Intestinal Epithelial Cell Proteome in IL-10 Deficient Mice and IL-10

Intestinal Epithelial Cell Proteome in IL-10 Deficient Mice and IL-10 Receptor Reconstituted Epithelial Cells: Impact on Chronic Inflammation Tanja Werner, Anna Shkoda, and Dirk Haller* Technical University of Munich, Departments of Food and Nutrition Science and Medicine, Experimental Nutritional Medicine, Else-Kroener-Fresenius Center, Am Forum 5, Freising-Weihenstephan, Germany Received April 20, 2007

The interaction of nonpathogenic enteric bacteria with intestinal epithelial cells (IEC) in the absence of host-derived Interleukin 10 (IL-10) may contribute to the development of chronic inflammation. Functional proteome analysis of primary IEC from Enterococcus faecalis-monoassociated WT and IL10-/- mice as well as IL-10 receptor reconstituted IEC revealed expression changes of proteins that are involved in endoplasmic reticulum stress, energy metabolism, and apoptosis, suggesting a protective role for IL-10 at the epithelial cell level. Keywords: IBD • Experimental colitis • Interleukin 10 • IL-10-/- mice • Intestinal epithelial cells • Epithelial cell proteome • Colitogenic bacteria • Enterococcus faecalis • Galectin 3

Introduction Inflammatory bowel diseases (IBDs) including ulcerative colitis (UC) and Crohn’s disease (CD) are spontaneously relapsing, immunologically mediated disorders of the distal gastrointestinal tract. Previous studies showed increased association of luminal enteric bacteria with the intestinal epithelium1,2 and a loss of immunologic tolerance in patients with active IBD.3,4 Current etiologic theories assume an involvement of the normal enteric microbiota in this idiopathic disorder,5 suggesting that the interaction of nonpathogenic enteric bacterial species with the epithelium may contribute to the initiation and/or perpetuation of chronic intestinal inflammation in the genetically susceptible host. Selective colonization of germ-free rodent models for experimental colitis with defined commensal bacterial species confirmed these clinical observations regarding specific colitogenic mechanisms of certain bacteria at the epithelial cell level.6,7 Indeed, the presence of colitogenic Enterococcus faecalis transiently induced toll-like receptor (TLR) 2-mediated RelA phosphorylation and NF-κB-dependent gene expression in native intestinal epithelial cells (IEC) from wild type (WT) mice but persistent activation of the TLR/NF-κB pathway in Interleukin-10 (IL-10) deficient mice (IL-10-/-), suggesting a pathological role for bacteria-epithelial cell signaling under chronic inflammation.8 The absence of colitis and pathologic immune responses in E. faecalis-colonized WT mice demonstrated the nonpathogenic nature of this Gram-positive enteric bacterial species and, most importantly, suggests that normal hosts develop immunosuppressive mechanisms that control mucosal * 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. 10.1021/pr070222x CCC: $37.00

 2007 American Chemical Society

immune responses to the constant challenge of commensal bacterial antigens. IL-10 signals through JAK1/STAT3- and p38-MAPK-mediated pathways to trigger anti-inflammatory mechanisms dependent on suppressor of cytokine signaling (SOCS)9,10 and heme oxygenase (HO)-1.11 In addition to these well-described inhibitory mechanisms in leukocytes, we found that IL-10 modulates the overexpression of the endoplasmic reticulum (ER)-derived glucose-regulated protein (grp)-78 in the intestinal epithelium under conditions of chronic inflammation.12 The intestinal epithelium not only forms an effective barrier, but is also considered to be a constitutive component of the mucosal immune system.7,13,14 Indeed, IEC contribute to the initiation and regulation of innate and adaptive defense mechanisms by integrating numerous signals from both enteric bacteria and underlying immune cells. Interestingly, we showed that the expression of the ER chaperon grp-78 was increased in primary IEC from chronically inflamed E. faecalis-monoassociated IL10-/- mice as well as CD and UC patients,12 supporting the hypothesis that the failure of ER homeostasis contributes to the loss of epithelial cell function and disease progression in IBD. In addition, the oral administration of genetically engineered Lactococcus lactis strains that locally secrete the immunosuppressive cytokine IL-10 have already shown the potential for future therapeutic approaches in the treatment of IBD.15-17 The preliminary feasibility to translate the experimental animal studies into a clinically relevant IBD treatment regiment was recently shown in a phase I clinical trial.16 Although these findings identified novel therapeutic approaches for the anti-inflammatory effects of IL-10, the molecular understanding of IL-10-mediated protective mechanisms at the epithelial cell level remain to be characterized. To better define epithelial cell responses under the pathological conditions of chronic intestinal inflammation in the Journal of Proteome Research 2007, 6, 3691-3704

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research articles absence of IL-10, we characterized the protein expression profile (proteome) in primary IEC from E. faecalis-monoassociated WT and IL-10-/- mice at early (2 weeks) and late stages (14 weeks) of bacterial colonization. Additionally we characterized protein expression profile in IL-10 receptor reconstituted (IL-10R) Mode-K cells after the stimulation of the epithelial cell line with E. faecalis and IL-10. In total, we identified 76 target proteins with significantly altered steady-state expression levels in primary and IL-10R reconstituted IEC lines using 2D-gel electrophoreses (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS). The bibliometric data analysis generated a protein-protein network tree from cell culture experiments and animal model systems and identified three main clusters of interrelated proteins with cytoskeletal functions and catalytic activity, protein metabolism as well as transport functions.

Materials and Methods Animals and Bacterial Monoassociation. Germ-free 129 SvEv TAC mice and germ-free IL-10 gene deficient (-/-) 129 SvEv TAC mice were monoassociated at 12-16 weeks of age with the colitogenic E. faecalis strain OG1RF. In collaboration with Dr. R. Balfour Sartor (University of North Carolina, Chapel Hill, NC) the mice were maintained at the National Gnotobiotic Rodent Resource Center at the University of North Carolina at Chapel Hill. Bacterial monoassociation and absence of contamination by other bacterial species were confirmed by culturing samples from the small and large intestine at necropsy and culturing serial fecal samples as previously described.12,18 Animal use protocols were approved by the Institutional Animal Care and Use Committee (IACUC), University of North Carolina. Mice were killed 1, 2, and 14 weeks after initial bacterial colonization. Germ-free mice were used as controls. Sections of the ileum, cecum, proximal, and distal colon were fixed in 10% neutral buffered formalin (Sigma Aldrich). The fixed tissue was embedded in paraffin. Histology scoring was analyzed by blindly assessing the degree of lamina propria mononuclear cell infiltration, crypt hyperplasia, goblet cell depletion, and architectural distortion as previously described.19 Isolation of Primary Mouse Intestinal Epithelial Cells. Primary IEC from the cecal and colonic epithelium of germfree and E. faecalis-monoassociated wild type as well as IL10-/- mice were purified as previously described.8 Briefly, the cecal and colonic tissue was cut into pieces and incubated at 37 °C in DMEM containing 5% FCS (Gibco, Invitrogen, Auckland, New Zealand) and 1 mM dithiothreitol (DTT; Roth, Karlsruhe, Germany) for 30 min. The remaining tissue was incubated in 30 mL of PBS (1×) (Gibco, Invitrogen, Auckland, New Zealand) containing 1.5 mM EDTA (Roth, Karlsruhe, Germany) for additional 10 min. The supernatants were filtered and centrifuged for 5 min at 400g, and the cell pellet was resuspended in DMEM containing 5% FCS. Finally, the primary IEC suspension was purified by centrifugation through a 20%/ 40% discontinuous Percoll gradient (GE Healthcare, Uppsala, Sweden) at 600g for 30 min. Generation of IL-10R Reconstituted Mode-K Cells. The mouse IEC line Mode-K (passage 10-30) was grown in tissue culture plates as previously described.8 Mode-K cells were transfected with the expression vector for murine IL-10R (a generous gift from Dr. Heinz Baumann, Rosewell Park Cancer Institute, Buffalo, NY) at 50-80% confluency using 2 µg of the 6505 bp plasmid (MSCVneoEB) and 6 µL of FuGENE tranfection 3692

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reagent (Roche Diagnostics, Mannheim, Germany). Stable transfected Mode-K cells were selected for their neomycin (1 µg/mL; Roth, Karlsruhe, Germany) resistance. Where indicated, the confluent epithelial cell monolayers were treated with TNF (R&D Systems, Heidelberg, Germany) and IL-10 (R&D Systems, Heidelberg, Germany). Sample Preparation for 2D-PAGE and Gel Analysis. Purified primary IEC from E. faecalis-monoassociated WT and IL-10-/mice were lysed in 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 used for further analysis or 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 a GE Healthcare Ettan IPGphor 3 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; all from Roth, Karlsruhe, Germany) 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; Sigma Aldrich). SDS-PAGE gel electrophoresis was performed by a Bio-Rad PROTEAN plus Dodeca Cell with 54 mA per gel for 2 h followed by 13 mA per gel using 12.5% SDS-polyacrylamide gels (1 mmthick). For protein staining, gels were fixed in 40% ethanol and 10% acetic acid (both from Roth, Karlsruhe, Germany) 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 (all from Roth, Karlsruhe, Germany). Destaining of the gels was performed in aqua bidest until the background was completely clear. All 12 gels from germ-free (pooled control) and E. faecalis-monoassociated WT (N ) 5) and IL-10-/- (N ) 5) mice were simultaneously subjected to all steps of 2D-gel electrophoresis including IEF, SDS-PAGE, Coomassie Brillant Blue (CBB) staining, and quantitative analysis to minimize variability between samples. The detection limit of CBB stains is in the range of 10-200 ng/mm2. Coomassie-stained gels were scanned (GS-800 Calibrated Densitometer, Bio-Rad) and analyzed by ProteomWeaver software (Definiens, Munich, Germany) including background subtraction and volume normalization. Reference gels from the pooled samples of all 5 different germ-free WT and IL-10-/mice were generated and compared with the IEC samples gels from of E. faecalis-monoassociated wild type and IL-10-/- mice, respectively. The average number of Coomassie-stained protein spots that could be resolved on the 2D SDS-PAGE gels ranged between

IEC Proteome in IL-10- Mice and IL-10 Receptor Reconstituted IEC

400 and 600 spots. Protein spots differentially expressed in at least 3 of 5 animals and with 2-fold change in the average steady-state expression level were submitted to MALDI-TOF MS. Trypsin Digestion of Protein Spots and MALDI-TOF MS. Coomassie-stained spots were picked, washed alternately in acetonitrile and 50 mM NH4HCO3 (both from Roth, Karlsruhe, Germany), dried, and then digested using 0.1 µg of sequencing grade modified trypsin (Promega, Mannheim, Germany) in 50 mM NH4HCO3. The dried spots were rehydrated for 1 h at 4 °C by adding 6 µL of 0.02 µg/µL trypsin and incubated overnight at 37 °C. Finally, 7 µL of 1% trifluouroacetic acid (TFA, Sigma, Germany) was added, and the tryptic peptide fragments were extracted using ultrasonication for 15 min at room temperature. Supernatants were stored at -80 °C or directly used for MALDITOF-MS analysis. Mass spectrometric analysis was performed according to the method of Bruker Daltonics (Leipzig, Germany) using the Autoflex Control software and the mass spectrometer from Bruker Daltonics. Briefly, 2-3 µL of the extracted protein sample, together with 2 µL of 0.1% TFA, was spotted onto the target using the thin-layer affinity HCCA AnchorChip preparation by Bruker Daltonics. Proteins were identified by using the Mascot Server 1.9 (Bruker Daltonics) based on mass searches within murine sequences only. The search parameters allowed the carboxyamidomethylation of cystein and one missing cleavage. The minimum score of 61 and a mass accuracy of (100 ppm were selected as criteria for positive identification of proteins. The protein identification and accession number, the molecular mass (MrM), their isoelectric point (pI), the sequence coverage for the peptide fingerprints (SCov), the mean fold changes (MFC) ( standard deviation (SD) and the frequency (F) of changes are compiled in Tables 1-7. Western Blot Analysis. Purified primary IEC or Mode-K cells were lysed in 1× Laemmli buffer, and 20-50 µg of protein was subjected to electrophoresis on 10% SDS-PAGE gels. Antiphospho-RelA (Ser536), anti-RelA, anti-phospho-p38 (Thr180/ Tyr182), anti-p38, phospho-STAT3 (Tyr705), STAT-3 (all antibodies from Cell Signaling, Beverly, MA), and anti-β-actin (ICN, Costa Mesa, CA) were used to detect immunoreactive phosphoRelA, RelA, phospho-p38, p38, phopsho-STAT-3 and STAT-3, and β-actin, respectively, using an enhanced chemiluminescence light-detecting kit (Amersham, Arlington Heights, IL). Small Interference RNA and Cell Transfection. Synthetic galectin-3 (NM_010705.2) specific siRNA was purchased from Qiagen (Hilden, Germany). The target sequence for galectin-3 was 5′-AGGGAACTATGTAATTATCAA-3′. The sequences of galectin-3-specific double-stranded ribo-oligonucleotides was as follows: Sense, r(UUGAUAAUUACAUAGUUCC)d(TT); Reverse, r(GGAACUAUGUAAUUAUCAA) d(TT). The annealed doublestranded ribo-oligonucleotides including negative control siRNA (Qiagen, Hilden, Germany) were heat-treated for 1 min at 90 °C and then incubated for additional 60 min at 37 °C. Mode-K cells were grown in 24-well culture plates to 50-80% confluency followed by the transfection with 1.5 µg of singlestranded galectin-3-specific siRNA in 13.5 µL of RNAi transfection reagent according to the protocol of the manufacturer (Qiagen, Hilden, Germany). RNA Isolation and Real-Time Reverse-Transcription PCR. RNA from purified native IEC was extracted using Trizol Reagent (Invitrogen Life Technologies, Karlsruhe, Germany) according to the manufacturer’s instructions. Extracted RNA was dissolved in 20 µL of water containing 0.1% diethyl-

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Figure 1. (A) Differential induction of phospho-p38, phosphoRelA and phospho-STAT3 in IEC of WT and IL-10-/- mice colonized with E. faecalis. Induction of p38 phosphorylation and absence of permanent RelA phosphorylation in primary IEC from WT but not in IL-10-/- mice after the colonization with E. faecalis. Germ-free WT and IL-10-/- mice were monoassociated for 1 and 14 weeks with E. faecalis. Western blot analysis was performed with 50 µg of total protein derived from pooled IEC samples from 5 germ-free and E. faecalis monoassociated mice, using immunoreactive phospho-RelA, RelA, phospho-p38, p38, phosphoSTAT3, and STAT3. (B) Differential IP-10 expression in primary IEC of WT (grey bars) and IL-10-/- (black bars) mice colonized with E. faecalis. Total RNA was isolated from large intestine of WT and IL-10-/- germ-free and colonized animals (1, 2, and 14 weeks), cDNA was generated, and Light Cycler RT-PCR was performed using specific primer for murine IP-10 and GAPDH. Germ-free animals were taken as control for appropriate calculations of fold-induction.

pyrocarbonate. Reverse transcription was performed from 1 µg of total RNA. Real-time PCR was performed from 1 µL of reverse-transcribed cDNA in glass capillaries using a Light Cycler system (Roche Diagnostics, Mannheim, Germany) as previously described.8 Primer sequences were as follows: IP10 sense, 5′-TCCCTCTCGCAAGGAC-3′ and reverse, 5′-TTGGCTAAACGCTTTCAT-3′; GAPDH sense 5′-ATCCCAGAGCTGAACG-3′ and reverse, 5′-AAGT-CGCAGGAGACA-3′. The amplified product was detected by the presence of a SYBR green fluorescent signal. Melting curve analysis and gel electrophoresis were used to document the amplicon specificity. The crossing point (Cp) of the log-linear portion of the amplification Journal of Proteome Research • Vol. 6, No. 9, 2007 3693

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Table 1. Differentially Regulated Proteins in IEC of WT Mice after 2 Weeks of Colonization with E. faecalis no.

accession number

protein

MrM

pI

SC

MFC

SD

F

1 2 3 4 5 6 7 8 9 10 11 12 13 14

AAH49104 JC4398 BAB22060 AAC63098 S29170 S60028 BAC401585 AJMSRS BAC27697 Q8K0P6 Q8BSR6 Q8BFR5 AAB03107 Q99K93

Oxoglutarate dehydrogenase Thiosulfate sulfotransferase Glyoxalase I Platelet-activating factor acetylhydratase 45 kDa subunit Annexin 7 Ferredoxin NADP reductase precursor Chloride channel ABP Argininosuccinate synthase Acetyl-CoA-acetyltransferase mitochondrial A430096B05 Rik fragment Valosin containing protein Elongation factor TU mitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase Elfin (PDZ and LIM domain 1)

117298 33673 20967 44972 50162 54568 27338 48840 45129 133116 89998 49876 34641 36208

6.51 7.71 5.24 6.95 5.91 8.90 5.09 8.36 8.71 4.98 5.11 7.23 8.70 6.38

7 18 45 24 17 18 38 15 20 9 10 21 28 28

5.32 2.61 2.52 0.30 0.40 0.27 0.42 0.32 0.44 0.43 0.42 0.40 0.31 0.23

1.66 0.13 0.52 0.10 0.03 0.06 0.10 0.03 0.03 0.04 0.06 0.09 0.04 0.13

5/5 3/5 3/5 5/5 4/5 4/5 4/5 4/5 4/5 3/5 3/5 3/5 3/5 3/5

Table 2. Differentially Regulated Proteins in IEC of IL-10-/- after 2 Weeks of Colonization with E. faecalis no.

accession number

protein

MrM

pI

SC

MFC

SD

F

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

Q9Z2A3 BAB22842 Q8CDP8 Q99C98 JQ0028 T30173 DEMSG BAC37447 AAH08174 CAD20433 AAH06749 ILBP_ MOUSE Q9CYW4 S55921 Q8CDX3 Q8BFR5 B55729

Glial cell line derived neurotrophic factor family member receptor alpha 2b Succinate dehydrogenase complex, subunit B Ankyrin repeat domain-containing SOCS box protein Asb15 Voltage dependent anion channel 2 Cytokeratin 19, fragment Zink finger protein Pw1 Glyceraldehydes-3-phosphate gehydrogenase, phosphorylated Destrin Peroxiredoxin 5 Sequence 17 from patent WO0185986 UDP-glucose dehydrogenase Gastrotropin Ileal lipid binding protein Hypothetical Haloacid dehalogenase Pyruvate kinase M2 Hypothetical protein, fragment Elongation factor TU mitochondrial Hydroxymethylglutaryl-CoA-synthase, mitochondrial.

41043 32591 66440 32310 44515 158815 36072 18852 22226 30374 55482 14403 28237 58394 29523 49876 53115

7.69 8.96 5.67 7.44 5.28 5.12 8.44 8.14 9.10 6.61 7.49 5.93 6.31 7.58 11.80 7.23 7.46

14 20 10 20 26 2 47 33 24 21 24 44 27 12 39 21 11

2.19 3.06 2.97 3.70 2.81 2.25 2.6 2.53 2.96 0.42 0.35 0.41 0.41 0.36 0.41 0.34 0.39

0.10 0.50 0.34 0.77 0.74 0.18 0.46 0.38 0.74 0.06 0.09 0.07 0.05 0.06 0.05 0.07 0.06

5/5 4/5 4/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 3/5 4/5 4/5 4/5 3/5

curve was determined. The relative induction of gene mRNA expression was calculated using the following equation: E∆Cp(control samples - treated samples) and normalized for the expression of GAPDH. All 5 samples from E. faecalis-monoassociated mice were measured and blotted as fold increase over the 5 samples from germ-free control mice. ELISA Analysis. Protein concentrations were determined in spent culture supernatants of IEC cultures using an ELISA technique. IFN-inducible protein-10 (IP-10) production was determined by mouse-specific ELISA kits, according to the manufacturer’s instructions (R&D Systems).

Results Differential Signal Transduction in Native IEC from GermFree WT and IL-10-/- Mice in Response to Colonization with E. faecalis. Germ-free WT and IL-10-/- mice were monoassociated with E. faecalis and killed after 1, 2 and 14 weeks after bacterial colonization. Histopathological changes (score 0-4) in the cecal and distal colon were observed only in IL-10-/(3.6 ( 0.2) but not in WT mice (0.5 ( 0.1) after 14 weeks of bacterial colonization.18 To better understand the cellular mechanisms of the disease progression, we characterized the induction of pro-inflammatory and protective signaling pathways in these animals at early (1 and 2 weeks) and a late (14 weeks) time points of bacterial colonization. Transcription factor subunits of the NF-κB family control gene expression were involved in inflammation, cell proliferation, and apoptosis.20 Paradoxically, the p38 MAPK and STAT3 cascades are targeted through mediators that trigger proinflammatory as well as protective signals. 3694


To better define the dynamic changes in pro- and antiinflammatory signaling at the epithelial cell level, we performed Western blot analysis for NF-κB RelA, p38 MAPK, and STAT3 (Figure 1A). We found a transient induction of NF-κB RelA phosphorylation after 1 week of bacterial colonisation in noninflamed WT mice and a persistent NF-κB RelA phosphorylation in primary IEC from chronically inflamed IL-10-/mice.18 In contrast to the NF-κB activation pattern, phosphop38 was continuously present in IEC from noninflamed WT mice. Transient activation of the p38 MAPK pathway was only detected after 1 week of E. faecalis monoassociation in IL-10-/mice. Phospho-STAT3 was only measured in primary IEC from IL-10-/- mice at 1 and 14 weeks of bacterial colonization. The expression of the Interferon-gamma inducible protein10 (IP-10) is strongly associated with Th1-type of colitis and is mediated by activation of NF-κB.21 Real-time quantitative RTPCR from isolated primary IEC showed a transient increase of IP-10 mRNA expression in WT and IL10-/- mice after 1 and 2 weeks of bacterial monoassociation (Figure 1B). After 14 weeks, IP-10 mRNA expression was down-regulated in primary IEC from WT mice (2.5-fold) but further increased in IL-10-/- mice (18.9-fold). Protein Expression Profiling in Primary IEC from WT and IL-10-/- Mice Colonized with E. faecalis. We next sought to better characterize the protein expression changes in activated primary IEC at early and late stages of bacterial colonization. Primary IEC of germ-free WT and IL-10-/- mice were purified after 2 and 14 weeks of E. faecalis-monoassociation. Protein expression profiling was performed using 2D-SDS-PAGE and MALDI-TOF MS. Reference gels were generated from pooled


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Figure 2. Coomassie stained 2D-gels with differentially regulated proteins in IEC from WT mice after 2 weeks of colonization with E. faecalis. Native IEC were isolated and purified from large intestine of WT mice, and 500 µg of total protein extract was subjected to IEF and 2D-SDS-PAGE. Gels were stained with Coomassie Blue and analyzed by Proteomweaver software. Indicated protein spots were picked and analyzed by MALDI-TOF-MS as described in Materials and Methods. Reference gel from germ-free mice and the corresponding regions from IEC of single colonized mice are shown. (A) Up-regulated proteins; (B) down-regulated proteins.

samples of germ-free WT and IL-10-/- mice (N ) 5). For the expression analysis, the reference gels were compared with the separate samples of all colonized WT and IL-10-/- mice (N ) 5). As shown in Tables 1 and 2, we identified 14 and 17 protein spots in germ-free WT and IL-10-/- mice, respectively, after 2 weeks of E. faecalis monoassociation. Representative gels from germ-free WT and IL-10-/- mice after 2 weeks of monoassociation with E. faecalis are shown in Figures 2 and 3. Interestingly, the protein expression profiles substantially differed between both experimental groups except for one common, down-regulated protein, the mitochondrial elongation factor TU (accession number Q8BFR5). These results clearly demon-

strate that, even in the presence of similar activation patterns and no signs of histopathology in the colonic tissue, the protein expression profile differed dramatically in WT and IL-10-/- IEC. Consistent with the down-regulated activation profile in IEC at the level of signal transduction and gene expression, we identified only 2 differential regulated proteins in primary IEC from WT mice after 14 weeks of E. faecalis monoassociation (Table 3). In addition, we detected 14 differentially regulated proteins in chronically inflamed IL-10-/- mice after 14 weeks of bacterial colonization (Table 4). The expression of pyruvate kinase M2, hydroxymethylglutaryl-CoA-synthase, and UDPglucose dehydrogenase was down-regulated at 2 and 14 weeks of colonisation in IL-10-/- mice. Journal of Proteome Research • Vol. 6, No. 9, 2007 3695

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Figure 3. Coomassie stained 2D-gels with differentially regulated proteins in IEC from IL-10-/- mice after 2 weeks of colonization with E. faecalis. Native IEC were isolated and purified from large intestine of IL-10-/- mice, and 500 µg of total protein extract was subjected to IEF and 2D-SDS-PAGE. Gels were stained with Coomassie Blue and analyzed by Proteomweaver software. Indicated protein spots were picked and analyzed by MALDI-TOF-MS as described in Materials and Methods. Reference gel from germ-free mice and the corresponding regions from IEC of single colonized mice are shown. (A) Up-regulated proteins; (B) down-regulated proteins. Table 3. Differentially Regulated Proteins in IEC of WT Mice after 14 Weeks of Colonization with E. faecalis no.

accession number

protein

MrM

pI

SC

MFC

SD

F

1 2

Q8VDY6 Q76G10

F-box protein 9 DnaJ (Hsp40) homologue, subfamily A member 1

51153 45581

6.38 6.65

17 41

3.11 2.76

1.04 0.52

5/5 3/5

Functional Characterization of Galectin-3 in IEC Using siRNA-Mediated Knock-Down. Galectin-3 is a multifunctional protein detected in the nucleus, cytoplasm, and extracellular matrix of various cells in the tissue with pathophysiologic relevance for IBD.22 We next investigated galectin-3 protein and gene expression in primary IEC from E. faecalis-monoassociated WT and IL-10-/- mice after 14 weeks of bacterial colonization. First, we used pooled samples from all 5 mice per experimental group and performed 2D gel electrophoreses and MALDI-TOF MS. As shown in Figure 4A, we identified the 3696


galectin-3 protein spot as down-regulated in IL-10-/- IEC, confirming our initial proteome analysis (MFC ) 0.39 ( 0.02). Of note, the decreased galectin-3 protein expression was present in IEC from E. faecalis but not from germ-free IL-10-/mice, suggesting that galectin-3 is selectively regulated under conditions of chronic inflammation. Second, we performed Western blot analysis in primary IEC, demonstrating reduced galectin-3 expression in E. faecalis-monoassociated IL-10-/mice (Figure 4B). Densitometric analysis revealed a 2.7-fold decrease in galectin-3 expression between E. faecalis-mono-

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Table 4. Differentially Regulated Proteins in IEC of IL-10-/- Mice after 14 Weeks of Colonization with E. faecalis no.

accession number

protein

MrM

pI

SC

MFC

SD

F

1 2 3 4 5 6 7 8 9 10 11 12 13 14

A37048 Q922N3 Q6ZQH1 S31975 BAB27292 CAA65761 B55729 AAH06749 S24612 Q9D154 Q99J99 JQ1004 A45983 Q9QYS0

DnaK type molecular chaperone grp78 Propionyl-CoA-carboxylase, alpha chain MKIAA0186 14-3-3 protein epsilon Tubulin beta 5 Pyruvate kinase M2 Hydroxymethyl-glutaryl-CoA--synthase mitochondrial UDP-glucose dehydrogenase Creatine kinase, mitochondrial Serin protease inhibitor clade B 3-mercapto-pyruvate sulfotransferase Aldehyde dehydrogenase NAD precursor Lactose-binding lectin Mac-2, Galectin-3 Sulfurylase/APS kinase isoform SK2

72491 80517 19922 29326 50064 58448 53115 55482 47373 42719 33231 55131 27455 70991

5.12 7.00 8.65 4.63 4.78 7.18 7.46 7.49 8.39 5.85 6.11 7.89 8.57 7.31

27 15 41 24 28 38 14 37 43 16 21 41 20 13

2.42 2.73 2.74 2.85 3.43 0.34 0.28 0.35 0.40 0.37 0.40 0.30 0.39 0.39

0.50 0.28 0.41 0.76 0.50 0.08 0.08 0.06 0.06 0.08 0.08 0.07 0.02 0.01

5/5 3/5 3/5 3/5 3/5 5/5 5/5 5/5 5/5 4/5 4/5 3/5 3/5 3/5

associated WT and IL-10-/- mice and a 2.1-fold decrease between germ-free and E. faecalis-monoassociated IL-10-/mice. Finally, we confirmed proteome and Western blot analysis at the mRNA expression level (Figure 4C), demonstrating a 2.1-fold decrease of galectin-3 mRNA expression in IL10-/- IEC (black bar) compared to WT controls (gray bar). To evaluate the biological significance of reduced galectin-3 protein expression under conditions of chronic inflammation, we next performed galectin-3-specific siRNA knock-down experiments in Mode-K cells. As shown in Figure 5A, we achieved significant galectin-3 protein knock-down after 72 h of transfection. The loss of galectin-3 protein expression in IEC was associated with a dramatic increase in caspase-3 cleavage, suggesting an anti-apoptotic function of galectin-3 in IEC. Parallel to the in vitro analysis, we measured cleaved caspase-3 in primary IEC, demonstrating increased expression of cleaved caspase-3 in E. faecalis-monoassociated IL-10-/- mice (Figure 5B). IL-10-Mediated Proteome Changes in Cultured IL-10 R Reconstituted IEC in the Absence and Presence of E. faecalis. Since IL-10-mediated p38 MAPK signaling was associated with protective mechanisms in IEC, we next investigated the IL-10mediated proteome in IL-10R reconstituted Mode-K cells. The functional expression of the IL-10 receptor complex in Mode-K cells was confirmed by measuring IL-10-mediated phosphorylation of p38 and STAT3 (Figure 6A). There was no induction of p38 and STAT3 phosphorylation/activation in Mode-K cells in the absence of the IL-10 receptor complex (data not shown). Since IP-10 mRNA expression was differentially regulated in primary IEC from E. faecalis-monoassociated WT and IL-10-/mice, we next stimulated IL-10R reconstituted Mode-K cells for 12 h with recombinant (r)IL-10 and E. faecalis. As shown in Figure 6B, E. faecalis significantly triggered IP-10 protein expression, but IL-10 failed to inhibit IP-10 protein secretion, suggesting that IP-10 is not a direct target for the antiinflammatory mechanisms of IL-10. To better understand the functional consequences of IL-10mediated signaling, we next performed a proteome analysis in IL-10R reconstituted Mode-K cells after the stimulation for 12 h with rIL-10, E. faecalis, or the combination of rIL-10 and E. faecalis. In total, 52 proteins could be identified in all 3 experiments, 15 proteins with altered steady-state expression level after stimulation with rIL-10 (Table 5), 18 proteins after the stimulation with E. faecalis (Table 6), and 19 proteins after the combined stimulation with rIL-10 and E. faecalis (Table 7). A representative gel for the combined stimulation with rIL-10 and E. feacalis is shown in Figure 7.

Up-regulated FKBP65 binding protein, NADH dehydrogenase flavoprotein 2, and glutathione transferase mu1 were identified in all 3 in vitro experiments. Additionally, Fbxo11 protein was down-regulated in IL-10R reconstituted Mode-K cells stimulated with rIL-10 alone and with E. faecalis alone. Interestingly, the serin protease inhibitor clade B (serpin b1) was co-regulated in both the in vivo and the in vitro experiments. Serpin b1 was down-regulated in IL-10-/- mice after 14 weeks of E. faecaliscolonization and up-regulated in IL-10R reconstituted Mode-K cells after the combined stimulation with rIL-10 and E. faecalis. Bibliometric Analysis. On the basis of literature co-citation from NCBI Pubmed, we generated a protein-protein network tree using the data-mining program Bibliosphere software (Genomatix Software, Munich, Germany). As shown in Figure 8A, the network tree was compiled of 42 highly interrelated proteins. Although there is only one direct overlap of identified proteins between the in vivo and the in vitro experiment, there exists interrelation between these proteins according to molecular function or biological processes. The network tree was roughly divided in three functional clusters including cytoskeletal proteins (cluster I), protein metabolism (cluster II), and transport proteins (cluster III). We found an additional 18 proteins that were completely unrelated (Figure 8B).

Discussion In this study, we characterized the protein expression profile in primary and IEC lines under conditions of progressing chronic inflammation. To mimic the complex effects of luminal colitogenic bacteria in the presence and absence of hostderived IL-10 at the epithelial cell level, we used E. faecalismonoassociated WT and IL-10-/- mice at early (1 and 2 weeks) and late stages (14 weeks) of bacterial colonization as well as IL-10R reconstituted Mode-K cells after the stimulation with E. faecalis and rIL-10. We identified 76 target proteins with significantly altered steady-state expression levels in primary and IL-10R reconstituted IEC lines using 2D-gel electrophoreses and peptide mass fingerprinting via MALDI-TOF MS. The bibliometric data analysis generated a protein-protein network tree from cell culture experiments and animal model systems and identified three main clusters of interrelated proteins including cytoskeletal functions and catalytic activity, protein metabolism, as well as transport functions. It seems important to understand that, although tissue pathology is selectively present in E. faecalis-monoassociated IL-10-/- mice after 14 weeks of bacterial colonization, IEC activation was demonstrated at early stages of bacterial colonization (1 and 2 weeks) in both WT and IL-10-/- mice. Thus, Journal of Proteome Research • Vol. 6, No. 9, 2007 3697

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Figure 4. (A) Down-regulated galectin-3 protein spot in E. faecalis-monoassociated IL-10-/- IEC after 14 weeks of colonization. Proteome analysis was performed for pooled samples from all 5 mice per experimental group. Galectin-3 protein spot was down-regulated in bacterial colonized IL-10-/- with a MFC of 0.39 ( 0.02, but no decrease could be detected in germ-free IL-10-/-. (B) Reduced galectin-3 protein expression in E. faecalis-monoassociated IL-10-/- IEC after 14 weeks of colonization. Western blot analysis was performed with 50 µg of total protein derived from pooled IEC samples from 5 germ-free and E. faecalis monoassociated WT and IL-10-/- mice, respectively, using immunoreactive galectin-3 and β-actin. Densitometric analysis revealed 2.7-fold decrease in galectin-3 expression between E. feacalis-monoassociated WT and IL-10-/- mice and 2.1-fold decrease between germ-free and E. feacalis-monoassociated IL-10-/- mice. (C) Decreased mRNA expression levels of galectin-3 in E. faecalis-monoassociated IL-10-/- mice. Light Cycler RT-PCR was performed using specific primer for murine galectin-3 and GAPDH revealing a 2.1-fold decrease of galectin-3 mRNA expression in IL-10-/- IEC (black bar) compared to WT controls (gray bar).

the induction of NF-κB signaling (phospho-RelA) and NF-κBdependent IP-10 expression as cellular markers of activated inflammatory processes preceded any signs of histopathological changes in the colon. Interestingly, the composition but not quantity of differentially regulated proteins was completely different in primary IEC from WT and IL-10-/- mice, suggesting that at the proteome level the absence of IL-10 may have primed the intestinal epithelium towards a completely different response pattern after bacterial colonization. Consistent with 3698


the persistent cellular activation process in IL-10-/- but not WT IEC, proteome analysis identified 14 differentially regulated proteins in E. faecalis-monoassociated IL-10-/- mice and only 2 proteins in WT mice after 14 weeks of bacterial colonization. Proteome analysis revealed 14 differentially regulated proteins in IEC from E. faecalis-monoassociated WT mice after 2 weeks of colonization. Interestingly, oxoglutarate dehydrogenase (R-ketoglutarate dehydrogenase) was 5.32-fold up-regulated in IEC of E. faecalis-monoassociated WT mice at these


Figure 5. (A) siRNA knock down of galectin-3 revealed an increase in caspase 3 cleavage in transfected Mode-K cells. Mode-K cells were transfected with control and galectin-3-specific siRNA for 0-72 h, and 20 µg of total protein was subjected to SDS-PAGE followed by immunoblotting with specific antibodies to galectin-3, cleaved caspase-3, and β-actin. The loss of galectin-3 protein expression in IEC was associated with a dramatic increase in caspase 3 cleavage. (B) Increased expression of cleaved caspase-3 in E. faecalis-monoassociated IL-10-/- mice. Pooled samples containing 50 µg of protein lysate were generated using IEC of germ-free and E. faecalis-monoassociated WT and IL-10-/- (14 weeks of colonization) and subjected to Western blot analysis using monoclonal antibodies to cleaved caspase-3 and β-actin. Induction of cleaved caspase-3 was only observed in IEC of IL-10-/- but not in WT mice after 14 weeks of bacterial colonization.

early stages of bacterial colonization, supporting the hypothesis that WT mice exhibit protective mechanisms to resist bacterial or cytokine-induced oxidative stress responses in the tissue. Oxoglutarate dehydrogenase is a key enzyme in the citrate cycle and an early target of oxidative stress. Inhibition of the R-ketoglutarate dehydrogenase complex leads to metabolic alteration (e.g., energy metabolism) and mitochondrial dysfunction (cell death), and affects Ca2+-homeostasis between the cytosolic compartment and the endoplasmatic reticulum.23-25 In addition, it may be of interest to know that proteins involved in detoxification processes including thiosulfate sulfurtransferase and glyoxalase I were up-regulated with 2.61- and 2.52fold, respectively. Glycation of proteins, nucleotides, and basic phospholipids can be toxic for cells,26 and the decrease in glyoxalase activity during aging and oxidative stress responses leads to increased protein glycation and to tissue damage.27,28 Finally, after 14 weeks of monoassociation with E. faecalis, only 2 up-regulated proteins could be identified. This includes the F box protein 9 and the DnaJ (Hsp40) protein homologue. For example, Hsp40 is a co-chaperone and stimulates hsp70 expression to assist a large variety of protein folding processes.33 Taken together, these findings may suggest that, in the presence of IL-10, the initial activation processes and protein expression changes are normalized and normal gut homeostasis was acquired at later stages of bacterial colonization. In contrast, IL-10 deficient mice fail to counteract the colitogenic effect of E. faecalis, and opposite to the tightly regulated response in WT mice, the colonization of IL-10-/- mice with E. faecalis revealed persistant NF-κB activation and IP-10 gene expression

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Figure 6. (A) IL-10 triggers p38 and STAT3 phosphorylation in IL-10R reconstituted Mode-K cells. Mode-K cells were reconstituted with the murine IL-10R complex followed by stimulation with IL-10 for up to 3 h. Western blot analysis was performed with 20 µg of total protein using specific antibodies for phosphop38, p38, phospho-STAT3, and STAT3. (B) IL-10 failed to inhibit E. faecalis-induced IP-10 expression on protein level. IL-10R Mode-K cells were stimulated with rIL-10, E. faecalis and in combination with rIL-10 and E. faecalis for 24 h. IP-10 protein expression was measured in cell culture supernatant using ELISA technique. Bars represent the combined value ( standard deviation of triplicate stimulation from 3 independent experiments.

in primary IEC. Interestingly, the protein expression profiling in primary IEC of IL-10-/- and WT mice identified a completely different set of regulated proteins. For example, the expression of zinc finger protein Pw1, up-regulated in IL-10-/- mice after 2 weeks of bacterial colonization, is associated with the induction of apoptosis and shows NF-κB-activating mechanisms. The NF-κB-activation via Pw1 is mediated by IκB dissociation and acts synergistically with TRAF2 in TNF-R response.34 On the other hand, studies revealed a direct involvement of Pw1 in p53-mediated apoptosis, showing that mutant and anti-sense Peg3/Pw1 were able to prevent hypoxiainduced cell death.35-37 The protein expression of the succinate dehydrogenase (SDHC) subunit B was up-regulated in IL-10-/mice. It has been shown that a mutated succinate dehydrogenase gene in the transgenic mouse cell line SDHC E69 leads to increased stress oxidative rates and apoptosis conciliated by an increase in caspase-3 activity.41 Proteins of the peroxiredoxin familiy show strong anti-oxidative properties to protect cells from ROS-mediated tissue damage.42 The up-regulation of succinate dehydrogenase and peroxiredoxin 5 in IL-10-/- mice may suggest that especially at early stages of bacterial colonization a set of regulated proteins protects the cell from oxidative stress, especially at the mitochondrial level.43-45 The expression of several metabolic enzymes involved in energy metabolism such as pyruvate kinase, UDP-glucose dehydrogenase, and Journal of Proteome Research • Vol. 6, No. 9, 2007 3699

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Table 5. Differentially Regulated Proteins in IL-10R Reconstituted Mode-K Cells, Stimulated with rIL-10 for 12 h no.

accession number

protein

MrM

pI

SC

MFC

SD

F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Q8VCQ8_MOUSE AAH04061 Q5FWK2_MOUSE Q6P2L2_MOUSE Q8C2A0_MOUSE S33860 ATP5H_MOUSE Q8K2L0_MOUSE Q5FWJ3_MOUSE I49669 Q8BRB8_MOUSE Q91ZR9_MOUSE Q5M7B4_MOUSE ANXA3_MOUSE S54774

Caldesmon 1 Filamin, alpha Hnrpf protein Khsrp protein Hnrp A2 Glutathione transferase mu 1 ATP synthase D chain, mitochondrial NADH dehydrogenase flavoprotein 2 Vimentin FKBP65 binding protein Hypothetical PDZ domain containing protein Hnrp A2/B1 Fbxo11 protein Annexin 3 High mobility group 2 protein

60531 54429 43943 32728 32497 26067 18664 27610 53712 65142 54105 36014 103418 36389 24318

6.97 6.52 5.30 6.16 8.74 7.71 5.52 7.00 5.06 5.38 5.53 8.67 6.52 5.33 6.88

41 35 35 25 26 34 59 41 30 23 40 37 5 40 45

2.01 2.06 2.06 2.03 2.02 2.24 2.22 2.10 2.02 2.79 2.06 0.37 0.34 0.44 0.48

0.11 0.25 0.23 0.16 0.74 0.15 1.45 0.51 1.31 2.31 0.88 0.13 0.16 0.04 0.33

6/6 6/6 6/6 6/6 6/6 5/6 6/6 6/6 6/6 6/6 6/6 6/6 6/6 5/6 5/6

Table 6. Differentially Regulated Proteins in IL-10R Reconstituted Mode-K Cells, Stimulated with E. faecalis for 12 h no.

accession number

proteins

MrM

pI

SC

MFC

SD

F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

I49669 Q8K2L0_MOUSE Q5HZK3_MOUSE I49259 Q6P071_MOUSE S26434 Q6P2L2_MOUSE S33860 AAH03432 S31485 Q5M7B4_MOUSE Q80ZZ9_MOUSE BAB22767 Q811L7_MOUSE AAH27206 B48124 TAGL_MOUSE Q8VIJ6_MOUSE

FKBP65 binding protein NADH dehydrogenase flavoprotein 2 Proteasome 28 subunit Cellular nucleic acid binding protein Cellular retinoid acid binding protein 1 Interferon-induced protein 15 K Khsrp protein Glutathione transferase mu 1 Electron transferring flavoprotein, alpha peptide Calponin H2 Fbxo11 protein D130059P03 Rik protein, fragment Cyclophilin-40 Hnrp H1 Hnrp L Core-binding factor, beta subunit p22 Transgelin (actin associted protein p27) PTB-associated splicing factor

65142 27610 28826 20833 7841 17749 32728 26067 35360 33590 103418 112636 41116 49454 60712 22245 22487 75508

5.38 7.00 5.70 7.76 9.20 7.74 6.61 7.71 8.62 7.53 6.52 9.44 7.08 5.89 6.65 5.59 8.86 9.45

23 41 41 40 72 27 25 34 36 33 5 9 25 32 24 50 44 18

2.09 3.02 2.15 2.57 2.07 2.15 5.08 2.35 2.52 2.02 0.37 0.41 0.30 0.46 0.47 0.39 0.43 0.44

0.98 1.60 0.50 2.13 0.89 0.08 0.75 1.03 0.03 0.67 0.25 0.17 0.23 0.05 0.43 0.04 0.28 0.07

5/6 6/6 5/6 5/6 5/6 5/6 6/6 5/6 6/6 6/6 6/6 5/6 6/6 5/6 6/6 5/6 5/6 6/6

Table 7. Differentially Regulated Proteins in IL-10R Reconstituted Mode-K Cells, Stimulated with rIL-10 and E. faecalis for 12 h no.

accession number

protein

MrM

pI

SC

MFC

SD

F

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

I49669 Q8VHP7_MOUSE BAC27981 BAB25754 Q8K2L0_MOUSE I49259 Q6P071_MOUSE S26434 Q5HZK3_MOUSE S33860 Q6P2L2_MOUSE Q7TMW6_MOUSE AAH03432 JQ0448 BAB22767 Q99JW2_MOUSE BAC33407 S11390 Q8R4N2_MOUSE

FKBP65 binding protein Serpin 1b, clade B Nuclear distribution gene C homologue Inorganic pyrophosphatase NADH dehydrogenase flavoprotein 2 Cellular nucleic acid binding protein Cellular retinoid acid binding protein 1 Interferon-induced protein 15 K Proteasome 28 subunit Glutathione transferase mu 1 Khsrp protein Nuclear prelamin A recognition factor like Electron transferring flavoprotein, alpha peptide CArG-binding factor A Cyclophilin-40 Aminoacylase 1 ADP-ribosylation factor like protein 3 Tropomyosin 5 Titin L1 fusion protein

65142 43202 38334 33102 27610 20833 7841 17749 28826 26067 32728 53674 35360 30926 41116 45980 20645 29231 25725

5.38 6.28 5.17 5.37 7.00 7.76 9.20 7.74 5.70 7.71 6.61 6.05 8.62 7.68 7.08 5.89 6.74 4.75 9.68

23 20 22 42 41 40 72 27 41 34 25 11 36 18 25 28 53 18 18

2.09 2.85 2.73 2.04 2.64 3.50 3.23 2.40 2.50 3.48 4.60 2.12 3.80 2.11 0.24 0.41 0.21 0.25 0.25

0.98 1.72 1.72 0.23 1.74 1.25 2.15 0.40 1.30 1.90 0.85 0.38 2.40 0.47 0.08 0.11 -

6/6 5/6 6/6 6/6 6/6 5/6 5/6 6/6 6/6 5/6 6/6 6/6 5/6 6/6 6/6 5/6 4/6 4/6 4/6

mitochondrial hydroxmethylglutaryl-CoA-synthase (HMG-CoAS) was down-regulated in IEC from IL-10-/- mice after 2 weeks of bacterial colonization, supporting the hypothesis that chronic intestinal inflammation represents an energy-deficiency disease with alterations in the oxidative metabolism of epithelial cells.46,47 Another indicator for the loss of cell homeostasis and induction of pro-apoptotic mechanisms is the down-regulation 3700


of galectin-3. Since this family of proteins plays an important role in diverse biological processes such as adhesion, proliferation, apoptosis, and mRNA splicing, as well as modulation of the immune response, and galactin 3-specific immunoreactive antibodies are available for Western blot analysis, we have validated our proteome findings at the protein and mRNA expression level. In addition, and most important for the molecular function of this protein in IEC, we used siRNA-


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Figure 7. Coomassie stained 2D- gels with up-regulated proteins in IL-10R reconstituted Mode-K cells stimulated with the combination of rIL-10 and E. faecalis after 12 h. IL-10R Mode-K cells were stimulated in combination with rIL-10 and E. faecalis for 12 h, and cells were collected. A total of 500 µg of total protein extract was subjected to IEF and 2D-SDS-PAGE. Gels were stained with Coomassie Blue and analyzed by Proteomweaver software. Indicated protein spots were picked and analyzed by MALDI-TOF-MS as described in Materials and Methods. Reference gel from unstimulated IL-10R reconstituted Mode-K cells and the corresponding regions from the stimulated cells are shown.

mediated knock-gown approaches to perform functional studies in vitro. We showed that the loss of galectin-3 in Mode-K cells was associated with the activation of caspase-3, a major executive caspase of apoptosis.49,50 Accordingly, the downregulation of galectin-3 correlates with increased caspase-3 cleavage in IL-10-/- IEC compared to WT controls. Interestingly, recent clinical studies from Muller et al. demonstrated a down-regulation of galectin-3 expression in the intestinal epithelium of inflamed IBD patients,22 confirming the pathophysiological relevance of our findings. Thus, the lack and/or failure of regulatory mechanisms under the developing conditions of chronic inflammation in IL-10 deficient mice may prime the intestinal epithelium toward uncontrolled oxidative and ER stress responses, energy deficiency and apoptosis leading to functional disturbances and loss of tissue homeostasis. In addition to the work in primary IEC, we recently showed that IL-10R reconstituted IEC regain IL-10-mediated p38 phosphorylation, supporting the hypothesis that IL-10-mediated p38 signaling directly confers protective effects at the epithelial cell level.12 To further characterize the role of IL-10 at the epithelial cell level, we used proteome analysis in IL-10R reconstituted IEC after the stimulation with E. faecalis and/or rIL-10 and identified 52 differentially regulated proteins. The protein expression profile of IL-10R reconstituted Mode-K cells stimulated with rIL-10 revealed up-regulated proteins with cytoskeletal function (caldesmon 1, filamin alpha and vimentin), energy metabolism (ATP synthase, NADH dehydrogenase flavoprotein 2) and proteins involved in regulatory processes (Hnrpf protein, Khsrp protein, and FKBP65). For example, FKBP65 is an ER-localized peptidy-prolyl cis-trans isomerase that regulates the secretion of extracellular matrix proteins, linking ER response mechanisms to the regulation of tissue

repair and wound healing.52 The stimulation of IL-10R reconstituted Mode-K cells with E. faecalis showed up-regulation of proteins that belong to regulators of cell proliferation and antigen presentation (proteasome 28 subunit, interferoninduced protein), suggesting an onset of immune response toward bacterial recognition. Finally, the proteome analysis of simultaneously treated IL-10R reconstituted IEC with rIL-10 and E. faecalis identified a similar expression pattern compared to the stimulation with E. faecalis alone. Nevertheless, the additional stimulation with rIL-10 induced an enhanced expression of regulatory proteins including prelamin A and serpin b1. It has been shown that the deficiency of the nuclear protein prelamin A in knock-out mice leads to slow growth and an early death by 6 weeks of age.53 Serpin b1, also known as serin (or cysteine) protease inhibitor clade B, was identified in both in vivo and in vitro experiment. Since clade B serpins (ov-serpins) lack the signal peptide necessary for secretion, this class of proteins function as intracellular mediators. Preliminary studies assign mammalian serpins to important cellular processes including inflammation, apoptosis, microbial and viral infection and tumor invasion as well as hormone transport.54 Because of the biological pertinence of serpines, the down-regulation of serpin b1 in IL-10-/- mice after 14 weeks of bacterial monoassociation could be associated with a loss of protective mechanisms in IEC. Accordingly, the up-regulation of serpin in Mode-K cells stimulated with rIL-10 and E. faecalis suggests a direct role of IL-10 on serpin protein expression under proinflammatory conditions. Several cytoplasmic serpins have been shown to have anti-apoptotic properties through the inhibition of the caspase activation cascade, thereby protecting the cell from their own proteases. Serpin-mediated inhibition of granzym B and cathepsin G was observed in granules of neutrophils, cytotoxic and NK-cells, allowing bacterial degradaJournal of Proteome Research • Vol. 6, No. 9, 2007 3701

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Figure 8. Bibliometric data analysis. Bibliometric analysis was performed on the base of literature co-citation from NCBI Pubmed. The data-mining program Bibliosphere software (Genomatix) was used to generate protein-protein network trees from cell culture experiments (blue boxes) and animal model systems (green boxes). (A) In total, 42 proteins are integrated in the highly interrelated network tree. (B) Additional 18 proteins remain unconnected to the large network tree.

tion and digestion by macrophages without host cell destruction during degranulation.55 Interestingly, the serpin promoter structure revealed NF-κB-binding sites and predicted STATbinding sequences, supporting the observation that bacterial products and cytokines activate serpin gene-expression.56 It seems important to define functional clusters of interrelated proteins and to identify complex protein networks in order to characterize their specific contribution in maintaining epithelial cell homeostasis. The bibliometric data analysis including all 60 differentially regulated proteins (16 proteins not in Bibliosphere Database) from in vivo and in vitro experiments generated a complex network with at least 42 3702


highly interrelated proteins (Figure 7A). Major functions were associated with these proteins in clusters including cluster 1 with cytoskeletal functions and catalytic activity (12 from 22 proteins), cluster 2 with protein metabolism (6 from 12 proteins) and cluster 3 with transport functions (4 from 8 proteins). We found additional 18 proteins that were completely unrelated to any other protein. Although there is only one direct overlap between the different experiments (serpin 1b), the network analysis revealed proteins from in vivo and in vitro experiments that share the same biological function. This includes Hspa5, Dnaja1, Sfpq, and Isg15 that all represent stress response signaling mechanisms.


In conclusion, these data strongly suggest that bacteria- and host-derived signals are tightly controlled by a complex network of proteins that involve many different cellular functions. The proteome analysis from primary and IEC lines revealed changes in expression levels of proteins involved in ER stress, energy metabolism and apoptosis, suggesting a protective role for IL10 at the interface between luminal enteric and host-derived mediators. Abbreviations: Acat1, acetyl-Coenzyme A acetyltransferase 1; Acy1, aminoacylase 1; Ahnak, AHNAK nucleoprotein (desmoyokin); Aldh3a1, aldehyde dehydrogenase family 3, subfamily A1; Anxa3, annexin A3; Anxa7, annexin A7; Ass1, argininosuccinate synthetase 1; Cald1, caldesmon 1; Cbfb, core binding factor; Ckmt1, creatine kinase, mitochondrial 1, ubiquitous; Clic1, chloride intracellular channel 1; Cnbp1, cellular nucleic acid binding protein 1; Cnn2, calponin 2; Crabp1, cellular retinoic acid binding protein I; Dnaja1, DnaJ (Hsp40) homolog, subfamily A, member 1; Dstn, Destrin; Etfa, electron transferring flavoprotein; Fabp6, fatty acid binding protein 6, ileal (gastrotropin); Fdrx, ferredoxin reductase; Fkbp10, FK506 binding protein 10 (FKBP65); Flna, filamin, alpha; Gfra2, glia cell line derived neurotrophic factor family receptor alpha 2; Glo1, glyoxalase 1; Gstm1, glutathione S-transferase, mu1; Hmgb2, high mobility group box 2; Hmgcl, 3-hydroxy-3-methylglutarylCoenzym A lyase; Hmgcs2, 3-hydroxy-3-methylglutaryl-Coenzym A synthase 2; Hnrpa2b1, heterogeneous nuclear ribonucleoprotein A2/B1; Hnrpab, heterogeneous nuclear ribonucleoprotein A/B; Hnrpf, heterogeneous nuclear ribonucleoprotein F; Hnrph1, heterogeneous nuclear ribonucleoprotein H1; Hspa5, heat shock 70kDa protein 5 (glucose-regulated protein); Isg15, ISG15 ubiquitin-like modifier; Khsrp, KH-type splicing regulatory protein; Krt1-19, keratin complex 1, acidic, gene 19; Lgals3, lectin, galactoside-binding, soluble, 3 (galectin-3); Mpst, mercaptopyruvate sulfurtransferase; Ndufv2, NADH dehydrogenase (ubiquinone) flavoprotein 2; Nudc, nuclear distribution gene C homologue (Aspergillus); Ogdh, oxoglutarate dehydrogenase; Pafahl1b1, platelet-activating factor acetylhydrolase, isoform 1b; Papss2, 3′-phosphoadenosine 5′-phosphosulfate synthase 2; Pcca, propionyl-Coenzyme A carboxylase; Pkm2, pyruvate kinase, muscle; Ppa1, pyrophosphatase (inorganic) 1; Ppid, peptidylprolyl isomerase D (cyclophilin D); Prdx5, peroxiredoxin 5; Psme1, proteasome activator subunit 1 (PA28 alpha); Sdhb, succinate dehydrogenase complex, subunit B; Serpinb1a, serpin (or cysteine) peptidase inhibitor, clade B, member 1a; Sfpq, splicing factor proline/glutamine rich; Tagln, transgelin; Tpm3, tropomyosin 3, gamma; Tst, thiosulfate sulfurtransferase, mitochondrial; Ttn, titin; Ugdh, UDP-glucosedehydrogenase; Vcp, valosin containing protein; Vdac2, voltage-dependent anion channel 2; Vim, vimentin; Ywhae tyrosine 3-/tryptophan 5-monooxygenase activation protein.

References (1) Swidsinski, A.; Weber, J.; Loening-Baucke, V.; Hale, L. P.; Lochs, H. Spatial organization and composition of the mucosal flora in patients with inflammatory bowel disease. J. Clin. Microbiol. 2005, 43, 3380-9. (2) Swidsinski, A.; Ladhoff, A.; Pernthaler, A.; Swidsinski, S.; LoeningBaucke, V.; Ortner, M.; Weber, J.; Hoffmann, U.; Schreiber, S.; Dietel, M.; Lochs, H. Mucosal flora in inflammatory bowel disease. Gastroenterology 2002, 122, 44-54. (3) Kraus, T. A.; Toy, L.; Chan, L.; Childs, J.; Mayer, L. Failure to induce oral tolerance to a soluble protein in patients with inflammatory bowel disease. Gastroenterology 2004, 126, 17718.

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(4) Duchmann, R.; Kaiser, I.; Hermann, E.; Mayet, W.; Ewe, K.; Meyer zum Buschenfelde, K. H. Tolerance exists towards resident intestinal flora but is broken in active inflammatory bowel disease (IBD). Clin. Exp. Immunol 1995, 102, 448-55. (5) Sartor, R. B. Mechanisms of disease: pathogenesis of Crohn’s disease and ulcerative colitis. Nat Clin. Pract. Gastroenterol. Hepatol. 2006, 3, 390-407. (6) Kim, S. C.; Tonkonogy, S. L.; Albright, C. A.; Tsang, J.; Balish, E. J.; Braun, J.; Huycke, M. M.; Sartor, R. B. Variable phenotypes of enterocolitis in interleukin 10-deficient mice monoassociated with two different commensal bacteria. Gastroenterology 2005, 128, 891-906. (7) Haller, D. Intestinal epithelial cell signalling and host-derived negative regulators under chronic inflammation: to be or not to be activated determines the balance towards commensal bacteria. Neurogastroenterol. Motil. 2006, 18, 184-99. (8) Ruiz, P. A.; Shkoda, A.; Kim, S. C.; Sartor, R. B.; Haller, D. IL-10 gene-deficient mice lack TGF-beta/Smad-mediated TLR2 degradation and fail to inhibit proinflammatory gene expression in intestinal epithelial cells under conditions of chronic inflammation. Ann. N. Y. Acad. Sci. 2006, 1072, 389-94. (9) Alexander, W. S.; Hilton, D. J. The role of suppressors of cytokine signaling (SOCS) proteins in regulation of the immune response. Annu. Rev. Immunol. 2004, 22, 503-29. (10) Moore, K. W.; de Waal Malefyt, R.; Coffman, R. L.; O’Garra, A. Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 2001, 19, 683-765. (11) Ricchetti, G. A.; Williams, L. M.; Foxwell, B. M. Heme oxygenase 1 expression induced by IL-10 requires STAT-3 and phosphoinositol-3 kinase and is inhibited by lipopolysaccharide. J. Leukocyte Biol. 2004, 7, 7. (12) Shkoda, A.; Ruiz, P. A.; Daniel, H.; Kim, S. C.; Rogler, G.; Sartor, R. B.; Haller, D. Interleukin-10 blocked endoplasmic reticulum stress in intestinal epithelial cells: impact on chronic inflammation. Gastroenterology 2007, 132, 190-207. (13) Zaph, C.; Troy, A. E.; Taylor, B. C.; Berman-Booty, L. D.; Guild, K. J.; Du, Y.; Yost, E. A.; Gruber, A. D.; May, M. J.; Greten, F. R.; Eckmann, L.; Karin, M.; Artis, D. Epithelial-cell-intrinsic IKK-beta expression regulates intestinal immune homeostasis. Nature 2007, 446, 552-6. (14) Nenci, A.; Becker, C.; Wullaert, A.; Gareus, R.; van Loo, G.; Danese, S.; Huth, M.; Nikolaev, A.; Neufert, C.; Madison, B.; Gumucio, D.; Neurath, M. F.; Pasparakis, M. Epithelial NEMO links innate immunity to chronic intestinal inflammation. Nature 2007, 446, 557-61. (15) Steidler, L.; Hans, W.; Schotte, L.; Neirynck, S.; Obermeier, F.; Falk, W.; Fiers, W.; Remaut, E. Treatment of murine colitis by Lactococcus lactis secreting interleukin- 10 [see comments]. Science 2000, 289, 1352-5. (16) Braat, H.; Rottiers, P.; Hommes, D. W.; Huyghebaert, N.; Remaut, E.; Remon, J. P.; van Deventer, S. J.; Neirynck, S.; Peppelenbosch, M. P.; Steidler, L. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin. Gastroenterol. Hepatol. 2006, 4, 754-9. (17) Vandenbroucke, K.; Hans, W.; Van Huysse, J.; Neirynck, S.; Demetter, P.; Remaut, E.; Rottiers, P.; Steidler, L. Active delivery of trefoil factors by genetically modified Lactococcus lactis prevents and heals acute colitis in mice. Gastroenterology 2004, 127, 502-13. (18) Ruiz, P. A.; Shkoda, A.; Kim, S. C.; Sartor, R. B.; Haller, D. IL-10 gene-deficient mice lack TGF-beta/Smad signaling and fail to inhibit proinflammatory gene expression in intestinal epithelial cells after the colonization with colitogenic Enterococcus faecalis. J. Immunol 2005, 174, 2990-9. (19) Rath, H. C.; Herfarth, H. H.; Ikeda, J. S.; Grenther, W. B.; Hamm, T. E., Jr.; Balish, E.; Taurog, J. D.; Hammer, R. E.; Wilson, K. H.; Sartor, R. B. Normal luminal bacteria, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLAB27/human beta2 microglobulin transgenic rats. J. Clin. Invest. 1996, 98, 945-53. (20) Baldwin, A. S., Jr. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 1996, 14, 64983. (21) Neville, L. F.; Mathiak, G.; Bagasra, O. The immunobiology of interferon-gamma inducible protein 10 kD (IP-10): a novel, pleiotropic member of the C-X-C chemokine superfamily. Cytokine Growth Factor Rev. 1997, 8, 207-19. (22) Muller, S.; Schaffer, T.; Flogerzi, B.; Fleetwood, A.; Weimann, R.; Schoepfer, A. M.; Seibold, F. Galectin-3 modulates T cell activity and is reduced in the inflamed intestinal epithelium in IBD. Inflammatory Bowel Dis. 2006, 12, 588-97.

Journal of Proteome Research • Vol. 6, No. 9, 2007 3703

research articles (23) Gibson, G. E.; Zhang, H.; Xu, H.; Park, L. C.; Jeitner, T. M. Oxidative stress increases internal calcium stores and reduces a key mitochondrial enzyme. Biochim. Biophys. Acta 2002, 1586, 177-89. (24) Huang, H. M.; Zhang, H.; Xu, H.; Gibson, G. E. Inhibition of the alpha-ketoglutarate dehydrogenase complex alters mitochondrial function and cellular calcium regulation. Biochim. Biophys. Acta 2003, 1637, 119-26. (25) Nulton-Persson, A. C.; Starke, D. W.; Mieyal, J. J.; Szweda, L. I. Reversible inactivation of alpha-ketoglutarate dehydrogenase in response to alterations in the mitochondrial glutathione status. Biochemistry 2003, 42, 4235-42. (26) Furber, J. D. Extracellular glycation crosslinks: prospects for removal. Rejuvenation Res. 2006, 9, 274-8. (27) Thornalley, P. J. The enzymatic defence against glycation in health, disease and therapeutics: a symposium to examine the concept. Biochem. Soc. Trans. 2003, 31, 1341-2. (28) Thornalley, P. J. Glyoxalase I-structure, function and a critical role in the enzymatic defence against glycation. Biochem. Soc. Trans. 2003, 31, 1343-8. (29) Schmidt, A. M.; Yan, S. D.; Yan, S. F.; Stern, D. M. The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses. J. Clin. Invest. 2001, 108, 949-55. (30) Wang, Q.; Song, C.; Li, C. C. Molecular perspectives on p97-VCP: progress in understanding its structure and diverse biological functions. J. Struct. Biol. 2004, 146, 44-57. (31) Wojcik, C.; Yano, M.; DeMartino, G. N. RNA interference of valosin-containing protein (VCP/p97) reveals multiple cellular roles linked to ubiquitin/proteasome-dependent proteolysis. J. Cell. Sci. 2004, 117, 281-92. (32) Asai, T.; Tomita, Y.; Nakatsuka, S.; Hoshida, Y.; Myoui, A.; Yoshikawa, H.; Aozasa, K. VCP (p97) regulates NFkappaB signaling pathway, which is important for metastasis of osteosarcoma cell line. Jpn. J. Cancer. Res. 2002, 93, 296-304. (33) Laufen, T.; Mayer, M. P.; Beisel, C.; Klostermeier, D.; Mogk, A.; Reinstein, J.; Bukau, B. Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5452-7. (34) Relaix, F.; Wei, X. J.; Wu, X.; Sassoon, D. A. Peg3/Pw1 is an imprinted gene involved in the TNF-NFkappaB signal transduction pathway. Nat. Genet. 1998, 18, 287-91. (35) Johnson, M. D.; Wu, X.; Aithmitti, N.; Morrison, R. S. Peg3/Pw1 is a mediator between p53 and Bax in DNA damage-induced neuronal death. J. Biol. Chem. 2002, 277, 23000-7. (36) Deng, Y.; Wu, X. Peg3/Pw1 promotes p53-mediated apoptosis by inducing Bax translocation from cytosol to mitochondria. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 12050-5. (37) Yamaguchi, A.; Taniguchi, M.; Hori, O.; Ogawa, S.; Tojo, N.; Matsuoka, N.; Miyake, S.; Kasai, K.; Sugimoto, H.; Tamatani, M.; Yamashita, T.; Tohyama, M. Peg3/Pw1 is involved in p53mediated cell death pathway in brain ischemia/hypoxia. J. Biol. Chem. 2002, 277, 623-9. (38) Chandra, D.; Choy, G.; Daniel, P. T.; Tang, D. G. Bax-dependent regulation of Bak by voltage-dependent anion channel 2. J. Biol. Chem. 2005, 280, 19051-61. (39) Premkumar, A.; Simantov, R. Mitochondrial voltage-dependent anion channel is involved in dopamine-induced apoptosis. J. Neurochem. 2002, 82, 345-52. (40) De Pinto, V.; Messina, A.; Accardi, R.; Aiello, R.; Guarino, F.; Tomasello, M. F.; Tommasino, M.; Tasco, G.; Casadio, R.; Benz, R.; De Giorgi, F.; Ichas, F.; Baker, M.; Lawen, A. New functions of an old protein: the eukaryotic porin or voltage dependent anion selective channel (VDAC). Ital. J. Biochem. 2003, 52, 1724. (41) Ishii, T.; Yasuda, K.; Akatsuka, A.; Hino, O.; Hartman, P. S.; Ishii, N. A mutation in the SDHC gene of complex II increases oxidative

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Werner et al.

(42) (43)

(44)

(45)

(46)

(47) (48)

(49)

(50) (51)

(52)

(53)

(54)

(55)

(56)

stress, resulting in apoptosis and tumorigenesis. Cancer Res. 2005, 65, 203-9. Fujii, J.; Ikeda, Y. Advances in our understanding of peroxiredoxin, a multifunctional, mammalian redox protein. Redox Rep. 2002, 7, 123-30. Paron, I.; D’Elia, A.; D’Ambrosio, C.; Scaloni, A.; D’Aurizio, F.; Prescott, A.; Damante, G.; Tell, G. A proteomic approach to identify early molecular targets of oxidative stress in human epithelial lens cells. Biochem. J. 2004, 378, 929-37. Kang, S. W.; Chae, H. Z.; Seo, M. S.; Kim, K.; Baines, I. C.; Rhee, S. G. Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha. J. Biol. Chem. 1998, 273, 6297-302. Banmeyer, I.; Marchand, C.; Verhaeghe, C.; Vucic, B.; Rees, J. F.; Knoops, B. Overexpression of human peroxiredoxin 5 in subcellular compartments of Chinese hamster ovary cells: effects on cytotoxicity and DNA damage caused by peroxides. Free Radical Biol. Med. 2004, 36, 65-77. Fukushima, K.; Fiocchi, C. Paradoxical decrease of mitochondrial DNA deletions in epithelial cells of active ulcerative colitis patients. Am J. Physiol.: Gastrointest. Liver Physiol. 2004, 286, G804-13. Roediger, W. E. The colonic epithelium in ulcerative colitis: an energy-deficiency disease. Lancet 1980, 2, 712-5. Dolder, M.; Wendt, S.; Wallimann, T. Mitochondrial creatine kinase in contact sites: interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage. Biol. Signals Recept. 2001, 10, 93111. Matarrese, P.; Tinari, N.; Semeraro, M. L.; Natoli, C.; Iacobelli, S.; Malorni, W. Galectin-3 overexpression protects from cell damage and death by influencing mitochondrial homeostasis. FEBS Lett. 2000, 473, 311-5. Yang, R. Y.; Hsu, D. K.; Liu, F. T. Expression of galectin-3 modulates T-cell growth and apoptosis. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 6737-42. Won, J.; Kim, D. Y.; La, M.; Kim, D.; Meadows, G. G.; Joe, C. O. Cleavage of 14-3-3 protein by caspase-3 facilitates bad interaction with Bcl-x(L) during apoptosis. J. Biol. Chem. 2003, 278, 19347-51. Patterson, C. E.; Abrams, W. R.; Wolter, N. E.; Rosenbloom, J.; Davis, E. C. Developmental regulation and coordinate reexpression of FKBP65 with extracellular matrix proteins after lung injury suggest a specialized function for this endoplasmic reticulum immunophilin. Cell Stress Chaperones 2005, 10, 285-95. Fong, L. G.; Ng, J. K.; Lammerding, J.; Vickers, T. A.; Meta, M.; Cote, N.; Gavino, B.; Qiao, X.; Chang, S. Y.; Young, S. R.; Yang, S. H.; Stewart, C. L.; Lee, R. T.; Bennett, C. F.; Bergo, M. O.; Young, S. G. Prelamin A and lamin A appear to be dispensable in the nuclear lamina. J. Clin. Invest. 2006, 116, 743-52. Silverman, G. A.; Bird, P. I.; Carrell, R. W.; Church, F. C.; Coughlin, P. B.; Gettins, P. G.; Irving, J. A.; Lomas, D. A.; Luke, C. J.; Moyer, R. W.; Pemberton, P. A.; Remold-O’Donnell, E.; Salvesen, G. S.; Travis, J.; Whisstock, J. C. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem. 2001, 276, 33293-6. Hamerman, J. A.; Hayashi, F.; Schroeder, L. A.; Gygi, S. P.; Haas, A. L.; Hampson, L.; Coughlin, P.; Aebersold, R.; Aderem, A. Serpin 2a is induced in activated macrophages and conjugates to a ubiquitin homolog. J. Immunol. 2002, 168, 2415-23. Bird, C. H.; Sutton, V. R.; Sun, J.; Hirst, C. E.; Novak, A.; Kumar, S.; Trapani, J. A.; Bird, P. I. Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway. Mol. Cell. Biol. 1998, 18, 6387-98.

PR070222X

Intestinal Epithelial Cell Proteome in IL-10 Deficient Mice and IL-10

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