2. Academic Validation
  3. METTL3/METTL14 Transactivation and m6A-Dependent TGF-β1 Translation in Activated Kupffer Cells

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.
Yue Feng 1,  Haibo Dong 1,  Bo Sun 1,  Yun Hu 1,  Yang Yang 1,  Yimin Jia 1,  Longfei Jia 2,  Xiang Zhong 3,  Ruqian Zhao 4
Affiliations

Affiliations

  • 1 MOE Joint International Research Laboratory of Animal Health & Food Safety, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China; Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China.
  • 2 Division of Nutritional Sciences, Cornell University, Ithaca, New York.
  • 3 College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China.
  • 4 MOE Joint International Research Laboratory of Animal Health & Food Safety, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China; Key Laboratory of Animal Physiology and Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China. Electronic address: [email protected].
PMID: 33992834 DOI: 10.1016/j.jcmgh.2021.05.007
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.

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