Tumour necrosis factor alpha converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease - PubMed (original) (raw)

Tumour necrosis factor alpha converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease

J Brynskov et al. Gut. 2002 Jul.

Abstract

Background: Anti-tumour necrosis factor alpha (TNF-alpha) antibodies are effective in Crohn's disease and perhaps ulcerative colitis but antigenicity and the high cost have raised interest in other strategies to block TNF-alpha. These include the TNF-alpha converting enzyme (TACE) which releases soluble TNF-alpha from transmembrane pro-TNF-alpha.

Aim: To investigate whether TACE activity is present in human colonic mucosa.

Materials and methods: Detergent extracts of cell membranes from colonic biopsies were obtained from 12 controls and 28 patients with inflammatory bowel disease. Enzyme activity was measured by hydrolysis assays using pro-TNF-alpha or oligopeptide substrates spanning the known pro-TNF-alpha cleavage site at Ala(76)-Val(77). Cleavage products were identified by western blotting, high pressure liquid chromatography, or mass spectrometry. TACE protein was localised by immunohistochemistry and identified by western blotting of detergent extracts from purified lamina propria mononuclear cells (LPMNC) or epithelial cells.

Results: Detergent extracts released TNF-alpha from pro-TNF-alpha and cleaved a model oligopeptide as predicted. Substrate hydrolysis was sensitive to known TACE/matrix metalloproteinase (MMP) inhibitors, but not trocade which has low activity against TACE. The median TACE level was increased in active ulcerative colitis (147 arbitrary units (AU)/mg; p<0.01) but not in Crohn's disease (81 AU/mg) compared with controls (79 AU/mg). Both the full length proform and the active form of TACE protein were expressed in LPMNC cells and epithelial cells.

Conclusions: Functional TACE activity is ubiquitously expressed in the human colon and increased in ulcerative colitis, raising interest in MMP inhibitors targeting TACE.

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Figures

Figure 1

Figure 1

Sodium dodecyl sulphate electrophoresis showing hydrolysis of glutathione-S-transferase-pro-tumour necrosis factor α (GST-pro-TNF-α) by recombinant TNF-α converting enzyme (TACE) or detergent extract of cell membranes from human colonic mucosa. Bands were visualised by western blotting using antibodies against GST (A) or TNF-α (B, C). Lane 1: recombinant TACE, t=0 minutes; lane 2: recombinant TACE, t=60 minutes; lane 3: recombinant TACE, t=60 minutes + inhibitor (EDTA 5 mM (A, B), CH4474 1 μg/ml (C)); lane 4: detergent extract, t=0 minutes; lane 5: detergent extract, t=60 minutes; lane 6: detergent extract, t=60 minutes + inhibitor (EDTA 5 mM (A, B), CH4474 1μg/ml (C)); lane 7: purified standards. Similar results were obtained in two further independent experiments.

Figure 2

Figure 2

Hydrolysis of an unlabelled oligopeptide substrate mimicking pro-tumour necrosis factor α (pro-TNF-α) cleavage by detergent extracts of cell membranes from human colonic mucosa. Substrate (S) hydrolysis was analysed by reverse phase high pressure liquid chromatography and the cleavage products (P1, P2) were identified by synthetic standards (A). (B) Magnification of product formation. (C, D) Inhibition by EDTA (5 mM) and CH4474 (1 μg/ml). Similar results were obtained in five further independent experiments.

Figure 3

Figure 3

Effect of protease inhibitors on tumour necrosis factor α (TNF-α) converting enzyme (TACE) activity in detergent extracts of cell membranes from human colonic mucosa, as measured by hydrolysis of a dinitrophenol labelled oligopeptide substrate mimicking the pro-TNF-α cleavage site. TACE activity was quantitated by high pressure liquid chromatography and expressed relative to protein concentration. Mean values of three individual experiments of material pooled from individual patients are shown. Samples contained dimethylsulphoxide (DMSO) 0.05% (*) when necessary for solubilisation of the inhibitors. Controls with (*) and without DMSO are shown. TLCK, _N_α-p-tosyl-l-lysine chloromethyl ketone.

Figure 4

Figure 4

Hydrolysis of a dinitrophenol (dnp) labelled peptide substrate mimicking the pro-tumour necrosis factor α (pro-TNF-α) cleavage site by detergent extracts of cell membranes from human colonic mucosa. Substrate (S) hydrolysis was analysed by reverse phase high pressure liquid chromatography and the dnp labelled cleavage product (P) was identified by synthetic standard (A). (B) Product formation was inhibited by CH4474 (1 μg/ml).

Figure 5

Figure 5

Sodium dodecyl sulphate (SDS) electrophoresis showing hydrolysis of glutathione-S-transferase-pro-tumour necrosis factor α (GST-pro-TNF-α) by recombinant TNF-α converting enzyme (TACE). Samples were analysed on continuous SDS polyacrylamide gels (20%) using silver staining. Lane 1: GST-pro-TNF-α substrate; lane 2: recombinant TACE, t=0 minutes; lane 3: recombinant TACE, t=60 minutes; lane 4: recombinant TACE, t=60 minutes + _N_α-p-tosyl-l-lysine chloromethyl ketone (TLCK 10 μg/ml). Similar results were obtained in two further independent experiments.

Figure 6

Figure 6

Immunohistochemistry of tumour necrosis factor α (TNF-α) converting enzyme (TACE) protein expression in normal human colonic mucosa. (A) (×20) and (C) (×80) show that TACE was widely expressed in lamina propria mononuclear cells and in crypt epithelial cells. (B) (×20) and (D) (×80) show the corresponding background staining when the primary antibody was substituted with unspecific goat immunoglobulins. Examples of cells regarded as positive for TACE are indicated by arrows (C). Similar results were obtained in two independent experiments.

Figure 7

Figure 7

Western blotting of tumour necrosis factor α (TNF-α) converting enzyme (TACE) protein immunoreactivity in detergent extract of membranes from intact biopsies (lane A), isolated epithelial cells (lane B), or purified lamina propria mononuclear cells (lane C). The arrows indicate the proform (p) and active (a) forms of TACE. Lanes D and E show the absence of immunoreactivity when an epitope mimicking blocking peptide was present during incubation. The TACE antibody was the same as that used in fig 6 ▶. Similar results were obtained in three independent experiments.

Figure 8

Figure 8

Tumour necrosis factor α (TNF-α) converting enzyme (TACE) activity in detergent extracts of cell membranes from colonic mucosa from patients with ulcerative colitis (UC) or Crohn's disease (CD) and healthy controls. TACE activity is expressed as specific activity (SA) in arbitrary units per mg of protein based on high pressure liquid chromatography analysis of hydrolysis of a dinitrophenol labelled oligopeptide assay substrate mimicking the pro-TNF-α cleavage. Horizontals lines indicate median values. **p<0.01.

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