Suppression of epithelial syndecan-1 expression by tumour necrosis factor-a

Abstract

Background: Syndecan-I, a transmembrane heparan sulphate proteoglycan, is expressed on the basolateral surface of columnar epithelium where it functions as a cell-cell, cell-matrix adhesion molecule. Syndecan-I binds to a variety of matrix and heparin-binding growth factor molecules and is thought to play an important role during wound repair, where its expression is usually upregulated, by facilitating increased growth factor binding. In inflammatory bowel disease (ffiD) syndecan-l expression is reduced in areas of ulcerated mucosa, particularly in reparative epithelium. The mechanism causing reduced syndecan-l expression in ffiD is unknown. Aim: To investigate the regulatory effect of the pro-inflammatory cytokine tumour necrosis factor-a (TNF-a) on syndecan-I expression in the HT29 colonic epithelial cell line. Methods: Confluent monolayers of HT29 cells were stimulated with TNF-a (0.625, 0.125, 2.5, 5.0, 10, 25, 50 ng/ml) for 24 hours. Flow cytometry (FACS) and immunofluorescent microscopy using indirect immunostaining procedures were used to analyze syndecan-l protein expression. The mean fluorescent intensity (MFI) of syndecan-I staining in viable antibody labeled treated cells was compared with that of untreated control cells. All experiments were performed in triplicate. Modulation of syndecan-I expression at the RNA level was determined using RT-PCR. Results: Stimulation of HT29 monolayers with TNF-a resulted in down-regulation of syndecan-I at both the protein and RNA levels as revealed by FACS analysis and RT-PCR. Reduction of MFI ranged from 10% at 0.625 ng/ml to 64% at 50 ng/ml. Time course studies revealed that cells treated with 5 ng/ml TNF-a had a 2% reduction in MFI at 3-5 hours and a maximum reduction (-35%) at 13 hours. Immunofluorescence microscopy revealed a visible progressive reduction in membrane staining with increasing concentrations of TNF-a. No increase in cytoplasmic staining was observed at any concentration of TNF-a. Conclusions: Reduced syndecan-l expression is likely to impair mucosal healing due to diminished anti-ulcerogenic growth factor binding. These data show that TNF-a, a pro-inflammatory cytokine thought to have an etiological role in Crohn's disease, has a strong effect on the downregulation of syndecan-l expression. Such an effect may account for the apparently unique reduction of syndecan-I expression in ffiD.