Effect of Endoplasmic Reticulum Stress on the Phenotype of Macrophages Derived from THP-1 Monocytes

In addition to its role in protein synthesis and folding, the endoplasmic reticulum (ER) plays a critical role in sensing stress and maintaining cellular homeostasis. When an accumulation of unfolded proteins is detected due to impaired protein synthesis or folding, specific intracellular signaling pathways are activated. These pathways are collectively known as the unfolded protein response (UPR). Prolonged stress, when a cell is unable to maintain homeostasis, can lead to apoptosis. The aim of this study was to investigate the effect of endoplasmic reticulum (ER) stress induced by the RIP-II protein, viscumin, on the phenotype of THP-1-derived macrophages. Viscumin hydrolyzes the N-glycosidic bond of adenosine at position A4324 in the 28S RNA of the eukaryotic 60S ribosomal subunit, resulting in translation inhibition and accumulation of unfolded proteins in the ER lumen. To determine the half maximal inhibitory concentration (IC50) of viscumin, we performed a cytotoxicity assay (MTT). We found that ~5 nM viscumin concentration inhibited 50% of macrophage cell viability. At a concentration of 1 nM, more than 80% of the cells remained viable, but only 1% of the ribosomes were inactivated. These results suggest that translation inhibition is not the primary reason for the high sensitivity of macrophages to viscumin and other RIP-II proteins. Using RT-PCR, we found that viscumin induces the unfolded protein response in macrophages by activating three ER stress sensors, IRE1a, PERK and ATF6. However, bioinformatic sequencing analysis revealed that only one pathway activated by the PERK sensor (the PERK-eIF2a-ATF4-CHOP pathway) increases the level of inflammation in macrophages and plays a critical role in inducing apoptosis.