SYNTHETIC MATRIX METALLOPROTEINASE INHIBITORS AND TISSUE INHIBITOR OFMETALLOPROTEINASE (TIMP)-2, BUT NOT TIMP-1, INHIBIT SHEDDING OF TUMOR-NECROSIS-FACTOR-ALPHA RECEPTORS IN A HUMAN COLON ADENOCARCINOMA (COLO205) CELL-LINE

The solubilization of plasma membrane receptors through proteolytic cl eavage of the ligand binding domain at the cell surface is an importan t mechanism for regulating cytokine function and receptor signaling. T he inhibition of the shedding of a variety of receptors by synthetic i nhibitors of the matrix metalloproteinases (MMPs) implicates metallopr oteinases in this regulatory event. We examined the effects of two nat urally occurring tissue inhibitors of metalloproteinases, TIMP-1 and T IMP-2, and several synthetic MMP inhibitors (MMPIs) on the shedding of both tumor necrosis factor alpha receptor type I(TNF alpha-RI; M-r 55 ,000) and TNF alpha-RII (M-r 75,000) by the Cole 205 human colon adeno carcinoma cell line. Culture of Cole 205 cells for 48 h resulted in th e shedding of both TNF alpha-RI and TNF alpha-RII, as determined by EL ISA. The shedding of TNF alpha receptors was not affected by TIMP-1 or protease inhibitors aprotinin, pepstatin, or leupeptin but was inhibi ted in a dose-dependent manner by the following synthetic MMPIs: batim astat and marimastat (BB-94 and BB-2516, respectively, British Biotech , Inc.); CT1418 (Celltech Therapeutics); CGS27023A (Novartis Pharmaceu ticals); and RO31-9790 (Roche), with IC(50)s ranging from 3.2 to 38.0 mu M. Similarly, TIMP-2 from two different sources reproducibly inhibi ted the shedding of both TNF alpha-RI and TNF alpha-RII in a dose-depe ndent manner (IC50 = 286 +/- 33 nM for TNF alpha-RI shedding and 462 /- 52 nM for shedding of TNF alpha-RII). The inhibition of TNF alpha-R I shedding was confirmed in the SW626 human ovarian adenocarcinoma cel l line, The synthetic MMPIs and TIMP-2, but not TIMP-1, also caused a dose-dependent increase in the number of TNF alpha receptors retained on the surface of Cole 205 cells, as determined by flow cytometry. Inh ibition of TNF alpha receptor shedding with TIMP-2 occurs at molar con centrations 10-100 times less than those required with low molecular w eight, synthetic MMPIs but at concentrations greater than those requir ed to inhibit collagen degradation. Modulation of TNF alpha receptor s hedding by TIMP-2 could have important implications for the pleiotropi c effects of TNF alpha in both normal and malignant cells and for the pharmacological activity of synthetic MMPIs.