PRMT1-mediated Extracellular Matrix Deposition And Mechanism Of Alleviation By Vitamin D₃

Chronic respiratory disease has become a major infectious disease in poultry.After infection,the rates of weak chicks is increased,the laying rates of laying hens,fertilization rates and hatching rates of laying eggs are significantly decreased.broiler has a pathological damage on the respiratory system,which causes an increase in the ratio of feed to meat and a decrease in production performance.Due to the poor protective and therapeutic effect of vaccines and antibiotics on poultry chronic respiratory diseases,it has brought huge economic losses to the chicken industry.Changes in the structure of the extracellular matrix not only enhance the infection of pathogens,but also cause airway remodeling,which can lead to disease progression.As an epigenetic modification,protein arginine methyltransferases（PRMTs）are mainly responsible for protein arginine methylation and can activate or inhibit gene expression according to the cell environment.Protein arginine methylation may regulate the changes of extracellular matrix structure and participate in the process of pulmonary inflammatory diseases.Therefore,this study explores the particular mechanism of PRMT1 in the collagen deposition process and the mechanism of attenuation by vitamin D₃ on the technical platform of lung fibroblast culture in vitro,and provides an effective molecular target and method for the treatment of chronic respiratory diseases in poultry.The experiment consists of three parts:Experiment 1 The role of PRMTs in fibroblast collagen depositionAfter TSLP stimulated fibroblasts（0 h,6 h,12 h,24 h and 48 h）,the mRNA level of TSLP receptor in fibroblasts was increased（P<0.01）.and the collagen-1A1（COL1A1）was up-regulated at mRNA（P<0.01）and protein levels（P<0.05）.However,the mRNA expression of collagen-3A1（COL3A1）in fibroblasts was not affected by TSLP treatment（P>0.05）.Meanwhile,PRMT1 and PRMT6 in the PRMTs family were increased in a time-dependent manner（P<0.01）,and lasting for 48 hours.PRMT4 and PRMT7 were increased after 12 hours of TSLP stimulation（P<0.05）,and the expression decreased rapidly at 24 hours.The mRNA expression of PRMT5（P<0.01）and PRMT9（P<0.05）were up-regulated after 12 hours,and there was no significant difference on its expression at 24and 48 hours.It can be seen that PRMT4,PRMT7 and PRMT5 may play a regulatory role within 24 h by TSLP stimulation.In addition,there was no significant difference in the expression of PRMT2,PRMT3 and PRMT8.Furthermore,we examined the protein expression of PRMT1,and found that the protein expression of PRMT1 was also increased in a time-dependent manner under the induction of TSLP（P<0.01）.PRMT1 siRNAs were used to knockdown the PRMT1 expression in fibroblasts.The results showed si-PRMT1completely suppressed COL1A1 expression（P<0.05）.fibroblasts were treated with overexpression plasmid of PRMT1.We observed that The expression levels of COL1A1 was incresed.These results indicate that TSLP induced collagen-1A1 deposition by binding to TSLP receptor.Protein arginine methylation modification is involved in collagen deposition,and PRMT1 can regulate collagen expression.Experiment 2 Molecular mechanism of collagen deposition in fibroblasts during chronic inflammation of the lungThe expression of the enzyme（MMP1）which mainly degraded type I interstitial collagen was detected at different time points（0 h,6 h,12 h,24 h and 48 h）after TSLP stimulation.The results showed MMP1 was up-regulated at mRNA and protein levels（P<0.01）,the mRNA level of MMP1 was starting at 12 hour and lasting for 48 hours,the protein level was up-regulated from 12 h and maintained for 48 hours.Furthermore,the expression of MMP2 in fibroblasts was not affected by TSLP stimulation.PRMTs-specific inhibitor AMI-1 or siRNAs were used to knockdown the PRMT1 expression in fibroblasts.The results showed 10nM and 20nM si-PRMT1 completely suppressed MMP1 expression,as did pretreatment with 10uM AMI-1.Fibroblasts were treated with overexpression plasmid of PRMT1.We observed that The expression levels of MMP1 was incresed.After fibroblasts were stimulated with 10 ng/mL TSLP for 5 min,15 min,30 min,1 h and 3 h,the results showed that STAT3 was phosphorylated after 30 min of TSLP stimulation and disappeared 1h after TSLP stimulation.At the same time,we also used the STAT3 inhibitor S3I-201 to block the phosphorylation of STAT3,and found that 10μM S3I-201 inhibited the mRNA and protein expression of PRMT1 and MMP1（P<0.05）.After inhibiting the ERK1/2 pathway,the protein expression of PRMT1 and MMP1 were decreased,and ERK1/2 was phosphorylated after 5 min of TSLP stimulation（P<0.05）.These findings confirm that MMP1 is involved in the process of collagen deposition,and PRMT1 can directly regulate the expression of MMP1.TSLP up-regulates the expression of PRMT1 and MMP1 in fibroblasts through ERK1/2 MAPK and sub-sequent STAT3 activation.Experiment 3 Effects of vitamin D₃ supplementation on collagen deposition in fibroblastsTo investigate the effects of continued vitamin D₃ supplementation on the production of collagen from fibroblasts.Fibroblasts were treated with 10 nM calcitriol continued for three generation,then take out of calcitriol when changing serum-free medium.Lung fibroblasts were treated with 10 ng/ml TSLP for 0,5 min,15 min,30 min,1 h,3 h,6 h,12 h,24 h and48 h.We observed down-regulated of STAT3 phosphorylation level beganing at 5 min,and the expression of ERK followed by 15 min began to be inhibited until 3 h.With the prolongation of stimulation time,the expression of PRMT1 was decreased after 6 h of TSLP treatment,and recovered after 12 h.The expression of MMP1 precursor and COL1A1 were decreased,starting at 12 hour and lasting for 24 hours.These findings suggest that Continuous and early vitamin D supplementation has a protective effect on collagen deposition induced by TSLP.In summary,we found a novel signalingmechanism for collagen deposition.In lung fibroblasts,TSLP binds to TSLP receptors,which induces the phosphorylation of ERK1/2MAPK and sub-sequent STAT3,and the expression of PRMT1 and MMP1 is up-regulated,thereby regulating collagen deposition.In addition,continued early supplementation of vitamin D₃ can alleviate collagen deposition by reducing phosphorylation of STAT3.It can be seen that PRMT1 and vitamin D₃ play a key role in the process of collagen deposition.These findings provide an effective molecular target and approach for the treatment of chronic respiratory diseases.