FTO O-GlcNAcylation promotes TRIM21-mediated FTO ubiquitination degradation to sustain the negative feedback control of macrophage inflammation.

INTRODUCTION

The fat mass and obesity-associated protein (FTO), a key RNA N-methyladenosine (mA) demethylase, has been highlighted for its important role in inflammatory response. Emerging evidences link the O-GlcNAcylation to numerous human diseases, particularly inflammation. However, the specific role and underlying mechanism of FTO O-GlcNAcylation in inflammation remain elusive.

METHODS

The FTO O-GlcNAcylation modification was determined by co-immunoprecipitation (Co-IP) assay, metabolic glycan labeling combined with click reaction, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP)-qPCR and dual-luciferase reporter assay were used to determine FOXO1 binding to the 2 promoter and the 2 promoter activity during LPS stimulation. The FTO ubiquitination modification and the interaction between FTO and TRIM21 were detected by confocal microscopy, pull-down, and mass spectrometry analysis. The effects of FTO O-GlcNAcylation on the ubiquitination degradation of FTO were assessed by Co-IP and protein stability assays. The 1 mRNA mA methylation levels were detected by mA-RNA-immunoprecipitation (RIP)-qPCR. The myeloid-specific deletion (mye ) mice, the macrophage depletion and reconstitution experiments, and enzyme-linked immunosorbent assays (ELISA) were used to evaluate inflammatory responses in () or bacterial endotoxin (lipopolysaccharide, LPS) induced sepsis mouse models.

RESULTS

We demonstrate that FTO undergoes O-GlcNAcylation specifically at the serine 95 (Ser95) site. LPS enhances FTO O-GlcNAcylation modification levels by increasing the FOXO1-regulated GFAT2 expression. O-GlcNAcylation of FTO promotes TRIM21-mediated K48-ubiquitination degradation of FTO, which further induces suppressor of cytokine signaling () 1 mA methylation, thus sustains SOCS1 protein expression and suppresses multiple inflammatory cytokines IL-1β/IL-6/TNF-α production in LPS-stimulated macrophages. FTO O-GlcNAcylation mutation (S95A) aggravates or LPS-induced sepsis, while FTO O-GlcNAcylation suppresses the hyperinflammatory phenotype in mice. Promotion of O-GlcNAcylation by an OGA inhibitor Thiamet-G alleviates LPS-induced inflammatory responses and septic shock in mice.

CONCLUSION

These findings reveal a mechanism that FTO O-GlcNAcylation promotes its ubiquitination degradation, and thus induces mA methylation and downregulates LPS-mediated inflammatory response, which maintains the negative feedback control of macrophage inflammatory cytokine storm in sepsis. Regulation of FTO O-GlcNAcylation may offer a potential therapeutic strategy for combating endotoxin-induced inflammatory disease and other FTO abnormal expression-associated diseases.