Tumor-necrosis factor (TNF), a pleiotropic cytokine, triggers physiological and pathological responses in several organs. Here we show that deletion of the mouse gene Timp3 resulted in an increase in TNF-alpha converting enzyme activity, constitutive release of TNF and activation of TNF signaling in the liver. The increase in TNF in Timp3(-/-) mice culminated in hepatic lymphocyte infiltration and necrosis, features that are also seen in chronic active hepatitis in humans. This pathology was prevented when deletion of Timp3 was combined with Tnfrsf1a deficiency. In a liver regeneration model that requires TNF signaling, Timp3(-/-) mice succumbed to liver failure. Hepatocytes from Timp3(-/-) mice completed the cell cycle but then underwent cell death owing to sustained activation of TNF. This hepatocyte cell death was completely rescued by a neutralizing antibody to TNF. Dysregulation of TNF occurred specifically in Timp3(-/-), and not Timp1(-/-) mice. These data indicate that TIMP3 is a crucial innate negative regulator of TNF in both tissue homeostasis and tissue response to injury.

Membrane-type matrix metalloproteinases (MT-MMPsTIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.Matrix metalloproteinases (MMPs) 2 are fundamental to biological processes because of their ability to cleave and remodel the extracellular matrix (ECM). MT1-MMP is one of six cell surface membrane-type MMPs (MT-MMP). Its activity and regulation have been widely studied in the context of cell surface MMP activation, cell migration, and invasion (1-3), independently and in conjunction with other cell adhesion molecules (4 -10). A key function of MT1-MMP is to process pro-MMP-2, whose activity also correlates with an invasive propensity in several cancers and is predictive of poor survival (reviewed in . Understanding the regulation of pro-MMP-2 processing by MT1-MMP and other members of the MT-MMP family is central to defining their role in cancer biology.The classical model for the cell surface activation of MMP-2 is through the formation of a trimolecular complex comprising MT1-MMP, TIMP-2, and pro-MMP-2 (15). The transmembrane MT1-MMP interacts via its N-terminal domain to the N terminus of TIMP-2, forming a "receptor" onto which pro-MMP-2 (72 kDa) binds. Pro-MMP-2 is initially cleaved to its intermediate form (64 kDa) by an adjacent active MT1-MMP. The second stage of MMP-2 processing, resulting in its fully active form (62 kDa), involves an autocatalytic event that requires an MMP-2 molecule in trans (16). It is known that of the six MT-MMPs, MT2-, 3-, 5-, and 6-MMP (17-21) also have the capacity to activate pro-MMP-2. Alternative mechanisms of MMP-2 processing at the plasma membrane, such as the urokinase plasminogen system (22, 23) or TIMP-independent processing involving MT2-MMP, have also been suggested (24). Although the dynamics of the trimolecular complex have been well studied, new insights into its regulation and control are continually being discovered. More recently, cell surface processing of pro-MMP-2 is reported to occur through formation of a trimolecular complex comprised of MT3-MMP, TIMP-3, and pro-MMP-2 (25), although TIMP-3 is known not to form similar complexes with MT1-MMP (26).TIMPs are the naturally occurring inhibitors of metalloproteinase activity. There are four members of the TIMP family, and each has a specific niche with respect to function. Studies have focused on the dual role of TIMP-2 in regulating the processing of pro-MMP-2. A threshold level of TIMP-2 is required in relation to MT1-MMP to construct the trimolecular complex, which still leaves sufficient MT1-MMP uninhibited to cleave pro-MMP-2. At higher concentrations, TIMP-2 prevents MMP-2 processing by inhibiting all free MT1-MMP (27-29). The presence of TIMP-2 was initially considered necessary to achieve any form of MMP-2 processing (30, 31), but recently Timp-2 Ϫ/Ϫ cells were shown capable of some pro...

To test if proteolysis is involved in tumor cell extravasation, we developed an in vitro model where tumor cells cross an endothelial monolayer cultured on a basement membrane. Using this model we classified the ability of the cells to transmigrate through the endothelial cell barrier onto the underlying matrix, and scored this invasion according to the stage of passage through the endothelium. Metalloproteinase inhibitors reduced tumor cell extravasation by at least 35%. Visualization of protease and cell adhesion molecules by confocal microscopy demonstrated the cell surface localization of MMP-2, MMP-9, MT1-MMP, furin, CD44 and αvβ3, during the process of transendothelial migration. By the addition of inhibitors and bio-modulators we assessed the functional requirement of the aforementioned molecules for efficient migration. Proteolytic digestion occurred at the cell-matrix interface and was most evident during the migratory stage. All of the inhibitors and biomodulators affected the transition of the tumor cells into the migratory stage, highlighting the most prevalent use of proteolysis at this particular step of tumor cell extravasation. These data suggest that a proteolytic interface operates at the tumor cell surface within the tumor-endothelial cell microenvironment.