Dataset Information

NF-kappaB-dependent synergistic regulation of CXCL10 gene expression by IL-1beta and IFN-gamma in human intestinal epithelial cell lines.

ABSTRACT: BACKGROUND AND AIMS: Little is known about the intestinal epithelial expression and secretion of CXCL10 (IP-10), a chemokine involved in recruiting T cells and monocytes. We aimed to study CXCL10 gene expression and regulation by the pro-inflammatory cytokines interleukin (IL)-1beta, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in intestinal epithelial cell lines. MATERIALS AND METHODS: CXCL10 expression and secretion kinetics were assessed in Caco-2, HT-29 and DLD1 human colon epithelial cells, treated with IL-1beta, TNF-alpha, IFN-gamma alone or in combination with each other by real-time polymerase chain reaction (PCR), Northern blotting and enzyme-linked immunoabsorbent assay (ELISA). Transient transfections with TGL-IP10 (CXCL10 promoter) and TGL-IP10-kappaB2 mutant promoter and gelshifts and supershifts for nuclear factor (NF)-kappaB were also performed. RESULTS: Real-time PCRs and ELISA experiments revealed that IL-1beta was the strongest and earliest inducer of CXCL10 messenger ribonucleic acid (mRNA) expression and protein secretion in Caco-2 cell line, whereas INF-gamma had a delayed kinetics. There was a strong synergistic effect of either TNF-alpha or IL-1beta with IFN-gamma both on CXCL10 mRNA expression and protein secretion in all three cell lines. Real-time PCR and ELISA experiments using a specific NF-kappaB inhibitor and transfection experiments with a NF-kappaB-binding defective CXCL10 promoter construct revealed that the induction of CXCL10 by IL-1beta and its synergism with IFN-gamma is NF-kappaB dependent. CONCLUSION: These data demonstrate that in colonic epithelial cells, depending on the cellular context and utilizing the NF-kappaB pathway, IL-1beta alone and/or in synergism with IFN-gamma may play a major role in the induction of CXCL10.

SUBMITTER: Yeruva S

PROVIDER: S-EPMC2225996 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Platelet-derived growth factors are a family of potent mitogens and chemoattractants for fibroblasts and other cells of mesenchymal origin. Platelet-derived growth factor (PDGF) dimeric ligands (composed of A-, B-, C-, and D-chains) exert their biological activity through high affinity interactions with cell surface receptor subunits (alpha and beta). PDGF-receptor-alpha is widely implicated in the pathogenesis of hyperplastic fibrotic disease, yet the molecular mechanisms controlling its expression in response to injury are poorly understood. Here we show that PDGF-R alpha expression is induced in fibroblasts by mechanical injury and interleukin (IL)-1beta, which was abolished by neutralizing IL-1beta antibodies in the culture supernatant or inhibitors of NF-kappaB. Chromatin immunoprecipitation and electrophoretic mobility shift assays revealed the existence of a new NF-kappaB binding site at -531/-521 bp in the PDGF-R alpha promoter. We have recently shown that ATF-4 is also induced by injury (Malabanan, K. P., Kanellakis, P., Bobik, A., and Khachigian, L. M. (2008) Circ. Res. 103, 378-387), and we demonstrate here that ATF-4 binds a novel element -259/-254 and stimulates PDGF-R alpha transcription. ATF-4 and NF-kappaB interact, occupy the PDGF-R alpha promoter, and induce PDGF-R alpha transcription in a cooperative manner. IL-1beta facilitates the dissociation of histone deacetylase (HDAC)-1/2 from the PDGF-R alpha promoter, whereas the HDAC inhibitors suberoylanilide hydroxamic acid and trichostatin A potentiate IL-1beta induction of PDGF-R alpha transcription. These findings, taken together, demonstrate that injury stimulates IL-1beta secretion by fibroblasts, which activates NF-kappaB and ATF-4 and stimulates interaction with the PDGF-R alpha promoter, triggering PDGF-R alpha transcription. Physical and functional interactions between NF-kappaB and ATF-4 have not been reported in any gene. This is also the first report of HDAC regulation of PDGF-R alpha transcription.

Project description:Platelet-activating factor (PAF), a potent proinflammatory phospholipid mediator, has been implicated in inducing intestinal inflammation in diseases such as inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). However, its mechanisms of inducing inflammatory responses are not fully understood. Therefore, studies were designed to explore the mechanisms of PAF-induced inflammatory cascade in intestinal epithelial cells.Nuclear factor kappa B (NF-kappaB) activation was measured by luciferase assay and enzyme-linked immunosorbent assay (ELISA), and interleukin 8 (IL-8) production was determined by ELISA. B-cell lymphoma 10 (Bcl10), caspase recruitment domain-containing membrane-associated guanylate kinase protein 3 (CARMA3), and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) mRNA and protein levels were assessed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. siRNA silencing of Bcl10 was used to examine its role in PAF-induced NF-kappaB activation and IL-8 production. The promoter region of the Bcl10 gene was cloned with the PCR method and promoter activity measured by luciferase assay.The adaptor protein Bcl10 appeared to play an important role in the PAF-induced inflammatory pathway in human intestinal epithelial cells. Bcl10 was required for PAF-induced I kappaB alpha phosphorylation, NF-kappaB activation, and IL-8 production in NCM460, a cell line derived from normal human colon, and Caco-2, a transformed human intestinal cell line. PAF also stimulated Bcl10 interactions with CARMA3 and MALT1, and upregulated Bcl10 expression in these cells via transcriptional regulation.These findings highlight a novel PAF-induced inflammatory pathway in intestinal epithelial cells, requiring Bcl10 as a critical mediator and involving CARMA3/Bcl10/MALT1 interactions. The proinflammatory effects of PAF play prominent roles in the pathogenesis of IBD and this pathway may present important targets for intervention in chronic inflammatory diseases of the intestine.

Dataset Information

NF-kappaB-dependent synergistic regulation of CXCL10 gene expression by IL-1beta and IFN-gamma in human intestinal epithelial cell lines.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets