NLRP3 is the most crucial member of the NLR family, as it detects the existence of pathogen invasion and self-derived molecules associated with cellular damage. Several studies have reported that excessive NLRP3 inflammasome-mediated caspase-1 activation is a key factor in the development of diseases. Recent studies have reported that Syk is involved in pathogen-induced NLRP3 inflammasome activation; however, the detailed mechanism linking Syk to NLRP3 inflammasome remains unclear. In this study, we showed that Syk mediates NLRP3 stimuli-induced processing of procaspase-1 and the consequent activation of caspase-1. Moreover, the kinase activity of Syk is required to potentiate caspase-1 activation in a reconstituted NLRP3 inflammasome system in HEK293T cells. The adaptor protein ASC bridges NLRP3 with the effector protein caspase-1. Herein, we find that Syk can associate directly with ASC and NLRP3 by its kinase domain but interact indirectly with procaspase-1. Syk can phosphorylate ASC at Y146 and Y187 residues, and the phosphorylation of both residues is critical to enhance ASC oligomerization and the recruitment of procaspase-1. Together, our results reveal a new molecular pathway through which Syk promotes NLRP3 inflammasome formation, resulting from the phosphorylation of ASC. Thus, the control of Syk activity might be effective to modulate NLRP3 inflammasome activation and treat NLRP3-related immune diseases.
Toll-like receptors (TLRs) are a major family of pattern recognition receptors, and they play a crucial role in innate immune responses. Activation of TLR4 signaling at the plasma membrane by its ligand lipopolysaccharide (LPS) stimulates a proinflammatory pathway dependent on the E3 ubiquitin ligase TRAF6 (tumor necrosis factor receptor-associated factor 6) and the kinase TAK1 (transforming growth factor β-activated kinase 1), whereas TLR4 signaling at endosomes stimulates the production of type I interferons (IFNs) through a pathway that depends on TRAF3 and the kinase TBK1 (TANK-binding kinase-1). We found that the nonreceptor tyrosine kinase Syk partially mediated the endocytosis of TLR4, but it also played a dual role in TLR4-mediated signaling. LPS-dependent stimulation of TLR4 in Syk-deficient macrophages led to enhanced activation of TAK1 and increased production of proinflammatory cytokines compared to that in wild-type macrophages. In contrast, Syk-deficient macrophages exhibited decreased TLR4-dependent activation of TBK1 signaling and production of type I IFNs. We found that Syk was present in both TRAF6- and TRAF3-containing signaling complexes; however, the LPS-dependent, lysine 63-linked ubiquitination of TRAF6 and TRAF3 was oppositely regulated by Syk. We identified the domains of Syk that interacted with TRAF3, TRAF6, TAK1, and TBK1, factors activated by multiple TLRs, which suggests that Syk may act as a common regulator of various TLR responses. Together, our results demonstrate the opposing regulatory roles of Syk in TLR-mediated TRAF6 and TRAF3 signaling pathways, which suggests that Syk may fine-tune the innate immune response to lessen inflammation.
Gfi-1B (growth factor independence-1B) gene is an erythroid-specific transcription factor, whose expression plays an essential role in erythropoiesis. Our laboratory has previously defined the human Gfi-1B promoter region and shown that GATA-1 mediates erythroid-specific Gfi-1B transcription. By further investigating the regulation of the Gfi-1B promoter, here we report that (i) Gfi-1B transcription is negatively regulated by its own gene product, (ii) GATA-1, instead of Gfi-1B, binds directly to the Gfi-1-like sites in the Gfi-1B promoter and (iii) Gfi-1B suppresses GATA-1-mediated stimulation of Gfi-1B promoter through their protein interaction. These results not only demonstrate that interaction of GATA-1 and Gfi-1B participates in a feedback regulatory pathway in controlling the expression of the Gfi-1B gene, but also provide the first evidence that Gfi-1B can exert its repression function by acting on GATA-1-mediated transcription without direct binding to the Gfi-1 site of the target genes. Based on these data, we propose that this negative auto-regulatory feedback loop is important in restricting the expression level of Gfi-1B, thus optimizing its function in erythroid cells.
Expression of Gfi (growth factor-independence)-1B, a Gfi-1-related transcriptional repressor, is restricted to erythroid lineage cells and is essential for erythropoiesis. We have determined the transcription start site of the human Gfi-1B gene and located its first non-coding exon approximately 7.82 kb upstream of the first coding exon. The genomic sequence preceding this first non-coding exon has been identified to be its erythroid-specific promoter region in K562 cells. Using gel-shift and chromatin immunoprecipitation (ChIP) assays, we have demonstrated that NF-Y and GATA-1 directly participate in transcriptional activation of the Gfi-1B gene in K562 cells. Ectopic expression of GATA-1 markedly stimulates the activity of the Gfi-1B promoter in a non-erythroid cell line U937. Interestingly, our results have indicated that this GATA-1-mediated trans-activation is dependent on NF-Y binding to the CCAAT site. Here we conclude that functional cooperation between GATA-1 and NF-Y contributes to erythroid-specific transcriptional activation of Gfi-1B promoter.
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