EIF2α Phosphorylation And ATF6 Are Involved In Benzo(a)pyrene-7, 8-dihydrodiol-9, 10-epoxide Induced Cellular Stress Response

Benzopyrene(BaP) is a representative polycyclic aromatic hydrocarbon(PAH) that is generated as a result of combustion and is found in significant concentrations in tobacco smoke.BaP is a procarcinogen requiring metabolic activation by cytochrome P450-dependent oxidations and epoxide hydrolysis to form the ultimate carcinogen benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide(BPDE).BPDE covalently reacts with the exocyclic amino groups of deoxyguanosine to form bulky adducts and causes DNA damage,leading to mutagenesis and carcinogesis.BPDE induced-DNA damage can block the cell cycle progression and induce apoptosis.On the other hand,cells can elicit a series of defensive and protective mechanisms,such as base-excision repair, translesion synthesis(TLS),and ER stress to eliminate the damages.Our microarray data show that BPDE induced expression of genes involved in multiple functions including cell survive,cell cycle regulation,apoptosis and so on.We used Promoter Analysis Pipeline(PAP, http://bioinformatics.wustl.edu/webTools/PromotorSearch.do) to analyse the 0.5μM BPDE induced up-regulated genes,and found that some of them have relationship with stress related transcriptional factors such as AP-1 family(AP-1,c-FOS and ATF2:c-Jundimer),CREB/ATF family(CREB,ATF4,ATF6 and ATF3),NF-κB family(p50,p65 and c-REL),Elkl,and p53.Among these transcriptional factors,the activation of CREB/ATF family members hasn't been reported before.Therefore,in this study,we examined the activation of CREB/ATF family members ATF4,ATF6 and the upstream regulator of ATF4,theα-subunit of the Eukaryotic Initiation Factor-2(eIF2α) after BPDE exposure,and analysed the possible function of these changes in the cellular stress response to BPDE.eIF2αis an important contributor for eukaryotic cells to recognize and process diverse stress signaling,which elicits programmes of gene expression that are designed to alleviate cellular damage or alternatively induce apoptosis.Four different eIF2αkinases have been identified so far in mammals,and each actives distinct stress signaling pathways,eIF2αis required for the transfer of the initiator Met-tRNA_i^Met (aminoacylated initiator methionyl-tRNA) to the small ribosomal subunit. Phosphorylation at Ser 51 abrogates the function of eIF2αand leads to a shutdown of global mRNA translation,along with increased translation of a few selected mRNA such as ATF4,ATF5,and consequently mobilizes stress-induced gene expression involved in cell growth,differentiation and apoptosis.ATF6(ATF6αand ATF6β) is a transcriptional factor activated by ER stress.It is translocated from ER to Golgi apparatus and cleaved by the site-1 protease(SP1) and the site-2 protease(SP2) during ER stress.The N-terminal fragments will be released from membrane and enter into nuclei and activate the transcription of their target genes.ATF6αand ATF6βshare significant sequence homology and both are proteolytically processed during ER stress, suggesting they may be regulated in a similar way.However,recent evidence has shown that ATF6βis a poor transcriptional activator and can inhibit activation of ATF6α.The experiments described here are based on ATF6α.In this study,we found that phosphorylation of eIF2αwas induced in normal human FL amnion epithelial cell line,and the expression of ATF4,a conserved downstream transcriptional factor of eIF2αphosphorylation,was up-regulated after BPDE exposure.However,the four known kinases for eIF2αphosphorylation were not found activated.While BPDE induced severe cell cycle arrest and apoptosis and decreased cell viability,salubrinal,a selective inhibitor of eIF2αdephosphorylation to maintain the eIF2αphosphorylation,attenuated cell cycle arrest and apoptosis and promoted cell survival.The full-length ATF6αreduced,indicating the involvement of ATF6 in BPDE induced cellular response and the possibility of ATF6 cleavage after BPDE exposure.We conclude that eIF2αphosphorylation and the activation of ATF4 and ATF6 are integral to cellular pathways induced by BPDE.When BPDE causes cellular damages,it induces eIF2αphosphorylation as well,which produces a pro-survival and anti-apoptotic effect to alleviate the cellular damages.Thus,the present study proposes a new cellular defensive mechanism during the environmental mutagen and carcinogen attack.