The key functional molecules involved in inflammatory bowel disease (IBD) and IBD-induced colorectal tumorigenesis remain unclear. In this study, we found that the apoptosis repressor with caspase recruitment domain (ARC) protein plays critical roles in IBD. ARC-deficient mice exhibited substantially higher susceptibility to dextran sulfate sodium (DSS)-induced IBD compared with wild-type mice. The inflammatory burden induced in ARC-deficient conditions was inversely correlated with CCL5 and CXCL5 levels in immune cells, especially CD4-positive T cells. Pathologically, ARC expression in immune cells was significantly decreased in clinical biopsy specimens from patients with IBD compared with normal subjects. In addition, ARC levels inversely correlated with CCL5 and CXCL5 levels in human biopsy specimens. ARC interacted with TNF receptor associated factor (TRAF) 6, regulating ubiquitination of TRAF6, which was associated with NF-κB signaling. Importantly, we identified a novel ubiquitination site at lysine 461, which was critical in the function of ARC in IBD. ARC played a critical role in IBD and IBD-associated colon cancer in a bone marrow transplantation model and azoxymethane/DSS-induced colitis cancer mouse models. Overall, these findings reveal that ARC is critically involved in the maintenance of intestinal homeostasis and protection against IBD through its ubiquitination of TRAF6 and subsequent modulation of NF-κB activation in T cells.

Significance:

This study uncovers a crucial role of ARC in the immune system and IBD, giving rise to a novel strategy for IBD and IBD-associated colon cancer therapy.

Inflammatory bowel disease (IBD), which includes ulcerative colitis and Crohn disease, is a chronic inflammatory disease of the gastrointestinal tract (1, 2). IBD can cause various complications, such as abscesses, fistulas, colitis-associated neoplasias, and cancer (3). Approximately 1.6 million patients suffer from the disease in the United States and 2.5–3 million in Europe (4, 5). IBD is associated with an immunologic imbalance of the intestinal mucosa, mainly related to cells in the adaptive immune system leading to chronic inflammation conditions in patients. The pathophysiologic mechanisms of IBD are still not clear, even though these diseases were discovered several decades ago (6–8).

The protein apoptosis repressor with caspase recruitment domain (ARC), also referred to as Nol3, plays an important role in suppressing apoptotic responses (9, 10). ARC was believed to exert its function through multiple protein–protein interactions and transcriptional regulation (11). ARC was primarily discovered as an endogenous inhibitor of cell death and is highly expressed in cardiomyocytes, skeletal muscle cells, and neurons under physiologic conditions (12–14). It was independently identified in later studies as having other functions, including posttranslational modifications like phosphorylation, calcium binding, and ubiquitination (15, 16). ARC has increased expression in solid tumors and in patients with acute myeloid leukemia to mediate the response of cells to the induction of pharmacologic apoptosis (17–20). Intriguingly, studies also revealed that ARC might perform its function as a tumor suppressor in renal cell carcinoma cells and myeloid tumors (21, 22). These findings suggest dual roles for ARC in oncogenesis that may be cell type dependent. The CARD family participates in the regulation of apoptosis, inflammation, and NF-κB signaling pathway activation. However, the function of ARC in the inflammation reaction is still not clear (23, 24). Recently, ARC was shown to play a pivotal role in the pathogenesis of acute kidney injury and ARC knockout (KO) markedly accelerated the expression levels of inflammatory factors (25). Moreover, ARC has been reported to suppress NF-κB pathway activation and to interact directly with p53 to disrupt its transcriptional activity (23, 26). These findings suggest that ARC plays potential roles in the inflammatory response and cancer development. In addition, ARC is highly expressed in almost all primary colon cancers compared with corresponding controls, suggesting that ARC is a novel marker for human colon cancer (27). However, the function of ARC in IBD and colon cancer has not yet been assessed.

In this study, we examined the function of ARC in IBD and IBD-associated colon cancer. We demonstrated a role for ARC in regulation of inflammatory response in an ARC-deficient mouse model and clinical biopsy specimens. We identified ARC as a protector for IBD in immune cells and also aimed to ascertain the mechanisms of ARC related to the inflammatory response in IBD development. Furthermore, the bone marrow transplantation and azoxymethane/dextran sulfate sodium (DSS) mouse models were used to study the effect of ARC on IBD and IBD-associated colorectal tumorigenesis. Our findings uncover a critical role for ARC in IBD development and give rise to a potential new strategy for IBD therapy.

Reagents and antibodies

Cell culture media, gentamicin, penicillin, and l-glutamine were all obtained from Invitrogen. FBS was from Gemini Bio-Products and Tris, NaCl, and SDS for molecular biology and buffer preparation were purchased from Sigma-Aldrich. Antibodies to detect β-actin (sc-47778), NF-κB (p50) (sc-7178), Lamin B (sc-6216), CD4 (sc-13573), ARC (sc-11435), ENA-78 (sc-377026), RANTES (sc-514019), TRAF1 (sc-271683), TRAF4 (sc-10776), TRAF5 (sc-74502), and TRAF6 (sc-8409) were from Santa Cruz Biotechnology, Inc. His-HRP (R94125) was from Invitrogen. Anti-HA (901503) was obtained from Covance and anti-HA-HRP (12013819001) was purchased from Roche. Anti-Flag (F-3165) was from Millipore Sigma. Anti-CCL5 (ab9679) and anti-CXCL5 (ab9802) were purchased from Abcam. NF-κB (p65) (#3034), TRAF2 (#14712), PE-ARC (#89210), and TRAF3 (#4729T) antibodies were purchased from Cell Signaling Technology. PE-CCL5 (149104), FITC-CD4 (557307), FITC-CD8 (553030), FITC-CD49b (561067), and FITC-CD19 (557398) were purchased from BioLegend.

Construction of expression vectors

Expression constructs, including ARC and HA-Lys-63-ubiquitin, were obtained from Addgene. The packaging vector, pcDNA4/His Max Vector, was obtained from Invitrogen. To construct His-tagged expression vector of ARC, we amplified the DNA sequences corresponding to HA-ARC by PCR and the HotStarTaq Master Mix Kit (Qiagen) was used. We designed specific primers for His-ARC, forward: 5′AT GGATCC ATGGGCAACGCGCAGGAG-3′ and reverse, 5′AT CTCGAG CTATCCAGCATGGGCGGG-3′. The His-ARC PCR product was digested with BamHI/XhoI following the instructions provided by the manufacturer, and then inserted into the corresponding sites of pcDNA4/His Max (Invitrogen) to generate the expression plasmids encoding His-ARC.

In addition, the lentivirus plasmids shARC (#1 V2LHS_47055 and #2 V2LHS_47054) were purchased from GE Healthcare Dharmacon (OpenBioSystem). The pLKO.1-puro Non-Target shRNA Control Plasmid DNA (shNT) was purchased from Sigma-Aldrich Co. LLC. All constructs were confirmed by restriction enzyme mapping, DNA sequencing, and Blast.

Cell culture and transfection

All cells were purchased from the ATCC. The cells were routinely screened to confirm Mycoplasma-negative status and to verify the identity of the cells by short tandem repeat profiling before being frozen. Each vial was thawed and maintained for a maximum of 2 months. Enough frozen vials of each cell line were available to ensure that all cell-based experiments were conducted on cells that had been tested and in culture for 8 weeks or less. Cells were cultured at 37°C in a 5% CO2 humidified incubator following the ATCC protocols. Jurkat and MOLT3 cells were grown in RPMI1640 medium supplemented with 10% FBS and 1% antibiotics. HEK293T cells (stably expressing the SV40 large T antigen in HER293 cells) were purchased from the ATCC and cultured at 37°C in a humidified incubator with 5% CO2 in DMEM (Corning) supplemented with 10% FBS (Corning) and 1% penicillin–streptomycin (Gen DEPOT). When cells reached 60% confluence, transfection was performed using iMFectin DNA Transfection Reagent (GenDEPOT) following the manufacturer's instructions. The cells were cultured for 36–48 hours and proteins were extracted for further analysis.

Immunofluorescence, immunoprecipitation, and Western blot analysis

Protein samples were extracted with Nonidet P-40 lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.5% Nonidet P-40, and protease inhibitor mixture). For immunoblotting, 40 μg of proteins were detected with specific antibodies and an alkaline phosphatase–conjugated secondary antibody. Proteins were visualized by chemiluminescence (Amersham Biosciences). The immunoprecipitation assay was performed as described previously (28). The extractions were precleared with 10 μL Protein G Agarose Beads (GenDEPOT) by rocking for 30 minutes at 4°C. The precleared supernatant fractions were combined with fresh Protein A/G Agarose Beads (Santa Cruz Biotechnology) and appropriate 2 μg of antibodies were added and rocked overnight at 4°C. The immunoprecipitates were washed four times with the above lysis buffer. Immunoprecipitates were suspended in SDS sample buffer and subjected to SDS-PAGE and Western blotting. For immunoprecipitation under denaturing conditions, proteins were extracted using regular immunoprecipitation lysis buffer plus 1% SDS and heated at 95°C for 5 minutes. Samples were diluted 10 times by using regular immunoprecipitation lysis buffer before immunoprecipitation. The beads were washed, mixed with SDS sample buffer, boiled, and then resolved by SDS-PAGE. Signals were visualized by immunoblotting.

Equal amounts of protein were determined by using a Protein Assay Kit (Bio-Rad Laboratories). Lysates were resolved by SDS-PAGE and then transferred onto Polyvinylidene Difluoride Membranes (Millipore) and blocked with 5% nonfat milk for 1 hour at room temperature. Blots were probed with appropriate primary antibodies (1:1,000) overnight at 4°C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000) for hybridization. Protein bands were visualized with a Chemiluminescence Reagent (GE Healthcare Biosciences).

Inflammation array

Colon tissue lysates from DSS-induced mice were subjected to a Mouse Inflammation Array C1 (RayBiotech) following the manufacturer's instructions. Density scores were obtained using ImageJ software.

Measurement of ARC, CCL5, and CXCL5 in clinical human samples

The leukocytes (buffy coat) and plasma samples with EDTA from normal subjects or Crohn disease or ulcerative colitis patient samples were obtained after informed consent as Mayo Clinic Institutional Review Board (Rochester, MN) protocol #622-00.The baseline demographics are shown in Supplementary Table S1. The measurement of ARC, CCL5, and CXCL5 was performed using Immunoassay Kits (ARC: MBS95000636; CCL5: MBS773187; and CXCL5: MBD825081) from MyBioSource following the manufacturer's instructions.

Ubiquitination assays

The ubiquitination assay was performed as described previously (28, 29). HEK293T cells were transfected with combinations of expression vectors Flag-TRAF6, His-ARC, and HA-Lys63-ubiquitin. The proteins were extracted using lysis buffer (50 mmol/L Tris-HCl, pH 8.0, 0.5 mmol/L EDTA, 1% SDS, and 1 mmol/L dithiothreitol) and boiled for 10 minutes before cellular debris were removed by centrifugation. The lysates were immunoprecipitated as described above, and immunoprecipitation was conducted with anti-Flag. Bound proteins were eluted in SDS sample loading buffer and subjected to immunoblotting.

For the in vitro ubiquitination assay (30), Flag-TRAF6 was cotransfected with HA-ubiquitin into HEK293T cells. At 24 hours later, the transfected cells were stimulated with IL1β for 15 minutes. The cells were harvested using RIPA buffer (100 mmol/L Tris/HCl pH 7.4, 30 mmol/L NaCl, 2.5% sodium deoxycholate, 2 mmol/L EDTA, and 2% nonidet P40) containing protease inhibitor cocktail and N-ethylmaleimide. The cell lysates were incubated with Flag M2 beads overnight with rotation at 4°C. After extensive washing with TBS (25 mmol/L Tris/HCl, pH 7.4, 150 mmol/L NaCl, and 3 mmol/L KCl), Flag-TRAF6 was eluted with elution buffer (1 × PBS, pH 7.4) containing 3 × Flag peptide. Then, 15 μL of the eluted Flag-TRAF6 was incubated with purified His-ARC in DUB assay buffer (50 mmol/L HEPEs/NaOH, pH 8.0, 10% glycerol, and 3 mmol/L dithiothreitol) at 37°C for 4 hours. Ubiquitination was analyzed by Western blotting using anti-His, anti-Flag, or anti-HA.

Liquid chromatography/mass spectrometry-mass spectrometry analysis to identify ubiquitination sites of TRAF6

Flag-TRAF6 was coexpressed, with or without His-ARC, along with HA-Ub-K63 into HEK293T cells. After cell lysis, samples were immunoprecipitated with anti-Flag, then denatured with urea buffer [7 mol/L urea, 2 mol/L thiourea, 2% 3-[(3-cholamidopropyl dimethylammonio)] propanesulfonate], reduced with 4 mmol/L dithiothreitol for 1 hour at 37°C, and alkylated with 14 mmol/L iodoacetamide for 45 minutes at room temperature under dark conditions. Excess iodoacetamide was quenched with excess dithiothreitol to provide a final concentration of 7 mmol/L. Subsequently, the sample was diluted with 25 mmol/L ammonium bicarbonate to ensure less than 1 mol/L urea content, and digested with trypsin (Promega) at an enzyme content of 2% (w/w) for 16 hours at 37°C. The tryptic peptides were dried by vacuum evaporation using a speed vacuum and then cleaned up with a Sep-Pak C18 Cartridge (Waters). The Sciex TripleTOF 5600 System (Sciex) coupled with Eksigent 1D+ Nano LC System (Sciex) was used to identify TRAF6 ubiquitination. The mass spectrometry was automatically calibrated by acquisition of β-galactosidase peptides (25 fmole/μL) in the same acquisition batch. The raw data were processed and searched with ProteinPliot Software (version 4.5; Sciex) using the Paragon algorithm. Proteins were identified by searching the UniProtKB human database (www.uniprot.org).

qRT-PCR

TRizol Reagent (Invitrogen) was used for total RNA extraction from mouse colon tissue and Jurkat and MOLT3 cells. The CCL5 and CXCL5 genes expression was analyzed with 100 ng of total RNA. Primers included mouse CCL5-specific real-time primers: F: 5′CCATGAAGGTCTCCGCGGCAC-3′; R: 5′-CCTAGCTCATCTCCAAAGAG-3′; mouse CXCL5-specific real-time primers: F: 5′-TGGCCCCTTTCACAGAGTAG-3′; R: 5′-CTAAAAACCCGACAGGCATC-3′; and mouse glyceraldehyde 3-phosphate dehydrogenase–specific real-timer primers: F: 5′-CTTCACCACCATGGAGGAGGC-3′; R: 5′-GGCATGGACTGTGGTCATGAG-3′; human CCL5-specific real-time primers: F: 5′-GCTGTCATCCTCATTGCTACTG-3′; R: 5′-TGGTGTAGAAATACTCCTTGATGTG-3′; human CXCL5-specific real-time primers: F: 5′-TGGACGGTGGAAACAAGG-3′; R: 5′-CTTCCCTGGGTTCAGAGAC-3′; and human a glyceraldehyde 3-phosphate dehydrogenase–specific real-timer primers: F: 5′-GCCCAATACGACCAAATCC-3′; R: 5′-CTCTGCTCCTCCTGTTCGAC-3′. Quantitative one-step real-time PCR using the TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems) was performed following the manufacturer's suggested protocols. The CT values of CCL5 and CXCL5 genes expression were normalized with the CT values of glyceraldehyde 3-phosphate dehydrogenase as an internal control to monitor equal RNA utilization.

Flow cytometry analysis

Single-immune cell populations in spleens were cultured with PMA (5 ng/mL, Sigma-Aldrich) for 6 hours, and then harvested for surface and intracellular staining. In addition, single-immune cell populations in spleens were isolated from 7 days DSS-treated or vehicle-treated C57BL/6 mice. Flow cytometry data were collected with a FACSCalibur Flow Cytometer (BD Biosciences). Data analyses were performed with FlowJo Software (Tree Star). The antibodies used for cell staining included anti-CD4, anti-CD8, anti-CD19 anti-CD49b, anti-CCL5, and anti-ARC.

Protein–protein docking of ARC and TRAF6

First, the three-dimensional (3-D) structures of ARC and TRAF6 were derived from the Protein Data Bank (PDB ID:1LB5 and 4UZ0; ref. 31). The 3-D fast Fourier transform–based protein docking algorithm of HEX 8.00 (32) was then used for docking experiments to assess the possible binding mode between ARC and TRAF6. We selected 100 sorted docked configuration possibilities for further analysis.

DSS colitis and bone marrow cell transplantation mouse models

All animal studies were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). The ARC-KO mice were obtained from Albert Einstein College of Medicine of Yeshiva University (New York, NY). The C57BL/6 ARC wild-type (WT) mice were purchased from The Jackson Laboratory. The animals were housed in climate-controlled quarters with a 12-hour light/12-hour dark cycle. The mice were maintained and bred under virus- and antigen-free conditions.

For the DSS colitis model (protocol ID: 1807-36163), C57BL/6 ARC WT and ARC-KO mice (8–10 weeks old, 10 mice/vehicle group, 16 mice/DSS group, the male:female ratio was 1:1) were used. DSS (1.5%; molecular weight: ca 4000; Alfa Aesar) was added to drinking water for 7 days, then changed to fresh water for 7 days. Mice in the control group were given normal water during the experiment. The mice were euthanized with CO2, and colon tissues were collected for further analysis.

For the bone marrow cell transplantation (protocol ID: 1701-34463A), 1 week before irradiation, the recipient mice were given acidified, antibiotic water. Water was first adjusted to pH 2.6 with concentrated hydrochloric acid and autoclaved in 1 L bottles. Then, 10 mL of 10 mg/mL neomycin in saline and 400 μL of 25 mg/mL polymyxin B sulfate were added in water, sterilized by filtration, and then diluted into 1 L of acidic water. Transplantation of bone marrow cells was performed using female WT or ARC-KO mice as recipients and male ARC-KO mice as donors. For isolation of bone marrow cells, male WT or ARC-KO mice (10–12 weeks old) were euthanized, and their limbs were removed. Bone marrow cells were flushed from the medullary cavities of the tibias and femurs. For the preparation of TRAF6-mutated bone marrow cells, the isolated bone marrow cells (2 × 107 cells) were electroporated with a linearized Flag-TRAF6 (WT or K201R or K461R mutant) plasmid (20 μg) at 230 V and 500 μF using the Gene Pulser X (Bio-Rad Laboratories). Female ARC-KO mice (10–12 weeks old) were sublethally irradiated (950 rad) using an X-ray generator and bone marrow cells (1 × 106 cells) were transplanted intravenously within 3 hours after irradiation. At 4 weeks after irradiation, the mice were examined by the method described previously (33). One 544-bp product representing the IL3 gene and one 402-bp band representing the Y-specific Sry gene were identified. These were the presumptive males. Only the IL3-associated 544-bp band were the presumptive females. Then, the successfully transplanted mice were transferred to the DSS colitis model (protocol ID: 1807-36163) for the further study.

Azoxymethane-initiated and DSS-promoted colon carcinogenesis

The azoxymethane-initiated and DSS-promoted colon carcinogenesis model is described in our previous study (34) and followed an approved protocol (ID: 1807-36163A). Briefly, WT and ARC-KO mice (8–10 weeks old; 6 mice/vehicle group; 24 mice per azoxymethane/DSS group; male:female ratio was 1:1) were injected once subcutaneously with azoxymethane (Sigma, 10 mg/kg bodyweight), followed by exposure to two cycles of 3% DSS (Alfa Aesar) in drinking water. All animal care and experimental procedures were performed under the guidelines of the University of Minnesota IACUC. After 50 days, the mice were euthanized with CO2, and the colon tissue was flushed with PBS. Colon lesions were measured and snap-frozen for further analysis. The fixed colon tissue was processed by the Swiss-roll technique and embedded in paraffin for histopathologic examination in a subset of animals.

IHC analysis

Human colon tissues were obtained from the Mayo Clinic (Rochester, MN). The baseline demographics are shown in Supplementary Table S1. The human and mice colon tissues were supplied for IHC analysis. A Vectastain Elite ABC Kit obtained from Vector Laboratories was used for IHC staining according to the protocol recommended by the manufacturer. Mice colon tissues were embedded in paraffin for examination. Sections were stained with hematoxylin and eosin (H&E) and analyzed by IHC. Briefly, all specimens were deparaffinized and rehydrated. To expose antigens, samples were unmasked by submerging into boiling sodium citrate buffer (10 mmol/L, pH 6.0) for 10 minutes, and then treated with 3% H2O2 for 10 minutes. The slide was blocked with 10% goat serum albumin in 1 × PBS in a humidified chamber for 1 hour at room temperature. Then, the slide of human tissue array sections with ARC antibody (1:100) and the mouse colon tissue sections were hybridized with proliferating cell nuclear antigen (PCNA, 1:3000), CCL5 (1:100), or CXCL5 (1:100) at 4°C in a humidified chamber overnight. The slides were washed and hybridized with the secondary antibodies from Vector Laboratories (anti-rabbit 1:150, anti-mouse 1:150, or anti-rat 1:150) for 1 hour at room temperature. Slides were stained using the Vectastain Elite ABC Kit (Vector Laboratories, Inc.). After developing with 3,3′-diaminobenzidine, the sections were counterstained with hematoxylin and observed under microscope (200×), and analyzed by Image-Pro PLUS (v.6) computer software program (Media Cybernetics, Inc.).

Statistical analysis

All quantitative data are expressed as mean values ± SD of at least three independent experiments or samples. Significant differences were determined by a Student t test or one-way ANOVA

Susceptibility of ARC-deficient mice to DSS-induced IBD

We established a DSS-induced experimental mouse model of IBD, in which mice were exposed to DSS (1.5%) in drinking water for 7 days and then given normal tap water for another 7 days (Fig. 1A). We found that ARC-KO mice were more susceptible to DSS exposure. ARC-KO mice exhibited more severe clinical symptoms such as loss of body weight and shortening of colon length (Fig. 1B–D), indicating a greater extent of tissue damage in ARC-deficient mice. Moreover, ARC-KO mice had a more severe intestinal inflammatory response and epithelial injury. The tissue lysates were prepared from pooled colon tissue from each mouse of each group. Three sets were prepared for each group and each lane shows one set of pooled samples subjected to Western blotting. There was no ARC expression in ARC-KO mice groups (Fig. 1E). ARC-KO mice exhibited higher expression of cytokines, especially CCL5 and CXCL5 compared with WT mice in the DSS-induced IBD model as determined by a mouse inflammation array (Fig. 1F). There was no marked change in CXCL13, TNFSF8, CCL11, MPIF-2, TNFSF6, CX3XL1, GCSF, GM-CSF, IFNγ, IL1 F1, IL1 F2, IL2, IL3, IL4, IL6, IL9, IL10, IL12 p40/p70, IL12 p70, IL13, IL17A, CXCL11, CXCL1, leptin, XCL1, CCL2, M-CSF, CXCL19, CCL3, MIP-1 gamma, CXCL12a, TCA-3, CCL25, TIMP-1, TIMP-2, TNFα, TNFRSF1A, and TNFRSF1B expression. Significantly higher expression of mRNA and protein levels of CCL5 and CXCL5 was also confirmed by RT-PCR and IHC analysis, respectively (Fig. 1G and H). Notably, H&E staining showed that the colons of ARC-KO mice following DSS treatment exhibited more severe inflammation in the mucosa, muscularis propria, and submucosa with the entire loss of the crypts and partial loss of the surface epithelia compared with WT mice (Fig. 1H). In addition, we found DSS treatment increased the expression level of ARC in CD4-, CD19-, and CD49b-positive cells. Especially, DSS treatment markedly decreased ARC expression in CD4-positive cells (Supplementary Fig. S1). These data demonstrate a novel role for ARC in protecting against inflammation and colonic injury.

Figure 1.

Increased susceptibility of ARC-deficient mice to DSS-induced IBD. A, WT and ARC-KO mice were administered 1.5 % DSS for 7 days and then given normal tap water for another 7 days (n = 8). Some mice were only administered normal tap water and served as the vehicle control group (n = 5). B, Representative images of colons at day 14 from WT or ARC mice in vehicle-treated (healthy) or DSS-treated (DSS) group are shown. C, Body weight of vehicle- and DSS-treated mice over the course of acute colitis, and a percentage of original weight. D, Colon length of healthy and DSS-treated animals. E, Protein levels of ARC and β-actin were detected in colon of WT and ARC-KO mice. The tissue lysates were prepared from pooled colon tissue from each mouse of each group. Three sets were prepared for each group and each lane shows one set of pooled samples subjected to Western blotting. F, Cytokine expression levels in colon tissue were measured using a mouse inflammation array. Density scores were obtained using ImageJ software. G, mRNA expression level of CCL5 and CXCL5 was measured by RT-PCR. H, Representative H&E-stained sections of colon from vehicle- and DSS-treated mice. CCL5 and CXCL5 expression levels were detected by IHC (day 14). Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between ARC-KO and WT mice in vehicle- or DSS-treated groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 1.

Increased susceptibility of ARC-deficient mice to DSS-induced IBD. A, WT and ARC-KO mice were administered 1.5 % DSS for 7 days and then given normal tap water for another 7 days (n = 8). Some mice were only administered normal tap water and served as the vehicle control group (n = 5). B, Representative images of colons at day 14 from WT or ARC mice in vehicle-treated (healthy) or DSS-treated (DSS) group are shown. C, Body weight of vehicle- and DSS-treated mice over the course of acute colitis, and a percentage of original weight. D, Colon length of healthy and DSS-treated animals. E, Protein levels of ARC and β-actin were detected in colon of WT and ARC-KO mice. The tissue lysates were prepared from pooled colon tissue from each mouse of each group. Three sets were prepared for each group and each lane shows one set of pooled samples subjected to Western blotting. F, Cytokine expression levels in colon tissue were measured using a mouse inflammation array. Density scores were obtained using ImageJ software. G, mRNA expression level of CCL5 and CXCL5 was measured by RT-PCR. H, Representative H&E-stained sections of colon from vehicle- and DSS-treated mice. CCL5 and CXCL5 expression levels were detected by IHC (day 14). Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between ARC-KO and WT mice in vehicle- or DSS-treated groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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The expression of ARC is inversely correlated with CCL5 and CXCL5 levels in human biopsy specimens

To evaluate the role ARC in human IBD, human biopsy specimens were supplied for further analysis. Initially, we found that ARC is expressed in both colon crypts and immune cells in normal colon; whereas the expression level of ARC is substantially decreased in immune cells, but not in colon crypts (Fig. 2A). Forty IBD and 40 normal subjects were used. Three different fields were randomly selected from each sample slide to count positive immunostained and total inflammatory cells. All fields were added together for each sample. Then, from the average number of cells, the percentage was determined. Interestingly, ARC was less expressed in the leukocytes (buffy coat) of patients with IBD compared with normal subjects (Fig. 2B–E). There was no difference between male and female human biopsy specimens (Supplementary Fig. S2A and S2B). Not surprisingly, the expression levels of CCL5 and CXCL5 were significantly higher in plasma from patients with IBD (n = 40) compared with normal subjects (n = 40). Notably, ARC expression was inversely correlated with the expression of CCL5 and CXCL5, but ARC was more closely correlated with CCL5 expression (Fig. 2F–I).

Figure 2.

The expression of ARC in inflammatory cells is inversely correlated with CCL5 and CXCL5 expression levels in human biopsy specimens. A, H&E staining of colon from normal and IBD tissues. Expression of ARC in normal colon or in colon from patients with IBD was measured by IHC. Scale bar, 100 μm. B, ARC expression level in leukocytes (buffy coat) of normal or IBD biopsy specimens was detected using an immunoassay kit. Heatmap across all the samples shows the ARC levels. C and D, The level of CCL5 (C) and CXCL5 (D) in plasma from normal or IBD biopsy specimens was detected using immunoassay kits. Heatmap across all the samples shows the CCL5 or CXCL5 levels. E–G, The analysis of ARC (E), CCL5 (F), and CXCL5 (G) levels in human biopsy specimens. H and I, On the basis of the results from B–D, the correlation between ARC and CCL5 (H) or CXCL5 (I) is shown. Statistical significance was determined by Student t test. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between normal and IBD biopsy specimens. ***, P < 0.001.

Figure 2.

The expression of ARC in inflammatory cells is inversely correlated with CCL5 and CXCL5 expression levels in human biopsy specimens. A, H&E staining of colon from normal and IBD tissues. Expression of ARC in normal colon or in colon from patients with IBD was measured by IHC. Scale bar, 100 μm. B, ARC expression level in leukocytes (buffy coat) of normal or IBD biopsy specimens was detected using an immunoassay kit. Heatmap across all the samples shows the ARC levels. C and D, The level of CCL5 (C) and CXCL5 (D) in plasma from normal or IBD biopsy specimens was detected using immunoassay kits. Heatmap across all the samples shows the CCL5 or CXCL5 levels. E–G, The analysis of ARC (E), CCL5 (F), and CXCL5 (G) levels in human biopsy specimens. H and I, On the basis of the results from B–D, the correlation between ARC and CCL5 (H) or CXCL5 (I) is shown. Statistical significance was determined by Student t test. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between normal and IBD biopsy specimens. ***, P < 0.001.

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ARC performs its function in CD4-positive immune cells

On the basis of our findings, CCL5 is a critical cytokine associated with the function of ARC in IBD. Thus, flow cytometry analysis was conducted to identify the immune cells in which ARC performs its function. Our data showed that CCL5 expression was significantly increased in CD4-, CD19-, and CD49b-positive cells, but not in CD8-positive cells, in spleens of ARC-KO mice compared with WT mice (Fig. 3A–D). Especially, CCL5 was markedly increased in CD4-positive cells (Fig. 3A).

Figure 3.

CCL5 is upregulated in ARC-KO mice. Flow cytometry analysis of the percentage of CCL5/CD4-positive (A), CCL5/CD8-positive (B), CCL5/CD19-positive (C), or CCL5/CD49b (D) cells in primary cells isolated from mouse spleen. Statistical significance was determined by Student t test. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between WT and ARC-KO specimens. *, P < 0.05; ***, P < 0.001.

Figure 3.

CCL5 is upregulated in ARC-KO mice. Flow cytometry analysis of the percentage of CCL5/CD4-positive (A), CCL5/CD8-positive (B), CCL5/CD19-positive (C), or CCL5/CD49b (D) cells in primary cells isolated from mouse spleen. Statistical significance was determined by Student t test. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between WT and ARC-KO specimens. *, P < 0.05; ***, P < 0.001.

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ARC blocks TRAF6 Lys63-linked ubiquitination regulating the TRAF6-associated NF-κB signaling pathway

TNF receptor-associated factors (TRAF) have been identified as adapters controlling signaling pathways (35). TRAF molecules are essential in the regulation of inflammation and inflammatory diseases (36, 37). Initially, we found that ARC binds with TRAF6, but not with other TRAF members in T cells (Fig. 4A). In addition, we found that ARC does not bind with RSK2, NF-κB, IκB-α, β-catenin, and EGFR (Supplementary Fig. S3). The relationship between ARC and TRAF6 was studied by exogenously expressing these proteins in HEK293T cells. The results showed that ARC could bind with TRAF6 (amino acid 288–522; Fig. 4B–D). In addition, the protein-docking model also showed that ARC could bind with TRAF6 in a similar area (Fig. 4E). As we know, Lys63-linked polyubiquitination chains are critical for protein activation and Lys48-linked polyubiquitination chains are critical for protein degradation (38). Intriguingly, ARC reduces TRAF6 Lys63-linked ubiquitination in exogenous systems, including in vitro ubiquitination assay and mouse embryo fibroblasts (MEF) of WT and ARC-KO mice. ARC deficiency decreased IκB-α expression in IL1-treated MEFs (Fig. 4F–H). However, knockdown of ARC did not change TRAF6 expression (Supplementary Fig. S4A), indicating that ARC did not affect Lys48-linked ubiquitination. To identify the sites of TRAF6 that were ubiquitinated, we generated cells expressing mutants of TRAF6 (K201R and K461R) based on mass spectrometry analysis (Table 1) and evaluated the susceptibility of these TRAF6 mutants to ubiquitination. We overexpressed WT-TRAF6 only, WT-TRAF6, or TRAF6 (K201R and K461R) with ARC in 293T cells. We found that ubiquitination level of TRAF6 was reduced by ARC, and it was disrupted by the TRAF6 K461 mutation, but not K201 mutation (Fig. 4I). These results indicate that ubiquitination site of K461 is important for ARC for regulating TRAF6 ubiquitination. Moreover, we also found that knocking down ARC increased the expression levels of CCL5 and CXCL5, and also enhanced NF-κB nuclear translocalization in Jurkat and MOLT3 cells (Supplementary Fig. S4B–S4D). Overall, ARC binds with TRAF6, and regulates TRAF6 Lys63-linked ubiquitination and the NF-κB signaling pathway (Fig. 4J).

Figure 4.

ARC interacts with TRAF6 and blocks TRAF6 Lys63-linked ubiquitination regulating the TRAF6-associated NF-κB signaling pathway. A, ARC can bind with TRAF6 in Jurkat and MOLT3 cells as visualized by an endogenous immunoprecipitation (IP) assay. B, To confirm the binding between TRAF6 and ARC, the Flag-TRAF6 construct was cotransfected with HA-ARC into 293T cells. After culturing for 48 hours, cells were disrupted with lysis buffer and immunoprecipitated with an HA antibody. The coimmunoprecipitated Flag-TRAF6 or HA-ARC was detected using a specific antibody. C, Identification of the domain of ARC that binds with TRAF6. ARC deletion constructs were individually cotransfected with Flag-TRAF6 into 293T cells. After culturing for 48 hours, cells were disrupted with lysis buffer and immunoprecipitated with a Flag-TRAF6 mAb. Coimmunoprecipitated Flag-TRAF6 was detected by Western blotting. Flag-TRAF6 or ARC was detected in whole-cell lysates. D, To identify the domain of TRAF6 binding to ARC, six Flag-TRAF6 deletion constructs were individually cotransfected with Flag-ARC into 293T cells. After culturing for 48 hours, cells were disrupted with lysis buffer and immunoprecipitated with a Flag-ARC mAb. The coimmunoprecipitated Flag-ARC was detected by Western blotting. HA-TRAF6 or Flag-ARC was detected in whole-cell lysates.E, Modeling of TRAF6 (blue) binding with ARC (red). F, ARC blocks ubiquitination of TRAF6. Flag-TRAF6 was cotransfected with or without His-ARC. The ubiquitination of TRAF6 was measured by using an in vivo ubiquitination assay. The precipitates and whole-cell extracts were analyzed by Western blotting by using anti-Flag, anti-HA, or anti-His. G,In vitro, ARC deubiquitinates TRAF6. 293T cells were transfected with Flag-TRAF6 and HA-Ub-K63. Cell lysates were immunoprecipitated with anti-Flag and then incubated with purified His-ARC proteins at 37°C for 4 hours. Ubiquitination of TRAF6 was determined by Western blotting with Flag and His antibodies. H, Kinetics of TRAF6 ubiquitination. WT and ARC-KO MEFs were stimulated with IL1β for the indicated times. Proteins from lysates were immunoprecipitated with antibody to detect TRAF6, followed by Western blotting with an Ub-k63- or TRAF6-specific antibody to examine Ub-K63 or TRAF6 expression. Lysates were subjected to Western blotting with antibodies to detect IκBα, ARC, and β-actin. I, WT Flag-TRAF6, mutant Flag-TRAF6 (K201R), or Flag-TRAF6 (K461R) was cotransfected with HA-Ub-K63 and His-ARC as indicated. After immunoprecipitation with Flag, HA-Ub-K63 or Flag-TRAF6 was detected by Western blotting. J, ARC binds with TRAF6, regulating the TRAF6 Lys63-linked ubiquitination and NF-κB signaling pathway. IB, immunoblot.

Figure 4.

ARC interacts with TRAF6 and blocks TRAF6 Lys63-linked ubiquitination regulating the TRAF6-associated NF-κB signaling pathway. A, ARC can bind with TRAF6 in Jurkat and MOLT3 cells as visualized by an endogenous immunoprecipitation (IP) assay. B, To confirm the binding between TRAF6 and ARC, the Flag-TRAF6 construct was cotransfected with HA-ARC into 293T cells. After culturing for 48 hours, cells were disrupted with lysis buffer and immunoprecipitated with an HA antibody. The coimmunoprecipitated Flag-TRAF6 or HA-ARC was detected using a specific antibody. C, Identification of the domain of ARC that binds with TRAF6. ARC deletion constructs were individually cotransfected with Flag-TRAF6 into 293T cells. After culturing for 48 hours, cells were disrupted with lysis buffer and immunoprecipitated with a Flag-TRAF6 mAb. Coimmunoprecipitated Flag-TRAF6 was detected by Western blotting. Flag-TRAF6 or ARC was detected in whole-cell lysates. D, To identify the domain of TRAF6 binding to ARC, six Flag-TRAF6 deletion constructs were individually cotransfected with Flag-ARC into 293T cells. After culturing for 48 hours, cells were disrupted with lysis buffer and immunoprecipitated with a Flag-ARC mAb. The coimmunoprecipitated Flag-ARC was detected by Western blotting. HA-TRAF6 or Flag-ARC was detected in whole-cell lysates.E, Modeling of TRAF6 (blue) binding with ARC (red). F, ARC blocks ubiquitination of TRAF6. Flag-TRAF6 was cotransfected with or without His-ARC. The ubiquitination of TRAF6 was measured by using an in vivo ubiquitination assay. The precipitates and whole-cell extracts were analyzed by Western blotting by using anti-Flag, anti-HA, or anti-His. G,In vitro, ARC deubiquitinates TRAF6. 293T cells were transfected with Flag-TRAF6 and HA-Ub-K63. Cell lysates were immunoprecipitated with anti-Flag and then incubated with purified His-ARC proteins at 37°C for 4 hours. Ubiquitination of TRAF6 was determined by Western blotting with Flag and His antibodies. H, Kinetics of TRAF6 ubiquitination. WT and ARC-KO MEFs were stimulated with IL1β for the indicated times. Proteins from lysates were immunoprecipitated with antibody to detect TRAF6, followed by Western blotting with an Ub-k63- or TRAF6-specific antibody to examine Ub-K63 or TRAF6 expression. Lysates were subjected to Western blotting with antibodies to detect IκBα, ARC, and β-actin. I, WT Flag-TRAF6, mutant Flag-TRAF6 (K201R), or Flag-TRAF6 (K461R) was cotransfected with HA-Ub-K63 and His-ARC as indicated. After immunoprecipitation with Flag, HA-Ub-K63 or Flag-TRAF6 was detected by Western blotting. J, ARC binds with TRAF6, regulating the TRAF6 Lys63-linked ubiquitination and NF-κB signaling pathway. IB, immunoblot.

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Table 1.

Mass spectrometry analysis was used to identify the ubiquitination sites of ARC.

Conf.aScbPrec m/zczdSequenceTheor MWeΔMassfSite
61 946.6122 QVSC*VNC*AVSMAYEEK-ubEIHD 4728.1099 −0.0852 K201 
    QSC*PLAN§IIC*EYC*GTILIR    
99 454.0724 QNHEEVMDAK-ubPELLAFQRPT 2718.3547 0.0361 K461 
Conf.aScbPrec m/zczdSequenceTheor MWeΔMassfSite
61 946.6122 QVSC*VNC*AVSMAYEEK-ubEIHD 4728.1099 −0.0852 K201 
    QSC*PLAN§IIC*EYC*GTILIR    
99 454.0724 QNHEEVMDAK-ubPELLAFQRPT 2718.3547 0.0361 K461 

Abbreviation: MW, molecular weight.

aThe confidence for the peptide identification.

bThe score for the peptide.

cPrecursor m/z.

dThe charge for the fragmented ion.

eTheoretical precursor MW for peptide sequence.

fThe difference between theoretical MW and experimental MW of the matching peptide sequence.

*Carbamidomethyl.

§Deamidated (N).

ARC in bone marrow–derived cells attenuates DSS-induced IBD

To address whether ARC deficiency principally affects blood-derived immune cells, reciprocal bone marrow transplantations were performed. Female ARC-KO mice served as recipients and were randomly assigned to groups as follows: (i) mice injected with bone marrow cells from donor male ARC-KO mice, (ii) mice injected with bone marrow cells from donor male WT mice, (iii) mice injected with bone marrow cells with TRAF6-K201 mutation from donor male WT mice, (iv) mice injected with bone marrow cells with TRAF6-K461 mutation from donor male WT mice, (v) mice injected with vehicle medium, and (vi) mice with no treatment. At 4 weeks later, we examined the mice to confirm the success of transplantation (Supplementary Fig. S5A). The successfully transplanted mice were transferred to the DSS colitis model for further study (Fig. 5A). Results indicated that ARC-KO recipients of WT cells displayed less body weight loss and increased colon length compared with ARC-KO recipients of ARC-KO cells (Fig. 5B–D). In addition, bone marrow from WT donors significantly suppressed the DSS-induced increase in CCL5 and CXCL5 levels in colon tissue (Fig. 5E–G; Supplementary Fig. S5B). Importantly, WT mice exhibited reduced loss of crypts and surface epithelia compared with ARC-KO mice suffering from DSS-induced IBD. Notably, TRAF6-K461, but not the TRAF6-K201 mutation, disrupted the rescue function of WT on DSS-induced IBD in ARC-KO mice (Fig. 5B–G).

Figure 5.

Bone marrow transplantation overcomes the detrimental effects of ARC deficiency in colitis. A, Generation of bone marrow chimeric mice where recipients (female) were tail vein–injected with donor bone marrow (male, ARC-KO, or WT mice) or medium as a control. The chimeric recipients were subjected to acute colitis induction by DSS. An untreated group served as a negative control. B, Representative images of colons from ARC-KO recipients in vehicle- or DSS-treated mice are shown at day 14. C, Body weight of ARC-KO recipients during the course of acute colitis and a percentage of original weight. D, Colon length of ARC-KO recipients. E and F, mRNA expression level of CCL5 and CXCL5 was measured by RT-PCR. G, Representative H&E-stained sections of colon from ARC-KO recipients or vehicle control. CCL5 and CXCL5 expression levels were detected by IHC (day 14). Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between ARC-KO and WT mice in vehicle or DSS treatment groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

Bone marrow transplantation overcomes the detrimental effects of ARC deficiency in colitis. A, Generation of bone marrow chimeric mice where recipients (female) were tail vein–injected with donor bone marrow (male, ARC-KO, or WT mice) or medium as a control. The chimeric recipients were subjected to acute colitis induction by DSS. An untreated group served as a negative control. B, Representative images of colons from ARC-KO recipients in vehicle- or DSS-treated mice are shown at day 14. C, Body weight of ARC-KO recipients during the course of acute colitis and a percentage of original weight. D, Colon length of ARC-KO recipients. E and F, mRNA expression level of CCL5 and CXCL5 was measured by RT-PCR. G, Representative H&E-stained sections of colon from ARC-KO recipients or vehicle control. CCL5 and CXCL5 expression levels were detected by IHC (day 14). Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between ARC-KO and WT mice in vehicle or DSS treatment groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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ARC deficiency enhances colorectal tumorigenesis after azoxymethane/DSS treatment

Colitis-associated tumorigenesis is closely associated with the duration and severity of colonic inflammation. Earlier results (Fig. 1) demonstrated that ARC-deficient mice exhibited substantially higher susceptibility to DSS-induced IBD compared with WT mice. Here, we evaluated the protective effect of ARC in colitis-associated tumorigenesis. WT and ARC-KO mice were injected with azoxymethane, followed by two cycles of a 7-day administration of 3% DSS (days 7–14 and 28–35), followed by normal tap water for additional 15 days (Fig. 6A). At day 50, we found that the tumor number in ARC-KO mice was significantly higher compared with WT, and the colon length of ARC-KO mice was significantly decreased compared with WT mice in the azoxymethane/DSS treatment groups (Fig. 6B–D). No significant difference was observed between the vehicle-treated groups (Fig. 6B–D). In addition, the ARC-KO mice exhibited significant body weight loss and had a higher mortality rate compared with WT mice (Fig. 6E and F). The tissue lysates were prepared from pooled colon tissue from each mouse of each group. Three sets were prepared for each group and each lane shows one set of pooled samples subjected to Western blotting. There was no ARC expression in ARC-KO mice groups (Fig. 6G). ARC KO significantly enhanced the expression of mRNA level of CCL5 and CXCL5 by RT-PCR (Fig. 6H and I). Furthermore, we found that ARC KO enhanced expression of PCNA in colon tissue of KO mice compared with WT mice. The expression levels of CCL5 and CXCL5 were significantly enhanced in colon of ARC compared with WT mice (Fig. 6J; Supplementary Fig. S6). These results confirmed that ARC suppresses colitis-associated tumor development.

Figure 6.

ARC deficiency enhances IBD and IBD-induced tumorigenesis. A, WT and ARC-KO mice were injected once subcutaneously with azoxymethane (AOM), followed by exposure to two cycles of 3% DSS in drinking water (1 cycle: 5 days of DSS and 16 days of fresh water). B, Representative images of colon from WT or ARC mice in vehicle- or azoxymethane/DSS-treated (AOM/DSS) mice are shown at day 50. C, The number of tumors in WT or ARC mice in vehicle- or azoxymethane/DSS-treated mice. D, Colon length of healthy and DSS-treated animals. E, Body weight of vehicle- and azoxymethane/DSS-treated mice over the course of acute colitis and a percentage of original weight. F, The survival rate of WT and ARC-KO mice in healthy or azoxymethane/DSS-treated animals. G, Protein levels of ARC and β-actin were detected in colon of WT and ARC-KO mice. The tissue lysates were prepared from pooled colon tissue from each mouse of each group. Three sets were prepared for each group and each lane shows one set of pooled samples subjected to Western blotting. H and I, mRNA expression level of CCL5 and CXCL5 was measured by RT-PCR. J, Representative H&E-stained sections of colon from vehicle- and azoxymethane/DSS-treated mice. PCNA, CCL5, and CXCL5 expression levels were detected by IHC (day 50). Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between ARC-KO and WT mice in vehicle- or DSS-treated groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001. i.p., intraperitoneal.

Figure 6.

ARC deficiency enhances IBD and IBD-induced tumorigenesis. A, WT and ARC-KO mice were injected once subcutaneously with azoxymethane (AOM), followed by exposure to two cycles of 3% DSS in drinking water (1 cycle: 5 days of DSS and 16 days of fresh water). B, Representative images of colon from WT or ARC mice in vehicle- or azoxymethane/DSS-treated (AOM/DSS) mice are shown at day 50. C, The number of tumors in WT or ARC mice in vehicle- or azoxymethane/DSS-treated mice. D, Colon length of healthy and DSS-treated animals. E, Body weight of vehicle- and azoxymethane/DSS-treated mice over the course of acute colitis and a percentage of original weight. F, The survival rate of WT and ARC-KO mice in healthy or azoxymethane/DSS-treated animals. G, Protein levels of ARC and β-actin were detected in colon of WT and ARC-KO mice. The tissue lysates were prepared from pooled colon tissue from each mouse of each group. Three sets were prepared for each group and each lane shows one set of pooled samples subjected to Western blotting. H and I, mRNA expression level of CCL5 and CXCL5 was measured by RT-PCR. J, Representative H&E-stained sections of colon from vehicle- and azoxymethane/DSS-treated mice. PCNA, CCL5, and CXCL5 expression levels were detected by IHC (day 50). Scale bar, 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean values ± SD from triplicate experiments. The asterisks indicate a significant difference between ARC-KO and WT mice in vehicle- or DSS-treated groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001. i.p., intraperitoneal.

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In this study, we provide functional evidence showing that ARC mediates the inflammatory processes in the intestine and plays a protective role in IBD and IBD-associated colorectal tumorigenesis. Systemic deficiency of ARC exacerbated DSS-induced colitis and increased azoxymethane/DSS-induced tumorigenesis. We found that ARC performs this protective function in immune cells. Notably, ARC interacts with TRAF6 and mediates ubiquitination of TRAF6 in IBD and IBD-associated tumorigenesis.

ARC is a potent inhibitor of cell death and has the unique ability to antagonize both the intrinsic and extrinsic pathways of apoptosis (39). ARC exhibits increased expression in solid tumors and in patients with acute myeloid leukemia, and mediates the response of cells to the induction of pharmacologic apoptosis (17–20). A previous study showed that ARC is present at high levels in most colon cancer cell lines and in almost all primary colon cancers compared with corresponding controls, suggesting that ARC is a novel marker for human colon cancer (27). These results indicate that ARC might be a potential target in colon cancer development. Unexpectedly, our data revealed that ARC instead has a protective role in DSS-induced IBD and in an azoxymethane/DSS-induced colorectal tumorigenesis mouse model (Figs. 1 and 6), and is not an enhancer for IBD or oncogene in cancer development. This study showed that ARC has low expression in leukocytes (buffy coat) of patient with IBD compared with a normal person (Fig. 2). There might be the link between the increased expression of ARC in primary tumors and yet decreased expression in the leukocytes (buffy coat). Further experiments should be conducted to study the relationship. Previous reports also showed that ARC might act as a tumor suppressor in renal cancer and myeloid tumor development (21, 22). Moreover, ARC suppresses inflammatory responses associated with inhibition of NF-κB activation (23, 25, 26). These interesting results led us to uncover the mystery of ARC in the development of IBD and IBD-associated colorectal tumorigenesis.

Our findings demonstrated that the expression level of ARC is decreased in immune cells of patients with IBD (Fig. 2A, B, and E). CCL5 and CXCL5 are highly expressed in IBD patient plasma and in ARC-deficient mice in an IBD mouse model. Notably, the expression of ARC is inversely correlated with CCL5 and CXCL5 expression (Figs. 1 and 2). CCL5 and CXCL5 are critical cytokines, which play a crucial role in IBD (40–42). CCL5 belongs to the C-C chemokine family and is a target gene of NF-κB activity and is expressed by T lymphocytes, macrophages, platelets, synovial fibroblasts, tubular epithelium, and certain types of tumor cells (43). CXCL5 is a member of a proangiogenic subgroup of the CXCtype chemokine family of small, secreted proteins. Recently, evidence showed that CXCL5 is involved in carcinogenesis and cancer progression and is associated with NF-κB activity (44, 45). CCL5 is produced by T lymphocytes, epithelial cells, fibroblasts, and platelets (46). Several cell types activated by CCL5 are directly involved in antiviral response, including natural killer (NK) cells (47), and CCL5 also shows immunomodulatory functions in B cells (48). In this study, we found that ARC performs its function by regulating CCL5 expression in T cells, B cells, and NK cells, but especially in CD4-positive T cells (Fig. 3). These results indicate ARC should play a critical role in immune system. ARC interacts with TRAF6, mediating the TRAF6–NF-κB signaling pathway by regulating TRAF6 Lys63-linked ubiquitination (Fig. 4). TRAFs were identified as adapters controlling signaling pathways (35). The activation of TRAF6 in immune cells plays an essential role in patients with IBD (49). The TRAF6-dependent signaling pathway leads to activation of NF-κB and MAPKs, which are critical regulators of the immune response (50). TRAf6 K63 ubiquitination is known to activate IKK in the NF-κB signaling pathway, inducing proinflammatory cytokine expression (51). Intriguingly, our data indicated that TRAF6 Lys461 could play a critical role in ARC regulation of TRAF6 Lys63-linked ubiquitination (Figs. 4I and 5). Our results suggest that ARC is a key protector against IBD and IBD-associated colorectal tumorigenesis. ARC blocks TRAF6 Lys63-linked ubiquitination and mediates the NF-κB signaling pathway.

Therapy for IBD is most often implemented in a stepwise fashion, progressing through amino salicylates, corticosteroids, immunosuppressive medications, including ciclosporin and tacrolimus, and finally biological therapy (52). A proportion of patients with IBD suffer with an aggressive disease course despite pharmacologic treatments. Moreover, surgical intervention is not always a viable option due to the location or extent of the disease. Fortunately, autologous hematopoietic stem cell transplantation and mesenchymal stem cells have been regarded as a salvage therapy for patients with severe immune-mediated diseases, including IBD (53–56). Notably, by using ARC-deficient mice, we found that ARC in bone marrow–derived cells could successfully attenuate colitis (Fig. 5), suggesting that ARC might be a novel therapeutic target for IBD treatment.

In summary, our study reveals a novel role of ARC in IBD and IBD-associated colorectal tumorigenesis. We found that ARC in immune cells, especially CD4-positive T cells, interacts with TRAF6 and regulates TRAF6 Lys63-linked ubiquitination, mediating the NF-κB signaling pathway. These data identify ARC as a potential target for IBD treatment.

No potential conflicts of interest were disclosed.

Q. Wang: Conceptualization, resources, data curation, software, formal analysis, investigation, visualization, methodology, writing-original draft, writing-review and editing. T. Zhang: Conceptualization, resources, data curation, software, formal analysis, investigation, visualization, methodology, writing-original draft, project administration. X. Chang: Software, investigation. D.Y. Lim: Data curation, investigation. K. Wang: Investigation, visualization. R. Bai: Data curation. T. Wang: Data curation, investigation. J. Ryu: Data curation, software, methodology. H. Chen: Software. K. Yao: Methodology. W.-Y. Ma: Resources, methodology. L.A. Boardman: Resources. A.M. Bode: Writing-review and editing. Z. Dong: Supervision, funding acquisition.

The authors thank Todd Schuster for supporting experiments and Tara Adams for supporting animal experiments (The Hormel Institute, University of Minnesota, Austin, MN). This work was supported by the Hormel Foundation (to Z. Dong). This work was also supported by the Clinical Core of the Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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