RNA m⁶A-mediated post-transcriptional repression of glucocorticoid receptor in LPS-activated Kupffer cells on...

Glucocorticoid receptors are distributed in various cells of the body and participate in the regulation of metabolism and immunity in response to glucocorticoids. RNA m⁶A methylation participates in various metabolic and inflammatory responses, yet it remains elusive whether m⁶A is involved in GR regulation during immune activation. Here, we observed uncoupled GR responses with increased mRNA yet suppressed protein levels in the LPS-challenged broilers chicken liver, in association with global elevation of RNA m⁶A methylation, especially in the expression of METTL3 and YTHDF2. Further analysis using isolated primary hepatocytes and Kupffer cells revealed that such uncoupled GR responses and m⁶A hypermethylation occurred specifically in KCs. The same GR and m⁶A responses were reproduced in LPS-activated KC cell line, implying a possible role of m⁶A in the post-transcriptional suppression of GR in KC. Indeed, m⁶A inhibitor cycloleucine alleviated LPS-induced GR protein suppression, whilst GR antagonist RU486 had no effect on global m⁶A methylation in KC. We observed that YTHDF2 siRNA can alleviate LPS-induced GR mRNA stability decrease. Subsequently, specific m⁶A sites on GR were predicted and verified. Mutation m⁶A sites and luciferase reporter assay was also applied to validate these findings. Mechanistically, m⁶A methylation on the transcripts of GR impairs its mRNA stability in a YTHDF2-dependent manner, which leads to the decrease of its protein. Our study indicates successive roles of RNA m⁶A modification in the down regulation of GR expression, which provides new drug targets for epigenetic therapy of liver inflammation.