Differential Protein Expression Profile in the Intestinal Epithelium from

The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of inflammatory bowel disease (IBD). ...
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Differential Protein Expression Profile in the Intestinal Epithelium from Patients with Inflammatory Bowel Disease Anna Shkoda,†,# Tanja Werner,†,# Hannelore Daniel,‡ Manuela Gunckel,§ Gerhard Rogler,§ and Dirk Haller*,† Else-Kroener-Fresenius Center for Experimental Nutritional Medicine, Molecular Nutrition, Technical University of Munich, Freising-Weihenstephan, Germany, and Department of Internal Medicine, University of Regensburg, Germany Received August 25, 2006

The loss of intestinal epithelial cell (IEC) function is a critical component in the initiation and perpetuation of chronic intestinal inflammation in the genetically susceptible host. We applied proteome analysis (PA) to characterize changes in the protein expression profile of primary IEC from patients with Crohn’s disease (CD) and ulcerative colitis (UC). Surgical specimens from 18 patients with active CD (N ) 6), UC (N ) 6), and colonic cancer (N ) 6) were used to purify primary IEC from ileal and colonic tissues. Changes in protein expression were identified using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS) as well as Western blot analysis. PA of primary IEC from inflamed ileal tissue of CD patients and colonic tissue of UC patients identified 21 protein spots with at least 2-fold changes in steady-state expression levels compared to the noninflamed tissue of control patients. Statistical significance was achieved for 9 proteins including the Rho-GDP dissociation inhibitor R that was up-regulated in CD and UC patients. Additionally, 40 proteins with significantly altered expression levels were identified in IEC from inflamed compared to noninflamed tissue regions of single UC (N ) 2) patients. The most significant change was detected for programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase). PA in primary IEC from IBD patients revealed significant expression changes of proteins that are associated with signal transduction, stress response as well as energy metabolism. The induction of Rho GDI R expression may be associated with the destruction of IEC homeostasis under condition of chronic intestinal inflammation. Keywords: inflammatory bowel disease • ulcerative colitis • Crohn’s disease • intestinal epithelial cells • MALDITOF mass spectrometry • chronic intestinal inflammation • epithelial cell proteome • Rho-GDP dissociation inhibitor R • programmed cell death protein 8 • annexin 2A

Introduction Inflammatory bowel diseases (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD) are spontaneously relapsing, immunologically mediated disorders of the distal gastrointestinal tract. The current etiologic theories of these idiopathic disorders imply the presence of environmental triggers including enteric microbial factors in the genetically susceptible host.1-3 It seems apparent from rapidly evolving studies that the lack of innate immune defense mechanisms may actually contribute to the development of adaptive immune pathologies. Clinical studies showed an increased association of luminal enteric bacteria with the intestinal epi* To whom correspondence should be addressed. Dirk Haller, Ph.D., Associate Professor, Experimental Nutritional Medicine, Technical University of Munich, Am Forum 5, 85350 Freising-Weihenstephan, Germany. E-mail, [email protected]; phone, ++49-8161-71-2026; fax, ++49-8161-71-2097. † Else-Kroener-Fresenius Center for Experimental Nutritional Medicine, Technical University of Munich. # Both authors contributed equally to the manuscript. ‡ Molecular Nutrition, Technical University of Munich. § Department of Internal Medicine, University of Regensburg.

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thelium,4,5 an increased intestinal permeability,6-8 and a loss of immunologic tolerance including hyper-reactive T cell responses in patients with active IBD.9-12 The surface epithelium represents a highly selective barrier between the luminal gut environment and underlying lamina propria immune cells. Intestinal epithelial cells (IEC) constitutively express or can be induced to express co-stimulatory molecules13,14 and components of the human major histocompatibility complex (MHC) including class II, classical I, and nonclassical class Ib MHC molecules;15,16 pattern recognition receptors;17,18 inflammatory and chemoattractive cytokines19 as well as antimicrobial peptides.20,21 There is also accumulating evidence that IEC contribute to the initiation and regulation of innate and adaptive defense mechanisms by directly interacting with lamina propria dendritic cells and macrophages, lamina propria lymphocytes, and intraepithelial lymphocytes and are therefore considered to be a constitutive component of the mucosal immune system.11,22-27 The selective colonization of germ-free rodent models for experimental colitis support clinical observations regarding specific colitogenic mechanisms 10.1021/pr060433m CCC: $37.00

 2007 American Chemical Society

Intestinal Epithelial Cell Proteome in IBD

of certain bacteria at the epithelial cell level.28-32 It seems an attractive hypothesis that bacteria- and host-derived signals challenge the homeostatic mechanisms of the intestinal epithelium under conditions of chronic inflammation in the genetically susceptible host.33 Although disease and tissue-specific proteomics has been used to study physiological and pathophysiological situations at the epithelial cell level,34,35 human studies to characterize the IBD-related changes of the protein expression profile in primary cells have not yet been performed. Since IEC are critical to the intestinal homeostasis,33 they are an excellent target cell type to assess tissue-specific changes that may contribute in perpetuating chronic intestinal inflammation in patients with IBD. In this study, we isolated and purified primary IEC from 18 patients with CD (N ) 6), UC (N ) 6), and colorectal carcinoma (N ) 6) and identified 61 different target proteins including Rho GDI R with significantly altered steady-state expression levels using 2D gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS). In addition, specific changes in protein expression were identified in inflamed versus noninflamed tissue regions for individual patients. The most significant differences were detected for the programmed cell death protein 8 (7.4-fold increase) and annexin 2A (7.7-fold increase) in individual UC patients. In conclusion, the epithelial cell proteome may help to generate novel mechanistic insights into the disease pathogenesis. The ultimate goal is to develop clinically relevant biomarkers of the specific disease status in individual patients and to identify potential targets for therapeutic intervention.

Materials and Methods Patients. Ileal or colonic tissue was obtained from 12 patients with active CD (N ) 6) and active UC (N ) 6) undergoing surgical resections. In addition, noninflamed and inflamed tissue regions of the surgical specimens were included for further analysis. The severity of inflammation was histologically graded in the resected tissue specimens (Institute of Pathology, University of Regensburg, Germany). Normal cells were isolated from colonic samples from patients with colorectal carcinoma (control patients) used as noninflammatory controls (N ) 6). The control tissue was taken at a distance of at least 5 cm from the tumor. The used mucosal areas from the control patients were macroscopically and histologically normal. The study was approved by the Ethics committee of the University of Regensburg and performed in accordance with the declaration of Helsinki. Isolation of Primary Human Intestinal Epithelial Cells. The isolation of primary human IEC from the resected ileal or colonic tissue sections was performed as previously described.36 Briefly, the mucosa was stripped from the submucosa within 30 min of intestinal resection and rinsed several times with phosphate-buffered saline (PBS). The mucus was removed by treatment for 15 min with 1 mM dithiothreitol (Serva, Heidelberg, Germany). After the washing process of the tissue, the mucosa was incubated with 1.5 mM EDTA in Hanks balanced salt solution without calcium and magnesium and tumbled for 10 min at 37 °C. The supernatant containing debris and mainly villus cells was discarded. The mucosa was incubated again with EDTA for 10 min at 37 °C. The supernatant of this isolation step was collected into a 15 mL tube. Then, the remaining mucosa was vortexed, and the supernatant was again collected containing complete crypts, some single cells, and a small

research articles amount of debris. To separate IEC from contaminating nonepithelial cells, the suspension was allowed to sediment for 15 min. The sediment containing mainly complete crypts was collected and washed twice with PBS. Primary IEC from the resected intestinal surgical specimens were combined and collected in sample buffer for subsequent Western blot and proteome analysis. The cell purity was assessed by determining the absence of CD3+ T-cell contamination using immunoreactive anti-CD3 antibodies for Western blot analysis. Sample Preparation for 2D-PAGE and Gel Analysis. Purified primary IEC from all 18 patients were lysed in 100-200 µL of buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 1% DTT (all from Roth, Karlsruhe, Germany), protease inhibitor (Roche Diagnostics, Mannheim, Germany), and 0.8% Pharmalyte (Amersham Biosciences, Freiburg, Germany). Homogenization of the cell extracts was achieved by ultrasonication (amplitude 35, cycle 0.5) using 10 impulses on ice. The lysed cells were then centrifuged for 30 min at 10 000g at 4 °C. The total protein concentrations of the solubilized proteins in the supernatants were determined using the Bio-Rad protein assay (Munich, Germany) and ranged between 4 and 18 µg/µL. The lysates were stored at -80 °C. Immobilized pH-gradient strips (IPG, pH 3-10, 18 cm, Amersham Biosciences) were rehydrated overnight in 350 µL of buffer (8 M urea, 0.5% CHAPS, 15 mM DTT, and 0.5% IPG buffer), and 500 µg of total protein was cup-loaded onto the strip. Isoelectric focusing (IEF) in the first dimension and sodium-dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension were performed. IEF was run by an Amersham IPGphor unit under the following conditions: 500 V (1 min, gradient), 4000 V (1.5 h, gradient), 8000 V (28 000 Vh, Step-and-hold). Subsequent to the first dimension and before loading onto SDS-PAGE gels, strips were incubated for 15 min in equilibration-buffer (1.5 M Tris-HCL, pH 8.8, 6 M urea, 26% glycerol, 2% SDS, and 1% DTT) followed by additional equilibration in buffer for another 15 min (1.5 M Tris-HCL, pH 8.8, 6 M urea, 26% glycerol, 2% SDS, and 4% iodoacetamide). SDS-PAGE gel electrophoresis was performed by an Amersham Biosciences Ettan-Dalt II System with 4 mA per gel for 1 h followed by 12 mA per gel using 12.5% SDS-polyacrylamide gels (1 mm-thick). For protein staining, gels were fixed in 40% ethanol and 10% acetic acid for 8 h followed by an overnight exposure to a Coomassie solution containing 10% (NH4)2SO4, 2% phosphoric acid, 25% methanol, and 0.625% Coomassie Brillant blue G-250. Destaining of the gels was performed in aqua bidest until the background was completely clear. All 12 gels from control (N ) 6) versus CD patients (N ) 6) and control (N ) 6) versus UC patients (N ) 6) were simultaneously submitted to all steps of 2D gel electrophoresis including IEF, SDS-PAGE, Coomassie Brillant Blue staining, and quantitative analysis in order to minimize variability between samples. Coomassie-stained gels were scanned (ImageScanner, Amersham Biosciences) and analyzed by ProteomWeaver software (Definiens, Munich, Germany) including background subtraction and volume normalization. Spots with at least 2-fold differences in protein intensity were picked for further MALDITOF-MS analysis. Trypsin Digests of Protein Spots and MALDI-TOF-MS. Coomassie-stained spots were picked, washed alternately in acetonitrile and 50 mM NH4HCO3, dried, and then digested using 0.1 µg of sequencing grade modified trypsin in 50 mM Journal of Proteome Research • Vol. 6, No. 3, 2007 1115

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Table 1. Proteome Changes in Intestinal Epithelial Cells from Patients with Ulcerative Colitis Compared to Control Patients spot no.

access. no.

1 2 3a 4 5a 6a 7 8a 9 10a 11 12a 13 14b 15a 16a 17

BAA37169 Q8WVXO_HUMAN A55927 Q96AGO_HUMAN LEG-3_HUMAN LDHA_HUMAN S65491 ACCOE CRHU2 MLRM_HUMAN A48043 Q53G17_HUMAN DHCA_HUMAN KRHU9 AAA36425 OWHU 1HJOA

a

protein name

Mr (Da)

IgG κ chain (fragment) Peroxisomale enoyl-coenzyme A hydratase-like protein nicotinamide phosphor-ribosyltransferase Staphylococcal nuclease domain containing 1 Galectin-3 L-lactate dehydrogenase A chain 26S proteasome regulatory chain 12 Rho gdp dissociation inhibitor (GDI) R chain  Carbonic Anhydrase II Myosin regulatory light chain 2, nonsarcomeric Ubiquinol-cytochrome c reductase core protein I NADH dehydrogenase (ubiquinone) Fe-S Protein 8 Carbonyl Reductase (NADPH) Keratine 19, type I Pancreatitis-associated protein Ornithine carbamoyl-transferase heat shock 70 kDa protein 42 kDa ATPase N-terminal fragment

23690 36078 55772 100294 26098 36819 36674 20571 29285 19707 53270 24232 30510 44065 19654 40057 41973

pI

S cov %

mean (fold)

MannWhitney test

6.92 8.47 6.69 6.52 8.61 8.46 6.16 6.73 6.87 4.67 5.94 5.53 8.55 5.04 8.16 8.75 6.69

51 24 47 22 34 53 28 40 43 47 26 36 47 38 40 25 37

2.07 2.09 2.13 2.12 2.66 2.04 2.52 2.20 2.37 2.33 0.45 0.43 0.49 0.43 0.25 0.22 0.34

0.0519 0.0571 0.0411 0.0931 0.0000 0.0000 0.1143 0.0303 0.0920 0.0469 0.2000 0.0000 0.0571 0.1000 0.0476 0.0000 0.2000

Significantly regulated protein spots (p-values 10 cm from tumor margin), and the tumor itself. When this study is compared with the protein expression of the control tissue used in our study, there is overlap in the modulation of proteins including β-actin, creatine kinase, cytokeratin 19 and 20, fatty acid binding protein, Rho GDI ly (not the R isoform) and 14-3-3 . This arises the question whether the differences of protein expression profile found in IBD are exclusively due to the inflammatory condition or are influenced by the cancer status of the patients used as a control. In particular, we compared the regulated proteins in tumor mucosa to our control tissue and found an overlap including β-actin, creatine kinase, and cytokeratin 19, but a different modulation regarding creatine kinase, which was shown to be down-regulated with a 3.1-fold change by Polley et al., but in our study this protein was 2.18-fold up-regulated. 1120

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Inflammation and cancer have been considered closely linked for many years, with colorectal cancer (CRC) being the most common malignant complication in patients with chronic intestinal inflammation (5% of all CRCs).49,50 Thus, it is difficult to say whether an aberrant protein profile expression is characteristic of cancer tissue or is a pathological factor of IBD. Our study shows a marginal overlap of 3 from 61 identified proteins (4.9%). In addition, our data showed that the regulation of protein expression in IBD patients is significantly in-/ decreased with respect to normal-appearing mucosa of cancer patients. Moreover, since IBD is a predisposing factor for CRC, it seems more likely that the regulated protein expression already found in inflamed tissue is more related to IBD than cancer even if still remaining in the subsequent malignant status. We next compared inflamed versus noninflamed regions from the same tissue sections including two UC patients and identified additional 40 proteins (UC1 ) 38, UC2 ) 3, 1 protein was detected twice) with significant changes in their protein

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Figure 3. (A-C) Differential expression of annexinA2 and programmed cell death protein 8 in inflamed versus noninflamed tissue regions of UC patient 1. 2D gel protein expression changes are shown for the programmed cell death protein 8 (PDCD8) and annexin A2 in noninflamed versus inflamed tissue regions from UC patient 1 (triplicate gels). The average fold changes are indicated (A). Representative peptide mass fingerprints are shown for PDCD8 and annexin A2 (B). Western blot analysis was performed with IEC from noninflamed (NI) versus inflamed (I) tissue regions from UC patient 1 and UC patient 2. Immunoblot was performed with 20 µg of the total protein using specific antibodies for PDCD8 and annexin A2 (C).

expression levels. Interestingly, the total number of differentially regulated proteins varied substantially between the two patients, confirming the heterogeneity of protein expression in patients with different genetic background, as well as the influence of environmental factors such as nutritional or smoking habits. In addition, UC patient number 1 is a 38-yearold male, whereas UC patient number 2 is female at the age of

52, with age and gender playing an important role in differential protein expression.51-53 Taken together, all these factors may account for the differences in protein expression found in both patients. This work presents the novelty of comparing inflamed and noninflamed tissue from the same patient, which offers the possibility to exclude the problem of noninflamed cancer tissue as healthy control and genetic heterogeneity. UnfortuJournal of Proteome Research • Vol. 6, No. 3, 2007 1121

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Figure 4. Bibliometric data analysis. Protein-protein network tree generation using the data-mining program Bibliosphere software. Colored proteins in the network tree are implicated in the energy metabolism (green), signal transduction (red), and cytoskeletal/ detoxification functions (black).

nately, it is very difficult to obtain tissue samples from single patients of inflamed and noninflamed areas to perform proteome analysis. The bibliometric data analysis including all 57 differentially regulated proteins (1 protein was hypothetical protein, 5 proteins were detected twice, 3 protein not in Bibliosphere Database) generated a complex network with at least 37 highly interrelated proteins (Figure 4, Group 1). The major functions that were associated with these proteins in Group 1 included signal transduction (16 from 27 proteins), energy metabolism (10 from 18 proteins), and cytoskeletal functions (11 from 12 proteins). Group 2 revealed 6 interrelated proteins that were associated with the energy metabolism (UQCRC2, UQCRC1, NDUFS1, CKMTB1) and proteasomal function (PSMD12, PSMC2). Group 3 revealed an additional 14 proteins that were completely unrelated to any other protein. The signalling proteins of Group 3 target the NF-κB (ETHEI), stress signalling (CCT7, HSPAIL), and G-protein-coupled signalling (GNAI2) pathways. Interestingly, 47% of all changes in the epithelial cell proteome were associated with signal transduction pathways (27 from 57) including proteins that were connected with Ca2+ signalling (MYLPF, ANXA2), hypoxia signalling (MYLPF, KHSRP), heat shock and stress response signalling (CCT7, HSPAIL), NF-κB nuclear export signalling (ETHE1), G-protein-mediated signalling (ARGHDIA, GNAI2, GAP1, PBP), proteasomal functions (PSME1, PSMD12, PSMC2), intra- and cell surface binding proteins (YWHAE, SELENBPI, FABP2), redox signalling (GSR, GSTP1), intracellular serine protease inhibitors (SERPINB1), and pro-apoptotic mechanisms (PDCD8). Interestingly, the programmed cell death protein 8 (PDCD8), also named as apoptosis inducing factor (AIF), was 7.4-fold up-regulated in in1122

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flamed versus noninflamed tissue regions in UC patient 1 (Figure 3A, protein spot 49, increase), supporting the hypothesis that defects in the epithelial cell barrier function contribute to conditions of chronic inflammation.54 The induction of apoptosis through AIF-mediated mechanisms was associated with oxidative stress-related changes in the mitochondria.55 Interestingly, the nuclear enzyme poly(ADP-ribose) polymerase (PARP) is associated with AIF-mediated apoptosis and,56 most importantly, has also been shown to be a substrate of the AMP kinase in IEC.57 The energy sensor AMP kinase is activated by the reduction of cellular ATP, suggesting that the regulation of the cellular energy status is tightly involved in cell survival by maintaining the chromatin structure, DNA repair, and cellular restoration.58 On the other hand, the strongest induction of protein expression was detected for annexin A2 (ANXA2) (7.7fold increase), a Ca2+ and RNA binding protein59 as well as a receptor for tissue-type plasminogen activator and plasminogen (Figure 3A, protein spot 40).60 Annexin A2 was shown to be sensitive to changes in the redox status of the cell modulated by oxidative stress, and most importantly, the decrease in annexin A2 expression was associated with the induction of cell death.61 Interestingly, the ANXA2 gene is growth-regulated, and its expression is stimulated by growth factors such as insulin, fibroblast growth factor, and epidermal growth factor,62 suggesting that the increase in annexin 2A protein expression in human IEC from IBD patients is a homeostatic consequence of the barrier disruption during chronic inflammation. Taken together, these two strongest regulated proteins in our proteome analysis (PDCD8 and ANXA2) illustrate strongly interrelated but also opposite biological processes, suggesting a dramatic disequilibrium in the cellular homeostasis of the epithelium. It seems also important to understand that the

Intestinal Epithelial Cell Proteome in IBD

induction of programmed cell death protein and annexin A2 protein expression was present in UC patient 1 but not in UC patient 2, suggesting individual differences in the disease status, disease mechanisms, genetic susceptibility, or medication. In addition, previous studies in human IBD showed that persistent inflammation was associated with increased nuclear factor κB (NF-κB) activity in lamina propria macrophages, and the epithelium.63-67 Experimental evidence for the importance of NF-κB in directing proinflammatory processes under conditions of chronic inflammation was elegantly shown in trinitrobenzene sulfonic acid (TNBS)-treated mice.68 The local administration of anti-sense NF-κB RelA oligonucleotides abrogated clinical and histological signs of TNBS-induced experimental colitis, suggesting a pivotal and mechanistic role for sustained NF-κB activity in the pathogenesis of chronic mucosal inflammation.68 Consistent with these observations, we showed an expression decrease of the RelA binding protein ETHE1 (ethylmalonic encephalopathy protein). The ETHE1 gene encodes a mitochondrial matrix protein involved in the development of the ethylmalonic encephalopathy69 but also capable of transcription factor shuttling between the cytoplasm and nucleus,70 suggesting that mitochondrial defects may be connected to the regulation of activated proinflammatory pathways under chronic inflammation. It has been suggested that chronic intestinal inflammation represents an energy-deficiency disease with alterations in the oxidative metabolism of epithelial cells.71,72 This assumption is further supported by the finding that ATP levels are decreased in the mucosa of patients with ulcerative colitis.73 We showed that 32% of all differentially regulated proteins (18 from 57) were associated with the energy metabolism, especially with changes in glycolosis and tricarboxylic cycle (LDHA, LDHC, ENO1, MDH1, ALDOA), as well as fatty acid β-oxidation and mitochondrial respiration (ECH1, CPT2, UQCRC1, UQCRC2, NDUFS1). The induction of L-lactate dehydrogenase in IBD patients may indicate a shift toward the anaerobic glycolosis for ATP generation, suggesting the presence of cellular stress and hypoxic conditions in the chronically inflamed tissue. In conclusion, we are the first to report proteome changes in the intestinal epithelium from chronically inflamed patients with IBD. It seems obvious from these results that follow-up studies require higher numbers of patients including IBD patient subsets with similarities in genetic polymorphisms, disease localization, and disease behavior in order to confirm and extend the number of significantly regulated proteins. The epithelial cell proteome may add significant insights for the disease pathology, and although the heterogeneity between IBD patients is high, we were able to show significant changes in the signal transduction regulator Rho GDI R. The epithelial cell proteome may help to generate novel mechanistic insights into the disease pathogenesis leading to identification of specific biomarkers according to the specific disease status, disease behavior, and individual genetic subgroup of the IBD patients.

Acknowledgment. This work was supported by Die Deutsche Forschungsgemeinschaft grant HA 3148/1-4 to D. Haller. References (1) Gaya, D. R; Russell, R. K.; Nimmo, E. R.; Satsangi, J. New genes in inflammatory bowel disease: lessons for complex diseases? Lancet 2006, 367, 1271-84. (2) Bouma, G.; Strober. W. The immunological and genetic basis of inflammatory bowel disease. Nat. Rev. Immunol. 2003, 3, 52133.

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