RESUMO
Ras Guanine Exchange Factor (RasGEF) domain family member 1b is encoded by a Toll-like receptor (TLR)-inducible gene expressed in macrophages, but transcriptional mechanisms that govern its expression are still unknown. Here, we have functionally characterized the 5' flanking Rasgef1b sequence and analyzed its transcriptional activation. We have identified that the inflammation-responsive promoter is contained within a short sequence (-183 to +119) surrounding the transcriptional start site. The promoter sequence is evolutionarily conserved and harbors a cluster of five NF-κB binding sites. Luciferase reporter gene assay showed that the promoter is responsive to TLR activation and RelA or cRel, but not RelB, transcription factors. Besides, site-directed mutagenesis showed that the κB binding sites are required for maximal promoter activation induced by LPS. Analysis by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) revealed that the promoter is located in an accessible chromatin region. More important, Chromatin Immunoprecipitation sequencing (ChIP-seq) showed that RelA is recruited to the promoter region upon LPS stimulation of bone marrow-derived macrophages. Finally, studies with Rela-deficient macrophages or pharmacological inhibition by Bay11-7082 showed that NF-κB is required for optimal Rasgef1b expression induced by TLR agonists. Our data provide evidence of the regulatory mechanism mediated by NF-κB that facilitates Rasgef1b expression after TLR activation in macrophages.
Assuntos
Macrófagos/metabolismo , NF-kappa B/metabolismo , Receptores Toll-Like/metabolismo , Fatores ras de Troca de Nucleotídeo Guanina/biossíntese , Animais , Células Cultivadas , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Fatores ras de Troca de Nucleotídeo Guanina/genética , Fatores ras de Troca de Nucleotídeo Guanina/metabolismoRESUMO
We present here the data to support the understanding of the implication of Rap2a GTPase in LPS-induced innate immune response and NF-κB activation. The data presented are related to molecular tools that were generated, acquired, optimized or validated to investigate Rap2a expression, activation and its effects in mammalian cells including RAW264.7 macrophages and THP-1 monocytes under inflammatory conditions. These data supplement important technical and biological information on immune function of Rap2a in macrophages activated by LPS, recently reported by us (Carvalho et al., 2019) [1].
RESUMO
Small Ras GTPases are key molecules that regulate a variety of cellular responses in different cell types. Rap1 plays important functions in the regulation of macrophage biology during inflammation triggered by toll-like receptors (TLRs). However, despite sharing a relatively high degree of similarity with Rap1, no studies concerning Rap2 in macrophages and innate immunity have been reported yet. In this work, we show that either way alterations in the levels of Rap2a hampers proper macrophages response to TLR stimulation. Rap2a is activated by LPS in macrophages, and although putative activator TLR-inducible Ras guanine exchange factor RasGEF1b was sufficient to induce, it was not fully required for Rap2a activation. Silencing of Rap2a impaired LPS-induced production of IL-6 cytokine and KC/Cxcl1 chemokine, and also NF-κB activity as measured by reporter gene studies. Surprisingly, overexpression of Rap2a did also lead to marked inhibition of NF-κB activation induced by LPS, Pam3CSK4 and downstream TLR signaling molecules. We also found that Rap2a can inhibit the LPS-induced phosphorylation of the NF-κB subunit p65 at serine 536. Collectively, our data suggest that expression levels of Rap2a in macrophages might be tightly regulated to avoid unbalanced immune response. Our results implicate Rap2a in TLR-mediated responses by contributing to balanced NF-κB activity status in macrophages.