The Mechanism Of Benzo[a]pyrene Facilitates SARS-CoV-2 Infection Via Trans-activating ACE2 And TMPRSS2 By Regulating NR4A2

BackgroundCoronavirus disease 2019(COVID-19),an emerging infectious disease caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),has become a global health concern and declared by the World Health Organization as pandemic.After multiple rounds of evolutionary mutations,its strain has stronger transmissibility and escape ability,and higher affinity for the novel coronavirus receptor.During SARS-CoV-2 infection,RBD of the S protein binds to the cellular receptor angiotensin-converting enzyme 2(ACE2)to facilitate viral entry Transmembrane protease serine 2(TMPRSS2),the priming activator of ACE2,is critical for priming the S protein to complex with ACE2 and bring SARS-CoV-2 to enter the recipient cells.External environmental stimulations such as smoking and internal environmental stimulations including aging-related inflammation,sexual hormone,and comorbidities up-regulate the expression of ACE2 and TMPRSS2.Most epidemiological surveys consider smoking to be a high risk factor for COVID-19 infection,It remains unknown if smoke activate the expression of ACE2 and TMPRSS2.The genetic predisposition may facilitate the transcriptional levels of ACE2 and TMPRSS2 induced by the environmental stimulations.The process by which SARSCo V-2 binds to cell surface receptors and enters cells is the focus of pathogenic mechanism research.Whether the effects of the above factors on ACE2 and TMPRSS2 affect viral infection remains to be clarified.ObjectiveThe purpose of this study was to elucidate the effects of exogenous exposure(smoking),and genetic factors(single nucleotide polymorphism)on the efficiency and severity of SARSCo V-2 infection,and to explore the effects and mechanisms of these factors on the expression of SARS-CoV-2 receptor ACE2 and TMPRSS2 in cells,mice and hamsters in vitro.We hope it will help to develop effective prevention and treatment programs for COVID-19,improve the ability to prevent novel coronavirus infection and better protect the high-risk population.MethodsThis study focuses on the mechanism of cigarette promoting SARS-CoV-2 receptor expression and virus infection,focusing on gene polymorphism and SARS-CoV-2 receptor,starting from two aspects of cell and animal model.1.The effects of cigarette smoke,benzopyrene,nicotine on the expression of ACE2 and TMPRSS2 were examined by q RT-PCR,WB。2.The SNPs of the upstream transcriptional regulatory region of ACE2 and TMPRSS2 genes was predicted by reading the literature and bioinformatics,and the corresponding plasmids were constructed.After transient cells,double luciferase reporter gene experiment was used to explore the effects of different genotypes and cigarette smoke extract,benzo pyrene and inflammatory factors on the transcriptional regulatory region of the receptor gene.3.The transcription factors binding to ACE2 and TMPRSS2 promoters were predicted by database,and the effects of cigarette smoke and its components on the expression level of selected transcription factors were confirmed by q RT-PCR,WB experiment.After the transcriptional molecules were screened,the si RNA of the transcription factor was designed based on double luciferase reporter genes,and the target gene expression was down-regulated by si RNA.The effect of knocking down the molecule on the activity of receptor promoter and the expression level of receptor was detected.4.The binding relationship between transcription factors and ACE2 and TMPRSS2 promoters was determined by chromatin co-immunoprecipitation,and the effects of cigarette smoke extracts and their components on their binding ability were determined.5.The lung tissue of mice was measured with sodium bisulfite pyrophosphate to compare the methylation rate of Cp G island upstream of transcriptional molecule promoter in different months.Combined with q RT-PCR,WB test,the expression of receptors and transcription factors in lung tissue was detected,and the effect of age on the expression of receptors and key transcription factors was explored.6.Pseudoviruses expressing GFP or luciferase were constructed by using virus S protein expression plasmid and different virus cytoskeleton.Flow cytometry and fluorescence intensity detection were used to verify the effects of cigarette smoke extract and its components and transcription factors on the level of virus infection.7.By injecting pseudoviruses expressing GFP into lung and testicular tissues of hamsters and mice,the effects of key components of cigarette smoke on virus infection in living tissues were identified in animal models by fluorescence development in vitro.ResultsCigarette smoke up-regulated the expression of ACE2 and TMPRSS2 via increasing their promoter activitiesThe transcription and protein levels of ACE2 and TMPRSS2 in lung cancer cell lines were measured 12 h after the stimulation with cigarette smoke extract(CSE)at different concentrations(10%,20%,and 50%).It was found that CSE at the concentration of 20% and50% significantly increased m RNA and protein levels of ACE2 and TMPRSS2 in Calu3 and H1650 cells.Our luciferase assays showed that CSE at the concentration of 20% significantly increased the activities of the ACE2 and TMPRSS2 promoters(-2000 bp to-1bp)in both Calu3 and H1650 cells.CSE significantly increased the activities of the ACE2 and TMPRSS2enhancers(-5000 bp to-2000bp)only in Calu3 cells not in H1650 cells.We further validated the above findings in 3 available transcriptome datasets from the Gene Expression Omnibus(GSE994,GSE8987,and GSE10718)and the dataset of The Cancer Genome Atlas lung adenocarcinoma(LUAD)cohort.The effects of exposure and duration of cigarette smoke on increased expression of TMPRSS2 were evident in GSE994,LUAD,GSE8987,and GSE10718 datasets.The effect of CSE on the transcription of ACE2 and TMPRSS2 was not influenced by functional genetic polymorphismsThe function of these five SNPs was confirmed by luciferase assays in Calu3 and H1650 cells.The activity of the ACE2 enhancer with rs112312217-G was significantly lower than that with rs112312217-A.rs112312217-G was mainly presented in African population rather than in East Asian and European populations.The activity of the ACE2 promoter with rs113208650-A was significantly higher than that with rs113208650-G.rs113208650-A was mainly presented in African population rather than in other races.The activity of the TMPRSS2 enhancer with rs9981570-G was significantly higher than that with rs9981570-A.The frequency of rs9981570-G was close to 40% in African,American,East Asian,and European populations.The activity of the TMPRSS2 promoter with rs12481984-G and rs812074-A was significantly higher than that with rs12481984-A and rs812074-A,whereas the activity of the TMPRSS2 promoter with rs12481984-A and rs812074-G was significantly lower than that with rs12481984-A and rs812074-A.rs8128074-G was more frequent in African population while rs12481984-G was less frequent in East Asian population.However,the genotypes of these SNPs did not influence overall effects of CSE on the transcriptions of ACE2 and TMPRSS2.Ba P is an active CSE ingredient that up-regulate the expression of ACE2 and TMPRSS2Ba P,nicotine,carbon monoxide,and tar are major harmful constituents of CSE.Carbon monoxide is hard to dissolve in water.Tar is a complex mixture of various compounds.The effects of nicotine and Ba P were therefore chosen for further study.Luciferase assays showed that Ba P(15μM,12h)significantly upregulated the activities of the ACE2 and TMPRSS2 promoters.The effects were unrelated to the genotypes at rs113208650,rs12481984,and rs8128074.The m RNA and protein levels of ACE2 and TMPRSS2 were also up-regulated by Ba P.Nicotine(12h,100μM)did not significantly affect the promoter activities and m RNA levels of ACE2 and TMPRSS2.Interestingly,nicotine significantly downregulated the protein level of TMPRSS2 but did not affect the protein level of ACE2.NR4A2 is an essential transcription factor upregulating the expression of ACE2 and TMPRSS2The promoter regions(-2000 bp to-1bp)of ACE2 and TMPRSS2 were applied to predict putative transcription factors.After the cross-validation of the data from JASPAR database and Regulome DB database,we identified 48 and 39 putative transcription factors binding to the ACE2 and TMPRSS2 promoters,respectively.The relationships between cigarette smoking and the levels of these transcription factors were investigated with the use of public databases of GSE994,GSE10718,GSE8987,and LUAD.The levels of NR4A2 and c AMP-responsive element modulator(CREM)were significantly elevated,while snail family transcriptional repressor 2(SNAI2)was decreased in samples exposed to cigarette smoke.These effects were repeated in more than one database.Our experiment showed that CSE significantly increased the transcription of NR4A2 in both Calu3 and H1650 cells and that the effects of CSE on the expression of CREM and SNAI2 were inconsistent in different cell lines in vitro.Therefore,NR4A2 was selected for further investigation.It was found that Ba P significantly increased the expression of NR4A2 in both Calu3 and H1650 cells.Knockdown of NR4A2 with si RNA significantly decreased the promoter activities,m RNA levels,and protein levels of ACE2 and TMPRSS2.Knocking-down NR4A2 significantly attenuated the Ba P-induced increase in the activities of the ACE2 and TMPRSS2 promoters.The effect of NR4A2 on the activities of ACE2 and TMPRSS2 promoters was not influenced by genotypes at rs113208650,rs12481984,and rs812074.Our Ch IP-q PCR experiments confirmed that NR4A2 was bound to the ACE2 and TMPRSS3 promoter regions in both Calu3 and H1650 cells and that the binding capacity was enhanced by Ba P.Thus,Ba P upregulates the activities of the ACE2 and TMPRSS2 promoters via upregulating the expression level and the binding capacity of NR4A2.NR4A2 is involved in age-and genetic polymorphism-mediated regulation of ACE2 and TMPRSS2 expressionUsing gene expression data for non-cancer lung tissues from the TCGA LUAD and LUSC cohorts,we identified significant positive correlations between age and expression level of NR4A2.Then,we investigated the expression levels of NR4A2 in lung tissues from healthy mice at different ages.Interestingly,the m RNA and protein levels of NR4A2 in the lung tissues of 20-month-old mice were significantly higher than those of 3-month-old mice.The expression levels of ACE2 and TMPRSS2 were also significantly higher in 20-month-old mice than in 3-month-old mice.The lung tissues were subjected to sodium bisulfite pyrosequencing to clarify the methylation rate of 8 Cp G islands within the NR4A2 promoter region in C57BL/6J mice(NC＿000068.8,GRCm39,Chr2:57006420-57006637).The average DNA methylation rates of the 8 Cp Gs decreased apparently in 20-month-old mice,as compared with those in 3-monthold mice.We identified a female COVID-19 patient who kept living with her husband and her daughter who were nevertheless all free of being infected.All the three family members were genotyped for the SNP by means of microarray analysis.No differences in genotypes of SNPs within the ACE2 and TMPRSS2 promoter regions were detected among them.Interestingly,this COVID-19 patient carried TT genotype at rs13428968,a SNP within 3’UTR of NR4A2,whereas her husband and her daughter carried rs13428968-TC genotype.Luciferase assays indicated that the m RNA of NR4A2 with rs13428968-T at the 3’UTR was more stable than that with rs13428968-C.The effects of CSE,Ba P,NR4A2 on the susceptibility to SARS-CoV-2 infectionEffects of CSE and Ba P on the susceptibility of cells to viral infection were first evaluated with a lentivirus-based pseudovirus expressing green fluorescence protein(GFP)-fused S protein of SARS-CoV-2.The level of pseudovirus infection was evaluated by detecting the percentage of GFP-positive cells via flow cytometry.the percentage of infected cells significantly increased in cells treated with CSE and Ba P for 12 h.We repeated these results in assays using herpesvirus-based pseudoviruses of wild-type SARS-Co V2 and Omicron BA.5,in which the luciferase expressed.Knockdown of NR4A2 and significantly inhibited the infection of pseudoviruses of wild-type SARS-Co V2 and Omicron BA.5,whereas nicotine did not affect the infection significantly.We further examined the function of Ba P in vivo.Hamster models received intrapleural and intrascrotal injections of Ba P(125mg/kg)and were then infected with pseudoviruses of Omicron BA.5 with the same approach three weeks later.The expression levels of NR4A2,ACE2,and TMPRSS2 proteins and the infection level of Omicron BA.5 pseudovirus were significantly higher in lung tissues from the Ba P-treated hamster group,as compared with DMSO-control hamster group.The protein levels of NR4A2,ACE2,and TMPRSS2 proteins and the infection level of Omicron BA.5 pseudovirus were also significantly increased in the testis tissues from the Ba P-treated hamster group,as compared with the control group.The pseudovirus infection test was repeated in lungs from mouse models,which demonstrated that Ba P significantly enhanced the susceptibility to SARS-Co V2 infection.Conclusion:This study shows that the expression of ACE2 and TMPRSS2 is not only affected by smoking,but also regulated by age and genetic polymorphism.It was confirmed that rs112312217、rs113208650、rs9981750、rs12481984 and rs812074 significantly affected the activity of ACE2 or TMPRSS2 transcriptional regulatory regions.Ba P is the major active CSE ingredient that facilitates the expression of SARS-Co V2 receptors and promotes the infection of SARS-Co V2 via upregulating an important transcript factor NR4A2.