The transcription factor NF-κB is an essential element of the TLR-mediated response in cells of the innate immune system. In macrophages, one of the physiological consequences of TLR stimulation is the release of inflammatory cytokines such as TNF, IL-1β and IL-6. The latent transcription factor NF-κB functions as an important bridge connecting TLR stimulation by endotoxins and the production of proinflammatory cytokines, in particular the TNF-α gene, which is a direct NF-κB target gene (1
). Here, we present evidence that in mouse macrophages, the zinc finger protein Gfi1 regulates the TLR signaling pathway by directly antagonizing NF-κB and preventing it from binding to its two NF-κB binding sites in target gene TNF-α promoter DNA after LPS stimulation. This finding may have important implications since a better understanding of how TLR signaling is regulated may facilitate the development of new strategies for controlling TLR-mediated inflammatory diseases.
We have reported that macrophages upregulate Gfi1 mRNA expression after stimulation with LPS (30
) and also upon treatment with other TLR ligands, such as CpG and PGN (41
). Our experiments with other cytokines and with TNF-deficient cells indicate that the induction of Gfi1 mRNA expression is elicited upon TLR4 signaling and not by inflammatory cytokines that are produced after TLR stimulation, for instance, through an autocrine loop. Our findings that TNF-deficient cells show Gfi1 mRNA induction similar to that in wild-type cells and the observation that TNF, IL-1β, and IL-6 cannot induce Gfi1 expression support this notion. Moreover, in contrast to what was found for other previously described regulators of TLR signaling, such as SHIP1 (50
), SOCS1 (13
), and IRAK-M (35
), the rapid induction of Gfi1 mRNA transcription seen within minutes of LPS stimulation in the present study is consistent with a role for Gfi1 as an immediate-early negative regulator of TLR signaling.
A second very important observation that we reported earlier was that Gfi1-deficient macrophages react with increased production of TNF-α and that Gfi1-deficient mice succumb quickly, with symptoms of septic shock, after LPS treatment (32
). In TNF−/−
double-deficient mice, the effect of Gfi1 deficiency is rescued (30
), suggesting that the high susceptibility of Gfi1 knockout mice toward LPS-induced septic shock is indeed mediated by heightened TNF-α production. In light of these findings, we had hypothesized that Gfi1 is induced by LPS through TLR signaling and acts as an upstream negative regulator of the TNF gene. Our new finding reported here, that in Gfi1−/−
macrophages the TNF-α mRNA level is increased severalfold over the wild-type level as early as 30 min after LPS stimulation, supports this conclusion and further emphasizes the role of Gfi1 in the regulation of TNF-α expression at the transcriptional level.
TLR activation is important for an infected host organism, since, on the one hand, it is essential for provoking the innate response and enhancing adaptive immunity against pathogens (2
), while, on the other hand, members of the TLR family are also involved in the pathogenesis of autoimmune, chronic inflammatory, and infectious diseases (6
). One of the most severe diseases is sepsis caused by LPS, an agonist of TLR4 (46
). A number of negative regulatory mechanisms that can dampen TLR-signaling pathways exist and have been described, suggesting multiple distinct types of safety mechanisms for controlling harmful inflammatory responses (5
). Many of the negative regulators involved act directly on proximal signaling events, for instance, at the level of adaptor molecules or upstream kinases (e.g., MyD88, IRAK, and TRAF6), which then affect more-distal events, such as the activation of MAP kinases and NF-κB. Our experiments suggest that Gfi1 does not interfere with the TLR signaling pathway at the proximal level, since the activation of cytoplasmic signaling molecules of the MAP kinase and PI3K pathways, as well as the cytoplasmic and nuclear components of the NF-κB pathway, appears unaltered in Gfi1-deficient cells after LPS stimulation when compared to the level in wild-type cells, although more-subtle effects cannot be entirely ruled out.
It is conceivable that Gfi1 dampens the physiological effects of the TLR4 response by downregulating TNF-α mRNA production through two mechanisms, first, by direct interference with the action of NF-κB, for instance, by blocking its binding to DNA, and second, by inhibiting its transactivation capacity. Our experiments presented here support the first mechanism, and we propose a model in which Gfi1 interacts with the p65 subunit of NF-κB and prevents it from binding to its cognate NF-κB binding sites present in the TNF-α promoter and very likely also the promoters of more than 50 other NF-κB target genes. Several lines of evidence support this hypothesis. First, electrophoretic mobility shift assays demonstrated that increased amounts of NF-κB-DNA complexes are present in LPS-stimulated BMDMs from Gfi1 knockout mice and decreased amounts in BMDMs from overexpressing vav-Gfi1 transgenic mice, compared to the amounts in wild-type macrophages. Second, reporter gene assays showed that Gfi1 blocks NF-κB-dependent transcription of synthetic target gene promoters appended to a luciferase gene. Third, when Gal4 fusions to the DNA binding or transactivation domains of p65 were tested, Gfi1 was found to inhibit only the activity of the fusion protein that retained the DNA binding domain of p65. Fourth, immunofluorescence experiments using transfected cells indicated that Gfi1 binds to the RHD domain of the p65 subunit of NF-κB, which contains sequences that contact DNA. Fifth, chromatin immunoprecipitation experiments with primers covering the TNF-α promoter demonstrated a higher rate of occupancy of the proximal NF-κB binding site by the p65 subunit in Gfi1-deficient macrophages than in wild-type cells. Last, PCR array analysis showed derepression of many NF-κB target genes in Gfi1-deficient BMDMs, compared to the level in wild-type BMDMs, suggesting that Gfi1 may play a role as a general negative regulator of NF-κB in inflammatory responses. Although these experiments are consistent with a direct interaction between Gfi1 and p65, it cannot be ruled out that other proteins act as intermediary factors or that the effect of Gfi1 on p65 and the NF-κB complex is mediated by a more indirect mechanism. More experimentation is required to resolve this question.
Our experiments show that 30 min after LPS stimulation, expression of the Gfi1 gene is induced, and Gfi1 protein is made and forms a complex with the NF-κB subunit p65 protein, which has translocated to the nucleus by this time. Also, at this time, TNF-α mRNA is upregulated and is present in Gfi1-deficient cells at a higher level than in wild-type cells. In addition, our ChIP experiments show that the TNF-α promoter is occupied by the NF-κB p65 subunit and that TNF-α mRNA is expressed as early as 30 min after LPS stimulation. When Gfi1 is absent, the TNF-α promoter shows a markedly higher p65 occupancy rate at two sites than at one site in wild-type cells and also a higher level of expression than in wild-type cells 30 min after LPS stimulation. This could be consistent with two different models: in the first model, the interaction between p65 and Gfi1 proteins directly precludes the access of p65 to the TNF-α promoter; in the second model, Gfi1 protein may act indirectly by masking a region in the RH domain of p65, which is responsible for the heterodimerization of p65 with p50 (which is required for promoter binding). The latter model is supported by gel shift experiments with in vitro-translated p65, p50, and Gfi1. Both models could explain how the binding of p65 to NF-κB target gene promoters is prevented by an interaction of Gfi1 with p65.
It remains to be shown how the interaction of p65 with Gfi1 precisely inhibits the DNA binding of NF-κB. One possibility is that Gfi1 simply competes with NF-κB binding sites for p65 and that in the absence of Gfi1, more p65 is free to access the TNF-α promoter, as reflected by a higher p65 occupancy rate as seen in our ChIP experiments. Since no known Gfi1 binding sites are present in the 1-kb proximal region of the TNF-α promoter tested here, and we were unable to detect Gfi1 by ChIP on the 1-kb 5′ upstream region of the TNF-α promoter (not shown), it seems unlikely that Gfi1 occupies the TNF-α promoter either in a direct way or through binding to p65. In addition, a model in which Gfi1 binds to NF-κB target sites through p65 seems unlikely since this model would be inconsistent with a number of findings reported here, such as the higher promoter occupancy rate of p65 in Gfi1-deficient cells, the increased level of p65-DNA complex formation in the absence of Gfi1 as detected by EMSA, and finally also data from our luciferase reporter assays using Gal4-p65 fusion proteins.
Since Gfi1 is a transcriptional repressor, another alternative explanation for the upregulation of NF-κB target gene expression could be that Gfi1 represses a coactivator of p65 or another transcriptional activator of p65 target genes. Again, this seems unlikely in light of our findings reported here. It is difficult to picture how such a model would lead to a higher level of p65-DNA complex formation or a higher rate of p65 occupancy at target gene promoters. Considering this, a more likely model is that after LPS stimulation, Gfi1 competes with NF-κB binding sites for p65 and simply titrates out p65 molecules able to bind to NF-κB target promoters rapidly. The facts that higher levels of p65-DNA complexes can be detected in Gfi1−/− cells and that a higher rate of promoter occupancy by p65 can be found in the absence of Gfi1 also argues for this hypothesis. This model would explain why a large number of NF-κB-responsive genes become upregulated by p65 in Gfi1 deficient cells and is consistent with the kinetics of the effects observed after TLR4 stimulation. However, it does not explain why some of the genes are not superinducible by Gfi1 deficiency. This may be due to a mechanism that restricts the action of Gfi1 to a subset of p65 target genes. A number of other p65-responsive promoters have to be tested similarly to the TNF promoter to answer this question and to confirm this hypothesis.
The evidence that we present here supports the view that the transcription factor Gfi1 acts as a general negative regulator for the TLR4 signaling pathway, which is critical for the transduction of inflammatory signals, for example, after exposure to bacterial cell wall antigens such as the endotoxin LPS. A clinical manifestation where this signaling pathway rapidly derails and is not under appropriate control is septic shock, and Gfi1-deficient mice treated with LPS indeed show symptoms reminiscent of septic shock. Our results suggest that Gfi1 prevents overactivity of the LPS-TLR4 pathway by dampening the effects of NF-κB at the downstream endpoint of TLR4 signaling in the nucleus. This represents an important advance in our understanding of the mechanisms that can lead to unregulated inflammatory reactions and may provide a theoretical basis for future interventional strategies designed to prevent death from septic shock.