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

Both DNA 5-methylcytosine (5mC) and RNA N6-methyladenosine (mA) 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 mA 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 mA hypermethylation in LPS-activated KCs. The suppression of 5mC demethylation or mA hypermethylation significantly alleviates the upregulation of PCK2 and proinflammatory cytokines in LPS-challenged KCs. Further reciprocal tests indicate 5mC demethylation is upstream of mA 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 mA-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.