Preparation and Characterization of Recombinant Tissue Inhibitor of Metalloproteinase 4 (TIMP-4)

Abstract

MMPs 1 and their inhibitors TIMPs play a critical role in ECM homeostasis. Controlled remodeling of the ECM is an essential aspect of normal development, and deregulated remodeling has been indicated to have a role in the etiology of diseases such as arthritis, periodontal disease, and cancer metastasis (1-5). Four mammalian TIMPs have been identified so far: TIMP-1 (6), TIMP-2 (7), TIMP-3 (8 -11), and the recently cloned TIMP-4 (12, 41). The proteins are classified based on structural similarity to each other, as well as their ability to inhibit metalloproteinases.TIMPs are secreted multifunctional proteins that have anti-MMP activity as well as erythroid-potentiating and cell growth-promoting activities. The stimulating effect on cell growth was initially recognized when TIMP-1 and TIMP-2 were identified as having erythroid-potentiating activities (14,15). It is now clear through several recent reports that TIMP-1 and TIMP-2 are mitogenic for non-erythroid cells, including normal keratinocytes (16), fibroblasts (17), lung adenocarcinoma cells (18), and melanoma cells (18). The involvement of TIMPs in the activation of pro-MMP has also been demonstrated (19). In addition, the recent evidence indicates that the TIMP family may be involved in steroidogenesis of rat testis and ovary indicating the potential role of TIMP in the reproduction (20).The most widely appreciated biological function of the TIMPs is their role in the inhibition of cell invasions in vitro (21-24) and tumorigenesis (25-29) and metastasis in vivo (25-31). Since the net MMP activity is the result of the balance between activated enzyme levels and TIMP levels, an increase in the amount of TIMPs relative to MMPs could function to block tumor cell invasion and metastasis. The tumor-suppressing activity of TIMP on primary tumor growth may be in part due to its anti-angiogenic activity. In fact, both TIMP-1 (32) and TIMP-2 (33, 34) have been demonstrated to have an antiangiogenic activity, and such inhibition of angiogenesis is mediated by inhibition of both endothelial cell proliferation (34) and migration (32). The underlying molecular mechanism for the tumor suppressing activities of TIMPs, nevertheless, is thought to depend on their anti-MMP activities.We had recently cloned and characterized a human TIMP-4 (12). Transfection of TIMP-4 into human breast cancer cells inhibited the invasion potential of the cells in the in vitro invasion assay (13). When injected orthotopically into nude mice, TIMP-4 transfectants were significantly inhibited in their tumor growth and axillary lymph node and lung metastasis as compared with controls (13). These results suggest the therapeutic potential of TIMP-4 in treating cancer malignant progression. These results suggest an important role of TIMP-4 in inhibiting primary tumor growth and progression leading to invasion and metastasis. In the present study, we have produced and purified rTIMP4p from baculovirus infected cells. rTIMP4p was shown to inhibit MMP activity and tumor cell invasion across reconstitute...

Coordinated peak expression of MMP-26 and TIMP-4 in preinvasive human prostate tumor

“…The therapeutic potential of TIMP-4 in the treatment of malignant progression has been examined, and the transfection of TIMP-4 cDNA into human MDA-MB-435 breast cancer cells inhibited tumor cell invasion across Matrigel, a barrier of reconstituted basement membrane components [20]. Incubation of these same cells with recombinant TIMP-4 protein displayed a similar inhibitory effect [21].…”

Inhibition of Wilms' tumor growth by intramuscular administration of tissue inhibitor of metalloproteinases-4 plasmid DNA

“…Unlike other TIMPs, tissue speci®c expression of TIMP-4 is limited to heart, pancreas, and kidney (Bigg et al, 1997;Greene et al, 1996;Liu et al, 1997). In this study we examined the expression patterns of TIMP-4 and MMP-2 in normal kidney vs Wilms' tumor and the eect of TIMP-4 on the growth of G401 cells.…”

Secreted and Membrane-Associated Matrix Metalloproteinases of IL-2-Activated NK Cells and Their Inhibitors

“…TIMP-2 from different species, i.e., mouse, rat, and bovine, have 97%, 98%, and 91% homology to human TIMP-2, respectively (11). TIMP-3 and TIMP-4 proteins have apparent molecular masses of 24 and 23 kDa, respectively (12,13). The balance between the production and activation of latent enzymes, and inhibition of active enzymes seems to play a critical role determining the invasive potential of many solid tumors and inflammation caused by tissueinfiltrating immune effector cells (3).…”