Intestinal Epithelial Cell Proteome from Wild-Type and TNF∆ARE/WT Mice: Effect of Iron on the Development of Chronic Ileitis Tanja Werner,† Gabriele Hoermannsperger,† Klaus Schuemann,‡ Gabriele Hoelzlwimmer,§ Shoutaro Tsuji,| and Dirk Haller*,† Chair for Biofunctionality, ZIEL - Research Center for Nutrition and Food Science, Technische Universita¨t Mu ¨nchen, Am Forum 5, Freising-Weihenstephan, Germany, Molecular Nutrition Unit, Research Center for Nutrition and Food Science, Technische Universita¨t Mu ¨nchen, Am Forum 5, Freising-Weihenstephan, Germany, Helmholtz Center Munich, Institute of Pathology, 85764 Munich, Germany, and Division of Cancer Therapy, Kanagawa Cancer Center Research Institute, 1-1-2 Nakao, Asahi-ku, Yokohama-shi Kanagawa 241-0815, Japan Received September 12, 2008
Environmental factors substantially contribute to the development of chronic intestinal inflammation in the genetically susceptible host. Nutritional components like iron may act as pro-oxidative mediators affecting inflammatory processes and cell stress mechanisms. To better characterize effects of dietary iron on epithelial cell responses under the pathological conditions of chronic intestinal inflammation, we characterized the protein expression profile (proteome) in primary intestinal epithelial cells (IEC) from iron-adequate and low-iron fed wild-type (WT) and TNF∆ARE/WT mice. We performed all possible comparisons between the 4 groups according to genotype or diet. Histological analysis of iron-adequate fed TNF∆ARE/WT mice (∼0.54 mg of iron/day) revealed severe ileal inflammation with a histopathology score of 8.3 ( 0.91 (score range from 0-12). Interestingly, low-iron fed mice (∼0.03 mg of iron/day) were almost completely protected from the development of inflammatory tissue destruction (histopathology score of 2.30 ( 0.73). In total, we identified 74 target proteins with significantly altered steady state expression levels in primary IEC using 2D-gel electrophoresis (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS). Interestingly, the overlap between the comparison of iron-adequate fed WT and TNF∆ARE/WT mice (inflamed conditions) and the comparison between the iron-adequate and iron-low fed TNF∆ARE/WT mice (absence of inflammation) revealed 4 contrarily regulated proteins including aconitase 2, catalase, intelectin 1 and fumarylacetoacetate hydrolase (FAH). These proteins are associated with energy homeostasis, host defense, oxidative and endoplasmic reticulum (ER) stress responses. In conclusion, the iron-low diet affected the epithelial cell proteome and inhibited the development of chronic intestinal inflammation, suggesting a critical role for nutritional factors in the pathogenesis of IBD. Keywords: IBD • Experimental ileitis • Iron • TNF∆ARE/WT mice • Intestinal epithelial cells • Epithelial cell proteome • Aconitase 2 • Catalase • Intelectin 1 • FAH
Introduction Oxidative stress is a critical process in the pathogenesis of inflammatory bowel disease (IBD) including Crohn’s disease (CD) and ulcerative colitis (UC). IBD patients show an imbalance between oxidative stress and antioxidative defense mechanisms in the mucosa.1,2 Nutritional components like iron may act as mediators for the induction of oxidative stress and are therefore considered as factors responsible for the initiation * To whom correspondence should be addressed. Dirk Haller, Ph.D., Professor, Biofunctionality, Technische Universita¨t Mu ¨ nchen, Am Forum 5, 85350 Freising-Weihenstephan, Germany. E-mail:
[email protected]. Phone: ++49-8161-71-2026. Fax: ++49-8161-71-2824. † Chair for Biofunctionality, ZIEL - Research Center for Nutrition and Food Science, Technische Universita¨t Mu ¨ nchen. ‡ Molecular Nutrition Unit, Research Center for Nutrition and Food Science, Technische Universita¨t Mu ¨ nchen. § Helmholtz Center Munich. | Kanagawa Cancer Center Research Institute.
3252 Journal of Proteome Research 2009, 8, 3252–3264 Published on Web 05/08/2009
and/or perpetuation of chronic intestinal inflammation in IBD patients. Anemia is a common problem in CD patients, but unfortunately, oral supplementation with iron enhances intestinal inflammation and oxidative stress-induced tissue damage in the intestine probably mediated through the production of reactive oxygen species (ROS).3-5 ROS accumulation at the epithelial level may lead to defects in the epithelial barrier function and enhances intestinal permeability which is observed in patients with IBD.6,7 Iron uptake is also discussed in association with changes in the intestinal structure like shortening of microvillus height and erosions of the microvilli in the duodenum8 and the development of colon cancer.3,9 Adverse environmental and/or metabolic conditions trigger cellular stress responses including endoplasmic reticulum (ER)specific mechanisms to ensure the transit of correctly folded proteins through the Golgi and cytoplasmic compartments.10 In addition to metabolic associated disorders, we recently 10.1021/pr800772b CCC: $40.75
2009 American Chemical Society
research articles
Effect of Iron on the Development of Chronic Ileitis a
Table 1. Overview of All Performed Comparisons
WT-comparison ARE-comparison ade WT-AREcomparison low WT-AREcomparison
control
treatment
iron-adequate fed wild-type mice iron-adequate fed ARE mice iron-adequate fed wild-type mice low-iron fed wild-type mice
low-iron fed wild-type mice low-iron fed ARE mice iron-adequate fed ARE mice low-iron fed ARE mice
a Regulation factors given in Table 2-5 refer to the treatment versus control group.
showed that the lack of immune-mediated control mechanisms at the epithelial cell level contributes to ER-associated cell stress responses and the development of chronic intestinal inflammation in animal models and human IBD.11 Additionally, Heazlewood et al. revealed mucin depletion by the loss of ERstressed goblet cells as component of the pathogenesis of human colitis leading to a depleted mucus barrier, increased epithelial permeability and ER stress triggered local mucosal inflammation.12 Consistent with the concept that cellular stress responses at the epithelial cell level contribute to the pathogenesis of IBD, Kaser et al. linked the loss of the ER-associated transcription factor XBP-1 to a Paneth cell dysfunction conferring to an increased risk for the development of IBD.13 The intestinal epithelium represents a highly selective barrier between gut lumen and underlying cells of the immune system. Intestinal epithelial cells (IEC) constitutively express, or can be induced to express, co-stimulatory molecules14,15 and components of the human major histocompatibility complex (MHC) including class II, classical I and nonclassical class Ib MHC molecules,16,17 Toll-like receptors (TLR) and nucleotide-binding oligomerization domain (NOD2) protein receptors,18,19 inflammatory and chemo-attractive cytokines20 as well as antimicrobial peptides.21,22 Of considerable importance, most of these molecules are at least in part transcriptionally regulated by the transcription factor NF-κB.23 In the intestinal tract, enhanced levels of the pro-inflammatory cytokine TNF-R, a known trigger for NF-κB activation,24 promote the pathogenesis of IBD.25,26 TNF∆ARE/WT mice, a murine model for experimental ileitis, show impaired regulation of TNF-R synthesis by deletion of a repeated octanucleotide AU-rich motif in the 3′-untranslated region of the TNF gene. This genetic variation results in enhanced mRNA stability and increased production of TNF-R accompanied by the development of a severe ileitis.27 Because iron can trigger oxidative stress, enhance intestinal inflammation and induce ER stress responses at the epithelial cell level (Werner and Haller, in progress), we investigated the effects of a low-iron diet on the development of chronic intestinal inflammation and characterized changes of the protein expression profile in primary epithelial cells from the ileum using the TNF∆ARE/WT mouse model. WT and TNF∆ARE/WT were fed with an iron-adequate and lowiron diet (N ) 20, 5 animals per group). To identify novel protein targets, we characterized the protein expression profile (proteome) from primary IEC of the iron-adequate and lowiron fed WT and TNF∆ARE/WT mice performing all possible comparison between the 4 groups. We identified 74 proteins with significantly altered steady-state expression levels in primary IEC using 2D-gel electrophoresis (2D SDS-PAGE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS). The bibliometric data analysis generated a protein-protein network tree from the different comparisons and identified
interrelated proteins with functions in energy metabolism, catalytic activity and antioxidative defense.
Material and Methods Animals. Conventionally raised TNF∆ARE/WT mice (a generous gift from Kollias, G., Institute for Immunology, Biomedical Sciences Research Center “Al. Fleming”, Greece) on C57BL/6 background and WT mice were fed with an iron-adequate (Altromin C1000, containing 180 mg of iron sulfate/kg) and lowiron diet (Altromin C1038, containing
