| BackgroundThe accumulation of senescent cells and chronic inflammation may lead to the development of aging and aging-associated diseases.Senescent cells can secrete a variety of senescence-associated secretory phenotype(SASP)factors,form a worsening microenvironment,and cause chronic inflammation.In addition,SASP factors can diffuse locally or even throughout the body by paracrine or humoral circulation.It has been known that some SASP factors can induce senescence in normal cells,and the diffusion of SASP factors will make tissues,organs and even the whole body in an environment prone to senescence.Therefore,the aims of this study are to screen and identify SASP factors that induce cellular senescence,and to explore the molecular mechanisms of SASP factors inducing cellular senescence.Methods(1)We built the screening library of SASP factors and used different concentrations of SASP factors to treat the cell line derived from human foetal lung fibroblast(IMR90).Senescence-associated heterochromatic foci(SAHF)detection and senescence-associated-?-galactosidase(SA-?-gal)staining experiments were completed to screen the SASP factors that induced cellular senescence.(2)We used different concentrations of SASP factors to treat human umbilical vein endothelial cells(HUVECs).SAHF detection and SA-?-gal staining experiments were performed to screen the SASP factors that induced cellular senescence.(3)The screening results of IMR90 and HUVEC cells were statistically analyzed to identify the SASP factors inducing cellular senescence in both cell lines.(4)SASP factors which can induce cellular senescence in normal cells were used to treat cancer cells.MTT,SAHF detection and SA-?-gal staining were used to investigate whether these SASP factors can induce senescence in cancer cells.(5)The models of quiescent IMR90 cells was constructed.SASP factors which can induce cellular senescence in normal cells were used to treat the quiescent cells.SAHF detection was used to investigate whether these SASP factors can induce senescence in quiescent cells.(6)Epidermal growth factor receptor(EGFR)signaling pathways were inhibited through EGFR inhibitors or si RNAs,and SASP factors which can induce cell senescence were also used to treat cells.SAHF detection and SA-?-gal staining were performed to investigate whether cellular senescence induced by SASP factors is through EGFR pathways.(7)SAHF detection,SA-?-gal staining,Brd U incorporation and other experiments were performed to investigate whether EGFR activation induced cellular senescence of IMR90 and HUVEC cells.(8)EGF were used to activate EGFR signaling pathways,and q RT-PCR and enzymelinked immunosorbent assays were performed to detect the changes of m RNA and protein levels of some classic SASP factors(Gro-?,IL-6,IL-8,MMP3),and to investigate whether EGFR activation promoted the secretion of SASP factors.(9)IMR90 cells and HUVEC cells were treated with different concentrations of EGF to activate EGFR signaling pathways.GST pull-down and MTT assays were used to explore the effects of EGFR activation on Ras activities and cell proliferation.(10)Western blotting and immunofluorescence were used to explore the effects of EGFR activation on Ras induced senescence(RIS)related signaling pathways.(11)ERK1/2 were inhibited with inhibitors or si RNAs,and EGF was also used to treat the cells.Then,SAHF detection,SA-?-gal staining,Brd U incorporation,cell growth curves,Western blotting,and immunofluorescence assays were performed to investigate whether EGFR activation induced cellular senescence through Ras-Erk1/2 pathways.Results(1)SASP factors which are most closely related to aging,aging-associated diseases and cellular senescence were selected for screening and study according to the literatures and important reviews.Twenty-one SASP factors were screened in human lung fibroblast cell line IMR90 cells.Results of SAHF detection and SA-?-gal staining showed that eleven SASP factors(Gro-?,IL-1?,IL-6,IL-8,IL-13,KGF,MCP-2,MCP-3,MIP-3?,SDF-1? and TGF-?1)induced cellular senescence of IMR90 cells.(2)Twenty-one SASP factors were screened in three primary HUVEC lines derived from different donors.SAHF detection and SA-?-gal staining demonstrated that seven SASP factors(IL-1?,IL-13,IL-15,MCP-2,MCP-3,MIP-3? and SDF-1?)induced cellular senescence of three primary HUVEC lines.(3)Summarizing the screening results of(1)and(2),we found that six SASP factors(IL-1?,IL-13,MCP-2,MCP-3,MIP-3? and SDF-1?)induced cellular senescence in all normal cell lines.(4)As an important mechanism for tumor suppression in vivo,cellular senescence is closely associated with the occurrence and development of cancer.The above SASP factors(IL-1?,IL-13,MCP-2,MCP-3,MIP-3? and SDF-1?)were investigated whether they can induce senescence in cancer cells.The results of MTT showed that SASP factors did not inhibit proliferation of cancer cells.The results of SAHF detection and SA-?-gal staining showed that SASP factor treatment did not induce senescence in cancer cells.These results suggest that these SASP factors are more closely associated with cellular senescence of normal cells.(5)Since most cells in the body are in quiescent state,it is interesting to study whether the SASP factors(IL-1?,IL-13,MCP-2,MCP-3,MIP-3? and SDF-1?)induce cellular senescence in quiescent phase.SAHF detection showed that SASP factors did not induce the formation of SAHF in quiescent cells.These results suggest that these SASP factors are more closely associated with cellular senescence of normal cells.(6)Ras is an important downstream effector of EGFR signaling pathway.Overactivation of Ras signaling pathway can induce cellular senescence,and is one of the classic cellular senescence signaling pathways.Therefore,whether SASP factors can induce cellular senescence through EGFR-Ras signaling pathway is an interesting question.Our results showed that inhibition of EGFR effectively inhibited cellular senescence induced by five SASP factors(IL-1?,IL-13,MCP-2,MIP-3? and SDF-1?),but did not inhibit senescence induced by MCP-3.(7)EGF is one of the most important ligands of EGFR.Activation of EGFR by EGF can study the effects of EGFR activation on cellular senescence.The formation of SAHF,the enhancement of SA-?-gal activities and the decrease of cell proliferation indicated that EGFR activation can indeed induce cellular senescence.(8)SASP is one of the important characteristics of cellular senescence.The senescent microenvironment can be formed by SASP factors and is closely associated with aging.Therefore,whether EGFR activation can induce expression and secretion of SASP factors and further amplify senescence signaling is a very interesting question.The results of q RT-PCR and enzyme-linked immunosorbent assays showed that EGFR activation up-regulated the m RNA levels of IL-8 and MMP-3 and enhanced the secretion of IL-8 and MMP-3 proteins.In addition,SASP factors also promoted the secretion of IL-8 and MMP-3.These results suggest that senescent cells induced by SASP factors through EGFR activation may induce cellular senescence in more normal cells by promoting the expression and secretion of SASP factors such as IL-8 and MMP-3 and amplifying the senescent microenvironment.(9)Ras is an important downstream effector of EGFR.Overactivation of Ras signaling pathway often induces cellular senescence.Therefore,the effects of EGFR activation on Ras and its downstream signaling pathways and the molecular mechanisms were investigated.The results of GST pull-down and MTT assays showed that Ras activities were increased with the increase of EGF concentration,and the effects of EGF changed from promoting cellular proliferation to inducing cellular senescence.(10)Ras activation can induce cellular senescence through RIS related signaling pathways.The up-regulation of proteins(p-p38,p38,p-p53,p53,p21 and p16)involved in RIS signaling pathways and biomarker proteins(53BP1 and ?H2AX)of DNA damage,and the increase of positive cells of 53 bp foci suggested that RIS related signaling pathway can be activated by EGFR signaling pathway.(11)Ras signaling depends on Ras-Erk1/2 signaling cascades,and Erk1/2 plays a key role in cellular senescence induced by Ras.The results of SAHF detection,SA-?-gal staining,Brd U incorporation,cell growth curve,Western blotting,and immunofluorescence assays showed that Erk1/2 inhibition(with inhibitor LY3214996 or Erk1/2 knockdown)effectively inhibited cellular senescence induced by EGFR activation.The above results confirmed that EGFR activation induced cellular senescence by activating Ras-Erk1/2 pathway.ConclusionIn this study,six SASP factors(IL-1?,IL-13,MCP-2,MCP-3,MIP-3? and SDF-1?)were found to induce cellular senescence in human normal cells by systematic screening.Five SASP factors(IL-1?,IL-13,MCP-2,MIP-3? and SDF-1?)were found to induce cellular senescence by activating EGFR and the downstream Ras-Erk1/2 signaling pathways in further studies.In addition,EGFR activation up-regulated the m RNA levels of IL-8 and MMP-3 and increased the secretion of IL-8 and MMP-3.These results suggest that cellular senescence induced by SASP factors through activating EGFR signaling pathways can expand the senescent microenvironment.This study revealed a novel mechanism of SASP factors promoting cellular senescence through EGFR activation,and suggested that growth signals can be transformed into senescence signals under certain circumstances.This study not only strongly promotes the theoretical research of cellular senescence induced by SASP factors,but also provides experimental foundation and theoretical guidance for a deeper understanding of development of aging and healthy aging. |
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