Análise dos níveis proteicos de RasGEF1b e do seu papel sobre a regulação da ativação de NF-kappaB em macrófagos estimulados com agonistas de receptores do tipo Toll (TLRs)

Abstract

RasGEF1b, a guanine nucleotide exchange factor, was described initially as the first example of a GEF whose expression is induced by inflammatory agonists of Toll- like receptors (TLRs). As RasGEF1b expression studies have been predominantly based on mRNA analysis, one of the aims of our study was to evaluate its protein expression in immune cells, especially in RAW264.7 cells, a mouse macrophage cell line. In addition, we also analyzed the mRNA and protein expression of RasGEF1b in THP-1 cells, a human promonocytic cell line. Our results show that expression of RasGEF1b at the mRNA and protein levels occurs in response to stimulation with inflammatory agonists of TLR2, TLR3 and TLR4 receptors. To investigate the role of RasGEF1b in the innate immune response, we have initiated functional studies in RAW264.7 cells, in order to provide evidence of the role of RasGEF1b in these cells. As RasGEF1b is an intracellular protein and no specific tools such as activators or inhibitors are available to carry out functional studies, procedures such as plasmid transfections should be employed that lead to increased or silencing of the expression in the cells of interest. However, macrophages are cells that have a very low permissiveness to transfection. In order to solve this problem, we carried out a study where some reagents that could be used in transfection assays in these cells were evaluated. Among the transfection reagents tested, we observed that Lipofectamine 2000 was the most effective reagent, and that caused the lowest toxicity to the cells. Current studies in our laboratory have demonstrated that RasGEF1b is a protein that exerts a negative regulatory function on the activation of NF-κB mediated by signaling pathway components of TLRs as well as by TLRs agonists, such as LPS and poly (I:C). However, these studies have been so far performed in human epithelial HEK293 cells, of little immune relevance, by assays with the overexpressing RasGEF1b. Therefore, as another objective in our study, we investigated the role of RasGEF1b in regulating the activation of NF-κB in murine macrophages. To do so, gene reporter assays were carried out in RAW264.7 cells to assess both gain and lossof-function of RasGEF1b. In the studies of gain-of-function, we observed that transfection of RasGEF1b in cells results in inhibition of NF-κB activation induced by LPS (TLR4 agonist). In the studies of loss-of-function performed with short hairpin RNA sequences, we observed that when the expression of RasGEF1b is knocked-down in cells, even transiently, the activation of NF-κB induced by LPS or Pam3CSK4 is increased. These results confirm that RasGEF1b exerts a negative regulatory effect on the activation of NF-κB mediated by TLRs agonists. Considering the difficulties of transfection procedures of murine macrophages and that functional studies could be carried out by aiming the knocking-down of RasGEF1b in a stable fashion and more effective obtained rather than by transient transfection, we initiated procedures to generate viral particles Lentivirus for transduction (infection) assays in RAW264.7 cells. However, at the end of the procedure to generate viral particles Lentivirus in HEK293T cells with subsequent infection of RAW264.7 macrophages with the particles, we have not been well succeeded in silencing the expression of RasGEF1b. Therefore, further studies are required to promote the silencing of RasGEF1b in a stable manner, so that its role can properly be better studied and understood in macrophages and other cell types. Finally, functional studies may contribute to understand the function of RasGEF1b in regulating the innate immune response and also in the establishment of preventive measures or therapeutic interventions in diseases whose pathogenesis is related with the exacerbated activation of NF-κB as, for example, inflammatory chronic diseases and cancer.