The Mechanism Of EGF Induced Cellular Senescence In Human Fetal Lung Fibroblasts

Background and purpose: Cellular senescence is a state that characterized by irreversible cessation of cell growth.The characteristics of senescent cells usually include increased ?-galactosidase activity,DNA damage accumulation,formation of senescence-associated heterochromatin foci(SAHF)and upregulation of senescence-related proteins and so on.Epidermal growth factor is an important mitogen.Currently,most studies have demonstrated that EGF inhibits cellular senescence and extends lifespan of cells.Only a few studies have proposed that EGF inhibits cell growth and induces senescence or apoptosis in cells,but the underlying mechanism has not been elucidated.In this research,it has been found that EGF treatment induces cellular senescence in IMR90 cells,also called human fetal lung fibroblasts,so the main purpose of this research is to investigate the underlying mechanism of EGF-induced cellular senescence in IMR90 cells.Methods and results: In our current study,we discovered that EGF treatment induces cellular senescence in IMR90 cells.At first,we found that EGF treatment inhibited the proliferation of IMR90 cells,and this finding was confirmed by cell growth curves and Brd U incorporation assay.Since we observed that cells presented the phenotypes of senescent cells after being treated with EGF,we proved that EGF induced senescence in IMR90 cells by SA-?-gal staining and DAPI staining.On this basis,the results of immunofluorescence and immunoblotting indicated that DNA damage response was activated as showed by formation of 53BP1 foci and upregulated protein levels of ?H2A.X and 53BP1 and senescence-related signaling pathways were activated as showed by upregulated protein levels of p38,p-p38,p53,p-p53,p21 and p16.Next,we investigated the mechanism underlying EGF-induced senescence in IMR90 cells.We proved that the Ras signaling pathway was activated after EGF treatment as showed by upregulated protein levels of Ras-GTP and p-ERK1/2,which is detected by Ras-GTP assay and Western blotting.Moreover,BRaf knockdown attenuated EGF-induced cellular senescence in IMR90 cells.Therefore,we concluded that EGF induces senescence in IMR90 cells through activating the Ras-Raf-MEK-ERK pathway.In addition,we found that EGF-induced cellular senescence is related to cell cycle and we demonstrated that IMR90 cells in G2/M phase are prone to be senescent after treatment with EGF.Conclusions and implications: In summary,we reported an interesting phenomenon that EGF induces cellular senescence in IMR90 cells through activating the Ras signaling pathway and then inducing the activation of DNA damage response and senescence-related signaling pathways.These results will be beneficial to enrich the knowledge of biological function of EGF and contribute to the researches on the mechanism of EGF-induced cellular senescence.