RESUMO
Macrophages play a pivotal role in the immune system through recognition and elimination of microbial pathogens. Toll-like receptors (TLRs) on macrophages interact with microbial substances and initiate signal transduction through intracellular adapters. TLR4, which recognizes the lipopolysaccharides (LPS) on Gram-positive and Gram-negative bacteria, triggers downstream signaling mediators and eventually activates IκB kinase (IKK) complex and mitogen-activated protein kinases (MAPKs) such as p38. Previous reports revealed that, in addition to NF-κB, a core transcription factor of the innate immune response, the induction of some LPS-induced genes in macrophages required another transcription factor whose activity depends on p38. However, these additional transcription factors remain to be identified. In order to identify p38-activated transcription factors that cooperate with NF-κB in response to LPS stimulation, microarrays were used to identify genes regulated by both NF-κB and p38 using wild-type, IKK-depleted, and p38 inhibitor-treated mouse bone marrow-derived macrophages (BMDMs). In silico analysis of transcription factor binding sites was used to predict the potential synergistic transcription factors from the co-expressed genes. Among these genes, NF-κB and C/EBPß, a p38 downstream transcription factor, were predicted to co-regulate genes in LPS-stimulated BMDMs. Based on the subsequent results of a chromatin immunoprecipitation assay and TNFAIP3 expression in C/EBPß-ablated macrophages, we demonstrated that Tnfaip3 is regulated by both NF-κB and p38-dependent C/EBPß. These results identify a novel regulatory mechanism in TLR4-mediated innate immunity.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Cisteína Endopeptidases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Ativação de Macrófagos , Macrófagos/imunologia , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células da Medula Óssea/citologia , Linhagem Celular , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Regiões Promotoras Genéticas/genética , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Transcrição Gênica/efeitos dos fármacos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa , Ubiquitina-Proteína Ligases/genéticaRESUMO
Far infrared (FIR) irradiation has been widely applied in health promotion. The aims of this study were to investigate the protective effect of FIR irradiation on stressed keratinocytes and the signaling pathways involved. HaCaT was subjected to sorbitol dehydration with or without 40min pretreatment with FIR radiation 4h earlier. Western blots of cell lysates were analyzed for caspase-3, HO-1, BCL2, Bax, ERK, and Akt. The incidence of apoptosis was also assessed by TUNEL staining. Evaluation of cell viability was determined using MTT. mRNAs were extracted and compared using Illumina Human Ref-8 v2 BeadChips. Hyperosomotic injury of HaCaT cells caused by sorbitol resulted in increased cleaved caspase-3 expression and this effect was decreased by FIR pretreatment; these findings were confirmed by TUNEL staining and MTT tests. Pre-treatment with FIR irradiation before sorbitol-induced dehydration significantly upregulated phosphorylated Akt (p-Akt) levels and A6730, an Akt kinase inhibitor (5µM), attenuated the protective effect of FIR irradiation. A microarray study showed FIR irradiation had far less effect at the transcriptional level. FIR pretreatment attenuates apoptosis and cell death in dehydration-stressed cultured keratinocytes through the PI-3K/Akt pathway, this protective effect of FIR irradiation is not at the transcriptional level.
