The Study Of JNK/c-Jun Pathway And Its Target Gene Bim And PUMA On Oxidative Stress Induced Apoptosis Of Human Lens Epithelial Cells

1PrefaceA cataract is any opacity in the lens. Aging is the most common cause of cataract. Cataract is one of the most common causes of visual impairment. The mechanism of the etiology and pathogenesis of cataract is unclear. Many studies have been focused on cataract in order to explore the effective cure for cataract.Many factors can induce the development of cataract. Oxidative Stress is believed the initial factor that can induce the development of cataract. Recently, studies on the mechanism of cataract focus on the cellular levels and molecular biology. Human lens epithelial cells are important to keep the transparency of lens and the internal environment homeostasis. Study shows that the apoptosis of human lens epithelial cells is the cellular basis of all cataracts except congenital cataract, so the key point of preventing congenital and acquired cataract is how to regulate the apoptosis of human lens epithelial cells.PUMA and Bim are important members of BH3-only Bcl-2family which play an important role in many diseases and the initial period of programmed cell death (apoptosis) induced by various factors. The effect of Bim and PUMA on apoptosis of human lens epithelial cells (HLEC-B3) induced by oxidative stress has not been reported so far. To Investigate the effect of PUMA and Bim on apoptosis of human lens epithelial cells (HLEC-B3) induced by oxidative stress and to clarify the mechanism of cataract further can set a preliminary theoretical basis for gene therapy.JNK signal pathway is involved in many physiological and pathological processes. Activated JNK signal pathway can activate lots of effector proteins including transcription factors which could play biological action. C-jun is the most important downstream factor of JNK, and its product proteins can develop to AP-1. AP-1is mainly target of JNK/SAPK in cells. In the activation conditions, JNK can phosphorylate c-jun and increase its transcriptional activity. Whether protein c-jun induce apoptosis or inhibit apoptosis depent on cell types. JNK/c-jun signal pathway in apoptosis of human lens epithelial induced by oxidative stress has not been reported.The regulation of PUMA and Bim differ greatly in different cells, such as transcription levels^post-translational modifications and the regulation of protein levels. Moreover, Bim can be regulated by many signal pathways, such as PI3K/Akt、 MLK/JNK, RAS/MAPK and so on.In the process of the apoptosis of human lens epithelial, it is unclear that how to increase the expression of PUMA and Bim. It is a new way to cure cataract that to know the mediated way further and to inhibit the expression of PUMA and Bim by inhibiting the activity of special transcription factor.2PurposeTo investigate the effect of Bim and PUMA on oxidative stress induced apoptosis of human lens epithelial cells (HLEC-B3); To investigate the relationship between the activation of JNK/c-Jun pathway and the upregulation of its target gene Bim and PUMA in oxidative stress-induced apoptosis of human lens epithelial cells (HLEC-B3), so we can get a further illustration of molecular mechanism of apoptosis in human lens epithelial cells.3Methods3.1Cell culture The HLEC-B3is cultured in DMEM with10%FBS in a37℃incubator with5%CO2atmosphere;3.2The apoptosis of cellsHuman lens epithelial cells cultured in vitro were treated with hydrogen peroxide. Apoptosis was quantified by counting the cells with pyknotic nuclei after staining with Hoechst33258.;3.3Western blot analysisCaspase-3and JNK were detected by western blot, c-Jun、Bim and PUMA were detected by western blot;3.4RT-PCRRT-PCR was applied to detect the mRNA level of c-Jun、Bim and PUMA;3.5Small interferingThe ability of JNK/c-Jun pathway inhibitor CEP11004and SP600125to protect the HLEC apoptosis was observed; the ability of Bim or PUMA knockdown to protect the oxidative stress-induced apoptosis of lens epithelial cells was observed.4Results4.1The oxidative stress induced apoptosis of human lens epithelial cells (HLEC-B3).After treated with hydrogen peroxide at50μM concentration for4h、8h and12h, a high fraction of cells exhibited apoptosis-like changes, such as detachment and cytoplasmic-condensation leading to rounding. Apoptosis happened in4.30%±1.15%、27.08%±0.74%、46.59%±0.91%of the cells, and the expression of apoptosis related protein Caspase-3increased with the time increasing. To summarize, the oxidative stress can induce typical apoptosis of human lens epithelial cells (HLEC-B3).4.2In the apoptosis of human lens epithelial cells induced by oxidative stress, the JNK/c-jun signal pathway is activated, and the expression of PUMA and Bim is increased.The phosphorylation of JNK is increased significantly in a time-dependent manner and the JNK protein levels remained unchangeably. The JNK signal pathway is activated and then to phosphorylate downstream substrate c-jun to increase the transcriptional activity of c-jun. The level of c-jun phosphorylation is upregulated remarkably at2h after treated with H2O2,and it is consistent with the tendency of the phosphorylation of JNK. The results proved that the JNK/c-jun signal pathway is activated during the apoptosis of human lens epithelial cells (HLEC-B3) induced by oxidative stress.We examined the expression of PUMA and Bim at the protein levels during the apoptosis of cells. The protein of PUMA and Bim increased significantly at4h which is earlier than the moment when the morphologic changes of HLEC-B3cells happened. The mRNA expression of PUMA and Bim is increased dramatically at2h. To sum up, oxidative stress can induce the apoptosis of HLEC-B3cells and also induce the upregulation of apoptosis related protein PUMA and Bim.4.3The JNK/c-jun signal pathway mediated the upregulation of PUMA and Bim in the apoptosis of human lens epithelial cells induced by oxidative stress.The human lens epithelial cells were treated with H2O2. H2O2and CEP11004(MLK inhibitor). H2O2and SP600125(JNK inhibitor) for four hours. The increased expression of c-jun protein induced by oxidative stress can be inhibited by CEP11004and SP600125which can block the activity of JNK signal pathway. The upregulation levels of PUMA and Bim can be inhibited significantly by CEP11004and SP600125. The results suggested that the upregulation of PUMA and Bim is mediated by JNK signal pathway, and the inhibitor of JNK signal pathway can reduce the apoptosis of human lens epithelial induced by oxidative stress from43.49%±1.05%to13.29%±0.57%(CEP11004) and15.89%±1.03%(SP600125)4.4siPUMA and siBim protect human lens epithelial cells from apoptosis induced by oxidative stress.The effectiveness of siRNA is need to be verified. To the most effective siRNA,24hours after transfected into human lens epithelial cells, the cells were treated with H2O2for8hours. We examined the protein expression of PUMA and Bim by western blot. The results proved that siPUMA and siBim effectively inhibited the upregulation of PUMA and Bim protein induced by oxidative stress separately. After verifying the effectiveness of siRNA, we observed the apoptosis of human lens epithelial cells (HLEC-B3) induced by oxidative stress after knocking out PUMA or Bim. Apoptosis happened in47.70%±0.42%after treated with hydrogen peroxide for12h, and decreased to25.50%±1.02%(siBim1) and30.39%±0.62%(siBim2) when Bim was knocked out; Similarly, apoptosis of human lens epithelial cells (HLEC-B3) decreased from44.50%±0.63%to20.51%±0.78%(siPUMA1)and23.23%±0.58%(siPUMA2) respectively after PUMA was knocked out. These results suggest that knocking out Bim or PUMA have the protective effect on HLEC-B3, and PUMA/Bim is involved in apoptosis of human lens epithelial cells (HLEC-B3) induced by the oxidative stress.5ConclusionsPUMA、Bim were increased in the apoptosis of human lens epithelial cells induced by hydrogen peroxide which is related with JNK/c-Jun pathway. So JNK/c-Jun pathway and its target gene Bim and PUMA were involved in the apoptosis of human lens epithelial cells induced by hydrogen peroxide, and maybe play an important role in the development of cataract. The study further discuss the apoptosis mechanism of human lens epithelial cells and provide more theoretical basis for the prevention and therapy of cataract.