Inibição da função redox de APE1/REF-1 e sua influência na regulação transcricional em um modelo de estimulação inflamatória

Abstract

The human apurinic/apyrimidinic endonuclease 1 (APE1/REF-1) is a multifunctional enzyme that plays a role in both DNA repair and transcription pathways, being considered a hub protein, controlling key pathways in cell homeostasis and survival. This study aimed to investigate the mechanism behind the inhibition of APE1/REF-1 redox activity using an inflammation model. As the experimental model, U937 cells were incubated with LPS at 1 μg/ml and submitted to two different treatments with 100 μM of E3330, which inhibits specifically the APE1/REF-1 redox function. Inflammation stimulation kinetics was performed with LPS for 1 hour, followed by treatment with E3330 for 2, 4, 6, 24 and 48 hours. In the second model the cells were stimulated with LPS for 24 hours and a subsequent treatment with E3330 for 4 hours. After the treatments, parameters such as cell viability; the expression patterns of inflammatory markers; apoptosis and mitochondrial membrane potential were assessed. The transcriptome analysis was performed using the 454 GS-FLX Titanium platform, followed by functional enrichment and protein-protein interaction networks (PPI) design based on the differentially expressed genes list. In parallel, to validate the transcriptome data and the cellular mechanisms proposed, qPCR, western blotting, rRNA degradation and AP site incision assays were carried out. In addition, searching for putative transcription factors (TFs) binding sites in target genes, the iRegulon tool was used. Our results showed that after stimulation with LPS for 24 hours, the treatment with E3330 for 4 hours did not cause significant variations in cell viability and apoptosis ratio. However, the results obtained using the inflammatory kinetics model showed that cell viability was reduced after treatment with E3330 for 48 hours. Regarding to PPI networks, it was noticed that the most enriched functional categories in the downregulated network were associated to gene expression, immune response, rRNA processing and ribosome biogenesis. On the other hand, biological processes such as signal transduction, stress response, chromatin modification, DNA damage response and positive regulation of apoptosis were the most representative pathways in the network unique to upregulated genes. The protein quantification showed that E3330 lead to the decrease of c-Myc and NF- κB(p65) levels, concomitantly to the increase in MDM2 60 kDa isoform levels. The rRNA integrity assay indicated a decrease of 12 and 20% in the 28S/18S ratio in samples treated with E3330 or LPS respectively. It is noteworthy that all of this finds resulted from the specific inhibition of APE/REF-1 redox activity, since the AP site incision assay confirmed that E3330 at the concentration tested was not able to inhibit APE/REF-1 repair function. In general, regarding to downregulated genes, the TFs are modulated by redox regulation and inhibition of this function by E3330 might alter the activity of these factors, leading to the noticed decrease in the expression of key genes. In this work, it was possible to determine which cellular events are regulated by APE1/REF-1 redox function and it was suggested that inhibition of this function by E3330 reduces the inflammatory response as well as promotes the inhibition of pathways associated to ribosome biogenesis and rRNA processing, without causing any disturbance in its APendonucleases function.