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

Cell Mol Gastroenterol Hepatol. 2021;12(3):839-856. doi: 10.1016/j.jcmgh.2021.05.007. Epub 2021 May 13.

Abstract

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.

Keywords: N6-Methyladenosine; NASH; NF-κB; TGF-β1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5' Untranslated Regions
  • Adenosine / analogs & derivatives*
  • Adenosine / metabolism
  • Animals
  • Base Sequence
  • Diet, High-Fat
  • Disease Models, Animal
  • Gene Expression Regulation
  • Gene Knockdown Techniques
  • Kupffer Cells / metabolism*
  • Liver Cirrhosis / etiology
  • Liver Cirrhosis / metabolism
  • Liver Cirrhosis / pathology
  • Methylation
  • Methyltransferases / deficiency
  • Methyltransferases / metabolism*
  • Models, Biological
  • Protein Biosynthesis*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Transcription Factor RelA / metabolism
  • Transcriptional Activation*
  • Transforming Growth Factor beta1 / genetics*
  • Transforming Growth Factor beta1 / metabolism

Substances

  • 5' Untranslated Regions
  • RNA, Messenger
  • Transcription Factor RelA
  • Transforming Growth Factor beta1
  • N-methyladenosine
  • Methyltransferases
  • Adenosine