Metoprolol restrains EPAC1 expression to reinforce eEF2K-mediated translation brake. (A) Kyoto Encyclopedia of Genes and Genomes analysis of RNA-downregulated genes in metoprolol prophylactically treated monocytes. The top eight items in Kyoto Encyclopedia of Genes and Genomes were shown by the gene ratio. (B and C) CD14⁺ human monocytes were cultured with KS for indicated time points and treated with PBS or metoprolol (100 μM) prophylactically (B) and conditionally (C). RAPGEF3 mRNA levels were measured by qPCR. (D) Schematic depiction of EPAC1-related signaling pathway and the subsequent experimental approaches. (E–H) CD14⁺ human monocytes were cultured with KS for indicated time points and treated with DMSO, EPAC1 inhibitors (CE3F4 [5 μM], HJC0197 [5 μM]; E and F) or EPAC1 activator (8-CPT [50 μM]; G and H) 24 h before sample analysis. Intracellular IL-6 levels were measured by ELISA (E and G) and IL6 mRNA levels were measured by qPCR (F and H). (I–L) CD14⁺ human monocytes were transfected with negative control (siNC) or RAPGEF3 specific short interfering RNA (siRAPGEF3). 2 d after transfection, cells were re-plated and cultured with KS for indicated time points. Supernatant and intracellular IL-6 levels were measured by ELISA (J and K). RAPGEF3 and IL6 mRNA levels were measured by qPCR (I and L). (M–O) SiNC or siRAPGEF3 transfected CD14⁺ human monocytes were cultured with KS and treated with PBS or metoprolol (100 μM) for indicated time points. Supernatant IL-6 levels were measured by ELISA (N). RAPGEF3 and IL6 mRNA levels were measured by qPCR (M and O). (P–R) CD14⁺ human monocytes were treated with EPAC1 inhibitors (P), activator (R), or transfected with siRAPGEF3 (Q), and the protein levels of p-eEF2K (Ser366), total eEF2K, p-eEF2 (Thr56) and total eEF2 were detected by Western blotting. Representative data of three independent experiments. *P < 0.05, **P < 0.01, ns, not significant; two-tailed paired t test in B, C, and E–O. Independent experiments in B, C, and E–O were performed with cells from one healthy donor for each experiment. Source data are available for this figure: SourceData F4.