Role Of MsrB1(SelR) In Cytoprotection Against Peroxynitrite-induced Damage In Human Lens Epithelial Cells

Cataract is a visible opacity in the lens substance, which poses a substantial economicand public health burden and is the leading cause of blindness worldwide. Peroxynitrite(ONOO) is the reaction product of superoxide (O2) and nitric oxide (NO). The moleculerapidly breaks down at physiological pH to yield the hydroxyl radical (·OH) and nitrogendioxide radical (·NO2). As a potent oxidant and nitrating species, ONOO is proposed tobe an effector of cell damage in selenite cataract and diabetic cataract, by oxidizing and/ornitrating biological molecules including proteins, lipids, and DNA. Previous studies haveproved that ONOO can oxidize Met residues of proteins to MetO, and the formation ofnitrotyrosine is considered a footprint of oxidative damage mediated by ONOO. Otherselenoproteins, such as glutathione peroxidases, thioredoxin reductase and selenoprotein Phave been shown to play a potential role in protection against ONOO. However, the roleof MsrB1, which is a selenoprotein named selenoprotein R, in cellular protection againstONOO-induced hLE cells damage remains to be evaluated, and a role in regulatingcertain cell signaling molecules and certain proteins remain unclear. So the role of MsrB1in cataract formation and development needs a further study.In this paper, the role and mechanism of MsrB1in protection againstONOO-induced hLE cells damage were investigated. The main results are as follows:(1) Involvement of MsrB1in the regulation of redox balance and inhibition ofperoxynitrite-induced apoptosis in hLE cells.In an attempt to shed light on the roles of MsrB1, known as selenoprotein R, inprotecting hLE cells against peroxynitrite damage, and contribution of loss of its normalactivity to cataract, the influences of MsrB1gene silencing on peroxynitrite-inducedapoptosis in hLE cells were studied by real time RT-PCR analysis, MTT assay, Westernblotting, transmission electron microscopy, fluorescence microscopy, flow cytometry,ROS and MDA measurement kit. The results showed that low concentrations of ONOO markedly stimulated the proliferation of hLE cells, while the viabilities of hLE cellstreated with high concentrations of ONOO were significantly decreased; both exogenous peroxynitrite and MsrB1gene silencing by short interfering RNA (siRNA) independentlyresulted in oxidative stress, endoplasmic reticulum (ER) stress, activation of caspase-3aswell as an increase of apoptosis in hLE cells; moreover, when MsrB1-gene-silenced cellswere exposed to300μM peroxynitrite, these indexes were further aggravated at the sameconditions and DNA strand breaks occurred. The results demonstrate that in hLE cellsMsrB1may play important roles in regulating redox balance and mitigating ER stress asinduced by oxidative stress under physiological conditions; MsrB1may also protect hLEcells against peroxynitrite-induced apoptosis by inhibiting the activation of caspase-3andoxidative damage of DNA under pathological conditions. Our results imply that loss of itsnormal activity is likely to contribute to cataract.(2) MsrB1protects human lens epithelial cells against peroxynitrite-induced F-actindisruption.Previous studies have demonstrated that MsrB1can resist to oxidative stress and mayact as a ROS scavenger, as well as have a potential role in protection against ONOO. Inthe present study, the role of MsrB1in protecting hLE cells against ONOO-inducedF-actin disruption was studied by Western blotting, immunoprecipitation, U0126inhibitortreatment and immunofluorescence. The results showed that low concentrations ofONOO might induce assembly of F-actin, while high concentrations of ONOO mightdiminish assembly of F-actin, and when MsrB1-gene-silenced hLE cells were exposed toONOO, F-actin protein levels were further decreased; Low concentrations of ONOO activated ERK1/2and high concentrations of ONOO inhibited ERK1/2activation, whichwas consistent with the changes of assembly or disassembly of F-actin by low or highconcentrations of ONOO; The results of immunoprecipitation showed that disruption ofF-actin was accompanied by increase of F-actin nitration. Furthermore, bFGF-activatedERK1/2phosphorylation occurs via a MEK-dependent pathway, whereasONOO-activated ERK1/2phosphorylation occurs via a MEK-independent pathway. Insummary, both high concentrations of ONOO and MsrB1gene silencing by siRNAindependently increased disassembly of F-actin and induced inactivation of ERK in hLEcells; moreover, MsrB1gene silencing could significantly aggravate disassembly ofF-actin by ONOO through increase of nitration of F-actin and inactivation of ERK, suggesting that MsrB1may play important roles in protecting hLE cells againstONOO-induced F-actin disruption by inhibiting nitration of F-actin and inactivation ofERK. These results imply that loss of its normal activity is likely to influence the lens cellelongation and differentiation.(3) Role of MsrB1in cytoprotection against BCS-induced oxidative stress in hLEcells.In the present study, the role of MsrB1in protecting hLE cells against BCS-inducedapoptosis and oxidative stress were studied by MTT assay, Cu/Zn-SOD and ROSmeasurement kit，fluorescence microscopy and Western blotting. The results showed thatboth BCS (1000μM) and MsrB1gene silencing by short interfering RNA (siRNA)independently resulted in oxidative stress, accumulation of ROS levels and an increase ofapoptosis in hLE cells; moreover, when MsrB1-gene-silenced cells were exposed to1000μM BCS, ROS levels and cell apoptosis were further aggravated at the same conditions.Whereas the Cu/Zn-SOD activity in MsrB1-gene-silenced cells was significantlyincreased, implying that MsrB1gene silencing by siRNA induces an increase of ROSlevels and than stimulate the increase of Cu/Zn-SOD activity. Moreover, the results ofWestern blotting showed that BCS and MsrB1gene silencing induced hLE cell apoptosisaccompanied by the inhibition of ERK1/2activation, since the formation of ONOO induced ERK nitration. In addition, the protein nitration detection results also confirmedthe generation of ONOO. In summary, both BCS and MsrB1gene silencing resultedincrease of apoptosis in hLE cells by accumulation of ROS levels and inhibition ofERK1/2activation. Our results suggest that MsrB1may protect hLE cells againstBCS-induced apoptosis by regulation of ERK1/2phosphorylation and nitration viascavenging ROS directly or indirectly.