Mediation by NF-κB of cytokine induced expression of intercellular adhesion molecule 1 (ICAM-1) in an intestinal epithelial cell line, a process blocked by proteasome inhibitors

Abstract

Background/aims—The gene promoter for the intercellular adhesion molecule ICAM-1 possesses binding sites for several transcriptional factors, including nuclear factor κB (NF-κB). The role of NF-κB in ICAM-1 gene regulation was therefore examined by using different proteasome inhibitors in tumour necrosis factor α (TNF-α) stimulated IEC-6 rat intestinal epithelial cells. 
Methods—ICAM-1 expression was analysed by enzyme linked immunosorbent assay (ELISA), reverse transcriptase polymerase chain reaction, and immunohistochemistry. Steady state levels of cytoplasmic IκB protein were evaluated by western blot, and nuclear translocation of NF-κB was determined by electrophoretic mobility shift assay and immunofluorescence staining. Cell adhesion was assayed by measuring the binding of fluorescence labelled MOLT-4 cells. 
Results—TNF-α induced ICAM-1 mRNA and protein expression in IEC-6 cells, which was followed by increased adhesion of MOLT-4 lymphocytes. Blocking TNF-α induced IκBα degradation with proteasome inhibitors reduced TNF-α induced NF-κB activation and ICAM-1 gene induction and notably decreased MOLT-4 cell adhesion without affecting Jun N-terminal kinase (JNK/SAPK) activity or de novo protein synthesis. 
Conclusion—TNF-α induction of ICAM-1 expression is mediated by the transcription factor NF-κB and can be inhibited by blocking IκBα degradation. Thus the IκB/NF-κB system is a promising target for pharmacological modulation of the expression of adhesion molecules and other inflammatory genes in the intestine. 



Keywords: adhesion molecules; ICAM-1; cytokines; tumour necrosis factor α; intestinal inflammation; NF-κB.

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Figure 1 .

Effect of tumour necrosis factor α (TNF-α) stimulation and selective proteasome blockade on steady state IκBα concentrations (A), NF-κB binding activity (B), and RelA (p65) subcellular localisation (C). (A) Cells were pretreated with MG-132 (5 µg/ml; left panel), ALLN (50 µg/ml; right panel), or medium alone for 30 minutes and then stimulated for 0 to 60 minutes with TNF-α (10 ng/ml). Total protein was extracted and 20 µg subjected to SDS/PAGE followed by immunoblotting of IκBα using the ECL technique as described in the Methods section. Note that the IκBα standard used as a control contains seven extra amino acids (IκB-tag) compared with the endogenous epithelial IκBα. (B) Cells were treated as described above or infected for 12 hours with the Ad5IκB virus and then stimulated for 30 minutes with TNF-α (10 ng/ml). Nuclear extracts (5 µg) were tested for κB binding activity by electrophoretic mobility shift assay. Antibody supershifting is indicated by arrowheads. (C) Cells were treated as described in (A) and RelA localisation was visualised using an anti-RelA antibody followed by a rhodamine conjugated detection antibody. 


Figure 2 .

ALLN does not interfere with tumour necrosis factor α (TNF-α) induced JNK/SAPK activity in IEC-6 cells. Cells were pretreated with ALLN (50 µg/ml) or medium alone for 30 minutes and then stimulated with TNF-α (10 ng/ml) for 30 minutes. Phosphorylated glutathione S-transferase (GST)-c-Jun was visualised after protein fractionation using SDS/PAGE (12.5% gel) and quantified using PhosphorImager analysis. Coomassie staining was used to confirm equal protein loading. A representative result of three independent experiments is shown. 


Figure 3 .

Inhibition of ICAM-1 gene expression by proteasome inhibitors in IEC-6 cells as measured by RT-PCR (A) and ELISA (B) techniques. (A) Cells were pretreated with ALLN (50 µg/ml; upper panel), MG-132 (5 µg/ml; lower panel), or medium alone for 30 minutes and then stimulated with tumour necrosis factor α (TNF-α) (10 ng/ml) or medium alone for four hours. Total RNA was extracted, reverse transcribed, and amplified using specific ICAM-1 or actin primers. PCR products were run on a 2% agarose gel and stained with ethidium bromide. These results are representative of three different experiments. (B) Cells were stimulated with TNF-α (10 ng/ml) or medium alone for 16 hours in the presence or absence of ALLN (50 µg/ml) or aprotinin (10 µg/ml). Immunoreactive ICAM-1 protein synthesis was measured as described in the Methods section. Data represent mean (SE) from four samples. *p<0.05 compared with control samples. This result is representative of three different experiments. 


Figure 4 .

Tumour necrosis factor α (TNF-α) induces ICAM-1 protein systhesis in IEC-6 cells as measured by immunohistochemical analysis. Cells were grown on glass coverslips and then stimulated with TNF-α (10 ng/ml) (B) or cultured in medium alone (A) for 16 hours. The cells were stained for ICAM-1 using anti-ICAM-1 antibody as described in the Methods section. Similar results were obtained in two other experiments. 


Figure 5 .

Effect of MG-132 on IEC-6 cellular protein synthesis. Cells were starved for 60 minutes in medium without methionine and then labelled for 12 hours with 150 µCi [³⁵S]methionine in the presence or absence of MG-132 or cycloheximide (CHX). Total protein was extracted and 20 µg subjected to SDS/PAGE. (A) Gel exposed to autoradiography or (B) stained with Coomassie blue as described in the Methods section. Similar results were observed in two other independent experiments. 


Figure 6 .

ALLN blocks the tumour necrosis factor α (TNF-α) mediated adhesion of IEC-6 to MOLT-4 lymphocytes. Fluorescence labelled MOLT-4 cells were added to TNF-α stimulated IEC-6 cells pretreated with ALLN (50 µg/ml), aprotinin (10 µg/ml), or medium alone. Fluorescence emission intensity in cell lysates after washing was measured as described in the Methods section. Similar inhibition was observed in two other independent experiments.