Synthetic Matrix Metalloproteinase Inhibitors and Tissue Inhibitor of Metalloproteinase (TIMP)-2, but not TIMP-1, Inhibit Shedding of Tumor Necrosis Factor-α Receptors in a Human Colon Adenocarcinoma (Colo 205) Cell Line

Abstract

The solubilization of plasma membrane receptors through proteolytic cleavage of the ligand binding domain at the cell surface is an important mechanism for regulating cytokine function and receptor signaling. The inhibition of the shedding of a variety of receptors by synthetic inhibitors of the matrix metalloproteinases (MMPs) implicates metalloproteinases in this regulatory event. We examined the effects of two naturally occurring tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, and several synthetic MMP inhibitors (MMPIs) on the shedding of both tumor necrosis factor α receptor type I (TNFα-RI; M_r 55,000) and TNFα-RII (M_r 75,000) by the Colo 205 human colon adenocarcinoma cell line. Culture of Colo 205 cells for 48 h resulted in the shedding of both TNFα-RI and TNFα-RII, as determined by ELISA. The shedding of TNFα receptors was not affected by TIMP-1 or protease inhibitors aprotinin, pepstatin, or leupeptin but was inhibited in a dose-dependent manner by the following synthetic MMPIs: batimastat and marimastat (BB-94 and BB-2516, respectively, British Biotech, Inc.); CT1418 (Celltech Therapeutics); CGS27023A (Novartis Pharmaceuticals); and RO31-9790 (Roche), with IC₅₀s ranging from 3.2 to 38.0 µm. Similarly, TIMP-2 from two different sources reproducibly inhibited the shedding of both TNFα-RI and TNFα-RII in a dose-dependent manner (IC₅₀ = 286 ± 33 nm for TNFα-RI shedding and 462 ± 52 nm for shedding of TNFα-RII). The inhibition of TNFα-RI shedding was confirmed in the SW626 human ovarian adenocarcinoma cell line. The synthetic MMPIs and TIMP-2, but not TIMP-1, also caused a dose-dependent increase in the number of TNFα receptors retained on the surface of Colo 205 cells, as determined by flow cytometry. Inhibition of TNFα receptor shedding with TIMP-2 occurs at molar concentrations 10–100 times less than those required with low molecular weight, synthetic MMPIs but at concentrations greater than those required to inhibit collagen degradation. Modulation of TNFα receptor shedding by TIMP-2 could have important implications for the pleiotropic effects of TNFα in both normal and malignant cells and for the pharmacological activity of synthetic MMPIs.