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Ymer Acts as a Multifunctional Regulator in Nuclear Factor-κB and Fas Signaling Pathways

Molecular Medicine volume 18, pages 587–597 (2012)Cite this article

Abstract

The nuclear factor (NF)-κB family of transcription factors regulates diverse cellular functions, including inflammation, oncogenesis and apoptosis. It was reported that A20 plays a critical role in the termination of NF-κB signaling after activation. Previously, we showed that Ymer interacts and collaborates with A20, followed by degradation of receptor-interacting protein (RIP) and attenuation of NF-κB signaling. Here we show the function of Ymer in regulation of several signaling pathways including NF-κB on the basis of results obtained by using Ymer transgenic (Ymer Tg) mice. Ymer Tg mice exhibited impaired immune responses, including NF-κB and mitogen-activated protein kinase (MAPK) activation, cell proliferation and cytokine production, to tumor necrosis factor (TNF)-α, polyI:C or lipopolysaccharide (LPS) stimulation. Ymer Tg mice were more resistant to LPS-induced septic shock than wild-type mice. Transgene of Ymer inhibited the onset of glomerulonephritis in lpr/lpr mice as an autoimmune disease model. In contrast to the inflammatory immune response to LPS, Fas-mediated cell death was strongly induced in liver cells of Ymer Tg mice in which Ymer is abundantly expressed. These findings suggest that Ymer acts as a regulator downstream of several receptors and that Ymer functions as a positive or negative regulator in a signaling pathway-dependent manner.

Introduction

Nuclear factor (NF)-κB is a ubiquitously expressed transcription factor that induces the expression of target genes and regulates diverse cellular functions including inflammation, immunity, cell growth and apoptosis in response to various stimuli (1). NF-κB is an important molecule downstream of the tumor necrosis factor receptor (TNFR) and Toll-like receptor (TLR). Tumor necrosis factor (TNF)-α stimulation results in recruitment of receptor-interacting protein (RIP) to the intracellular region of TNFR, and then RIP undergoes lysine (K) 63-linked polyubiquitination by ubiquitin-conjugating enzeme 13 (Ubc13)/ubiquitin-conjugating enzyme E2 variant 1 (Uev1) A and TNFR-associated factor 2 (TRAF2) as E2 and E3, respectively. The K63-linked polyubiquitin chain on RIP activates transforming growth factor (TGF)-β-associated kinase (TAK)/TAK1 binding protein (TAB) kinase complex. Activated TAK/TAB complex phosphorylates and activates the IκB kinase (IKK) complex, which consists of IKKα, IKKβ and IKKγ/Nemo. Activated IKKβ phosphorylates IκB, and then phosphorylated IκB is recognized and ubiquitinated by a ubiquitin ligase complex, SCFFbw1 (Skp1-Cul1-F-box complex containing Fbw1), followed by degradation of IκB via a proteasome-dependent pathway and activation of NF-κB (2,3).

However, termination of NF-κB activation is usually required after transient activation, because sustained activation of NF-κB causes excess inflammatory cytokine secretion, by which systemic and lethal shock such as a cytokine storm may be induced. A unique intracellular enzyme, A20, is induced by NF-κB activation and plays an important role in attenuating NF-κB signaling (4). A20 hydrolyzes the K63-linked polyubiquitin chain on RIP and then conjugates the K48-linked polyubiquitin chain. K48-linked polyubiquitinated RIP is degraded by proteasome, resulting in termination of the NF-κB signal (5). Taken together, the results indicate that induction of A20 after activation of NF-κB functions as a negative feedback loop to return to a resting state. It was reported that A20 is a unique and key molecule that negatively regulates NF-κB signaling, as indicated by experiments using A20-disrupted mice, in which drastic systemic inflammation occurs a few weeks after birth followed by early death (4). We have shown that Ymer is a novel A20-interacting protein by yeast two-hybrid screening (6). Ymer is a cytoplasmic protein containing a coiled-coil region and two motifs interacting with ubiquitin (MIU) domains. Overexpression of Ymer diminishes NF-κB signaling, whereas knockdown of endogenous Ymer upregulates NF-κB signaling, suggesting that Ymer functions as a negative regulator for NF-κB signaling.

To examine the physiological function of Ymer in NF-κB signaling, we generated CAG promoter-driven Ymer transgenic (Ymer Tg) mice. Ymer Tg mice were viable and fertile, but they exhibited impaired immunoreactions and apoptosis in response to TNFα and lipopolysaccharide (LPS) stimulation in vitro and in vivo. In contrast to TNFα and LPS stimulation, Fas-mediated apoptosis of hepatocytes was greatly enhanced in Ymer-expressing Tg mice. These observations suggest that Ymer functions as a receptor-dependent multidirectional regulator that suppresses TNFR or TLR3/4-dependent NF-κB signaling but enhances Fas-dependent signaling.

Materials and Methods

Transgenic Mice

The BamHI/XhoI fragment of FLAG-tagged human Ymer cDNA from pCR-FLAG-Ymer was ligated into the BglII/XhoI sites of pCAGI-puro, and then the KpnI/NotI fragment from it was transferred into the KpnI/NotI sites of pCAGGS-p7 (7). The SalI/BamHI fragment (3.6 kb) from this plasmid was microinjected into the pronuclei of fertilized mouse zygotes derived from BDF1 (C57BL/6 × DBA) × BDF1 crosses. The injected zygotes were implanted into the uterus of pseudo-pregnant ICR mice, and the progeny were screened by polymerase chain reaction (PCR) with human Ymer-specific internal primers: forward, 5′-TTTTT AATGT TTGTA ATGAA AACCT TTATG-3′; reverse, 5′-GGTAT GGCTG AAGTC AGCAT CGACC AGTCC-3′. Transgenic mouse lines were propagated by sequential backcrossing to C57BL/6 mice. The F4-5 mice were used for the experiments at 6–8 wks of age. Southern blot analysis was performed to confirm genetic integration of the transgene with the XhoI/NotI fragment of the transgenic construct, which contains the full length of human Ymer cDNA. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8) and were approved by the animal care and use committee of Hokkaido University.

Cell Culture, Reagents, Transfection and Reporter Assay

Mouse thymocytes, splenocytes or macrophages were cultured in RPMI 1640 (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA, USA). Mouse embryonic fibroblasts (MEFs) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen). All of these primary cells were incubated under an atmosphere of 5% CO2 at 37°C. Luciferase reporter assay for NF-κB activities was carried out using the NAT reporter system (9). Briefly, wild-type (WT) and Ymer Tg MEFs (1 × 105 cells) were seeded into 24-well plates and were transfected with NAT-EGFP:Luc2 reporter plasmids (100 ng/well) and Renilla reporter gene plasmids (2 ng/well) with FuGENE HD Transfection Reagent (Roche, Mannheim, Germany). Twelve hours after transfection, cells were further stimulated for 12 h with TNFα (20 ng/mL; Sigma). The cells were then harvested and lysed with lysis buffer (100 µL). Luciferase activities were measured using 20 µL lysate and 100 luciferase assay substrates (Promega, Madison, WI, USA). The luminescence was quantified with a luminometer (Promega).

Flow Cytometry

Single splenocyte suspensions were stained with phycoerythrin-Cy5 (PE-Cy5)-conjugated anti-B220 (RA3-6B2; eBioscience, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-Mac-1a (M1/70; eBioscience) and PE-conjugated anti-T-cell receptor β (anti-TCRβ) (H57-597; eBioscience) antibodies (Abs). Purified macrophages or thymocytes were stained with Abs against PE-conjugated anti-TNFR1 (55R-286; BioLegend, San Diego, CA, USA), anti-TLR4 (MTS510; eBioscience) or anti-Fas (Jo2; BD Pharmingen, San Jose, CA, USA). All analyses were performed with a FAC-SCalibur flowcytometer and CellQuest software (Becton Dickinson, San Jose, CA, USA).

In Vitro or In Vivo Splenocyte Proliferation in Response to LPS or TNFα

Isolated splenocytes were stimulated with LPS (100 ng/mL) from Escherichia coli 055:B5 (Sigma), and cell proliferation was measured with CellTiter 96 Aqueous One Solution Reagent (Promega) and MULTISKAN JX (ThermoFisher, Waltham, MA, USA) at the indicated day according to the manufacturer’s protocol. Splenocyte proliferation in vivo was assessed by intraperitoneal injection with TNFα (50 µg/kg; Sigma) or LPS (100 µg/kg) in 0.9% saline. Three days after injection, TNFα or LPS-injected spleens were isolated and analyzed with a flowcytometer. Survival study against endotoxin shock was performed by intraperitoneal injection of LPS (50 mg/kg) in 0.9% saline.

Macrophage Purification and Stimulation

Three days after intraperitoneal injection of 3% thioglycollate, peritoneal macrophages were isolated from WT and Ymer Tg mice and then macrophages were seeded into a six-well plate (1 × 106 cells/well). One hour after culture, the plates with seeded cells were washed with phosphate-buffered saline (PBS) three times to remove cells other than macrophages. The purity of isolated macrophages was confirmed by flowcytometric analysis with anti-Mac-1 monoclonal antibody (Supplementary Figure 3). The percentages of isolated macrophages were >85%. Purified macrophages were stimulated with LPS (1 µg/mL; Sigma).

RNA Isolation, Reverse Transcription-PCR and Quantitative Real-Time PCR

Cells and tissues stimulated with LPS or polyI:C (high molecular weight [HMW], 50 µg/mL; Invitrogen) were homogenized in Isogen reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Reverse transcription-polymerase chain reactions (RT-PCRs) were performed in a total volume of 20 µL containing total RNA (400 ng) at 50°C for 30 min using Rever-Tra-Plus (Toyobo, Osaka, Japan). PCR was carried out as follows: 25–28 cycles of denaturalization at 94°C for 20 s, annealing at 60°C for 20 s and extension at 72°C for 30 s. The primer sequences were obtained from the PrimerBank database (https://doi.org/pga.mgh.harvard.edu/primer-bank) as follows: Ppia, forward: 5′-GAGCT GTTTG CAGAC AAAGT TC-3′; Ppia, reverse: 5′-CCCTG GCACA TGAAT CCTGG-3′; IL-1β, forward: 5′-GACAG TGATG AGAAT GACCT-3′; IL-1β, reverse: 5′-CTAAT GGGAA CGTCA CACAC C-3′; IL-6, forward: 5′-GAGAG GAGAC TTCAC AGAGG ATAC-3′; IL-6, reverse: 5′-GTACT CCAGA AGACC AGAGG-3′; TNFα, forward: 5′-GACGT GGAAC TGGCA GAAGA G-3′; TNFα, reverse: 5′-TTGGT GGTTT GTGAG TGTGA G-3′; interferon (IFN)-α, forward: 5′-CTTCC ACAGG ATCAC TGTGT ACCT-3′; IFN-α, reverse: 5′-TTCTG CTCTG ACCAC CTCCC-3′; inducible nitric oxide synthase (iNOS), forward: 5′-GTTCT CAGCC CAACA ATACA AGA-3′; and iNOS, reverse: 5′-GTGGA CGGGT CGATG TCAC-3′. Quantitative real-time PCRs and analyses were performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) using a StepOne Real-Time PCR System (Applied Biosystems).

Quantification of Secreted IL-6 by Enzyme-Linked Immunosorbent Assay

Sera from the mice treated with LPS or culture supernatant of LPS-stimulated macrophages were used for mouse IL-6 enzyme-linked immunosorbent assay (ELISA) Ready-Set-Go (eBioscience) and MULTISKAN JX according to the manufacturer’s protocol.

Fas-Induced Apoptosis and Histological Analysis

Apoptosis of thymocytes was induced with anti-Fas Ab (Jo2, 10 ng/mL). Briefly, thymocytes (1 × 107 cells) were stimulated with anti-Fas Ab for 5 min at 37°C, washed and then incubated for the indicated periods. Apoptosis of hepatocytes was induced by intraperitoneal injection of anti-Fas Ab (20 ng/mice). Four or two hours after injection, livers were isolated from two mice of each genotype and then used for hematoxylin-eosin (HE) staining or for immunoblot analysis, respectively. Livers freshly isolated from mice were fixed overnight in 10% formalin/PBS, dehydrated in methanol and embedded in paraffin. Serial sections (10 nm) were stained with HE and analyzed using BX51 and DP71 microscopic systems (Olympus, Tokyo, Japan).

Immunoblot Analysis

Cells or tissues isolated from mice were lysed with a solution containing 50 mmol/L Tris-HCl (pH 7.6), 300 mmol/L NaCl, 0.5% Triton X-100, aprotinin (10 µg/mL), leupeptin (10 µg/mL), 10 mmol/L iodoacetamide, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 0.4 mmol/L Na3VO4, 0.4 mmol/L EDTA, 10 mmol/L NaF and 10 mmol/L sodium pyrophosphate. The lysates were incubated on ice for 20 min and then centrifuged at 16,000g for 20 min at 4°C. After determination of protein concentrations with the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA), cell lysates (20 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 8% or 10% gel, and separated proteins were transferred to an Immobilon-P membrane (Millipore, Bedford, MA, USA). The membranes were probed with Abs for poly(ADP-ribose) polymerase (PARP) (9532; Cell Signaling Technology [CST], Danvers, MA, USA), phospho-IKK (2681; CST), IKK (2684; CST), phospho-JNK (c-Jun N-terminal kinase) (9251; CST), JNK (9252; CST), phospho-extracellular signal-regulated kinase (phospho-ERK) (9101; CST), ERK (9102; CST), phospho-p38 (9211; CST), p38 (9212; CST), FLAG (M5; Sigma), Ymer (6), α/β-tubulin (2148; CST), HSP70 (heat shock protein 70) (7; BD Pharmingen) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (6C5; Applied Biosystems). Immune complexes were detected with horseradish peroxidase-conjugated Abs to mouse or rabbit IgG (Promega) and enhanced chemiluminescence (GE Healthcare Bioscience, Piscataway, NJ, USA) or Immobilon Western reagents (Millipore).

Quantification of Urinary Protein

Ymer545 Tg mice, which are back-crossed with C57BL/6 mice five times, were further crossed with B6-lpr mice to obtain B6-lpr;Ymer Tg545 hybrid mice. Urinary protein from B6-lpr;Ymer Tg545 hybrid mice was measured every 2 wks using an albustick (Siemens Healthcare Diagnostics K.K., Tokyo, Japan) during 4–18 wks of age.

Histological Analysis of Glomerulonephritis

Tissues were dissected out at 7–8 months of age and fixed in 10% formalin for 24 h at 4°C. Paraffin sections (4 µm) were stained with the periodic acid Schiff (PAS) procedure and evaluated by light microscopy. The frequencies of inflammatory glomeruli, which exhibit the proliferation of mesangium cells (containing more than four cells in a mesangial region), were scored by observation from 20 glomeruli that were randomly selected in each kidney.

Statistical Analysis

The Student t test (Figures 1A, 2C, 3C, E and Supplementary Figure 4), Welch t test (Figures 1C, 2D, 4C) and Kaplan-Meier test (Figures 2B and 5A and Supplementary Figure 1D) were used to measure differences among samples.

Figure 1
figure 1

Impaired immune response of Ymer Tg mice to TNFα and LPS stimulations. (A) Splenocytes (1 × 106) isolated from WT and Ymer Tg mice were stimulated with LPS (100 ng/mL), and cell proliferation was measured by an MTS assay. (B) Four days after injection with TNFα (50 µg/kg) or LPS (100 µg/kg), spleens were dissected out from WT and Ymer Tg mice. (C) Cell numbers in LPS-stimulated spleen in Ymer Tg mice. Cell numbers of splenocytes in (B) were counted (saline-treated WT, n = 4; saline-treated Tg413, n = 4; saline-treated Tg545, n = 3; LPS-treated WT, n = 5; LPS-treated Tg413, n = 3; LPS-treated Tg545, n = 4). (D) Flowcytometric analysis of LPS-treated splenocytes in Ymer Tg mice. Splenocytes in (B) were analyzed by flowcytometry using anti-B220, anti-TCRβ and anti-Mac-1 Abs. Percentages of each population are indicated in the panels.

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Figure 2
figure 2

Resistance to septic shock in Ymer Tg mice. (A) Immunoblot analysis of Ymer Tg mice treated with TNFα or LPS. Three days after intraperitoneal injection with TNFα or LPS, cell lysates from the livers of WT and Ymer Tg mice were used for immunoblot analysis with Abs against PARP,RIP,IKK and P-IKK. (B) Survival study of Ymer Tg mice by LPS. WTand Ymer Tg mice were injected with LPS (50 mg/kg) and then observed for 24 h (WT, n = 4; Tg413, n = 4; Tg545, n = 5). (C) Production of inflammatory cytokines in Ymer Tg mice. Expression levels of IL-1β, IL-6, TNFα, IFNα and iNOS were measured by qRT-PCR at 4 h after LPS injection. (D) Secretion of IL-6 in sera of Ymer Tg mice. Four hours after LPS injection, IL-6 concentrations of sera from WTand Ymer Tg mice were measured by ELISA (WT, n = 3; Tg413, n = 4; Tg545, n = 5).

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Figure 3
figure 3

Impaired activation after treatment with LPS or TNFα in Ymer Tg mice. (A) Thioglycollate-induced peritoneal macrophages isolated from WT and Ymer Tg mice were stimulated with LPS (1 ng/mL) for the indicated times, and the cell lysates were analyzed by immunoblotting to detect the activation of IKK and MAPK. The percentages of isolated macrophages were >85%. (B) Cytokine production in thioglycollate-induced peritoneal macrophages from Ymer Tg mice after treatment with LPS. mRNA expression levels of cytokines (IL-6, TNFα and IL-1 β) were analyzed by reverse transcription-polymerase chain reactions (RT-PCRs). (C) IL-6 production by thioglycollate-induced peritoneal macrophages from Ymer Tg mice after treatment with LPS. ELISA was performed to measure IL-6 production in the culture supernatant at the indicated times. (D) MEFs isolated from WT and Ymer Tg mice were stimulated with TNFα (10 ng/mL) for the indicated times, and the cell lysates were analyzed by immunoblotting to detect the activation of IKK and JNK. (E) NF-κB luciferase reporter assay using MEFs from Ymer Tg mice. Eight hours after TNFα (10 ng/mL) stimulation, NF-κB-dependent transcriptional activities were measured.

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Figure 4
figure 4

Ymer inhibits glomerulonephritis in lpr/lpr mice. (A) The levels of urinary protein in B6-lpr/+;Ymer545, B6-lpr/lpr and B6-lpr/lpr;Ymer545 mice. Urinary proteins were measured during 4–18 wks of age (B6-lpr/+;Ymer545, n = 9; B6-lpr/lpr, n = 10; B6-lpr/lpr;Ymer545, n = 7). (B) Histochemical analysis of glomerulonephritis. Renal tissues were dissected out at 7–8 months of age and used for histochemical analysis with PAS staining. Arrows indicate the proliferation of mesangium cells in the glomerulus. Scale bars indicate 50 µm. (C) Quantification of glomerulonephritis in Ymer Tg mice crossed with lpr mice. The frequency of inflammatory glomerulus in which cells were proliferated in the mesangial region was quantified using B6-lpr;Ymer hybrid mice (B6-lpr/+;Ymer545, n = 7; B6-lpr/lpr, n = 8; B6-lpr/lpr;Ymer545, n = 5). Each dot indicates the score of glomerulonephritis obtained from individual mice. The Welch t test was used to measure differences among samples.

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Figure 5
figure 5

Acceleration of Fas-induced apoptosis in livers of Ymer Tg mice. (A) Survival study for Fas-induced cell death was performed with anti-Fas Ab (10 ng/mouse) intraperitoneal injection into WT and Ymer Tg mice (WT, n = 5; Tg413, n = 6; Tg545, n = 6). Survival of the mice was observed for 24 h after injection. Statistical analysis was performed by using the Kaplan-Meier test. (B) Livers treated with anti-Fas Ab were dissected out at 6 h after injection. (C) Livers treated with anti-Fas Ab were dissected out at 4 h after injection and used for histological analysis with HE staining. Scale bars indicate 100 nm in low magnification and 20 nm in high magnification (boxed). (D) Immunoblot analysis of apoptosis-related proteins in anti-Fas Ab-treated livers from Ymer Tg mice. Two hours after injection with anti-Fas Ab, cell lysates from livers were probed with the indicated Abs.

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All supplementary materials are available online at https://doi.org/www.molmed.org.

Results

Impaired Response of Splenocytes to TNFα and LPS Stimulations in Ymer Tg Mice

To examine the physiological function of Ymer, we established two strains of CAG promoter-driven Ymer Tg mice (Ymer Tg413 and Ymer Tg545) (Supplementary Figure 1). Immunoblot analysis with anti-Ymer and anti-FLAG Abs showed that the two stains have slightly different expression patterns. Tg545 mice highly expressed exogenous Ymer in the liver and macrophages, whereas Tg413 mice had low expression levels of exogenous Ymer in the liver and macrophages. In contrast, Tg413 mice highly expressed exogenous Ymer in MEFs (Figures 3A, 5D and Supplementary Figure 1).

We then examined the immune responses of splenocytes isolated from WT and Ymer Tg mice to LPS stimulation. Although Ymer Tg mice were healthy and displayed a normal lifespan and development of immune cells, including T cells, B cells and macrophages, splenocytes isolated from Ymer Tg mice exhibited impaired proliferation after LPS stimulation compared with WT splenocytes (Figure 1 and Supplementary Figures 1D, 2). Splenomegaly and increase in total splenocyte number were induced in WT mice by in vivo LPS administration (100 µg/kg), whereas splenomegaly and increase in total splenocyte number were inhibited in both lines of Ymer Tg mice (Figures 1B, C). Flowcytometric analysis showed that proliferation of splenic T-cell, B-cell and macrophage subsets in Ymer Tg mice was inhibited after injection of LPS, suggesting that excess expression of Ymer attenuates immune response via TLR4 to LPS in vivo (see Figure 1C). However, the ratio of cell number of each subset was not changed (Figure 1D).

Resistance to LPS-Induced Septic Shock in Ymer Tg Mice

It was reported that LPS or TNFα induces activation of caspase 3 in hepatocytes, followed by cleavage of poly(ADP-ribose) polymerase (PARP) and apoptosis (10,11). To examine whether cleavage of PARP after administration of LPS or TNFα is affected by overexpression of Ymer, immunoblot analysis with anti-PARP Abs was performed using Ymer Tg mice. The cleavage product of PARP at 50 kDa, which has been reported as evidence of necrotic cell death (12), was observed in WT mice with LPS or TNFα treatment, but cleavage products were not observed in either line of Ymer Tg mice (Figure 2A). These findings suggest that overexpression of Ymer causes decrease in the NF-κB signal via TLR4 or TNFR in hepatocytes as well as splenocytes. To confirm activation of NF-κB signaling in hepatocytes stimulated with LPS or TNFα, we examined the expression level of RIP and activation of IKKα/β in Ymer Tg mice. Stimulation with LPS or TNFα caused a decrease in expression level of RIP and phosphorylation of IKKα/β in hepatocytes of WT mice, whereas stimulation with LPS or TNFα did not cause a decrease in expression level of RIP and phosphorylation of IKKα/β in hepatocytes of Ymer Tg mice (see Figure 2A). It was reported that the expression level of RIP and phosphorylation of IKKα/β after NF-κB activation were inhibited by negative feedback machinery including A20 (4). Taken together, because NF-κB activation is inhibited in Ymer Tg mice, negative feedback to downregulate RIP may be suppressed. These findings suggest that overexpression of Ymer protein in splenocytes and hepatocytes causes impaired response via TNFR and TLR4 and that negative feedback after NF-κB activation is inhibited in Ymer Tg mice.

In addition to low-dose stimulation with LPS (50 µg/kg i.p.) (see Figure 2A), we also examined viability of Ymer Tg mice with a high dose of LPS (50 mg/kg i.p.). Statistical analysis showed that Ymer Tg545 mice were more resistant to LPS than WT mice (WT versus Tg514: P = 0.321; WT versus Tg545: P < 0.05) (Figure 2B). These findings suggest that Ymer Tg mice have resistance to LPS-induced septic shock. To clarify at the molecular level why Ymer Tg mice are resistant to LPS, mRNA levels of LPS-induced target genes including several proinflammatory cytokines and iNOS were examined by quantitative real-time polymerase chain reaction (qRT-PCR) (13,14). The gene expressions of IL-1 β, IL-6, TNFα and iNOS were downregulated in both Ymer Tg lines with LPS, whereas the expression of IFNα was upregulated in Ymer Tg mice (Figure 2C). In addition, we observed a similar effect of Ymer on cytokine production in polyI:C-stimulated splenocytes, suggesting that Ymer also regulates TLR3-mediated signal transduction (15,16) (Supplementary Figure 4). ELISA was also performed to confirm the levels of IL-6 in sera from LPS-treated mice, and the results showed that secretion of IL-6 in sera with LPS stimulation was inhibited in Ymer Tg mice (Figure 2D). These findings suggest that cytokine production by NF-κB activation was inhibited in Ymer Tg mice, and consequently LPS-induced septic shock was suppressed in Ymer Tg mice.

Impaired NF-κB Response of Macrophages in Ymer Tg Mice

Next, to determine whether downstream molecules are affected by LPS stimulation using macrophages from Ymer Tg mice, immunoblot analysis was performed with Abs to signaling molecules including the IKK and mitogen-activated protein kinase (MAPK) families. Immunoblot analysis showed that activations of p38 MAPK and JNK were attenuated in Tg545 mice, in which exogenous FLAG-Ymer is abundantly expressed (Figure 3A). Similar to MAPK signaling, IKK phosphorylation, which reflects activity of NF-κB signaling, was inhibited in Ymer Tg mice compared with that in WT mice. Taken together, the results indicate that activation of both MAPK and NF-κB signaling with LPS stimulation is suppressed in macro phages from Ymer Tg mice. To confirm suppression of NF-κB signaling by overexpression of Ymer, we examined the expression levels of NF-κB target genes by reverse transcription-polymerase chain reactions (RT-PCRs) and IL-6 production by ELISA using LPS-stimulated macrophages (Figures 3B, C). mRNA levels of several cytokines (IL-6, TNFα and IL-1β; Figure 3B)and the secretion of IL-6 protein in Ymer Tg mice, especially in Tg545 mice, were lower than those in WT mice (Figures 3C). We further analyzed activation of NF-κB signaling using MEFs derived from Ymer Tg mice. Activation of IKKα/β and JNK with TNFα was inhibited in MEFs from Ymer Tg413 mice, in which exogenous FLAG-Ymer is highly expressed, whereas activation of both IKKα/β and JNK were observed after TNFα stimulation in MEFs from WT and Tg545 mice (Figure 3D). To examine NF-κB-mediated transcription, a luciferase reporter assay for NF-κB signaling was performed using MEFs. The luciferase assay showed that NF-κB-mediated transcription with TNFα was suppressed in MEFs from Ymer Tg mice and that overexpression of Ymer suppressed NF-κB signaling in a dose-dependent manner (Figure 3E). These findings suggest that excess Ymer expression causes inhibition of downstream signaling and production of inflammatory cytokines.

Ymer Negatively Regulates Inflammations Induced in lpr/lpr Mice

We showed that Ymer inhibits inflammatory signaling including NF-κB with several stimulations. It is well known that lpr/lpr mice that lack Fas expression exhibit systemic lupus erythematosus-like autoimmune disease and that the inflammatory phenotypes induced by the lpr/lpr genotype are different in a genetic background-dependent manner (17). To evaluate the physiological function of Ymer in vivo, we introduced Ymer Tg545 transgene into B6-lpr/lpr mice; these mice exhibit more mild inflammation than MRL-lpr/lpr mice and show a slight increase in urinary protein level and glomerulonephritis in the kidney (17). B6-lpr/lpr mice exhibited increasing levels of proteinuria after 10 wks of age, as previously reported. However, the levels of urinary protein in B6-lpr/lpr;Ymer Tg545 mice were almost normal and similar to those of B6-lpr/+;Ymer Tg545 mice (Figure 4A). To confirm the inhibitory effect of Ymer on autoimmune-mediated inflammation, we performed histochemical analysis for renal inflammation using B6-lpr/lpr;Ymer Tg545 mice. Histochemical analysis with PAS staining showed that B6-lpr/lpr mice caused proliferation of cells in the mesangial region of the glomerulus, in which mesangial cell proliferation as glomerulonephritis is weaker than MRL-lpr/lpr mice, as previously reported (17). However, mesangial cell proliferation was not observed in the kidney from B6-lpr/lpr;Ymer Tg545 mice as well as B6-lpr/+;Ymer Tg545 mice (Figure 4B). Quantitative analysis showed that the frequency of mesangial proliferation in the glomerulus in B6-lpr/lpr;Ymer Tg545 mice was significantly lower than B6-lpr/lpr mice (Figure 4C). These results indicated that Ymer inhibits inflammation induced in lpr autoimmune model mice.

Ymer Enhances Fas-Mediated Apoptosis of Hepatocytes

We previously reported that Ymer interacts with RIP and accelerates degradation of RIP, which regulates the NF-κB signal, and others have reported that RIP functions as a downstream molecule of Fas, which regulates cell death (6,18). Although Figure 4 demonstrated that Ymer inhibits the inflammation induced in mice lacking the Fas-mediated signal, it is not clear whether Ymer affects the Fasmediated apoptotic pathway. It was reported that anti-Fas Ab administration induces drastic apoptosis in liver cells. Therefore, to examine the effect of Ymer in the Fas-mediated apoptotic pathway, we analyzed Fas-mediated liver injury (fulminant hepatitis) with anti-Fas Ab using Ymer Tg mice. Surprisingly, anti-Fas Ab killed Ymer Tg545 mice from 6 h after injection, whereas it killed both WT and Tg413 mice from 10 h (Figure 5A). The survival rate of Ymer Tg545 mice was lower than that of WT and Tg413 mice at 24 h after anti-Fas Ab treatment, but the difference was not statistically significant (WT versus Tg545: P = 0.227). Anti-Fas Ab treatment caused hemorrhage in the liver of Ymer Tg545 mice at 6 h after injection, but it did not cause liver hemorrhage in WT and Tg413 mice (Figure 5B). Histological analysis showed that anti-Fas Ab treatment induced partial apoptosis in the liver of WT and Ymer Tg413 mice at 4 h but that it caused massive apoptosis with hemorrhage in the liver of Tg545 mice (Figure 5C).

To determine whether apoptosis-related proteins in Ymer Tg545 mice are affected by anti-Fas Ab treatment, we performed immunoblot analysis with Abs against apoptosis-related proteins using lysates prepared from the liver at 2 h after anti-Fas Ab administration. Immunoblot analysis showed that exogenous FLAG-Ymer was highly expressed in the liver of Ymer Tg545 mice (Supplementary Figure 1C), and reduction of the intact form of PARP (116 kDa) and increase in the cleaved form of PARP (89 kDa) as apoptotic markers were observed in Ymer Tg545 mice with anti-Fas Ab treatment. Only a slight increase in cleaved products was observed in WT and Tg413 mice, suggesting that Tg545 mice are sensitive to Fas-mediated apoptosis (Figure 5D) (10). However, these results were not consistent with results presented in Figure 2A, showing TNFα-and LPS-induced cell death, suggesting that the function of Ymer in Fas signaling is different from that in TNFR or TLR4. To clarify the effect of Ymer on Fas-mediated apoptosis, we also tested caspase-independent apoptotic signaling through RIP or JNK. Immunoblot analysis showed reduction of RIP in Ymer Tg545 mice (Figure 5D). It has been reported that caspase 8 and caspase 10 bind to and cleave RIP (19). These findings indicate that overexpression of Ymer causes activation of caspase-mediated apoptotic cascades in hepatocytes by anti-Fas Ab treatment. Immunoblot analysis showed that JNK in Ymer Tg545 mice was activated more than that in WT or Tg413 mice. It is known that Fas-induced JNK activation through the Fas-Daxx-Ask1-JNK pathway leads the Bcl2 phosphorylation and consequently causes apoptosis, suggesting that excessive activation of JNK in Tg545 mice may reduce the antiapoptotic activity of Bcl2 (20,21). Taken together, these results indicate that abundant expression of Ymer may enhance sensitivity to Fas-mediated apoptosis in the liver via caspase and JNK activation.

Ymer Maintains Long-Term Activation of NF-κB Signaling in Thymocytes

We showed that Ymer accelerates apoptotic cell death and that JNK is activated after Fas stimulation in hepatocytes from Ymer Tg mice (Figure 5). It was previously reported that NF-κB signaling is required for survival of hepatocytes (22–24). Considering these findings, we hypothesized that attenuation of NF-κB signaling activity by Ymer causes acceleration of Fas-mediated hepatocyte apoptosis in Ymer Tg mice. To confirm this hypothesis, we examined Fasmediated activation of NF-κB signaling in thymocytes of WT and Ymer Tg545 mice. Immunoblot analysis showed increased cleavage of PARP in Ymer Tg545 mice compared with that in WT mice with anti-Fas Ab treatment, indicating that thymocytes in Ymer Tg545 mice are also sensitive to anti-Fas Ab treatment (Figures 6A, B). Transient phosphorylation of IKKα/β was observed between 10 and 40 min and was then attenuated after 160 min in WT mice. However, phosphorylation of IKKα/β was maintained for a long time (>160 min) in Ymer Tg545 mice (Figure 6C). Immunoblot analysis showed that the phosphorylation level of IKKα/β in Ymer Tg545 mice was already higher than that in WT mice in a resting state and that IKKα/β was quickly and robustly activated in Ymer Tg545 mice after Fas stimulation. These findings indicate that attenuation of NF-κB signaling activity is not the reason for acceleration of Fas-mediated apoptosis in Ymer Tg mice and that Ymer may directly affect Fas-mediated apoptotic signal pathways.

Figure 6
figure 6

Ymer accelerates the caspase-dependent signal and maintains long-term activation of NF-κB signaling in Fas-stimulated thymocytes. (A) Thymocytes isolated from WT and Ymer545 Tg mice were stimulated with anti-Fas Ab (10 µg/mL) and then probed with the indicated Abs to detect the activation of downstream signaling after stimulation. (B) Time course of cleaved form of PARP in thymocytes from WT and Ymer545 Tg mice after anti-Fas Ab treatment. A relative amount of the cleaved form of PARP at each time in (A) is shown. The value of the chase period at 0 h was defined as 1. (C) Time course of phosphorylated IKKα/β in thymocytes from WT and Ymer545 Tg mice after anti-Fas Ab treatment. Relative amount of phosphorylated IKKα/β at each time in (A) is shown. The value of the chase period at 0 h was defined as 1. (D) Model for biological roles of Ymer. Ymer may have multi-functions for several signaling pathways including TLR4, TNFR and Fas. Ymer attenuates NF-κB signaling downstream of TLR4 and TNFR, whereas it accelerates LPS-induced IFN production and Fas-induced NF-κB and JNK activation for apoptotic cell death.

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Discussion

In this study, to clarify the physiological function of Ymer, we established transgenic mouse strains expressing Ymer. Ymer Tg mice showed attenuated activation of NF-κB signaling in response to TNFα, polyI:C and LPS stimulation, similar to results of in vitro experiments showing that Ymer enhances RIP degradation with A20, followed by suppression of NF-κB signaling. This study also showed that abundant Ymer expression enhances Fas-mediated JNK phosphorylation and LPS- and polyI:C-induced IFN production and prolongs Fas-induced IKK activation. These findings are in contrast to the results obtained by TNFα-, polyI:C- or LPS-induced NF-κB activation. Ymer may serve as an activator for the Fas-mediated signaling pathway, whereas it functions as a negative regulator for the TNFα- or LPS-mediated signaling pathway (Figure 6D). Hence, it is important to clarify how Ymer functions as a multifunctional signal regulator downstream of different receptors.

It has been reported that endocytosis of receptors is important to regulate signal activation and that low-temperature treatment of HeLa cells suppressed internalization of TNFR, resulting in inhibition of NF-κB signaling (25). In addition to this report, monodansylcadaverin, a transglutaminase inhibitor, was shown to prevent endocytosis and suppress TNFα-dependent cell death in U937 cells (26). Others have reported that cytoplasmic translocation of the assembled signaling complex from the cell surface is required for activation of cytokine receptor signaling (27). These findings suggest that internalization of TNFR after TNFα binding is required for the activation of signals including NF-κB signaling. Furthermore, internalization of Fas or TNFR causes activation of the caspase cascade and sequentially apoptotic cell death (28–30). Taken together, the results suggest that Ymer functions as a general regulator for receptor internalization and that impaired endocytosis of receptors by abundant Ymer expression causes inhibition of NF-κB signaling via TNFR and TLR4 and causes activation of NF-κB and JNK via Fas signaling.

It was reported that Ymer interacts with epidermal growth factor receptor (EGFR) to downregulate the receptor localization on the cell surface, suggesting that Ymer inhibits internalization and degradation of EGFR after EGF stimulation (31). It was also reported that an E3 ubiquitin ligase, Cbl, ubiquitinates the cytoplasmic domain of EGFR in a phosphorylation-dependent manner and regulates EGFR internalization (32,33) and that E2 ubiquitin-conjugating enzymes Ubc4/5 and Cbl have the ability to conjugate the K63-linked polyubiquitin chain, suggesting that the K63-linked polyubiquitin chain regulates internalization of EGFR (34–36). Moreover, we showed that Ymer selectively interacts with K63-linked polyubiquitin (6). Taken together, the results suggest that Ymer likely regulates internalization of receptors via K63-linked ubiquitination for EGFR, TNFR, TLR4 and Fas. However, flowcytometric analysis using an antibody to each receptor showed that there was no difference in receptor internalization of TLR4, TNFR or Fas between Ymer Tg and WT mice, suggesting that Ymer does not affect receptor internalization and internalization-dependent switching of signaling pathways (Supplementary Figure 5). These findings suggest that Ymer functions at a stage downstream of receptor internalization after stimulus and upstream of IKKα/β activation.

We showed that activation of NF-κB is inhibited in Ymer Tg mice, whereas IFN expression is induced by LPS in Ymer Tg mice. It was reported that LPS-induced activation of macrophages is regulated by two different signaling mechanisms: one is Myd88-dependent NF-κB and MAPK activation and the other is TRIF-dependent IFN induction via TANK-binding kinase (TBK)/IKKε and interferon regulatory factor 3 (IRF3) (37–39). We examined whether Ymer overexpression affects polyI:C-induced IFNα expression through TLR3, which does not recruit MyD88 but depends solely on TRIF. Our results showed that IFNα induction was enhanced in Ymer Tg mice with polyI:C stimulation, suggesting that Ymer affects the TRIF-dependent pathway via a mechanism different from that in Myd88-dependent signaling.

It is important to clarify how Ymer accelerates IKK activation and JNK phosphorylation via Fas-mediated signaling. Our results indicate that Ymer may have reciprocal roles between TNFR and Fas. It was reported that Fas signaling involves RIP to activate NF-κB and that RIP is independent of Fas-mediated signaling (40,41). Therefore, although Ymer interacts and inhibits the function of RIP, it is not clear whether Ymer affects Fasmediated signals via modulation of RIP.

Conclusion

In summary, our results indicate that Ymer is a novel multifunctional signal regulator in several signaling pathways for inflammation, cell death and proliferation. It is important to determine the functions of Ymer and the binding partners to Ymer in each signaling pathway in the future. Therefore, a genetic approach using knockout mice is needed to clarify physiological functions of Ymer. Further analysis of Ymer may prove to provide therapeutic benefits not only for the suppression in inflammation and autoimmune diseases but also for the inhibition of growth of cancer cells.

Disclosure

The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.

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Acknowledgments

We thank Jun Miyazaki for the plasmids, Akihiro Ishizu for histological analysis and Yuri Soida for helping prepare the manuscript.

This work was supported in part by KAKENHI from the Ministry of Education, Culture, Sports, Science and Technology (18076001, 21390087 to S Hatakeyama).

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  1. Department of Biochemistry, Hokkaido University Graduate School of Medicine, Kita15, Nishi7, Kita-ku, Sapporo, 060-8638, Japan

    Tadasuke Tsukiyama, Mayuko Matsuda-Tsukiyama, Miyuki Bohgaki, Sayuri Terai & Shigetsugu Hatakeyama

  2. Department of Pathology, Hokkaido University Graduate School of Medicine, Sapporo, Hokkaido, Japan

    Shinya Tanaka

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Tsukiyama, T., Matsuda-Tsukiyama, M., Bohgaki, M. et al. Ymer Acts as a Multifunctional Regulator in Nuclear Factor-κB and Fas Signaling Pathways. Mol Med 18, 587–597 (2012). https://doi.org/10.2119/molmed.2011.00435

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