METTL3/METTL14 Transactivation and m6A-Dependent TGF-β1 Translation in Activated Kupffer Cells

BACKGROUND AND AIMS:Transforming growth factor β1 (TGF-β1) secreted from activated Kupffer cells (KC) promotes the progression of nonalcoholic steatohepatitis (NASH) to liver fibrosis. N6-methyladenosine (m6A) RNA modification participates in various cell stress responses, yet it remains unknown whether it plays a role in TGF-β1 upregulation in activated KCs. METHODS:Western blot, dot blot, and liquid chromatography with tandem mass spectrometry were used to determine the expression of m6A methyltransferase, METTL3, and METTL14, as well as global m6A modification. RNA-sequencing and m6A-seq were employed to screen differentially expressed genes and responsive m6A peaks. Nuclear factor κB (NF-κB)-mediated METTL3/METTL14 transactivation were validated with chromatin immunoprecipitation polymerase chain reaction and dual-luciferase reporter system, and the role of m6A in TGF-β1 upregulation was further verified in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice. RESULTS:Serum lipopolysaccharide (LPS) concentration is increased in high-fat diet-induced NASH rats. TGF-β1 upregulation is closely associated with METTL3/METTL14 upregulation and global m6A hypermethylation, in both NASH rat liver and LPS-activated KCs. LPS-responsive m6A peaks are identified on the 5' untranslated region (UTR) of TGF-β1 messenger RNA (mRNA). NF-κB directly transactivates METTL3 and METTL14 genes. LPS-stimulated TGF-β1 expression is abolished in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice. Mutation of m6A sites on the 5'UTR of TGF-β1 mRNA blocks LPS-induced increase of luciferase reporter activity. CONCLUSIONS:NF-κB acts as transcription factor to transactivate METTL3/METTL14 genes upon LPS challenge, leading to global RNA m6A hypermethylation. Increased m6A on the 5'UTR of TGF-β1 mRNA results in m6A-dependent translation of TGF-β1 mRNA in a cap-independent manner. We identify a novel role of m6A modification in TGF-β1 upregulation, which helps to shed light on the molecular mechanism of NASH progression.