BACKGROUND Expression and enzymatic activity of gelatinases were examined in biopsy specimens from patients with colon and rectal neoplasms. The objective of this study was to determine whether the activity of these enzymes is altered between tumor areas compared with areas of noninvolved, normal mucosa and between colon and rectal carcinoma. METHODS Matrix metalloproteinase (MMP) production was analyzed by Western immunoblot analysis and gelatin zymography. mRNA was determined by quantitative, real‐time polymerase chain reaction analysis. RESULTS Patients with colon carcinoma (n = 20 patients) showed a significant increase in levels of MMP‐9 (92 kDa and 88 kDa) and MMP‐2 (72 kDa and 62 kDa) in tumor areas compared with noninvolved regions. In contrast, patients with rectal carcinoma (n = 10 patients) had revealed the same high activity of MMP‐9 in tumor regions and corresponding healthy tissue. Confirming activity measurements, in colon tumors, but not in rectal tumors, there was significant up‐regulation of MMP‐9 transcription compared with healthy tissue in the same patients. There were no significant changes in the tissue inhibitor of metalloproteinase‐1 protein when colon and rectal tumor tissues were compared with the corresponding noninvolved regions. Cell culture experiments revealed fibroblasts as the cellular origin of MMPs. The findings showed that the secretion and activation of gelatinases depend on soluble factors secreted by tumor cells and are influenced by extracellular matrix components. CONCLUSIONS This is the first report showing differences in MMP‐9 activity between rectal carcinoma and colon carcinoma. Previous results indicating an active involvement of stromal cells in the generation of MMPs during tumor invasion are extended. Because the abundance of gelatinases increases in colorectal carcinoma, inhibitors of these proteases may be of therapeutic value. Cancer 2001;92:2680–91. © 2001 American Cancer Society.

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Unbalanced expression of tissue inhibitors of metalloproteinases (TIMP) such as TIMP‐1 relative to matrix metalloproteinases (MMPs) promotes progression of fibrosis in liver, lung, and kidney. As to therapeutic strategies, it would be advantageous to antagonize TIMP‐1. MMP‐9 binds TIMP‐1 with high affinity (Ki < 50 pM). Three histidine residues constitute the active site of the enzyme by binding the catalytic zinc. MMP‐9 mutants were generated in which the histidine residues at position 401, 405 or 411, respectively, were replaced by alanine. All mutants were enzymatically inactive as demonstrated by gelatin zymography. In immuno‐precipitation experiments, the MMP‐9 mutant H401A bound 2‐fold more efficiently to TIMP‐1 than H405A, whereas H411A did not bind at all. The mutant H401A was approximately 3‐fold less active in TIMP‐1 binding than wild‐type MMP‐9. Recently, we established cell lines secreting TIMP‐1 constitutively (HepG2‐TIMP‐1 cells). In this cell line, increased expression of TIMP‐1 resulted in suppressed migration and enhanced cell‐cell contact. In co‐cultures of HepG2‐TIMP‐1 cells with HepG2 cells overexpressing the H401A mutant of MMP‐9, the TIMP‐1‐associated phenotype was completely neutralized. These results suggest that a catalytically inactive metalloproteinase (e.g., MMP‐9‐H401A) that still binds TIMP‐1 can be used as a specific antagonist of TIMP‐1 activity in vivo.

The influence of transforming growth factor-beta (TGF-beta) on the expression of different forms of small proteoglycans was investigated in human skin fibroblasts and in a human osteosarcoma cell line. TGF-beta was not found to act as a general stimulator of small proteoglycan biosynthesis. In both cell types, an increased expression of the core protein of proteoglycan I was found. However, there was a profound decrease in the expression of a 106 kDa core protein, and either no alteration or a small decrease in the biosynthesis of the collagen-binding small proteoglycan II core protein. These results show that the production of individual members of the small proteoglycan family is differentially regulated.