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. Author manuscript; available in PMC: 2017 Feb 2.
Published in final edited form as: Cell Rep. 2016 Jan 14;14(4):737–749. doi: 10.1016/j.celrep.2015.12.069

The 11S Proteasome Subunit PSME3 Is a Positive Feedforward Regulator of NF-κB and Important for Host Defense against Bacterial Pathogens

Jinxia Sun 1,5, Yi Luan 1,5, Dong Xiang 1, Xiao Tan 1, Hui Chen 1, Qi Deng 1, Jiaojiao Zhang 1, Minghui Chen 1, Hongjun Huang 1, Weichao Wang 1, Tingting Niu 1, Wenjie Li 4, Hu Peng 4, Shuangxi Li 1, Lei Li 1, Wenwen Tang 2, Xiaotao Li 1,*, Dianqing Wu 3,*, Ping Wang 1,2,*
PMCID: PMC4740229  NIHMSID: NIHMS753071  PMID: 26776519

SUMMARY

The NF-κB pathway plays important roles in immune responses. Although its regulation has been extensively studied, here, we report an unknown feedforward mechanism for the regulation of this pathway by Toll-like receptor (TLR) ligands in macrophages. During bacterial infections, TLR ligands upregulate the expression of the 11S proteasome subunit PSME3 via NF-κB-mediated transcription in macrophages. PSME3, in turn, enhances the transcriptional activity of NF-κB by directly binding to and destabilizing KLF2, a negative regulator of NF-κB transcriptional activity. Consistent with this positive role of PSME3 in NF-κB regulation and importance of the NF-κB pathway in host defense against bacterial infections, the lack of PSME3 in hematopoietic cells renders the hosts more susceptible to bacterial infections, accompanied by increased bacterial burdens in host tissues. Thus, this study identifies a substrate for PSME3 and elucidates a proteolysis-dependent, but ubiquitin-independent, mechanism for NF-κB regulation that is important for host defense and innate immunity.

Graphical Abstract

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INTRODUCTION

Bacterial infections are still threatening public health worldwide, even though various types of antibiotics are available (Scallan et al., 2011). A better understanding of the mechanisms by which bacterial pathogens are sensed by host immune systems may contribute to new approaches for developing antimicrobials. Bacterial pathogens can elicit host responses by interacting with cell-surface Toll-like pattern recognition receptors (Creagh and O’Neill, 2006; Kawai and Akira, 2010; Kumar et al., 2009; Skaug et al., 2009) to activate signaling pathways and gene transcription of various proinflammatory cytokines, bactericidal agents, and enzymes for protection of the hosts from pathogenic infection (Kolls et al., 2008; Medzhitov and Horng, 2009). Tight regulation of these key signaling pathways is essential for immune system homeostasis. Otherwise, inappropriate activation or overactivation of immune responses may result in inflammatory disorders such as septic shock and autoimmunity (Gordon and Martinez, 2010; Karin et al., 2006; Liew et al., 2005). Thus, identification of the regulators that control the inflammatory responses is vital.

The protein degradation pathways have important roles in bacterial infections, host defense, and immune responses (Jiang and Chen, 2012). The degradation of cellular proteins is mediated primarily by the ubiquitously expressed multi-subunit proteasome, which is involved in the regulation of almost every biological process, including the cell cycle, apoptosis, development, and major histocompatibility complex class I (MHC I) antigen presentation (Kloetzel, 2001; Rock et al., 2002). The proteasome is composed of a 20S catalytic subunit and one of three classes of regulatory subunits: 19S (PA700), 11S (PSME, PA28, or REG), and PA200 (Demartino and Gillette, 2007; Löwe et al., 1995; Ustrell et al., 2002). The 26S proteasome, composed of the 19S and 20S subunits, is responsible for selective degradation of most intracellular substrates in an ATP- and ubiquitin-dependent manner (Murata et al., 2009). The 11S-activated proteasome mediates protein degradation in a ubiquitin- and ATP-independent manner. Three members of the PSME family—PSME1-3 (a.k.a. PA28α, -β, and -γ or REGα, -β, and -γ)—have been characterized. In contrast to PSME1 and PSME2, which form hetero-oligomers, PSME3 forms a homo-oligomer (Dubiel et al., 1992). In addition, PSME3 is found mainly in the nucleus, whereas PSME1/2 is distributed throughout the cell (Wójcik et al., 1998).

Myriad amount of evidence indicates that the ubiquitin-proteasome pathway plays important roles in host defense (Sun, 2008). However, the role of the PSME-proteasome pathway in the inflammatory response remains largely unknown. Previous studies have shown that interferon γ (IFNγ)-inducible PSME1 and PSME2 are part of the immunoproteasomes, which are involved in the generation of MHC-I-presented peptides, whereas PSME3 is not involved (Früh and Yang, 1999; Rechsteiner et al., 2000; Sun et al., 2002). PSME3-knockout (KO) mice have a smaller body size (Murata et al., 1999). In agreement with this, growth arrest and increased apoptosis are observed in mouse embryonic fibroblasts (MEFs) (Mao et al., 2008; Murata et al., 1999; Rechsteiner and Hill, 2005). Furthermore, PSME3-KO mice show slightly decreased numbers of CD8+ T cells and impaired clearance of pulmonary fungal infections (Barton et al., 2004). PSME3 also speeds degradation of hepatitis C virus core protein and is essential for the nuclear retention of the hepatitis C virus (HCV) core protein (Moriishi et al., 2003). Increased class switching in B cells can also be observed from PSME3-deficient mice, since PSME3 can target nuclear AID for degradation (Uchimura et al., 2011). However, the role of PSME3 in innate immunity and host defense against infections by bacterial pathogens remains unknown.

Lung Krüppel-like factor 2 (KLF2) (Anderson et al., 1995), which belongs to the Krüppel-like factor (KLF) family of zinc-finger transcription factors, has been implicated in the regulation of many cellular processes, including T cell activation and migration, atherosclerosis, and monocyte differentiation (Cao et al., 2010; Carlson et al., 2006; Shaked and Ley, 2012). In myeloid cells, KLF2 functions as a negative regulator of myeloid cell activation by suppressing the activation of the necrosis factor κB (NF-κB) pathway and has an important role in the host response to polymicrobial infections (Das et al., 2006; Mahabeleshwar et al., 2011, 2012).

In this study, we identified KLF2 as a substrate for PSME3 and characterized a feedforward mechanism for the regulation of the NF-κB pathway in macrophages. In this mechanism, Toll-like receptor (TLR) ligands upregulate PSME3 expression via NF-κB, and PSME3, in turn, enhances the transcriptional activity of NF-κB through destabilizing KLF2. This mechanism has an important role in host defense against bacterial infections.

RESULTS

Upregulation of PSME3 Expression by TLR Ligands through NF-κB

Query of PSME3 in the gene expression database BioGPS (http://www.biogps.org) revealed that the expression of PSME3 is significantly upregulated in peripheral blood cells of human patients with sepsis (Wong et al., 2009). We confirmed these microarray results by quantitative real-time PCR analysis of an independent group of patients with sepsis in comparison to normal individuals (Figure 1A). This result raises a possibility that PSME3 expression may be regulated by bacteria or TLR ligands. Indeed, the mRNA and protein levels of PSME3 significantly increased in mouse peritoneal macrophages (PEMs) treated with Escherichia coli (E. coli) (Figures 1B and 1C) or lipopolysaccharide (LPS) (Figures 1D and 1E). LPS could also stimulate the expression of PSME3 in a mouse macrophage cell line (RAW264.7) (Figures S1A and S1B). In addition to LPS, which activates TLR4, ligands to other TLRs could also stimulate the expression of PSME3 in PEMs (Figure 1F). Consistent with this conclusion, Listeria monocytogenes (L. monocytogenes), which primarily activates TLR2, could also upregulate PSME3 expression (Figures S1C and S1D).

Figure 1. NF-κB Dependent Upregulation of PSME3 Expression.

Figure 1

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(A) The mRNA expression level of PSME3 in peripheral white blood cells from healthy human controls (HC) or patients with sepsis as determined by quantitative real-time PCR. ***p < 0.001 (Student’s t test); n = 20.

(B and C) Effects of E. coli on PSME3 expression in PEMs. The mRNA (B) and protein (C) levels of PSME3 in PEMs incubated with E. coli were determined by quantitative real-time PCR and western blot analysis, respectively. *p < 0.05 (Student’s t test).

(D and E) Effects of LPS on PSME3 expression in PEMs. The mRNA (D) and protein (E) levels of PSME3 in PEMs incubated with LPS were determined by quantitative real-time PCR and western blot analysis, respectively. *p < 0.05 (Student’s t test).

(F) Effects of Pam3Csk4, poly(I:C), LPS, or CpG-ODN on PSME3 expression in PEMs. PEMs were treated with the ligands for 4 hr, and cell lysates were analyzed for expression of PSME3 by immunoblotting. Ctrl, control.

(G) Effects of MAPK and NF-κB inhibitors on PSME3 expression in PEMs. PEMs were pretreated with or without the inhibitors for 2 hr before LPS treatment for 4 hr. Cell lysates were analyzed by immunoblotting.

(H) Effects of p65 knockdown on PSME3 expression in PEMs. PEMs were transfected with control siRNA or p65-specific siRNA for 72 hr and then treated with LPS for 4 hr. Cell lysates were analyzed by immunoblotting.

(I) Schematic representation of the PSME3 promoter and the putative NF-κB cis-element sequence.

(J) Effect of LPS on binding of p65 and its coactivator to the cis-element on the PSME3 promoter. PEMs were treated with or without LPS, and a ChIP assay was performed using an anti-p65 or anti-p300 antibody for immunoprecipitation (IP), followed by PCR with PSME3 promoter-specific primers.

Error bars indicate mean ± SD.

See also Figure S1.

Next, we investigated the mechanism by which PSME3 expression is regulated by TLR signaling. TLRs signal through both NF-κB and MAPK (mitogen-activated protein kinase) pathways (Krachler et al., 2011; Neish and Naumann, 2011). Therefore, we tested a number of MAPK and NF-κB inhibitors: SP600125 (a JNK inhibitor), SB20358 (a p38 inhibitor), U0126 (a MEK inhibitor), BMS-345541, BAY-11-7082, and SN-50 (NF-κB inhibitors). We found that inhibition of NF-κB, but not of MAPKs, impeded LPS-induced PSME3 upregulation in PEMs (Figure 1G). Consistent with the pharmacological results, small interfering RNA (siRNA)-mediated knockdown of p65, a key NF-κB isoform in macrophages (Li and Verma, 2002), in PEMs inhibited LPS-induced upregulation of PSME3 expression (Figure 1H). Furthermore, overexpression of IKKβ, which activates NF-κB signaling in 293T cells, could also increase PSME3 expression (Figure S1E). To determine whether PSME3 is a direct target gene of NF-κB, we analyzed the PSME3 promoter region using TFSEARCH software; we also used UCSC (University of California, Santa Cruz) Genome Bioinformatics and rVista 2.0 (Chan et al., 2010) and identified a potential NF-κB cis-element sequence (GGAAAGCCCGGTGC). This sequence is located at 1.3 kb upstream of the first exon of the PSME3 gene (Figure 1I). A chromatin immunoprecipitation (ChIP) assay revealed that p65, as well as its transcriptional coactivator p300, bound to this NF-κB cis-element sequence in response to LPS treatment (Figure 1J). Thus, these data, together with the results that protein synthesis inhibitor cycloheximide (CHX) abrogated LPS-induced upregulation of PSME3 protein (Figure S1F), indicate that TLR ligands upregulate PSME3 expression via NF-κB-mediated gene transcription.

Hematopoietic PSME3 Is Important for Host Defense against Bacterial Infections

NF-κB signaling in macrophages plays important roles in host defense against bacterial infections, including the L. monocytogenes infection (Rahman and McFadden, 2011). We used mice receiving the transfer of either PSME3-KO or wild-type (WT) bone marrow (BM) to assess the role of PSME3 in host defense to the L. monocytogenes infection to avoid complications that may result from PSME3 expression in non-hematopoietic cells. The BM-transplanted mice were inoculated with a lethal dose of L. monocytogenes, and those that received the PSME3-KO BM were significantly more susceptible to L. monocytogenes-induced lethality than those receiving the WT BM (Figure 2A). Concordantly, PSME3-KO BM-transplanted mice showed a greater loss of average body weight than that of WT BM-transplanted ones after L. monocytogenes infection (Figure 2B).

Figure 2. PSME3 Has an Important Role in Bacterial Clearance.

Figure 2

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(A and B) Mice lacking PSME3 in their hematopoietic cells are more susceptible to L. monocytogenes infection. Mice were lethally irradiated and transplanted with either WT or PSME3-KO BM cells. Two months after transplantation, the mice were infected with L. monocytogenes (5 × 105 CFU per mouse). Their survival (A) and body weight (B) were recorded.

(C and D) Effect of PSME3-deficiency on L. monocytogenes clearance. BM-transplanted mice were infected with L. monocytogenes, and the liver and spleen were collected on day 3. The enumerations of bacterial CFUs were performed through the serial dilution plating assay. ***p < 0.001 (Student’s t test); n = 6.

(E–F) Effect of PSME3 deficiency on bacterial clearance in a CLP model. Sham or CLP procedure was performed on the BM-transplanted mice, and the blood, lungs, livers, and kidneys were collected 12 hr later for assessment of bacterial loads. **p < 0.01 and ***p < 0.001 (Student’s t test); n = 6.

Next, we assessed the role of PSME3 in bacterial clearance. The lack of PSME3 resulted in significantly higher bacterial loads in the liver and spleen in mice inoculated with L. monocytogenes (Figures 2C and 2D). We also examined the importance of PSME3 in bacterial clearance using a polymicrobial infection model—the cecal ligation and puncture (CLP) model. The CLP has been adopted to model human sepsis (Buras et al., 2005). Enumeration of bacterial colonies from the blood and homogenates of the liver, spleen, and lung revealed that the lack of PSME3 in the hematopoietic cells resulted in significantly increased numbers of colony-forming units (CFUs) (Figures 2E–2H) over the controls. All of the results together indicate that PSME3 expression in hematopoietic cells plays an important role in effective bacterial clearance and host defense against bacterial infections.

PSME3 Deficiency Impairs Bactericidal Activities of Macrophages

The defect in bacterial clearance can be caused by an impairment of the bactericidal capability of macrophages. Therefore, we examined the role of PSME3 in bacterial clearance by macrophages using an in vitro assay—the antibiotic protection assay (APA). In this assay, PEMs lacking PSME3 showed significant impairment in bactericidal activity compared to the WT PEMs (Figure 3A). Consistent with this observation, knockdown of the endogenous PSME3 by its specific siRNA in RAW264.7 cells also significantly attenuated bactericidal activity (Figure S2A).

Figure 3. PSME3 Deficiency Impairs Bactericidal Activity of Macrophages.

Figure 3

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(A) Effect of PSME3 deficiency on bactericidal activity in vitro. PEMs were incubated with E. coli at an MOI of 4, and bacterial killing capability was analyzed by APA. ***p < 0.001 (Student’s t test).

(B–D) Effect of PSME3 deficiency on LPS-induced expression of iNOS and CRAMP. PEMs were treated with L. monocytogenes (B), E. coli (C), or LPS (D) for 6 hr. The relative mRNA levels of iNOS and CRAMP was determined by quantitative real-time PCR. *p < 0.05; **p < 0.01 (Student’s t test). Ctrl, control.

(E) Effect of PSME3 deficiency on the protein level of iNOS. PEMs were stimulated with LPS for indicated hours. Cell lysates were analyzed by immunoblotting.

(F) Effect of PSME3 deficiency on NO production. Conditioned media of LPS-treated PEMs were analyzed for nitrite by the Griess assay. ***p < 0.001 (Student’s t test).

Error bars indicate mean ± SD.

See also Figures S2 and S3.

Because PSME3 deficiency in PEMs or PSME3 knockdown in RAW264.7 cells did not affect phagocytosis (Figures S2B and S2C), we suspected that PSME3 deficiency may affect the expression of antimicrobial substances by macrophages. Indeed, PSME3-deficient macrophages expressed significantly lower levels of CRAMP and iNOS mRNAs when they were stimulated by L. monocytogenes, E. coli, or LPS (Figures 3B–3D). Consistent with the reduction in the iNOS mRNA in macrophages lacking PSME3, PSME3 deficiency also reduced the iNOS protein levels and nitric oxide (NO) production in macrophages upon LPS stimulation (Figures 3E and 3F). These observations could also be recapitulated in RAW264.7 cells; knockdown of PSME3 by its siRNA resulted in reduction in LPS-induced expression of CRAMP and iNOS mRNAs and iNOS protein, as well as production of NO (Figures S3A–S3D).

PSME3 Regulates the Assembly of NF-κB Transcriptional Complex at Its Promoters

To understand how PSME3 regulates the expression of antimicrobial genes, we first examined whether PSME3 deficiency affected LPS-stimulated MAPK and NF-κB signaling in macrophages, which regulates the expression of the iNOS and CRAMP genes (Li et al., 2009; Rahman and McFadden, 2011). PSME3 deficiency did not significantly affect the phosphorylation levels of JNK, ERK, and p38 in BM-derived macrophages (BMDMs) stimulated with LPS (Figure 4A), nor did it affect the degradation rate of IκBα (Figure 4A). PSME3 deficiency also did not exert a noticeable effect on LPS-induced nuclear accumulation of the NF-κB subunit p65 or its phosphorylation (Figures 4B–4D). These results suggest that PSME3 may not be involved in MAPK and NF-κB signaling. Therefore, we examined the effect of PSME3 on the binding of p65 to the promoters of its downstream target genes using the ChIP assay. We found that the binding of p65 to the iNOS, TNFα, IL1β, and CRAMP promoters was markedly attenuated in PSME3-deficient macrophages (Figure 4E). We also found that the binding of p300, a p65 coactivator that is required for transcriptional activity of the NF-κB pathway, to the iNOS, TNFα, IL1β, and CRAMP promoters was impaired in PSME3-KO macrophages (Figure 4E). These data suggest that PSME3 regulates the expression of NF-κB downstream target genes by affecting the binding of NF-κB and its transcriptional coactivator to its promoter, and this may be a general mechanism for PSME3 to regulate of NF-κB-mediated transcription of its target genes. Supporting this hypothesis, PSME3 deficiency in PEMs or PSME3 knockdown in RAW cells attenuated LPS-induced expression of two other NF-κB target genes, IL-1β and TNFα (Figure S4). Therefore, the data we have gathered thus far indicate that PSME3, whose expression is upregulated by the TLR ligands via the NF-κB pathway, facilitates NF-κB-mediated transcriptional activity. In other words, PSME3 functions in a positive feedforward mechanism for LPS-induced NF-κB signaling.

Figure 4. PSME3 Deficiency Inhibits the Binding of p65 and Its Transcriptional Coactivators to NF-κB Target Gene Promoters.

Figure 4

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(A) Effect of PSME3 deficiency on LPS-induced MAPK and NF-κB signaling. BMDMs were stimulated with LPS followed by western blotting analysis.

(B–D) Effect of PSME3 deficiency on LPS-induced p65 nuclear translocation and phosphorylation. BMDMs were stimulated with LPS. They were directly analyzed by western blot for phosphorylated p65 (D), fractionated into the nuclear and cytosolic fractions followed by western blotting (B), or stained with an anti-p65 antibody (green) followed by observation under a fluorescence microscope (C). Ctrl, control.

(E) Effect of PSME3 deficiency on LPS-induced binding of p65 and its coactivator to the promoters of NF-κB downstream target genes. PEMs were treated with or without LPS, and the ChIP assay was performed with an anti-p65, anti-p300, or IgG antibody for immunoprecipitation (IP), followed by PCR with the iNOS, TNFα, IL1β, CRAMP promoter-specific primers.

See also Figure S4.

PSME3 Promotes the Degradation of KLF2

To understand how PSME3 regulates NF-κB-mediated transcriptional activity, we sought to identify binding partners or substrates for PSME3 that may link PSME3 to the NF-κB pathway. We immunoprecipitated PSME3 from H1299 cells expressing Flag-tagged PSME3 and subjected precipitated proteins to mass spectrometry (MS) analysis. Among the proteins identified by MS with high confidence, there was one known to be related to NF-κB signaling, which is KLF2 (Figure S5A). KLF2 inhibits NF-κB signaling by suppressing the binding of p65 coactivators to the promoters of the NF-κB target genes (Das et al., 2006). We subsequently confirmed the interaction of PSME3 with KLF2, using both coimmunoprecipitation and pull-down assays (Figures 5A and 5B). We also found that PSME3 was colocalized with KLF2 in the nuclei of co-transfected HeLa cells (Figure S5B) and that endogenous PSME3 interacted with endogenous KLF2 in PEMs (Figure 5C). Importantly, PSME3 could target KLF2 for degradation, because expression of PSME3 reduced the protein levels of coexpressed KLF2 in HEK293T cells (Figure 5D). By contrast, expression of the other PSME3 family members (PSME1, PSME2, or PSME1/2) had no effect on the KLF2 protein level (Figure 5D). To determine whether KLF2 is directly degraded by the PSME3 proteasome, we used a rabbit reticulocyte-coupled transcription/translation system (Promega) to produce the KLF2 protein, and incubation of PSME3 and the 20S proteasome resulted in the degradation of KLF2, suggesting that KLF2 might be a direct substrate of PSME3 (Figure 5E). Furthermore, the finding that PSME3 reduced the half-life of KLF2 (Figures 5F and 5G) supports the idea that PSME3 regulates the protein stability of KLF2, and the observation that PSME3-mediated destabilization of KLF2 is sensitive to the 20S proteasome inhibitor MG132 (Figure S5C) further confirms that the action of PSME3 is proteasome mediated. Next, we found that the inactivated mutants of PSME3, including N151Y, P245Y, and G105S, that failed to destabilize p21 as previously reported (Chen et al., 2007; Li et al., 2007) also failed to destabilize KLF2 (Figure 5H). Together with the observation that the activated PSME3 mutant K188D showed enhanced KLF2 degradation (Figure 5H), we conclude that PSME3 most likely promotes KLF2 destabilization via their direct interaction in a proteasome activity-dependent manner.

Figure 5. PSME3 Binds to and Destabilizes KLF2.

Figure 5

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(A) Interaction of PSME3 with KLF2. GFP-PSME3 was cotransfected with an empty vector or Flag-KLF2 into 293T cells. The transfected cells were lysed after treatment with MG132 for 6 hr, followed by immunoprecipitation with an anti-Flag antibody. The immunoprecipitates (IPs) and the original whole-cell extracts (WCEs) were analyzed by immunoblotting.

(B) Direct interaction between PSME3 and KLF2. Purified GST-PSME3 protein was incubated with purified His-KLF2. After GST pull-down, proteins were analyzed by immunoblotting.

(C) Interaction of endogenous proteins. BMDMs were stimulated with or without LPS for 4 hr, and immunoprecipitation was performed with an anti-KLF2 antibody or IgG control. The proteins were analyzed by immunoblotting.

(D) Effect of PSME3 expression on the protein levels of coexpressed HA-KLF2. HA-KLF2 was expressed in 293T cells with PSME1, PSME2, PSME3, or PSME1/2. The KLF2 protein was detected by immunoblotting.

(E) KLF2 is is a direct substrate of PSME3. In vitro proteolytic analysis was performed using 10 μl purified KLF2, 0.25 μg purified 20S proteasome, and 1 μg PSME3 heptamers for the indicated times in 50 μl reaction volume at 30°C. An aliquot of the reaction was analyzed by western blotting.

(F and G) Effect of PSME3 expression on the protein stability of coexpressed HA-KLF2. HA-KLF2 was coexpressed with PSME3 in 293T cells. The half-life of HA-KLF2 was detected by the CHX chase assay.

(H) Effect of PSME3 mutant expression on the protein levels of coexpressed HA-KLF2. HA-KLF2 was coexpressed with PSME3 mutants including N151Y, P245Y, G105S, and K188D in 293T cells.

(I and J) Effect of PSME3 knockdown on the protein levels of HA-KLF2. HA-KLF2 and PSME3 siRNA were cotransfected into HeLa cells, and the half-life of KLF2 was assessed by the CHX chase assay.

(K) Effect of PSME3 deficiency on LPS-induced upregulation of the KLF2 protein levels. BMDMs were stimulation with LPS, and the KLF2 protein was detected by immunoblotting.

(L and M) Effect of PSME3 deficiency on KLF2 protein stability. The KLF2 protein stability in BMDMs was assessed by the CHX chase assay. See also Figure S5.

Next, we examined the importance of endogenous PSME3 in the stability of the KLF2 protein. Knockdown of PSME3 increased the half-life of the KLF2 protein in HeLa cells (Figures 5I and 5J). More importantly, the levels of endogenous KLF2 protein were higher in the PSME3 null PEMs than in the WT PEMs (Figure 5K), whereas the mRNA levels of KLF2 were no different in these cells (Figure S5D). The same results were observed in PSME3 knockdown of RAW264.7 cells (Figures S5E and S5F). In addition, the half-life of the KLF2 protein was increased in the PSME3-deficient macrophages compared to that in the WT cells (Figures 5L and 5M). Previous studies demonstrated that three E3 ligases (FBW7, WWP1, and Smurf1) could target KLF2 for degradation via the 26S proteasome (Wang et al., 2013; Xie et al., 2011; Zhang et al., 2004). However, siRNA-mediated depletion of FBW7, WWP1, or Smurf1 in HeLa cells did not affect PSME-mediated degradation of KLF2 (Figures S5G–S5I), suggesting that these E3 ligases may not be involved in the action of PSME. Additionally, the KLF2 4A mutant, which is resistant to FBW7-mediated degradation (Wang et al., 2013), could still be degraded by PSME3 (Figures S5J and S5K). These results together clearly support a conclusion that PSME3 directly targets KLF2 for degradation.

PSME3 Regulates Bactericidal Activities via KLF2

Consistent with the role of KLF2 in inhibiting NF-κB-dependent gene expression (Mahabeleshwar et al., 2011), expression of KLF2 markedly inhibited tumor necrosis factor α (TNFα) and IKKβ-activated NF-κB reporter gene activity (Figure 6A). Importantly, coexpression of WT PSME3, but not the N151Y mutant, was able to reverse the inhibitory effect of KLF2, indicating that PSME3 can regulate NF-κB transcriptional activity by relieving KLF2-mediated inhibition (Figure 6A; Figure S6A). To determine whether KLF2 is involved in the regulation of bactericidal activities by PSME3, peritoneal macrophages were transfected with the siRNAs against PSME3 and/or KLF2, which worked efficiently in these cells (Figure S6B). KLF2 depletion not only significantly enhanced the macrophages’ bactericidal activities as previously reported (Mahabeleshwar et al., 2011) but also abrogated the inhibitory effect of PSME3 knockdown on bactericidal activities (Figure 6B). The same results were observed for LPS-induced NO production and expression of iNOS, IL-1β, and TNFα; knockdown of KLF2 increased the production of NO and expression of iNOS, IL-1β, and TNFα and abrogated the effects of PSME3 knockdown (Figures 6C–6F). These results support the conclusion that PSME3 regulates NF-κB and bactericidal activities in a largely KLF2-dependent manner.

Figure 6. PSME3 Regulates Bactericidal Activities via KLF2.

Figure 6

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(A) PSME3 expression abrogates KLF2-mediated suppression of NF-κB reporter gene activity. KLF2 and PSME3 or PSME3 N151Y were cotransfected with a NF-κB promoter reporter gene into 293T cells. The reporter gene activity was analyzed by dual-luciferase assay after stimulation with TNFα (10 ng/ml) for 3 hr. *p < 0.05 (Student’s t test).

(B) PSME3 fails to impact bactericidal effects of macrophages in the absence of KLF2. PEMs were transfected with control siRNA or PSME3- and KLF2-specific siRNA and incubated with E. coli at an MOI of 4. The bacterial killing activity was analyzed by APA. *p < 0.05; **p < 0.01; and ***p < 0.001 (Student’s t test).

(C) PSME3 fails to impact NO production in macrophages in the absence of KLF2. PEMs were transfected with control siRNA or PSME3- and KLF2-specific siRNA and stimulated with LPS for 12 hr. Nitrite levels in the culture media were determined by the Griess assay. **p < 0.01 (Student’s t test).

(D–F) PSME3 fails to impact NF-κB target genes expression in macrophages in the absence of KLF2. PEMs were transfected with control siRNA or PSME3- and KLF2-specific siRNA and stimulated with LPS for 6 hr. The mRNA levels of iNOS (D), IL-1β (E), and TNFα (F) were determined by quantitative real-time PCR. *p < 0.05; **p < 0.01 (Student’s t test).

Error bars indicate mean ± SD.

See also Figure S6.

DISCUSSION

In the present work, we describe a previously unknown positive feedforward mechanism for the regulation of NF-κB by TLR ligands via the 11S proteasome regulatory subunit PSME3 (Figure 7). In this mechanism, PSME3 expression is upregulated by TLR ligands via NF-κB, and PSME3, in turn, promotes NF-κB transcriptional activity by destabilizing a NF-κB transcriptional suppressor KLF2. This mechanism has an important role in effective clearance of bacterial infection in mice.

Figure 7. A Model for the Role of PSME3 in NF-κB Signaling.

Figure 7

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PSME3 regulates host defense against bacterial pathogens by regulating the stability of the transcription factor KLF2. After bacterial infection or TLR ligands stimulation, PSME3, whose expression is upregulated via the NF-κB pathway, directly binds to and destabilizes KLF2, leading to augmented activation of NF-κB signaling.

Studies have demonstrated that ubiquitination underlies important mechanisms for the regulation of diverse immune responses (Liu and Chen, 2011; Sun, 2008). A number of E3 ubiquitin ligases have been identified for their roles in immune functions, including the development and activation of immune cells, immune tolerance, and innate immune responses (Liu and Chen, 2011; Sun, 2008). For example, ubiquitin-mediated protein degradation and different types of polyubiquitin chains, such as K63-linked ubiquitination, are involved in the activation of NF-κB (Skaug et al., 2009; Wertz and Dixit, 2010). Our present study provides an important insight into the role of the ubiquitin-independent 11S proteasome subunit PSME3 in the regulation of TLR-NF-κB signaling in macrophages and host defense against bacterial infections. Of note, a recent report suggests that PSME3 is downregulated in sepsis and negatively regulates NF-κB signaling (Yan et al., 2014). The reason for the discrepancies is unclear, which are likely due to the use of different techniques, animal models, sample collections, and/or cell types. For instance, Yan et al. relied solely on siRNA in their study of the function of PSME3 in macrophages without rigorous controls for possible “off-target” effects of this approach.

NF-κB represents a family of structurally related transcription factors that are regulated by a wide range of extracellular stimuli. They play pivotal roles in regulation of innate and adaptive immune responses. TLR ligands are potent activators of the NF-κB pathway. The core mechanism for this activation centered on the IKK complex has been well understood. In addition, NF-κB signaling is modulated by other regulations at various steps of the pathway. KLF2 is a transcription regulator that inhibits NF-κB transcriptional activity by directly binding to the NF-κB transcriptional coactivator p300 (Ahmad and Lingrel, 2005; Das et al., 2006; Huddleson et al., 2005). KLF2 has been shown to have functions in macrophages that are opposite to what we have shown for PSME3 (Das et al., 2006; Mahabeleshwar et al., 2011), which is consistent with the conclusion of this study that PSME3 regulates macrophage functions by destabilizing KLF2. Although both the protein level and the mRNA level of KLF2 in macrophages decrease upon treatment of bacterial products (Mahabeleshwar et al., 2012), the regulation of KLF2 at the protein level in macrophage remains largely uncharacterized. Previously, we showed that the E3 ligase FBXW7 can target KLF2 for degradation in endothelial cells through a mechanism that depends on GSK3-mediated phosphorylation of KLF2 (Wang et al., 2013). In the present study, we demonstrated that KLF2 stability is regulated by an ubiquitin-independent mechanism via the 11S proteasome subunit PSME3 in macrophages. It remains to be determined whether KLF2 stability can also be regulated by ubiquitin-dependent mechanisms in macrophages.

PSME3 deficiency resulted in significant reductions in a number of TLR-mediated transcriptional responses despite the magnitudes of these reductions being modest, i.e., at about 50%. We believe the significant in vivo phenotypes in lethality and bacterial burden are probably the results of summation of these modest reductions. We postulate that this PSME3-mediated positive feedforward mechanism may be needed by the hosts to mount more effective defense against bacterial infection. Although our present study focuses on the macrophages, it is possible that this feedforward mechanism plays important roles in NF-κB signaling in other immune and non-immune cells. Further investigation is warranted to investigate this possibility.

EXPERIMENTAL PROCEDURES

Mice

Mice were purchased from the National Rodent Laboratory Animal Resources, Shanghai Branch, and maintained in a laminar airflow cabinet under specific pathogen-free conditions according to the NIH standards established in the Guide for the Care and Use of Laboratory Animals. PSME3-KO mice were described previously (Li et al., 2015). All the protocols were approved by East China Normal University.

Cell Culture and Transfection

HEK293T cells, HeLa cells, and RAW264.7 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin. To obtain BMDMs, BM cells were collected from mouse femurs and tibias and cultured for 7 days in DMEM supplemented with 10% FBS, penicillin-streptomycin, and 20% conditioned medium (a 7-day supernatant from an L-929 cell line stably expressing macrophage colony-stimulating factor [M-CSF]) (Lombardo et al., 2007). To isolate primary mouse peritoneal macrophages, mice were injected with 3% sterile thioglycollate (Sigma, 70157) intraperitoneally (3 ml per mouse). Three days later, the mice were euthanized, and RPMI medium was injected intraperitoneally and then retrieved. The cells were then treated with ACK (ammonium-chloride-potassium) red blood cell (RBC) lysis buffer (0.15 M NH4Cl, 10.0 mM KHCO3, and 0.1 mM EDTA) and washed with PBS. The purity of the macrophages was confirmed by flow cytometry.

Transfections were performed using calcium phosphate-DNA coprecipitation (for 293T cells), Lipofectamine 2000 (for HeLa cells and PEMs; Invitrogen), and FUGEN HD (for RAW264.7 cells; Roche) according to the manufacturers’ instructions and with the following siRNA sequences: PSME3, GAATCAA TATGTCACTCTA; KLF2, GCAUGGAUGAGGACCUAAAUUTT.

BM Transplantation

BM cells were flushed from the tibias and femurs of C57BL/6 mice using RPMI supplemented with 2% FBS and penicillin-streptomycin. After RBC lysis, the cells were washed and resuspended in 1 ml PBS containing 107 cells, after which 200 μl of BM cells was injected intravenously into lethally irradiated (10 Gy) recipients.

CLP

All mice were maintained under specific pathogen-free conditions. C57BL/6 mice between 6 and 8 weeks old were given general anesthesia. A midline laparotomy was performed, and the cecum was exteriorized, ligated, and punctured twice with a 21-gauge needle. A small amount of the bowel contents was extruded to ensure wound patency. The cecum was replaced in its original location, and the abdomen was closed using a two-layer technique. Control surgeries (sham) were performed in the same manner, but the cecum was neither ligated nor punctured. The mice were resuscitated while under a 37°C heating blanket. These mice were euthanized 12 hr after CLP, and their blood, lung, liver, heart, and kidney tissues were isolated and homogenized. Bacterial CFUs were enumerated by serial dilution and plated on soya agar plates.

APA

Bacteria were grown to the logarithmic phase in Luria broth (LB) medium (for E. coli) to an optical density at 600 nm (OD600) ≈ 108 CFU/ml. These bacteria were pelleted and washed with PBS and then resuspended and diluted to the desired concentration in non-antibiotic-containing DMEM supplemented with 10% FBS. The bacteria were added to monolayers of RAW264.7 cells or PEMs at an MOI of four (for E. coli) bacteria per cell, and then the culture plates were incubated for 2 hr at 37°C after centrifugation at 500 × g for 10 min. The cells were washed three times with PBS and incubated with antibiotic-containing media for 30 min to kill surface-associated bacteria. The macrophages were cultured for an additional 4 hr and lysed with trypsin-EDTA and 0.025% Triton X-100. The cell lysates containing intracellular bacteria were serially diluted and spread on agar plates to enumerate bacterial CFUs.

Reagents

LPS (from E. coli serotype 055:B5) was purchased from Sigma-Aldrich. Pam3Csk4, poly(I:C), and CpG-ODN were from InvivoGen. The inhibitors SP600125, BAY-11-7082, SB20358, and U0126 were obtained from Biyuntian Biotechnology, and SN-50 was from Merck Millipore. Antibodies specific for p65 and p300 were obtained from Santa Cruz Biotechnology. Antibodies specific for ERK1/2 (Thr202/Tyr204), JNK1/2 (Thr183/Tyr185), p38 (Thr180/Tyr182), and iNOS were purchased from Cell Signaling Technology. Anti-PSME3 antibody was from Invitrogen. Anti-HA (hemagglutinin) and anti-Flag antibodies were purchased from Sigma-Aldrich. Rabbit anti-KLF2 polyclonal antibody was generated by GL Biochem (Shanghai), using recombinant protein as the antigen.

Flow Cytometry

To assay bacterial uptake, GFP-labeled heat-inactive E. coli were added to monolayers of RAW264.7 cells or BMDMs at an MOI of 8 and cocultured with these cells for 2 hr at 37°C, after which the cells were washed three times and treated with Trypan blue for 10 min to quench cell surface fluorescence. The data were collected using a FACSCalibur flow cytometer (BD Biosciences) and analyzed using FlowJo software.

Nitrite Determination

Conditioned cell culture media were collected after 12 hr of LPS treatment and then incubated with Griess reagent (Biyuntian Biotechnology) in 96-well plates. The absorbance was measured at 540 nm using a microplate spectrophotometer according to the manufacturer’s protocol.

Real-Time PCR

Total RNA was isolated from primary macrophages and RAW264.7 cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions and subjected to reverse transcription with random hexanucleotide primers. Real-time PCR was performed with Universal SYBR Green PCR Master Mix and an Applied Biosystems StepOnePlus Real-Time PCR System using specific primers with the following sequences: TNFα forward, 5′-GACCCTCA CACTCAGATCAT-3′; TNFα reverse, 5′-TTGAAGAGAACCTGGGAGTA-3′; IL-1β (interleukin-1β) forward, 5′-CAACCAACAAGTGATATTCTCCATG-3′; IL-1β reverse, 5′-GATCCACACTCTCCAGCTGCA-3′; iNOS forward, 5′-CCTCCTC CACCCTACCAAGT-3′; iNOS reverse, 5′-CACCCAAAGTGCTTCAGTCA-3′; CRAMP forward, 5′-CTTCAACCAGCAGTCCCTAGACA-3′; CRAMP reverse, 5′-TCCAGGTCCAGGAGACGGTA-3′; PSME3 forward, 5′-TCCTCACCAATAGCCACG-3′; and PSME3 reverse, 5′-CTCGATCAGCAGCCGAAT-3′.

ChIP Assay

PEMs were stimulated with or without LPS and then crosslinked with 1% formaldehyde at room temperature for 10 min. Nuclear extracts from the PEMs were subjected to immunoprecipitation with anti-IgG (immunoglobulin G), anti-p65, and anti-p300 antibodies and protein A/G beads at 4°C overnight. The precipitates were then washed and eluted, followed by heating at 65°C to reverse the formaldehyde crosslinking. The specific primers used in the ChIP assay were as follows: PSME3 forward, 5′-GATAAGCCACGAG GCTGTTTTG-3′; PSME3 reverse, 5′-GTCCCTCGGGCTAGTGTCAAAC-3′; iNOS forward, 5′-CACCACAGAGTGATGTAATC-3′; iNOS reverse 5′-GTC TTCAACTCCCTGTAAAG –3′; IL1β forward 5′-CTACCTTTGTTCCGCACA TCC-3′; IL1β reverse, 5′-CCCTCCCTTGTTTTATAGTCC-3′; TNFα forward, 5′-GAAAACTCACTTGGGAGCAGG-3′; TNFα reverse, 5′-GTGCTTCTGAAAGCTGGGTG-3′; CRAMP forward, 5′-CACTAAATCTAGGCTCACTCC-3′; and CRAMP reverse, 5′-GCCTTGGCTTCTCCTGGGCCC-3′.

Immunoprecipitation and Immunoblotting

Cells were treated with the proteasome inhibitor MG132 (20 μM; Sigma) for 4 hr before harvesting, lysed in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1 mM EDTA, and 0.5% Nonidet P-40) supplemented with a mixture of protease inhibitors and cleared by centrifugation. The cleared cell lysate was incubated with the indicated antibody and 15 μl of protein A/G beads (Santa Cruz Biotechnology) for 3 hr at 4°C. The immunoprecipitates were washed at least three times in lysis buffer. These protein samples were resolved by SDS-PAGE and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% nonfat milk and then incubated with the indicated primary antibodies. The immunoblot bands were detected using the Odyssey system (LI-COR Biosciences).

GST Pull-Down Assays

Bacteria-expressed glutathione S-transferase (GST) or GST-PSME3 proteins were purified using Glutathione-Sepharose 4B beads (Amersham Biosciences) and then incubated with His-KLF2 for 1 hr at 4°C. Bound KLF2 was detected by immunoblotting.

MS Experiment

H1299 cells were transfected with the plasmid for Flag-PSME3 expression. Cells were treated with the proteasome inhibitor MG132 (20 μM; Sigma) for 6 hr before harvesting and were lysed in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1 mM EDTA, and 0.5% Nonidet P-40) supplemented with a mixture of protease inhibitors. After clearance by centrifugation, the cell lysate was incubated with the M2 anti-Flag beads (Sigma) for 3 hr at 4°C. The immunoprecipitate was washed at least three times in lysis buffer. The samples were then separated on the 10% SDS-PAGE gel and stained with Coomassie blue. After extensive destaining, the gel was excised into approximately 1-mm bands and digested with sequencing grade modified trypsin (Promega). Peptides were extracted and analyzed using a liquid chromatography (LC) system (Dionex UltiMate 3000 Nano/Cap Pump, Thermo Fisher Scientific) coupled with an electrospray ionization quadruple time-of-flight (ESI-Q-TOF) mass spectrometer (Maxis Impact, Bruker Daltonik).

Statistical Analysis

Values are presented as the mean ± SD unless otherwise indicated. The statistical significance of differences between two groups was determined using Student’s t test analysis. Survival data were analyzed by constructing Kaplan-Meier plots using the log-rank test in Prism software. A difference with p < 0.05 was determined to be statistically significant.

Supplementary Material

supplementary figures
supplementary text

Highlights.

  • PSME3 is a target gene of NF-κB during bacterial infections

  • Hematopoietic PSME3-deficient mice are more susceptible to bacterial infections

  • PSME3-deficiency impairs macrophage functions

  • PSME3 activates NF-κB by degrading KLF2

Acknowledgments

We thank Dr. Hongyan Wang (SIBS, CAS) for kind assistance and comments. We also thank all the members of P.W. laboratory for encouragement and comments. This work was supported by grants from the National Basic Research Program of China (973 program 2012CB910404), the National Natural Science Foundation of China (91440104, 81402417, 31500735, and 31171338), the Doctoral Fund of the Ministry of Education of China (20130076110022), and the Science and Technology Commission of Shanghai Municipality (11DZ2260300).

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes six figures and can be found with this article online at http://dx.doi.org/10.1016/j.celrep.2015.12.069.

AUTHOR CONTRIBUTIONS

J.S., Y.L., D.W., and P.W. conceived the project and designed experiments. J.S., Y.L., D.X., X.T, H.C., Q.D, J.Z., M.C., H.H., W.W., T.N., W.L., H.P., S.L., and L.L. performed experiments. J.S, Y.L., D.X., X.T., W.T., X.L., D.W, and P.W. performed data analysis. J.S., Y.L., D.W., and P.W. wrote the manuscript.

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supplementary figures
NIHMS753071-supplement-supplementary_figures.pdf (681.9KB, pdf)
supplementary text
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