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

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.

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.

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.

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.