Papel do fator de troca de nucleotídeos guanina RASGEF1B na regulação da expressão de SCHLAFEN-4 e SERPINB2 em macrófagos

Abstract

Slfn4 and Serpinb2 are genes expressed in macrophages that can have their mRNA levels increased in response to stimulation by inflammatory agonists of Toll-like receptors. In analyzes of global sequencing of RNAs, a cluster containing Slfn4 and Serpinb2 indicates that their expression is reduced in the absence of the guanine nucleotide exchange factor RasGEF1b in stimulated mouse bone marrow-derived macrophages with LPS. However, it is not fully established how Slfn4 expression is regulated in macrophages at rest or under inflammatory conditions, while Serpinb2 lacks conclusive evidence. Furthermore, the characterization of the Slfn4 promoter with its binding sites for transcription factors and its consequent transcriptional activation, and the role of RasGEF1b on the transcriptional regulation of Serpinb2 remains to be established. Based on these questions, we tested the hypothesis that RasGEF1b regulates the expression of Slfn4 and Serpinb2 in macrophages. RT-qPCR analyzes confirm that Slfn4 and Serpinb2 levels are reduced in untreated or LPS-treated RasGEF1b-KO macrophages, and the effect of the absence of RasGEF1b on gene expression observed in unstimulated cells is maintained after LPS treatment. Expression of Slfn4, Serpinb2, and cluster genes were also reduced from baseline levels in LPS-treated and untreated RAW264.7 macrophages silenced to RasGEF1b. However, at Ch25h the reverse effect was identified. Analysis of the binding motifs for specific transcription factors in the putative promoter region of Slfn4, Serpinb2 and genes from the same cluster revealed the presence of motifs for the interferon regulatory factors IRFs, AP-1, STATs, ATF and GATA. Accordingly, Slfn4 and Serpinb2 levels in RasGEF1b-KO macrophages activated with LPS and IFN remained at low levels, comparable to those of unstimulated cells. Overexpression of RasGEF1b induced transcriptional activity of Serpinb2, whereas it was reduced in RAW264.7 macrophages silenced for RasGEF1b. Mutation in the binding sites for the CEBP and AP-1 transcription factors reduced the luciferase activity of the Serpinb2 promoter in HEK293 cells overexpressing RasGEF1b for 24 hours, but not at 48 hours for the mutated CEBP site. Our results suggest that RasGEF1b facilitates gene transcription of Slfn4 and Serpinb2 at sufficient levels to allow their expression at maximum levels after cellular activation by inflammatory agents.