TNF-α and IFN-γ regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells

doi:10.1053/gast.2003.50157

Gastroenterology

Volume 124, Issue 4, April 2003, Pages 1001-1009

https://doi.org/10.1053/gast.2003.50157 Get rights and content

Abstract

Background & Aims: NOD2, a member of the NOD1/Apaf-1 family, was recently identified as the first susceptibility gene for Crohn's disease. The aim of this report was to describe the regulation and functional significance of NOD2 expression in intestinal epithelial cells. Methods: Expression of NOD2 messenger RNA was determined by reverse-transcription polymerase chain reaction (RT-PCR); NOD2 protein was detected by Western blot. Promoter activity was assessed by reporter gene assays and DNA-binding of NF-κB by electrophoretic mobility shift assays. IL-8 production was investigated by RT-PCR and enzyme-linked immunosorbent assay. Results: TNF-α induced an up-regulation of NOD2 in epithelial cell lines (HT-29, SW620, SW948, HeLa S3) and in primary colonic epithelial cells. A synergism was seen by cotreatment with IFN-γ. Two NF-κB binding sites were identified in the promoter. Deletion of either site or overexpression of dominant negative IκBα led to reduced levels of TNF-α/IFN-γ-stimulated reporter gene activity. The identified κB3 site was bound by NF-κB as determined by gelshift assays. Elevated amounts of NOD2 protein were also found in colonic epithelial cells from patients with IBD. LPS induced high levels of IL-8 production in SW620 cells overexpressing NOD2. Conclusions: TNF-α(/IFN-γ) treatment up-regulates the expression of the NOD2 gene in intestinal epithelial cells and subsequently increases their LPS susceptibility. Together with the mutation-derived truncation and functional change of the NOD2 protein, this could be part of the complex pathophysiology of barrier disruption as it is observed in inflammatory bowel diseases.

Section snippets

Cell culture and transfection

Human epithelial HeLa S3 cells (ACC 161), intestinal epithelial HT-29 cells (ACC 299), and Caco-2 cells (ACC 169) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). Two other colonic epithelial cell lines, SW620 and SW948 cells were kindly provided by Holger Kalthoff (UKK, Kiel, Germany). All cells were cultured in RPMI + 10% fetal calf serum. One day before transfection the cells were seeded at a density of 5 × 10⁵ cells/2 mL on 6-well

NOD2 mRNA and protein expression is synergistically up-regulated in intestinal epithelial cell lines by TNF-α and IFN-γ

Different intestinal epithelial cell lines (HT-29, SW620, SW948) and HeLa S3 cells were stimulated for different durations with TNF-α (10 ng/mL), and total RNA was isolated as described. NOD2 complementary DNA was amplified as outlined and analyzed on an agarose gel (Figure 1A).

. Detection of NOD2 mRNA by RT-PCR. (A) Stimulation of HT-29 and HeLa S3 cells for different durations with 10 ng/mL TNF-α for 24 hours. (B) Treatment of primary intestinal epithelial cells (IEC) for 12 hours with TNF-α

Conclusions

Epithelial cells are an integral part of the primary mechanical and immunological barrier against the invasion of microorganisms. The up-regulation of the NOD2 gene in IECs by TNF-α and IFN-γ could be part of the regulatory cascade of events during primary inflammatory processes. NOD2 has been shown to interact with RIP-like interacting CLARP kinase and overexpression of NOD2 in HEK293T cells led to activation of NF-κB.⁵ Interestingly, expression of NOD2 has been mainly presented in monocytic

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^☆: Address requests for reprints to: Dirk Seegert, Ph.D., Conaris Research Institute AG, Schauenburgerstr. 116, D-24118 Kiel, Germany. e-mail: [email protected]; fax: (49) 431-5606-822.

^☆☆: Supported by grants from the “Deutsche Forschungsgemeinschaft” (SFB 415) and through a Competence Network from the German Ministry for Education and Research (BMBF) as well as from the National Genome Research Network.

^★: The authors thank the patients, the endoscopy staff, and the physicians (S. Nikolaus, U. Helwig, J. Grebe). The authors also thank G. Nunez for the pcDNA3NOD2wt plasmid. Ilka Woywod and Tanja Kaacksteen are gratefully acknowledged for their technical assistance.

^★★: P. Rosenstiel and M. Fantini contributed equally to this work. D. Seegert and S. Schreiber share senior authorship.

^♢: 0016-5085/03/$30.00

View full text

Basic-Alimentary TractTNF-α and IFN-γ regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells☆,☆☆,★,★★,♢

Abstract

Section snippets

Cell culture and transfection

NOD2 mRNA and protein expression is synergistically up-regulated in intestinal epithelial cell lines by TNF-α and IFN-γ

Conclusions

Cell

J Biol Chem

J Biol Chem

J Biol Chem

Lancet

Trends Biochem Sci

Curr Opin Microbiol

Trends Cell Biol

Lancet

Lancet

Cell

Gastroenterology

J Biol Chem

J Biol Chem

J Biol Chem

Gastroenterology

The Caenorhabditis elegans cell death gene ced-4 encodes a novel protein and is expressed during the period of extensive programmed cell death

Development

The NOD: a signaling module that regulates apoptosis and host defense against pathogens

Oncogene

The NB-ARC domain: a novel signalling motif shared by plant resistance gene products and regulators of cell death in animals

Curr Biol

Plant pathogens and integrated defence responses to infection

Nature

A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease

Nature

Association of NOD2 leucine-rich repeat variants with susceptibility to Crohn's disease

Nature

Interferons and their role in inflammation

Curr Pharm Des

Interferon expression in Crohn's disease patients: increased interferon-gamma and -alpha mRNA in the intestinal lamina propria mononuclear cells

J Interferon Res

Basic-Alimentary Tract
TNF-α and IFN-γ regulate the expression of the NOD2 (CARD15) gene in human intestinal epithelial cells☆,☆☆,★,★★,♢