title: [Roles of specific serines phosphorylation of αA crystallin on human lens epithelial cells].
abstract: OBJECTIVE
  To investigate the impacts of specific serines phosphorylation of αA Crystallin on anti-oxidation and anti-apoptotic ability of human lens epithelial cells (HLEC).
  
  
  METHODS
  Experimental Study. Using site-directed mutagenesis, serines on specific sites (Ser45, Ser66, Ser122) were replaced respectively by aspartic acid (D) to mimic phosphorylation. Target genes were inserted into pEGFP-C1 plasmid and transfected into HLEC by Lipofectamine™ 2000. HLEC transfected with blank plasmid and plasmid over-expressing wide-type (WT) αA Crystallin were defined as control group and WT group. After H2O2 induction, Dihydroethidium (DHE) and Annexin V-PE/7AAD staining were used to detect the reactive oxygen species level and apoptotic rate. T-test, Wilcoxon-two sample test, analysis of variance and Kruskal-Wallis test were used for statistical analysis.
  
  
  RESULTS
  The DHE intensity of three phosphorylation groups were 167.53±18.21, 143.54±4.78 and 143.40±30.19, which reduced significantly comparing to control group (274.42±3.71, t=9.963, P=0.001; t=34.961, P<0.01; Z=1.993, P=0.046); The same tendency were found when compared with cell group (WT) over-expressing wide-type αA Crystallin (192.67±1.47, Z=1.964, P=0.049; t=17.887, P<0.01; Z=1.964, P=0.049). After H2O2 induction for apoptosis, early apoptotic rates for three phosphorylation groups were 15.08%±0.90%, 16.59%±5.37% and 15.36%±2.62% respectively, which significantly decreased comparing with control group (41.28%±2.70%; t=21.632, P<0.01; t=8.855, t=15.450, P<0.01) and WT group (31.40%±1.64%, t=15.117, P<0.01; t=4.524, P=0.006; t=8.988, P=0.001). No significant differences were found in both measurements between the three phosphorylation groups (F=1.08, P=0.407; x2=4.345, P=0.114).
  
  
  CONCLUSIONS
  The Ser45, Ser66 and Ser122 phosphorylation enhanced the anti-oxidative stress and anti-apoptotic capability of HLEC.
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
authors.forename: Hongfei
authors.initials: H
authors.lastname: Ye
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Zhou
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
authors.forename: Xiangjia
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Cai
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
authors.forename: Keke
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Fan
authors.affiliation: Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China; Email: luyi_eent@126.com.
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000704:Analysis of Variance; D017209:Apoptosis; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide; D007908:Lens, Crystalline / Q000166:cytology*; D016297:Mutagenesis, Site-Directed; D018384:Oxidative Stress; D010766:Phosphorylation; D010957:Plasmids; D017382:Reactive Oxygen Species / Q000032:analysis; D012694:Serine / Q000378:metabolism*; D018709:Statistics, Nonparametric; D014162:Transfection; D038202:alpha-Crystallin A Chain / Q000378:metabolism*
chemical_list: D017382:Reactive Oxygen Species; D038202:alpha-Crystallin A Chain; D012694:Serine; D006861:Hydrogen Peroxide