FEBS Letters 588 (2014) 4513–4519
journal homepage: www.FEBSLetters.org
The transcription factor GFI1 negatively regulates NLRP3 inflammasome activation in macrophages Liuluan Zhu a,b,1, Qingcai Meng a,b,1, Shuntao Liang a,b, Yaluan Ma c, Rui Li a,b, Guoli Li a,b, Hui Zeng a,b,⇑ a
Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China c Institute of Basic Medical Theory of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China b
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Article history: Received 5 August 2014 Revised 13 October 2014 Accepted 22 October 2014 Available online 4 November 2014 Edited by Michael R. Bubb Keywords: Gfi1 NLRP3 Inflammasome IL-1b Macrophage
a b s t r a c t Interleukin-1b (IL-1b) secretion downstream of Toll-like receptor (TLR) activation is tightly controlled at the transcriptional and post-translational levels. NLRP3 inflammasome is involved in the maturation of pro-IL-1b, with NLRP3 expression identified as the limiting factor for inflammasome activation. Previously, we had demonstrated that the zinc-finger protein GFI1 inhibits pro-IL-1b transcription. Here, we show that GFI1 inhibits NLRP3 inflammasome activation and IL-1b secretion in macrophages. GFI1 suppressed Nlrp3 transcription via two mechanisms: (1) by binding to the Gli-responsive element 1 (GRE1) in the Nlrp3 promoter; and (2) by antagonizing the nuclear factor-jB (NF-jB) transcriptional activity. Thus, GFI1 negatively regulates TLR-mediated IL-1b production at both transcriptional and post-translational levels. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Innate immune cells initiate an immediate response to pathogens by generating abundant pro-inflammatory cytokines, including interleukin-1b (IL-1b), which exerts a wide range of inflammatory and antimicrobial activities [1–3]. Production of IL-1b is under tight regulation and requires a two-step process: (1) Toll-like receptor (TLR) and NF-jB signaling-dependent transcription and translation resulting in an inactive precursor, pro-IL-1b, and (2) inflammasome-mediated cleavage of pro-IL-1b into mature biologically active IL-1b [4,5]. Inflammasome is a multiprotein complex critical for IL-1b maturation and release [6,7]. The complex is generally composed of pro-caspase-1, adaptor protein apoptosis-associated speck-like protein (ASC) and one of the NOD-like receptor (NLR) family members, such as NLRP1 (NOD-like receptor containing pyrin 1), NLRP3 and NLRC4 (NOD-like receptor containing CARD) [8–10]. Upon stimulation with a myriad of microbial and endogenous signals, NLR is activated to assemble the inflammasome and sequentially primes auto-catalysis of pro-caspase-1 into active caspase-1, ⇑ Corresponding author at: Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, No. 8, Jing Shun East Street, Beijing 100015, China. Fax: +86 10 84322606. E-mail address: [email protected] (H. Zeng). 1 These authors contributed equally to this work.
which, in turn, proteolytically cleaves pro-IL-1b to release mature IL-1b [11–14]. Among the NLR family members, NLRP3 is the most sensitive receptor that can be activated by a broad range of pathogenassociated molecular patterns [13,15,16]. The NLRP3 inflammasome is particularly important for IL-1b production. Although several stimuli effectively trigger NLRP3 [17,18], one established mechanism for regulating its expression involves the TLR/MyD88 pathway, which is tightly controlled by signals culminating into activation of NF-jB [19,20]. Since NLRP3 expression is a critical checkpoint for inflammasome activation [19,21], negative regulatory mechanisms that suppress NLRP3 are necessary to control excessive IL-1b production during sepsis [22,23]. Recently, miR-223 was shown to suppress NLRP3 expression. However, since miR-223 itself is not regulated by pro-inflammatory signals, it may act like a rheostat to tightly control the NLRP3 message for basal protection [24]. Growth factor independence 1 (Gfi1) encodes a 55 kDa nuclear zinc finger transcription factor with an N-terminal SNAG domain and a six C-terminal zinc finger motifs linked by an intermediary region [25,26]. Gfi1 acts as a transcriptional repressor by binding to a DNA recognition sequence in target gene promoters [26–28]. Transcriptional repression activity of Gfi1 is dependent on the zinc finger motifs required for DNA binding as well as the intact N-terminal SNAG domain responsible for nuclear localization [26,28]. In addition, Gfi1 harbors functions independent of its
http://dx.doi.org/10.1016/j.febslet.2014.10.025 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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activity as a transcriptional regulator. Gfi1 is reported to interact with the STAT3 (signal transducer and activator of transcription 3) inhibitor PIAS3 (protein inhibitor of activated STAT 3) and the p65 subunit of NF-jB via its intermediary region [29,30]. Earlier studies revealed that Gfi1-deficient mice exhibit defects in development of hematopoietic and immune systems as well as excessive endotoxin-initiated innate immune responses [31–35]. Gfi1-deficient mice are highly susceptible to lipopolysaccharide (LPS)-induced septic shock due to overproduction of proinflammatory cytokines [31]. Recently, Gfi1 was shown to inhibit TLR-mediated production of TNF-a through binding p65 of NF-jB and consequently interfering with p65 binding to target gene promoter DNA [30]. In addition, the finding that Gfi1-deficient macrophages display higher levels of IL-1b mRNA than their wild-type counterparts after LPS stimulation suggests that Gfi1 negatively regulates IL-1b production at the transcriptional level [30]. In the present study, we investigated whether Gfi1 regulates TLR-mediated production of IL-1b at the post-transcriptional stages. The promoter sequence of Nlrp3 contains two NF-jB binding elements [20]. Screening of the promoter sequence revealed two putative Gfi1 binding sites containing the consensus motif AATC. Our data suggest that Gfi1 inhibits NLRP3 expression via two different pathways, either by binding directly to the promoter cis-element or antagonizing NF-jB p65 transcriptional activity, leading to subsequent effects on IL-1b maturation. 2. Materials and methods 2.1. Mice Gfi1/ mice were kindly provided by Professor Tarik Möröy (Institut de recherches cliniques de Montréal, Canada). Mice were housed under specific pathogen-free conditions at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College. Mouse treatments were approved by the local animal research committee at the Institute of Laboratory Animal Sciences. 2.2. Plasmids WT and NRE mutant Nlrp3 promoters were a kind gift from Professor Chengjiang Gao, including the full-length Nlrp3 promoter (nt 3033/+116), functional promoter (nt 1434/1113), mutNRE1 (nt 1434/1113, referred to as mutA in their report), mutNRE2 (nt 1434/1113, referred to as mutB) and mutNRE1/2 (nt 1434/1113, referred to as mutAB) [20]. The mutGRE1, mutGRE2 and mutGRE1/2 promoters were constructed by mutation of GRE1 and/or GRE2 sequence based on the full-length Nlrp3 promoter (nt 3033/+116). The mutNRE1/GRE1 promoter (nt 1434/1113) was generated by mutation of GRE1 (AATC to GGAG) based on the mutNRE1 promoter using the GBclonart Seamless Cloning and Assembly Kit (Genebank Biosciences Inc). All promoters were cloned into the pGL3-basic reporter vector. NF-jB p65 and various Gfi1 expression plasmids were kindly provided by Professor Tarik Möröy, including Gfi1-FL (full-length), DSNAG (SNAG domain deletion), DIM (intermediate region deletion) and DZF (zinc finger motif deletion). All plasmids were confirmed by DNA sequencing. 2.3. Stimulation of bone marrow-derived macrophages Bone marrow-derived macrophages (BMDM) were differentiated from bone marrow cells of 6- to 8-week-old C57BL/6 Gfi1-deficient mice and their wild-type (WT) littermates, as described previously [30]. Unless otherwise indicated, cells were primed for 4 h with 100 ng/ml LPS (Sigma–Aldrich) before
treatment with 5 mM ATP (Sigma–Aldrich) for a further 45 min. Supernatant fractions were collected for ELISA, and the cells lysed for RNA and protein isolation. 2.4. Cell culture, transfection and dual-luciferase reporter assay 293ET cells were cultured in DMEM with 10% FBS and 1% penicillin–streptomycin. For the luciferase reporter assay, 5 104 cells were cultured on a 24-well plate and subsequently transfected with a mixture of the indicated expression plasmids, promoter firefly luciferase reporters and Renilla luciferase plasmid (pRL-TK) using VigoFect reagent (Vigorous Biotechnology). After 24 h, luciferase assays were performed using a dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions. Relative luciferase activity was calculated by normalizing the ratio of firefly/Renilla luciferase. 2.5. Electrophoretic mobility shift assay (EMSA) In total, 2 106 293ET cells were cultured in a 60 mm dish and transfected with 5 lg Gfi1 expression plasmids or the vehicle control for 48 h. WT and Gfi1-deficient BMDMs were stimulated with 100 ng/ml LPS for 4 h before nuclear protein extraction. Nuclear extracts were prepared from 293ET cells or BMDMs using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce). EMSA was conducted with Light Shift Chemiluminescent EMSA Kit (Pierce) using the manufacturer’s protocol. Oligonucleotide probes were labeled with biotin. The sequences of the GRE1 and NRE1 probes were 50 -CAGATGAGTAACTGCCAATCCGTCTTTGACAGTG-30 and 50 -AGGCTTCTGCAGGGAACCCCCGGGAATTCT-30 , respectively. In competitive binding assays, unlabeled oligonucleotides were added at 200-fold molar excess. For supershift assay, 3 lg anti-Gfi1 antibody (Santa Cruz) was added. 2.6. Chromatin immunoprecipitation assay and PCR (ChIP–PCR) BMDMs were stimulated with 100 ng/ml LPS for 1 h and ChIP– PCR assay performed as described previously [36]. Input samples corresponded to 0.5% (v/v) of the total solution before immunoprecipitation. Purified chromatin was immunoprecipitated with 4 lg anti-Gfi1 antibody (Santa Cruz) or IgG. After DNA purification, the presence of specific DNA sequences was assessed using PCR. The primers used were 50 -CTCCACACACTGGTTCTGCTTG-30 (forward) and 50 -AGCAGCCTTGGGTACAAATAAAG-30 (reverse) for the GRE1 element (proximal region), and 50 -TTCTTCAAGGTCATTA GTGGCTTC-30 (forward) and 50 -AGTGCTAAAGGCCTAAGCAGATC-30 (reverse) for the negative control (distal region). The intensities of bands were quantified with Image J software (National Institutes of Health, Maryland, USA). 2.7. Real-time PCR Total RNA was isolated with TRIzol reagent (Life Technology) according to the manufacturer’s instructions. RNA (2 lg) was reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Life Technology), and specific transcripts quantified using the Applied BiosystemsÒ 7500 Real-Time PCR System (Life Technology). Quantitative real-time PCR was performed using TaqManÒ Gene Expression Master Mix and TaqManÒ probes according to the manufacturer’s instructions (Life Technology). 2.8. Western blot Whole cell extracts (20 lg) were used for Western blot analysis with primary antibodies against IL-1b (Abcam), NLRP3 (AdipoGen),
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Fig. 1. Gfi1 inhibits NLRP3 inflammasome activity and NLRP3 expression in macrophages. WT and Gfi1/ macrophages were primed with 100 ng/ml LPS for 4 h and stimulated with or without 5 mM ATP for 45 min. (A) IL-1b secretion in supernatants was measured using ELISA. (B) Whole-cell proteins were subjected to Western blot for pro-IL-1b, mature IL-1b, pro-caspase-1, cleaved caspase-1 (p10), ASC and NLRP3. b-Actin was detected as the loading control. (C) The mRNA levels of Nlrp3, Nlrp1a and Asc were quantified with real-time PCR. Values were normalized to those of Gadph and expressed relative to WT control cells (Ctrl). Representative results from three independent experiments are shown. ⁄P < 0.05; ⁄⁄⁄P < 0.001.
NF-jB p65 (CST), Flag (Sigma), caspase-1, ASC, Gfi1 and b-Actin (Santa Cruz). 2.9. Measurement of cytokine production BMDMs were seeded in 96-well plates and cultured overnight. After priming with 100 ng/ml LPS for 4 h and stimulating with 5 mM ATP for another 45 min, supernatant fractions were collected and IL-1b concentrations measured using the mouse IL-1b ELISA set (BD Biosciences Pharmingen), according to the manufacturer’s instructions. 2.10. Statistical analysis Data are presented as means ± S.D. of three independent experiments. Statistical differences were determined with the Student’s t-test, and P values < 0.05 considered statistically significant. 3. Results and discussion
increase in IL-1b production in culture medium than WT BMDMs in response to LPS stimulation alone, and a more dramatic release of IL-1b when stimulated with LPS and ATP (Fig. 1A). Moreover, Western blot analysis revealed a significant increase in mature IL-1b in Gfi1-deficient BMDMs (Fig. 1B and Suppl. 1). The data collectively suggest that Gfi1 inhibits maturation of IL-1b. 3.2. Gfi1 inhibits inflammasome activation and NLRP3 expression Since caspase-1 activity is essential for cleavage of pro-IL-1b into the mature form, we assessed cleavage of caspase-1 upon LPS/ATP stimulation in WT and Gfi1-deficient macrophages. Compared with WT, Gfi1/ BMDMs exhibited increased levels of caspase-1 after LPS/ATP stimulation, indicating elevated autocatalysis of pro-caspase-1. Consistent with enhanced caspase-1 cleavage, we detected higher levels of NLRP3, but unaltered ASC expression in Gfi1/ BMDMs (Fig. 1B). Real-time PCR revealed increased Nlrp3 mRNA levels in Gfi1-deficient macrophages (Fig. 1C), implying that Gfi1 affects expression of Nlrp3 at the transcriptional level.
3.1. Secretion of IL-1b is increased in Gfi1-deficient macrophages Our previous experiments disclosed significantly higher levels of IL-1b in sera of Gfi1-deficient mice treated with LPS [31], indicating that Gfi1 inhibits cytokine production upon endotoxin challenge. To confirm the potential regulatory activity of Gfi1 on IL-1b production, WT and Gfi1/ BMDMs were primed with LPS and subsequently stimulated with ATP. Consistent with previous in vivo findings [31], Gfi1-deficient BMDMs exhibited a slight
3.3. Gfi1 inhibits Nlrp3 transcription through direct binding to promoter DNA In view of the finding that Gfi1 represses target genes by binding the response element in the promoter region, we further examined whether Gfi1 acts as a transcriptional regulator of Nlrp3 through binding to its promoter DNA. 293ET cells were transfected with the mouse Nlrp3 promoter reporter, Gfi1 and activator NF-jB
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Fig. 2. Gfi1 inhibits Nlrp3 transcription by binding the GRE1 element in the promoter. (A) 293ET cells were transfected with 3 ng pRL-TK, 150 ng full-length Nlrp3 promoter reporter (nt 3033/+116) or pGL3-basic vehicle vector, 300 ng Gfi1 expression plasmid or pcDNA3.1 and 150 ng p65 expression plasmid for 24 h. Luciferase activities were measured and normalized to that of Renilla luciferase. (B) Alignment of the Gfi1 binding site consensus with the two potential GRE sites. The core sequence is in uppercase. Schematic of GRE1 and GRE2 location within the Nlrp3 promoter. (C) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 plasmid, 300 ng Gfi1 expression plasmid or pcDNA3.1 and 150 ng full-length Nlrp3 promoter or the mutants (nt 3033/+116) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. (D) Nuclear extracts were prepared with Gfi1-transfected 293ET cells (left) and LPS-stimulated BMDMs (right). EMSA was performed with biotin-labeled Nlrp3 oligonucleotides containing the Gfi1 binding site, GRE1. In competitive binding assays, 200-fold molar excess cold probe was added. The supershift bands indicate the complexes of Gfi1 antibody, Gfi1 protein and GRE1 element. (E) ChIP–PCR analysis of the promoter region of GRE1. BMDMs were stimulated with 100 ng/ml LPS for 1 h, protein–DNA complexes were immunoprecipitated with anti-Gfi1 or control IgG antibody. The 1434/1113 region harboring GRE1 element was amplified by PCR assay. An irrelevant distal region (nt 2269/2067) was amplified as the negative control. The relative intensities of the PCR bands were quantified and normalized to input control. ⁄P < 0.05; ⁄⁄⁄P < 0.001.
p65 expression plasmids. As expected, the dual luciferase report assay revealed significant suppression of Nlrp3 transcription by Gfi1 (Fig. 2A). Screening of the Nlrp3 promoter sequence further disclosed two putative binding sites for Gfi1 located at positions 1210/1207 and 408/405 of the transcription start site. To validate the functions of these two putative binding sites, mutant reporter promoters (mutGRE1, mutGRE2 and mutGRE1/2) were constructed and compared with the Gfi1 consensus binding site [27], as depicted in Fig. 2B. Notably, mutation of GRE1 reversed the inhibitory effect of Gfi1 while mutation of GRE2 had no influence on Nlrp3 transcription (Fig. 2C).
To further establish the effects of binding between GRE1 and Gfi1, EMSA was performed with 293ET nuclear extracts and a biotin-labeled probe containing GRE1. Compared with the Gfi1deficient nuclear protein (lane 2 in the left panel and lane 3 in the right panel), a shift band was observed in the Gfi1-expressing nuclear protein (lane 3 in the left panel and lane 2 in the right panel). The protein–DNA complex formation was remarkably reduced with the unlabeled probe competition (Lane 4). The binding specificity was further verified by the supershift band with anti-Gfi1 antibody (Lane 5). Consistent with this finding, a binding shift was observed with nuclear extracts from BMDMs (Fig. 2D).
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Fig. 3. Gfi1 suppresses Nlrp3 transcription by antagonizing the binding of NF-jB p65 and NRE1 elements. (A) Schematic of NRE1 and NRE2 positions in the Nlrp3 promoter. (B) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 expression plasmid or pcDNA3.1 and 150 ng functional Nlrp3 promoter mutants (nt 1434/1113) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. (C) 293ET cells were transfected with p65 and Gfi1 plasmids, and nuclear extracts identified via Western blot. EMSA was performed with biotin-labeled Nlrp3 oligonucleotides containing the NF-jB binding site, NRE1. In competitive binding assays, 200-fold molar excess cold probe was added. ⁄⁄⁄P < 0.001.
Fig. 4. Functions of different domains in Gfi1. (A) Schematic diagram depicts the deletion of various regions in the Gfi1 protein. All of the mutants were recombinant with a Flag-tag, and the expressions of them were detected by Western blot. (B–D) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 expression plasmid, 300 ng full-length or mutant Gfi1 expression plasmids and 150 ng WT Nlrp3 promoter (nt 3033/+116) (B), 150 ng mutNRE1 promoter (nt 1434/1113) (C) or 150 ng mutGRE1 promoter (nt 3033/+116) (D) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
These results suggest a combination of Gfi1 and GRE1 elements. The ChIP–PCR assay was additionally performed to determine whether Gfi1 occupies the endogenous promoter site at GRE1. The Nlrp3 promoter sequence containing GRE1 (proximal region) was successfully amplified from precipitates with the anti-Gfi1 antibody, but not the IgG control sample. For the negative control, the distal promoter region without GRE1 was not amplified from any of the immunoprecipitates (Fig. 2E). Our results collectively validate interactions between Gfi1 and GRE1 elements, both
in vivo and in vitro, and support direct transcriptional repression of Nlrp3 by Gfi1. 3.4. Gfi1 inhibits Nlrp3 transcription indirectly by interfering with NF-
jB p65 activity As specified earlier, mutation of both GRE1 and GRE2 did not counteract inhibition by Gfi1 completely (Fig. 2C), signifying the existence of other regulatory mechanisms. Accordingly, we further
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mutation of NRE1 almost completely suppressed activation of NFjB in the Nlrp3 promoter, mutation of NRE2 had no effect, and mutation of both NRE1 and 2 induced a similar level of inhibition as mutNRE1 (Fig. 3A and B). Our data indicate that NF-jB interacts specifically with NRE1 for activation in this system. To determine whether Gfi1 interferes with NF-jB binding to NRE1, EMSA was performed with nuclear NF-jB p65 and Gfi1. As shown in Fig. 3C, NF-jB p65 strongly interacted with the labeled probe (lane 2). This binding was competed by Gfi1 (lane 3) and excess cold probes (lane 4), suggesting that Gfi1 interferes with NF-jB p65 binding to the Nlrp3 promoter. 3.5. SNAG domain and zinc-finger motif of Gfi1are indispensable for regulation of Nlrp3
Fig. 5. Both GRE1 and NRE1 elements are involved in negative regulation by Gfi1. (A) A map of GRE1/NRE1 mutations in the Nlrp3 promoter region. (B) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 plasmid, 300 ng Gfi1 plasmid or pcDNA3.1 and 150 ng Nlrp3 promoter mutants for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. ⁄P < 0.05.
investigated whether Gfi1 modulates Nlrp3 transcription by interfering with NF-jB p65. Earlier, Qiao et al. identified two NF-jB binding sites in the Nlrp3 promoter [20]. To avoid endogenous influence, the dual luciferase reporter assay was performed with 293ET cells instead of macrophages. The Nlrp3 promoter was transfected along with NF-jB binding site mutants (mutNRE1, mutNRE2 and mutNRE1/2) into 293ET cells. Surprisingly, although
To clarify the specific domains that function in regulation of Nlrp3 transcription, a series of Gfi1 mutants (depicted in Fig. 4A) were adopted for the dual luciferase reporter assay. Significantly, all three domains were involved in the regulation of Nlrp3 transcription (Fig. 4B). With the mutNRE1 promoter containing only the GRE1 element, suppression by Gfi1 was abolished upon mutation of either SNAG (Gfi1 DSNAG) or zinc finger (Gfi1 DZF) domains, suggesting that intact SNAG domain and zinc finger motifs are indispensable for GRE1-mediated regulation (Fig. 4C). In contrast, with the mutGRE1 promoter containing only NRE1, the inhibitory effect of Gfi1 was counteracted by deletion of the intermediate region (Gfi1 DIM), indicating that this region is necessary for NF-jB-dependent inhibition (Fig. 4D). 3.6. Gfi1 regulates the NLRP3 inflammasome via a ‘‘dual repression model’’ mechanism Next, we constructed an Nlrp3 promoter reporter with a NRE1/ GRE1 double mutant (Fig. 5A). As shown in Fig. 5B, upon mutation of NRE1 and GRE1 separately, Gfi1 suppressed NF-jB-mediated transcription of Nlrp3. Notably, maximal inhibition was attained
Fig. 6. Model for regulation of the NLRP3 inflammasome in macrophages. (A) LPS/ATP induce robust activation of the NLRP3 inflammasome. The inflammatory response may be unregulated in the absence of Gfi1. (B) Gfi1 plays an essential role in controlling NLRP3 expression and inflammasome activation by binding the GRE1 element directly and blocking the transcriptional activity of NF-jB p65.
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upon mutation of both NRE1 and GRE1 elements. Our results collectively imply that Gfi1 inhibits Nlrp3 transcription through dual mechanisms: (1) direct binding of promoter DNA and (2) antagonizing NF-jB transcriptional activity. Upon mutation of the NRE1 element in the Nlrp3 promoter, the active signal of NF-jB was disabled so that promoter activity was reduced to the fundamental level. Our data suggest that Gfi1 is able to control Nlrp3 transcription at both active and basal levels. In summary, Gfi1 mediates regulation of NLRP3 expression through a ‘‘dual repression mode’’ mechanism. Upon activation by LPS, NF-jB promotes NLRP3 expression by binding the cis-element, NRE1, and subsequently induces assembly of the NLRP3 inflammasome to generate biologically active IL-1b (Fig. 6A). Meanwhile, Gfi1 is induced to control LPS-induced inflammation [30]. Gfi1 inhibits Nlrp3 transcription directly by binding the GRE1 site with C-terminal zinc-finger motifs or blocks NF-jB mediated Nlrp3 transcription through binding NF-jB p65 with the intermediate region. Both modes of action reduce IL-1b release from macrophages in response to TLR stimulation (Fig. 6B). It is worth mentioning that in response to LPS stimulation alone, Gfi1-deficient BMDMs exhibited a slight increase in IL-1b production in culture medium than WT BMDMs (Fig. 1A). A possible explanation is that Gfi1 might inhibit the basal secretion of IL-1b in an NLRP3 inflammasome-independent manner. Data from the present study, in conjunction with previous reports, suggest that Gfi1 restricts pro-IL-1b transcription and plays pivotal roles in regulating IL-1b production at both the transcriptional and post-translational levels. Our novel findings provide an ideal platform for the development of effective anti-inflammatory therapies to prevent IL-1b-induced tissue injury and mortality during sepsis.
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Funding This work was supported by the National Natural Science Foundation of China (No. 81101446) and Funding for Young Scientists in Beijing (No. 2012A003034000037). Acknowledgments We thank Professor Chengjiang Gao at Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University Medical School, Jinan, Shandong, China, for providing Nlrp3 promoter reporter genes. We are additionally grateful to Professor Tarik Möröy at Institut de recherches cliniques de Montréal, Canada, for p65 and Gfi1 expression plasmids, Gfi1/ mice, and critical comments on the manuscript.
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The transcription factor GFI1 negatively regulates NLRP3 inflammasome activation in macrophages Liuluan Zhu a,b,1, Qingcai Meng a,b,1, Shuntao Liang a,b, Yaluan Ma c, Rui Li a,b, Guoli Li a,b, Hui Zeng a,b,⇑ a
Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China Beijing Key Laboratory of Emerging Infectious Diseases, Beijing, China c Institute of Basic Medical Theory of Chinese Medicine, China Academy of Chinese Medical Sciences, Beijing, China b
a r t i c l e
i n f o
Article history: Received 5 August 2014 Revised 13 October 2014 Accepted 22 October 2014 Available online 4 November 2014 Edited by Michael R. Bubb Keywords: Gfi1 NLRP3 Inflammasome IL-1b Macrophage
a b s t r a c t Interleukin-1b (IL-1b) secretion downstream of Toll-like receptor (TLR) activation is tightly controlled at the transcriptional and post-translational levels. NLRP3 inflammasome is involved in the maturation of pro-IL-1b, with NLRP3 expression identified as the limiting factor for inflammasome activation. Previously, we had demonstrated that the zinc-finger protein GFI1 inhibits pro-IL-1b transcription. Here, we show that GFI1 inhibits NLRP3 inflammasome activation and IL-1b secretion in macrophages. GFI1 suppressed Nlrp3 transcription via two mechanisms: (1) by binding to the Gli-responsive element 1 (GRE1) in the Nlrp3 promoter; and (2) by antagonizing the nuclear factor-jB (NF-jB) transcriptional activity. Thus, GFI1 negatively regulates TLR-mediated IL-1b production at both transcriptional and post-translational levels. Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Innate immune cells initiate an immediate response to pathogens by generating abundant pro-inflammatory cytokines, including interleukin-1b (IL-1b), which exerts a wide range of inflammatory and antimicrobial activities [1–3]. Production of IL-1b is under tight regulation and requires a two-step process: (1) Toll-like receptor (TLR) and NF-jB signaling-dependent transcription and translation resulting in an inactive precursor, pro-IL-1b, and (2) inflammasome-mediated cleavage of pro-IL-1b into mature biologically active IL-1b [4,5]. Inflammasome is a multiprotein complex critical for IL-1b maturation and release [6,7]. The complex is generally composed of pro-caspase-1, adaptor protein apoptosis-associated speck-like protein (ASC) and one of the NOD-like receptor (NLR) family members, such as NLRP1 (NOD-like receptor containing pyrin 1), NLRP3 and NLRC4 (NOD-like receptor containing CARD) [8–10]. Upon stimulation with a myriad of microbial and endogenous signals, NLR is activated to assemble the inflammasome and sequentially primes auto-catalysis of pro-caspase-1 into active caspase-1, ⇑ Corresponding author at: Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, No. 8, Jing Shun East Street, Beijing 100015, China. Fax: +86 10 84322606. E-mail address: [email protected] (H. Zeng). 1 These authors contributed equally to this work.
which, in turn, proteolytically cleaves pro-IL-1b to release mature IL-1b [11–14]. Among the NLR family members, NLRP3 is the most sensitive receptor that can be activated by a broad range of pathogenassociated molecular patterns [13,15,16]. The NLRP3 inflammasome is particularly important for IL-1b production. Although several stimuli effectively trigger NLRP3 [17,18], one established mechanism for regulating its expression involves the TLR/MyD88 pathway, which is tightly controlled by signals culminating into activation of NF-jB [19,20]. Since NLRP3 expression is a critical checkpoint for inflammasome activation [19,21], negative regulatory mechanisms that suppress NLRP3 are necessary to control excessive IL-1b production during sepsis [22,23]. Recently, miR-223 was shown to suppress NLRP3 expression. However, since miR-223 itself is not regulated by pro-inflammatory signals, it may act like a rheostat to tightly control the NLRP3 message for basal protection [24]. Growth factor independence 1 (Gfi1) encodes a 55 kDa nuclear zinc finger transcription factor with an N-terminal SNAG domain and a six C-terminal zinc finger motifs linked by an intermediary region [25,26]. Gfi1 acts as a transcriptional repressor by binding to a DNA recognition sequence in target gene promoters [26–28]. Transcriptional repression activity of Gfi1 is dependent on the zinc finger motifs required for DNA binding as well as the intact N-terminal SNAG domain responsible for nuclear localization [26,28]. In addition, Gfi1 harbors functions independent of its
http://dx.doi.org/10.1016/j.febslet.2014.10.025 0014-5793/Ó 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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activity as a transcriptional regulator. Gfi1 is reported to interact with the STAT3 (signal transducer and activator of transcription 3) inhibitor PIAS3 (protein inhibitor of activated STAT 3) and the p65 subunit of NF-jB via its intermediary region [29,30]. Earlier studies revealed that Gfi1-deficient mice exhibit defects in development of hematopoietic and immune systems as well as excessive endotoxin-initiated innate immune responses [31–35]. Gfi1-deficient mice are highly susceptible to lipopolysaccharide (LPS)-induced septic shock due to overproduction of proinflammatory cytokines [31]. Recently, Gfi1 was shown to inhibit TLR-mediated production of TNF-a through binding p65 of NF-jB and consequently interfering with p65 binding to target gene promoter DNA [30]. In addition, the finding that Gfi1-deficient macrophages display higher levels of IL-1b mRNA than their wild-type counterparts after LPS stimulation suggests that Gfi1 negatively regulates IL-1b production at the transcriptional level [30]. In the present study, we investigated whether Gfi1 regulates TLR-mediated production of IL-1b at the post-transcriptional stages. The promoter sequence of Nlrp3 contains two NF-jB binding elements [20]. Screening of the promoter sequence revealed two putative Gfi1 binding sites containing the consensus motif AATC. Our data suggest that Gfi1 inhibits NLRP3 expression via two different pathways, either by binding directly to the promoter cis-element or antagonizing NF-jB p65 transcriptional activity, leading to subsequent effects on IL-1b maturation. 2. Materials and methods 2.1. Mice Gfi1/ mice were kindly provided by Professor Tarik Möröy (Institut de recherches cliniques de Montréal, Canada). Mice were housed under specific pathogen-free conditions at the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences, and Peking Union Medical College. Mouse treatments were approved by the local animal research committee at the Institute of Laboratory Animal Sciences. 2.2. Plasmids WT and NRE mutant Nlrp3 promoters were a kind gift from Professor Chengjiang Gao, including the full-length Nlrp3 promoter (nt 3033/+116), functional promoter (nt 1434/1113), mutNRE1 (nt 1434/1113, referred to as mutA in their report), mutNRE2 (nt 1434/1113, referred to as mutB) and mutNRE1/2 (nt 1434/1113, referred to as mutAB) [20]. The mutGRE1, mutGRE2 and mutGRE1/2 promoters were constructed by mutation of GRE1 and/or GRE2 sequence based on the full-length Nlrp3 promoter (nt 3033/+116). The mutNRE1/GRE1 promoter (nt 1434/1113) was generated by mutation of GRE1 (AATC to GGAG) based on the mutNRE1 promoter using the GBclonart Seamless Cloning and Assembly Kit (Genebank Biosciences Inc). All promoters were cloned into the pGL3-basic reporter vector. NF-jB p65 and various Gfi1 expression plasmids were kindly provided by Professor Tarik Möröy, including Gfi1-FL (full-length), DSNAG (SNAG domain deletion), DIM (intermediate region deletion) and DZF (zinc finger motif deletion). All plasmids were confirmed by DNA sequencing. 2.3. Stimulation of bone marrow-derived macrophages Bone marrow-derived macrophages (BMDM) were differentiated from bone marrow cells of 6- to 8-week-old C57BL/6 Gfi1-deficient mice and their wild-type (WT) littermates, as described previously [30]. Unless otherwise indicated, cells were primed for 4 h with 100 ng/ml LPS (Sigma–Aldrich) before
treatment with 5 mM ATP (Sigma–Aldrich) for a further 45 min. Supernatant fractions were collected for ELISA, and the cells lysed for RNA and protein isolation. 2.4. Cell culture, transfection and dual-luciferase reporter assay 293ET cells were cultured in DMEM with 10% FBS and 1% penicillin–streptomycin. For the luciferase reporter assay, 5 104 cells were cultured on a 24-well plate and subsequently transfected with a mixture of the indicated expression plasmids, promoter firefly luciferase reporters and Renilla luciferase plasmid (pRL-TK) using VigoFect reagent (Vigorous Biotechnology). After 24 h, luciferase assays were performed using a dual-Luciferase Reporter Assay System (Promega), according to the manufacturer’s instructions. Relative luciferase activity was calculated by normalizing the ratio of firefly/Renilla luciferase. 2.5. Electrophoretic mobility shift assay (EMSA) In total, 2 106 293ET cells were cultured in a 60 mm dish and transfected with 5 lg Gfi1 expression plasmids or the vehicle control for 48 h. WT and Gfi1-deficient BMDMs were stimulated with 100 ng/ml LPS for 4 h before nuclear protein extraction. Nuclear extracts were prepared from 293ET cells or BMDMs using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Pierce). EMSA was conducted with Light Shift Chemiluminescent EMSA Kit (Pierce) using the manufacturer’s protocol. Oligonucleotide probes were labeled with biotin. The sequences of the GRE1 and NRE1 probes were 50 -CAGATGAGTAACTGCCAATCCGTCTTTGACAGTG-30 and 50 -AGGCTTCTGCAGGGAACCCCCGGGAATTCT-30 , respectively. In competitive binding assays, unlabeled oligonucleotides were added at 200-fold molar excess. For supershift assay, 3 lg anti-Gfi1 antibody (Santa Cruz) was added. 2.6. Chromatin immunoprecipitation assay and PCR (ChIP–PCR) BMDMs were stimulated with 100 ng/ml LPS for 1 h and ChIP– PCR assay performed as described previously [36]. Input samples corresponded to 0.5% (v/v) of the total solution before immunoprecipitation. Purified chromatin was immunoprecipitated with 4 lg anti-Gfi1 antibody (Santa Cruz) or IgG. After DNA purification, the presence of specific DNA sequences was assessed using PCR. The primers used were 50 -CTCCACACACTGGTTCTGCTTG-30 (forward) and 50 -AGCAGCCTTGGGTACAAATAAAG-30 (reverse) for the GRE1 element (proximal region), and 50 -TTCTTCAAGGTCATTA GTGGCTTC-30 (forward) and 50 -AGTGCTAAAGGCCTAAGCAGATC-30 (reverse) for the negative control (distal region). The intensities of bands were quantified with Image J software (National Institutes of Health, Maryland, USA). 2.7. Real-time PCR Total RNA was isolated with TRIzol reagent (Life Technology) according to the manufacturer’s instructions. RNA (2 lg) was reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription kit (Life Technology), and specific transcripts quantified using the Applied BiosystemsÒ 7500 Real-Time PCR System (Life Technology). Quantitative real-time PCR was performed using TaqManÒ Gene Expression Master Mix and TaqManÒ probes according to the manufacturer’s instructions (Life Technology). 2.8. Western blot Whole cell extracts (20 lg) were used for Western blot analysis with primary antibodies against IL-1b (Abcam), NLRP3 (AdipoGen),
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Fig. 1. Gfi1 inhibits NLRP3 inflammasome activity and NLRP3 expression in macrophages. WT and Gfi1/ macrophages were primed with 100 ng/ml LPS for 4 h and stimulated with or without 5 mM ATP for 45 min. (A) IL-1b secretion in supernatants was measured using ELISA. (B) Whole-cell proteins were subjected to Western blot for pro-IL-1b, mature IL-1b, pro-caspase-1, cleaved caspase-1 (p10), ASC and NLRP3. b-Actin was detected as the loading control. (C) The mRNA levels of Nlrp3, Nlrp1a and Asc were quantified with real-time PCR. Values were normalized to those of Gadph and expressed relative to WT control cells (Ctrl). Representative results from three independent experiments are shown. ⁄P < 0.05; ⁄⁄⁄P < 0.001.
NF-jB p65 (CST), Flag (Sigma), caspase-1, ASC, Gfi1 and b-Actin (Santa Cruz). 2.9. Measurement of cytokine production BMDMs were seeded in 96-well plates and cultured overnight. After priming with 100 ng/ml LPS for 4 h and stimulating with 5 mM ATP for another 45 min, supernatant fractions were collected and IL-1b concentrations measured using the mouse IL-1b ELISA set (BD Biosciences Pharmingen), according to the manufacturer’s instructions. 2.10. Statistical analysis Data are presented as means ± S.D. of three independent experiments. Statistical differences were determined with the Student’s t-test, and P values < 0.05 considered statistically significant. 3. Results and discussion
increase in IL-1b production in culture medium than WT BMDMs in response to LPS stimulation alone, and a more dramatic release of IL-1b when stimulated with LPS and ATP (Fig. 1A). Moreover, Western blot analysis revealed a significant increase in mature IL-1b in Gfi1-deficient BMDMs (Fig. 1B and Suppl. 1). The data collectively suggest that Gfi1 inhibits maturation of IL-1b. 3.2. Gfi1 inhibits inflammasome activation and NLRP3 expression Since caspase-1 activity is essential for cleavage of pro-IL-1b into the mature form, we assessed cleavage of caspase-1 upon LPS/ATP stimulation in WT and Gfi1-deficient macrophages. Compared with WT, Gfi1/ BMDMs exhibited increased levels of caspase-1 after LPS/ATP stimulation, indicating elevated autocatalysis of pro-caspase-1. Consistent with enhanced caspase-1 cleavage, we detected higher levels of NLRP3, but unaltered ASC expression in Gfi1/ BMDMs (Fig. 1B). Real-time PCR revealed increased Nlrp3 mRNA levels in Gfi1-deficient macrophages (Fig. 1C), implying that Gfi1 affects expression of Nlrp3 at the transcriptional level.
3.1. Secretion of IL-1b is increased in Gfi1-deficient macrophages Our previous experiments disclosed significantly higher levels of IL-1b in sera of Gfi1-deficient mice treated with LPS [31], indicating that Gfi1 inhibits cytokine production upon endotoxin challenge. To confirm the potential regulatory activity of Gfi1 on IL-1b production, WT and Gfi1/ BMDMs were primed with LPS and subsequently stimulated with ATP. Consistent with previous in vivo findings [31], Gfi1-deficient BMDMs exhibited a slight
3.3. Gfi1 inhibits Nlrp3 transcription through direct binding to promoter DNA In view of the finding that Gfi1 represses target genes by binding the response element in the promoter region, we further examined whether Gfi1 acts as a transcriptional regulator of Nlrp3 through binding to its promoter DNA. 293ET cells were transfected with the mouse Nlrp3 promoter reporter, Gfi1 and activator NF-jB
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Fig. 2. Gfi1 inhibits Nlrp3 transcription by binding the GRE1 element in the promoter. (A) 293ET cells were transfected with 3 ng pRL-TK, 150 ng full-length Nlrp3 promoter reporter (nt 3033/+116) or pGL3-basic vehicle vector, 300 ng Gfi1 expression plasmid or pcDNA3.1 and 150 ng p65 expression plasmid for 24 h. Luciferase activities were measured and normalized to that of Renilla luciferase. (B) Alignment of the Gfi1 binding site consensus with the two potential GRE sites. The core sequence is in uppercase. Schematic of GRE1 and GRE2 location within the Nlrp3 promoter. (C) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 plasmid, 300 ng Gfi1 expression plasmid or pcDNA3.1 and 150 ng full-length Nlrp3 promoter or the mutants (nt 3033/+116) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. (D) Nuclear extracts were prepared with Gfi1-transfected 293ET cells (left) and LPS-stimulated BMDMs (right). EMSA was performed with biotin-labeled Nlrp3 oligonucleotides containing the Gfi1 binding site, GRE1. In competitive binding assays, 200-fold molar excess cold probe was added. The supershift bands indicate the complexes of Gfi1 antibody, Gfi1 protein and GRE1 element. (E) ChIP–PCR analysis of the promoter region of GRE1. BMDMs were stimulated with 100 ng/ml LPS for 1 h, protein–DNA complexes were immunoprecipitated with anti-Gfi1 or control IgG antibody. The 1434/1113 region harboring GRE1 element was amplified by PCR assay. An irrelevant distal region (nt 2269/2067) was amplified as the negative control. The relative intensities of the PCR bands were quantified and normalized to input control. ⁄P < 0.05; ⁄⁄⁄P < 0.001.
p65 expression plasmids. As expected, the dual luciferase report assay revealed significant suppression of Nlrp3 transcription by Gfi1 (Fig. 2A). Screening of the Nlrp3 promoter sequence further disclosed two putative binding sites for Gfi1 located at positions 1210/1207 and 408/405 of the transcription start site. To validate the functions of these two putative binding sites, mutant reporter promoters (mutGRE1, mutGRE2 and mutGRE1/2) were constructed and compared with the Gfi1 consensus binding site [27], as depicted in Fig. 2B. Notably, mutation of GRE1 reversed the inhibitory effect of Gfi1 while mutation of GRE2 had no influence on Nlrp3 transcription (Fig. 2C).
To further establish the effects of binding between GRE1 and Gfi1, EMSA was performed with 293ET nuclear extracts and a biotin-labeled probe containing GRE1. Compared with the Gfi1deficient nuclear protein (lane 2 in the left panel and lane 3 in the right panel), a shift band was observed in the Gfi1-expressing nuclear protein (lane 3 in the left panel and lane 2 in the right panel). The protein–DNA complex formation was remarkably reduced with the unlabeled probe competition (Lane 4). The binding specificity was further verified by the supershift band with anti-Gfi1 antibody (Lane 5). Consistent with this finding, a binding shift was observed with nuclear extracts from BMDMs (Fig. 2D).
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Fig. 3. Gfi1 suppresses Nlrp3 transcription by antagonizing the binding of NF-jB p65 and NRE1 elements. (A) Schematic of NRE1 and NRE2 positions in the Nlrp3 promoter. (B) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 expression plasmid or pcDNA3.1 and 150 ng functional Nlrp3 promoter mutants (nt 1434/1113) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. (C) 293ET cells were transfected with p65 and Gfi1 plasmids, and nuclear extracts identified via Western blot. EMSA was performed with biotin-labeled Nlrp3 oligonucleotides containing the NF-jB binding site, NRE1. In competitive binding assays, 200-fold molar excess cold probe was added. ⁄⁄⁄P < 0.001.
Fig. 4. Functions of different domains in Gfi1. (A) Schematic diagram depicts the deletion of various regions in the Gfi1 protein. All of the mutants were recombinant with a Flag-tag, and the expressions of them were detected by Western blot. (B–D) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 expression plasmid, 300 ng full-length or mutant Gfi1 expression plasmids and 150 ng WT Nlrp3 promoter (nt 3033/+116) (B), 150 ng mutNRE1 promoter (nt 1434/1113) (C) or 150 ng mutGRE1 promoter (nt 3033/+116) (D) for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. ⁄P < 0.05; ⁄⁄P < 0.01; ⁄⁄⁄P < 0.001.
These results suggest a combination of Gfi1 and GRE1 elements. The ChIP–PCR assay was additionally performed to determine whether Gfi1 occupies the endogenous promoter site at GRE1. The Nlrp3 promoter sequence containing GRE1 (proximal region) was successfully amplified from precipitates with the anti-Gfi1 antibody, but not the IgG control sample. For the negative control, the distal promoter region without GRE1 was not amplified from any of the immunoprecipitates (Fig. 2E). Our results collectively validate interactions between Gfi1 and GRE1 elements, both
in vivo and in vitro, and support direct transcriptional repression of Nlrp3 by Gfi1. 3.4. Gfi1 inhibits Nlrp3 transcription indirectly by interfering with NF-
jB p65 activity As specified earlier, mutation of both GRE1 and GRE2 did not counteract inhibition by Gfi1 completely (Fig. 2C), signifying the existence of other regulatory mechanisms. Accordingly, we further
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mutation of NRE1 almost completely suppressed activation of NFjB in the Nlrp3 promoter, mutation of NRE2 had no effect, and mutation of both NRE1 and 2 induced a similar level of inhibition as mutNRE1 (Fig. 3A and B). Our data indicate that NF-jB interacts specifically with NRE1 for activation in this system. To determine whether Gfi1 interferes with NF-jB binding to NRE1, EMSA was performed with nuclear NF-jB p65 and Gfi1. As shown in Fig. 3C, NF-jB p65 strongly interacted with the labeled probe (lane 2). This binding was competed by Gfi1 (lane 3) and excess cold probes (lane 4), suggesting that Gfi1 interferes with NF-jB p65 binding to the Nlrp3 promoter. 3.5. SNAG domain and zinc-finger motif of Gfi1are indispensable for regulation of Nlrp3
Fig. 5. Both GRE1 and NRE1 elements are involved in negative regulation by Gfi1. (A) A map of GRE1/NRE1 mutations in the Nlrp3 promoter region. (B) 293ET cells were transfected with 3 ng pRL-TK, 150 ng p65 plasmid, 300 ng Gfi1 plasmid or pcDNA3.1 and 150 ng Nlrp3 promoter mutants for 24 h. Luciferase activity was measured and normalized to that of Renilla luciferase. ⁄P < 0.05.
investigated whether Gfi1 modulates Nlrp3 transcription by interfering with NF-jB p65. Earlier, Qiao et al. identified two NF-jB binding sites in the Nlrp3 promoter [20]. To avoid endogenous influence, the dual luciferase reporter assay was performed with 293ET cells instead of macrophages. The Nlrp3 promoter was transfected along with NF-jB binding site mutants (mutNRE1, mutNRE2 and mutNRE1/2) into 293ET cells. Surprisingly, although
To clarify the specific domains that function in regulation of Nlrp3 transcription, a series of Gfi1 mutants (depicted in Fig. 4A) were adopted for the dual luciferase reporter assay. Significantly, all three domains were involved in the regulation of Nlrp3 transcription (Fig. 4B). With the mutNRE1 promoter containing only the GRE1 element, suppression by Gfi1 was abolished upon mutation of either SNAG (Gfi1 DSNAG) or zinc finger (Gfi1 DZF) domains, suggesting that intact SNAG domain and zinc finger motifs are indispensable for GRE1-mediated regulation (Fig. 4C). In contrast, with the mutGRE1 promoter containing only NRE1, the inhibitory effect of Gfi1 was counteracted by deletion of the intermediate region (Gfi1 DIM), indicating that this region is necessary for NF-jB-dependent inhibition (Fig. 4D). 3.6. Gfi1 regulates the NLRP3 inflammasome via a ‘‘dual repression model’’ mechanism Next, we constructed an Nlrp3 promoter reporter with a NRE1/ GRE1 double mutant (Fig. 5A). As shown in Fig. 5B, upon mutation of NRE1 and GRE1 separately, Gfi1 suppressed NF-jB-mediated transcription of Nlrp3. Notably, maximal inhibition was attained
Fig. 6. Model for regulation of the NLRP3 inflammasome in macrophages. (A) LPS/ATP induce robust activation of the NLRP3 inflammasome. The inflammatory response may be unregulated in the absence of Gfi1. (B) Gfi1 plays an essential role in controlling NLRP3 expression and inflammasome activation by binding the GRE1 element directly and blocking the transcriptional activity of NF-jB p65.
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upon mutation of both NRE1 and GRE1 elements. Our results collectively imply that Gfi1 inhibits Nlrp3 transcription through dual mechanisms: (1) direct binding of promoter DNA and (2) antagonizing NF-jB transcriptional activity. Upon mutation of the NRE1 element in the Nlrp3 promoter, the active signal of NF-jB was disabled so that promoter activity was reduced to the fundamental level. Our data suggest that Gfi1 is able to control Nlrp3 transcription at both active and basal levels. In summary, Gfi1 mediates regulation of NLRP3 expression through a ‘‘dual repression mode’’ mechanism. Upon activation by LPS, NF-jB promotes NLRP3 expression by binding the cis-element, NRE1, and subsequently induces assembly of the NLRP3 inflammasome to generate biologically active IL-1b (Fig. 6A). Meanwhile, Gfi1 is induced to control LPS-induced inflammation [30]. Gfi1 inhibits Nlrp3 transcription directly by binding the GRE1 site with C-terminal zinc-finger motifs or blocks NF-jB mediated Nlrp3 transcription through binding NF-jB p65 with the intermediate region. Both modes of action reduce IL-1b release from macrophages in response to TLR stimulation (Fig. 6B). It is worth mentioning that in response to LPS stimulation alone, Gfi1-deficient BMDMs exhibited a slight increase in IL-1b production in culture medium than WT BMDMs (Fig. 1A). A possible explanation is that Gfi1 might inhibit the basal secretion of IL-1b in an NLRP3 inflammasome-independent manner. Data from the present study, in conjunction with previous reports, suggest that Gfi1 restricts pro-IL-1b transcription and plays pivotal roles in regulating IL-1b production at both the transcriptional and post-translational levels. Our novel findings provide an ideal platform for the development of effective anti-inflammatory therapies to prevent IL-1b-induced tissue injury and mortality during sepsis.
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Funding This work was supported by the National Natural Science Foundation of China (No. 81101446) and Funding for Young Scientists in Beijing (No. 2012A003034000037). Acknowledgments We thank Professor Chengjiang Gao at Key Laboratory for Experimental Teratology of the Ministry of Education, Shandong University Medical School, Jinan, Shandong, China, for providing Nlrp3 promoter reporter genes. We are additionally grateful to Professor Tarik Möröy at Institut de recherches cliniques de Montréal, Canada, for p65 and Gfi1 expression plasmids, Gfi1/ mice, and critical comments on the manuscript.
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