Cigarette Smoke Induced Urocystic Epithelial-mesenchymal Transition In SV-HUC-1 Cells Via ERK5 Pathway

Objectives Bladder cancer is universally acknowledged as a significant public health problem worldwide. Abundant evidence shows that cigarette smoke is the primary risk factor for bladder cancer. However, the mechanism of cigarette smoke-induced bladder cancer has not been fully elucidated. Cigarette smoke(CS)-induced epithelial-mesenchymal transition(EMT) is critically involved in cell malignant transformation. The role of ERK5, the lesser studied member of MAPKs family, in regulating CS-triggered EMT has not been investigated. The objective of this study was to investigate the regulatory role of ERK5 in CS-induced urocystic EMT.Methods SV-40 immortalized normal human urothelial cell(SV-HUC-1 cell) was used as in vitro CS exposure models. EMT phenotypic alterations were assessed by changes in cell morphology, invasive capacities, as well as epithelial and mesenchymal markers’ expression. Protein and m RNA expressions were analyzed by Western blotting, and quantitative reverse transcriptase–polymerase chain reaction(q RT-PCR).ERK5 inhibition studies were performed with XMD8-92,which is a specific inhibitor of ERK5.Results(1)We screened 0%,0.25%,0.5%,1% of CSE as experimental dose by MTT assay. Meanwhile, we found that Treatment of SV-HUC-1 cells with CSE for 5 days resulted in significant morphological change from urothelial oblate-shaped to spindle-like mesenchymal form. Additionally,the cells became dispersive and some SV-HUC-1 cells even generated tail-like change after treatment with CSE.(2) transwell assays were carried out to analyze SV-HUC-1 cell invasive capacities in response to CSE. invasion of cells through reconstituted matrigel matrices was enhanced by CSE.(3) Exposure of SV-HUC-1 cells to CSE resulted in decreased protein expression of the epithelial markers E-cadherin and ZO-1 in both protein and m RNA level. In contrast, mesenchymal markers Vimentin and N-cadherin were increased.(4) Exposure of SV-HUC-1 cells to CSE for 5 days dose-dependently activated phosphorylated ERK5(p-ERK5) level and simultaneously restrained total ERK5 level,suggesting the stimulative effect of CS on ERK5 activity. Meanwhile, CS increased the activation of AP-1 protein in SV-HUC-1 cells, as indicated by elevated levels of phosphorylated-c-Jun(p-c-jun) and phosphorylated c-Fos(p-c-fos).(5) SV-HUC-1 cells were treated 5 days with 5 μM XMD8-92, a highly specific ERK5 inhibitor. As expected, XMD8-92 down-regulated p-ERK5 level and levels of p-c-jun and p-c-fos in SV-HUC-1 cells. XMD8-92 treatment reversed the mensenchymal-like morphological change of the cells. Moreover, XMD8-92 also restrained the invasive capacities of SV-HUC-1 cells, as determined with transwell assays. Furthermore, inhibition of ERK5 by XMD8-92 resulted in up-regulation of the protein and m RNA levels of the epithelial markers E-cadherin and ZO-1, as well as down-regulation of the mesenchymal markers Vimentin and N-cadherin. Therefore, these results suggested that ERK5 activity may play an important role in CS-induced EMT in SV-HUC-1 cells.Conclusion In summary, CS induced EMT in SV-HUC-1 cells.ERK5 positively regulated CS-induced urocystic EMT in in vitro settings. These novel findings indicated the important role of ERK5 activity in cigarette smoke-related carcinogenesis and provide a plausible strategy for the search of potential interventional target of cigarette smoke-associated bladder tumorigenesis.