In Vitro Study Of The Effects Of Cell Stress On Caveolin Distribution And Expression In Human Lens Epithelial Cells

The mechanisms of cataract formation are very complicated. Oxidative stress is believed to be an important contributing factor in maturity onset of cataract formation. Reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), superoxide anion are postulated to contribute to this process. ROS causes a number of biochemical changes, which lead to an increase in water-insoluble lens proteins and the appearance of lens opacity.The plasma membrane of the lens is extremely rich in cholesterol. This high level of cholesterol is proposed to preserve lens membranestructure in the physiological state. Disruption of cholesterol has been shown to destroy subdomains of lipid rafts, and methyl-β-cyclodextrin (MpCD) is known to deplete cholesterol from cell membranes.Caveolae are non-clathrin-coated plasma membrane microdomains rich in cholesterol and glycosphingolipid. They invaginate to form 50-100 nm vesicles in the plasma membrane that have been found in many cell types. Caveolae contain the signature marker proteins termed caveolins, which have been implicated in vesicular trafficking and signal transduction. The mammalian caveolin family members include three isoforms: caveolin-1, caveolin-2 and caveolin-3.Some studies showed that caveolin and caveolae exits in lens and lens epithelial cells, and samples from cataract lens were not exhibited nonsedimenting bands which included abundant caveolin after sucrose linear density gradient centrifiigation. But the reports on study of human lens epithelial cells line are very rare, and no paper in China at present. Oxidative stress induced by H₂O₂ is a good model for study on cataract, so we select this model for our study on caveolin and cell stress and to further explore the mechanism of cataract.Purpose: Oxidative stimulation induced by hydrogen peroxide on human lens epithelial cells (HLECs) was performed to observe the effects on cell proliferation, caveolin expression, caveolin-1 phosphorylation, and cholesterol depletion in HLECs caused by MβCD was also studied. The expression and distribution of caveolin in HLECs under H₂O₂ stimulation and cholesterol depletion were determined to assess the possible roles of caveolin in cell signal transduction and cholesterol trafficking. And the possible relationship between the caveolin and cataract formation are also explored.Methods: SRA01/04 HLECs were exposed to H₂O₂ or MβCD of various concentrations and durations. We used a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrozolium bromide (MTT) assay to measure the effect of H₂O₂ on the proliferation of SRA01/04 HLECs. The distributions of caveolin after oxidative stimulation were probed by fluorescence microscopy and laser scanning confocal microscopy. Western blot was performed to analyze the alterations of caveolin expression and caveolin-1 phosphorylation. And also, we observed the caveolae distribution in HLECs by electron microscopy.Results: We observed that the proliferation of SRA01/04 HLECs under 0.1 mM H₂O₂ for 10 min or longer, or 1.0 mM for 5 min or longer was significantly reduced (p<0.05, F=11.63; or p<0.05,F=185.984, respectively). Laser scanning microscopy showed immunofluorescent caveolin in SRA01/04 HLECs under 1.0 mM H₂O₂ for 10 min or longer, caveolin were largely confined to intracellular domains. SRA01/04 HLECs under 10 mM MβCD for 5 min or longer became remarkably smaller in size under fluorescence microscopy. Western blot 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 0.1mM (p<0.05, F=6.843; or p<0.05, F=7.944, respectively) and 1.0 mM (p<0.05, F=6.242; p<0.05, F=5.457, respectively) H₂O₂ for different durations also down regulated. Western blot showed that the membrane and total caveolin protein level down regulated when treated with 10mM MβCD. The membrane caveolin under 10mM M(3CD for 15min or longer showed significant down regulation (p<0.05, F=9.890), whereas the total caveolin showed no significant down regulation (p>0.05, F=1.480). Western blot also showedcaveolin-1 was phosphorylated on tyrosine 14 in SRA01/04 HLECs after stimulated with 1.0 mM H₂O₂ for 60 min. Fluorescence microscopy also showed that phosphorylated caveolin-1 was distributed near the focal adhesions of the cells. On electron microscopy, the surface of untreated HLECs exhibited plasma membrane invaginated to form omega- or flask-shaped caveolae in a small amount.Conclusions: This study concludes that the responses of HLECs to oxidative stress may include the suppression of the proliferation of HLECs, down regulation of caveolin and tyrosine 14 phosphorylation of caveolin-Land MβCD also down regulates caveolin while depleting cholesterol in HLECs. These results show that HLECs contain caveolae and caveolin/caveolin-1. Abundant cholesterols are necessary for maintenance the function of caveolae. Caveolin might involve in the signal transduction of HLECs. And tyrosine 14 phosphorylation of caveolin-1 is a key factor that caveolin participate in the signal transduction. Oxidative stress down regulating caveolin might be one of the many mechanisms, which result in the formation of cataract.