Differential expression of matrix metalloproteinases and their tissue inhibitors in colon mucosa of patients with inflammatory bowel disease

Abstract

Background/aims: Alterations in synthesis and breakdown of extracellular matrix components are known to play a crucial role in tissue remodelling during inflammation and wound healing. Degradation of collagens is highly regulated by a cascade of matrix metalloproteinases (MMPs). The current study was therefore designed to determine gene expression patterns of MMPs and their tissue inhibitors (TIMPs) in single endoscopic biopsies of patients with inflammatory bowel disease (IBD).

Patients/methods: mRNA expression was measured by quantitative competitive polymerase chain reaction (PCR) in biopsies from patients with ulcerative colitis (n=21) and Crohn's disease (n=21). Protein expression was analysed by western blotting and immunohistochemistry.

Results: MMP-2, MMP-14, and TIMP-1 mRNAs were marginally increased in inflamed, but 9-12-fold increased in ulcerated colonic mucosa in IBD whereas TIMP-2 mRNA expression remained unchanged. MMP-1 and MMP-3 mRNA expression correlated well with the histological degree of acute inflammation, resulting in more than 15-fold increased MMP-1 and MMP-3 mRNA levels in inflamed versus normal colon samples from patients with ulcerative colitis and Crohn's disease.

Conclusion: Profound overexpression of MMP-1 and MMP-3 mRNA transcripts suggests an important role for these enzymes in the process of tissue remodelling and destruction in inflammatory bowel disease.

Figure 1

Evaluation of equal amplification efficiencies of the MMP-1 PCR product and the MMP-1 competitor construct. Purified MMP-1 PCR product (+; 247 bp) and the MMP-1 competitor construct (#; 222 bp) were mixed in an OD ratio of 1:1 (lane 1) and 1:5 (lane 6). These solutions were diluted 1:200 000 and used as templates for PCR for 27 cycles (lanes 2, 7) and 35 cycles (lanes 3, 8). An inverted image of an ethidium bromide stained gel is shown demonstrating equal density ratios before (lanes 1, 6) and after (lanes 2, 3, 7, 8) reamplification. Lanes 4 and 5 contain a DNA mass standard used for calibration of the densitometry. Numbers on the right indicate the DNA size in base pairs (bp).

Figure 2

Expression of MMP-1, -2, -3, -14, the tissue inhibitors TIMP-1 and TIMP-2, collagen type III, and GAPDH mRNA in colon biopsies from non-inflamed areas and inflamed areas of patients with Crohn's disease (CD) and ulcerative colitis (UC). Expression was measured by a competitive PCR technique and is expressed as the molar ratio of gene specific to β-actin copies. Scattergrams of all measurements in a logarithmic scale are shown. Bars represents the medians of the groups. Degree of statistical significance calculated by the Kruskal-Wallis test is given in each upper left corner. Statistically significant differences between the three groups (*p<0.05, **p<0.01, ***p<0.001; NS, not significant) were calculated using Dunn's post hoc test.

Figure 3

Increased protein expression of MMP-1, MMP-2, and MMP-3 in inflamed (i) versus non-inflamed (n) colon biopsies from patients with IBD. Endoscopic biopsies from macro- and microscopically normal (lanes 1, 3, 5) and inflamed (lanes 2, 4, 6) colon of one patient with Crohn's disease (lanes 1, 2) and two patients with ulcerative colitis (lanes 3-6) were lysed in a buffer containing a cocktail of protease inhibitors. Proteins were separated by polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were probed with antibodies against MMP-1 (A), MMP-2 (B), and MMP-3 (C) and bound antibody was detected by enhanced chemiluminescence. Membranes were reprobed with an antibody against β-actin (D) as a loading control. Estimated molecular weights of the bands detected are given on the right.

Figure 4

Molar ratios of matrix metalloproteinase to TIMP-1 specific cDNA derived from reverse transcribed mRNA isolated from colon biopsies of non-inflamed areas and inflamed areas of patients with Crohn's disease (CD) and ulcerative colitis (UC). Competitive PCR measurements are displayed as scattergrams in a logarithmic scale. Bars represent the medians of the groups. Statistical significance, calculated using the Kruskal-Wallis test, is given in each upper left corner. Statistically significant differences between the three groups (*p<0.05, **p<0.01, ***p<0.001; NS, not significant) were calculated using Dunn's post hoc test.

Figure 5

Relationship between histological degree of acute inflammation in colon biopsies from IBD patients and mRNA expression of MMPs, TIMP-1, and collagen type III measured by competitive PCR. Scattergrams show the ratio of gene specific transcripts to β-actin transcripts in a logarithmic scale and bars mark the medians of each group. The histological degree of acute inflammation was graded according to Truelove and Richards on a four point scale: no (0), mild (1), moderate (2), and severe (3) inflammation, as outlined in the methods section. Correlation between histological score and mRNA expression was computed using the Spearman rank test and the correlation coefficient r is given in the upper left corner. Statistical significant differences between probes with and without histological confirmed acute inflammation were calculated (*p<0.05, **p<0.01,***p<0.001).

Figure 6

Immunohistochemical staining of MMP-1 in inflamed and non-inflamed colon of a patient with ulcerative colitis. Section of paraffin embedded biopsies from non-inflamed (A, C) and inflamed (B, D-F) colon were stained with haematoxylin/eosin (A, B) and anti-MMP-1 polyclonal antibody using the APAAP technique (C, D). Double immunostaining with anti-MMP-1 (stained red) and anti-CD68 (stained brown) is shown in (E) and a magnified view is given in (F). Dark arrows indicate macrophages expressing CD68 and MMP-1; open arrows indicate extracellular MMP-1 staining along the basal membrane of a blood vessel.