| Background and ObjectivePosterior capsule opacification (PCO) is the most common postoperative complication. The development of PCO is a combination of the processes of proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs) on the lens capsule triggered by many cytokines. Among these, tumor necrosis factor alpha (TNF-α), an important inflammatory mediator, has been detected at a higher concentration after cataract extraction and was reported to have consequences for PCO. Treatment with COX-2 inhibitors appeared to prevent PCO formation in both animal and ex vivo models. However, the molecular mechanisms of PCO and pharmacological effects of COX-2 inhibitors are still unclear.The aims of the present study were to determine the regulation system of COX-2 induced by TNF-α, to investigate the role of COX-2 in TNF-α-induced cell proliferation and EMT and to explore the potential mechanism in hLEC-B3.MethodsThe hLEC-B3 cells were divided into two groups:group exposed to 10 ng/ml TNF-a and control group. The mRNA and protein expression levels of COX-2 in hLEC-B3 exposed to TNF-α for 6 h,24 h and 48 h were detected by RT-qPCR and western blotting assay, respectively. The expression levels of cyclin D1 and vimentin, the biomarkers of proliferation and EMT respectively, were also detected in hLEC-B3 exposed to 10 ng/ml TNF-a for 24 h. Then the cell proliferation level was measured by Cell Count Kit-8 kit (CCK-8) and the migration ability of cells in vitro was examined by transwell assay. Moreover, the morphological change of hLEC-B3 exposed to TNF-a was observed under bright field microscopy. To further identify the role of COX-2 in TNF-a-induce cell proliferation and EMT, the COX inhibitor, bromfenac, was used at 24 h before TNF-a exposure.The expression levels of phosphorylated JNK and the transcriptional factor c-Jun in hLEC-B3 exposed to 10 ng/ml TNF-a were detected by western blotting assay to observe the time-effect. The immunofluorescence assay and nucleus protein analysis were used to exhibit the nuclear translocation of p-c-Jun. To study the role of JNK pathway in regulating the expression of COX-2, the cells were pretreated with JNK specific inhibitor SP600125 or transfected with c-Jun siRNA, and the direct interaction between p-c-Jun and DNA sequence of COX-2 promoter region was identified by chromatin immunoprecipitation (ChIP) assay.Further, the effects of TNF-a on the activation of GSK-3β/β-catenin signaling and β-catenin-driven transcriptional activity were studied by western blotting assay and dual luciferase reported gene assay. Then the cells were pre-treated with bromfenac or SP600125 to inhibit COX-2 or JNK pathway, exploring the association of JNK/COX-2 with GSK-3β/β-catenin signaling.ResultsThe expression levels of COX-2 mRNA and protein were both elevated with time in TNF-a-stimulated hLEC-B3 cells. The cell proliferation level and migration ability also increased with higher expression of Cyclin Dl and Vimentin in hLEC-B3 cells exposed to TNF-a for 24 h. Moreover, the morphology of cells changed significantly, becoming elongated with irregular arrangement among cells after exposure to TNF-a for 24 h and 48 h. Blockage of COX-2 with pretreatment of bromfenac inhibited the increased expression of Cyclin Dl and Vimentin induced by TNF-a. Also, the cell proliferation level and EMT features were attenuated.TNF-a rapidly activated JNK pathway with the phosphorylation of JNK and c-Jun. Either inhibition of JNK pathway by SP600125 or transfection with c-Jun siRNA inhibited the increased expression of COX-2 induced by TNF-a. ChIP-qPCR results showed a direct interaction between p-c-Jun and COX-2 promoter region.TNF-a could phosphorylate GSK-3β and increase the nuclear accumulation of β-catenin, resulting in an elevation of β-catenin-driven transcriptional activity. Blockage of COX-2 or inhibition of JNK pathway attenuated the GSK-3β/β-catenin signaling and β-catenin-driven transcriptional activity. Knockdown of β-catenin suppressed the pharmacological effect of bromfenac on the inhibition of increased expression of Cyclin D1 and Vimentin induced by TNF-α.ConclusionTNF-a up-regulates the expression of COX-2 through an activation of JNK pathway in hLEC-B3. JNK/COX-2 contributes to TNF-α-induced cell proliferation-and EMT in hLEC-B3 via GSK-3β/β-catenin signaling. |
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