Mechanism Of Susceptibility Gene TMEM232 In Atopic Dermatitis

Background and objective:Atopic dermatitis(AD)is a chronic,relapsing inflammatory skin disease associated with a genetic predisposition to allergy.It is characterized by pruritus,polymorphic skin lesions and exudative tendency.In recent years,the incidence of AD has been increasing year by year,and the current incidence of children in the world is as high as 15%-20%.According to statistics,about a quarter of children will develop into adult AD after adulthood,and about a fifth of children with AD have the possibility of further developing asthma.Since there is currently no complete cure,symptomatic treatment is the main clinical treatment.Medical practice has shown that AD often occurs repeatedly,and its severe itching,sleep disturbance and social difficulties caused by extensive skin lesions cause great distress to the physical and mental health of patients,and also bring a heavy burden to the society,but so far there is still a lack of effective treatment methods and preventive measures.Genome-wide association studies(GWAS)have identified several loci associated with AD risk,and these studies of AD susceptibility loci have repeatedly suggested that AD susceptibility genes are associated with disease phenotypes,risks,and mechanisms.Although genomic variation is the evidence-based source of the pathogenesis of AD,there is still a lack of research on the downstream mechanism of many susceptibility genes.Revealing the mechanism of susceptibility genes in the pathogenesis of AD can inhibit the development of the disease from the source and provide a more sufficient basis for targeted therapy.Our previous study found that transmembrane protein 232(TMEM232)gene was associated with AD through GWAS study and fine-mapping study,which was subsequently validated in a Japanese GWAS population.However,its function is currently unknown.The purpose of this study was to explore the role and mechanism of TMEM232 in the pathogenesis of AD.Methods:The expression and localization of TMEM232 in the skin lesions of AD patients were detected by immunofluorescence,and the expression of TMEM232 in the skin lesions of AD patients was confirmed by real-time quantitative PCR(qRT-PCR)and Western blotting(WB)experiments.The expression of TMEM232 in AD skin lesions induced by MC903 was detected by qRT-PCR and WB experiments.Human primary keratinocytes were isolated and human immortalized keratinocytes(HaCaT)were used to detect the expression of TMEM232 with different inflammatory stimulations.After determining the expression of TMEM232 in AD,TMEM232 overexpression and knockdown HaCaT cell lines were constructed.The expression levels of AD-related inflammatory factors and chemokines were detected by qRT-PCR and enzyme linked immunosorbent assay(ELISA)after knockdown of TMEM232 followed by the administration of TNF-α/IFN-y mixed stimulation.In order to find the potential downstream pathways,a series of AD-related signaling pathways were screened and verified by WB experiment.The specific inhibitors of the corresponding signaling pathways were given to the cells overexpressing TMEM232 to verify the expression levels of downstream related inflammatory factors and chemokines.In order to identify the transcription factors that regulate the expression of TMEM232,the national center for biotechnology information(NCBI)was used to make predictions.After identifying the transcription factors that bind to the TMEM232 promoter,The Transcription factor motif database(JASPAR)and the Human transcription factors database(Human DFTB)were used to predict the binding sites of transcription factor and TMEM232 promoter,and the binding sites were mutated for dual luciferase reporter gene assay.In vivo,Tmem232 knockout(Tmem232-/-)mice were established.Calcipotriol(MC903)was used to induce inflammation in the ear tissue and back skin.The changes of T cell subsets in cervical lymph nodes were detected by flow cytometry.In order to find out the mechanism of Tmem232,we further examined the expression levels of various inflammatory factors in MC903-induced skin lesions of wild-type and homozygous mice using immunohistochemistry,WB and qRT-PCR.Combined with the results of in vitro experiments,the downstream pathways were also verified in vivo experiments.To verify the effect of deletion of Tmem232 in local skin on AD,Tmem232-siRNA liposome hydrogel was applied to the skin lesions of a mouse AD model,and the therapeutic effect of Tmem232 as a target for AD was evaluated by qRT-PCR and WB experiments.Results:1.Correlation analysis of TMEM232 expression in AD patients or AD models(1)The expression of TMEM232 in skin lesions of AD patients was examined.The results of immunofluorescence showed that the expression of TMEM232 in AD lesions was significantly higher than that in healthy controls,and it was mainly expressed in the epidermal layer.qRT-PCR and WB results showed that the expression of TMEM232 in skin lesions of AD patients was significantly higher than that of healthy controls.(2)The expression of Tmem232 in the skin lesions of AD mouse model was detected.MC903 induced AD-like dermatitis in mice,and the expression levels of Tmem232 mRNA and protein in skin lesions were detected by qRT-PCR and WB,respectively.The results showed that Tmem232 was up-regulated in MC903-induced AD skin lesions of mice.(3)Primary human keratinocytes and HaCaT cells were cultured,and the expression of TMEM232 was analyzed after stimulation with IL-17A,IFN-γ/TNF-α,and IL-4.WB results showed that the expression of TMEM232 was significantly increased in the keratinocyte stimulated by IFN-γ,TNF-α and IL-4,but not by IL-17A,which was further confirmed by qRT-PCR results.These results indicate that T helper cell(Th)type 1 and Th2 cytokines promote TMEM232 expression.2.Functional studies of TMEM232 were performed by in vitro cell experiments(1)After the expression of TMEM232 in AD was determined,we constructed stable HaCaT cell lines with TMEM232 overexpression or knockdown.Quantitative PCR and ELISAresults showed that the expression levels of AD-related inflammatory cytokines(IL-1β,IL-6,IL-8,IL-31,IL-33 and TSLP)and chemokines(CCL17 and CCL22)were significantly decreased after TMEM232 knockdown combined with TNF-α/IFN-γstimulation.However,overexpression of TMEM232 increased the secretion of the above-mentioned inflammatory cytokines and chemokines.(2)In order to further explore the mechanism of action of TMEM232,we also screened a series of AD-related signaling pathways and found that TMEM232 regulates inflammatory response through NF-κB and STAT3 signaling pathways through WB experiments.To clarify the relevant transcription factors that regulate TMEM232 expression,we performed projections using the NCBI website,where STAT6 transcription factors appear in the transcriptionally active region and at higher frequency,and STAT6 is a key molecule downstream of IL-4,we found that TMEM232 was accompanied by an increase in phosphorylated STAT6 after administration of IL-4 stimulation,and TMEM232 expression was significantly decreased after administration of inhibition of the STAT signaling pathway.Then we used JASPAR and Human DFTB databases to predict the binding sites of STAT6 and TMEM232 promoters,and the activity of dual-luciferase reporter gene was significantly decreased after the binding site was mutated,suggesting that IL-4/STAT6 axis regulated the expression of TMEM232.3.Functional studies of TMEM232 were performed by in vitro experiments(1)To verify the in vivo function of TMEM232,Tmem232-/-mice were generated.In the MC903-induced AD model,homozygous mice showed significantly lower morphological manifestations(including erythema,edema,scaling and scab),clinical scores and ear thickness compared with WT mice.Homozygous mice showed mild epidermal thickening,parkeratosis and acanthosis,and less inflammatory cell infiltration.(2)Flow cytometry analysis of T cell subsets in cervical lymph nodes showed that the percentage of Th2 and Th1 positive cells in homozygous mice was significantly lower than that in wild-type mice.Immunohistochemistry,WB and qRT-PCR were used to detect Th1(IL-1β,TNF-α),Th2(IL-4,IL-5,IL-13)and Th17 inflammatory cytokines in the skin lesions of wild-type mice and homozygous mice induced by MC903,and it was found that the secretion of Thl and Th2 inflammatory cytokines was decreased,especially Th2 inflammatory cytokines.(3)Tmem232-siRNA liposome hydrogel was applied to the site 24 hours after MC903 topical application.The results showed that local knockdown of Tmem232 in the skin reduced the clinical scores and ear thickness after MC903 model was established.The results of WB and qRT-PCR confirmed that the topical application of Tmem232-siRNA liposome hydrogel could reduce the secretion of Thl and Th2 inflammatory factors and the activation of NF-κB and STAT3 signaling pathways in the skin tissue of mice.These results suggested that local knockdown of Tmem232 could ameliorate MC903-induced AD-like lesions.Conclusion:Our study is the first to explore the function of TMEM232 gene.The expression of TMEM232 is up-regulated in AD patients.TMEM232 promotes AD inflammation by activating NF-κB and STAT3 pathways and is regulated by the IL-4/STAT6 axis to form a positive feedback.This may provide new targets and therapeutic basis for the treatment of AD.