We recently reported that NF-κB–mediated inflammation caused by breakpoint cluster region (BCR) is dependent on the α subunit of casein kinase II (CK2α) complex. In the current study, we demonstrate that presenilin 1 (Psen1), which is a catalytic component of the γ-secretase complex and the mutations of which are known to cause familial Alzheimer disease, acts as a scaffold of the BCR–CK2α–p65 complex to induce NF-κB activation. Indeed, Psen1 deficiency in mouse endothelial cells showed a significant reduction of NF-κB p65 recruitment to target gene promoters. Conversely, Psen1 overexpression enhanced reporter activation under NF-κB responsive elements and IL-6 promoter. Furthermore, the transcription of NF-κB target genes was not inhibited by a γ-secretase inhibitor, suggesting that Psen1 regulates NF-κB activation in a manner independent of γ-secretase activity. Mechanistically, Psen1 associated with the BCR–CK2α complex, which is required for phosphorylation of p65 at serine 529. Consistently, TNF-α–induced phosphorylation of p65 at serine 529 was significantly decreased in Psen1-deficient cells. The association of the BCR–CK2α–p65 complex was perturbed in the absence of Psen1. These results suggest that Psen1 functions as a scaffold of the BCR–CK2α–p65 complex and that this signaling cascade could be a novel therapeutic target for various chronic inflammation conditions, including those in Alzheimer disease.

Although inflammation is a biological defense mechanism, chronic inflammation leads to several pathological conditions, such as autoimmune diseases, metabolic syndrome, and neurodegenerative diseases including Alzheimer disease (AD) (14). As a molecular mechanism for inflammation development, we previously discovered the inflammation amplifier, which is a chemokine hyperproduction mechanism operated in nonimmune cells such as fibroblasts and endothelial cells (5, 6). In this system, coactivation of NF-κB and STATs in nonimmune cells leads to a synergistic activation of NF-κB to express inflammatory mediators, including chemokines, growth factors, and cytokines, that contribute to the development of various inflammatory diseases (5, 711). Not only animal models but also patients showed that the coactivation of NF-κB and STAT3 is observed in lung inflammation with bronchiolitis obliterans (8, 12). Furthermore, NF-κB/STAT3–induced soluble target molecules are found at high levels in serum from patients with rheumatoid arthritis and multiple sclerosis (13, 14). Using genome-wide screening, we identified 1289 genes that are positive regulators of the synergistic activation of NF-κB, and many genes associated with human diseases were highly enriched in these regulators (13). The gene list also includes genes involved in NF-κB activation with previously unidentified function. For example, we recently reported that breakpoint cluster region (BCR), which forms the oncogenic fusion protein BCR-Abl (15), is associated with the α subunit of casein kinase II (CK2α), leading to the phosphorylation of BCR at Y177 and establishment of a NF-κB p65 binding site after TNF-α stimulation (16). The binding of p65 to the BCR–CK2α complex phosphorylates serine 529 of p65, which is required for the association of p65 with p300 histone acetyltransferase, chromatin opening, and the transcription of NF-κB target genes (16). In the current study, we selected presenilin 1 (Psen1) from the aforementioned screening and investigated its role in inflammation development.

Psen1 is a transmembrane protein that acts as a critical component of the γ-secretase complex catalytic core (17, 18). There are many substrates for γ-secretase, including amyloid precursor protein (APP) and Notch (19, 20). More than 185 mutations have been identified in Psen1 (18). These mutations increase the ratio of the 42- to 40-residue amyloid-β (Aβ) protein to promote Aβ aggregation, which is considered a potential cause of AD (1719). The Aβ aggregates trigger a variety of inflammatory pathways, and inflammation has been suggested to significantly contribute to the pathogenesis of AD (1, 4, 21). Because some Psen1 mutants of familial AD do not increase the production of neurotoxic Aβ42 or increase the Aβ42/Aβ40 ratio (22), we considered other mechanisms through which Psen1 could promote inflammation. In fact, many γ-secretase–independent systems have been reported (2326). For example, Psen1 downregulates insulin signaling by inhibiting the transcription of insulin receptor (25). It is also reported that Psen1 modulates the turnover of β-catenin by associating with glycogen synthase kinase 3β and protein kinase A for cell proliferation (23, 24). However, a direct contribution by Psen1 in NF-κB signaling and inflammation development has not been established.

In the current study, we show that Psen1 is involved in BCR–CK2α–p65 complex formation. Although a chemical inhibitor of γ-secretase did not have an inhibitory effect, we found that short hairpin RNA (shRNA)–mediated deficiency of Psen1 decreased the phosphorylation of CK2α and BCR at Y177 and the association between BCR and p65. These results suggest that Psen1 acts as a scaffold for BCR, CK2α, and p65, allowing efficient NF-κB activation. Therefore, the Psen1–BCR–CK2α–p65 cascade could be a novel therapeutic target for diseases that show chronic inflammatory such as AD.

C57BL/6 mice were purchased from Japan SLC (Shizuoka, Japan). F759 mice were back-crossed with C57BL/6 mice for more than 10 generations (7). All mice were maintained under specific pathogen-free conditions according to the protocols of Osaka and Hokkaido Universities. The protocols for animal experiments were approved by the Institutional Animal Care and Use Committees of Osaka University and Hokkaido University.

A mouse type 1 collagen–positive BC1 endothelial cell line was obtained from Dr. M. Miyasaka (Osaka University) (27). BC1 cells were cultured on day 1 in a 96-well flat-bottom plate (1000 cells per well) in 100 μl of DMEM containing 10% FBS. The medium was replaced on day 2 with DMEM containing 1 μl of lentivirus carrying shRNA (nontarget shRNA, Sigma Mission SHC002V; Psen1 shRNA, TRCN0000030520; Sigma-Aldrich), 10% FBS, and 8 μg/ml Polybrene. On day 3, 200 μl of DMEM containing 10% FBS and 5 μg/ml puromycin was added to each well.

H4 neuroglioma cells were obtained from American Type Culture Collection (Sumitomo Pharma, Japan). Small interfering RNAs (siRNAs) for human Psen1 (s224428; Thermo Fisher Scientific), p65 (Ambion Silencer Select RELA siRNA; Thermo Fisher Scientific), and nontarget (Ambion Negative Control no. 1 siRNA; Thermo Fisher Scientific) were transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific).

Nontarget shRNA, p65 shRNA, or Psen1 shRNA were injected into the ankle joints of F759 mice on days 0, 1, and 2, and then IL-6 and IL-17 (100 ng each) were injected into the ankle joints on days 6, 7, and 8. Clinical scores of the arthritis were evaluated as reported previously (7).

Psen1-deficient BC1 cells (2 × 104) cells were seeded in a 96-well flat-bottom plate and incubated at 37°C, 5% CO2 overnight. The next day, the cells were starved in 100 μl Opti-MEM (Thermo Fisher Scientific) for 3 h and stimulated with human IL-6 (Toray) and human soluble IL-6R (sIL-6R; R&D Systems), mouse IL-17 (R&D Systems), IL-6 plus IL-17 (i.e., human IL-6, human sIL-6R and mouse IL-17), or TNF-α (R&D Systems) for 6 h. Total RNA was extracted and treated with DNase I using the Isospin cell and tissue RNA kit (Nippon Gene), and cDNA was synthesized by Moloney murine leukemia virus reverse transcriptase (Promega). KAPA SYBR Fast qPCR Kit (KAPA BIOSYSTEMS) and ABI Prism 7300 apparatus (Applied Biosystems) were used for real-time PCR. mRNA expression levels were normalized to the levels of Hprt mRNA expression. The following primers were used. Mouse HPRT, 5′-GAAGCGAGAGAACCAGG-3′ and 5′-CCCCCACCCCAGACA-3′; mouse IL-6, 5′-GAGGAACCACCCCAACAGACC-3′ and 5′-AAGGCACACGGCAACA-3′; mouse lipocalin 2 (LCN2), 5′-CCACCGGCAGGGAC-3′and 5′-GGCCCAACAGGG-3′; mouse SOCS3, 5′-GCGGACCGCGGAG-3′ and 5′-GAGACGCCGGGACA-3′; and mouse STAT3, 5′-CACCTTGGATTGAGAGTCAAGAC-3′ and 5′-AGGAATCGGCTATATTGCTGGT-3′.

IL-6 levels in culture supernatant were detected by ELISA kits. Because human IL-6 acts on mouse cells, we could detect IL-6 production from mouse cells stimulated with human IL-6 using ELISA specific for mouse IL-6 (BD Biosciences, Tokyo, Japan).

Cell growth was determined with thiazolyl blue tetrazolium bromide according to the manufacturer’s instruction (Sigma-Aldrich).

Full-length mouse Psen1 cDNA was cloned into pEF-BOS expression vector (28). pGL4.32 (luc2P/IL-6-RE/Hygro), pRL-TK (Promega), and pEF-BOS Psen1 were transiently cotransfected into 293T cells by using polyethylenimine. Twenty-four hours after transfection, the cells were stimulated with 50 ng/ml TNF-α for 6 h. Luciferase activities of total cell lysates were measured using the Dual-Luciferase Reporter Assay System (Promega).

Control and Psen1-knockdown cells were stimulated with 50 ng/ml TNF-α for 0, 90, and 180 min. These cells were fixed with 1% PFA and lysed with cell lysis buffer (10 mM Tris-HCl [pH 7.5], 140 mM NaCl, 1% Triton X-100, 1 mM EDTA, and 1% SDS). The cell lysate was sonicated to prepare chromatin DNA. Immunoprecipitation was performed using Dynabeads protein G (Life Technologies, Tokyo, Japan) and anti-p65, anti-p300, anti–acetyl-H3K27 Abs, or rabbit IgG or mouse IgG. The DNA was purified with 10% Chelex 100 (Bio-Rad, Tokyo, Japan). Real-time PCR was performed with IL-6 or LCN2 promoter primer that included a p65 binding site. The following primer sequences were used for the PCR. Mouse LCN2, 5′-ACCAAAGCCCGGGAAGC-3′ and 5′-GGGAGCCACCACCAA-3′; and mouse IL-6, 5′-CGAGCAAACGACGCAC-3′ and 5′-GAGCACAGACACCCCAG-3′. Relative to the transcription start site, the p65 binding site in IL-6 promoter is −26 to −17 bp, and the forward and reverse primers start at −131 bp and +27 bp, respectively. The p65 binding site in LCN2 promoter is −261 to −252 bp, and the forward and reverse primers start at −273 bp and −74 bp, respectively (29).

Chromatin accessibility assay was performed with Chromatin Accessibility Assay Kit (EpiGentek). PCR primers for IL-6 and LCN2 promoters were the same as those used for the chromatin immunoprecipitation assay.

Nontarget control and Psen1-deficient BC1 cells were stimulated with TNF-α for 0, 15, and 30 min. The stimulated cells were fixed in 4% paraformaldehyde for 20 min, permeabilized with Perm/Wash solution (BD Biosciences Cytofix/Cytoperm Kit), and incubated with rabbit anti-p65 (1/50; Santa Cruz Biotechnology) for 1 h. After washing, the cells were incubated with anti-rabbit Alexa Fluor 488–conjugated secondary Ab (1/200; Life Technologies) and Hoechst 33342 nuclear stain (1/10,000; Life Technologies) for 1 h. Cells were then observed by confocal microscopy (30).

HEK293T cells were cotransfected with pEF-BOS containing full-length wild-type (WT) or mutant Psen1, BCR, and/or CK2α cDNA. Mutant cDNAs for mouse BCR were described in our recent paper (16). Mouse Psen1 mutant cDNA lacking aa 1–73 (Δ1–73) or 271–376 (Δ271–376), in which a large part of the hydrophilic cytoplasmic loop was deleted, was prepared by an inverse PCR method using full-length Psen1 cDNA. A CK2α mutant cDNA that lacks 35 N-terminal amino acids (ΔN-ter) was also generated by inverse PCR. Some molecules were tagged with Flag, Myc, or hemagglutinin (HA). These transfected cells were suspended in lysis buffer (50 mM Tris-HCl [pH 7.4], 500 mM NaCl, 1% NP40, and 3 mM EDTA) and precleared with 30 μl protein G–Sepharose (Pharmacia, Tokyo, Japan). These samples were centrifuged at 9000 rpm at 4°C for 3 min, and the supernatants were collected. The samples were mixed with 30 μl anti-FLAG M2 beads slurry (Sigma-Aldrich) and incubated for 2 h at 4°C with gentle agitation. The samples were centrifuged at 9000 rpm at 4°C for 3 min, and the supernatants were discarded. Anti-FLAG M2 beads were washed five times with 800 μl HEPES-buffered saline plus Triton X-100. The immunoprecipitates were eluted with 3× flag peptide (Sigma-Aldrich), separated by SDS–PAGE, and transferred to a PVDF membrane followed by Western blotting.

WT or Psen1-knockdown cells were washed three times with cold PBS, scraped from the bottom of the dish, and lysed with HEPES-buffered saline plus Triton X-100 lysis buffer (15 mM NaCl, 1 mM HEPES [pH 7.4], 0.5% Triton X-100) containing protease and phosphatase inhibitor mixtures (Sigma-Aldrich). The concentration of protein was measured with a protein assay kit (Promega). The cell lysates were separated by SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Tokyo, Japan). Immunoblotting was performed using Can Get Signal Immunoreaction Enhancer Solution (Toyobo) according to the manufacturer’s protocol. The Abs used were mouse anti-Flag Ab (1/5000; Sigma-Aldrich), rabbit anti-HA Ab (1/4000; Sigma-Aldrich), rabbit anti-BCR Ab (1/1000; Cell Signaling Technology), rabbit anti-Psen1 (1/2000; Cell Signaling Technology), rabbit anti-Na, K ATP-ase (1/2000; Cell Signaling Technology), goat anti–lamin B (1/4000; Santa Cruz Biotechnology), rabbit anti–phospho-CK2α (1/1000; Sigma-Aldrich), rabbit anti-CK2α (1/1000; Cell Signaling Technology), rabbit anti–phospho-BCR (1/2000; Cell Signaling Technology), mouse anti-tubulin (1/8000; Sigma-Aldrich), anti-mouse IgG HRP (1/5000; Southern Biotech), and anti-rabbit IgG HRP (1/10,000; Southern Biotech). The proteins were visualized by ECL (Chemi-Lumi One L, nacalai tesque) according to the manufacturer’s instructions.

WT and Psen1-deficient BC1 cells were stimulated with TNF-α for 5 min and washed three times with cold PBS. The cells were then fractionated into cytosol, membrane, and nuclear fractions using EzSubcell Extract (ATTO). Successful separation of these fractions was confirmed by immunoblotting with anti-tubulin, anti-Na/K–ATPase and anti–lamin B Abs.

Student t (two-tailed) and ANOVA tests were used for the statistical analysis. The p values <0.05 were considered significant.

In our previous study (13), shRNA-based genome-wide screening identified Psen1 as a regulatory gene of the inflammation amplifier. To confirm this result, we established Psen1-deficient cells using a mouse endothelial cell line and lentivirus carrying shRNA against Psen1 (Fig. 1A). The protein and mRNA levels of IL-6, which is a NF-κB target gene, were significantly decreased in Psen1-deficient cells stimulated with IL-6 and IL-17 or with TNF-α alone (Fig. 1B, 1C). The reduced expression of IL-6 was rescued by overexpression of Psen1, excluding the possibility of off-target effects by the shRNA (Fig. 1D). In contrast, cell viability was not significantly changed by the deficiency of Psen1 (Fig. 1B, 1D). Suppressing IL-6 mRNA expression by the transient knockdown of Psen1 using siRNA in H4 neuroglioma cells gave similar results. The mRNA levels of other NF-κB–dependent genes such as IκBα and Cxcl2 were also suppressed in Psen1-knockdown cells (Supplemental Fig. 1A, 1B). A γ-secretase inhibitor, Compound E, did not suppress IL-6 production (Supplemental Fig. 1C), suggesting the function of Psen1 in the NF-κB pathway does not significantly depend on γ-secretase activity. Next, to investigate whether Psen1 acts as a positive regulator of the NF-κB pathway in vivo, we employed cytokine-induced arthritis in F759 mice (7, 13, 14, 16). Lentivirus carrying Psen1 shRNA or p65 shRNA was injected into the ankle joints of F759 mice followed by injections of IL-6 and IL-17 (coactivation of STAT3 and NF-κB) into the ankle joints to induce NF-κB–mediated arthritis development. The arthritis score was significantly suppressed in the Psen1-knockdown and p65-knockdown (positive control) groups compared with the negative control group, which was injected with nontarget shRNA (Fig. 1E). These results suggest that Psen1 is a positive regulator of the NF-κB pathway in vitro and in vivo.

FIGURE 1.

Psen1 regulates the inflammation amplifier in vitro and in vivo. (A) Psen1 mRNA and protein levels in nontarget and Psen1-knockdown cells. (B and C) Psen1-deficient and control (nontarget) BC1 cells were stimulated with IL-6, IL-17, IL-6 plus IL-17, or TNF-α, and protein (B) and mRNA (C) levels for IL-6 were measured. (D) IL-6 production from Psen1-deficient and Nontarget cells with (Psen1) or without (mock) overexpression of Psen1. (E) Clinical arthritis scores of F759 mice after ankle joint injections of Psen1 or control shRNA followed by IL-6 plus IL-17 (cytokine) injections. Saline injections without cytokines did not induce arthritis. Closed squares indicate relative living cell numbers (right y-axis) to assess cytotoxicity by knockdown. Data represent the mean + SD (A–D) or SEM. (E) *p < 0.05, **p < 0.01.

FIGURE 1.

Psen1 regulates the inflammation amplifier in vitro and in vivo. (A) Psen1 mRNA and protein levels in nontarget and Psen1-knockdown cells. (B and C) Psen1-deficient and control (nontarget) BC1 cells were stimulated with IL-6, IL-17, IL-6 plus IL-17, or TNF-α, and protein (B) and mRNA (C) levels for IL-6 were measured. (D) IL-6 production from Psen1-deficient and Nontarget cells with (Psen1) or without (mock) overexpression of Psen1. (E) Clinical arthritis scores of F759 mice after ankle joint injections of Psen1 or control shRNA followed by IL-6 plus IL-17 (cytokine) injections. Saline injections without cytokines did not induce arthritis. Closed squares indicate relative living cell numbers (right y-axis) to assess cytotoxicity by knockdown. Data represent the mean + SD (A–D) or SEM. (E) *p < 0.05, **p < 0.01.

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The simultaneous activation of NF-κB and STAT3 is important for the inflammation amplifier (5, 6). To elucidate which of these signaling pathways is regulated by Psen1, the expression levels of target genes for the NF-κB or STAT3 pathways were examined. In addition to the suppression of IL-6 levels (Fig. 1A–C), the expression of Lcn2, which is another NF-κB target gene, was significantly suppressed in Psen1-deficient cells, whereas expression of STAT3 and SOCS3, which are targets of the STAT3 pathway, were unaffected (Fig. 2). These results suggest that Psen1 regulates the NF-κB pathway in nonimmune cells. Therefore, in the following experiments, we mainly used TNF-α for cell stimulation.

FIGURE 2.

Psen1 mainly regulates the NF-κB pathway. (AC) Psen1-deficient or control (nontarget) BC1 cells were stimulated with IL-6, IL-17, IL-6 plus IL-17, or TNF-α, and mRNA levels for Lcn2 (A), STAT3 (B), and SOCS3 (C) were measured. Data represent the mean + SD. **p < 0.01.

FIGURE 2.

Psen1 mainly regulates the NF-κB pathway. (AC) Psen1-deficient or control (nontarget) BC1 cells were stimulated with IL-6, IL-17, IL-6 plus IL-17, or TNF-α, and mRNA levels for Lcn2 (A), STAT3 (B), and SOCS3 (C) were measured. Data represent the mean + SD. **p < 0.01.

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Multiple events such as phosphorylation, nuclear translocation, and promoter binding are involved in NF-κB activation (31). We first investigated the nuclear translocation of NF-κB p65 in Psen1-deficient cells. The localization of endogenous p65 was examined by confocal microscopy before and after TNF-α stimulation. p65 nuclear translocation was not affected by Psen1 deficiency (Fig. 3A, 3B). We then investigated the nuclear events of NF-κB activation. Chromatin immunoprecipitation revealed that the occupancies of p65, p300, and acetylated H3K27 in the IL-6 and Lcn2 promoters were significantly reduced in Psen1-deficient cells (Fig. 3C–E). In addition, chromatin accessibility assessed by DNase I digestion was also reduced at these promoters (Fig. 3F). Consistently, luciferase activities were significantly increased by the forced expression of Psen1 in the presence of TNF-α (Fig. 3G). These results suggest that Psen1 is involved in a signaling pathway responsible for the binding of NF-κB p65 to target promoter regions.

FIGURE 3.

Psen1 deficiency impairs the promoter binding ability of NF-κB p65. (A) Psen1-knockdown or control (nontarget) cells were stimulated with TNF-α for 0, 15, and 30 min, and the localization of p65 (green) was observed by confocal microscopy. The nucleus was stained with Hoechst 33342 (purple). Representative images are shown. The images were taken using a 63× lens. (B) Quantitative analysis of (A). C > N, cells with more p65 localized in the cytoplasm than nucleus; C = N, cells with equal localization of p65 in the cytoplasm and nucleus; C < N, cells with more p65 localized in the nucleus than cytoplasm. (CE) p65 (C), p300 (D), and acetyl-H3K27 (E) recruitment to the IL-6 (left) or Lcn2 (right) promoter were assessed by chromatin immunoprecipitation in nontarget and Psen1-deficient BC1 cells stimulated with TNF-α for the indicated time periods. Chromatin immunoprecipitation values relative to 10% of input are shown. Relative occupancy by control IgG is shown in Supplemental Fig. 2. (F) Chromatin accessibility of the IL-6 (left) or Lcn2 (right) promoter was assessed in nontarget and Psen1-deficient BC1 cells with TNF-α stimulation at 0 and 60 min. (G) Luciferase assay using artificial tandem NF-κB binding elements (left) or IL-6 promoter (right) was performed in HEK293T cells with (Psen1) or without (mock) overexpression of Psen1 in the presence or absence of TNF-α stimulation. Data represent the mean + SD (C–G). *p < 0.05, **p < 0.01.

FIGURE 3.

Psen1 deficiency impairs the promoter binding ability of NF-κB p65. (A) Psen1-knockdown or control (nontarget) cells were stimulated with TNF-α for 0, 15, and 30 min, and the localization of p65 (green) was observed by confocal microscopy. The nucleus was stained with Hoechst 33342 (purple). Representative images are shown. The images were taken using a 63× lens. (B) Quantitative analysis of (A). C > N, cells with more p65 localized in the cytoplasm than nucleus; C = N, cells with equal localization of p65 in the cytoplasm and nucleus; C < N, cells with more p65 localized in the nucleus than cytoplasm. (CE) p65 (C), p300 (D), and acetyl-H3K27 (E) recruitment to the IL-6 (left) or Lcn2 (right) promoter were assessed by chromatin immunoprecipitation in nontarget and Psen1-deficient BC1 cells stimulated with TNF-α for the indicated time periods. Chromatin immunoprecipitation values relative to 10% of input are shown. Relative occupancy by control IgG is shown in Supplemental Fig. 2. (F) Chromatin accessibility of the IL-6 (left) or Lcn2 (right) promoter was assessed in nontarget and Psen1-deficient BC1 cells with TNF-α stimulation at 0 and 60 min. (G) Luciferase assay using artificial tandem NF-κB binding elements (left) or IL-6 promoter (right) was performed in HEK293T cells with (Psen1) or without (mock) overexpression of Psen1 in the presence or absence of TNF-α stimulation. Data represent the mean + SD (C–G). *p < 0.05, **p < 0.01.

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We recently reported a role for the BCR gene in NF-κB activation via its formation of a complex with CK2α (16). CK2α phosphorylates p65 at a specific serine residue, 529, which establishes a binding site for histone acetyltransferase p300 (16). In the absence of BCR, the transcription of NF-κB target genes, but not of STAT3 target genes, is repressed, and chromatin opening and NF-κB binding to the target promoter regions are significantly reduced in nonimmune cells, whereas the nuclear translocation of p65 remains intact (16). These phenotypes are similar to those observed in Psen1-deficient cells (Figs. 13). Therefore, we hypothesized that Psen1 is involved in the BCR–CK2α–p65 pathway. Consistent with this theory, the phosphorylation of p65 at serine 529 was attenuated in Psen1-deficient cells (Fig. 4A). In contrast, phosphorylation of p65 at serine 536, which is mediated by multiple kinases including IKK (32, 33), was normal upon cytokine stimulation (Fig. 4A). BCR and CK2α were phosphorylated followed by NF-κB activation after TNF-α stimulation (16). The phosphorylation states of BCR and CK2α were reduced in Psen1-deficient cells (Fig. 4B). Consistently, association of p65 with BCR, CK2α, and their phosphorylated forms at endogenous protein levels was at least slightly reduced in Psen1-deficient cells (Supplemental Fig. 3). Together, these results suggest that Psen1 is involved in the BCR–CK2α–p65 pathway.

FIGURE 4.

Psen1 is required for BCR–CK2α-mediated NF-κB p65 activation. (A) Nontarget control and Psen1-deficient BC1 cells were stimulated with TNF-α for 5 min, and the phosphorylation of p65 at serine 529 or serine 536 was detected by Western blotting after immunoprecipitation of p65. (B) Nontarget and Psen1 knockdown BC1 cells were stimulated with TNF-α for 0, 5, 15, and 30 min, and the phosphorylation of CK2α and BCR at Y177 was detected by Western blotting.

FIGURE 4.

Psen1 is required for BCR–CK2α-mediated NF-κB p65 activation. (A) Nontarget control and Psen1-deficient BC1 cells were stimulated with TNF-α for 5 min, and the phosphorylation of p65 at serine 529 or serine 536 was detected by Western blotting after immunoprecipitation of p65. (B) Nontarget and Psen1 knockdown BC1 cells were stimulated with TNF-α for 0, 5, 15, and 30 min, and the phosphorylation of CK2α and BCR at Y177 was detected by Western blotting.

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Based on our previous findings (16), trimolecular complexes that consist of BCR, CK2α, and p65 promote the phosphorylation of p65 at serine 529. Because Psen1 is a membrane protein and could be a new player in the BCR–CK2α–p65 pathway, we checked the interaction of Psen1 with these three subunits of the complex and their cellular localization. Consistent with Supplemental Fig. 3, coimmunoprecipitation assays revealed that Psen1 clearly associated with BCR and CK2α and to a lesser extent with p65 (Fig. 5A), suggesting the possibility that Psen1 directly binds to BCR and CK2α and most likely to p65 indirectly via BCR and CK2α. We then sought the binding regions of Psen1 to CK2α and BCR using two Psen1 mutants, in which the N-terminal region (aa 1–73) or a large part of the hydrophilic cytoplasmic loop (34) (aa 271–376) of Psen1 was deleted (Supplemental Fig. 4). The binding of Psen1 mutant Δ271–376 to CK2α and BCR was decreased compared with WT Psen1 (Fig. 5B, 5C), suggesting the importance of the hydrophilic cytoplasmic loop of Psen1 for the associations. We then prepared mutant molecules of CK2α and BCR (Supplemental Fig. 4) and examined their binding regions for Psen1. The N-terminal region of CK2α and the Rho/GEF domain of BCR were important for the association with Psen1 (Fig. 5D, 5E). These results indicate that the hydrophilic cytoplasmic loop of Psen1, Rho/GEF domain of BCR, and N-terminal domain of CK2α are critical regions for association with Psen1 and formation of the BCR–CK2α complex, which is required for p65 activation. Consistently, the association between p65 and BCR became weaker under Psen1 deficiency (Fig. 5F). To examine the cellular localization, we prepared membrane, cytosol, and nuclear fractions, and immunoblotted them for p65, BCR, and CK2α in control and Psen1-deficient cells. p65, BCR, and CK2α were detected in a Psen1-dependent manner in the membrane fraction even before TNF-α stimulation, and their localization was not significantly changed after TNF-α stimulation (Fig. 5G). These results suggest that the membrane protein Psen1 can serve as a scaffold for the complex formation and subsequent activation of the BCR–CK2α–p65 axis for NF-κB–mediated inflammation development.

FIGURE 5.

Psen1 associates with BCR and CK2α. (A) HEK293T cells overexpressing Flag–Psen1 were immunoprecipitated with Flag beads, followed by the detection of p65 (left), CK2α (center), or BCR (right) by Western blotting. In the case of CK2α detection, HA-tagged CK2α was co-overexpressed with Flag–Psen1, and anti-HA Ab was used for the immunoblotting. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (B) HEK293T cells co-overexpressing HA-CK2α and Flag-tagged WT Psen1 or its mutants were immunoprecipitated with Flag beads, followed by the detection of CK2α using anti-HA Ab. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (C) HEK293T cells overexpressing Flag-tagged WT Psen1 or Psen1 mutants were immunoprecipitated with Flag beads, followed by the detection of BCR using anti-BCR Ab. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (D) HEK293T cells co-overexpressing Flag-Psen1 and HA-tagged WT or N-terminal–deleted mutant of CK2α (ΔN-ter) were immunoprecipitated with Flag beads, followed by the detection of CK2α using anti-HA Ab. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (E) HEK293T cells co-overexpressing HA-Psen1 and Flag-tagged WT BCR or its mutants (oligomerization domain deletion [ΔOLI], putative serine/threonine kinase domain deletion [ΔS/T], and Rho/GEF domain deletion [ΔRho/GEF]) were immunoprecipitated with Flag beads, followed by the detection of Psen1 using anti-HA Ab. BCR expression levels detected by anti-Flag Ab are shown in the bottom. (F) Nontarget control and Psen1-deficient BC1 cells stimulated with TNF-α were immunoprecipitated with anti-p65 Ab, followed by immunoblotting for p65 (top) or BCR (bottom). (G) Nontarget control and Psen1-deficient BC1 cells were stimulated with or without TNF-α. Membrane, cytosol, and nuclear fractionations were prepared, and immunoblotting of p65, BCR, CK2α, tubulin (cytosolic marker), Na/K-ATPase (membrane marker), and lamin B (nuclear marker) were performed.

FIGURE 5.

Psen1 associates with BCR and CK2α. (A) HEK293T cells overexpressing Flag–Psen1 were immunoprecipitated with Flag beads, followed by the detection of p65 (left), CK2α (center), or BCR (right) by Western blotting. In the case of CK2α detection, HA-tagged CK2α was co-overexpressed with Flag–Psen1, and anti-HA Ab was used for the immunoblotting. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (B) HEK293T cells co-overexpressing HA-CK2α and Flag-tagged WT Psen1 or its mutants were immunoprecipitated with Flag beads, followed by the detection of CK2α using anti-HA Ab. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (C) HEK293T cells overexpressing Flag-tagged WT Psen1 or Psen1 mutants were immunoprecipitated with Flag beads, followed by the detection of BCR using anti-BCR Ab. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (D) HEK293T cells co-overexpressing Flag-Psen1 and HA-tagged WT or N-terminal–deleted mutant of CK2α (ΔN-ter) were immunoprecipitated with Flag beads, followed by the detection of CK2α using anti-HA Ab. Psen1 expression levels detected by anti-Flag Ab are shown in the bottom. (E) HEK293T cells co-overexpressing HA-Psen1 and Flag-tagged WT BCR or its mutants (oligomerization domain deletion [ΔOLI], putative serine/threonine kinase domain deletion [ΔS/T], and Rho/GEF domain deletion [ΔRho/GEF]) were immunoprecipitated with Flag beads, followed by the detection of Psen1 using anti-HA Ab. BCR expression levels detected by anti-Flag Ab are shown in the bottom. (F) Nontarget control and Psen1-deficient BC1 cells stimulated with TNF-α were immunoprecipitated with anti-p65 Ab, followed by immunoblotting for p65 (top) or BCR (bottom). (G) Nontarget control and Psen1-deficient BC1 cells were stimulated with or without TNF-α. Membrane, cytosol, and nuclear fractionations were prepared, and immunoblotting of p65, BCR, CK2α, tubulin (cytosolic marker), Na/K-ATPase (membrane marker), and lamin B (nuclear marker) were performed.

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Our previous study using genome-wide screenings identified Psen1 as a positive regulator of the inflammation amplifier, a molecular mechanism that hyperactivates NF-κB signaling in nonimmune cells (13). In the current study, we performed a mechanistic study on how Psen1 contributes to the activation of NF-κB. Our data indicate that Psen1 positively regulates NF-κB activation by participating in the BCR–CK2α–p65 pathway, which we recently reported (16). Indeed, Psen1-deficient cells phenocopied many aspects of BCR-deficient cells including 1) reduced p65 phosphorylation at serine 529, 2) impaired histone acetylation at H3K27 due to insufficient p300 accumulation at the promoter regions of NF-κB targets, 3) reduced promoter binding of p65, and 4) decreased levels of the transcription of NF-κB target genes. Importantly, Psen1 deficiency in joints suppressed cytokine-induced arthritis development in vivo. In contrast, p65 phosphorylation at serine 536, its nuclear translocation, and the transcription of STAT3 target genes were largely intact in Psen1-deficient cells just like in BCR-deficient cells (16). Therefore, we concluded that Psen1 is critical for BCR–CK2α-mediated p65 phosphorylation and subsequent inflammation development.

How does Psen1 contribute to the BCR–CK2α-mediated p65 phosphorylation? We found that Psen1 clearly interacts with BCR and CK2α via its hydrophilic cytoplasmic loop and that Psen1 deficiency abrogated the phosphorylation of BCR and CK2α, as well as the association of p65 with BCR. Based on these findings, we propose that Psen1 acts as a scaffold protein for the BCR–CK2α complex formation to phosphorylate p65 at serine 529, which allows NF-κB activation through a p300-mediated chromatin opening. Consistent with this notion, the role of Psen1 as a scaffold protein has been described during β-catenin phosphorylation (23, 24). Indeed, glycogen synthase kinase 3β and protein kinase A in complex with Psen1 facilitate the phosphorylation of β-catenin, which is required for the rapid turnover of β-catenin, preventing aberrant cell proliferation and tumorigenesis (23, 24). Our current study identified BCR, CK2α, and p65 as new scaffold partner proteins for Psen1 and found that Psen1 is required for NF-κB–induced inflammation development by facilitating the phosphorylation of p65 at serine 529.

Psen1 is known to be the catalytic component of γ-secretase enzyme, which cleaves APP to generate Aβ and has nine transmembrane domains with a large hydrophilic loop (34, 35). We showed that the scaffold role of Psen1 is dependent on the hydrophilic cytoplasmic loop but not on its γ-secretase enzyme activity, as a γ-secretase inhibitor, Compound E, did not inhibit NF-κB activation in nonimmune cells. Many mutations of Psen1 are found in familial AD (17, 18, 36), and some of them are present in the hydrophilic cytoplasmic loop (36). Because accumulating evidence indicates that chronic inflammation and proinflammatory cytokines such as IL-6 and TNF-α contribute to the pathogenesis of AD (1, 4, 37), it is possible that certain familial AD mutation(s) in the hydrophilic cytoplasmic loop of Psen1 have a gain-of-function effect on the BCR–CK2α complex formation and subsequent NF-κB–driven inflammation, thereby contributing to the pathogenesis of AD.

In summary, we identified a novel γ-secretase-independent role for Psen1 in the regulation of the NF-κB pathway. Psen1 acts as a scaffold for formation of the BCR, CK2α, and p65 complex, allowing efficient NF-κB activation. Our current study therefore suggests that the Psen1–CK2α–BCR cascade is a novel therapeutic target for various inflammatory diseases including AD.

We appreciate the excellent technical assistances provided by Ezawa and Nakayama and thank Fukumoto for her excellent assistance. We thank Dr. P. Karagiannis (Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan) for carefully reading the manuscript.

This work was supported by grants from KAKENHI (to D.K., Y.A., and M.M.), the Takeda Science Foundation (to M.M.), the Institute for Fermentation Osaka (to M.M.), the Mitsubishi Foundation (to M.M.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to D.K.), the Suzuken Memorial Foundation (to D.K. and Y.A.), the Japan Prize Foundation (to Y.A.), the Ono Medical Research Foundation (to Y.A.), the Kanzawa Medical Research Foundation (to Y.A.), the Kishimoto Foundation (to Y.A.), the Nagao Takeshi Research Foundation (to Y.A. and Y.T.), the Japan Multiple Sclerosis Society (to Y.A.), the Kanae Foundation (to Y.A.), The Uehara Memorial Foundation (to Y.A.), the Japan Brain Foundation (to Y.A.), The Kao Foundation for Arts and Sciences (to Y.A.), and the Tokyo Medical Research Foundation (to M.M. and Y.A.).

The online version of this article contains supplemental material.

Abbreviations used in this article:

amyloid-β

AD

Alzheimer disease

BCR

breakpoint cluster region

CK2α

α subunit of casein kinase II

HA

hemagglutinin

LCN2

lipocalin 2

Psen1

presenilin 1

shRNA

short hairpin RNA

siRNA

small interfering RNA

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data