DNA 5mC and RNA m⁶A Collaborate to Upregulate Phosphoenolpyruvate Carboxykinase 2 for Kupffer Cell Activation

Both DNA 5-methylcytosine (5mC) and RNA N6-methyladenosine (m⁶A) modifications are reported to participate in cellular stress responses including inflammation. Phosphoenolpyruvate carboxykinase 2 (PCK2) is upregulated in Kupffer cells (KCs) to facilitate the proinflammatory phosphorylation signaling cascades upon LPS stimulation, yet the role of 5mC and m⁶A in PCK2 upregulation remain elusive. Here, we report that the significantly augmented PCK2 mRNA and protein levels are associated with global 5mC demethylation coupled with m⁶A hypermethylation in LPS-activated KCs. The suppression of 5mC demethylation or m⁶A hypermethylation significantly alleviates the upregulation of PCK2 and proinflammatory cytokines in LPS-challenged KCs. Further reciprocal tests indicate 5mC demethylation is upstream of m⁶A hypermethylation. Specifically, CpG islands in the promoters of PCK2 and RNA methyltransferase (METTL3 and METTL14) genes are demethylated, while the 3'UTR of PCK2 mRNA is m6A hypermethylated, in LPS-stimulated KCs. These modifications contribute to the transactivation of the PCK2 gene as well as increased PCK2 mRNA stability and protein production via a m⁶A-mediated mechanism with IGF2BP1 as the reader protein. These results indicate that DNA 5mC and RNA m6A collaborate to upregulate PCK2 expression, respectively, at the transcriptional and post-transcriptional levels during KC activation.