epithelial cells.It has been reported that PIAS1 can mediate activation of JNK1/2 to promote apoptosis in human osteosarcoma U2 OS cells.Western blot analysis revealed that PIAS1 does not regulate the apoptosis of lens epithelial cells by activating JNK1/2.Subsequently,this thesis examined if PIAS1 can mediate sumoylation of P53,and affect its downstream gene to regulate the apoptosis of lens epithelial cells.Co-IP experiments demonstrated that PIAS1 mediates sumoylation of P53 at K386 residue in lens epithelial cells.PIAS1 positively regulates expression of the P53 downstream pro-apoptotic protein,Bax.Double fluorescence reporter gene activity analysis and ChIP assay revealed that sumoylated P53 directly bind to the promoter of Bax gene to positively regulate its expression.Furthermore,both the live/dead assay and ATP loss analysis results showed that the sumoylated P53 enhances oxidative stress-induced apoptosis of lens epithelial cells.Finally,to prove that PIAS1-mediated up-regulation of Bax through P53 sumoylation plays a major role in promoting oxidative stress-induced apoptosis of lens epithelial cells,we transfected the expression plasmids of Flag vector,or Flag-PIAS1 into mock knockout cells or Bax knockout cells.Cell viability was examined by ATP loss analysis.The results indicated that absence of Bax significantly attenuated PIAS1-induced apoptosis under treatment of oxidative stress.These results clearly demonstrated that PIAS1-mediated P53 sumoylation at K386 residue specifically promotes expression of Bax,enhancing oxidative stress-induced apoptosis of lens epithelial cells and leading to cataractogenesis.In summary,this thesis reveals the following conclusions:(1)PIAS1mediates P53 sumoylation at the K386 residue in lens epithelial cells;(2)PIAS1 promotes expression of Bax in lens epithelial cells;(3)PIAS1-mediated P53 sumoylation enhances expression of Bax and promotes oxidative stress-induced apoptosis of lens epithelial cells;(4)Knockout of endogenous Bax inhibits the stress-induced apoptosis of the lens epithelial cells expressing exogenous PIAS1.Together,this thesis provides novel information regarding the mechanism mediating stress-induced apoptosis of lens epithelial cells and non-genetic cataractogenesis.