Theresa Pesch

Protein tyrosine phosphatase N2 regulates TNFα-induced signalling and cytokine secretion in human intestinal epithelial cells

Objective The Crohns disease (CD) susceptibility gene, protein tyrosine phosphatase N2 (PTPN2), regulates interferon γ (IFNγ)-induced signalling and epithelial barrier function in T84 intestinal epithelial cells (IECs). The aim of this study was to investigate whether PTPN2 is also regulated by tumour necrosis factor α (TNFα) and if PTPN2 controls TNFα-induced signalling and effects in IECs. Methods T84 IECs were used for all cell studies. Protein levels were assessed by western blotting, mRNA levels by reverse transcription–PCR (RT–PCR) and cytokine levels by ELISA. PTPN2 knock-down was induced by small interfering RNA (siRNA). Imaging was performed by immunohistochemistry or immunofluorescence. Results TNFα treatment elevated PTPN2 mRNA as well as nuclear and cytoplasmic protein levels and caused cytoplasmic accumulation of PTPN2. Biopsy specimens from patients with active CD showed strong immunohistochemical PTPN2 staining in the epithelium, whereas samples from patients with CD in remission featured PTPN2 levels similar to controls without inflammatory bowel disease (IBD). Though samples from patients with active ulcerative colitis (UC) revealed more PTPN2 protein than non-IBD patients and patients with UC in remission, their PTPN2 expression was lower than in active CD. Samples from patients with CD in remission and responding to anti-TNF treatment also showed PTPN2 levels that were similar to those in control patients. Pharmacological inhibition of nuclear factor-κB (NF-κB) by BMS-345541 prevented the TNFα-induced rise in PTPN2 protein, independent of apoptotic events. PTPN2 knock-down revealed that the phosphatase regulates TNFα-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 phosphorylation, without affecting c-Jun N-terminal kinase (JNK), inhibitor of κB (IκB) or NF-κB phosphorylation. Loss of PTPN2 potentiated TNFα-induced secretion of interleukin 6 (IL-6) and IL-8. In TNFα- and IFNγ-co-treated cells, loss of PTPN2 enhanced protein expression of inducible nitric oxide synthase (iNOS). Conclusions TNFα induces PTPN2 expression in IECs. Loss of PTPN2 promotes TNFα-induced mitogen-activated protein kinase signalling and the induction of inflammatory mediators. These data indicate that PTPN2 activity could play a crucial role in the establishment of chronic inflammatory conditions in the intestine, such as CD.

Protein tyrosine phosphatase nonreceptor type 2 regulates autophagosome formation in human intestinal cells

Background: Autophagy is a process of central importance for maintaining cell homeostasis, survival, and the regulation of inflammation. Recent studies associated variants within the gene loci, encoding protein tyrosine phosphatase nonreceptor type 2 (PTPN2), and autophagy genes, such as autophagy‐related 16‐like 1 (ATG16L1), with chronic inflammatory disorders, such as Crohns disease (CD). We show that PTPN2 regulates autophagy in human intestinal epithelial cells (IEC) and primary colonic lamina propria fibroblasts (CLPF). Methods: Protein analysis in IEC and CLPF was performed by western blotting. Autophagososme formation was assessed by LC3B immunofluorescence or immunohistochemistry. Human intestinal tissue samples were obtained from noninflammatory bowel disease (IBD) control or from CD patients and genotyped for disease‐associated PTPN2 or ATG16L1 variations. Results: Knockdown of PTPN2 causes impaired autophagosome formation and dysfunctional autophagy resulted in increased levels of intracellular Listeria monocytogenes (LM) and elevated IEC apoptosis in response to tumor necrosis factor (TNF) and interferon gamma (IFN‐&ggr;). Similar findings were observed in primary CLPF derived from CD patients carrying the CD‐associated PTPN2 variant. Presence of the ATG16L1 variant prevented the cytokine‐induced rise in PTPN2 protein, finally resulting in impaired LC3B‐II levels in IEC. Actively inflamed intestinal biopsies from CD patients carrying either ATG16L1 or PTPN2 genetic variants revealed aberrant LC3B expression patterns when compared with samples from non‐IBD control patients. Conclusions: Our results demonstrate that PTPN2 regulates autophagosome formation in human intestinal cells. We provide a model of how a dysfunction of the CD susceptibility genes, PTPN2 and/or ATG16L1, may contribute to the onset and perpetuation of chronic intestinal inflammation. (Inflamm Bowel Dis 2011;)

Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.

Background: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non‐receptor type 2 (PTPN2) has been associated with Crohns disease (CD), ulcerative colitis (UC), type‐I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen‐activated protein kinase (MAPK) signaling and cytokine secretion in human THP‐1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide‐oligomerization domain 2 (NOD2) ligand, muramyl‐dipeptide (MDP). Materials and Methods: Genomic DNA samples from 343 CD and 663 non‐IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2‐variant rs1893217 was introduced into T84 IEC or THP‐1 cells using a lentiviral vector. Results: We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP‐1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T‐bet transcription factor and secretion of interferon‐&ggr; in response to the bacterial wall component, MDP. In contrast, secretion of interleukin‐8 and tumor necrosis factor were reduced. In both, T84 IEC and THP‐1 monocytes, autophagosome formation was impaired. Conclusions: We identified a novel CD‐associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease. (Inflamm Bowel Dis 2011;)

Interleukin-13 and transforming growth factor β synergise in the pathogenesis of human intestinal fistulae

Objective Epithelial to mesenchymal transition (EMT) seems to play an important role in the pathogenesis of fistulae, a common clinical complication of Crohns disease (CD). TGFβ and interleukin-13 (IL-13) have been correlated with the onset of EMT-associated organ fibrosis and high levels of TGFβ have been shown in transitional cells (TCs) lining CD fistula tracts. This study investigated whether IL-13 could be involved in the pathogenesis of CD-associated fistulae. Design Protein or mRNA levels in HT29 intestinal epithelial cells (IECs) or colonic lamina propria fibroblasts (CLPFs) were studied by western blotting or real-time PCR. CLPFs were isolated from non-inflammatory disease controls or patients with CD with or without fistulae and IL-13 levels were analysed in surgically removed fistula specimens by immunohistochemistry. Results TGFβ induced IL-13 secretion in CLPFs from patients with fistulising CD. In fistula specimens high levels of IL-13 were detected in TCs covering fistula tracts. In HT29 IEC monolayers, IL-13 induced SLUG and β6-integrin mRNA, which are associated with cell invasion. HT29 spheroids completely disintegrated when treated with TGFβ for 7 days, whereas IL-13-treated spheroids did not show morphological changes. Here, TGFβ induced mRNA expression of SNAIL1 and IL-13, whereas IL-13 elevated SLUG and β6-integrin mRNA. An anti-IL-13 antibody was able to prevent IL-13-induced SLUG expression in HT29 IECs. Conclusions TGFβ induces IL-13 expression and an EMT-like phenotype of IECs, while IL-13 promotes the expression of genes associated with cell invasion. These findings suggest that TGFβ and IL-13 play a synergistic role in the pathogenesis of fistulae and inhibition of IL-13 might represent a novel therapeutic approach for fistula treatment.

Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines

BackgroundCholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro.MethodsExpression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations.ResultsEGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation.ConclusionCC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

Potential role for SNAIL family transcription factors in the etiology of Crohn's disease‐associated fistulae

Background: Fistulae represent an important clinical complication of Crohns disease (CD). The fistula tracts are covered by flat, myofibroblast‐like cells with an epithelial origin (transitional cells, TC). We recently demonstrated a role of epithelial mesenchymal transition (EMT) in the pathogenesis of CD‐associated fistulae. EMT is associated with an increased migratory and invasive potential of epithelial cells in different tissues. Here we investigated whether cytokines or growth factors as well as EMT‐associated SNAIL family transcription factors are expressed in CD fistulae. Methods: By immunohistochemistry we analyzed seven perianal fistulae from seven CD and two perianal fistulae from two non‐inflammatory bowel disease (IBD) control patients. Hematoxylin and eosin staining or immunohistochemistry for the expression of tumor necrosis factor (TNF), TNF‐receptor I (TNF‐RI), SNAIL1, SLUG, fibroblast growth factors (FGF) 1, 2, 4, 7, epidermal growth factor (EGF), and TWIST were performed using standard techniques. Results: Immunohistochemical staining of surgical specimens from CD patients revealed a strong expression of TNF and TNF‐RI in and around fistula tracts. While SNAIL1 was also heavily expressed in the nuclei of TC, indicative of transcriptionally active protein, SLUG, FGF‐1, and FGF‐2 were detected rather in the fibrotic periphery of CD fistulae than in TC. In contrast, we did not detect considerable protein staining for FGF‐4 and FGF‐7 nor of EGF or the transcription factor, TWIST. Conclusions: Our data demonstrate that SNAIL1 and TNF are strongly expressed in TC of CD‐associated fistulae. These observations support our previous data and indicate the onset of EMT‐associated events in the pathogenesis of CD fistulae. (Inflamm Bowel Dis 2010;17:1907–1916)

Hypoxia induces the expression of transketolase-like 1 in human colorectal cancer.

Background and Aims: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. Methods: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. Results: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1α protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. Conclusions: TKTL1 expression correlated with HIF-1α protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances.

A role for tumor necrosis factor and bacterial antigens in the pathogenesis of Crohn's disease-associated fistulae

Background:Intestinal fistulae represent a severe complication of Crohn’s disease (CD). The authors have demonstrated that epithelial-to-mesenchymal transition plays a pivotal role in their pathogenesis. High levels of interleukin-13 and tumor necrosis factor (TNF) are detected in myofibroblast-like transitional cells covering the fistula tracts. Here, a functional role was investigated for the transcription factor Ets-1, TNF, and the bacterial wall component (muramyl dipeptide [MDP]) in the pathogenesis of CD-associated fistulae. Methods:Perianal fistulae from CD patients were analyzed by immunohistochemistry. Primary colonic lamina propria fibroblasts (CLPFs) were isolated from CD patients with or without fistulizing disease. Messenger RNA (mRNA) levels were assessed by real-time polymerase chain reaction in CLPF or HT29 intestinal epithelial cells (IECs) grown as monolayers or spheroids. Results:Strong expression of Ets-1 transcription factor was demonstrated in transitional cell covering the fistula tracts by immunohistochemistry. TNF induced mRNA expression of ETS-1 and &bgr;6-integrin in HT29 IEC and in CLPF from fistulizing CD patients. These effects could be fully blocked by administration of anti-TNF antibodies. In HT29 cells, TNF further induced mRNA levels of TNF and transforming growth factor beta by treatment for 24 hours. In fistula CLPF derived from CD patients, TNF induced expression of &bgr;6-integrin, TNF, and transforming growth factor beta. Of note, the bacterial wall component, MDP, induced mRNA levels of ETS-1, transforming growth factor beta, interleukin-13, SNAIL1, and &bgr;6-integrin in HT29 IEC monolayers and fistula CLPF by treatment for 24 hours. Conclusions:TNF and MDP induce the expression of factors associated with epithelial-to-mesenchymal transition and invasion in IEC and fistula CLPF. Our findings indicate that TNF and MDP might synergize in the pathogenesis of CD-associated fistulae.

The role for dickkopf-homolog-1 in the pathogenesis of Crohn's disease-associated fistulae.

Background One of the most challenging conditions in Crohn’s disease (CD) patients is the treatment of perianal fistulae. We have recently shown that epithelial-to-mesenchymal transition (EMT) plays a crucial role during CD-fistulae development. Dickkopf-homolog 1 (DKK-1) is known to play a key role during EMT. Here, we investigated a role for DKK-1 in the pathogenesis of CD-associated fistulae. Methods Dkk-1 protein expression in CD-fistula specimens were investigated by immunohistochemistry. Colonic lamina propria fibroblasts (CLPF) were obtained from either non-IBD control patients or patients with fistulizing CD. HT-29 intestinal epithelial cells (IEC) were either grown as monolayers or spheroids. Cells were treated with either TNF-α, TGF-β or IL-13. Knock-down of DKK-1 or β-Catenin was induced in HT-29-IEC by siRNA technique. mRNA expression was determined by real-time-PCR. Results Dkk-1 protein was specifically expressed in transitional cells lining the fistula tracts. TGF-β induced DKK-1 mRNA expression in HT-29-IEC, but decreased it in fistula CLPF. On a functional level, DKK-1 knock-down prevented TGF-β-induced IL-13 mRNA expression in HT-29-IEC. Further, loss of β-Catenin was accompanied by reduced levels of DKK-1 and, again, IL-13 in IEC in response to TGF-β. In turn, treatment of HT-29-IEC as well as fistula CLPF with IL-13 resulted in decreased levels of DKK-1 mRNA. Treatment with TNF-α or the bacterial wall component, muramyl-dipeptide, decreased DKK-1 mRNA levels in HT-29-IEC, but enhanced it in fistula CLPF. Discussion We demonstrate that DKK-1 is strongly expressed in cells lining the CD-fistula tracts and regulates factors involved in EMT initiation. These data provide evidence for a role of DKK-1 in the pathogenesis of CD-associated perianal fistulae.

Cytosolic Ca2+ shifts as early markers of cytotoxicity

The determination of the cytotoxic potential of new and so far unknown compounds as well as their metabolites is fundamental in risk assessment. A variety of strategic endpoints have been defined to describe toxin-cell interactions, leading to prediction of cell fate. They involve measurement of metabolic endpoints, bio-energetic parameters or morphological cell modifications. Here, we evaluated alterations of the free cytosolic Ca2+ homeostasis using the Fluo-4 dye and compared results with the metabolic cell viability assay Alamar Blue. We investigated a panel of toxins (As2O3, gossypol, H2O2, staurosporine, and titanium(IV)-salane complexes) in four different mammalian cell lines covering three different species (human, mouse, and African green monkey). All tested compounds induced an increase in free cytosolic Ca2+ within the first 5 s after toxin application. Cytosolic Ca2+ shifts occurred independently of the chemical structure in all tested cell systems and were persistent up to 3 h. The linear increase of free cytosolic Ca2+ within the first 5 s of drug treatment correlates with the EC25 and EC75 values obtained in Alamar Blue assays one day after toxin exposure. Moreover, a rise of cytosolic Ca2+ was detectable independent of induced cell death mode as assessed by caspase and poly(ADP-ribose) polymerase (PARP) activity in HeLa versus MCF-7 cells at very low concentrations. In conclusion, a cytotoxicity assay based on Ca2+ shifts has a low limit of detection (LOD), is less time consuming (at least 24 times faster) compared to the cell viability assay Alamar Blue and is suitable for high-troughput-screening (HTS).