NNMT Facilitates Pulmonary Fibrosis Via Promoting Fibroblast Differentiation

BackgroundIdiopathic pulmonary fibrosis(IPF)is a chronic,progressive and fatal interstitial pulmonary disease,with undetermined etiology,poor prognosis and high morbidity.The clinical manifestations are progressively dyspnea,persistent decline in lung function,and grid-like cellular lung changes on pathology and HRCT imaging.The pathological features of IPF are alveolar epithelial mesenchymal transformation and myofibroblasts differentiation,accompanied by excessive deposition of extracellular matrix(ECM)and remodeling of pulmonary tissue structure.Nicotinamide N-methyltransferase(NNMT)is a metabolic enzyme that catalyzes the methylation of nicotinamide(NAM)using the methyl donor S-adenosylmethionine(SAM)to generate 1-methylnicotinamide(1-MNA)and S-adenosyl-L-homocysteine(SAH).NNMT is involved in the regulation of SAM,acting as universal methyl donor in cellular activities,as well as the SAM/SAH ratio,directly linking cell’s methylation balance.In recent years,increasing number of studies have shown that methylation has been associated with fibrosis,for example,histone methylation regulated epigenetic processes leading to damage to the endoplasmic reticulum,which promoted the development of alcoholic liver fibrosis.Additionally,there are findings that suggest that DNA methylation contributes to the development of idiopathic pulmonary fibrosis.Recently,it has been reported that NNMT maintains low methylation level by reducing the binding of repressed histones H3K27me3 and H3K9me3 to the promoter of fibrosis markers,resulting in normal human fibroblast activation;Correspondingly,knocking down NNMT reversed the differentiation of IPF fibroblasts.However,whether NNMT regulates fibrosis through methylation and the role of NNMT in IPF remain unclear.ObjectThis study aims to explore the potential role of NNMT in IPF.MethodsWe first analyzed the expression of NNMT in bleomycin(BLM)-induced mice fibrosis models as well as control lung tissues through proteomics.Secondly,we analyzed the expression difference of NNMT in IPF patients and donor controls through the GEO public database.Then,we collected fibroblasts as well as lung tissues from clinical IPF patients and donors.NNMT expression was detected by q PCR,Western blot,immunohistochemistry and immunofluorescence,respectively.The expression of NNMT in mouse lung tissues and its fibroblasts were detected by Western blot.Subsequently,we monitored NNMT expression in fibroblasts stimulated by TGF-β.To further explore the effect of an increased NNMT in fibrosis,we constructed normal lung fibroblasts with stable overexpression of NNMT and IPF fibroblasts with silencing of NNMT by lentivirus,and examined fibrosis-related genes by qPCR and Western blot.Besides,the capability of cells’ migration and proliferation were detected by Edu and Wound healing,respectively.Finally,we treated BLM-induced mice with NNMT inhibitor(NNMTi),Western blot was performed to examine the expression of fibrosisrelated biomarkers;Hematoxylin-eosin(HE)staining was performed to investigate pathologic change;Sirius Red staining and Masson’s Trichrome staining were performed to examine the production of collagen Ⅰ;Micro-CT was performed to analyze the degree of fibrosis.ResultsWe first identified NNMT significantly elevated in lung tissue in BLM-induced mice fibrosis models through proteomics.Then we found that NNMT significantly elevated in cells in IPF patients through the GEO public database as well.Subsequent validation in lung tissue and fibroblasts derived from IPF patients,as well as in BLMinduced mice fibrosis models also revealed significantly elevated NNMT in those compared with the controls.In our work,we demonstrated the important role of NNMT in the process of pulmonary fibrosis in vitro and vivo.NNMT was induced in fibroblasts following TGF-β stimulation in dose-and time-dependent manner.Next,we overexpressed and knocked down NNMT,respectively using normal human and IPF patient fibroblasts.A series of functional experiments were performed to prove that overexpression of NNMT not only promoted the development of pulmonary fibrosis,but also enhanced the capability of cell’s migration and proliferation.Knocking down NNMT suppressed myofibroblasts differentiation of IPF fibroblasts and inhibited the capability of cell’s migration and proliferation.In BLM-induced mice fibrosis models,Western blot results showed that the depression of NNMT could improve pulmonary fibrosis;the results of Masson’s trichrome staining and Sirius red staining showed that the depression of NNMT could reduce the production of collagen Ⅰ;HE staining and CT analysis confirmed that the depression of NNMT could reduce inflammation and structure damage in lung.ConclusionsThis study identified the role of NNMT in idiopathic pulmonary fibrosis for the first time.NNMT facilitates idiopathic pulmonary fibrosis by promoting fibroblast differentiation into myofibroblast.