Mettl3/Ythdf2 regulate macrophage inflammation and ROS generation by controlling Pyk2 mRNA stability

As one of the most prevalent modifications on RNA, N6-methyladenosine (m⁶A) has been recently found implicated in various pathological processes. Emerging studies have demonstrated the role of m⁶A and its writer Mettl3 in fine-tuning the immune response, which now becomes a research hotspot owing to its potential therapeutic value. However, the results are inconsistent and even contradictory, suggesting that there might be multiple Mettl3 target genes involved in different pathways. To delve deeper into the function of Mettl3 in the cellular inflammatory response, we first conducted bioinformatics analysis using RNA-seq in Mettl3 ablation macrophages, and found that Mettl3 might attenuate LPS-induced proinflammatory pathways and reactive oxygen species (ROS) generation process. Mettl3 knockdown significantly increased the LPS-induced IL-6, TNF-α, NOXs (Nox1, Nox2, Ncf1, and Ncf2) expression, ROS generation, and the phosphorylation of MAPKs and AKT signaling. Combining the results of RNA-seq and m⁶A mapping, we found that Pyk2 might be the target gene of Mettl3 affecting the inflammatory response. Mettl3 and Ythdf2 depletion increased the expression and mRNA stability of Pyk2, and RIP-PCR showed that Ythdf2 directly targeting Pyk2 was Mettl3 dependent. Moreover, the upregulated expression of TNF-α, IL-6, NOXs, ROS generation, and the phosphorylation of MAPKs and AKT signaling were downregulated by Pyk2 inhibitor in Mettl3 knockdown cells. All of these results suggest that Mettl3 regulates the mRNA stability and expression of Pyk2 in a Ythdf2-dependent way, which consequently triggers the activation of MAPKs and AKT signaling and upregulation of NOXs, thus promoting the generation of proinflammatory cytokines and ROS.