[Hydrogen peroxide (H2O2) and methyl-beta-cyclodextrin (MPCD) down regulate caveolin expression in human lens epithelial cells (HLECs)].
Oxidative stimulation induced by hydrogen peroxide (H2O2) on human epithelial cells (HLECs) was performed to observe the effects on cell viability, caveolin expression, and cholesterol depletion in HLECs caused by methyl-beta-cyclodextrin (MbetaCD) was also studied. SRA01/04 HLECs were exposed to H2O2 or MbetaCD of various concentrations and durations. We used a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to measure the effect of H2O2 on the viability of SRA01/04 HLECs. The distributions of caveolins after oxidative stimulation were probed by fluorescence microscopy and laser scanning confocal microscopy. Immunoblotting was performed to analyze alterations of caveolins expression. We observed that the viability of SRA01/04 HLECs under 0.1 mM H2O2 for 10 min or longer was significantly reduced (*p < 0.05, F = 11.63). Laser scanning microscopy showed immunofluorescent caveolins in SRA01/04 HLECs under 0.1 mM H2O2 for 10 min or longer, caveolins were largely confined to intracellular domains. Western blots showed both membrane and total caveolin protein (22 kDa) levels in SRA01/04 HLECs treated with 0.1, 0.2, 0.5 or 1.0 mM H2O2 for 30 min were significantly reduced, compared with the untreated (*p < 0.05, F = 6.149, or *p < 0.05, F = 14.489 respectively). In addition, the membrane and total caveolin protein level after treated with 0.1 mM (*p < 0.05, F = 6.843, or *p < 0.05, F = 7.944 respectively) H2O2 for different durations also down regulated. Fluorescence microscopy also showed that phosphorylated caveolin-1 was distributed near the focal adhesions of the cells. This study concludes that the responses of HLECs to oxidative stress may include down regulation of caveolin and phosphorylation of caveolin-1 on Tyr14, and that MbetaCD also down regulates caveolin while depleting cholesterol in HLECs.
Jun
Hong-ying
HY
Yao
Ke
K
Ma
Jin
J
Li
Hong-wu
HW
Tang
Xia-jing
XJ
D051242:Caveolin 1 / Q000096:biosynthesis / Q000378:metabolism; D022461:Caveolins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002784:Cholesterol / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D047392:beta-Cyclodextrins / Q000494:pharmacology*
C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol; D006861:Hydrogen Peroxide