ATF3-Associated Pathways Proinflammatory Signals in Two Convergent Inactivation on Epithelial Nod2-Linked Novel Regulatory Actio

Novel Regulatory Action of Ribosomal Inactivation on Epithelial Nod2-Linked Proinflammatory Signals in Two Convergent ATF3-Associated Pathways This information is current as of September 27, 2021. Seong-Hwan Park, Kee Hun Do, Hye Jin Choi, Juil Kim, Ki-Hyung Kim, Jiyeon Park, Chang Gyu Oh and Yuseok Moon J Immunol 2013; 191:5170-5181; Prepublished online 4 October 2013; Downloaded from doi: 10.4049/jimmunol.1301145 http://www.jimmunol.org/content/191/10/5170 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/10/04/jimmunol.130114 Material 5.DC1 References This article cites 74 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/191/10/5170.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 © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Novel Regulatory Action of Ribosomal Inactivation on Epithelial Nod2-Linked Proinﬂammatory Signals in Two Convergent ATF3-Associated Pathways

Seong-Hwan Park,*,† Kee Hun Do,*,† Hye Jin Choi,*,† Juil Kim,*,† Ki-Hyung Kim,‡ Jiyeon Park,*,† Chang Gyu Oh,*,† and Yuseok Moon*,†

In response to excessive nucleotide-binding oligomerization domain–containing protein 2 (Nod2) stimulation caused by mucosal bacterial components, gut epithelia need to activate regulatory machinery to maintain epithelial homeostasis. Activating transcription factor 3 (ATF3) is a representative regulator in the negative feedback loop that modulates TLR-associated inﬂammatory responses. In the current study, the regulatory effects of ribosomal stress-induced ATF3 on Nod2-stimulated proinﬂammatory

signals were assessed. Ribosomal inactivation caused persistent ATF3 expression that in turn suppressed proinflammatory chemo- Downloaded from kine production facilitated by Nod2. Decreased chemokine production was due to attenuation of Nod2-activated NF-kB and early growth response protein 1 (EGR-1) signals by ATF3. However, the underlying molecular mechanisms involve two convergent regulatory pathways. Although ATF3 induced by ribosomal inactivation regulated Nod2-induced EGR-1 expression epigenetically through the recruitment of histone deacetylase 1, NF-kB regulation was associated with posttranscriptional regulation by ATF3 rather than epigenetic modification. ATF3 induced by ribosomal inactivation led to the destabilization of p65 mRNA caused by nuclear entrapment of transcript-stabilizing human Ag R protein via direct interaction with ATF3. These findings demonstrate http://www.jimmunol.org/ that ribosomal stress-induced ATF3 is a critical regulator in the convergent pathways between EGR-1 and NF-kB, which contributes to the suppression of Nod2-activated proinflammatory gene expression. The Journal of Immunology, 2013, 191: 5170–5181.

uman intestinal epithelium is a frontline defense against immune responses (2, 3). Acute Nod2 stimulation by MDP spe- luminal commensal bacteria. Contact between the in- cifically activates proinflammatory NF-kB and MAPK cascades H testinal epithelium and microorganisms is facilitated that mediate the expression of proinflammatory mediators, in- through interactions between the pattern recognition receptors cluding cytokines (4, 5). Although Nod2 usually triggers proin- by guest on September 27, 2021 (PRRs) and bacterial Ags (1). In response to N-acetylmuramyl-L- flammatory responses, Nod2-induced signals are also crucial for alanyl-D-isoglutamine (MDP), a minimal peptidoglycan moiety intestinal homeostasis because various Nod2 gene mutations are of both Gram-positive and Gram-negative bacteria, nucleotide- found in patients with Crohn’s disease, a type of inflammatory binding oligomerization domain–containing protein 2 (Nod2) is bowel disease (IBD) characterized by severe damage to intes- activated and initiates a wide spectrum of innate recognition and tinal tissues (6–8). Mechanistically, Nod2 increases the expression of defensin, a factor that contributes to gut protection against harmful bacteria. *Laboratory of Mucosal Exposome and Biomodulation, Department of Microbiology To maintain gut homeostasis in the presence of luminal bacteria, and Immunology, Medical Research Institute, Pusan National University School of the intestinal epithelia are hyporesponsive to bacterial inflamma- † Medicine, Yangsan 626-870, Korea; Immunoregulatory Therapeutics Group in Brain, gens via regulatory mechanisms (9). As a representative negative Busan 21 Project, Busan 609-735, Korea; and ‡Department of Obstetrics and Gynecol- ogy, School of Medicine and Medical Research Institute, Pusan National University regulator of the PRR-linked signals, the peroxisome proliferator– Hospital, Busan 602-739, Korea activated receptor g (PPARg) mediates immune tolerance to Received for publication April 30, 2013. Accepted for publication September 4, bacteria-stimulated inflammation associated with colitis (10–12). 2013. Genetic ablation of PPARg is observed in patients with ulcerative This work was supported by the Basic Science Research Program through the Na- colitis, which is associated with severe chronic inflammatory tional Research Foundation of Korea, funded by Ministry of Education, Science and Technology Grant 2012R1A1A2005837, and a grant from the Korean Health Tech- outcomes (10). As another regulatory transcription factor, ac- nology Research and Development Project, Ministry of Health and Welfare, Republic tivating transcription factor 3 (ATF3) is induced as part of the of Korea (HI13C0259). negative feedback loop that modulates PRR-stimulated inflam- Address correspondence and reprint requests to Prof. Yuseok Moon, Department of matory responses (13–15). In particular, ATF3 regulates the Microbiology and Immunology, Pusan National University School of Medicine, Yangsan 626-870, Korea. E-mail address: [email protected] expression of proinflammatory cytokine genes, such as ones The online version of this article contains supplemental material. encoding MIP1-b, IL-6, and IL-12. Not surprisingly, ATF3- deficient macrophages produce elevated levels of IL-6 and IL- Abbreviations used in this article: 15-AcDON, 15-acetyl deoxynivalenol; ANS, anisomycin; ATF3, activating transcription factor 3; CXCL-1, chemokine (C-X-C motif) 12p40 cytokines in response to TLR 4 activator, a bacterial LPS ligand 1; DON, deoxynivalenol; EGR-1, early growth response protein 1; HDAC, (13, 15). Mechanistically, the negative regulation of transcription histone deacetylase; HSP, heat shock protein; HuR, human Ag R; IBD, inflammatory d bowel disease; IP, immunoprecipitation; MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine; by ATF3 may occur indirectly via inhibition of C/EBP , a positive Nod2, nucleotide-binding oligomerization domain–containing protein 2; PPARg, per- regulator of cytokine gene induction (16). In addition, recent oxisome proliferator–activated receptor g; PRR, pattern recognition receptor; shRNA, studies have shown that ATF3 mediates epigenetic regulation of short hairpin RNA. proinflammatory cytokines (13, 17). This finding suggests that Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 ATF3 can potentially function as a therapeutic target of immune www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301145 The Journal of Immunology 5171 regulation and tolerance in patients with chronic inflammatory was confirmed by expression of a pMX-GFP vector. For transient ex- diseases, including IBD. pression of ATF3-SC, shEGR-1, shATF3, and shHDAC-1, cells were On the basis of the assumption that epithelial ATF3 can promote transfected using jetPRIME or OmicsFect according to the manufacturer’s instructions. At 5 h after transfection with jetPRIME, the media were tolerance to Nod2-associated proinflammatory activation in the changed and the cells were incubated for another 48 h. For OmicsFect intestine, specific ribosome-directed xenobiotics were used as po- transfection, the cell culture media were changed after 24 h. tent ATF3 modulators and assessed for their regulatory actions on Western blot analysis Nod2-stimulated proinflammatory signals. Ribosome-inactivating xenobiotics such as anisomycin (ANS), UV radiation, ricin, and Levels of protein expression were compared by Western blot analysis. Cells a variety of sesquiterpenoid trichothecene fungal metabolites can were washed with ice-cold phosphate buffer, lysed by boiling in lysis buffer [1% (w/v) SDS, 1.0 mM sodium ortho-vanadate, and 10 mM Tris (pH 7.4)], disrupt 28S rRNA during gene translation (18, 19). This disruption and sonicated for 5 s. Proteins in the lysates were quantified using a BCA elicits a ribotoxic stress response that activates intracellular senti- Protein Assay Kit (Pierce, Rockford, IL). A total of 50 mg protein was nel signaling pathways. Ribosomal inactivation results in the ex- separated by electrophoresis using a Bio-Rad mini gel apparatus (Bio-Rad, pression of genes, such as ATF3, that are important for cellular Hercules, CA). The proteins were transferred onto polyvinylidene difluoride membrane (Pall Corporation, New York City, NY), and the blots homeostasis and of genes essential for a variety of processes in- were blocked for 1 h with 5% skimmed milk in TBS plus 0.1% Tween 20 volved in cell survival, proliferation, and death, as well as in- (TBST) before being probed with primary Abs for 2 h at room temperature flammatory responses (20, 21). In the current study, ribosomal or overnight at 4˚C. The primary Abs used were rabbit polyclonal human inactivation was used as the inducer of prolonged ATF3 expression anti-actin, rabbit polyclonal anti-ATF3, rabbit polyclonal anti–EGR-1, and assessed for its usefulness in epithelial hyporesponsiveness rabbit polyclonal anti–NF-kB p65, and mouse monoclonal anti-hnRNP Abs (Santa Cruz Biotechnology, Santa Cruz, CA) along with rabbit to Nod2 stimulation, and the underlying molecular mechanism of polyclonal anti-phosphorylated (p)-p65 and mouse monoclonal anti– Downloaded from ATF3-linked action was addressed. HDAC-1 Abs (Cell Signaling Technology, Beverly, MA). After washing three times with TBST, the blots were incubated with horseradish- conjugated secondary Ab for 1 h and then washed again three times Materials and Methods with TBST. Ab binding was detected with pico enhanced peroxidase de- Cell culture and reagents tection (ELPIS Biotech, Daejon, South Korea). For immunoprecipitation (IP), primary 1 mg Ab (anti-ATF3, anti-HDAC1, or anti-HuR) was added Human epithelial cell lines, including HCT-8, HCT-116, and intestinal 407

to the sample and incubated overnight at 4˚C. Protein G PLUS-agarose http://www.jimmunol.org/ cells, were purchased from the American Type Culture Collection (Man- (30 ml per sample; Santa Cruz Biotechnology) was then added to the Ab– assas, VA). All cells were maintained in RPMI 1640 medium (Invitrogen, nuclear extracts, and the solution was rotated at 4˚C for another 3 h. Next, Carlsbad, CA) supplemented with 10% (v/v) heat-inactivated FBS (Sigma- the bound Ab–nuclear extracts were washed three times with IP lysis Aldrich, St. Louis, MO), 50 U/ml penicillin (Sigma-Aldrich), and 50 mg/ml buffer(50mMTris,pH7.2,150mMNaCl,1mMEDTA,400mM streptomycin (Sigma-Aldrich) in a 5% CO2 humidified incubator (Sanyo, Na3VO4, and 2.5 mM phenylmethanesulfonylfluoride) and 63 SDS sam- Tokyo, Japan) at 37˚C. Medium for the intestinal 407 cells was addition- ple buffer (60% glycerol, 300 mM Tris pH 6.8, 12 mM EDTA, 12% SDS, ally supplemented with 1% nonessential amino acids (Invitrogen). More- 864 mM 2-ME, and 0.05% bromophenol blue) was added. The immuno- over, we isolated the mouse enterocytes to validate the cell line–based precipitates were boiled at 95˚C for 5 min and collected by centrifugation results. Briefly, 6-wk-old male C57BL/6 mice were anesthetized and (12,000 rpm; 10 min and 4˚C) and subjected to SDS-PAGE. exsanguinated, and the entire small intestine was opened longitudinally and washed in 20 ml HBSS several times until luminal matter was com- Isolation of cellular fractions by guest on September 27, 2021 pletely removed by inverting. The intestine was then cut into 0.5-mm-long fragments and incubated in 5 ml prewarmed 0.05% trypsin for 20 min at For nuclear protein isolation, cells were scraped into ice-cold PBS. The cell 37˚C. After incubation, the detached enterocytes were passed through the pellet was resuspended in lysis buffer containing 10 mM HEPES, 10 mM cell strainer and collected in RPMI 1640 medium. Cells were separated by KCl, 1.5 mM MgCl2, 0.5 mM DTT, 0.5 mM PMSF, 0.1% Nonidet P-40, centrifugation in the 25/40% discontinuous Percoll (Sigma-Aldrich) gra- and protease inhibitor mixture (Sigma-Aldrich); incubated for 10 min on dient at 600 g 3 10 min. Small-intestinal epithelial cells were collected ice; and centrifuged. Supernatant (the cytosolic fraction) was collected from the interface between two different Percoll gradients and resuspended while the pellet was resuspended in the buffer containing 20 mM HEPES, in RPMI 1640 media. Cell number was determined by trypan blue (Sigma- 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 0.2 mM Aldrich) dye exclusion using a hemocytometer. Deoxynivalenol (DON) PMSF, 25% glycerol, and a protease inhibitor mixture (Sigma-Aldrich). (97.6 6 2.4% pure) isolated from Fusarium graminearum was obtained After incubating for 10 min on ice, the samples were centrifuged, and from Sigma-Aldrich. Bay 11-7082 was purchased from Calbiochem (San the supernatants (nuclear proteins) were collected, aliquoted, and stored 2 Diego, CA). All other chemicals were purchased from Sigma-Aldrich. at 80˚C before analysis. Construction of plasmids Conventional and real-time RT-PCR Total RNA was extracted with RiboEx (GeneAll Biotech, Seoul, South The cDNA containing the entire coding region of ATF3, including the Korea) according to the manufacturer’s instructions. RNA (300 ng) from TATAA region, was generated by RT-PCR using RNA from HCT-8 cells each sample was transcribed to cDNA using Prime RT premix (Genetbio, with the following primers: 59-CGTGAGTCCTCGGTGCTC-39 (forward) Nonsan, South Korea). cDNA amplification was performed with Takara and 59-GACAGCTCTCCAATGGCTTC-39 (reverse). The resulting 721-bp HS ExTaq DNA polymerase (Takara Bio, Shiga, Japan) in a MyCycler construct was cloned using a TopCloner TA Kit (Enzynomics, Daejeon, Thermal Cycler (Bio-Rad) using the following parameters: denaturation at South Korea) followed by excision at the HindIII/NotI sites, and then 94˚C for 2 min followed by 25 cycles of denaturation at 98˚C for 10 s, transferred in the sense orientations into the expression plasmid pcDNA3.1 annealing at 59˚C for 30 s, and elongation at 72˚C for 45 s. An aliquot of Zeo(+) (Invitrogen) using T4 DNA ligase (NEB, Beverly, MA). This vector each PCR product was subjected to 1.2% (w/v) agarose gel electrophoresis was named ATF3-SC. CMV-driven small interfering RNA was generated by and visualized by staining with ethidium bromide. The following 59 for- inserting short hairpin RNA (shRNA) templates into a pSilencer 4.1-CMV- ward and 39 reverse-complement PCR primers were used for amplification: neo vector (Ambion, Austin, TX). The vectors containing early growth human IL-8 (59-ATG ACT TCC AAG CTG GCC GTG GCT-39 and 59- response protein 1 (EGR-1), ATF3, histone deacetylase (HDAC1) 1, or TCT CAG CCC TCT TCA AAA ACT TCT C-39), human MCP-1 (59-TCT human Ag R (HuR) shRNA inserts were named shEGR-1, shATF3, GTG CCT GCT GCT CATAG-39 and 59-TGG AAT CCT GAA CCC ACT shHDAC1, or shHuR, respectively. EGR-1, ATF3, HDAC-1, and HuR TC-39), human chemokine (C-X-C motif) ligand 1(CXCL-1) (59-CTG shRNA targeted the sequences 59-GTT ACT ACC TCT TAT CCA T-39,59- CTC CTG CTC CTG GTA G-39 and 59-TGG ATT TGT CAC TGT TCA TGA AGA AGA TGA AAG GAA A-39,59-GCA GAT GCA GAG ATT GCA-39), human EGR-1 (59-CAG TGG CCT AGT GAG CAT GA-39 and CAA C-39,and59-GTG CAA AGG GTT TGG CTT T-39, respectively (22). 59-CCG CAA GTG GAT CTT GGT AT-39), human ATF3 (59-CTC CTG Transient transfection GGT CAC TGG TGT TT-39 and 59-AGG CAC TCC GTC TTC TCC TT- 39), human GAPDH (59-TCA ACG GAT TTG GTC GTA TT-39 and 59- Cells were transfected with a mixture of plasmids using jetPRIME (Polyplus CTG TGG TCA TGA GTC CTT CC-39), mouse CXCL-1 (59-CCC GCT Transfection, New York City, NY) or OmicsFect (Omicsbio, Taipei City, CGC TTC TCT GTG-39 and 59-ACT TGG GGA CAC CTT TTA GCA Taiwan) according to the manufacturer’s protocol. Transfection efficiency -39), mouse CXCL-2 (59-GTC CTG CTG CTG CTG CT-39 and 59-CAG 5172 REGULATION OF Nod2-LINKED PROINFLAMMATORY SIGNALS

TTA GCC TTG CCT TTG TTC A-39), mouse MCP-1 (59-CAG ATG CAG ELISA TTA ACG CCC CA-39 and 59-TGG AAT CCT GAA CCC ACT TC-39), and mouse GAPDH (59-TCA ACG GAT TTG GCC GTATT-39 and 59-CTG IL-8 in cell supernatants was quantified using an ELISA. HCT-8 cells were 3 4 TGG TCA TGA GCC CTT CC-39). For real-time PCR, FAM was used as plated at a density of 5 10 cells in each well of a 24-well plate. After a fluorescent reporter dye and conjugated to the 59 ends of the probes used pretreatment with DON, ANS, 15-acetyl DON (15-AcDON), or vehicle, to detect the amplified cDNA. Real-time PCR was performed with an the cell culture medium was removed and the cells were treated with ve- iCycler Thermal Cycler (Bio-Rad), using the following parameters: de- hicle or 10 mg/ml MDP for 18 h. Cell debris was removed from the naturation at 94˚C for 2 min followed by 40 cycles of denaturation at 98˚C culture media by centrifugation. IL-8 concentrations were measured for 10 s, annealing at 59˚C for 30 s, and elongation at 72˚C for 45 s. Each with an ELISA using an OptEIA Human IL-8 ELISA Kit (BD Biosciences, sample was tested in triplicate to ensure statistical significance. Relative Franklin Lakes, NJ) according to the manufacturer’s instructions. Briefly, quantification of gene expression was performed using the comparative Ct the ELISA plates were coated with capture Ab overnight at 4˚C. After method. For this, the Ct value is defined as the point at which a statistically washing with PBS containing Tween 20 and blocking overnight with PBS significant increase in fluorescence is observed. The number of PCR cycles supplemented with 10% (v/v) FBS at 4˚C, the plates were incubated with (Ct) required for the FAM intensities to exceed a threshold value just above serial dilutions of the samples and IL-8 standards. After treatment with background was calculated for the test and reference reactions. In all experi- detection Ab and tetramethylbenzidine substrate, absorbance was mea- ments, GAPDH was used as the internal control. Results were analyzed in sured at 405 nm, using an ELISA reader. The assay detection limit was a relative quantitation study with the vehicle treated. 3.1 pg/ml IL-8. Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 1. Effects of ATF3 on Nod2-induced IL-8 expression in human enterocytes. (A) After HCT-8 cells transfected with ATF3-SC or an empty vector were pretreated with a ribosomal inactivator (500 ng/ml DON) for 9 h, the cells were then treated with the vehicle (PBS) or 10 mg/ml MDP for 18 h. The concentration of IL-8 secreted into the culture media was then measured using an ELISA. Blots to the left in the box show ATF3 expression in the transfected HCT-8 cells. Blots to the right in the box show ATF3 expression induced by ribosomal-inactivating DON in the presence of MDP. Total cell lysates were subjected to Western blot analysis. *, A significant difference between two groups (p , 0.05). (B) HCT-8 cells pretreated with 500 ng/ml DON for 9 h, ANS for 24 h, or 15-AcDON for 9 h were incubated with the vehicle (PBS) or 10 mg/ml MDP for 18 h. IL-8 secreted into the culture medium was quantitated using an ELISA. *A significant difference from the group treated with MDP alone (p , 0.05). (C) HCT-8 cells transfected with an empty vector or shATF3 were pretreated with DMSO or 500 ng/ml DON for 9 h. The cells were then treated with the vehicle (PBS) or 10 mg/ml MDP for 18 h. IL-8 secreted into the culture medium was quantitated using an ELISA. Different letters over each bar with the SD represent significant differences between the two groups (p , 0.05). (D) HCT-8 cells transfected with an empty vector or shATF3 were pretreated with DMSO or 500 ng/ml ANS for 24 h. The cells were then treated with the vehicle (PBS) or 10 mg/ml MDP for 18 h. IL-8 secreted into the medium was quantitated using an ELISA. The figures in the box represent the relative levels of ATF3 mRNA in HCT-8 cells transfected with an empty vector or shATF3. *A significant difference between two groups (p , 0.05). The Journal of Immunology 5173

Confocal microscopy After centrifugation, each sample was washed twice in dilution buffer. The chromatin was resuspended in a dilution buffer:lysis buffer (9:1) solution Cells were incubated in a glass-bottom culture dish. After pretreatment with and incubated at 37˚C with proteinase K and RNase A (500 mg/ml per DMSO as vehicle or ribotoxins, the cells were treated with 10 mg/ml MDP. sample) for 1 h. Chromatin was purified using a MEGAquick-spin kit The cells were fixed with 4% paraformaldehyde (Biosesang, Sungnam, (iNtRON, Sungnam, South Korea). The recovered chromatin was amplified South Korea). The fixed cells were permeabilized with 0.2% Triton X-100 with HS Prime Taq DNA Polymerase (Genet Bio, Nonsan, South Korea). in PBS for 10 min. After 2-h blocking with 3% BSA in PBS, the cells were Amplification was performed with Takara HS ExTaq DNA Polymerase incubated at room temperature for 2 h with target Ab diluted 1:200 in (Takara Bio) in a Mycycler Thermal Cycler (Bio-Rad). The following 59 a buffer (3% BSA in PBS). Next, the cells were repeatedly washed with forward and 39 reverse-complement PCR primers were used for amplifi- PBS and incubated with Alexa Fluor 488 goat anti-rabbit IgG (H + L), cation: human EGR-1 promoter (59-CTAGGGTGCAGGATGGAGGT-39 Alexa Fluor 546 goat anti-mouse IgG (H + L), or Texas Red (SurModics, and 59-GAA CAC TGA GAA GCG TGC AG-39). Eden Prairie, MN) for 2 h at room temperature. The cells were repeatedly washed in PBS and subsequently stained with 100 ng/ml DAPI (absor- RNA IP bance at 405 nm) in PBS for 10 min. Confocal images were obtained with an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan) using Immunoprecipitation of protein–RNA complexes was performed using single-line excitation (488 nm for anti-rabbit Abs or 546 nm or 596 nm for a modified protocol for chromatin IP (23). Briefly, HCT-8 cells were 6 anti-mouse Abs) or multitrack sequential excitation (488 nm and 633 nm). seeded at a density of 2.5 3 10 cells per 100-mm dish in complete RPMI Images were acquired and processed with FV10-ASW software. 1640 and grown for 24 h. The cells were pretreated with 500 ng/ml DON (ribotoxin), and then incubated with 10 mg/ml MDP for 2 h. After treat- Chromatin IP assay ment, protein and RNA were cross-linked with 1% formaldehyde for 10 min at room temperature. The cytoplasmic extract was incubated overnight Cells were cross-linked for 10 min in 1% formaldehyde. The reaction was at 4˚C with 5 mg of either goat anti-mouse IgG (nonspecific control) or stopped by the addition of glycine at a final concentration of 125 mM, and 3 anti-HuR Ab. The Ab-bound complexes were precipitated with protein G– Downloaded from the cells were washed twice with 1 PBS. Chromatin was fragmented to Sepharose beads, and then sequentially washed in low-salt, high-salt, LiCl, a size of 1000–2000 bp by sonication for 10 s of nine times in lysis buffer and TE buffers (5 min per wash). The protein–RNA complexes were eluted [1% (w/v) SDS, 10 mM EDTA (pH 8.0), 50 mM Tris-HCl (pH 8.0), and from the protein G–Sepharose beads with 250 ml elution buffer at 37˚C for a protease inhibitor mixture] using a Vibra-Cell sonicator (Sonics and 15 min. RNA in the immunoprecipitated complexes was released by re- Materials, Newtown, CT). The soluble chromatin was immunoprecipitated versing the cross-linkage by incubating at 65˚C for 4–5 h in 200 mM NaCl with 2 mg target Ab in a solution of nine parts dilution buffer [1% Triton and 20 mg proteinase K. RNA was then extracted with TRIzol reagent and X-100, 150 mM NaCl, 2 mM EDTA (pH 8.0), 20 mM Tris (pH 8.0), and subjected to RT-PCR.

protease inhibitor mixture] and one part lysis buffer. After rotating over- http://www.jimmunol.org/ night at 4˚C, protein G PLUS-agarose (30 ml; Santa Cruz Biotechnology) Statistical analysis was added to each chromatin/Ab sample in a 100-mL volume containing a 9:1 mixture of dilution buffer and lysis buffer with 100 mg/ml BSA Data were analyzed using SigmaStat for Windows (Jandel Scientiﬁc, San (Promega, Madison, WI) and 500 mg/ml salmon sperm DNA (Invitrogen). Rafael, CA). To compare two groups of data, the Student t test was used. To by guest on September 27, 2021

FIGURE 2. Effects of ATF3 on Nod2-induced chemokine gene expression in human enterocytes. (A) HCT-8 cells pretreated with 500 ng/ml DON for 9 h, ANS for 24 h, or 15-AcDON for 9 h were exposed to the vehicle (PBS) or 10 mg/ml MDP for 2 h. The mRNA expression was measured using real-time RT-PCR. *A significant difference from the group treated with MDP alone (p , 0.05). (B) HCT-8 cells transfected with an empty vector or ATF3-SC were incubated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. The mRNA expression was measured using conventional RT-PCR. (C and D) HCT-116 (C)or intestine 407 (D) cells pretreated with DMSO or 500 ng/ml DON for 9 h were incubated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. The mRNA expression was measured using real-time RT-PCR. *A significant difference compared with the group treated with MDP alone (p , 0.05). 5174 REGULATION OF Nod2-LINKED PROINFLAMMATORY SIGNALS compare multiple groups, data were subjected to an ANOVA, and pairwise cecum of the human small intestine, is one of the organs most comparisons were made using the Student–Newman–Keuls method. Data susceptible to the biological effects of ribosomal inactivation (30, not meeting the normality assumptions were subjected to Kruskal–Wallis 31). In addition to DON, increased ATF3 expression promoted by ANOVA by ranks, and pairwise comparisons were then made with the Student–Newman–Keuls method. other ribosome-inactivating agents, such as 15-AcDON and ANS, also suppressed MDP-induced IL-8 production in a dose-dependent manner (Fig. 1B). The direct involvement of ATF3 in the sup- Results pression of IL-8 production by ribosomal inactivation was assessed Nod2-induced proinflammatory chemokines are suppressed by usingATF3shRNA.AblationofATF3expressionlessenedthe induced ATF3 in human enterocytes suppressive effect of ribosome-inactivating agents (DON and ANS) Intracellular accumulation of bacterial products such as MDP on Nod2-activated IL-8 production (Fig. 1C, 1D). In addition, triggers signals that lead to the initiation of intestinal inflammatory ribosomal inactivation or ATF3 overexpression downregulated responses via NF-kB signaling, particularly during the devel- MDP-induced mRNA expression of other chemokines (Fig. 2A, opment of IBD (24, 25). The basic hypothesis is that ATF3 can 2B), including MCP-1 and CXCL-1. Similar suppressive effects attenuate MDP-linked proinflammatory responses in human in- on chemokine expression by ribosomal inactivation were also ob- testinal epithelial cells. In the current study, induction of ATF3 served in other intestinal epithelial cells, such as HCT-116 and by ribosomal inactivation or plasmid transfection suppressed IL-8 INT-407 (Fig. 2C, 2D). Moreover, the chemokine suppression was production promoted by Nod2 ligand in HCT-8 human intestinal validated in the primary mouse enterocytes as well (Supplemental epithelial cells (Fig. 1A). The blots shown in Fig. 1A (box) con- Fig. 1A). Finally, the suppression of ATF3 expression using shRNA

firmed findings from our previous report demonstrating that ATF3 was shown to attenuate the action of ribosome-inactivating agents Downloaded from expression is induced by the ribosomal inactivator DON in human (DON and ANS) in human enterocytes (Fig. 3A, 3B), demon- enterocytes (26). In the current study, the effects of ATF3 or ATF3- strating the direct involvement of ATF3 in the reduction of che- inducing ribosomal inactivation on chemokine expression were mokine expression by ribosomal inactivation. Taken together, assessed in cultured HCT-8 intestinal epithelial cells, which are these data suggest that ribosomal inactivation suppresses Nod2- frequently used as a model of microbial infection and inflammatory induced proinflammatory chemokine expression via ATF3 in human

diseases (27–29). Moreover, the source of HCT-8 cells, the ileo- enterocytes. http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Effect of ATF3 on the expression of proinflammatory chemokines altered by ribosomal inactivation. (A) HCT-8 cells transfected with an empty vector or shATF3 were pretreated with DMSO or 500 ng/ml DON for 9 h and subsequently treated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. The mRNA expression was measured using real-time RT-PCR. (B) HCT-8 cells transfected with an empty vector or shATF3 were pretreated with DMSO or 500 ng/ml ANS for 24 h and then incubated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. The mRNA expression was measured using real-time PCR. The figures in the box represent the relative levels of ATF3 mRNA in HCT-8 cells transfected with an empty vector or shATF3. *A significant difference compared with the group treated with MDP alone (p , 0.05). #A significant difference from the group of empty vector-transfected cells under the treatment with MDP/ribosomal inactivation (p , 0.05). The Journal of Immunology 5175

ATF3 induction suppresses proinflammatory transcription phosphorylation (Fig. 4B). However, pre-exposure to ATF3- factors NF-kB and EGR-1 promoted by epithelial Nod2 inducing ribosomal inactivation using DON attenuated EGR-1 Among the various proinflammatory transcription factors, NF-kB and NF-kB activation in a dose-dependent manner. In contrast plays central roles in Nod2-activated proinflammatory responses to the suppression of EGR-1 and NF-kB, ATF3 expression was in human enterocytes (32–35). Moreover, the expression of some gradually enhanced by increasing doses of ribosome-inactivating NF-kB–associated proinflammatory transcription factors such as DON, indicating that ATF3 expression may be inversely associ- EGR-1 can be induced by a range of stimuli, including NF-kB ated with that of EGR-1 and NF-kB in response to Nod2 stimu- activators (36–38). As expected, both NF-kBandEGR-1were lation in HCT-8 epithelial cells. Similar patterns of Nod2-activated positively involved in chemokine induction by Nod2 activation in transcription factor were also observed in cells subjected to other the current study (Fig. 4A). This finding was observed using EGR- ribosomal insults, including ANS and 15AcDON (Fig. 4C). 1 shRNA and Bay 11-7082, a specific NF-kB inhibitor. Epithelial Moreover, these were validated in the primary mouse enterocytes Nod2 activation also enhanced EGR-1 expression as well as p65 (Supplemental Fig. 1B). The effects of ATF3 overexpression on Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Effects of increased ATF3 expression via ribosomal inactivation on Nod2-induced NF-kB and EGR-1 expression in human enterocytes. (A) HCT-8 cells transfected with an empty vector or shEGR-1 were pretreated with DMSO or 20 mM Bay 11-7082 and then incubated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. The mRNA expression was measured using RT-PCR. The figures in the box represent the relative levels of EGR-1 mRNA in HCT-8 cells transfected with an empty vector or shEGR-1. (B) After HCT-8 cells were pretreated with different concentrations of DON for 9 h, the cells were incubated with 10 mg/ml MDP for 1 or 2 h. Cell lysates were then subjected to Western blot analysis. (C) HCT-8 cells pretreated with 500 ng/ml DON for 9 h, ANS for 24 h, or 15-AcDON for 9 h were then incubated with 10 mg/ml MDP for the indicated time. Cell lysates were subjected to Western blot analysis. (D) HCT-8 cells transfected with an empty vector or ATF3-SC were treated with the vehicle (PBS) or 10 mg/ml MDP for 1 h to detect EGR-1 or for 2 h to detect phosphorylated (p)-p65. Image in the box (right) indicates ATF3 expression in HCT-8 cells expressing either the empty vector or ATF3-SC. Original magnification 31800. (E) After pretreatment with DMSO or 500 ng/ml DON for 9 h, HCT-8 cells were treated with the vehicle (PBS) or 10 mg/ml MDP for 1 h to detect EGR-1 and p-p65. The cells were then stained with anti–EGR-1 Ab, anti–p-p65 Ab, and DAPI before being examined by confocal microscopy (original magnification 31800). The right panel shows the relative quantitative values of nuclear EGR-1 and p-p65. The ratio is the measured nuclear densities of EGR-1 or p-p65 signals corresponding to the DAPI-stained area. *A significant difference compared with the group treated with MDP alone (p , 0.05). (F and G) HCT-8 cells transfected with an empty vector or shATF3 were pretreated with 500 ng/ml DON for 9 h (F) or 500 ng/ml ANS for 24 h (G). The cells were then treated with 10 mg/ml MDP for the indicated time. Cell lysates were subjected to Western blot analysis. 5176 REGULATION OF Nod2-LINKED PROINFLAMMATORY SIGNALS

Nod2-activated EGR-1 and NF-kB were also confirmed with con- result was partly decreased by Nod2 activation (Fig. 6A). Con- focal microscopy (Fig. 4D). Nod2 activation enhanced the ex- focal microscopy demonstrated that ribosomal inactivation en- pression and nuclear localization of EGR-1 and p65 in human hanced ATF3 expression and its nuclear interaction with HDAC1, enterocytes. This result was attenuated by elevated ATF3. Ribosome- and Nod2 activation itself had little effect on this interaction (Fig. inactivating compounds that increased ATF3 expression also caused 6B). Our IP assay also confirmed that ATF3 and HDAC1 inter- similar reductions of Nod2-activated EGR-1 and p65 in the nuclei acted in cells subjected to ribosomal inactivation (Fig. 6C). In (Fig. 4E). These findings indicated that ATF3 is negatively associ- summary, these data show that ribosomal inactivation increased ated with proinflammatory transcription factors such as EGR-1 and the recruitment of HDAC1 by ATF3. This accounted for the epi- NF-kB in response to Nod2 stimulation. Finally, the direct in- genetic regulation of EGR-1 transcription in response to Nod2 volvement of ATF3 in the regulation of Nod2-activated proin- activation in the epithelial cells. In contrast to the previously flammatory EGR-1 and NF-kB was assessed. Ablation of ATF3 known mechanism of epigenetic regulation of NF-kBbyATF3, expression with shRNA lessened the suppressive effects of ribosome- HDAC1-mediated epigenetic regulation by ATF3 did not con- inactivating agents (DON or ANS) on proinflammatory NF-kB tribute to the inhibition of NF-kB expression by ribosomal inac- and EGR-1 activated by Nod2 ligand in human enterocytes (Fig. tivation. 4F, 4G). Taken together, results of these experiments demon- Ribosomal inactivation–induced ATF3 suppresses strated that increased ATF3 expression induced by ribosomal Nod2-activated NF-kB through p65 mRNA destabilization inactivation mediated the suppression of Nod2-activated NF-kB and EGR-1 expression, which contributed to reduced chemokine Because NF-kB signal was not epigenetically regulated by ribo- production in Nod2-activated enterocytes. somal inactivation, other gene regulation modes such as tran- Downloaded from scription and posttranscription were assessed. Along with reduced Ribosomal inactivation–induced ATF3 epigenetically regulates phosphorylation of NF-kB, total expression of p65, a central Nod2-induced EGR-1 expression via HDAC1 subunit of canonical NF-kB, was also decreased by ribosomal Because epigenetic modification is a well-known ATF3-associated inactivation (Fig. 7A). Moreover, exogenous ATF3 overexpression regulatory mechanism of NF-kB activation by bacterial LPS, subsequently suppressed both p65 expression and phosphorylation

we assessed the effect of HDAC inhibition on ATF3-mediated (Fig. 7B). However, because ribosomal inactivation did not affect http://www.jimmunol.org/ regulation of Nod2-stimulated NF-kB and EGR-1 expression. p65 transcriptional activity (data not shown), it was therefore as- Contrary to our expectations, chemical HDAC inhibition using sumed that ribosomal inactivation may lead to alterations in p65 trichostatin A did not attenuate the reduction of NF-kB by ribo- mRNA stability via ATF3. Measurement of p65 mRNA stability somal inactivation (Fig. 5A). Instead, HDAC inhibition restored demonstrated that ribosomal inactivation signiﬁcantly destabilized EGR-1 expression in response to Nod2 activation. Similar patterns p65 mRNA (Fig. 7C). This stability was restored by suppressing were also observed in cells deﬁcient in HDAC1, using HDAC1 ATF3 expression using shRNA (Fig. 7C). Moreover, the destabi- shRNA (Fig. 5B). Therefore, ATF3-mediated epigenetic regulation lization of p65 mRNA by ribosomal inactivation was also validated appears to be more critical for EGR-1 expression than in NF-kB in the primary mouse enterocytes (Supplemental Fig. 1C). activated by Nod2 ligand, indicating the presence of convergent The mechanism underlying ATF3-mediated posttranscriptional by guest on September 27, 2021 regulatory pathways for NF-kB and EGR-1 signaling. regulation was assessed in enterocytes treated with Nod2 ligand. As It was next determined whether ribosomal inactivation–induced mediators of posttranscriptional regulation, RNA-binding proteins ATF3 translocates into the nuclei and can interact with the ATF/ may play important roles in ATF3-associated mRNA destabilization. CREB element of the EGR-1 promoter. Ribosomal inactivation Although most RNA-binding proteins destabilize targeted mRNA, strongly enhanced ATF3 bindingtotheEGR-1promoter;this a few of these proteins, including HuR, bind to 39 untranslated

FIGURE 5. Involvement of HDAC1 in ATF3- regulated EGR-1 expression. (A) After HCT-8 cells were pretreated to 500 ng/ml DON, with or without 80 nM trichostatin A (TSA), for 9 h, the cells were treated with 10 mg/ml MDP for the indicated time. Cell lysates were subjected to Western blot analysis. (B) HCT-8 cells transfected with an empty vector or shHDAC1 were pretreated with 500 ng/ml DON for 9 h. The cells were then incubated with 10 mg/ml MDP for the indicated time. Cell lysates were subjected to Western blot analysis. The ﬁgures in the box represent the relative levels of HDAC1 mRNA in HCT-8 cells transfected with an empty vector or shHDAC1. The mRNA expression was measured using RT-PCR. The Journal of Immunology 5177

FIGURE 6. Epigenetic regulation of EGR-1 transcription mediated by ATF3– HDAC1 complexes. (A)AfterHCT-8cells were pretreated with 500 ng/ml DON for 9 h, the cells were incubated with 10 mg/ml MDP for 1 h. Fragmented cellular chromatin was immunoprecipitated with anti-ATF3 Ab, and the collected chromatin fragments were subjected to PCR. The lower panel shows the quantitation of ATF3-bound chromatin fragment using real-time PCR. (B) After HCT-8 cells were pretreated with DMSO or 500 ng/ml DON for 9 h, the cells were treated with 10 mg/ml MDP for 2 h. The cells were then stained with anti-ATF3 Ab, anti-HDAC1 Ab, or DAPI and examined with a confocal microscope (original magniﬁcation 31800). (C)After HCT-8 cells were pretreated with DMSO Downloaded from or 500 ng/ml DON for 9 h, the cells were treated with 10 mg/ml MDP for 2 h. Nuclear fractions of the cell lysate were subjected to IP with anti-ATF3 or anti- HDAC1 Ab. Immunoprecipitated proteins were then analyzed by Western blotting. http://www.jimmunol.org/ regions with AU-rich elements and stabilize the transcripts of genes, were only marginally altered, Nod2 activation enhanced the cy- including ones encoding growth factors or proinﬂammatory cyto- tosolic translocation of HuR protein (Fig. 8C). This result was kines (33, 39). In the current study, the impact of HuR on p65 suppressed by ATF3 expression or ribosomal inactivation, indi- mRNA stabilization was assessed. In addition to ATF3 over- cating that ATF3 may inhibit the cytosolic release of HuR protein. expression and ATF3-inducing ribosomal inactivation, ablation of Interaction between ATF3 and HuR protein in the nuclei was also HuR expression using shRNA suppressed p65 protein expression assessed (Fig. 8D). Most ATF3 entered the nuclei and bound to

(Fig. 8A), and the blocking of HuR expression, using its shRNA, nuclear HuR through direct interaction visualized with confocal by guest on September 27, 2021 decreased the half-life of p65 mRNA (Fig. 8B), indicating that microscopy (Fig. 8E). This interaction may limit HuR-mediated HuR mediates p65 mRNA stabilization. In further examination of functions in the cytoplasm, including the stabilization of p65 HuR function, we determined whether ATF3 can modulate the transcript. As expected, although cytosolic p65 mRNA binding to behavior of the HuR protein. Although total levels of HuR protein HuR protein was enhanced by Nod2 activation, increased ATF3

FIGURE 7. Suppression of p65 mRNA stability by ribosomal inactivation. (A) After HCT-8 cells were pretreated with each dose of DON for 9 h, the cells were incubated with 10 mg/ml MDP for 2 h. Total cell lysates were subjected to Western blot analysis (B) HCT-8 cells transfected with an empty vector or ATF3-SC were treated with 10 mg/ml MDP for the indicated time. Total cell lysates were subjected to Western blot analysis. (C) HCT-8 cells transfected with an empty vector or shATF3 were treated with the vehicle control or 500 ng/ml DON for 9 h. The cellular transcription was then arrested with 5 mM actinomycin D in the presence of 10 mg/ml MDP for the indicated time. The level of p65 mRNA was measured at different time points after actinomycin D treatment, using real-time RT-PCR. *A signiﬁcant difference from the ribosome-inactivated group (s) at each indicated time (p , 0.05). 5178 REGULATION OF Nod2-LINKED PROINFLAMMATORY SIGNALS Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. Nuclear sequestration of HuR by ATF3 in response to Nod2 activation. (A) HCT-8 cells transfected with an empty vector, ATF3-SC, or shHuR were treated with DMSO or 500 ng/ml DON for 9 h. Total cell lysates were subjected to Western blot analysis. (B) The cellular transcription of HCT-8 cells transfected with an empty vector or shHuR was arrested with 5 mM actinomycin D in the presence of 10 mg/ml MDP for the indicated time. The level of p65 mRNA was measured at different time points after actinomycin D treatment, using real-time RT-PCR. *A significant difference from the control group (d) at each indicated time (p , 0.05). (C) HCT-8 cells transfected with an empty vector or ATF3-SC were pretreated with DMSO or 500 ng/ ml DON for 9 h. The cells were then treated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. Total, cytosolic, or nuclear fractions were subjected to Western blot analysis. (D) HCT-8 cells transfected with an empty vector, ATF3-SC, or shHuR were treated with DMSO or 500 ng/ml DON for 9 h. The cells were incubated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. Nuclear fractions were subjected to IP with anti-HuR or anti-ATF3 Ab. Immunopre- cipitated proteins were analyzed by Western blotting. (E) HCT-8 cells transfected with an empty vector or ATF3-SC were treated with DMSO or 500 ng/ml DON for 9 h. Cells were examined with a confocal microscope after immunostaining (original magnification 32400). (F) HCT-8 cells transfected with an empty vector or ATF3-SC were pretreated with DMSO or 500 ng/ml DON for 9 h. The cells were then exposed to the vehicle (PBS) or 10 mg/ml MDP for 2 h. Cytosolic RNA-bound HuR protein was immunoprecipitated, and the level of HuR-bound p65 mRNA was measured using real-time RT-PCR. *A significant difference from the group treated with MDP alone (p , 0.05). (G) HCT-8 cells transfected with an empty vector or ATF3-SC were pretreated with DMSO or 500 ng/ml DON for 9 h. The cells were then incubated with the vehicle (PBS) or 10 mg/ml MDP for 2 h. The cells were examined with a confocal microscope (original magnification 32400) after immunostaining. Quantitation of p65 protein localized in the nuclei is shown in the lower panel. Relative density of the nuclear p65 protein was measured in the DAPI-stained areas. *A significant difference from the group treated with MDP alone (p , 0.05). expression by ribosomal inactivation reduced the levels of HuR- Discussion bound p65 mRNA (Fig. 8F). As a result, increased ATF3 levels The present study suggested new mechanisms underlying ATF3- led to a reduction of nuclear and total p65 protein levels (Fig. mediated regulation of Nod2-activated proinflammatory signals 8G). Taken together, results from these experiments indicated that in enterocytes. In particular, ATF3 overexpression or ATF3- ATF3 destabilized p65 mRNA by inhibiting HuR cytosolic trans- inducing ribosomal inactivation suppressed the production of location through direct interaction in the nuclei of human enterocytes proinflammatory chemokines owing to decreased expression of (Fig. 9). NF-kB and EGR-1. ATF3 expression can be also induced by TLR The Journal of Immunology 5179

FIGURE 9. A putative scheme for the molecular regulation of Nod2-linked proinﬂammatory signaling by ATF3 expression induced by ribosomal inactivation in human enterocytes. In response to Nod2 stimulation, chemokine gene expression is induced via upregulated EGR-1 and NF-kB signaling. Although p65 expression can be activated by Nod2 in various ways, posttranscriptional stabilization of p65 mRNA is enhanced by HuR exported from the nucleus following Nod2 stimulation in human intestinal epithelial cells. However, increased ATF3 expression by ribosomal inactivation can inhibit translocation of HuR into the cytosol, which contributes to p65 destabilization. Moreover, ATF3 can recruit HDAC1 and epigenetically regulate EGR-1 expression. In summary, disrupted EGR-1 expression and p65 mRNA destabilization contribute to attenuated proinﬂammatory Downloaded from chemokine production in human enterocytes.

activation by bacterial infection, ischemia-reperfusion injury, or in many cases also serve as NF-kB activators (46–48). NF-kB and

stress-induced inflammatory responses (13, 40, 41). These previ- EGR-1 have been shown to cooperatively stimulate chemokine http://www.jimmunol.org/ ous studies have indicated that ATF regulates NF-kB expression gene transcription (49). Moreover, the EGR-1 promoter includes through epigenetic or transcriptional mechanisms (13, 14). Instead an NF-kB binding motif that is important for the regulation of of epigenetic or transcriptional regulation, the current study dem- EGR-1 expression (50). Results from the current study suggested onstrated that Nod2-activated p65 was rather regulated by ATF3 that ATF3 epigenetically regulated EGR-1 expression by recruit- at the posttranscriptional level. Stabilization of the p65 transcript ing HDAC1 protein (Fig. 9). Another recent study also demon- was decreased because the RNA-binding protein HuR was se- strated that the HDAC1 inhibitory complex binds to the NF-kB questered in the nuclei via direct interaction with ATF3 (Fig. 9). binding motif in the EGR-1 promoter and prevents EGR1 tran- Compared to other factors that promote ATF3 expression, ribo- scription (51). In the current study (Fig. 6A), an inhibitory somal inactivation induced the expression of ATF3, which was complex consisting of ATF3 and HDAC1 bound to the EGR-1 by guest on September 27, 2021 prolonged for a relatively long time (24–48 h). Such an extended promoter, but this binding was partly attenuated in the presence of period of ATF3 expression thus triggered pathways that regulate Nod2 ligand. Because the ATF3–HDAC1 complex can bind to the p65 expression in response to Nod2 ligand. According to our NF-kB binding motif, Nod2-activated NF-kB could competitively previous study, ribosomal inactivation can enhance the transient inhibit the binding of the ATF3–HDAC1 complex to the EFG-1 cytosolic release of HuR protein (42). However, chronically in- promoter. Therefore, the current study showed that the HDAC1– duced ATF3 expression by ribosomal inactivation contributed to ATF3 complex could regulate NF-kB–activated EGR-1 expres- the nuclear entrapment of HuR protein via direct interaction with sion, although HDAC1 does not appear to directly affect NF-kB ATF3, which in turn caused the destabilization of p65 mRNA in activation by the Nod2 ligand. In contrast, EGR-1 as an upstream the current study. regulator of NF-kB signaling can indirectly inhibit chemokine So far, only a few studies have reported on the regulation of p65 gene expression via other extracellular mediators such as heat expression at the posttranscriptional level (43, 44). Although the shock protein (HSP). EGR-1 mediates the synthesis and release ubiquitously encoded housekeeping p65 gene does not contain of HSP70 into the extracellular environment (46). In response to NF-kB binding motifs in its promoter region (45), the p65 tran- stressors such as myocardial infarction, HSP70 is released into the script has three AU-rich elements in the 39 untranslated region, circulation and promotes the increase of TLR4 levels in patients, which would make the gene susceptible to regulation by RNA- thereby triggering NF-kB signaling and proinflammatory cytokine stabilizing HuR protein. Therefore, when HuR protein is con- production (52, 53). Further studies are thus needed to eluci- fined in the nuclear region through binding to ATF3, the balance date the roles of extracellular mediators such as HSP in EGR-1– between synthesis and degradation of p65 transcript in the cy- associated proinflammatory chemokine production in response to toplasm would be disrupted. Prolonged presence of ATF3 in the Nod2 activation. nuclei would thus lead to depletion of cytosolic p65 transcript, ATF3-mediated anti-inflammatory responses are not always which would contribute to the decreased induction of proin- beneficial to the host because these can weaken the defense against flammatory chemokine expression observed in the current study. infectious agents. Whereas chronic induction of ATF3 expression As a result, ribosomal inactivation can trigger epithelial tolerance by ribosomal inactivation can offer protection against inflam- to Nod2-linked proinflammatory activation by inducing ATF3 ex- matory insults, ATF3 increases susceptibility to secondary infections pression. These findings suggest that ribosomal inactivation may during sepsis-associated immune suppression (40). The precise be a potent modulator of anti-inflammatory responses to NF-kB– effects of ribosomal inactivation on the human immune system associated epithelial inflammatory insults. remain unknown, but both up- and downregulation of systemic In addition to NF-kB, Nod2-induced EGR-1 was found to be and mucosal immune responses have been observed in different critical for chemokine gene expression in the current study. The animal experiments (54–59). In various exposure models, ribo- expression of EGR-1 can be induced by a range of stimuli, which somal inactivation was found to decrease host immune responses, 5180 REGULATION OF Nod2-LINKED PROINFLAMMATORY SIGNALS including lymphocyte proliferation responses, monocyte matura- References tion, and host defense against pathogens (60–63). Another recent 1. Abreu, M. T. 2010. 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