The American Journal of Pathology, Vol. 182, No. 3, March 2013

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TUMORIGENESIS AND NEOPLASTIC PROGRESSION

Death-Associated Protein Kinase Controls STAT3 Activityin Intestinal Epithelial CellsSaritha Chakilam,*y Muktheshwar Gandesiri,*y Tilman T. Rau,y Abbas Agaimy,y Mahadevan Vijayalakshmi,z Jelena Ivanovska,*y

Ralph M. Wirtz,x Jan Schulze-Luehrmann,*y Natalya Benderska,*y Nadine Wittkopf,{ Ajithavalli Chellappan,z Petra Ruemmele,k

Michael Vieth,** Margret Rave-Fränk,yy Hans Christiansen,yyzz Arndt Hartmann,y Clemens Neufert,{ Raja Atreya,{

Christoph Becker,{ Pablo Steinberg,xx and Regine Schneider-Stock*y

From Experimental Tumor Pathology* the Institute of Pathology,y and Medical Clinic 1,{ University of Erlangen-Nuremberg, Erlangen, Germany; the Schoolof Chemical & Biotechnology,z SASTRA University, Thanjavur, India; STRATIFYER Molecular Pathology GmbH,x Cologne, Germany; the Department ofPathology,k University of Regensburg, Regensburg, Germany; the Institute of Pathology,** Bayreuth, Germany; the Department of Radiation Oncology,yy

University Hospital Goettingen, Goettingen, Germany; the Department of Radiotherapy and Oncology,zz Medical School, University of Hanover, Hannover,Germany; and the Institute for Food Toxicology and Analytical Chemistry,xx University of Veterinary Medicine Hannover, Hannover, Germany

Accepted for publication

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November 15, 2012.

Address correspondence toRegine Schneider-Stock, Ph.D.,Head of Experimental TumorPathology, Institute ofPathology, University ofErlangen-Nürnberg,Universitätsstr. 22, 91054Erlangen, Germany. E-mail:Regine.Schneider-stock@uk-erlangen.de.

opyright ª 2013 American Society for Inve

ublished by Elsevier Inc. All rights reserved

ttp://dx.doi.org/10.1016/j.ajpath.2012.11.026

The TNFeIL-6eSTAT3 pathway plays a crucial role in promoting ulcerative colitis-associated carcinoma(UCC). To date, the negative regulation of STAT3 is poorly understood. Interestingly, intestinalepithelial cells of UCC in comparison to ulcerative colitis show high expression levels of anti-inflammatory death-associated protein kinase (DAPK) and low levels of pSTAT3. Accordingly, epithelialDAPK expression was enhanced in STAT3IEC-KO mice. To unravel a possible regulatory mechanism, weused an in vitro TNF-treated intestinal epithelial cell model. We identified a new function of DAPK insuppressing TNF-induced STAT3 activation as DAPK siRNA knockdown and treatment with a DAPKinhibitor potentiated STAT3 activation, IL-6 mRNA expression, and secretion. DAPK attenuated STAT3activity directly by physical interaction shown in three-dimensional structural modeling. This modelsuggests that DAPK-induced conformational changes in the STAT3 dimer masked its nuclear localizationsignal. Alternatively, pharmacological inactivation of STAT3 led to an increase in DAPK mRNA andprotein levels. Chromatin immunoprecipitation showed that STAT3 restricted DAPK expression bypromoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negativeregulation principle might balance TNF-induced inflammation and seems to play an important role inthe inflammation-associated transformation process as confirmed in an AOMþDSS colon carcinogenesismouse model. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatmentof ulcerative colitis and UCC. (Am J Pathol 2013, 182: 1005e1020; http://dx.doi.org/10.1016/j.ajpath.2012.11.026)

Supported by a research grant of the Deutsche Forschungsgemeinschaft(SCHN477-9-2 to R.S.S) and partly by the Interdisciplinary Centre for Clin-ical Research (IZKF-D18) at the University of Erlangen-Nürnberg (R.S.S.).

Disclosures: R.M.W. is employed by STRATIFYERMolecular PathologyGmbH, which produces molecular methods for analysis of RNA, microRNA,and DNA, products unrelated to the content of this report. A patent is beingfiled related to the content of this work.

Tumor necrosis factor-a (TNF-a) is a pleiotropic cytokinethat participates in several biological functions, includinginflammation, apoptosis, growth, and differentiation.1,2 Itactivates the inflammatory pathway via nuclear factor-kB(NFkB) or apoptosis via caspases, which depends on theparticular proteins recruited to the receptors.3,4 Moreover,TNF has been implicated in the pathogenesis of variousinflammatory diseases such as ulcerative colitis (UC),Crohn’s disease, and rheumatoid arthritis.5,6

The etiology of UC still remains obscure; however,genetic, immunological, and environmental factors probably

stigative Pathology.

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contribute to disease pathogenesis.7 An imbalance betweenpro- and anti-inflammatory cytokines and a defect in intes-tinal barrier function cause chronic recurrent inflammationof the gut.8,9 As inflammation compromises gut homeostasis

Chakilam et al

and is also associated with cancer progression,10 it isimportant to understand the role of key molecules that areinvolved in the activation of the inflammatory cascade.

The death-associated protein kinase (DAPK) is a calcium/calmodulin-regulated serine/threonine kinase with a protec-tive role during chronic inflammation in UC and UC-associated carcinoma (UCC).11 Interestingly, DAPK canregulate inflammation either positively through NLRP3inflammasome formation12 or negatively through inhibitionof NFkB.13,14 TNF activates NFkB by phosphorylatingthe inhibitor of NFkB (IkBa), which is then degraded ina ubiquitin-mediated step. Activated NFkB initiates thetranscription of target genes including the proinflammatorycytokine IL-6.1,15,16 IL-6 is shown to be a major mediatorof inflammation through the activation of the signal trans-ducer and activator of transcription 3 (STAT3) pathway.17e19

Subsequent to the cytokine action, Janus kinases (JAK)phosphorylate and activate STAT3 at Y705.19,20 The acti-vation of STAT3 leads to its dimerization, followed bynuclear translocation and DNA binding to regulate targetgene expression.21 Until now, only a few negative regulatorsof STAT3 activity have been reported, such as SOCS3, PIAS,ERK, KAPI, and protein phosphatases.22 The TNF/NFkBand IL-6/STAT3 pathways are shown to play a crucial rolein promoting colitis-associated carcinoma formation.23e29

Until now, studies related to the pathogenesis of inflam-matory bowel disease (IBD) were performed using eitherimmune or cancer cells or mouse models, whereas nonim-mune cells, including epithelial cells, are considered to playa rather passive role.30 However, accumulating evidencesuggests that intestinal epithelial cells (IEC) are more than justa barrier and seem to be equally competent in IBD pathogen-esis.30 Therefore, we studied TNF-induced signaling in normalhuman colon epithelial cells (HCEC) and proved its in vivorelevance in UC tissues.

Our results demonstrate that DAPK and pSTAT3Y705

were activated under inflammation both in vitro and in vivo.We also report a novel negative regulation principlebetween DAPK and STAT3, which might balance TNF-induced inflammation. The divergent expression pattern ofthese proteins in UC and UCC emphasizes their importantrole in the inflammation-associated transformation process.

Materials and Methods

General cell culture reagents such as PBS, Trypsin, and BasalHCEC medium were obtained from PAN (PAN BiotechGmbH, Aidenbach, Germany). Other medium supplementsof HCEC medium were obtained from Sigma-Aldrich(St. Louis, MO). Human TNF (Immuno Tools GmbH, Frie-soythe, Germany), human IL-6, human IL-6 monoclonalantibodies (R&D Systems, Minneapolis, MN), DAPKinhibitor (4Z)-2-phenyl-4-(pyridine-3-ylmethylidene)-4,5-dihydro-1,3-oxazol-5-one (MolPort, Riga, Latvia), JAKinhibitor Tyrphostin AG 490 (Sigma Aldrich), and Stattic

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(Calbiochem, Darmstadt, Germany) were obtained from thesources mentioned.

Cell Culture

HCEC cells were kindly provided by Professor PabloSteinberg (Institute for Food Toxicology and AnalyticalChemistry, University of Veterinary Medicine Hannover,Germany) and maintained as previously described.31 After 24hours of seeding, cells were either stimulated with 0.66 ng/mLTNF (ImmunoTools) for various time points. For inhibitorexperiments, cells were pre-incubated for 1 to 2 hours with thecorresponding inhibitors.

Patient Samples

Gut specimens were obtained from UC or non-IBD controlpatients and analyzed by immunohistochemistry. The UCgroup included 140 samples from 120 patients (average age:51 � 32 years) with inactive UC (n Z 49), low-active UC(n Z 41), highly active UC (n Z 15), dysplasia-associatedlesion or mass (DALM; n Z 11), and UCC (n Z 24). Thecontrol group consisted of patients that underwent controlcolonoscopy for cancer prevention (n Z 11; average age:64 � 21 years). IEC preparations from gut specimens of UCpatients (n Z 4) were assessed by Western blotting. Detailssuch as age, sex, and histological/pathological activity aregiven in Supplemental Tables S1, S2, and S3). The DiseaseActivity Score was calculated as previously described32 foravailable cases. The present study was performed followingapproval by our local ethical committee.

IHC and Histological Score

Immunohistochemistry (IHC) was used to detect the expres-sion of DAPK, pSTAT3Y705, and TNF in the formalin-fixed,paraffin-embedded tissue microarrays. Sections (2 to 4 mmthick) were dewaxed at 72�C for 30 minutes and then incu-bated in fresh xylene 2� 5 minutes. Tissue sections wererehydrated in descending concentrations of ethanol (96% to70%). Antigen was retrieved by heating in a pressure cooker(1 mmol/L Tris-EDTA buffer, 120�C, 5 minutes). Endoge-nous peroxidases and nonspecific biding sites were blockedby incubating the slices with blocking solution (Dako,Glostrup, Denmark). All slices were then incubated withprimary antibodies anti-DAPK (1:100), anti-pSTAT3Y705

(1:50), and anti-TNF (1:300) at room temperature for 30minutes. After washing with washing buffer (Dako), sectionswere incubated with secondary antibody at room temperaturefor 30minutes. Secondary antibodies were EnVisionþSystemhorseradish peroxidase-linked (goat anti-mouse or goat anti-rabbit; Dako), and positive immunoreactivity was detectedusing diaminobenzidineþ (Dako) or Fast Red (Dako) aschromogen substrate. Nuclei were counterstained withhematoxylin (Dako). Appropriate positive and negativecontrols were included in each run of IHC. Histological

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evaluation was performed by reviewing the H&E-stainedtissue sections. The percentage of epithelial cells that stainedpositive (immunoreactivity above the background) wasquantified/scored in a blinded manner (T.T.R., A.A., A.H.).

RNA Isolation and Real-Time RT-PCR

Expression of IL-6, IL-8, and DAPKmRNAwas analyzed byreal-time RT-PCR. RNA isolation (mRNeasy RNA IsolationKit) and cDNA synthesis (Quantitect Reverse TranscriptaseKit) were performed according to the manufacturer’sinstructions (Qiagen, Hilden, Germany). One microliter ofcDNA was amplified in a thermal cycler (Bio-Rad CFX-96;BioRad Laboratories, Hercules, CA) with correspondingprimers in a total volume of 25 mL using Quantifast SYBRgreen kit (Qiagen) under the following conditions: 95�C for 5minutes followed by 26 to 40 cycles of 95�C for 10 secondsand 60�C for 30 seconds. The following primers were used,forward and reverse, respectively: DAPK, 50-CCTTGCAA-GACTTCGAAAGGATA-30 and 50-GATCCCGAGTGGC-CAAA-30; IL-6, 50-ATGAACTCCTTCTCCACAAGCGC-30 and 50-CAGTCCAGCCTGAGGGCTCTTC-30; IL-8, 50-CCAAGGAAAACTGGGTGCAGAG-30 and 50-ACAAG-TCCTTGTTCCACTGTGCC-30; b2microglobulin (house-keeping gene), 50-CCAGCAGAGAATGGAAAGTC-30 and50-GATGCTGCTTACATGTCTCG-30; murine DAPK, 50-TGCACAACAGCTACACAGCA-30 and 50-GACCAGAC-GCTGGATGTCTT-30; murine glyceraldehyde-3-phosphatedehydrogenase (GAPDH; house-keeping gene), 50-TGTG-TCCGTCGTGGATCTGA-30 and 50-CCTGCTTCACCA-CCTTCTTGA-30. The results were expressed as foldinduction compared to unstimulated cells after normalizingto house-keeping gene. All primers were purchased frommetabion (Metabion International, Martinsried, Germany).

Western Blotting

Protein concentration was measured in duplicate using Bio-Rad DC Protein Assay. Equal amounts of protein were sep-arated by 10% or 12% SDS-PAGE using Laemmli buffersystem. Proteins were transferred electrophoretically to nitro-cellulose membrane (Millipore, Billerica, MA) and det-ected as recently described33 using the following antibodies:anti-DAPK (BD Transduction Laboratories, Lexington,NY), anti-pDAPKS308 (Sigma-Aldrich), anti-STAT3, anti-pSTAT3Y705, anti-caspase3 (Cell Signaling Technology,Danvers,MA), antieb-actin (Sigma-Aldrich), or anti-GAPDH(Abnova GmbH, Heidelberg, Germany).

ELISA

IL-6 and IL-8 secretion was analyzed by enzyme-linkedimmunosorbent assay (ELISA) according to the manufac-turer’s instructions (BD Biosciences, Heidelberg, Germany).Briefly,flat-bottom 96-well microtiter plates (BDBiosciences)were coated with 100 mL of capture antibody (1:250 diluted in

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Na2HCO3) and incubated at 4�C overnight. After blocking(300 mL of 3% BSA in PBS, 2 hours, room temperature) andwashing [0.1% Tween in PBS (PBS-T)], 100 mL of undilutedor diluted supernatant was added and incubated (2 hours atroom temperature or overnight 4�C). Thereafter, wells werewashed and incubated (1.5 hours at room temperature) withdetection antibody (1:250 diluted in 1% BSA in PBS-T) þenzyme reagent (streptavidinehorseradish peroxidase conju-gate; 1:250 diluted in detection antibody). After washing,TMB (1:1) substrate was added to each well and incubated inthe dark for 20 to 30 minutes. Reaction was stopped with stopsolution (100 mL of 1 mol/L H2SO4), and absorption wasmeasured using a spectrophotometer (Victor X3; PerkinElmer,Waltham, MA) at a wavelength of 450 nm with a wavelengthcorrection at 570 nm.

siRNA Transfection

Silencing of DAPK expression in HCEC cells was per-formed by the siRNA technique according to the manufac-turer’s instructions (Dharmacon, Chicago, IL). Briefly, theHCEC cells were grown to 60% confluence in a 6-welltissue culture plate. Transfection mixture was prepared ina final volume of 400 mL to achieve a final siRNAconcentration of 100 nmol/L. This mixture was incubatedfor 30 minutes at room temperature to allow complexformation and then added onto the cells drop by drop. After24 hours of incubation, the medium was replenished andsubsequently treated with 0.66 ng/mL TNF for 24 and 48hours. A nonspecific control siRNA SMARTpool (100nmol/L; Dharmacon) was used as a negative control. At theend of the incubation period, supernatants and cells wereharvested and stored at �80�C until analyzed further. Theknockdown efficiency was determined by Western blotting.

Immunoprecipitation

Immunoprecipitation (IP) was performed using the DynabeadsProtein G magnetic separation kit according the manufac-turer’s instructions (Invitrogen, Karlsruhe, Germany). Briefly,protein G magnetic Dynabeads were coated with DAPKantibody (1:500 to that of protein concentration) for 2 hourswith rotation at room temperature, and Dynabeads-antibodycomplexes were washed. 600-900 mg of protein lysate wasadded to the Dynabeads-antibody complex and gently res-uspended by pipetting. The Dynabeads-antibody-antigencomplex was incubated overnight at 4�C with rotation. TheDynabeads-antibody-antigen complexes were washed, andimmunoprecipitates were eluted in 20 mL of elution buffer.The proteins were separated by SDS-PAGE, andWestern blotanalysis was performed using anti-STAT3 antibody.

Structural Analysis of DAPK-STAT3 Complex

To understand the interactions between STAT3 and DAPK,the structures of JAK and STAT3 deposited in the Protein

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Data Bank (PDB) (JAK: 3EYGdcrystal structures of JAK1and JAK2 inhibitor complexes at 1.9 Å resolution; STAT3:1BG1dX-ray structure of the transcription factor STAT3B-DNA complex at 2.25 Å resolution) were considered. TheJAK2 inhibitor was removed from the structure 3EYG, andthe DNA was removed from the structure 1BG1 to facilitateanalysis of the JAK-STAT3 complex.

The catalytic domain of DAPK (PDB: 1JKSdX-raystructure of the catalytic domain of human DAPK at 1.5 Åresolution) was then docked to the STAT3 monomer, andranking was done using ClusPro server. The STAT3-STAT3dimer was then formed followed by the creation of a STAT3-DAPK-STAT3 complex through docking. The docking,energy filtering, and ranking of the complexes of thesestructures were done by the ClusPro server.34 In all cases, top1000 structures were chosen after energy filtering (electro-statics), clustered, and ranked according to cluster sizes.The hydrogen bond interactions in the STAT3-STAT3 dimerand in the STAT3-DAPK-STAT3 complex were analyzedusing HBOND Calculator (Hydrogen Bond Calculationversion 1.1; http://cib.cf.ocha.ac.jp/bitool/HBOND, last acc-essed January 18, 2013). The hydrophobic interactionsbetween these complexes were analyzed using the PIC Server(http://pic.mbu.iisc.ernet.in, last accessed January 21, 2013).All renderings were done using CHIMERA.35

Preparation of Cytoplasmic and Nuclear Lysates

Cell pellets were resuspended in 300 mL of cold Buffer A[10 mmol/L Tris (pH 7.9); 10 mmol/L KCl; 1.5 mmol/LMgCl2; 10% glycerol; 10 mmol/L K2HPO4; 1 mmol/LNa3VO4; 10 mmol/L NaF; 0.5 mmol/L dithiothreitol (DTT);1 mmol/L ABSF; 1�-protease inhibitors] with 0.125% NP-40 and incubated on ice for 5 minutes. The homogenate wascentrifuged for 10 minutes at 1,000 � g at 4�C, and thesupernatant containing cytoplasmic proteins was collectedinto a fresh tube. The nuclear pellet was washed once withBuffer A and then resuspended in 50 to 100 mL of Buffer C[20 mmol/L Tris (pH 7.9); 0.42 mmol/L NaCl; 1.5 mmol/LMgCl2; 2 mmol/L EDTA; 10% glycerol; 10 mmol/LK2HPO4; 1 mmol/L Na3VO4; 10 mmol/L NaF; 0.5 mmol/LDTT; 1 mmol/L ABSF; 1� protease inhibitors] and soni-cated. The nuclear extract was centrifuged for 10 minutes at12,000 � g at 4�C, and the supernatant with nuclear proteinswas transferred into a fresh tube.

Electrophoretic Mobility Shift Assay

STAT3 DNA binding activity was evaluated using nonra-dioactive electrophoretic mobility shift assay (EMSA), per-formed as recently described.36 For performing EMSA, 10 mgof nuclear protein was incubated with IRDye 700elabeleddouble-stranded STAT3 consensus or mutant oligonucleo-tides (0.5 mL of 50 nmol/L) in 20 mL of incubation buffercontaining 2 mL of binding buffer (100 mmol/L Tris; 500mmol/L NaCl; 100 mm EDTA; 10 mmol/L DTT; 50%

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glycerol), 1 mL of 1% NP-40, 1 mL of 2.5% Tween, 1 mL ofpoly dI-dC (2 mg/mL), 2 mL of BSA (10 mg/mL), 1 mL of2.5% Tween 20, and 1 mL of 1% NP-40. The sequence of theprobes usedwas as followswith bold type indicatingwild typeand italics indicating mutant, consensus sense 50-GATC-CTTCTGGGAATTCCTAGATC-30; and mutant sense 50-GATCCTTCTGGGCCGTCCTAGATC-30. After 30 minutesof incubation at 18�C, samples were loaded and run on a 4%Lipage gel at 150V, 4�C for 2 hours. DNA-protein complexeswere detected using Odyssey system (Li-Cor BiosciencesGmbH, Bad Homburg, Germany). The specificity of thecomplexes was also verified by competition experiments, co-incubating unlabeled consensus (100�) with labeledconsensus oligos.

Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) experiments wereperformed as previously described37 using the ChIP-ITexpress kit (Active Motif, Rixensart, Belgium). At the endof the incubation period, cells were treated with 1% form-aldehyde for 10 minutes at room temperature to cross-linkDNA and associated proteins. Chromatin was extractedand sonicated on ice 6� for 15 seconds at 30% power witha 01 01 pulse using an HTU Soni 130 (G. Heinemann,Schwäbisch Gmünd, Germany) sonicator to obtain DNAfragments of average size of 500 bp. Immunoprecipitationswere performed by incubating 60 mL of chromatin and 25mL of protein G magnetic beads with 15 mL of pSTAT3Y705

antibody (Cell Signaling Technology) or negative controlIgG of equivalent concentration overnight at 4�C ona rotating platform. The beads were washed, protein-DNAcross-links were reversed, and 5 mL of DNA from theinput and IP samples were subjected to real-time or end-point PCR using primers corresponding to two differentregions of the human DAPK promoter. The followingprimers were used region 1 (�1821/�1472) forward primer,50-TGCAGTGAGCCAAGATTTCA-30 and reverse primer,50-TTCCGATCCATACCGTTGTT-30 and region 2 (�631/�351) forward primer, 50-ATGAGGTACGCTCCCTTC-CT-30 and reverse primer, 50-TCGTCCCGAGATGTG-TACTG-30. PCR products were analyzed by agarose gelelectrophoresis in end-point PCR, and in real-time PCR,data were expressed as the fold increase over unstimulatedcells. All ChIP assays were performed four times.

Experimental Mouse Models and IEC Isolation

Mice carrying a loxP flanked Stat3 allele [Stat3 wild-type(wt)] were kindly provided by Shizuo Akira.38 C57BL/6mice carrying the sequence for the enzyme cre-recombinaseunder control of the Villin promoter (Villin-Cre mice) weredescribed earlier.39 Stat3 wt mice were crossbred with Villin-Cre mice. In this way, conditional knockout mice with IEC-specific deletion of Stat3 activity (Stat3IEC-KO) were gener-ated. We have previously shown that normal STAT3IEC-KO

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mice do not develop spontaneous colitis.40 Histology indi-cates that there is no underlying inflammation present inunchallenged mice. All mice were kept in individuallyventilated cages in compliance with the Animal Welfare Act.

Isolation of Intestinal Epithelial Cells

Intestinal epithelial cells were isolated by carefully removingthe entire intestine from the mouse corpse. The intestine wasinverted and washed free of stool in phosphate-bufferedsaline. Intercellular connections were destroyed by incu-bating the inverted gut tissue in pre-warmed isolation solu-tion [HBSS (PAA Laboratories, Linz, Austria), 1 mmol/LEGTA (Sigma-Aldrich), 2 mmol/L EDTA (Sigma-Aldrich),and 10% FCS (PAA Laboratories)] and shaking at 200rpm for 10 minutes at 37�C. Subsequently, the isolatedcells were pelleted at 250 � g and 4�C for 5 minutes, andwashed twice with phosphate-buffered saline, followed bycentrifugation.

Experimental Model of Intestinal Inflammation

To induce experimental colitis, mice were treatedwith dextransodium sulfate (DSS) (MP Biomedicals, Santa Ana, CA).DSS 2% to 3%was dissolved in sterile drinking water, and thesolution was given to the mice in drinking water bottles for 7days and renewed every second day. Mouse body weight wasmonitored regularly to determine the state of health of themice. Development of colitis was followed by regular colo-noscopy, and the severity of colitis in live mice was scoredas previously described.41,42 DSS administration at the spec-ified conditions caused moderate inflammation, and theweight loss per mouse was less than 10%.

Experimental Model of Colon Carcinogenesis

Experimental colitis-associated tumorigenesis was performedas previously described.43 In brief, 10 mg/kg azoxymethane(AOM) (Sigma-Aldrich) was injected intraperitoneally into6- to 8-week-old C57BL/6J mice, followed by three cyclesof DSS in drinking water. Each DSS cycle was composedof DSS [2.5% (w/v); MP Biomedicals] in drinking waterfor 7 days, followed by a recovery phase with regulardrinking water for 14 days. All tumors were harvested atday 65 to 70.

IEC Isolation from UC Patients

Intestinal tissue was obtained from patients with inflammatorybowel diseases who had to undergo surgery for variousreasons (eg, stenosis, fistulae, perforation, and therapy ref-ractory disease). The gut specimen was initially thoroughlywashed with sterile PBS, and the mesenteric fat tissue wascarefully removed. The intestinal mucosal layer was openedlongitudinally and removed from the underlying muscularlayer and thereafter cut into stripes (w1 cm � 4 cm). After

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incubation with 20 mL of PBS and 31 mg of DTT for 30minutes at 37�C at 200 rpm, the mucosa was again washedwith PBS. Next, the mucosal stripes were incubated in 20 mLof PBS with 80 mL of 0.5 mol/L EDTA (2 mmol/L) for 15minutes at 37�C at 200 rpm. Afterward, colonic epithelialcrypts were collected from this suspension, and the washingsteps with EDTAwere repeated until the resulting suspensionappeared clear of the isolated epithelial cells.

Isolated colonic epithelial crypts and/or cells were pel-leted and resuspended in 10 to 25 mL of DMEM mediumand further enriched using density gradient centrifugation.Three milliliters of cell suspension was overlaid on the topof the Percoll of 1.077 g/mL density and centrifuged at1750 � g for 20 minutes at room temperature. Cells bands atdensity level 1.077 g/mL were collected cautiously andwashed with PBS. A small fraction of the cell suspensionwas spread on a microscopic glass slide by centrifugationvia cytospin at 300 � g for 10 minutes and stained with pan-cytokeratin, cytokeratin-19, and CD34 antibodies. Anothersmall fraction of cell suspension was stained with EpCAM(CD326)-fluorescein isothiocyanate and assessed by flowcytometry. The remaining cell suspension was pelleted andused for protein extraction.

Apoptosis and Cell Viability Assay

Experimental procedures have previously been described.44

Apoptosis was measured using Annexin-V-FLUOS kit orM30 Cytodeath detection kit (Roche Diagnostic GmbH,Penzberg, Germany). At the end of the treatment, cells werestained with 100 mL of annexin V/PI solution (20 mL offluorescein isothiocyanateeconjugated annexin V reagent(20 mg/mL)þ 20 mL of propidium iodide reagent (50 mg/mL in1 mL of dilution/HEPES buffer) for 15 minutes at roomtemperature in dark. In case of M30 staining, cells were fixedwith ice-coldmethanol for30minutes at�20�C.Afterwashing,the cells were incubatedwithM30Cytodeath antibodyworkingsolution [1:250 in incubation buffer (PBS þ 1% BSA þ 0.1%Tween)] for 30 minutes at room temperature. In both cases,the cell suspension was diluted by adding an appropriateamount of dilution buffer and analyzed using FACSCaliburflow cytometer (BD Biosciences, Franklin Lakes, CA).

Cell viability was assessed by crystal violet staining. At theend of the incubation period, supernatants were discarded,cells were washed twice with pre-warmed PBS, and then cellswere stained with a crystal violet solution (0.5% crystal violetin 20%methanol) for 15 minutes. After removal of the crystalviolet solution, the plates were washed with tap water andthen air dried. The dye was eluted with methanol for 15minutes, and absorbance was measured at 595 nm usinga microtiter plate reader (Victor X3: PerkinElmer).

Statistical Analysis

Statistical analysis was performed using SPSS (SPSS,Chicago, IL). The Student’s t-test or the U-test was used for

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single comparisons and analysis of variance followed byTukey’s HSD, Dunnett’s t, and Student-Newman-Keuls posthoc tests were used for multiple comparisons. P values� 0.05were considered statistically significant. Scatter plots and theU-test were done by using GraphPad Prism version 7.1(GraphPad Software, La Jolla, CA).

Results

Expression of DAPK and pSTAT3Y705 Is Augmented inIEC of UC and UCC

We have previously shown an increase in DAPK expressionin UC-associated tumors11 and STAT3 activation in a colitismouse model.40 To better understand the role of thesetwo proteins in the inflammation-associated process, we

Figure 1 DAPK, pSTAT3Y705, and TNF expression pattern in the intestinal epicontrol and different stages of UC patients were analyzed by IHC. The pSTAT3Y705 eDAPK (FeJ), pSTAT3Y705 (KeO), and TNF (PeT) staining in non-IBD (n Z 11), inaUCC (n Z 24) are shown. Original magnification, �200.

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evaluated their immunohistochemical expression in IEC ofhuman gut specimens from non-IBD, inactive UC, low-active UC, highly-active UC, and UCC patients. H&Estaining depicts the inflammation grade of the sections(Figure 1, AeE). Up to 80% of the IEC in the active UCand UCC specimens expressed DAPK in the cytoplasm,a percentage that was significantly higher than that (less than20%) in non-IBD/inactive UC samples (Figure 1, FeJ, andFigure 2A). As in the case of DAPK, a strong pSTAT3Y705

expression was observed in up to 80% of IEC present in theactive UC samples, whereas less than 3% of IEC werepositive in non-IBD/inactive UC specimens. However, incontrast to DAPK, most of the carcinoma specimens lostpSTAT3Y705 expression and only up to 20% of the IEC inthe samples were scored positive, which was significantlylower if compared to the percentage observed in active UC

thelial cells of normal mucosa, UC, and UCC. Tissue sections derived fromxpression was predominantly nuclear. Representative images of H&E (AeE),ctive UC (n Z 49), low-active UC (n Z 41), highly-active UC (n Z 15), and

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Figure 2 Vertical scatter plots of (A) DAPK, (B) pSTAT3Y705, and (C) TNF scores grouped by sample type (non-IBD, inactive ulcerative colitis, low-active UC,highly active UC, and UC-associated carcinoma). A nonparametric Mann-Whitney test was performed to evaluate significance between two sample groups asdepicted (where P � 0.05) above the scatter plot.

Negative Regulation between DAPK/STAT3

specimens, but still significantly higher when compared tothat of non-IBD/inactive UC samples (Figure 1, KeO, andFigure 2B). The increase of epithelial TNF expression wassubstantial and significant only in UCC when compared tothe non-IBD/inactive UC/active UC specimens (Figure 1,PeT, and Figure 2C). The expression pattern of all of thethree markers (DAPK, pSTAT3Y705, and TNF) did not differfrom low-active UC to highly active UC.

Although DAPK as well as pSTAT3Y705 protein expres-sion increased with the severity of inflammation, thiscommon expression pattern seemed to be lost between UCand UCC. To clarify this issue, we analyzed the expression ofDAPK and pSTAT3Y705 in DALM samples, which representUC-associated intraepithelial neoplasia. A heterogeneouspattern of staining was observed in some of the DALMsamples, thereby showing all possible combinatory patternsfor both proteins (Figure 3A).

In the next step, human IEC were isolated from gut spec-imens of UC patients from macroscopically inflamed and

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noninflamed colonic mucosa (Supplemental Figure S1). Weobserved an inverse correlation between pSTAT3Y705 andDAPK: There were two UC patient samples showingenhanced STAT3 phosphorylation, but diminished DAPKexpression in IEC from the inflammatory region compared tonormal mucosa (patients 1 and 2; Supplemental Figure S2A).Vice versa, in two UCC patients (patients 3 and 4),STAT3Y705 phosphorylation appeared to be diminished, butDAPK expression was up-regulated in IEC from inflamedmucosa (Supplemental Figure S2A). In accordance with theobservation that the inactive pDAPKS308 form was almostcompletely lost in the course of inflammation, the DAPKlevel increased and kinase activity was enhanced in inflamedIEC (Supplemental Figure S2A). Obviously, DAPK resumesthe control during the malignant transformation process, andpSTAT3Y705 activation seems to be no longer necessary fortumor survival.

We further investigated the influence of STAT3 onDAPK expression in an in vivo mouse model. IEC were

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Figure 3 A: DAPK and pSTAT3Y705expression pattern in DALM. Repre-sentative images of DAPK and pSTAT3Y705 staining in DALM tissue sections(n Z 11) depicting heterogeneous pattern of expression are shown. Areaswith high DAPKehigh pSTAT3Y705 (filled arrowheads); high DAPKelowpSTAT3Y705 (open arrowheads); low DAPKehigh pSTAT3Y705 (asterisks);and low DAPKelow pSTAT3Y705 (arrows) expression are indicated. Note theserial sections for DAPK and pSTAT3Y705 allowing direct comparison ofexpression in the same region of interest. TNF-induced functions in normalhuman colon epithelial cells: HCEC cells were stimulated with 0.66 ng/mL ofTNF for various time points (1, 6, 24, 48, or 72 hours). DAPK (B) and STAT3(C) expression/activation were assessed by immunoblotting using thecorresponding antibodies. Representative Western blots of five indepen-dent experiments are shown. IL-6 (D) and IL-8 (E) secretion was measuredin cell culture supernatants by ELISA. Data were obtained from more thanfive independent experiments performed in duplicate or triplicate. *P <

0.05 versus untreated control cells.

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isolated from wt and STAT3IEC-KO mice. DAPK expressionwas significantly higher both at the mRNA (1.4-fold) andprotein (2.4-fold) level in STAT3IEC-KO mice than in wtmice, suggesting that DAPK expression might be negativelyregulated by STAT3 (Supplemental Figure S2, B and C).

Expression/Activation of DAPK/STAT3 Is Modulatedduring Colitis-Associated Carcinogenesis

Tissue extracts from colon of control and DSSmice as well asAOMþDSS tumors were assessed by Western blotting toevaluate the modulation of DAPK/STAT3 expression/activation following the transformation from inflammation tocancer (Figure 4A). Endoscopy images demonstrate theinduction of inflammation and tumor formation by treatmentwith AOMþDSS (Figure 4, BeE). No significant differences

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were observed in the levels of either DAPK or pDAPKS308

levels between control and DSS-treated mice. Although,the increase in total DAPK levels were moderate, thepDAPKS308 levels decreased drastically in AOMþDSS-treated mice indicating that DAPK is activated duringtransformation (Figure 4F). In case of STAT3 activation,STAT3Y705 phosphorylationwas increased byDSS treatment,whereas it decreased profoundly inAOMþDSSetreatedmice(Figure 4G), confirming the immunohistochemical obser-vations in human tissues of UC and UCC. These data impli-cate the importance of both proteins in the course ofinflammation-associated carcinogenesis.To find out whether this observation is a rather occasional

phenomenon or if the two molecules control the expressionof each other under inflammatory conditions, we developedan in vitro model to simulate the TNF-driven inflammatoryprocess using the normal intestinal epithelial cell lineHCEC. HCEC is an immortalized cell line developed bytransfection of the SV40 large T antigen cDNA into freshlyisolated human colon epithelial cells isolated from a nonetumor-carrying donor.45,46 HCEC cells differ from cancercells as they are not tumorigenic (do not develop tumors inSCID mice).31 We analyzed the immunohistochemicalexpression of cytokeratin to verify their epithelial origin. Asexpected, all of the cells were positive when stained withantiepan-cytokeratin (Supplemental Figure S3A). HCECcells were treated with TNF and the interaction betweenDAPK and STAT3 was characterized in detail.

TNF Induces an Inflammatory Pathway in HCEC Cells

To find out whether an inflammatory stimulus can modulatethe expression/activation of DAPK and STAT3, HCEC cellswere treated with TNF for various time points. Interestingly,TNF caused the dephosphorylation of DAPKS308 (inactiveform of DAPK) after 6 hours, reaching a maximum after 48hours. In addition, DAPK expression was enhanced after 48hours. A gradual increase in the DAPK/pDAPKS308 ratioindicated that DAPK is activated on TNF treatment(Figure 3B). In parallel, STAT3Y705 phosphorylation wassignificantly enhanced after a 24h-TNF treatment, whereastotal STAT3 levels did not change (Figure 3C). As TNF isknown to regulate the expression of other cytokines,19 IL-6and IL-8 secretion was measured by ELISA. As expected,TNF significantly induced the secretion of both pro-inflammatory cytokines IL-6 (w7.2-fold) and IL-8 (w12-fold) from 6 to 72 hours (Figure 3, D and E). This wasaccompanied by an increase in IL-6 and IL-8 mRNA levelsat earlier time points but was rather marginally increasedat 24 hours and later (Supplemental Figure S3, B and C).To find out whether STAT3 is activated by the releasedIL-6, HCEC cells were treated with TNF in the presenceor absence of antieIL-6 monoclonal antibodies. TNF-induced STAT3Y705 phosphorylation was diminished by30% after IL-6 neutralization (Supplemental Figure S3D). Inparallel, stimulation of cells with IL-6 induced STAT3Y705

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Figure 4 Expression/activation of DAPK/STAT3 through intestinalinflammation in the AOMþDSS-induced mouse model of CAC. A: Scheme forthe experimental course inducing colitis-associated tumors in wild-typemice using 10 mg/kg AOM and 3 cycles of DSS (2.5%). Endoscopicimages display mouse colon before treatment (B), after 7 days of DSS(2.5%) in the drinking water (C), before the start of the second cycle of DSSapplication at day 22 (d22) (D), and before tumor harvest at the end of theprotocol at day 65 (E). DAPK (F) and pSTAT3Y705 (G) expression/activationwas analyzed by Western blotting of tissue extracts from colon of control(n Z 2), DSS-treated (n Z 4), and AOMþDSS-treated (n Z 5) mice usingthe corresponding antibodies.

Negative Regulation between DAPK/STAT3

phosphorylation after 30 minutes at the earliest time pointand resumed after 48 hours (Supplemental Figure S3E).Other possible TNF-induced cellular functions such asapoptosis or cell viability were not altered (SupplementalFigure S4, AeD). Taken together, these results imply thatan inflammatory pathway was activated in HCEC cells inresponse to TNF.

TNF Activation of DAPK and STAT3 in Human ColonCancer Cells

To investigate whether TNF stimulation can alter theexpression/activation of DAPK and STAT3 in cancer cells,HT-29 and DLD1 colorectal cancer cells were treated withTNF for various time points and assessed byWestern blotting.In HT-29 cells, TNF treatment caused a slight enhancementin the DAPK/pDAPKS308 ratio and pSTAT3Y705 proteinlevel; whereas, pSTAT3Y705 was completely absent in thecontrol cells (Supplemental Figure S5, A and C). In DLD1cells, we also observed an increase in the DAPK/pDAPKS308

ratio (DAPKS308 phosphorylation decreased considerably),but the STAT3Y705 phosphorylation decreased (at 6 and24 hours) and reached the control levels at 48 hours(Supplemental Figure S5, B and D). When comparingpSTAT3Y705 protein level between both cell lines, the HT-29

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cells expressed a general lower protein level than DLD1 cellsbefore and after TNF treatment. Obviously there seem toexist major differences in TNF-induced signaling betweennormal and tumor epithelial cells in vitro.

DAPK Negatively Regulates the TNF-Induced STAT3Y705

Phosphorylation and IL-6 Secretion

To investigate the role of DAPK in TNF-induced inflamma-tion, a siRNA-mediated DAPK depletion experiment wasperformed. HCEC cells were transfected with DAPK siRNAor nonspecific control siRNA and subsequently treated withTNF for 24 and 48 hours. DAPK expression was depleted byup to 85% in cells transfectedwithDAPK siRNA (Figure 5A).Interestingly, TNF-induced STAT3Y705 phosphorylation wassignificantly enhanced after 48 hours (1.5-fold) followingDAPK knockdown when compared to nontransfected andcontrol siRNAetransfected cells (Figure 5A). Then, mRNAexpression of IL-6 was compared between TNF-treated and/orDAPK-silenced HCEC cells. DAPK siRNA knock downsignificantly induced IL-6 mRNA expression in untreated andTNF-treated cells at 24 and 48 hours in comparison to thecorresponding control siRNA transfected and nontransfectedcells (Figure 5B). In parallel, DAPK knockdown potentiatedTNF-induced secretion after 24 hours (1.7-fold) and 48 hours(3.4-fold), but not in untreated cells (Figure 5C). This suggeststhat DAPK per se has a clear effect on IL-6 mRNA expressionbut only TNF treatment triggers DAPK to influence thesecretion of this cytokine. These data further support theactive part of DAPK in inflammation of the mucosal micro-environment. Conversely, DAPK knockdown had no effecton IL-8 secretion (data not shown).

In a separate experiment, the requirement of DAPKkinase activity to suppress the TNF-induced inflammatoryprocess was assessed by treating the cells with TNF forvarious time points in the presence or absence of a specificDAPK kinase inhibitor.47 Inhibition of DAPK catalyticactivity significantly increased TNF-induced IL-6 secretionafter 6 and 24 hours, whereas STAT3Y705 phosphorylationwas enhanced later, ie, after 24, 48, and 72 hours (Figure 5,D and E). Notably, inhibition of DAPK catalytic activitypotentiated TNF-induced IL-6 secretion, but not to theextent of DAPK depletion, thereby indicating that not onlythe kinase activity, but also other functional domains of thekinase seem to be involved in regulating the TNF-inducedinflammatory response.

DAPK and STAT3 Interaction Is Increased under TNFTreatment

To further investigate whether DAPK interacts with STAT3,DAPK was immunoprecipitated and blotted with anti-STAT3 antibodies. Indeed, we demonstrated a physicalinteraction of DAPK with STAT3, which was elevatedunder TNF treatment (Figure 5F). To understand the role ofDAPK in this complex, we used a three-dimensional

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Figure 5 DAPK attenuates TNF-induced IL-6 secretion and STAT3Y705 phosphorylation in HCEC cells. AeC: HCEC cells were treated with 0.66 ng/mL TNF for24 and 48 hours in the presence (DAPK- or nonspecific siRNA) or absence of siRNA. A: DAPK knockdown and STAT3Y705 phosphorylation was assessed byWestern blotting of whole cell lysates. B: IL-6 mRNA expression was analyzed by real-time reverse transcription-PCR. Two similar experiments were performed.*P < 0.05; yP < 0.05 versus respective control. C: IL-6 was measured in cell culture supernatants by using an ELISA. Three similar experiments were performedin duplicate or quadruplicate. *P < 0.001; yP < 0.05 versus respective control. D and E: HCEC cells were treated with 0.66 ng/mL TNF for 6, 24, 48, or 72 hoursin the presence or absence of 10 mm DAPK inhibitor. DAPK and pSTAT3Y705 expression was analyzed by Western blotting (D). IL-6 was measured in cell culturesupernatants by using an ELISA (E). Two independent experiments were measured in triplicate. *P < 0.05; yP < 0.001, yyP < 0.001 versus TNF � DAPKinhibitor treatment. F: TNF-induced DAPK/STAT3 complex formation in normal human colon epithelial cells. HCEC cells were treated with 0.66 ng/mL TNF for 6,24, 48, or 72 hours. DAPK was immunoprecipitated and complexes were transferred to nitrocellulose membranes. The membranes were probed with anti-STAT3and anti-DAPK antibodies. Blots from a representative experiment (n Z 2) are shown.

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structural model to analyze DAPK-dependent conforma-tional changes. Activated STAT3 formed a butterfly-shapeddimer (Figure 6A). The distance between the phosphory-lated tyrosines (pY705) in the native STAT-STAT dimerwas 37.1 Å. This distance increased to 81.5 Å in the pres-ence of DAPK, and the original butterfly structure wasdistorted by DAPK docking (Figure 6B). Furthermore, theanalysis of hydrogen bond and hydrophobic interactionsshowed that DAPK seems to initiate new interactions(seven additional hydrogen bonds) between both mole-cules (Tables 1 and 2). Interestingly, the three-dimensionalmodeling showed that the DAPK binding region is nearlyoverlapping with that of the STAT3 upstream kinase JAK,thus suggesting a competition between both kinases forSTAT3 binding (Figure 6C).

TNF-Activated STAT3 Translocates into the Nucleus andBinds to the DAPK Promoter

To examine whether TNF induces the nuclear translocationof STAT3, nuclear extracts were analyzed by Western blot-ting. As shown in Figure 7A, increased levels of pSTAT3Y705

and STAT3 were observed in the nuclear fractions after24, 48, or 72 hours of TNF treatment. To investigate whetherTNF activated STAT3 transcriptional activity, EMSA assayswere performed using labeled and unlabeled oligonucleotides

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containing a consensus or mutated STAT3 binding motif.No complexes were detected in the case of untreated cells.Protein-DNA complexes were prominent after the TNFtreatment (after 24, 48, or 72 hours). This interaction wasdiminished when the extracts were incubated with an excessof cold unlabeled or mutant oligonucleotides (Figure 7B). Ahigher transcriptional transactivation is in agreement with thethree-dimensional structural model, in which the formation ofSTAT-STAT dimer showing an exposed nuclear localizationsignal (NLS) formed by the vital residues R414/R417 isessential for the shuttling of pSTAT3Y705 into the nucleus(Figure 6A). After docking of DAPK to the dimer, the NLSwhich favors the DNA binding is buried between the inter-face of the dimer (Figure 6B). Thus, the shuttling of thepSTAT3Y705 to the nucleus and subsequently the activationof target genes might be blocked, explaining the remarkableincrease in IL-6 mRNA expression after DAPK knock-down.Our sequence analysis of the DAPK promoter (Database

of Transcriptional StartSites: DBTSS: NM_004938) revealedthe presence of putative STAT3 binding motifs (TTN5AA orTTN6AA). The scheme of the DAPK promoter (Figure 7C)illustrates these putative STAT3 binding sites, five in Region1 (�1471 to �1821) and three in Region 2 (�351 to �631).Next, ChIP experiments were performed to verify whetherTNF induced the direct binding of pSTAT3Y705 to the DAPKpromoter in vivo. Two different primer pairs, specific for

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Figure 6 Structural analysis of the DAPK-STAT3 complex. A: Dockedcomplex of STAT3-STAT3 dimer. B: Docked complex of DAPK to the STAT3dimer. C: Superposed view of DAPK and JAK binding to the monomer ofSTAT3. DAPK (PDB ID: 1JKS) is represented in red, STAT3 (PDB ID: 1BG1) isrepresented in blue, and JAK (PDB ID: 3EYG) is represented in green.

Table 2 Analysis of Hydrophobic Interactions between STAT3-STAT3 Dimer and the STAT3-DAPK-STAT3 Complex

STAT3-STAT3 STAT3-DAPK-STAT3

STAT3 STAT3 STAT3 STAT3

Monomer 1 Monomer 2 Monomer 1 Monomer 2

Phe 710 Met 648 Leu 666 Ala 428Ala 703 Ala 578, Leu 577 Leu 706 Ala 376Pro 704 Leu 577 Phe 710 Leu 378

Pro 715 Phe 384, Val 432

Negative Regulation between DAPK/STAT3

each region, were designed. The analysis of precipitatedDNA using quantitative PCR and/or end-point PCR demon-strated that TNF augmented STAT3 binding to the DAPKpromoter in both regions when compared to untreated cells.Immunoprecipitated DNA with negative control IgG couldnot be amplified (Figure 7, D and E). These data suggest thatSTAT3 might regulate DAPK mRNA expression in responseto TNF stimulation in normal IEC and verified DAPK asa new transcriptional target of STAT3.

Table 1 Analysis of Hydrogen Bonding Interaction Patterns in the ST

STAT3-STAT3

STAT3 STAT3

Monomer 1 Monomer 2

Glu 638 Asn 664Asn 647 Lys 709Ser 649 Thr 708Ser 649 Leu 706Glu 652 Thr 708Arg 688 Leu 706Leu 706 Glu 652, Phe 710, Cys 712

*Arg414 and Arg417 are required for nuclear translocation of STAT3.

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DAPK and IL-6 Expression Are Regulated by STAT3

Previous studies have shown that STAT3 could eitherpromote or suppress the expression of its target genes.40,48 Tofurther evaluate STAT3 transcriptional regulation of DAPKexpression, HCEC cells were stimulated with TNF in thepresence or absence of AG490 (Janus kinase inhibitor) orStattic (inhibits STAT3 phosphorylation and dimeriza-tion). pSTAT3Y705 levels decreased significantly (by 35%)(Figure 8A) when cells were treated with AG490 before TNFtreatment. Whereas TNF induced the expression of DAPKmRNA after 48 or 72 hours only by 1.5-fold, the STAT3inactivation by AG490 pretreatment increased the DAPKmRNA expression after 72 hours by 3.3-fold, thus suggestinga transcriptional repression of DAPK by STAT3 (Figure 8A).Similarly, TNF-induced DAPK protein expression waselevated on STAT3 inactivation (Figure 8A).

TNF-induced STAT3Y705 phosphorylation was also down-regulated by Stattic pretreatment (up to 45%). Similar toAG490, DAPK protein (Figure 8B) as well as DAPK mRNAwas enhanced after 48 hours (1.5-fold) and 72 hours (2.8-fold)in the presence of Stattic (Figure 8B). These results reveal thatSTAT3 activation restricts the TNF-increased DAPK ex-pression and again suggests that STAT3 is a novel negativeregulator of DAPK expression.

In parallel,we studied the effect ofSTAT3 inactivation on theexpression of its already known target gene IL-6.Our data show

AT3-STAT3 Dimer and in the STAT3-DAPK-STAT3 Complex

STAT3-DAPK-STAT3

STAT3 STAT3

Monomer 1 Monomer 2

Arg 414* Pro 639, Glu 638, Gln 644Arg 417* Tyr 640, Thr 714, Cys 712, Val 713Glu 415 Tyr 640Ser 465 Pro 715Gln 416 Asn 647Asn 385 Gln 644, Asn 647Gln 469 Phe 716Arg 423 Leu 666Asp 374 Thr 708Asn 420 Glu 652, Lys 709, Met 655Arg 379 Lys 709

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Figure 7 TNF induces STAT3 nuclear translocation and DNA binding. HCEC cells were treated with 0.66ng/mL TNF for 24, 48, or 72 hours. A: Cytoplasmic and nuclear proteins were separated to analyze STAT3distribution by immunoblotting with anti-pSTAT3Y705, anti-STAT3, and anti-histone H1/anti-GAPDHantibodies. Western blots are representative of three independent experiments. B: DNA binding activity ofSTAT3 in nuclear extracts was evaluated by nonradioactive EMSA using infrared labeled oligonucleotidescontaining the STAT3 consensus sequence in the presence or absence of 100� competitor (Com) or mutant(Mut) oligos. Results are representative of two independent experiments. C: Theoretical image of the DAPKpromoter showing STAT3 bindingmotifs distributed in two regions. D and E: Normal human colon epithelialcells were treated with 0.66 ng/mL TNF for 48 hours. The ChIP assay was performed by using IgG or anti-pSTAT3Y705 antibodies. End-point PCR followed by agarose gel electrophoresis (D) and quantitative PCR (E)were used to analyze STAT3 binding to both regions of the DAPK promoter. Data are derived from fourindependent experiments performed in duplicate. *P < 0.05 versus the corresponding control, U-test.

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that treatment with AG490 significantly down-regulated TNF-induced IL-6 mRNA expression as well as secretion by 50%,thereby indicating that TNF-induced IL-6 expression is posi-tively regulated by STAT3 (Supplemental Figure S6, A and B).

Schematic Overview of TNF-Induced Signaling/Functions in HCEC

On thebasis of ourfindings,wepropose the followingworkingmodel (Figure 8C). TNF induces DAPK expression/activa-tion, which attenuates TNF-induced STAT3 activity eitherdirectly by physical interaction or indirectly by suppressingIL-6/STAT3 pathway. Vice versa, STAT3 represses DAPKexpression at the transcriptional level. Activated STAT3enhances IL-6 secretion, thereby forming a positive feedbackloop. Finally, DAPK and STAT3 negatively regulate eachother to promote their own expression/activation and mostprobably to balance the TNF-induced inflammatory signaling.

Discussion

Cellular response to the proinflammatory cytokine TNF variesdepending on the cellular setting.49 Our results demonstratethat DAPK expression and DAPK catalytic activity wereincreased in HCEC cells after TNF treatment. In parallel, TNFstimulation induced the IL-6/STAT3edependent inflam-matory pathway. This is in agreement with earlier reportsdemonstrating the induction of an inflammatory cascade byTNF in different cell types.4,17,19,50e52 For the first time, we

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show that both proteins, DAPK and STAT3, negativelyregulate each other.DAPK knockdown potentiated STAT3Y705 phosphoryla-

tion, IL-6 mRNA expression, and IL-6 secretion. Interest-ingly, DAPK knockdown enhanced IL-6 mRNA expressionirrespective of TNF treatment, whereas the increased IL-6secretion seems to be a clear TNF-dependent effect. Thesefindings are in line with the recently identified suppressivefunction of DAPK in TCR- and LPS-triggered NFkB acti-vation.13,14 Lungs and macrophages of DAPK�/� micesecreted higher levels of IL-6 and CXCL1 in response toLPS.14 However, the exact mechanism by which DAPKregulates inflammatory signaling remains unclear. Here, weshow that inhibiting DAPK kinase activity was less effectivethan DAPK knockdown in promoting TNF-induced IL-6/STAT3 activation. This suggests a structural involvement ofthe protein in suppressing inflammatory functions of TNF.Many DAPK interaction partners are phosphorylated byDAPK, and the catalytic activity of DAPK is required forfunctional consequences such as apoptosis or autophagy.53 Arecent paper by Chuang et al12 suggests a structural role ofDAPK in the assembly of the NLRP3 inflammasome. Wefound a physical interaction of DAPK with STAT3 byimmunoprecipitation. Because Y705 is not the DAPKconsensus motif (RxxS/T), we suggest a phosphorylation-independent mechanism by which DAPK can suppressTNF-induced inflammation. We can only speculate about therole of DAPK in STAT3 complex. It might either mask theNLS of STAT3 to impede its nuclear translocation or prevent

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Figure 8 STAT3 regulates the expression of DAPK. A: Normal human colon epithelial cells were treated with 0.66 ng/mL TNF for 24, 48, or 72 hours in thepresence or absence of 20 mm AG490. pSTAT3Y705 and DAPK expression were assessed by immunoblotting (upper panel). DAPK mRNA expression was analyzed byreal-time RT-PCR. Data represent the levels of DAPK mRNA after normalization to b2-microglobulin levels and expressed relative to the value of the untreatedcontrols. Results were obtained from two independent experiments performed in duplicate. *P < 0.001; yP < 0.05 versus untreated control; zP < 0.001 versus20 mmol/L AG490-treated cells (lower panel). B: HCEC cells were treated with 0.66 ng/mL TNF for 24, 48, and 72 hours in the presence or absence of 7 mmol/LStattic. pSTAT3Y705 and DAPK expression were assessed by immunoblotting of whole-cell lysates (upper panel). DAPK mRNA expression was analyzed by real-time RT-PCR. Data represent the levels of DAPK mRNA after normalization to b2-microglobulin levels and expressed relative to the value of TNF treatment at thecorresponding time point. Two similar experiments were performed, each in duplicate. *P < 0.001 (lower panel). C: Working model depicting TNF-inducedsignaling and functions in HCEC. TNF induces a dual signaling pro-inflammatory IL-6/STAT3 and anti-inflammatory-DAPK pathways. See the text for moredetails. Arrows depict proinflammatory signaling of TNF, and dashed lines show anti-inflammatory signaling of TNF.

Negative Regulation between DAPK/STAT3

the access of the upstream kinase JAK and the subsequentSTAT3 dimerization. Structural modeling supports boththeories. DAPK docking to the complex changes the con-formation of the STAT3 dimer in such a way that the NLSR414/417 is masked. The residues R414/417 are located inthe DNA binding domain of STAT3 and have been reportedto be required for the nuclear translocation of STAT3. Themutants of R214/215 or R414/417 failed to enter the nucleusin response to EGF or IL-6. Furthermore, mutations onR414/417 have been shown to destroy the DNA-bindingactivity of STAT3.54 The Y705 residues forming a cross-link in the dimer are separated from each other whenDAPK is associated with the complex. In addition, thebinding regions for DAPK and JAK are completely over-lapping, thereby suggesting a binding competition betweenboth kinases. Therefore, our data indicate that DAPK mightplay an essential role in equilibrating TNF-induced IL-6/STAT3 functions.

We show that TNF-activated STAT3 translocated to thenucleus, where its DNA binding activity was enhanced. Forthe first time using the ChIP assay, we identified DAPK asa transcriptional target of STAT3. STAT3 inhibition usingAG490 or Stattic elevated TNF-induced DAPK expression,thus demonstrating that STAT3 activation transcriptionallyrepresses DAPK. To date, the transcriptional regulation ofDAPK expression is only poorly understood. A recentreport shows that DAPK mRNA level is negatively regu-lated via the noncanonical Flt3lTD/NFkB pathway.55

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Another study reported that interferon geinduced DAPKexpression was dependent on C/EBP-b.56 Treatment ofmelanoma cells with 4-hydroxytamoxifen/oncostatin Minduced STAT3 activation and up-regulated DAPK mRNAtranscription.57 We have previously shown that promotermethylation leads to transcriptional silencing of DAPK inUC carcinogenesis and colorectal cancer.11,58 In our study,approximately 25% of UCC samples showed only a low ormoderate immunohistochemical DAPK protein expressionin the epithelium, thus suggesting an epigenetic regulationin these cases. Further studies are required to understand theassociation between methylation, inflammation, and IL-6/STAT3 signaling.

There is only one report showing a positive feedback loopbetween IL-6 and STAT3 in autophagic cancer cells inwhich STAT3 directly binds to the IL-6 promoter.59 Weobserved that blocking the IL-6/STAT3 pathway by IL-6neutralization or by addition of the JAK inhibitor AG490,led to a decreased TNF-induced STAT3Y705 phosphoryla-tion and IL-6 mRNA expression/secretion, thus indicatinga positive feedback loop after TNF treatment. We suggestthat IL-6 transduces the activation signal of STAT3, and inturn, IL-6eactivated STAT3 can contribute to IL-6production in the inflammatory milieu of the epithelium.These data are consistent with previous reports describinghow IL-6 and STAT3 co-operate with each other to enhancetheir activity.60e62 Our recent studies reported the involve-ment of IL-6/STAT3 in the disease perpetuation of UC.23

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Chakilam et al

By contrast, deficient gp130/IL-6/STAT3 signaling in IECincreased their sensitivity to DSS-induced colitis, showingthat the IL-6/STAT3 pathway is also important for regu-lating epithelial turnover and mucosal healing to maintaingastrointestinal homeostasis.27,28,40,63 Finally, the mecha-nism whereby IL6/STAT3 increases colitis severity stillremains unclear.64

As enhanced levels of proinflammatory cytokines mightcause instability in the balance of cell turnover leading tothe development of aberrant crypt architecture,29 the expres-sion of the inflammation-associated proteins, DAPK andpSTAT3Y705, was evaluated in UC tissues. IHC resultsdemonstrate that epithelial DAPK and pSTAT3Y705 expres-sion increased to the stage of active colitis and correlated withthe grade of inflammation as observed in our earlierstudies.11,65 Other reports show that epithelial STAT3 acti-vation correlates with the severity of colitis.29,66 Thus, DAPKand pSTAT3Y705 follow the same expression pattern from theinactive to the active colitis stage. However, the exact stepsthat follow the colitis-DALM-carcinoma sequence in bet-ween, have never been analyzed in detail for both proteins.In DALM samples, we found heterogeneity, allowing allpossible combinations (Figure 3A). In UCC specimens,DAPK levels remained high, but pSTAT3Y705 levelsdecreased in comparison to those in active UC samples.Interestingly, pSTAT3Y705 levels were also found to be less inAOMþDSS carcinomas when compared to DSS colitistissues. Both findings are in accordance to Wick et al,67 whoreported a decrease of pSTAT3Y705 expression in UCCsamples (0.75) when compared to UC samples (0.89) in theirscoring system, and to Li et al,29 who also showed thisdecrease of approximately 10%. Nevertheless, the limitedsample size in the available studies encourages conductingfurther studies using larger numbers of samples.

In summary, our findings provide a novel molecularinsight into the TNF-induced signaling network. TNF in-duced a dual signaling with simultaneous activation of ananti-inflammatory DAPK pathway and a proinflammatorySTAT3 pathway. We suggest that normal cells may havedeveloped mechanisms for reciprocal negative regulation ofpro- and anti-inflammatory proteins to balance the inflam-matory milieu. This is one of the very few reports showingthat normal mucosa is actively contributing to the devel-opment and maintenance of inflammatory conditions and inregulating the malignant transition in the gut. Furtherinvestigations will help to decipher the exact mechanism ofthis cross-regulation and to explore DAPK/STAT3 targetingin the treatment of UC and UCC.

Acknowledgments

We thank Jung Rudolf, Christa Winkelmann, ChristinaFuchs, Photini Drummer, and Adrian Koch for their excel-lent technical assistance and Prof. Reinhard Voll and Dr.Bettina Sehnert for their support to perform EMSA assay.

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Supplemental Data

Supplemental material for this article can be found athttp://dx.doi.org/10.1016/j.ajpath.2012.11.026.

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Negative Regulation between DAPK/STAT3

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