Efeitos do fármaco anti-inflamatório salicilato de sódio sobre a expressão e ativação do fator de transcrição ATF6: implicações sobre a resposta celular ao estresse do retículo endoplasmático

Abstract

Perturbation of the homeostasis of endoplasmic reticulum (ER) triggers the ER stress response, which is mediated by three ER transmembrane proteins: PKR-like ER kinase (PERK), activation transcription factor (ATF6), and inositol-requiring kinase (IRE1). Accumulating evidence indicates that non-steroidal anti-inflammatory drugs salicylates, such as Aspirin and sodium salicylate (NaSal), affect the activity of genes and proteins implicated in ER stress. NaSal treatment of cells leads to activation of PERK, but not IRE-1, resulting in eIF2alpha phosphorylation. Because the role of ATF6 in the cellular responses induced by salicylates remains elusive, we aimed to characterize the expression and activity of ATF6 after NaSal treatment of mouse embryonic fibroblasts (MEFs). Through RT-qPCR and western-blot studies, the expression of ATF6 was determined upon treatment of cells. In addition, mRNA and protein levels of ATF6 were evaluated in cells lacking eIF2alpha kinase PERK. Luciferase reporter gene assay was used to measure ATF6-dependent gene transcriptional activation in cells that were exposed only to NaSal or pre-treated with NaSal and further treated with tunicamycin. We found that NaSal induced an increase in ATF6 mRNA and protein expression in wild type cells. Interestingly, a robust expression of ATF6 mRNA was observed in PERK-deficient cells, although the protein did not accumulate in these cells. Intriguingly, an increase in ATF6 mRNA and protein levels was not accompanied by ATF6-mediated transcriptional activation, which was otherwise inhibited by NaSal in tunicamycin-treated cells. The analysis of the expression of ATF6 target genes revealed that only CHOP and GADD34 are induced by NaSal, but also that this expression is not ATF6-dependent. These results provide additional evidence of the effects of salicylates in ER stress components, and allow us to define ATF6 as a target for the biological actions of salicylates.