SKP2 attenuates NF-κB signaling by mediating IKKβ degradation through autophagy

SKP2 attenuates NF-κB signaling by mediating IKKβ degradation through autophagy Abstract NF-κB signaling controls a large set of physiological processes ranging from inflammatory responses to cell death. Its activation is tightly regulated through controlling the activity and stability of multiple signaling components. Here, we identify that NF-κB activation is suppressed by an F-box protein, S-phase kinase associated protein 2 (SKP2). SKP2 deficiency enhanced NF-κB activation as well as the production of inflammatory cytokines. In addition, SKP2 potently blocked the NF-κB activation at the IκB kinase (IKK) level. Mechanistic study further revealed that SKP2 functions as an adaptor to promote an interaction between active IKKβ and the autophagic cargo receptor p62 to mediate IKKβ degradation via selective autophagy. These findings identify a previously unrecognized role of SKP2 in NF-κB activation by which SKP2 acts as a secondary receptor to assist IKKβ delivery to autophagosomes for degradation in a p62-dependent manner. SKP2, IKKβ, NF-κB activation, autophagy, inflammation Introduction The nuclear factor-κB (NF-κB) is a critical transcription factor to mediate immediate responses to pathogens, and its activity is accurately controlled by a variety of regulators to prevent potential impairment from its excessive activation (Chan et al., 2015). Upon detection of invading pathogens, Toll-like receptors (TLRs), as well as other cytokines receptors, recruit their specific adaptor proteins for the association and subsequent activation of a variety of TRAF proteins. Activated TRAF2/6 or TRAF2/5 serve as a platform to recruit TAK1 and IκB kinase (IKK) complex through catalyzing lysine 63 (K63)-linked poly-ubiquitination on itself (Oeckinghaus et al., 2011). As the core of NF-κB signaling, the formation and activation of IKK complex in turn enable IKKβ to phosphorylate IκB protein and promote its degradation, thus allowing NF-κB to translocate into the nucleus to induce the transcription of a variety of pro-inflammatory genes (Hayden and Ghosh, 2008). NF-κB signaling is tightly regulated by multiple post-translational modifications of their signaling components (Bhoj and Chen, 2009). Aberrant activation of NF-κB results in physiologic disorders and diseases, such as cancer, inflammation, and autoimmune diseases (Baldwin, 2012). Due to its central role in NF-κB signaling, modulating IKK activity is the major way to control NF-κB activation (Chen, 2005). For example, Tripartite motif 13 (TRIM13) suppresses TNF-α-induced NF-κB activation through regulating the ubiquitination and turnover of NEMO, a regulatory sub-unit of IKK complex (Tomar and Singh, 2014). Previously, we have identified that nucleotide binding oligomerization domain (NOD)-like receptor (NLR) family member X1 (NLRX1) and NLRC5 negatively regulate TLR signaling through IKK inhibition (Cui et al., 2010; Xia et al., 2011). Furthermore, we found that NLRC5 shapes NF-κB signaling relied on its ubiquitination (Meng et al., 2015). Recently, our work demonstrated that LRRC14 attenuates TLR-mediated NF-κB signaling by blocking the formation of IKK complex (Wu et al., 2016). However, the regulation of IKK stability during NF-κB activation is not fully investigated yet. Recently, extensive studies have revealed that the innate immunity, including NF-κB activation, can be regulated by autophagy. Autophagy is a ubiquitous pathway in cells by which cytoplasmic material or organelles are delivered to lysosomes for degradation (Yang and Klionsky, 2010). In mice, knockout of Atg5, the key components of autophagy, in macrophages and neutrophils increases susceptibility to infection with bacteria, including L. monocytogenes and T. gondii (Kimmey et al., 2015). Besides non-selective bulk degradation via autophagosome, the selective autophagy is regarded as a degradative way of particular proteins or protein aggregates, in which the specificity of the substrates is determined by a variety of cargo receptors, such as p62, NBR1, or NDP5 (Moscat and Diaz-Meco, 2009). Increasing evidence demonstrates that selective autophagy plays a critical role in the regulation of innate immune signaling. We have reported that p62-mediated AIM2 degradation through selective autophagy inhibits the inflammasome activation during DNA virus infection (Liu et al., 2016). In addition, we found that p62-mediated cGAS degradation and NDP52-mediated MAVS degradation negatively regulate the activation of type I interferon signaling as well as the anti-virus response (Chen et al., 2016; Jin et al., 2017). Although it has been reported that IKKβ could undergo degradation via autophagy (Kim et al., 2010), it is unknown whether selective autophagy could regulate NF-κB activation via specifically mediating the stability of IKKβ. In the present study, we identify F-box protein SKP2 as a negative regulator of TLR or TNFα-induced NF-κB activation. Knockout of SKP2 significantly enhanced the expression and secretion of pro-inflammatory cytokines in THP-1 cells. SKP2 targeted to IKK complex upon LPS treatment, and specifically promoted the degradation of activated IKKβ. Furthermore, we found that SKP2 promoted the interaction between IKKβ and the autophagic cargo receptor p62 to mediate IKKβ for degradation via selective autophagy. Therefore, our findings provide an insight into the mechanisms of tight regulation of NF-κB through its crosstalk with autophagy. Results Identification of SKP2 as a negative regulator of NF-κB signaling To investigate the roles of F-box family proteins in NF-κB signaling, we screened a panel of Flag-tagged F-box proteins using MyD88-mediated NF-κB luciferase reporter activation assay in HEK293T cells. Of 24 candidate genes, we identified SKP2 (also known as FBXL1) as a negative regulator of MyD88-mediated NF-κB activation (Figure 1A). In order to confirm the function of SKP2, we constructed SKP2 plasmids with different tags or not, and found that all of them could inhibit MyD88-mediated NF-κB activation as the one without tag (Figure 1B), indicating that different tags do not affect the function of SKP2 in this study. Next, we tested whether SKP2 affects the NF-κB signaling induced by multiple stimuli, and found that SKP2 negatively regulated the activation of NF-κB induced by lipopolysaccharide (LPS), TNF-α, or IL-1β respectively (Figure 1C). The degradation of IκB-α is an important landmark of NF-κB activation. We found that SKP2 inhibited the degradation of endogenous IκB-α protein in the presence of TNF-α (Figure 1D). Consistently, SKP2 overexpression resulted in much lower expression of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β by TNF-α treatment (Figure 1E). Taken together, these results suggest that SKP2 inhibits NF-κB activation as well as the expression of pro-inflammatory cytokines. Figure 1 View largeDownload slide SKP2 inhibits NF-κB activation. (A) HEK293T cells were transfected with plasmids of 24 F-box proteins along with MyD88 and a reporter plasmid carrying the NF-κB promoter reporter (NF-κB-luc) plasmid and pRL-TK plasmid. The cells were analyzed for NF-κB activity by a reporter gene assay, protein expression levels were detected by immunoblot analysis. (B) Luciferase activity of HEK293T cells transfected with the plasmids encoding SKP2 with indicated tags or not, along with MyD88, NF-κB-luc, and pRL-TK, was determined 24 h after transfection. (C) HeLa cells were transfected with NF-κB-luc and pRL-TK plasmids, along with empty vector or increasing amounts of SKP2, and then treated with LPS (10 μg/ml), IL-1β (10 ng/ml), or TNF-α (10 ng/ml) for 10 h, the NF-κB activity was determined by a reporter gene assay. (D) HEK293T cells were transfected with Myc-SKP2 or empty vector, IκB-α turnover was monitored by immunoblot analysis using indicated antibodies after treated with TNF-α (10 ng/ml) for 2 h. Numbers underneath the blot represent the fold change of IκB-α band intensity compared with control group, using β-actin as a loading control. (E) HEK293T cells were transfected with an empty vector or different doses of Myc-SKP2 expression vector and then treated with TNF-α (10 ng/ml) for 2 h, the gene expressions of TNF-α, IL-6, and IL-1β were determined by real-time PCR. Data in A–C and E are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test). Figure 1 View largeDownload slide SKP2 inhibits NF-κB activation. (A) HEK293T cells were transfected with plasmids of 24 F-box proteins along with MyD88 and a reporter plasmid carrying the NF-κB promoter reporter (NF-κB-luc) plasmid and pRL-TK plasmid. The cells were analyzed for NF-κB activity by a reporter gene assay, protein expression levels were detected by immunoblot analysis. (B) Luciferase activity of HEK293T cells transfected with the plasmids encoding SKP2 with indicated tags or not, along with MyD88, NF-κB-luc, and pRL-TK, was determined 24 h after transfection. (C) HeLa cells were transfected with NF-κB-luc and pRL-TK plasmids, along with empty vector or increasing amounts of SKP2, and then treated with LPS (10 μg/ml), IL-1β (10 ng/ml), or TNF-α (10 ng/ml) for 10 h, the NF-κB activity was determined by a reporter gene assay. (D) HEK293T cells were transfected with Myc-SKP2 or empty vector, IκB-α turnover was monitored by immunoblot analysis using indicated antibodies after treated with TNF-α (10 ng/ml) for 2 h. Numbers underneath the blot represent the fold change of IκB-α band intensity compared with control group, using β-actin as a loading control. (E) HEK293T cells were transfected with an empty vector or different doses of Myc-SKP2 expression vector and then treated with TNF-α (10 ng/ml) for 2 h, the gene expressions of TNF-α, IL-6, and IL-1β were determined by real-time PCR. Data in A–C and E are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test). SKP2 deficiency enhances NF-κB activation To confirm the function of SKP2 in NF-κB signaling, we generated SKP2-knockout (KO) HEK293T and THP-1 cells using the CRISPR/Cas9 technology (Figure 2A). We treated wild type (control, ctrl) or SKP2-KO THP-1 cells with LPS, and found that knockout of SKP2 resulted in enhanced phosphorylation of IKKβ and IκB-α, and complementation of SKP2-KO cells with SKP2 decreased the level of p-IKKβ and p-IκB-α (Figure 2B). It is worth to note that SKP2 protein increased under LPS stimulation in control cells (Figure 2B). To conform it, we examined the mRNA and protein level of SKP2 after LPS, and found that LPS induced the expression of SKP2 at both mRNA and protein level (Supplementary Figure S1A and B). However, SKP2 protein did not enhance under LPS treatment in the presence of the protein synthesis inhibitor, cycloheximide (CHX) (Supplementary Figure S1C). These results suggest that SKP2 can be induced by LPS treatment. Since protein stability, mRNA stability, and translation efficiency are also important for the induction of immediate-response gene, how LPS induces SKP2 expression needs further study. Figure 2 View largeDownload slide SKP2 deficiency enhances IKK phosphorylation and the expression of NF-κB responsive cytokines. (A) The knockout efficiency of SKP2 was determined by immunoblot analysis with anti-SKP2 antibody. (B) Control (ctrl), SKP2-knockout (SKP2-KO) THP-1 cells, and SKP2-KO cells overexpressing ectopic SKP2 were treated with LPS (100 ng/ml) for the indicated time points, and indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of indicated band intensity compared with control, using β-actin as a loading control. (C) Control or SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at different time points. The nuclear translocation of p65 was determined by immunoblot analysis using indicated antibodies. (D) Wide type (WT) or SKP2-KO HeLa cells were treated with LPS (10 μg/ml) for 30 min, and then subjected to immunofluorescence analysis using p65 and SKP2-specific antibodies. DNA was stained by DAPI (blue). UT, untreated. Scale bar: 10 μm. (E) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml). The expression of TNF-α, IL-1β, and IL-6 was determined by quantitative RT-PCR. (F) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at the indicated time points. Cell supernatants were used for measuring the release of TNF-α, IL-6, and IL-1β by ELISA. Data in E and F are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. control cells with the same treatment, Student’s t-test). Figure 2 View largeDownload slide SKP2 deficiency enhances IKK phosphorylation and the expression of NF-κB responsive cytokines. (A) The knockout efficiency of SKP2 was determined by immunoblot analysis with anti-SKP2 antibody. (B) Control (ctrl), SKP2-knockout (SKP2-KO) THP-1 cells, and SKP2-KO cells overexpressing ectopic SKP2 were treated with LPS (100 ng/ml) for the indicated time points, and indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of indicated band intensity compared with control, using β-actin as a loading control. (C) Control or SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at different time points. The nuclear translocation of p65 was determined by immunoblot analysis using indicated antibodies. (D) Wide type (WT) or SKP2-KO HeLa cells were treated with LPS (10 μg/ml) for 30 min, and then subjected to immunofluorescence analysis using p65 and SKP2-specific antibodies. DNA was stained by DAPI (blue). UT, untreated. Scale bar: 10 μm. (E) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml). The expression of TNF-α, IL-1β, and IL-6 was determined by quantitative RT-PCR. (F) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at the indicated time points. Cell supernatants were used for measuring the release of TNF-α, IL-6, and IL-1β by ELISA. Data in E and F are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. control cells with the same treatment, Student’s t-test). It is known that activation of the NF-κB signaling pathway by LPS treatment induced the nuclear translocation of p65 from the cytoplasm. Our results revealed that SKP2 deficiency predominantly enhanced the nuclear translocation of p65 under LPS stimulation in THP-1 cells (Figure 2C). This result is further confirmed by the immunofluorescence analysis in HeLa cells (Figure 2D). Consistent with that, the mRNA levels of TNF-α, IL-6, and IL-1β in SKP2-KO cells were significantly increased after LPS treatment (Figure 2E). Meanwhile, knockout of SKP2 substantially increased the secretion of TNF-α, IL-6, and IL-1β after LPS treatment (Figure 2F). Taking together, these data suggest that SKP2 deficiency enhances NF-κB activity, by increasing nuclear accumulation of p65, as well as the secretion of NF-κB-dependent pro-inflammatory cytokines. SKP2 inhibits NF-κB signaling at IKK level To determine the molecular mechanisms by which SKP2 inhibits NF-κB signaling, we transfected HEK293T cells with MyD88, TRAF6, TAK1-TAB1, IKKβ, or p65 together with increasing amounts of SKP2 plus the NF-κB luciferase reporter and an internal control (renilla luciferase). We found that the activation of NF-κB by MyD88, TRAF6, TAK1-TAB1, and IKKβ was markedly inhibited by SKP2 (Figure 3A and Supplementary Figure S2). In contrast, SKP2 did not inhibit p65-mediated NF-κB activation (Figure 3A), suggesting that SKP2 inhibits the NF-κB pathway upstream of p65, most likely targeting the IKK complex. Consistent with these results, we found that knockout of SKP2 enhanced NF-κB luciferase activity induced by MyD88, TRAF6, TAK1-TAB1, IKKβ, but not p65 (Figure 3B). These results suggest that SKP2 inhibits NF-κB signaling upstream of p65, probably at the level of the IKK complex. Figure 3 View largeDownload slide SKP2 inhibits NF-κB signaling at the level of the IKK complex. (A) HEK293T cells were transfected with NF-κB-luc, pRL-TK, MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with increasing amounts of SKP2. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. (B) Control (Ctrl) and SKP2 knockout (SKP2-KO) HEK293T cells were transfected with MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with NF-κB-luc and pRL-TK plasmids. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. Data in A and B are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV or control cells with the same treatment, Student’s t-test). Figure 3 View largeDownload slide SKP2 inhibits NF-κB signaling at the level of the IKK complex. (A) HEK293T cells were transfected with NF-κB-luc, pRL-TK, MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with increasing amounts of SKP2. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. (B) Control (Ctrl) and SKP2 knockout (SKP2-KO) HEK293T cells were transfected with MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with NF-κB-luc and pRL-TK plasmids. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. Data in A and B are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV or control cells with the same treatment, Student’s t-test). SKP2 mediates autophagy-dependent degradation of IKKβ During the experiments, we observed a negative correlation between SKP2 and IKKβ in protein level upon NF-κB activation (Figure 2B and Supplementary Figure S2). Considering the inhibitory effect of SKP2 in NF-κB signaling occurs at IKK level, we wondered whether SKP2 could degrade IKK complex. To test this hypothesis, we overexpressed SKP2 in HeLa cells and detected IKK complex expression. We found that simply overexpression of SKP2 does not affect the protein level of the components from IKK complex (Supplementary Figure S3). However, SKP2 markedly reduced the protein level of IKKβ, but not IKKα or NEMO upon LPS treatment (Figure 4A). We next used a CHX-chase assay to determine the time course of SKP2-mediated IKKβ degradation, and found that SKP2 can accelerate the turnover rate of IKKβ (Figure 4B). Since overexpression of IKKβ could activate IKKβ via auto-phosphorylation, our results suggest that SKP2 could promote the degradation of active IKKβ. Figure 4 View largeDownload slide SKP2 mediates autophagy-dependent degradation of IKKβ. (A) HeLa cells were transfected with empty vector or increasing amount of Myc-SKP2, followed by LPS treatment (10 μg/ml, 3 h). The protein turnover of IKK complex was determined by immunoblot analysis with indicated antibodies. The mRNA levels of IKK complex were measured by quantitative RT-PCR. (B) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or empty vector, then treated with CHX at the indicated time points, and the indicated proteins were determined by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (C) HeLa cells were transfected with plasmid encoding Flag-IKKβ for 24 h. Before harvest, cells were treated with MG132 (10 μM), chloroquine (CQ, 50 μM), bafilomycin A1 (Baf A1, 100 nM), or 3-methyladenine (3-MA, 2.5 mM) for 8 h. Cell extracts were analyzed by immunoblotting. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (D) Immunoassay of extracts of HeLa cells transfected with Myc-SKP2, and then treated with LPS for 4 h in the presence of Baf A1 (8 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (E) Immunoassay of extracts of control (Ctrl) or ATG5 knockout (ATG5-KO) HeLa cells transfected with Myc-SKP2 for 24 h, followed by LPS treatment (4 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. Data in A are expressed as mean ± SEM of three independent experiments. Figure 4 View largeDownload slide SKP2 mediates autophagy-dependent degradation of IKKβ. (A) HeLa cells were transfected with empty vector or increasing amount of Myc-SKP2, followed by LPS treatment (10 μg/ml, 3 h). The protein turnover of IKK complex was determined by immunoblot analysis with indicated antibodies. The mRNA levels of IKK complex were measured by quantitative RT-PCR. (B) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or empty vector, then treated with CHX at the indicated time points, and the indicated proteins were determined by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (C) HeLa cells were transfected with plasmid encoding Flag-IKKβ for 24 h. Before harvest, cells were treated with MG132 (10 μM), chloroquine (CQ, 50 μM), bafilomycin A1 (Baf A1, 100 nM), or 3-methyladenine (3-MA, 2.5 mM) for 8 h. Cell extracts were analyzed by immunoblotting. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (D) Immunoassay of extracts of HeLa cells transfected with Myc-SKP2, and then treated with LPS for 4 h in the presence of Baf A1 (8 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (E) Immunoassay of extracts of control (Ctrl) or ATG5 knockout (ATG5-KO) HeLa cells transfected with Myc-SKP2 for 24 h, followed by LPS treatment (4 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. Data in A are expressed as mean ± SEM of three independent experiments. Next, we investigated the molecular mechanisms underlying SKP2-mediated IKKβ degradation. Three major systems of protein clearance in eukaryotic cells are the proteasome, lysosome, and auto-lysosome pathways (Kraft et al., 2010). We found that the autophagic-sequestration inhibitor 3-methyladenine (3-MA) or the lysosomal-acidification inhibitor Bafilomycin A1 (Baf A1), and chloroquine (CQ), but not the proteasome inhibitor MG132, stabilized IKKβ (Figure 4C). Meanwhile, we found that SKP2 could not degrade IKKβ anymore under Baf A1 treatment (Figure 4D), indicating that the SKP2-mediated IKKβ degradation is regulated by the auto-lysosome pathway. These results are further confirmed by using the ATG5-KO cells in which the autophagy is deficient (Figure 4E). Together, our results indicate that SKP2 promotes IKKβ degradation via auto-lysosome pathway. SKP2 interacts with activated IKKβ Since SKP2 specifically promotes IKKβ degradation (Figures 2B, 4A and B), we suggested that SKP2 might interact with the IKK complex. To test this hypothesis, we transfected HEK293T cells with SKP2 together with expressed plasmids of IKKα, IKKβ, or NEMO. Co-immunoprecipitation and immunoblot analysis revealed that SKP2 can interact with IKK complex (Figure 5A). As a kinase in NF-κB cascade, the activation of IKKβ occurs through dual phosphorylation on Ser177 and Ser181 (Mercurio et al., 1997). By substituting their activation sites (Ser177/Ser181) to alanine or glutamic acid, respectively, we constructed IKKβ constitutively active (IKKβ-SE) mutants or IKKβ enzymatically inactive (IKKβ-SA) mutants, and found that SKP2 specially interacted with active IKKβ (IKKβ-SE) (Figure 5B). Furthermore, SKP2 cannot interact with kinase activity-deficient form of IKKβ (IKKβ-K44A), which could not active itself via auto-phosphorylation (Figure 5B). Consistently, SKP2 promoted the degradation of active IKKβ (IKKβ-SE), but not inactive IKKβ (IKKβ-K44A or IKKβ-SA) (Supplementary Figure S4A–C). Moreover, we found that SKP2 interacted with active IKKα (Supplementary Figure S4D). We next performed endogenous immunoprecipitation of IKKβ, and detected stronger interaction between endogenous SKP2 and IKKβ under LPS treatment, indicating that SKP2 prefers to interact with active IKKβ (Figure 5C). Moreover, purified His-SKP2 was able to bind to Flag-tagged IKKβ under cell-free conditions (Figure 5D). Since SKP2 interacts with IKK complex, but only promotes IKKβ degradation, we hypotheses that it might release IKKβ from IKK complex for degradation. We transfected plasmids of IKKβ and NEMO, along with increasing amount of Myc-SKP2, and found that the interaction of NEMO and IKKβ was decreased by increasing amount of Myc-SKP2 (Figure 5E). To further determine which domain of IKKβ is responsible for interacting with SKP2, we generated deletion mutants encompassing the amino-terminal kinase domain (KD), leucine zipper domain (LZ), and a C-terminal helix-loop-helix (HLH) domain of IKKβ (Figure 5F). Co-immunoprecipitation results showed that the LZ domain of IKKβ interacted with SKP2 (Figure 5G). SKP2 contains one F-box domain and 10 leucine repeat regions (LRRs). To identify the functional domains of SKP2, we generated six domain deletion constructs of SKP2: ΔF-box, ΔLRR12, ΔLRR34, ΔLRR5, ΔLRR67, and ΔLRR8910 (Figure 5H). Co-immunoprecipitation results showed that ΔF-box and ΔLRR67 could still strongly interact with the IKKβ protein, but ΔLRR12 and ΔLRR8910 could only weakly bind with IKKβ (Figure 5I). Moreover, we found that three domain deletions ΔLRR12, ΔLRR67, and ΔLRR8910 could neither promote IKKβ degradation nor inhibit IKKβ-mediated NF-κB signaling (Figure 5J), suggesting that LRR12, LRR67, and LRR8910 are critical for the function of SKP2 in mediating NF-κB activity. Together, these results reveal that SKP2 specifically targets LZ domain of IKKβ and promotes active IKKβ degradation. Figure 5 View largeDownload slide SKP2 interacts with IKKβ. (A) HEK293T cells were transfected with Flag-IKKα, Flag-IKKβ, Flag-NEMO, and Myc-SKP2. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblotting. (B) Lysates of HEK293T cells transfected with Flag-IKKβ-WT, Flag-IKKβ-K44A, Flag-IKKβ-SA, and Flag-IKKβ-SE, together with Myc-SKP2 in the presence of Baf A1 (100 μM), were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (C) Lysates of HeLa cells in the presence of bafilomycin A1 (Baf A1, 100 μM) with or without LPS (10 μg/ml, 4 h) treatment were immunoprecipitated with anti-IgG or anti-IKKβ antibody, followed by immunoblot analysis to detect SKP2 protein. (D) Top: His-EV or His-SKP2 was retained on Ni IDA beads, incubated with Flag-IKKβ then immunoblotted with the antibody against Flag. Flag-IKKβ is purified by anti-Flag beads and then washed down by Flag peptides. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. (E) HEK293T cells were transfected with HA-IKKβ, Flag-NEMO, empty vector, or increasing amount of Myc-SKP2 for 24 h. The lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (F) A schematic diagram shows protein domain structures of the IKKβ deletions. KD, amino-terminal kinase domain; LZ, leucine zipper domain; HLH, C-terminal helix-loop-helix. (G) HEK293T cells were co-transfected with Myc-SKP2 with Flag-IKKβ or its deletion mutants. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblot analysis. (H) A schematic diagram shows protein domain structures of the SKP2 deletions. (I) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or its deletion mutants, followed by Baf A1 treatment. Myc-tagged proteins were immunoprecipitated with anti-Myc beads followed with immunoblot analysis. (J) HEK293T cells were transfected with NF-κB-luc, pRL-TK, Flag-IKKβ, along with Myc-SKP2 or it domain deletions. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. The indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. Data in J are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test. ns, no significant). Figure 5 View largeDownload slide SKP2 interacts with IKKβ. (A) HEK293T cells were transfected with Flag-IKKα, Flag-IKKβ, Flag-NEMO, and Myc-SKP2. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblotting. (B) Lysates of HEK293T cells transfected with Flag-IKKβ-WT, Flag-IKKβ-K44A, Flag-IKKβ-SA, and Flag-IKKβ-SE, together with Myc-SKP2 in the presence of Baf A1 (100 μM), were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (C) Lysates of HeLa cells in the presence of bafilomycin A1 (Baf A1, 100 μM) with or without LPS (10 μg/ml, 4 h) treatment were immunoprecipitated with anti-IgG or anti-IKKβ antibody, followed by immunoblot analysis to detect SKP2 protein. (D) Top: His-EV or His-SKP2 was retained on Ni IDA beads, incubated with Flag-IKKβ then immunoblotted with the antibody against Flag. Flag-IKKβ is purified by anti-Flag beads and then washed down by Flag peptides. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. (E) HEK293T cells were transfected with HA-IKKβ, Flag-NEMO, empty vector, or increasing amount of Myc-SKP2 for 24 h. The lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (F) A schematic diagram shows protein domain structures of the IKKβ deletions. KD, amino-terminal kinase domain; LZ, leucine zipper domain; HLH, C-terminal helix-loop-helix. (G) HEK293T cells were co-transfected with Myc-SKP2 with Flag-IKKβ or its deletion mutants. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblot analysis. (H) A schematic diagram shows protein domain structures of the SKP2 deletions. (I) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or its deletion mutants, followed by Baf A1 treatment. Myc-tagged proteins were immunoprecipitated with anti-Myc beads followed with immunoblot analysis. (J) HEK293T cells were transfected with NF-κB-luc, pRL-TK, Flag-IKKβ, along with Myc-SKP2 or it domain deletions. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. The indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. Data in J are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test. ns, no significant). SKP2 bridges IKKβ and p62 Increasing evidence supports that cargo receptors deliver cargoes to the autophagosome for selective degradation (Stolz et al., 2014). In order to find the cargo receptor of IKKβ, we performed co-immunoprecipitation between IKKβ and several major cargo receptors, and found that IKKβ mainly interacted with p62 rather than other receptors (Figure 6A). Further experiments revealed that SKP2 also strongly interacted with p62 than other cargo receptors (Figure 6B). Moreover, purified SKP2 was able to bind to p62 under cell-free conditions (Supplementary Figure S5A). Next, we found that all domains of SKP2 can strongly interact with p62 except ΔLRR67 and ΔLRR8910 (Supplementary Figure S5B), indicating the critical role of LRR67 and LRR8910 domains for SKP2–p62 interaction. In our previous study, TRIM11 functions as a co-receptor of AIM2 for autophagic degradation through linking p62 and AIM2 (Liu et al., 2016). We suggested that SKP2 might bridge IKKβ and p62. As expected, we observed that SKP2 promoted the interaction of p62 and IKKβ in a dose-dependent manner (Figure 6C). In addition, we found that purified SKP2 was able to enhance the interaction between p62 and IKKβ under cell-free conditions (Figure 6D). These results indicate SKP2 could bridge IKKβ and p62. To further confirm these results, we examined the endogenous interaction between IKKβ and p62. The results showed the association of IKKβ and p62 was enhanced after LPS treatment; however, the interaction was significantly impaired in SKP2-KO cells (Figure 6E). Moreover, we found that SKP2 failed to promote IKKβ degradation in p62-KO cells (Figure 6F). Collectively, these results indicate that SKP2 promotes autophagic degradation of IKKβ through bridging IKKβ–p62 interaction. Figure 6 View largeDownload slide SKP2 promotes p62-mediated selective autophagic degradation of IKKβ. (A) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, OPTN, Tollip, or NBR1, together with HA-IKKβ, followed by Baf A1 treatment. (B) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, NDP52, OPTN, Tollip, or NBR1, together with Myc-SKP2, followed by Baf A1 treatment. (C) HEK293T cells were co-transfected with Flag-IKKβ and HA-p62 together with increasing amounts of Myc-SKP2, then treated with chloroquine (CQ) for 8 h. Cell extracts were immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. Numbers underneath the blot represent the fold change of HA-p62 band intensity compared with control group, using immunoprecipitated Flag-IKKβ as a loading control. (D) The purified HA-p62 and Flag-IKKβ were incubated with His-EV or His-SKP2 and then immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. (E) WT or SKP2-KO THP-1 cells were treated with LPS (100 ng/ml) for indicated time points, and Baf A1 was used to treat the cells for 3 h before harvesting them. Whole-cell extracts were immunoprecipitated with anti-IKKβ antibody, followed by IB analysis with anti-p62 antibody. (F) Immunoassay of extracts of control (Ctrl) or p62 knockout (p62-KO) HeLa cells transfected with Myc-SKP2, followed by LPS treatment. Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (G) Work model of SKP2-mediated autophagic degradation of IKKβ. Figure 6 View largeDownload slide SKP2 promotes p62-mediated selective autophagic degradation of IKKβ. (A) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, OPTN, Tollip, or NBR1, together with HA-IKKβ, followed by Baf A1 treatment. (B) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, NDP52, OPTN, Tollip, or NBR1, together with Myc-SKP2, followed by Baf A1 treatment. (C) HEK293T cells were co-transfected with Flag-IKKβ and HA-p62 together with increasing amounts of Myc-SKP2, then treated with chloroquine (CQ) for 8 h. Cell extracts were immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. Numbers underneath the blot represent the fold change of HA-p62 band intensity compared with control group, using immunoprecipitated Flag-IKKβ as a loading control. (D) The purified HA-p62 and Flag-IKKβ were incubated with His-EV or His-SKP2 and then immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. (E) WT or SKP2-KO THP-1 cells were treated with LPS (100 ng/ml) for indicated time points, and Baf A1 was used to treat the cells for 3 h before harvesting them. Whole-cell extracts were immunoprecipitated with anti-IKKβ antibody, followed by IB analysis with anti-p62 antibody. (F) Immunoassay of extracts of control (Ctrl) or p62 knockout (p62-KO) HeLa cells transfected with Myc-SKP2, followed by LPS treatment. Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (G) Work model of SKP2-mediated autophagic degradation of IKKβ. Discussion Transcription factor NF-κB plays a crucial role in various physiological processes such as inflammation, adaptive immunity, and cell proliferation/death. It is fine-turned by various molecules at different levels. Numerous E3 ubiquitin ligases or deubiquitinases regulate the NF-κB activation by modulating the ubiquitinated levels of the distinct components of NF-κB signaling (Malynn and Ma, 2010). For example, we found that TRIM9 and USP18 can inhibit virus-induced and TLR-induced NF-κB signaling, respectively (Yang et al., 2015; Qin et al., 2016). F-box proteins belong to a large family of E3 ligases that contain a carboxy-terminal domain that interacts with substrates and a 42–48 amino-acid F-box motif, and play important regulatory roles in innate immune signaling, including inflammation. The F-box protein FBXL2 has been reported to serve as a sentinel inhibitor of NF-κB by mediating poly-ubiquitination and proteasomal degradation of TRAFs (Chen et al., 2013). Here, we screened the function of several F-box proteins and identified SKP2 (also named as FBXL1) as a negative modulator of TLR- and cytokine-mediated NF-κB activation by targeting IKKβ for autophagic degradation. IKK complex plays pivotal roles in the NF-κB signaling, and its activity is tightly regulated by multiple post-translational modifications. By recruiting phosphatase PP1 to dephosphorylate IKK, CUEDC2 deactivates IKK activity and further represses activation of NF-κB signaling (Li et al., 2008). Besides of controlling the phosphorylation and de-phosphorylation, modulation of IKKs stability is another way to regulate the activity of IKK complex. It has been revealed that p47 induces the lysosomal degradation of poly-ubiquitinated NEMO, the regulatory component of IKK complex (Shibata et al., 2012). Kelch-like ECH-associated protein 1 (KEAP1) facilitates IKKβ degradation through promoting its ubiquitination, and further mediates the down-regulation of NF-κB signaling via autophagy (Lee et al., 2009; Kim et al., 2010). Our results showed that SKP2 is a LPS-inducible gene, which inhibits NF-κB signaling through degrading active IKKβ, suggesting that SKP2 modulates NF-κB activation through a negative feedback loop. Although SKP2 interacts with the whole IKK complex, it selectively promotes the degradation of IKKβ. We found that SKP2 can reduce the interaction between IKKβ and NEMO, suggesting that SKP2 can release active IKKβ form IKK complex for degradation. Whether SKP2 affects the function of IKKα or NEMO needs further study. Furthermore, we found that blockage of autophagic flux by autophagic inhibitor or ATG5 deficiency prevents SKP2-promoted degradation of IKKβ, indicating the important role of autophagy in NF-κB activation. Autophagy was once considered as a non-selective bulk degradation pathway of cells, but it is now clear that autophagic degradation of proteins mediated by p62 can degrade substrates in a selective manner (Kraft et al., 2010). The autophagy pathway also functions in the control of inflammatory signaling. Autophagic gene deficiency increases the level of the adaptor protein p62, meanwhile reinforced NF-κB signaling and transcription of inflammatory factors through a mechanism involving TRAF6 oligomerization (Levine et al., 2011). Macrophages with deficiency of Atg16L1 or Atg7, which are the essential components of the autophagic process, enhanced TLR-3/4-mediated production of interleukin (IL)-1β and IL-18 (Saitoh et al., 2008). It is known that p62 negatively regulates the transcription of inflammatory factors in stimulated macrophages (Kim and Ozato, 2009). But the role of p62-mediated autophagic degradation in NF-κB signaling is still not fully investigated. Here, we demonstrated that SKP2 can be induced by NF-κB activation, binds active IKKβ. Finally, the complex of IKKβ-SKP2 is recognized and recruited by cargo receptor p62 to the autophagosome for degradation, subsequently dampening NF-κB signaling (Figure 6G). In this study, we found that three LRR domains of SKP2 might play essential roles to bridge IKKβ and p62: LRR12 and LRR8910 of SKP2 are important for its binding activity to IKKβ; while LRR67 and LRR8910 are critical for its binding activity to p62. Bafilomycin A1 efficiently stabilized the protein levels of IKKβ, which is consistent with the previous study that IKKs degradation requires autophagic flux (Kim et al., 2010). Considering of the critical role of p62 for directing autophagic uptake of proteins, our study demonstrated that SKP2 is a novel co-receptor of delivering IKKβ for selectively autophagic degradation. These results provide mechanistic evidence that autophagy selectively regulated NF-κB activation. As the negative regulator of NF-κB signaling, SKP2 might serve as a potential target for treating inflammation-associated diseases. Materials and methods Antibodies and reagents Horseradish peroxidase (HRP)-anti-Flag (M2) (#A8592) and anti-β-actin (#A1978) were purchased from Sigma; HRP-anti-hemagglutinin (#12013819001) and anti-Myc-HRP (#11814150001) were purchased from Roche Applied Science; Anti-phospho-IKKα/β (#2697S), anti-ATG5 (#12994S), anti-p62 (8025), anti-p65 (#6956), anti-IKKβ (#2684), anti-IκBα (#4814), and anti-phospho-IκBα (Ser32/36) (#9246) were purchased from Cell Signaling Technology; Goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, anti-SKP2 (#sc-74477), and anti-NEMO (#sc-8300) were purchased from Santa Cruz Biotechnology; Anti-IKKα (#07-1007) were purchased from Millipore; Protein A agarose and Protein G agarose were purchased from Pierce. Dimethyl sulfoxide (DMSO) (D2650), CHX (R750107), Puromycin (P9620), MG132 (C-2211-5MG), Chloroquine phosphate (CQ) (PHR1258), 3-methyladenine (3-MA) (M9281), and LPS (L4391) were purchased from Sigma. Bafilomycin A1 (S1413) was purchased from Selleck. Cell culture and transfection Human embryonic kidney 293T (HEK293T), HeLa, and THP-1 cells were cultured in humidified 5% CO2 at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) or RPMI-1640 (Gibco) medium supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, and 1× Penicillin/Streptomycin solution (Thermo). Lipofectamine 2000 (Invitrogen) and ViaFect™ Transfection Reagent (Promega) were used for transient transfection of HEK293T and HeLa cells, respectively. Luciferase reporter assays HEK293T or HeLa cells were transfected with luciferase reporter plasmids encoding NF-κB luciferase, pRL-TK Renilla luciferase, and SKP2 or control vectors, TNF-α, IL-1β, LPS as well as exogenous MyD88, TRAF6, TAK1/TAB1, IKKβ, and p65 were used as stimulators. Then, 24 h after transfection, cells were lysed. The luciferase activity was determined by a dual luciferase reporter assay kit (Promega) using a Luminoskan Ascent luminometer (Thermo Scientific) according to the manufacturer’s instructions. Reporter gene activity was determined by normalization of the firefly luciferase activity to renilla luciferase activity (Qin et al., 2017). Immunoprecipitation and immunoblot analysis Cells were washed with ice-cold PBS and then lysed in low-salt lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100) supplemented with 5 mg/ml cOmplete™ protease inhibitor cocktail tablets (Roche). Protein concentration was determined by BCA Protein Assay Kit (Pierce). After boiling, cell lysate was subjected to electrophoresis. For immunoprecipitation experiments, whole-cell lysates were prepared after transfection or stimulation and incubated with indicated antibodies together with protein A/G beads (Pierce) for overnight. For anti-flag or anti-myc immunoprecipitation, anti-flag or anti-myc agarose gels (Sigma) were used. Beads were then washed four times with lysis buffer, and immune-complexes were re-suspended in 2× SDS loading buffer. After boiling and electrophoresis, the proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and further incubated with the indicated antibodies. Protein detection was performed by a ChemiDoc™ XRS system (Bio-Rad) using enhanced chemiluminescence (Millipore). Immunofluorescence assay Cells grown on dishes were fixed with 4% paraformaldehyde for 15 min, and then permeabilized in methyl alcohol for 10 min at −20°C. After washing with PBS, cells were blocked in 5% fetal goat serum for 1 h, and then incubated with primary antibodies diluted in 10% bull serum albumin overnight. The cells were washed and followed by a fluorescently labeled secondary antibody. Nuclear DNA was stained using 5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI), a fluorescent DNA-intercalating dye. A radiance microscope system (Leica) was used to visualize the distribution of p65 or SKP2 (Liu et al., 2013). Generation of SKP2 knockout cell lines by CRISPR/Cas9 technology To generate SKP2-KO 293T cells, target sequences were cloned into pLentiCRISPRv2 by cutting with BsmBI as previous described (Jin et al., 2016). The sequences of target-related gene are as follows: Ctrl sgRNA: 5′-GGGCGAGGAGCTGTTCACCG-3′; SKP2 sgRNA 1#: 5′-AAGACTTTGTGATTGTCCGC-3′; SKP2 sgRNA 2#: 5′-GCAACGTTGCTACTCAGGTC-3′. Subcellular fractionation Cellular fractionation assay was carried out using the nuclear and cytoplasmic protein extraction kit (Beyotime) according the manufacturer’s protocol. Briefly, cells were harvested and washed twice with ice-cold PBS, and then were re-suspended with Buffer A. After incubated on ice for 15 min, cells were lysed with Buffer B. Next, cells were centrifuged for 5 min at 12000 g at 4°C. The supernatant (cytoplasm, membrane, and mitochondria) was collected. After washed the pellet twice with ice-cold PBS, sediment samples were re-suspended in Buffer C. Next, samples were incubated on ice for 30 min and centrifuged for 10 min at 12000 g at 4°C. The supernatant (nuclear extract) was collect. Real-time PCR analysis Total RNA was isolated directly from freshly collected THP-1 cells with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized by using 1 μg total RNA with HiScript II QRT-SuperMix (Vazyme). qRT-PCR reactions were carried out on LightCycler480 (Roche) by using of 2× Power SYBR green qPCR master mix (GenStar). All PCR products were checked by running an agarose gel to exclude the possibility of multiple products. RPL13A was used as an internal control. Comparative quantification of gene expression was evaluated using the 2−ΔΔCt method. All qRT-PCR assays were conducted in triplicates. Sequences of primers were listed as follows:SKP2: forward 5′-ACCTTTCTGGGTGTTCTGGA-3′, reverse 5′-ATTCAGCTGGGTGATGGTCT-3′; GAPDH: forward 5′-GCTGGTCATCAACGGGAAA-3′, reverse 5′-ACGCCAGTAGACTCCACGACA-3′; TNF-α: forward 5′-CCCAGGGACCTCTCTCTAATCA-3′, reverse 5′-GCTTGAGGGTTTGCTACAACATG-3′; IL-1β: forward 5′-AAATACCTGTGGCCTTGGGC-3′, reverse 5′-TTTGGGATCTACACTCTCCAGCT-3′; IL-6: forward 5′-AGAGGCACTGGCAGAAAACAAC-3′, reverse 5′-AGGCAAGTCTCCTCATTGAATCC-3′; IKK-α: forward 5′-CAGCCATTTACCTGGCATGAG-3′, reverse 5′-GAGGGTCCCAATTCAACATCAA-3′; IKK-β: forward 5′-ATCCCCGATAAGCCTGCCA-3′, reverse 5′-CTTGGGCTCTTGAAGGATACAG-3′; NEMO: forward 5′-GCGAGGAATGCAGCTGGAAG-3′, reverse 5′-GCCTGGAAGTCCGCCTTGTA-3′. Enzyme-linked immunosorbent assay THP-1 cells were simulated for 6 h with LPS (100 ng/ml). The supernatant medium of cells was collected. The concentration of TNF-α, IL-6, and IL-1β was determined by using of human specific ELISA Kit (BD Biosciences) according to the manufacturer’s instructions. Statistical analysis The results of all quantitative experiments are reported as mean ± SEM of three independent experiments. Statistical analysis was performed using GraphPad Prism 5.0. Student’s t-test was used for the comparison of two groups. For all tests, a P-value < 0.05 was considered statistically significant, and the level of significance was indicated as *P < 0.05, **P < 0.01, ***P < 0.001. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Funding This work was supported by grants from the National Natural Science Foundation of China (91629101, 31522018, 31601135, 81302197, 81700557, and 31071046), the National Key Basic Research Program of China (2015CB859800 and 2014CB910800), and the Guangdong Innovative Research Team Program (2011Y035). 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SKP2 attenuates NF-κB signaling by mediating IKKβ degradation through autophagy

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Oxford University Press
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© The Author(s) (2018). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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1674-2788
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1759-4685
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10.1093/jmcb/mjy012
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Abstract

Abstract NF-κB signaling controls a large set of physiological processes ranging from inflammatory responses to cell death. Its activation is tightly regulated through controlling the activity and stability of multiple signaling components. Here, we identify that NF-κB activation is suppressed by an F-box protein, S-phase kinase associated protein 2 (SKP2). SKP2 deficiency enhanced NF-κB activation as well as the production of inflammatory cytokines. In addition, SKP2 potently blocked the NF-κB activation at the IκB kinase (IKK) level. Mechanistic study further revealed that SKP2 functions as an adaptor to promote an interaction between active IKKβ and the autophagic cargo receptor p62 to mediate IKKβ degradation via selective autophagy. These findings identify a previously unrecognized role of SKP2 in NF-κB activation by which SKP2 acts as a secondary receptor to assist IKKβ delivery to autophagosomes for degradation in a p62-dependent manner. SKP2, IKKβ, NF-κB activation, autophagy, inflammation Introduction The nuclear factor-κB (NF-κB) is a critical transcription factor to mediate immediate responses to pathogens, and its activity is accurately controlled by a variety of regulators to prevent potential impairment from its excessive activation (Chan et al., 2015). Upon detection of invading pathogens, Toll-like receptors (TLRs), as well as other cytokines receptors, recruit their specific adaptor proteins for the association and subsequent activation of a variety of TRAF proteins. Activated TRAF2/6 or TRAF2/5 serve as a platform to recruit TAK1 and IκB kinase (IKK) complex through catalyzing lysine 63 (K63)-linked poly-ubiquitination on itself (Oeckinghaus et al., 2011). As the core of NF-κB signaling, the formation and activation of IKK complex in turn enable IKKβ to phosphorylate IκB protein and promote its degradation, thus allowing NF-κB to translocate into the nucleus to induce the transcription of a variety of pro-inflammatory genes (Hayden and Ghosh, 2008). NF-κB signaling is tightly regulated by multiple post-translational modifications of their signaling components (Bhoj and Chen, 2009). Aberrant activation of NF-κB results in physiologic disorders and diseases, such as cancer, inflammation, and autoimmune diseases (Baldwin, 2012). Due to its central role in NF-κB signaling, modulating IKK activity is the major way to control NF-κB activation (Chen, 2005). For example, Tripartite motif 13 (TRIM13) suppresses TNF-α-induced NF-κB activation through regulating the ubiquitination and turnover of NEMO, a regulatory sub-unit of IKK complex (Tomar and Singh, 2014). Previously, we have identified that nucleotide binding oligomerization domain (NOD)-like receptor (NLR) family member X1 (NLRX1) and NLRC5 negatively regulate TLR signaling through IKK inhibition (Cui et al., 2010; Xia et al., 2011). Furthermore, we found that NLRC5 shapes NF-κB signaling relied on its ubiquitination (Meng et al., 2015). Recently, our work demonstrated that LRRC14 attenuates TLR-mediated NF-κB signaling by blocking the formation of IKK complex (Wu et al., 2016). However, the regulation of IKK stability during NF-κB activation is not fully investigated yet. Recently, extensive studies have revealed that the innate immunity, including NF-κB activation, can be regulated by autophagy. Autophagy is a ubiquitous pathway in cells by which cytoplasmic material or organelles are delivered to lysosomes for degradation (Yang and Klionsky, 2010). In mice, knockout of Atg5, the key components of autophagy, in macrophages and neutrophils increases susceptibility to infection with bacteria, including L. monocytogenes and T. gondii (Kimmey et al., 2015). Besides non-selective bulk degradation via autophagosome, the selective autophagy is regarded as a degradative way of particular proteins or protein aggregates, in which the specificity of the substrates is determined by a variety of cargo receptors, such as p62, NBR1, or NDP5 (Moscat and Diaz-Meco, 2009). Increasing evidence demonstrates that selective autophagy plays a critical role in the regulation of innate immune signaling. We have reported that p62-mediated AIM2 degradation through selective autophagy inhibits the inflammasome activation during DNA virus infection (Liu et al., 2016). In addition, we found that p62-mediated cGAS degradation and NDP52-mediated MAVS degradation negatively regulate the activation of type I interferon signaling as well as the anti-virus response (Chen et al., 2016; Jin et al., 2017). Although it has been reported that IKKβ could undergo degradation via autophagy (Kim et al., 2010), it is unknown whether selective autophagy could regulate NF-κB activation via specifically mediating the stability of IKKβ. In the present study, we identify F-box protein SKP2 as a negative regulator of TLR or TNFα-induced NF-κB activation. Knockout of SKP2 significantly enhanced the expression and secretion of pro-inflammatory cytokines in THP-1 cells. SKP2 targeted to IKK complex upon LPS treatment, and specifically promoted the degradation of activated IKKβ. Furthermore, we found that SKP2 promoted the interaction between IKKβ and the autophagic cargo receptor p62 to mediate IKKβ for degradation via selective autophagy. Therefore, our findings provide an insight into the mechanisms of tight regulation of NF-κB through its crosstalk with autophagy. Results Identification of SKP2 as a negative regulator of NF-κB signaling To investigate the roles of F-box family proteins in NF-κB signaling, we screened a panel of Flag-tagged F-box proteins using MyD88-mediated NF-κB luciferase reporter activation assay in HEK293T cells. Of 24 candidate genes, we identified SKP2 (also known as FBXL1) as a negative regulator of MyD88-mediated NF-κB activation (Figure 1A). In order to confirm the function of SKP2, we constructed SKP2 plasmids with different tags or not, and found that all of them could inhibit MyD88-mediated NF-κB activation as the one without tag (Figure 1B), indicating that different tags do not affect the function of SKP2 in this study. Next, we tested whether SKP2 affects the NF-κB signaling induced by multiple stimuli, and found that SKP2 negatively regulated the activation of NF-κB induced by lipopolysaccharide (LPS), TNF-α, or IL-1β respectively (Figure 1C). The degradation of IκB-α is an important landmark of NF-κB activation. We found that SKP2 inhibited the degradation of endogenous IκB-α protein in the presence of TNF-α (Figure 1D). Consistently, SKP2 overexpression resulted in much lower expression of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β by TNF-α treatment (Figure 1E). Taken together, these results suggest that SKP2 inhibits NF-κB activation as well as the expression of pro-inflammatory cytokines. Figure 1 View largeDownload slide SKP2 inhibits NF-κB activation. (A) HEK293T cells were transfected with plasmids of 24 F-box proteins along with MyD88 and a reporter plasmid carrying the NF-κB promoter reporter (NF-κB-luc) plasmid and pRL-TK plasmid. The cells were analyzed for NF-κB activity by a reporter gene assay, protein expression levels were detected by immunoblot analysis. (B) Luciferase activity of HEK293T cells transfected with the plasmids encoding SKP2 with indicated tags or not, along with MyD88, NF-κB-luc, and pRL-TK, was determined 24 h after transfection. (C) HeLa cells were transfected with NF-κB-luc and pRL-TK plasmids, along with empty vector or increasing amounts of SKP2, and then treated with LPS (10 μg/ml), IL-1β (10 ng/ml), or TNF-α (10 ng/ml) for 10 h, the NF-κB activity was determined by a reporter gene assay. (D) HEK293T cells were transfected with Myc-SKP2 or empty vector, IκB-α turnover was monitored by immunoblot analysis using indicated antibodies after treated with TNF-α (10 ng/ml) for 2 h. Numbers underneath the blot represent the fold change of IκB-α band intensity compared with control group, using β-actin as a loading control. (E) HEK293T cells were transfected with an empty vector or different doses of Myc-SKP2 expression vector and then treated with TNF-α (10 ng/ml) for 2 h, the gene expressions of TNF-α, IL-6, and IL-1β were determined by real-time PCR. Data in A–C and E are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test). Figure 1 View largeDownload slide SKP2 inhibits NF-κB activation. (A) HEK293T cells were transfected with plasmids of 24 F-box proteins along with MyD88 and a reporter plasmid carrying the NF-κB promoter reporter (NF-κB-luc) plasmid and pRL-TK plasmid. The cells were analyzed for NF-κB activity by a reporter gene assay, protein expression levels were detected by immunoblot analysis. (B) Luciferase activity of HEK293T cells transfected with the plasmids encoding SKP2 with indicated tags or not, along with MyD88, NF-κB-luc, and pRL-TK, was determined 24 h after transfection. (C) HeLa cells were transfected with NF-κB-luc and pRL-TK plasmids, along with empty vector or increasing amounts of SKP2, and then treated with LPS (10 μg/ml), IL-1β (10 ng/ml), or TNF-α (10 ng/ml) for 10 h, the NF-κB activity was determined by a reporter gene assay. (D) HEK293T cells were transfected with Myc-SKP2 or empty vector, IκB-α turnover was monitored by immunoblot analysis using indicated antibodies after treated with TNF-α (10 ng/ml) for 2 h. Numbers underneath the blot represent the fold change of IκB-α band intensity compared with control group, using β-actin as a loading control. (E) HEK293T cells were transfected with an empty vector or different doses of Myc-SKP2 expression vector and then treated with TNF-α (10 ng/ml) for 2 h, the gene expressions of TNF-α, IL-6, and IL-1β were determined by real-time PCR. Data in A–C and E are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test). SKP2 deficiency enhances NF-κB activation To confirm the function of SKP2 in NF-κB signaling, we generated SKP2-knockout (KO) HEK293T and THP-1 cells using the CRISPR/Cas9 technology (Figure 2A). We treated wild type (control, ctrl) or SKP2-KO THP-1 cells with LPS, and found that knockout of SKP2 resulted in enhanced phosphorylation of IKKβ and IκB-α, and complementation of SKP2-KO cells with SKP2 decreased the level of p-IKKβ and p-IκB-α (Figure 2B). It is worth to note that SKP2 protein increased under LPS stimulation in control cells (Figure 2B). To conform it, we examined the mRNA and protein level of SKP2 after LPS, and found that LPS induced the expression of SKP2 at both mRNA and protein level (Supplementary Figure S1A and B). However, SKP2 protein did not enhance under LPS treatment in the presence of the protein synthesis inhibitor, cycloheximide (CHX) (Supplementary Figure S1C). These results suggest that SKP2 can be induced by LPS treatment. Since protein stability, mRNA stability, and translation efficiency are also important for the induction of immediate-response gene, how LPS induces SKP2 expression needs further study. Figure 2 View largeDownload slide SKP2 deficiency enhances IKK phosphorylation and the expression of NF-κB responsive cytokines. (A) The knockout efficiency of SKP2 was determined by immunoblot analysis with anti-SKP2 antibody. (B) Control (ctrl), SKP2-knockout (SKP2-KO) THP-1 cells, and SKP2-KO cells overexpressing ectopic SKP2 were treated with LPS (100 ng/ml) for the indicated time points, and indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of indicated band intensity compared with control, using β-actin as a loading control. (C) Control or SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at different time points. The nuclear translocation of p65 was determined by immunoblot analysis using indicated antibodies. (D) Wide type (WT) or SKP2-KO HeLa cells were treated with LPS (10 μg/ml) for 30 min, and then subjected to immunofluorescence analysis using p65 and SKP2-specific antibodies. DNA was stained by DAPI (blue). UT, untreated. Scale bar: 10 μm. (E) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml). The expression of TNF-α, IL-1β, and IL-6 was determined by quantitative RT-PCR. (F) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at the indicated time points. Cell supernatants were used for measuring the release of TNF-α, IL-6, and IL-1β by ELISA. Data in E and F are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. control cells with the same treatment, Student’s t-test). Figure 2 View largeDownload slide SKP2 deficiency enhances IKK phosphorylation and the expression of NF-κB responsive cytokines. (A) The knockout efficiency of SKP2 was determined by immunoblot analysis with anti-SKP2 antibody. (B) Control (ctrl), SKP2-knockout (SKP2-KO) THP-1 cells, and SKP2-KO cells overexpressing ectopic SKP2 were treated with LPS (100 ng/ml) for the indicated time points, and indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of indicated band intensity compared with control, using β-actin as a loading control. (C) Control or SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at different time points. The nuclear translocation of p65 was determined by immunoblot analysis using indicated antibodies. (D) Wide type (WT) or SKP2-KO HeLa cells were treated with LPS (10 μg/ml) for 30 min, and then subjected to immunofluorescence analysis using p65 and SKP2-specific antibodies. DNA was stained by DAPI (blue). UT, untreated. Scale bar: 10 μm. (E) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml). The expression of TNF-α, IL-1β, and IL-6 was determined by quantitative RT-PCR. (F) Control and SKP2-KO THP1 cells were treated with LPS (100 ng/ml) at the indicated time points. Cell supernatants were used for measuring the release of TNF-α, IL-6, and IL-1β by ELISA. Data in E and F are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. control cells with the same treatment, Student’s t-test). It is known that activation of the NF-κB signaling pathway by LPS treatment induced the nuclear translocation of p65 from the cytoplasm. Our results revealed that SKP2 deficiency predominantly enhanced the nuclear translocation of p65 under LPS stimulation in THP-1 cells (Figure 2C). This result is further confirmed by the immunofluorescence analysis in HeLa cells (Figure 2D). Consistent with that, the mRNA levels of TNF-α, IL-6, and IL-1β in SKP2-KO cells were significantly increased after LPS treatment (Figure 2E). Meanwhile, knockout of SKP2 substantially increased the secretion of TNF-α, IL-6, and IL-1β after LPS treatment (Figure 2F). Taking together, these data suggest that SKP2 deficiency enhances NF-κB activity, by increasing nuclear accumulation of p65, as well as the secretion of NF-κB-dependent pro-inflammatory cytokines. SKP2 inhibits NF-κB signaling at IKK level To determine the molecular mechanisms by which SKP2 inhibits NF-κB signaling, we transfected HEK293T cells with MyD88, TRAF6, TAK1-TAB1, IKKβ, or p65 together with increasing amounts of SKP2 plus the NF-κB luciferase reporter and an internal control (renilla luciferase). We found that the activation of NF-κB by MyD88, TRAF6, TAK1-TAB1, and IKKβ was markedly inhibited by SKP2 (Figure 3A and Supplementary Figure S2). In contrast, SKP2 did not inhibit p65-mediated NF-κB activation (Figure 3A), suggesting that SKP2 inhibits the NF-κB pathway upstream of p65, most likely targeting the IKK complex. Consistent with these results, we found that knockout of SKP2 enhanced NF-κB luciferase activity induced by MyD88, TRAF6, TAK1-TAB1, IKKβ, but not p65 (Figure 3B). These results suggest that SKP2 inhibits NF-κB signaling upstream of p65, probably at the level of the IKK complex. Figure 3 View largeDownload slide SKP2 inhibits NF-κB signaling at the level of the IKK complex. (A) HEK293T cells were transfected with NF-κB-luc, pRL-TK, MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with increasing amounts of SKP2. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. (B) Control (Ctrl) and SKP2 knockout (SKP2-KO) HEK293T cells were transfected with MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with NF-κB-luc and pRL-TK plasmids. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. Data in A and B are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV or control cells with the same treatment, Student’s t-test). Figure 3 View largeDownload slide SKP2 inhibits NF-κB signaling at the level of the IKK complex. (A) HEK293T cells were transfected with NF-κB-luc, pRL-TK, MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with increasing amounts of SKP2. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. (B) Control (Ctrl) and SKP2 knockout (SKP2-KO) HEK293T cells were transfected with MyD88, TRAF6, TAK1 + TAB1, IKKβ, or p65, along with NF-κB-luc and pRL-TK plasmids. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. Data in A and B are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV or control cells with the same treatment, Student’s t-test). SKP2 mediates autophagy-dependent degradation of IKKβ During the experiments, we observed a negative correlation between SKP2 and IKKβ in protein level upon NF-κB activation (Figure 2B and Supplementary Figure S2). Considering the inhibitory effect of SKP2 in NF-κB signaling occurs at IKK level, we wondered whether SKP2 could degrade IKK complex. To test this hypothesis, we overexpressed SKP2 in HeLa cells and detected IKK complex expression. We found that simply overexpression of SKP2 does not affect the protein level of the components from IKK complex (Supplementary Figure S3). However, SKP2 markedly reduced the protein level of IKKβ, but not IKKα or NEMO upon LPS treatment (Figure 4A). We next used a CHX-chase assay to determine the time course of SKP2-mediated IKKβ degradation, and found that SKP2 can accelerate the turnover rate of IKKβ (Figure 4B). Since overexpression of IKKβ could activate IKKβ via auto-phosphorylation, our results suggest that SKP2 could promote the degradation of active IKKβ. Figure 4 View largeDownload slide SKP2 mediates autophagy-dependent degradation of IKKβ. (A) HeLa cells were transfected with empty vector or increasing amount of Myc-SKP2, followed by LPS treatment (10 μg/ml, 3 h). The protein turnover of IKK complex was determined by immunoblot analysis with indicated antibodies. The mRNA levels of IKK complex were measured by quantitative RT-PCR. (B) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or empty vector, then treated with CHX at the indicated time points, and the indicated proteins were determined by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (C) HeLa cells were transfected with plasmid encoding Flag-IKKβ for 24 h. Before harvest, cells were treated with MG132 (10 μM), chloroquine (CQ, 50 μM), bafilomycin A1 (Baf A1, 100 nM), or 3-methyladenine (3-MA, 2.5 mM) for 8 h. Cell extracts were analyzed by immunoblotting. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (D) Immunoassay of extracts of HeLa cells transfected with Myc-SKP2, and then treated with LPS for 4 h in the presence of Baf A1 (8 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (E) Immunoassay of extracts of control (Ctrl) or ATG5 knockout (ATG5-KO) HeLa cells transfected with Myc-SKP2 for 24 h, followed by LPS treatment (4 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. Data in A are expressed as mean ± SEM of three independent experiments. Figure 4 View largeDownload slide SKP2 mediates autophagy-dependent degradation of IKKβ. (A) HeLa cells were transfected with empty vector or increasing amount of Myc-SKP2, followed by LPS treatment (10 μg/ml, 3 h). The protein turnover of IKK complex was determined by immunoblot analysis with indicated antibodies. The mRNA levels of IKK complex were measured by quantitative RT-PCR. (B) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or empty vector, then treated with CHX at the indicated time points, and the indicated proteins were determined by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (C) HeLa cells were transfected with plasmid encoding Flag-IKKβ for 24 h. Before harvest, cells were treated with MG132 (10 μM), chloroquine (CQ, 50 μM), bafilomycin A1 (Baf A1, 100 nM), or 3-methyladenine (3-MA, 2.5 mM) for 8 h. Cell extracts were analyzed by immunoblotting. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. (D) Immunoassay of extracts of HeLa cells transfected with Myc-SKP2, and then treated with LPS for 4 h in the presence of Baf A1 (8 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (E) Immunoassay of extracts of control (Ctrl) or ATG5 knockout (ATG5-KO) HeLa cells transfected with Myc-SKP2 for 24 h, followed by LPS treatment (4 h). Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. Data in A are expressed as mean ± SEM of three independent experiments. Next, we investigated the molecular mechanisms underlying SKP2-mediated IKKβ degradation. Three major systems of protein clearance in eukaryotic cells are the proteasome, lysosome, and auto-lysosome pathways (Kraft et al., 2010). We found that the autophagic-sequestration inhibitor 3-methyladenine (3-MA) or the lysosomal-acidification inhibitor Bafilomycin A1 (Baf A1), and chloroquine (CQ), but not the proteasome inhibitor MG132, stabilized IKKβ (Figure 4C). Meanwhile, we found that SKP2 could not degrade IKKβ anymore under Baf A1 treatment (Figure 4D), indicating that the SKP2-mediated IKKβ degradation is regulated by the auto-lysosome pathway. These results are further confirmed by using the ATG5-KO cells in which the autophagy is deficient (Figure 4E). Together, our results indicate that SKP2 promotes IKKβ degradation via auto-lysosome pathway. SKP2 interacts with activated IKKβ Since SKP2 specifically promotes IKKβ degradation (Figures 2B, 4A and B), we suggested that SKP2 might interact with the IKK complex. To test this hypothesis, we transfected HEK293T cells with SKP2 together with expressed plasmids of IKKα, IKKβ, or NEMO. Co-immunoprecipitation and immunoblot analysis revealed that SKP2 can interact with IKK complex (Figure 5A). As a kinase in NF-κB cascade, the activation of IKKβ occurs through dual phosphorylation on Ser177 and Ser181 (Mercurio et al., 1997). By substituting their activation sites (Ser177/Ser181) to alanine or glutamic acid, respectively, we constructed IKKβ constitutively active (IKKβ-SE) mutants or IKKβ enzymatically inactive (IKKβ-SA) mutants, and found that SKP2 specially interacted with active IKKβ (IKKβ-SE) (Figure 5B). Furthermore, SKP2 cannot interact with kinase activity-deficient form of IKKβ (IKKβ-K44A), which could not active itself via auto-phosphorylation (Figure 5B). Consistently, SKP2 promoted the degradation of active IKKβ (IKKβ-SE), but not inactive IKKβ (IKKβ-K44A or IKKβ-SA) (Supplementary Figure S4A–C). Moreover, we found that SKP2 interacted with active IKKα (Supplementary Figure S4D). We next performed endogenous immunoprecipitation of IKKβ, and detected stronger interaction between endogenous SKP2 and IKKβ under LPS treatment, indicating that SKP2 prefers to interact with active IKKβ (Figure 5C). Moreover, purified His-SKP2 was able to bind to Flag-tagged IKKβ under cell-free conditions (Figure 5D). Since SKP2 interacts with IKK complex, but only promotes IKKβ degradation, we hypotheses that it might release IKKβ from IKK complex for degradation. We transfected plasmids of IKKβ and NEMO, along with increasing amount of Myc-SKP2, and found that the interaction of NEMO and IKKβ was decreased by increasing amount of Myc-SKP2 (Figure 5E). To further determine which domain of IKKβ is responsible for interacting with SKP2, we generated deletion mutants encompassing the amino-terminal kinase domain (KD), leucine zipper domain (LZ), and a C-terminal helix-loop-helix (HLH) domain of IKKβ (Figure 5F). Co-immunoprecipitation results showed that the LZ domain of IKKβ interacted with SKP2 (Figure 5G). SKP2 contains one F-box domain and 10 leucine repeat regions (LRRs). To identify the functional domains of SKP2, we generated six domain deletion constructs of SKP2: ΔF-box, ΔLRR12, ΔLRR34, ΔLRR5, ΔLRR67, and ΔLRR8910 (Figure 5H). Co-immunoprecipitation results showed that ΔF-box and ΔLRR67 could still strongly interact with the IKKβ protein, but ΔLRR12 and ΔLRR8910 could only weakly bind with IKKβ (Figure 5I). Moreover, we found that three domain deletions ΔLRR12, ΔLRR67, and ΔLRR8910 could neither promote IKKβ degradation nor inhibit IKKβ-mediated NF-κB signaling (Figure 5J), suggesting that LRR12, LRR67, and LRR8910 are critical for the function of SKP2 in mediating NF-κB activity. Together, these results reveal that SKP2 specifically targets LZ domain of IKKβ and promotes active IKKβ degradation. Figure 5 View largeDownload slide SKP2 interacts with IKKβ. (A) HEK293T cells were transfected with Flag-IKKα, Flag-IKKβ, Flag-NEMO, and Myc-SKP2. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblotting. (B) Lysates of HEK293T cells transfected with Flag-IKKβ-WT, Flag-IKKβ-K44A, Flag-IKKβ-SA, and Flag-IKKβ-SE, together with Myc-SKP2 in the presence of Baf A1 (100 μM), were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (C) Lysates of HeLa cells in the presence of bafilomycin A1 (Baf A1, 100 μM) with or without LPS (10 μg/ml, 4 h) treatment were immunoprecipitated with anti-IgG or anti-IKKβ antibody, followed by immunoblot analysis to detect SKP2 protein. (D) Top: His-EV or His-SKP2 was retained on Ni IDA beads, incubated with Flag-IKKβ then immunoblotted with the antibody against Flag. Flag-IKKβ is purified by anti-Flag beads and then washed down by Flag peptides. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. (E) HEK293T cells were transfected with HA-IKKβ, Flag-NEMO, empty vector, or increasing amount of Myc-SKP2 for 24 h. The lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (F) A schematic diagram shows protein domain structures of the IKKβ deletions. KD, amino-terminal kinase domain; LZ, leucine zipper domain; HLH, C-terminal helix-loop-helix. (G) HEK293T cells were co-transfected with Myc-SKP2 with Flag-IKKβ or its deletion mutants. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblot analysis. (H) A schematic diagram shows protein domain structures of the SKP2 deletions. (I) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or its deletion mutants, followed by Baf A1 treatment. Myc-tagged proteins were immunoprecipitated with anti-Myc beads followed with immunoblot analysis. (J) HEK293T cells were transfected with NF-κB-luc, pRL-TK, Flag-IKKβ, along with Myc-SKP2 or it domain deletions. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. The indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. Data in J are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test. ns, no significant). Figure 5 View largeDownload slide SKP2 interacts with IKKβ. (A) HEK293T cells were transfected with Flag-IKKα, Flag-IKKβ, Flag-NEMO, and Myc-SKP2. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblotting. (B) Lysates of HEK293T cells transfected with Flag-IKKβ-WT, Flag-IKKβ-K44A, Flag-IKKβ-SA, and Flag-IKKβ-SE, together with Myc-SKP2 in the presence of Baf A1 (100 μM), were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (C) Lysates of HeLa cells in the presence of bafilomycin A1 (Baf A1, 100 μM) with or without LPS (10 μg/ml, 4 h) treatment were immunoprecipitated with anti-IgG or anti-IKKβ antibody, followed by immunoblot analysis to detect SKP2 protein. (D) Top: His-EV or His-SKP2 was retained on Ni IDA beads, incubated with Flag-IKKβ then immunoblotted with the antibody against Flag. Flag-IKKβ is purified by anti-Flag beads and then washed down by Flag peptides. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. Bottom: recombinant His-EV and His-SKP2 were purified from bacteria and analyzed by SDS-PAGE and Coomassie blue staining. (E) HEK293T cells were transfected with HA-IKKβ, Flag-NEMO, empty vector, or increasing amount of Myc-SKP2 for 24 h. The lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblot analysis. (F) A schematic diagram shows protein domain structures of the IKKβ deletions. KD, amino-terminal kinase domain; LZ, leucine zipper domain; HLH, C-terminal helix-loop-helix. (G) HEK293T cells were co-transfected with Myc-SKP2 with Flag-IKKβ or its deletion mutants. Flag-tagged proteins were immunoprecipitated with anti-Flag beads followed with immunoblot analysis. (H) A schematic diagram shows protein domain structures of the SKP2 deletions. (I) HEK293T cells were co-transfected with Flag-IKKβ with Myc-SKP2 or its deletion mutants, followed by Baf A1 treatment. Myc-tagged proteins were immunoprecipitated with anti-Myc beads followed with immunoblot analysis. (J) HEK293T cells were transfected with NF-κB-luc, pRL-TK, Flag-IKKβ, along with Myc-SKP2 or it domain deletions. NF-κB-dependent luciferase activity was analyzed after transfection for 24 h. The indicated proteins were measured by immunoblot analysis. Numbers underneath the blot represent the fold change of Flag-IKKβ band intensity compared with control, using β-actin as a loading control. Data in J are expressed as mean ± SEM of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the cells transfected with EV with the same treatment, Student’s t-test. ns, no significant). SKP2 bridges IKKβ and p62 Increasing evidence supports that cargo receptors deliver cargoes to the autophagosome for selective degradation (Stolz et al., 2014). In order to find the cargo receptor of IKKβ, we performed co-immunoprecipitation between IKKβ and several major cargo receptors, and found that IKKβ mainly interacted with p62 rather than other receptors (Figure 6A). Further experiments revealed that SKP2 also strongly interacted with p62 than other cargo receptors (Figure 6B). Moreover, purified SKP2 was able to bind to p62 under cell-free conditions (Supplementary Figure S5A). Next, we found that all domains of SKP2 can strongly interact with p62 except ΔLRR67 and ΔLRR8910 (Supplementary Figure S5B), indicating the critical role of LRR67 and LRR8910 domains for SKP2–p62 interaction. In our previous study, TRIM11 functions as a co-receptor of AIM2 for autophagic degradation through linking p62 and AIM2 (Liu et al., 2016). We suggested that SKP2 might bridge IKKβ and p62. As expected, we observed that SKP2 promoted the interaction of p62 and IKKβ in a dose-dependent manner (Figure 6C). In addition, we found that purified SKP2 was able to enhance the interaction between p62 and IKKβ under cell-free conditions (Figure 6D). These results indicate SKP2 could bridge IKKβ and p62. To further confirm these results, we examined the endogenous interaction between IKKβ and p62. The results showed the association of IKKβ and p62 was enhanced after LPS treatment; however, the interaction was significantly impaired in SKP2-KO cells (Figure 6E). Moreover, we found that SKP2 failed to promote IKKβ degradation in p62-KO cells (Figure 6F). Collectively, these results indicate that SKP2 promotes autophagic degradation of IKKβ through bridging IKKβ–p62 interaction. Figure 6 View largeDownload slide SKP2 promotes p62-mediated selective autophagic degradation of IKKβ. (A) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, OPTN, Tollip, or NBR1, together with HA-IKKβ, followed by Baf A1 treatment. (B) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, NDP52, OPTN, Tollip, or NBR1, together with Myc-SKP2, followed by Baf A1 treatment. (C) HEK293T cells were co-transfected with Flag-IKKβ and HA-p62 together with increasing amounts of Myc-SKP2, then treated with chloroquine (CQ) for 8 h. Cell extracts were immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. Numbers underneath the blot represent the fold change of HA-p62 band intensity compared with control group, using immunoprecipitated Flag-IKKβ as a loading control. (D) The purified HA-p62 and Flag-IKKβ were incubated with His-EV or His-SKP2 and then immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. (E) WT or SKP2-KO THP-1 cells were treated with LPS (100 ng/ml) for indicated time points, and Baf A1 was used to treat the cells for 3 h before harvesting them. Whole-cell extracts were immunoprecipitated with anti-IKKβ antibody, followed by IB analysis with anti-p62 antibody. (F) Immunoassay of extracts of control (Ctrl) or p62 knockout (p62-KO) HeLa cells transfected with Myc-SKP2, followed by LPS treatment. Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (G) Work model of SKP2-mediated autophagic degradation of IKKβ. Figure 6 View largeDownload slide SKP2 promotes p62-mediated selective autophagic degradation of IKKβ. (A) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, OPTN, Tollip, or NBR1, together with HA-IKKβ, followed by Baf A1 treatment. (B) Co-immunoprecipitation and immunoassay of extracts of HEK293T cells transfected with Flag-tagged p62, Nix, NDP52, OPTN, Tollip, or NBR1, together with Myc-SKP2, followed by Baf A1 treatment. (C) HEK293T cells were co-transfected with Flag-IKKβ and HA-p62 together with increasing amounts of Myc-SKP2, then treated with chloroquine (CQ) for 8 h. Cell extracts were immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. Numbers underneath the blot represent the fold change of HA-p62 band intensity compared with control group, using immunoprecipitated Flag-IKKβ as a loading control. (D) The purified HA-p62 and Flag-IKKβ were incubated with His-EV or His-SKP2 and then immunoprecipitated with anti-Flag beads followed by immunoblot analysis with indicated antibodies. (E) WT or SKP2-KO THP-1 cells were treated with LPS (100 ng/ml) for indicated time points, and Baf A1 was used to treat the cells for 3 h before harvesting them. Whole-cell extracts were immunoprecipitated with anti-IKKβ antibody, followed by IB analysis with anti-p62 antibody. (F) Immunoassay of extracts of control (Ctrl) or p62 knockout (p62-KO) HeLa cells transfected with Myc-SKP2, followed by LPS treatment. Numbers underneath the blot represent the fold change of IKKβ band intensity compared with control, using β-actin as a loading control. (G) Work model of SKP2-mediated autophagic degradation of IKKβ. Discussion Transcription factor NF-κB plays a crucial role in various physiological processes such as inflammation, adaptive immunity, and cell proliferation/death. It is fine-turned by various molecules at different levels. Numerous E3 ubiquitin ligases or deubiquitinases regulate the NF-κB activation by modulating the ubiquitinated levels of the distinct components of NF-κB signaling (Malynn and Ma, 2010). For example, we found that TRIM9 and USP18 can inhibit virus-induced and TLR-induced NF-κB signaling, respectively (Yang et al., 2015; Qin et al., 2016). F-box proteins belong to a large family of E3 ligases that contain a carboxy-terminal domain that interacts with substrates and a 42–48 amino-acid F-box motif, and play important regulatory roles in innate immune signaling, including inflammation. The F-box protein FBXL2 has been reported to serve as a sentinel inhibitor of NF-κB by mediating poly-ubiquitination and proteasomal degradation of TRAFs (Chen et al., 2013). Here, we screened the function of several F-box proteins and identified SKP2 (also named as FBXL1) as a negative modulator of TLR- and cytokine-mediated NF-κB activation by targeting IKKβ for autophagic degradation. IKK complex plays pivotal roles in the NF-κB signaling, and its activity is tightly regulated by multiple post-translational modifications. By recruiting phosphatase PP1 to dephosphorylate IKK, CUEDC2 deactivates IKK activity and further represses activation of NF-κB signaling (Li et al., 2008). Besides of controlling the phosphorylation and de-phosphorylation, modulation of IKKs stability is another way to regulate the activity of IKK complex. It has been revealed that p47 induces the lysosomal degradation of poly-ubiquitinated NEMO, the regulatory component of IKK complex (Shibata et al., 2012). Kelch-like ECH-associated protein 1 (KEAP1) facilitates IKKβ degradation through promoting its ubiquitination, and further mediates the down-regulation of NF-κB signaling via autophagy (Lee et al., 2009; Kim et al., 2010). Our results showed that SKP2 is a LPS-inducible gene, which inhibits NF-κB signaling through degrading active IKKβ, suggesting that SKP2 modulates NF-κB activation through a negative feedback loop. Although SKP2 interacts with the whole IKK complex, it selectively promotes the degradation of IKKβ. We found that SKP2 can reduce the interaction between IKKβ and NEMO, suggesting that SKP2 can release active IKKβ form IKK complex for degradation. Whether SKP2 affects the function of IKKα or NEMO needs further study. Furthermore, we found that blockage of autophagic flux by autophagic inhibitor or ATG5 deficiency prevents SKP2-promoted degradation of IKKβ, indicating the important role of autophagy in NF-κB activation. Autophagy was once considered as a non-selective bulk degradation pathway of cells, but it is now clear that autophagic degradation of proteins mediated by p62 can degrade substrates in a selective manner (Kraft et al., 2010). The autophagy pathway also functions in the control of inflammatory signaling. Autophagic gene deficiency increases the level of the adaptor protein p62, meanwhile reinforced NF-κB signaling and transcription of inflammatory factors through a mechanism involving TRAF6 oligomerization (Levine et al., 2011). Macrophages with deficiency of Atg16L1 or Atg7, which are the essential components of the autophagic process, enhanced TLR-3/4-mediated production of interleukin (IL)-1β and IL-18 (Saitoh et al., 2008). It is known that p62 negatively regulates the transcription of inflammatory factors in stimulated macrophages (Kim and Ozato, 2009). But the role of p62-mediated autophagic degradation in NF-κB signaling is still not fully investigated. Here, we demonstrated that SKP2 can be induced by NF-κB activation, binds active IKKβ. Finally, the complex of IKKβ-SKP2 is recognized and recruited by cargo receptor p62 to the autophagosome for degradation, subsequently dampening NF-κB signaling (Figure 6G). In this study, we found that three LRR domains of SKP2 might play essential roles to bridge IKKβ and p62: LRR12 and LRR8910 of SKP2 are important for its binding activity to IKKβ; while LRR67 and LRR8910 are critical for its binding activity to p62. Bafilomycin A1 efficiently stabilized the protein levels of IKKβ, which is consistent with the previous study that IKKs degradation requires autophagic flux (Kim et al., 2010). Considering of the critical role of p62 for directing autophagic uptake of proteins, our study demonstrated that SKP2 is a novel co-receptor of delivering IKKβ for selectively autophagic degradation. These results provide mechanistic evidence that autophagy selectively regulated NF-κB activation. As the negative regulator of NF-κB signaling, SKP2 might serve as a potential target for treating inflammation-associated diseases. Materials and methods Antibodies and reagents Horseradish peroxidase (HRP)-anti-Flag (M2) (#A8592) and anti-β-actin (#A1978) were purchased from Sigma; HRP-anti-hemagglutinin (#12013819001) and anti-Myc-HRP (#11814150001) were purchased from Roche Applied Science; Anti-phospho-IKKα/β (#2697S), anti-ATG5 (#12994S), anti-p62 (8025), anti-p65 (#6956), anti-IKKβ (#2684), anti-IκBα (#4814), and anti-phospho-IκBα (Ser32/36) (#9246) were purchased from Cell Signaling Technology; Goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, anti-SKP2 (#sc-74477), and anti-NEMO (#sc-8300) were purchased from Santa Cruz Biotechnology; Anti-IKKα (#07-1007) were purchased from Millipore; Protein A agarose and Protein G agarose were purchased from Pierce. Dimethyl sulfoxide (DMSO) (D2650), CHX (R750107), Puromycin (P9620), MG132 (C-2211-5MG), Chloroquine phosphate (CQ) (PHR1258), 3-methyladenine (3-MA) (M9281), and LPS (L4391) were purchased from Sigma. Bafilomycin A1 (S1413) was purchased from Selleck. Cell culture and transfection Human embryonic kidney 293T (HEK293T), HeLa, and THP-1 cells were cultured in humidified 5% CO2 at 37°C in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone) or RPMI-1640 (Gibco) medium supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, and 1× Penicillin/Streptomycin solution (Thermo). Lipofectamine 2000 (Invitrogen) and ViaFect™ Transfection Reagent (Promega) were used for transient transfection of HEK293T and HeLa cells, respectively. Luciferase reporter assays HEK293T or HeLa cells were transfected with luciferase reporter plasmids encoding NF-κB luciferase, pRL-TK Renilla luciferase, and SKP2 or control vectors, TNF-α, IL-1β, LPS as well as exogenous MyD88, TRAF6, TAK1/TAB1, IKKβ, and p65 were used as stimulators. Then, 24 h after transfection, cells were lysed. The luciferase activity was determined by a dual luciferase reporter assay kit (Promega) using a Luminoskan Ascent luminometer (Thermo Scientific) according to the manufacturer’s instructions. Reporter gene activity was determined by normalization of the firefly luciferase activity to renilla luciferase activity (Qin et al., 2017). Immunoprecipitation and immunoblot analysis Cells were washed with ice-cold PBS and then lysed in low-salt lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100) supplemented with 5 mg/ml cOmplete™ protease inhibitor cocktail tablets (Roche). Protein concentration was determined by BCA Protein Assay Kit (Pierce). After boiling, cell lysate was subjected to electrophoresis. For immunoprecipitation experiments, whole-cell lysates were prepared after transfection or stimulation and incubated with indicated antibodies together with protein A/G beads (Pierce) for overnight. For anti-flag or anti-myc immunoprecipitation, anti-flag or anti-myc agarose gels (Sigma) were used. Beads were then washed four times with lysis buffer, and immune-complexes were re-suspended in 2× SDS loading buffer. After boiling and electrophoresis, the proteins were transferred to polyvinylidene fluoride membrane (Bio-Rad) and further incubated with the indicated antibodies. Protein detection was performed by a ChemiDoc™ XRS system (Bio-Rad) using enhanced chemiluminescence (Millipore). Immunofluorescence assay Cells grown on dishes were fixed with 4% paraformaldehyde for 15 min, and then permeabilized in methyl alcohol for 10 min at −20°C. After washing with PBS, cells were blocked in 5% fetal goat serum for 1 h, and then incubated with primary antibodies diluted in 10% bull serum albumin overnight. The cells were washed and followed by a fluorescently labeled secondary antibody. Nuclear DNA was stained using 5 μg/ml 4′,6-diamidino-2-phenylindole (DAPI), a fluorescent DNA-intercalating dye. A radiance microscope system (Leica) was used to visualize the distribution of p65 or SKP2 (Liu et al., 2013). Generation of SKP2 knockout cell lines by CRISPR/Cas9 technology To generate SKP2-KO 293T cells, target sequences were cloned into pLentiCRISPRv2 by cutting with BsmBI as previous described (Jin et al., 2016). The sequences of target-related gene are as follows: Ctrl sgRNA: 5′-GGGCGAGGAGCTGTTCACCG-3′; SKP2 sgRNA 1#: 5′-AAGACTTTGTGATTGTCCGC-3′; SKP2 sgRNA 2#: 5′-GCAACGTTGCTACTCAGGTC-3′. Subcellular fractionation Cellular fractionation assay was carried out using the nuclear and cytoplasmic protein extraction kit (Beyotime) according the manufacturer’s protocol. Briefly, cells were harvested and washed twice with ice-cold PBS, and then were re-suspended with Buffer A. After incubated on ice for 15 min, cells were lysed with Buffer B. Next, cells were centrifuged for 5 min at 12000 g at 4°C. The supernatant (cytoplasm, membrane, and mitochondria) was collected. After washed the pellet twice with ice-cold PBS, sediment samples were re-suspended in Buffer C. Next, samples were incubated on ice for 30 min and centrifuged for 10 min at 12000 g at 4°C. The supernatant (nuclear extract) was collect. Real-time PCR analysis Total RNA was isolated directly from freshly collected THP-1 cells with TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized by using 1 μg total RNA with HiScript II QRT-SuperMix (Vazyme). qRT-PCR reactions were carried out on LightCycler480 (Roche) by using of 2× Power SYBR green qPCR master mix (GenStar). All PCR products were checked by running an agarose gel to exclude the possibility of multiple products. RPL13A was used as an internal control. Comparative quantification of gene expression was evaluated using the 2−ΔΔCt method. All qRT-PCR assays were conducted in triplicates. Sequences of primers were listed as follows:SKP2: forward 5′-ACCTTTCTGGGTGTTCTGGA-3′, reverse 5′-ATTCAGCTGGGTGATGGTCT-3′; GAPDH: forward 5′-GCTGGTCATCAACGGGAAA-3′, reverse 5′-ACGCCAGTAGACTCCACGACA-3′; TNF-α: forward 5′-CCCAGGGACCTCTCTCTAATCA-3′, reverse 5′-GCTTGAGGGTTTGCTACAACATG-3′; IL-1β: forward 5′-AAATACCTGTGGCCTTGGGC-3′, reverse 5′-TTTGGGATCTACACTCTCCAGCT-3′; IL-6: forward 5′-AGAGGCACTGGCAGAAAACAAC-3′, reverse 5′-AGGCAAGTCTCCTCATTGAATCC-3′; IKK-α: forward 5′-CAGCCATTTACCTGGCATGAG-3′, reverse 5′-GAGGGTCCCAATTCAACATCAA-3′; IKK-β: forward 5′-ATCCCCGATAAGCCTGCCA-3′, reverse 5′-CTTGGGCTCTTGAAGGATACAG-3′; NEMO: forward 5′-GCGAGGAATGCAGCTGGAAG-3′, reverse 5′-GCCTGGAAGTCCGCCTTGTA-3′. Enzyme-linked immunosorbent assay THP-1 cells were simulated for 6 h with LPS (100 ng/ml). The supernatant medium of cells was collected. The concentration of TNF-α, IL-6, and IL-1β was determined by using of human specific ELISA Kit (BD Biosciences) according to the manufacturer’s instructions. Statistical analysis The results of all quantitative experiments are reported as mean ± SEM of three independent experiments. Statistical analysis was performed using GraphPad Prism 5.0. Student’s t-test was used for the comparison of two groups. For all tests, a P-value < 0.05 was considered statistically significant, and the level of significance was indicated as *P < 0.05, **P < 0.01, ***P < 0.001. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Funding This work was supported by grants from the National Natural Science Foundation of China (91629101, 31522018, 31601135, 81302197, 81700557, and 31071046), the National Key Basic Research Program of China (2015CB859800 and 2014CB910800), and the Guangdong Innovative Research Team Program (2011Y035). 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Journal of Molecular Cell BiologyOxford University Press

Published: Apr 5, 2018

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