TGF- −β Activated Kinase 1 Signaling Maintains Intestinal Integrity by Preventing Accumulation of Reactive Oxygen Species in the Intestinal Epithelium This information is current as of September 25, 2021. Rie Kajino-Sakamoto, Emily Omori, Prashant K. Nighot, Anthony T. Blikslager, Kunihiro Matsumoto and Jun Ninomiya-Tsuji J Immunol 2010; 185:4729-4737; Prepublished online 20 September 2010; Downloaded from doi: 10.4049/jimmunol.0903587 http://www.jimmunol.org/content/185/8/4729 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/09/17/jimmunol.090358 Material 7.DC1 References This article cites 41 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/185/8/4729.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TGF-b–Activated Kinase 1 Signaling Maintains Intestinal Integrity by Preventing Accumulation of Reactive Oxygen Species in the Intestinal Epithelium

Rie Kajino-Sakamoto,* Emily Omori,* Prashant K. Nighot,† Anthony T. Blikslager,† Kunihiro Matsumoto,‡ and Jun Ninomiya-Tsuji*

The intestinal epithelium is constantly exposed to inducers of reactive oxygen species (ROS), such as commensal microorganisms. Levels of ROS are normally maintained at nontoxic levels, but dysregulation of ROS is involved in intestinal inflammatory diseases. In this article, we report that TGF-b–activated kinase 1 (TAK1) is a key regulator of ROS in the intestinal epithelium. tak1 gene deletion in the mouse intestinal epithelium caused tissue damage involving enterocyte apoptosis, disruption of tight junctions, and

inflammation. Disruption of TNF signaling, which is a major intestinal damage inducer, rescued the inflammatory conditions but Downloaded from not apoptosis or disruption of tight junctions in the TAK1-deficient intestinal epithelium, suggesting that TNF is not a primary inducer of the damage noted in TAK1-deficient intestinal epithelium. We found that TAK1 deficiency resulted in reduced expres- sion of several antioxidant-responsive genes and reduced the protein level of a key antioxidant transcription factor NF-E2–related factor 2, which resulted in accumulation of ROS. Exogenous antioxidant treatment reduced apoptosis and disruption of tight junctions in the TAK1-deficient intestinal epithelium. Thus, TAK1 signaling regulates ROS through transcription factor NF-E2– related factor 2, which is important for intestinal epithelial integrity. The Journal of Immunology, 2010, 185: 4729–4737. http://www.jimmunol.org/

ntegrity of the epithelial barrier is essential for preventing role in intestinal epithelial barrier function (3, 4). For example, invasion of microorganisms and the development of chronic depletion of commensal bacteria greatly increases sensitivity to I inflammatory conditions in the intestine. A single layer of en- stress-induced intestinal damage (4). Ablation of TLR signaling terocytes separates the lamina propria from the gut lumen, there- from commensal bacteria disrupts the integrity of tight junctions by functioning as a physical barrier. Enterocytes are derived from in enterocytes (5). Therefore, commensal bacteria-derived cell intestinal epithelial stem cells that are localized to the crypts. Pro- signaling is likely to play a crucial role in cell survival and reg- liferating enterocytes differentiate and migrate toward the vil- ulation of tight junctions in enterocytes. However, the intracellular lus tips in the small intestine (1). Enterocytes undergo apoptosis signaling pathways that maintain enterocyte survival and tight by guest on September 25, 2021 only at the apical component of the villi. Although the intestine is junctions remain elusive. constantly exposed to high levels of cell-death inducers, such as TGF-b–activated kinase 1 (TAK1) plays an important role in TNF and bacteria-derived stressors, enterocytes are resistant to several innate immune-signaling pathways. TAK1 is activated by those inducers and survive until they reach the tip of the villus. TLR ligands, the intracellular bacteria sensor NOD2, and cyto- Dysregulated apoptosis during the periods of proliferation and kines (e.g., TNF and IL-1) (6–8). The TAK1-signaling pathway migration disrupts the intestinal barrier. In addition to enterocyte leads to activation of two groups of transcription factors, AP-1 survival, tightly connected enterocyte–enterocyte junctions (tight and NF-kB, which are intimately involved in immune responses junctions) are essential to form the physical barrier (2). Recently, in immune cells (9). We recently reported that TAK1 deficiency it has become evident that commensal bacteria play a protective results in dysregulation of cell survival in two types of epithelial cells: keratinocytes and enterocytes (10, 11). These findings raised the possibility that commensal bacteria-induced TAK1 signaling *Department of Environmental and Molecular Toxicology, North Carolina State Uni- versity, Raleigh, NC 27695; †Department of Clinical Sciences, College of Veterinary regulates enterocyte survival. Medicine, North Carolina State University, Raleigh, NC 27606; and ‡Department of TNF is constitutively expressed in the intestine and is essential Molecular Biology, Graduate School of Science, Nagoya University, Nagoya, Japan for preventing the invasion of microorganisms (12). However, Received for publication November 5, 2009. Accepted for publication August 16, dysregulation in TNF signaling plays a major role in intestinal 2010. damage by inducing inflammation and apoptosis in inflammatory This work was supported by National Institutes of Health Grants RO1GM068812 and diseases, such Crohn’s disease, and inhibition of TNF signaling is RO1GM084406 and the Crohn’s and Colitis Foundation of America (to J.N.-T.). one of the most effective approaches to prevent intestinal damage Address correspondence and reprint requests to Dr. Jun Ninomiya-Tsuji, Department of Environmental and Molecular Toxicology, North Carolina State University, Cam- (13). TNF transcriptionally induces inflammatory genes through pus Box 7633, Raleigh, NC 27695. E-mail address: [email protected] TAK1–NF-kB and TAK1–AP-1 pathways in immune cells (7, 14– The online version of this article contains supplemental material. 16). TNF can activate caspase-dependent apoptosis through the Fas- Abbreviations used in this paper: BHA, butylated hydroxyanisole; CHX, cyclohex- associated death domain and procaspase 8 (also called FLICE) (17). imide; CM-H2DCFDA, 5-(and -6)-chloromethyl-2-dichlorodihydrofluorescein diac- TNF also activates NADPH oxidase, which generates reactive ox- etate; CT, control; Cyto, cytosolic fraction; IBD, inflammatory bowel disease; Nrf2, NF-E2–related factor 2; PD, potential difference; ROS, reactive oxygen species; ygen species (ROS) (18, 19). We recently reported that ablation of shRNA, short hairpin RNA; TAB, TGF-activated kinase 1-binding protein; TAK1, TAK1 causes hypersensitivity to TNF-induced apoptosis in kerati- TGF-b–activated kinase 1; tBHP, tert-butylhydroperoxide; TER, transepithelial elec- nocytes (20). In the epidermal-specific TAK1-deletion mice, the trical resistance. tissue damage in the skin is largely rescued by deletion of the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 TNFR1 gene (11). However, in the intestinal epithelium, although www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903587 4730 TAK1 REGULATES ROS IN THE INTESTINAL EPITHELIUM deletion of TNFR1 greatly reduces intestinal damage in neonatal 5 min. After removing the contents, the intestine was filled with 10 mM intestinal epithelial-specific TAK1-deletion mice, the mice sponta- EDTA in HBSS and incubated in PBS again for 10 min. The contents were neously develop ileitis and colitis at approximately postnatal day 15 collected into tubes and centrifuged at 1200 rpm for 5 min. The resulting pellets containing predominantly epithelial cells were washed twice in cold (10). Thus, ablation in enterocyte-derived TAK1 signaling results in PBS. TNF-dependent and -independent intestinal damage. In the current study, we investigated the mechanism by which TAK1 prevents Immunoblot analyses TNF-dependent and -independent intestinal epithelial damage. Nuclear and cytoplasmic extracts from enterocytes and keratinocytes were prepared using a Nuclear Extract Kit (Active Motif, Carlsbad, CA). Pro- Materials and Methods teins from cell lysates were electrophoresed on SDS-PAGE and transferred to Hybond-P (GE Healthcare). The membranes were immunoblotted with Mice polyclonal Abs against NF-E2–related factor 2 (Nrf2; Santa Cruz Bio- Mice carrying a floxed Map3k7 allele (TAK1FL/FL) (7) were backcrossed to technology, Santa Cruz, CA), TAK1 (8), TAK1-binding protein (TAB)2 C57BL/6 mice for at least five generations. TNFR1-deficient C57BL/6 (26), and mAbs against Lamin B1 (Zymed). Bound Abs were visualized mice Tnfrsf1atm1Mak (TNFR12/2) (21) and villin-Cre transgenic mice with HRP-conjugated Abs against rabbit or mouse IgG using the ECL (22) with a C57BL/6 background were from The Jackson Laboratory (Bar Western blotting system (GE Healthcare). T2 Harbor, ME). villin-CreER transgenic mice with a C57BL/6 background Quantitative real-time PCR were described previously (23). The backcrossed TAK1FL/FL mice were used FL/FL IE-KO T2 FL/FL to generate villin-CreTAK1 (TAK1 ), villin-CreER TAK1 Total RNA from the small intestine was isolated using RNeasy Mini IE-IKO T2 FL/FL 2/2 IE-IKO (TAK1 ), and villin-CreER TAK1 TNFR1 (TAK1 (Qiagen, Valencia, CA). cDNA was synthesized using TaqMan reverse 2/2 TNFR1 ) mice. In all experiments, littermates were used as controls. To transcription reagents (Applied Biosystems, Foster City, CA). mRNA levels induce TAK1 gene deletion, 4-wk-old mice were given i.p. injections of of NQO1 and b-actin were analyzed by real-time PCR with SYBR Green Downloaded from tamoxifen (1 mg/20 g body weight) for two to five consecutive days. Some (Applied Biosystems). NQO1 primers 59-CATTCTGAAAGGCTGGTT- mice were fed food containing 0.7% butylated hydroxyanisole (BHA) TGA-39 and 59-CTAGCTTTGATCTGGTTGTCAG, GST-M1 primers 59- beginning 1 wk prior to the tamoxifen treatment. The following primers CTCCCGACTTTGACAGAAGC-39 and 59-CAGGAAGTCCCTCAGGT- were used for genotyping: floxed TAK1: 59-CACCAGTGCTGGATTC- TTG-39, GST-A4 primers 59-GCCAAGTACCCTTGGTTGAA-39 and 59- TTTTTGAGGC-39 and 59-GGAACCCGTGGATAAGTGCACTTGAAT- AATCCTGACCACCTCAACA-39, and b-actin primers 59-CCCAGAG- T2 39; villin-CreER :59-CAAGCCTGGCTCGACGGCC-39 and 59-CGCG- CAAGAGAGGTATC-39 and 59-AGAGCATAGCCCTCGTAGAT-39 were AACATCTTCAGGTTCT-39; and TNFR1: 59-TGTGAAAAGGGCACC- used. Expression levels of Bcl2, BclxL, glutamylcysteine ligase catalytic TTTACGGC-39 and 59-GGCTGCAGTCCACGCACTGG-39. Mice were subunit, IL-1, IL-6, MIP2, Nrf2, and GAPDH were also analyzed by http://www.jimmunol.org/ bred and maintained under specific pathogen-free conditions. All animal TaqMan gene-expression assay (Applied Biosystems). Results were ana- experiments were done with the approval of the North Carolina State lyzed using the comparative threshold cycle method. Values were nor- University Institutional Animal Care and Use Committee. malized to the level of b-actin mRNA in SYBR Green and to the level of GAPDH mRNA in TaqMan gene-expression assays. Short hairpin RNA and cell culture Caco-2 cells (ATCC HTB-37) were cultured in DMEM with 10% bovine Transepithelial electrical resistance growth serum (Hyclone, Logan, UT) and penicillin-streptomycin at 37˚C in The ileal tissues were harvested immediately after euthanasia, cut longi- 2 5% CO2. TAK1 small interfering RNA target sequence, corresponding to tudinally, and placed on 0.12 cm -aperture Ussing chambers (27). Tissues nucleotides 88–106 of the TAK1-coding region, was used to generate a were bathed on the serosal and mucosal sides with Ringer solution. The retrovirus vector expressing short hairpin RNA (shRNA) against TAK1, serosal bathing solution contained 10 mM glucose, which was osmotically by guest on September 25, 2021 pSUPERRetro-puro-shTAK1 (24). Caco-2 cells were transfected with balanced on the mucosal side with 10 mM mannitol. Bathing solutions pSUPERRetro-puro vector or pSUPERRetro-puro-shTAK1 and selected were oxygenated (95% O2–5% CO2) and circulated in water-jacketed with puromycin for 2 wk. Pools of puromycin-resistant Caco-2 cells were reservoirs maintained at 37˚C. The spontaneous potential difference (PD) +/+ FL/FL used. TAK1 and TAK1D/D keratinocytes were isolated from TAK1 was measured using Ringer-agar bridges connected to calomel electrodes, FL/FL and K5-Cre TAK1 mice described previously (11). Spontaneously and the PD was short-circuited through Ag–AgCl electrodes using a volt- immortalized keratinocytes derived from the skin of postnatal day 0–2 age clamp that corrected for fluid resistance. Transepithelial electrical + mice were cultured in Ca2 -free MEM (Lonza, Walkersville, MD) sup- resistance (TER; V cm2) was calculated from the spontaneous PD and plemented with 4% Chelex-treated bovine growth serum (Hyclone), 10 ng/ short-circuit current. The Ussing-chamber experiments were run for up to ml human epidermal growth factor (Invitrogen, Carlsbad, CA), 0.05 mM 3 h after an initial equilibration period of 15 min. Duplicate tissues were calcium chloride, and penicillin-streptomycin at 33˚C in 8% CO2. studied from each animal in each Ussing-chamber experiment. Reagents used were BHA (Sigma-Aldrich, St. Louis, MO), tert-butylhy- droperoxide (tBHP; Sigma-Aldrich), MG-132 (Merck, Whitehouse Sta- Analysis of ROS production tion, NJ), and cycloheximide (Merck). Harvested small intestines were embedded and frozen in OCT compound, Histology and immunohistochemistry and frozen sections were prepared. Sections were stained with 5 mM 5-(and -6)-chloromethyl-2-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) Sections were stained with H&E for histological analysis. Sections were (Invitrogen) for 40 min at 37˚C. Images were taken using a fluorescent scored in a blinded fashion on a scale from 0 to 4, based on the degree of microscope (BX41; Olympus) controlled by IPlab (Scanalytics). Three to lamina propria mononuclear cell infiltration, crypt hyperplasia, goblet cell five randomly selected areas were photographed with the same exposure depletion, and architectural distortion, as previously described (25). To time. The images were processed using the same fixed threshold in all detect apoptotic cells, the TUNEL assay was performed on paraffin sec- samples by Photoshop software, and representative images are shown. tions using the DeadEnd Colorimetric TUNEL System (Promega, Madi- son, WI), according to the manufacturer’s instructions. Immunofluorescent Statistical analyses staining was performed on paraffin-embedded sections or cryosections using polyclonal Abs against claudin-3 (1:500; Zymed, San Francisco, Statistical comparisons were made using independent, Student t tests on CA), occludin (1:50; Zymed), ZO-1 (1:50; Zymed), Gr-1 (1:100; eBio- data with normal variance. science, San Diego, CA), and cleaved-caspase 3 (1:100; Cell Signaling Technology, Beverly, MA). Bound Abs were visualized by Cy3- or Cy2- Results conjugated secondary Abs against rabbit (1:500; GE Healthcare, Piscat- away, NJ). Nuclei were counterstained with DAPI. Images were visualized TAK1 prevents TNF-dependent and -independent intestinal using a microscope (BX41; Olympus, Melville, NY) controlled by the damage IPLab imaging software (Scanalytics, Fairfax, VA). Mice with intestinal epithelial-specific deletion of TAK1 (TAK1IE-KO) Isolation of enterocytes have a lethal defect within 24–48 h after birth due to severe dam- The small intestine was harvested and flushed with PBS to remove fecal age in the intestine (10). We previously demonstrated that TNFR1 contents. One end of the intestine was tied off, filled with HBSS (Sigma- deletion rescues this early lethality (10), indicating that the se- Aldrich) containing 10 mM EDTA, and incubated in a PBS bath at 37˚C for verity of damage is mainly caused by TNF. However, mice having The Journal of Immunology 4731 intestinal epithelial-specific TAK1 deletion, even on a TNFR12/2 epithelial damage, including extensive evidence of apoptosis in background, develop ileitis and colitis at postnatal day 15–17 (10). the crypts (Fig. 1A,1B). The TAK1-deficient epithelium also This suggests that TAK1 prevents TNF-dependent damage and exhibited extensive separation of villus epithelium from the lam- that TAK1 is also important for blockade of TNF-independent ina propria throughout the upper two thirds of the affected villi. epithelial dysregulation. In this study, we aimed to determine This damage was observed, regardless of TNFR1 status in the the mechanism by which TAK1 prevents TNF-dependent and ileum (Fig. 1A,1B). The numbers of apoptotic enterocytes were -independent epithelial dysregulation. Enterocyte survival and similar in the TAK1IE-IKO and TAK1IE-IKO TNFR12/2 ileum epi- tightly connected cell–cell junctions are essential for maintenance thelium. These results indicate that apoptosis was induced mainly of intestinal epithelial integrity. Therefore, we first examined ap- through a TNF-independent mechanism in the TAK1-deficient optosis and tight junctions in control and TAK1-deficient in- ileum. We noted that TAK1IE-IKO mice exhibited damage in the testinal epithelium on a wild type or TNFR12/2 background. To small intestine and colon, whereas damage in TAK1IE-IKTNFR12/2 compare the level of damage at the same age in the mouse mod- mice was observed primarily in the ileum (Supplemental Fig. 1). el, we generated intestinal epithelial-specific inducible TAK1- As reported previously, TAK1IE-IKO mice develop severe damage, deletion mice on a wild type background (TAK1IE-IKO) and which becomes lethal at 4–5 d after the initiation of tamoxifen TNFR12/2 background (TAK1IE-IKOTNFR12/2). In this system, injection (10). In contrast, the level of damage in TAK1IE-IKO we induce tak1 gene deletion by tamoxifen injection. We used 4- TNFR12/2 intestinal epithelium was not changed following 3 d of wk-old mice for all experiments and initially analyzed the small tamoxifen injection. We injected TAK1IE-IKOTNFR12/2 mice with intestine at day 3 of tamoxifen injection. TAK1 deletion caused tamoxifen for five consecutive days and maintained them without Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. TAK1 deletion causes TNF-dependent and -independent intestinal damage. A, H&E staining of ileal sections from littermate control (TAK1F/F, CT) and villin-CreERT2TAK1FL/FL (TAK1IE-IKO) mice and littermate control TNFR12/2 (TNFR12/2) and villin-CreERT2TAK1FL/FLTNFR12/2 (TAK1IE-IKO TNFR12/2) mice. Four-week-old mice were treated with tamoxifen for two consecutive days, and samples were prepared 1 d after the second injection (day 3). Scale bars, 50 mm. Arrows indicate examples of apoptotic enterocytes. B, TUNEL staining of sections in A. Scale bars, 20 mm. TUNEL+ cells were counted in $180 crypts of each ileum. Bar graph shows mean (6 SEM) TUNEL+ cells per crypt (n = 3). C, Immunofluorescence staining of claudin-3 using the sections prepared as described in A. Scale bars, 20 mm. D, Real-time PCR analysis of TAK1FL/FL (CT, open bars) and villin-CreERT2TAK1FL/FL (TAK1IE-IKO, filled bars) small intestine and TNFR12/2 (TNFR12/2, open bars) (upper panels) and villin-CreERT2TAK1FL/FLTNFR12/2 (TAK1IE-IKO TNFR12/2, filled bars) (lower panels) small intestine. Four-week-old mice were treated with tamoxifen for two consecutive days, and mRNA samples were prepared 1 d after the second injection (day 3). mRNA levels relative to GAPDH mRNA are shown. The data represent means 6 SEM (n = 7). pp , 0.05. 4732 TAK1 REGULATES ROS IN THE INTESTINAL EPITHELIUM

evidence of claudin-3 precisely at the region of the tight junction apical lateral membrane, rather it appeared to be within the cy- toplasm. The level of claudin-3 mislocalization was not noticeably different between TAK1IE-IKO and TAK1IE-IKOTNFR12/2 mice. To assess the levels of inflammation, we measured the mRNA levels of inflammatory cytokines that were expressed in the small intestine in TAK1IE-IKO and TAK1IE-IKOTNFR12/2 mice at day 3 of tamoxifen injection (Fig. 1D). TAK1IE-IKO mice had markedly IE-IKO 2/2 increased levels of inflammatory cytokines, whereas TAK1 FIGURE 2. TAK1 deficiency impairs intestinal barrier. TNFR1 (s) 2/2 2 2 TNFR1 mice did not exhibit a significant increase in those and villin-CreERT2TAK1FL/FLTNFR1 / (d) mice were treated with ta- cytokines. Collectively, TAK1 seems to be essential for enterocyte moxifen for five consecutive days. At 2 wk post-tamoxifen treatment, the ileum was isolated and TER was analyzed by using an Ussing chamber. survival and integrity of tight junctions, primarily in the ileum. This effect is independent of TNF signaling. When TNF signaling is intact, TAK1 deletion causes more severe tissue damage in the additional tamoxifen injection. We confirmed that the tak1 gene ileum as well as in other regions of the intestine. These results was deleted at 3 d and at 8 wk after the termination of tamoxifen suggest that TAK1 is primarily important for maintenance of injection. TAK1IE-IKOTNFR12/2 mice were viable for $6mo enterocyte survival and tight junction integrity and that TNF sig- without showing any clinical signs, but they exhibited tissue dam- naling amplifies TAK1 deficiency-induced damage by promoting age in the ileum at similar levels to those at 3 d after initiation of inflammation. Downloaded from tamoxifen injection. These results indicate that TAK1 prevents TNF-independent apoptosis in the ileum and that TNF is not a pri- TAK1 deficiency reduces TER mary mediator of intestinal damage, but it amplifies the damage. To verify whether the increased apoptosis and disruption of tight We examined whether the tight junctions were also affected by junctions impair intestinal barrier function, we measured TER in ablation of TAK1. We analyzed the localization of three major tight TAK1-deficient intestinal epithelium. We chose TNFR12/2 IE-IKO 2/2 junction-associated proteins in enterocytes: claudin-3, occludin, background (TAK1 TNFR1 ) mice to rule out the possi- http://www.jimmunol.org/ and tight junction plaque protein ZO-1. All three proteins were bility that inflammatory conditions could indirectly affect the diffusely localized in the ileum of TAK1IE-IKO and TAK1IE-IKO barrier function. TER was significantly lower in TAK1IE-IKO TNFR12/2 mice (Supplemental Fig. 2 and data not shown). Al- TNFR12/2 mice compared with control TNFR12/2 mice (Fig. 2). though localization of occludin and ZO-1 was marginally al- Thus, TAK1 is important for maintenance of the intestinal barrier tered, the mislocalization of claudin-3 was striking in TAK1- function through modulating enterocyte survival and tight junc- deficient intestinal epithelium (Fig. 1C); there was very little tions. by guest on September 25, 2021

FIGURE 3. Antioxidant-responsive genes are downregulated in TAK1-deficient intestinal epithelium. A, Real-time PCR analysis was used to quantify GST-M1, GST-A4, and NQO1 mRNA levels in the TAK1FL/FL (CT) and villin-CreERT2TAK1FL/FL (TAK1IE-IKO) small intestine and colon. The mice were treated with tamoxifen for three consecutive days, and mRNA samples were prepared 1 d after the third tamoxifen injection (day 4). Relative mRNA levels were determined using b-actin mRNA. The data represent means 6 SEM (n = 4). pp , 0.05; ppp , 0.01. B, Nrf2 mRNA levels were determined by real- time PCR analysis in the TAK1FL/FL (CT) and villin-CreERT2TAK1FL/FL (TAK1IE-IKO) small intestine (left panel). The mice were treated with tamoxifen for two consecutive days, and mRNA samples were prepared 1 d after the second tamoxifen injection (day 3). Relative mRNA levels were calculated using GAPDH mRNA. The data represent means 6 SEM (n = 7). Immunoblot analysis of Nrf2 in the nuclear extracts (Nuc) from TAK1FL/FL (CT) and villin- CreERT2TAK1FL/FL (TAK1IE-IKO) enterocytes (right panel). The enterocytes were prepared at day 3 of tamoxifen injection. Lamin B1 was used as a loading control. C, Nrf2 mRNA and protein levels of TNFR12/2 and villin-CreERT2TAK1FL/FLTNFR12/2 (TAK1IE-IKO TNFR12/2) small intestine were analyzed as described in B. The mRNA data are means 6 SEM (n = 7). The Journal of Immunology 4733

TAK1 deficiency downregulates the levels of the protein level of nuclear Nrf2 was greatly reduced in the TAK1- antioxidant-responsive genes deficient intestine. Nrf2 was not detectable in the cytoplasmic To determine the mechanism by which TAK1 mediates enterocyte fraction in intestinal epithelium (data not shown). Downregulation survival, we measured the mRNA levels of genes associated of Nrf2 was independent of TNFR1 status. Nrf2 regulation of with cell survival in control and TAK1-deficient intestine. We antioxidant-responsive genes plays an integral role in ROS me- initially analyzed samples from the intestine having control ge- tabolism (28, 29), which is critically involved in cell viability and notype and intestinal epithelial-specific constitutive deletion of tight junction integrity. We postulated that TAK1 might regulate TAK1 (TAK1IE-KO). The expression levels of antiapoptotic genes, the protein level of Nrf2, thereby modulating cell survival and including bcl2 and bclxL, were not altered (Supplemental Fig. 3). tight junctions. We found that several antioxidant-responsive genes (i.e., nqo1, gstm1, and gstm4), were downregulated in the constitutive and Ablation of TAK1 downregulates Nrf2 and sensitizes cells to inducible TAK1-deficient small intestine and the colon (Fig. 3A). oxidative stress These antioxidant-responsive genes are known to be regulated by To further investigate the mechanism by which TAK1 regulates a key antioxidant transcription factor, Nrf2. Therefore, we ex- Nrf2 and cell survival, we used two lines of cultured epithelial cells amined the levels of Nrf2 in inducible TAK1-deficient intestinal exhibiting TAK1 ablation: Caco-2 cells stably expressing an epithelium on wild type and TNFR12/2 background (TAK1IE-IKO shRNA targeted against TAK1 and TAK1-deficient skin epithelial and TAK1IE-IKOTNFR12/2) (Fig. 3B,3C). Although the mRNA cells (keratinocytes) that were isolated from the epidermal-specific levels of Nrf2 were not significantly altered by ablation of TAK1, TAK1-deletion mice (11). In both cell types, Nrf2 was not de- Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. TAK1 deficiency downregulates Nrf2 and causes hypersensitivity to oxidative stress in cultured epithelial cells. A, Caco-2 cells stably expressing a control vector or shRNA targeted against TAK1 were treated or not with 10 mM MG-132 for 5 h. The nuclear and cytosolic fractions were prepared from those cells. The levels of Nrf2 were analyzed by immunoblots in the nuclear fraction (Nuc), and the levels of TAK1 were analyzed in the cytosolic fraction (Cyto). Lamin B was used as a loading control. B, TAK1 wild type (+/+) and TAK1-deficient (D/D) keratinocytes were left untreated or were treated with 10 mM MG-132 for 5 h. The nuclear and cytosolic fractions were analyzed as described in A. TAK1 D/D keratinocytes expressed a truncated form of TAK1 (TAK1 D), and the expression level of TAK1 D was low compared with TAK1 wild type because of instability of the truncated form. C, 293 cells were transfected with expression vectors for 3 g Nrf2 or 0.33 mg Nrf2, 0.33 mg TAK1, and 0.33 mg TAB1. At 48 h posttransfection, cells were treated with 100 mg/ml cycloheximide (CHX) and harvested at 0, 2.5, and 5 h. Whole-cell extracts were analyzed by immunoblots. The amount of endogenous TAB2 is also shown as a loading control. D, Caco-2 cells stably expressing a control vector or shRNA targeted against TAK1 were treated with 1 mM tBHP for 15 h (left panel). Cells were analyzed by immunofluorescence staining with anti–ZO-1. TAK1 wild type (+/+) and TAK1-deficient (D/D) keratinocytes were treated with 0.3 mM tBHP for 24 h (right panel). Cells were analyzed by immunofluorescence staining with anti–ZO-1. Scale bars, 40 mm. E, Caco-2 cells stably expressing a control vector or shTAK1 and TAK1 wild type (+/+) and TAK1-deficient (D/D) keratinocytes were treated as described in C (upper panels). Cells were analyzed by immunofluorescence staining with anti-cleaved caspase-3 (lower panels). Cleaved caspase-3+ cells were counted in $300 cells of each sample. The data represent means 6 SEM (n = 5). Representative images of TAK1 wild type (+/+) and TAK1-deficient (D/D) keratinocytes with cleaved caspase-3 staining (red). DAPI staining (blue) was used to visualize nuclei. Scale bars, 40 mm. ppp , 0.01. 4734 TAK1 REGULATES ROS IN THE INTESTINAL EPITHELIUM tectable in the cytoplasmic fractions (data not shown), and the protein level of nuclear Nrf2 was lower in TAK1-deficient cells compared with control cells (Fig. 4A,4B). The protein level of Nrf2 is known to be primarily regulated by protein degradation through the proteasome pathway (30). Blockade of the proteasome pathway by MG-132 treatment greatly increased the levels of Nrf2 in control and TAK1-deficient cells (Fig. 4A,4B). This suggests that Nrf2 is always highly degraded through the proteasome pathway and that TAK1 might be involved, in part, in Nrf2 sta- bility. We asked whether activation of TAK1 could alter Nrf2 stability. Coexpression of TAK1 with TAB1 highly activates TAK1 (31). We determined the Nrf2 stability with and without coexpression of TAK1 and TAB1 in 293 cells (Fig. 4C). The protein level of Nrf2 was almost completely diminished within 5 h after blockade of protein synthesis when Nrf2 alone was expressed, whereas the level of Nrf2 decreased much more slowly in cells with coexpression of TAK1 and TAB1. These results sug- gest that TAK1 may participate in Nrf2 stability. Therefore, ab- lation of TAK1 might cause increased degradation of Nrf2. Downloaded from Nrf2 is important for preventing oxidative stress (29, 32). We next examined whether TAK1 deficiency could cause increased apoptosis and disruption of tight junctions in response to oxidative stress. We treated Caco-2 and keratinocytes with tBHP, a pro- FIGURE 5. TAK1 regulates ROS in the intestinal epithelium. A, FL/FL T2 FL/FL IE-IKO totypical organic oxidant, and the tight junctions and apoptotic TAK1 (CT) and villin-CreER TAK1 (TAK1 ) mice were fed a normal or BHA chow diet for 1 wk and were subsequently treated cells were observed by immunostaining with anti-cleaved caspase http://www.jimmunol.org/ 3 and anti–ZO-1, respectively (Fig. 4D,4E). We noted that with tamoxifen for two consecutive days. Unfixed cryosections were prepared from the ileum 1 d after the second injection (day 3) and were claudin-3 was less clearly detected in Caco-2 cells compared with incubated with 5 mM CM-H2-DCFDA for 40 min at 37˚C to detect ROS ZO-1, and we used ZO-1 to visualize the tight junctions in those 2/2 (green) (upper panels). CM-H2-DCFDA staining of TNFR1 and villin- cultured cells. Compared with control cells, the tight junctions CreERT2TAK1FL/FLTNFR12/2 (TAK1IE-IKOTNFR12/2) samples were were relatively more damaged, and apoptotic cells were signifi- prepared by the same procedure as above (lower panels). Scale bars, 20 cantly increased in TAK1-deficient cells. These results indicate mm. B, Unfixed cryosections were prepared from the ileum of villin- that TAK1 deficiency caused hypersensitivity to oxidative stress in CreERT2TAK1FL/FLTNFR12/2 (TAK1IE-IKOTNFR12/2) mice and stained cultured epithelial cells. Collectively, we postulated that ablation by CM-H2DCFDA (left panel). The sections were subsequently fixed, and of TAK1 causes dysregulation of Nrf2 stability and ROS, which cleaved caspase-3 was detected by immunofluorescent staining (middle by guest on September 25, 2021 m results in impaired tight junctions and increased apoptosis in the panel). Scale bars, 40 m. All images were photographed using the same intestinal epithelium. exposure time. The data shown are representatives of three or four separate experiments with similar results. ROS is the cause of TNF-dependent and -independent intestinal damage in TAK1-deficient intestinal epithelium reduced by BHA feeding in TAK1IE-IKO and TAK1IE-IKOTNFR12/2 We examined the hypothesis that TAK1 regulates ROS levels in the mice (Fig. 5A). Histological evaluation and TUNEL assays revealed intestinal epithelium. ROS were measured in the TAK1IE-IKO and that intestinal damage and apoptotic enterocytes were significantly TAK1IE-IKOTNFR12/2 intestinal epithelium at day 3 of tamoxifen reduced with BHA feeding (Fig. 6). BHA feeding was equally ef- +/+ injection. The unfixed fresh cryosections of the ileum were used to fective in TAK1-deficient intestinal epithelium on a TNFR1 2/2 detect ROS by CM-H2-DCFDA staining (Fig. 5A). The levels of or TNFR1 background. Furthermore, BHA feeding greatly ROS were greatly increased in TAK1-deficient intestinal epithe- improved tight-junction integrity (Fig. 7A). The mRNA levels of lium. The increased ROS might be generated from infiltrated mye- inflammatory cytokines were not upregulated in BHA-treated IE-IKO loid cells, because TAK1IE-IKO intestinal epithelium was highly in- TAK1 mice (Fig. 7B). These results indicate that ablation of flamed (Fig. 1). However, the levels of ROS were not different TAK1 causes enterocyte apoptosis and impairs barrier function, between highly inflamed TAK1IE-IKO mice and TAK1IE-IKO most likely as the result of increased ROS. TNFR12/2 mice, which did not exhibit significant inflammatory conditions. We detected some Gr-1+ cells in control TNFR12/2 Discussion and TAK1-deficient TAK1IE-IKOTNFR12/2 intestinal epithelium; In this study, we demonstrated that enterocyte-derived TAK1 however, the number of Gr-1+ cells was much smaller than that of signaling plays a critical role in ROS metabolism, possibly through ROS positive cells in TAK1IE-IKOTNFR12/2 intestinal epithelium transcription factor Nrf2 and its target genes. Ablation of this TAK1 (Supplemental Fig. 4). We found that the ROS positive cells were pathway caused accumulation of ROS, resulting in enterocyte highly overlapped with the cells having cleaved caspase 3 (Fig. apoptosis and disruption of tight junctions. We previously reported 5B), suggesting that ROS are produced in apoptotic intestinal that deletion of TNFR1 can rescue TAK1 deficiency-induced ap- epithelial cells. These results indicate that TAK1 signaling is optosis and inflammatory conditions in the intestinal epithelium essential for preventing ROS accumulation in the intestinal in neonatal mice (10) and in the epidermis of the skin (11). In epithelial cells. We next attempted to reduce ROS and tested cultured cells, we demonstrated that TNF greatly increases ROS whether reduction of ROS could rescue the apoptosis and tight- in TAK1-deficient keratinocytes, which causes TNF-induced ap- junction disruption in the TAK1-deficient epithelium. We fed the optosis (11, 20). Thus, we concluded that TAK1 signaling prin- mice a chow diet containing the antioxidant BHA for 1 wk prior to cipally reduces TNF-induced ROS and prevents TNF-induced ap- tak1 gene deletion. We found that the levels of ROS were greatly optosis in the intestine of neonatal mice and epidermis of the skin. The Journal of Immunology 4735 Downloaded from http://www.jimmunol.org/

FIGURE 6. Antioxidant prevents TNF-dependent and -independent intestinal damage in the TAK1-deficient intestinal epithelium. A, H&E staining of the ileal sections from control (TAK1F/F; CT), villin-CreERT2TAK1FL/FL (TAK1IE-IKO), TNFR12/2, and villin-CreERT2TAK1FL/FLTNFR12/2 (TAK1IE-IKO TNFR12/2) mice fed a normal or BHA chow diet for 1 wk and subsequently treated with tamoxifen for two consecutive days. Sections were prepared from the ileum 1 d after the second injection (day 3). Scale bars, 50 mm. Arrows indicate examples of apoptotic enterocytes. CT, TAK1IE-IKO, and TAK1IE-IKO +BHA, n = 3; TNFR12/2, TAK1IE-IKOTNFR12/2, and TAK1IE-IKOTNFR12/2 +BHA, n =4.B, Histological scores for samples shown in A. Data are the means 6 SEM. C, TUNEL staining of the sections shown in A. D, TUNEL+ cells were counted in $180 crypts of each small intestinal segment. The numbers shown are means (6 SEM) of TUNEL positive cells per crypt (n = 4). pp , 0.05; ppp , 0.01; pppp , 0.001. by guest on September 25, 2021

However, in the adult intestinal epithelium, we found that the bacteria in the adult intestines may be the major trigger of ROS. TAK1 deficiency-induced ROS was not altered by TNFR1 de- TAK1 signaling reduces those non-TNF–induced ROS, which is letion (Fig. 5A). This indicates that TNF is not the major inducer essential for enterocyte survival and integrity of tight junctions. of ROS in the adult intestinal epithelium. Mice are sterile in utero How does TAK1 regulate ROS? In this study, we showed that the and are inoculated with bacteria at birth, and populations of in- level of Nrf2 was downregulated in TAK1-deficient intestinal testinal commensal bacteria are known to be dramatically altered epithelium. Although Nrf2 knockout increases susceptibility to during postnatal development (33). We speculate that commensal intestinal injury, it alone does not increase enterocyte apoptosis or

FIGURE 7. BHA feeding blocks disruption of tight junctions and inflammation. A, Immunofluo- rescence analysis of claudin-3 of the ileum sections from control (CT; TAK1F/F), villin-CreERT2 TAK1FL/FL (TAK1IE-IKO), TNFR12/2, and villin- CreERT2TAK1FL/FLTNFR12/2 (TAK1IE-IKOTNFR12/2) mice fed a normal or BHA chow diet. Scale bars, 20 mm. B, Real-time PCR analysis was performed in the small intestine from control (CT; TAK1F/F) and villin-CreERT2TAK1FL/FL (TAK1IE-IKO) mice, with a normal or BHA chow diet. The mice were treated with the procedure described in A. mRNA levels relative to GAPDH mRNA are shown. The data are means 6 SEM. CT, TAK1IE-IKO, and TAK1IE-IKO + BHA, n = 7; CT + BHA, n =4. 4736 TAK1 REGULATES ROS IN THE INTESTINAL EPITHELIUM cause inflammatory conditions (34). Therefore, we would not 3. Artis, D. 2008. Epithelial-cell recognition of commensal bacteria and mainte- nance of immune homeostasis in the gut. Nat. Rev. Immunol. 8: 411–420. expect that ablation of Nrf2 alone would be sufficient to induce all 4. Rakoff-Nahoum, S., J. Paglino, F. Eslami-Varzaneh, S. Edberg, and R. Medzhitov. of the noted disruptions caused by intestinal epithelial-specific 2004. Recognition of commensal microflora by toll-like receptors is required deletion of TAK1. In our previous study, we found that an AP-1 for intestinal homeostasis. Cell 118: 229–241. 5. Cario, E., G. Gerken, and D. K. Podolsky. 2007. Toll-like receptor 2 controls family transcription factor c-Jun is downregulated in TAK1- mucosal inflammation by regulating epithelial barrier function. Gastroenterology deficient keratinocytes (20). Similar to Nrf2, AP-1 family tran- 132: 1359–1374. scription factors are critical to the transcriptional regulation of 6. Kim, J.-Y., E. Omori, K. Matsumoto, G. Nu´n˜ez, and J. Ninomiya-Tsuji. 2008. TAK1 is a central mediator of NOD2 signaling in epidermal cells. J. Biol. Chem. antioxidant-responsive genes (35–38). Overexpression of c-Jun 283: 137–144. partially blocks accumulation of TNF-induced ROS (20). In ad- 7. Sato, S., H. Sanjo, K. Takeda, J. Ninomiya-Tsuji, M. Yamamoto, T. Kawai, dition, TAK1 is an integral upstream kinase of IkB kinases, lead- K. Matsumoto, O. Takeuchi, and S. Akira. 2005. Essential function for the kinase k TAK1 in innate and adaptive immune responses. Nat. Immunol. 6: 1087–1095. ing to activation of transcription factor NF- B (11, 39), which is 8. Ninomiya-Tsuji, J., K. Kishimoto, A. Hiyama, J. Inoue, Z. Cao, and also a major transcription factor for several cellular antioxidant K. Matsumoto. 1999. The kinase TAK1 can activate the NIK-I kappaB as well as genes (40, 41). Taken together, we believe that TAK1 signaling re- the MAP kinase cascade in the IL-1 signalling pathway. Nature 398: 252–256. 9. Kawai, T., and S. Akira. 2006. TLR signaling. Cell Death Differ. 13: 816–825. gulates multiple antioxidant transcription factors, including Nrf2, 10. Kajino-Sakamoto, R., M. Inagaki, E. Lippert, S. Akira, S. Robine, K. Matsumoto, c-Jun, and possibly other unidentified factors that modulate the level C. Jobin, and J. Ninomiya-Tsuji. 2008. Enterocyte-derived TAK1 signaling pre- of ROS. vents epithelium apoptosis and the development of ileitis and colitis. J. Immunol. 181: 1143–1152. We showed that TAK1 deficiency downregulates the protein 11. Omori, E., K. Matsumoto, H. Sanjo, S. Sato, S. Akira, R. C. Smart, and levels of Nrf2 in the nucleus. Nrf2 is normally localized in the J. Ninomiya-Tsuji. 2006. TAK1 is a master regulator of epidermal homeostasis cytoplasm by its binding partner Keap1 and is constantly degraded involving skin inflammation and apoptosis. J. Biol. Chem. 281: 19610–19617. Downloaded from 12. Pasparakis, M., G. Courtois, M. Hafner, M. Schmidt-Supprian, A. Nenci, through the proteasome pathway (28). In the intestinal epithelium A. Toksoy, M. Krampert, M. Goebeler, R. Gillitzer, A. Israel, et al. 2002. TNF- and cultured Caco-2 and keratinocytes, Nrf2 was not detectable in mediated inflammatory skin disease in mice with epidermis-specific deletion of the cytoplasmic fraction. Antioxidants and oxidative stress oxidize IKK2. Nature 417: 861–866. 13. Rutgeerts, P., S. Vermeire, and G. Van Assche. 2009. Biological therapies for Keap1, which results in the release of Nrf2. Dissociation from inflammatory bowel diseases. Gastroenterology 136: 1182–1197. Keap1 stabilizes and translocates Nrf2 into the nucleus (28). We 14. Liu, H. H., M. Xie, M. D. Schneider, and Z. J. Chen. 2006. Essential role of TAK1 in thymocyte development and activation. Proc. Natl. Acad. Sci. USA 103: showed that TAK1 regulates Nrf2 stability; therefore, ablation of http://www.jimmunol.org/ 11677–11682. TAK1 downregulates the level of Nrf2. TAK1 signaling is likely to 15. Wan, Y. Y., H. Chi, M. Xie, M. D. Schneider, and R. A. Flavell. 2006. The kinase modulate Nrf2 or Keap1 and blocks Keap1-dependent Nrf2 deg- TAK1 integrates antigen and cytokine receptor signaling for T cell development, survival and function. Nat. Immunol. 7: 851–858. radation. Further studies are needed to define the mechanism by 16. Shim, J. H., C. Xiao, A. E. Paschal, S. T. Bailey, P. Rao, M. S. Hayden, which TAK1 modulates the Nrf2-Keap1 complex. K. Y. Lee, C. Bussey, M. Steckel, N. Tanaka, et al. 2005. TAK1, but not TAB1 or Intestinal epithelial-specific deletion of TAK1 causes increased TAB2, plays an essential role in multiple signaling pathways in vivo. Genes Dev. 19: 2668–2681. apoptosis and disruption of cell–cell tight junctions, primarily in 17. Aggarwal, B. B. 2003. Signalling pathways of the TNF superfamily: a double- the ileum. Those pathological conditions are very similar to the edged sword. Nat. Rev. Immunol. 3: 745–756. pathology noted in inflammatory bowel disease (IBD). Anti-TNF 18. Kim, Y. S., M. J. Morgan, S. Choksi, and Z. G. Liu. 2007. TNF-induced acti-

vation of the Nox1 NADPH oxidase and its role in the induction of necrotic cell by guest on September 25, 2021 therapy has recently been extensively used for the effective treat- death. Mol. Cell 26: 675–687. ment of IBD (13). In the TAK1-deficient intestinal epithelium, 19. Yazdanpanah, B., K. Wiegmann, V. Tchikov, O. Krut, C. Pongratz, M. Schramm, although the deletion of TAK1 causes sustained severe intestinal A. Kleinridders, T. Wunderlich, H. Kashkar, O. Utermo¨hlen, et al. 2009. Ribo- flavin kinase couples TNF receptor 1 to NADPH oxidase. Nature 460: 1159– damage, additional gene deletion of TNFR1 greatly reduces the 1163. inflammatory conditions, enabling the mice to survive. Thus, the 20. Omori, E., S. Morioka, K. Matsumoto, and J. Ninomiya-Tsuji. 2008. TAK1 effects of TNF downregulation have some similarities between regulates reactive oxygen species and cell death in keratinocytes, which is es- sential for skin integrity. J. Biol. Chem. 283: 26161–26168. IBD and the mouse model with intestinal epithelial-specific de- 21. Pfeffer, K., T. Matsuyama, T. M. Ku¨ndig, A. Wakeham, K. Kishihara, letion of TAK1. In the intestinal epithelial-specific TAK1-deletion A. Shahinian, K. Wiegmann, P. S. Ohashi, M. Kro¨nke, and T. W. Mak. 1993. mouse model, TNFR1 deletion did not block increased ROS, Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to en- dotoxic shock, yet succumb to L. monocytogenes infection. Cell 73: 457–467. apoptosis, or disruption of tight junctions. Although anti-TNF 22. Madison, B. B., L. Dunbar, X. T. Qiao, K. Braunstein, E. Braunstein, and therapy is effective in the treatment of select IBD patients, it D. L. Gumucio. 2002. Cis elements of the villin gene control expression in re- does not block all of the associated pathologic conditions. Our stricted domains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine. J. Biol. Chem. 277: 33275–33283. results raise the possibility that upregulation of the TAK1-Nrf2 23. el Marjou, F., K. P. Janssen, B. H. Chang, M. Li, V. Hindie, L. Chan, D. Louvard, pathway could reduce the level of ROS and enhance enterocyte P. Chambon, D. Metzger, and S. Robine. 2004. Tissue-specific and inducible survival and integrity of tight junctions. The TAK1 pathway Cre-mediated recombination in the gut epithelium. Genesis 39: 186–193. 24. Morioka, S., E. Omori, T. Kajino, R. Kajino-Sakamoto, K. Matsumoto, and may be a novel target involved in regulating intestinal barrier J. Ninomiya-Tsuji. 2009. TAK1 kinase determines TRAIL sensitivity by mod- function. ulating reactive oxygen species and cIAP. Oncogene 28: 2257–2265. 25. Sellon, R. K., S. Tonkonogy, M. Schultz, L. A. Dieleman, W. Grenther, E. Balish, D. M. Rennick, and R. B. Sartor. 1998. Resident enteric bacteria are Acknowledgments necessary for development of spontaneous colitis and immune system activation We thank Y. Tsuji for discussions and M. Mattmuler, K. Ryan, and B.J. in interleukin-10-deficient mice. Infect. Immun. 66: 5224–5231. 26. Takaesu, G., S. Kishida, A. Hiyama, K. Yamaguchi, H. Shibuya, K. Irie, Welker for support. J. Ninomiya-Tsuji, and K. Matsumoto. 2000. TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway. Mol. Cell 5: 649–658. Disclosures 27. Moeser, A. J., P. K. Nighot, K. J. Engelke, R. Ueno, and A. T. Blikslager. 2007. The authors have no financial conflicts of interest. Recovery of mucosal barrier function in ischemic porcine ileum and colon is stimulated by a novel agonist of the ClC-2 chloride channel, lubiprostone. Am. J. Physiol. Gastrointest. Liver Physiol. 292: G647–G656. References 28. Kobayashi, M., and M. Yamamoto. 2006. Nrf2-Keap1 regulation of cellular defense mechanisms against electrophiles and reactive oxygen species. Adv. 1. Crosnier, C., D. Stamataki, and J. Lewis. 2006. Organizing cell renewal in the Enzyme Regul. 46: 113–140. intestine: stem cells, signals and combinatorial control. Nat. Rev. Genet. 7: 349– 29. Jaiswal, A. K. 2004. Nrf2 signaling in coordinated activation of antioxidant gene 359. expression. Free Radic. Biol. Med. 36: 1199–1207. 2. Turner, J. R. 2006. Molecular basis of epithelial barrier regulation: from basic 30. Li, W., and A. N. Kong. 2009. Molecular mechanisms of Nrf2-mediated anti- mechanisms to clinical application. Am. J. Pathol. 169: 1901–1909. oxidant response. Mol. Carcinog. 48: 91–104. The Journal of Immunology 4737

31. Kishimoto, K., K. Matsumoto, and J. Ninomiya-Tsuji. 2000. TAK1 mitogen- 37. Yang, H., Y. Zeng, T. D. Lee, Y. Yang, X. Ou, L. Chen, M. Haque, R. Rippe, and activated protein kinase kinase kinase is activated by autophosphorylation within S. C. Lu. 2002. Role of AP-1 in the coordinate induction of rat glutamate- its activation loop. J. Biol. Chem. 275: 7359–7364. cysteine ligase and glutathione synthetase by tert-butylhydroquinone. J. Biol. 32. Kensler, T. W., N. Wakabayashi, and S. Biswal. 2007. Cell survival responses to Chem. 277: 35232–35239. environmental stresses via the Keap1-Nrf2-ARE pathway. Annu. Rev. Pharma- 38. Karin, M., and E. Shaulian. 2001. AP-1: linking hydrogen peroxide and col. Toxicol. 47: 89–116. oxidative stress to the control of cell proliferation and death. IUBMB Life 52: 33. Hooper, L. V. 2004. Bacterial contributions to mammalian gut development. 17–24. Trends Microbiol. 12: 129–134. 39. Hayden, M. S., and S. Ghosh. 2008. Shared principles in NF-kappaB signaling. 34. Khor, T. O., M. T. Huang, K. H. Kwon, J. Y. Chan, B. S. Reddy, and A. N. Kong. Cell 132: 344–362. 2006. Nrf2-deficient mice have an increased susceptibility to dextran sulfate 40. Pham, C. G., C. Bubici, F. Zazzeroni, S. Papa, J. Jones, K. Alvarez, S. Jayawardena, sodium-induced colitis. Cancer Res. 66: 11580–11584. E. De Smaele, R. Cong, C. Beaumont, et al. 2004. Ferritin heavy chain upregu- 35. Tsuji, Y. 2005. JunD activates transcription of the human ferritin H gene through an lation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing re- antioxidant response element during oxidative stress. Oncogene 24: 7567–7578. active oxygen species. Cell 119: 529–542. 36. Gerald, D., E. Berra, Y. M. Frapart, D. A. Chan, A. J. Giaccia, D. Mansuy, 41. Kamata, H., S. Honda, S. Maeda, L. Chang, H. Hirata, and M. Karin. 2005. J. Pouysse´gur, M. Yaniv, and F. Mechta-Grigoriou. 2004. JunD reduces tumor Reactive oxygen species promote TNFalpha-induced death and sustained JNK angiogenesis by protecting cells from oxidative stress. Cell 118: 781–794. activation by inhibiting MAP kinase phosphatases. Cell 120: 649–661. Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021