2.4. Matrix metalloproteinases (MMPs)

Matrix metalloproteinases form a continuously growing family of zinc-dependent endopeptidases. MMPs are classified into subfamilies based on their substrate preferences. Those include gelatinases, collagenases, stromelysins, elastases, MT-MMPs (membrane type-MMPs) and a group of unnamed members. The members and substrates of MMPs are shown in Table 1.

All MMPs are synthesized in the latent form. They are secreted as proenzymes and require extracellular activation. They can be activated in vitro by many mechanisms including organomercurials, chaotropic agents and other proteases (Woessner 1991, Murphy et al. 1992, Birkedal-Hansen 1995). MMPs need Zn2+ to be active. MMP activity is regulated at many levels. The messenger RNA (mRNA) is transcriptionally regulated by biologically active agents such as hormones, oncogenes, growth factor and tumor ­promoters (Overall et al. 1991, Mauviel 1993, Vincenti et al. 1994, Birkedal-Hansen 1995, Chambers & Matrisian 1997). The activation processes consist of three different mechanisms: stepwise activation, activation on the cell surface and intracellular activation. In addition, two progelatinases (proMMP-2 and proMMP-9) can bind to endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs) (Nagase 1997).

These enzymes are synthesized and secreted in low amounts by normal cells associated with physiologic tissue remodeling such as wound healing, implantation, trophoblastic invasion and angiogenesis (Puistola et al. 1989, Liotta et al. 1991, Turpeenniemi-Hujanen et al. 1992, Salo et al. 1994, Ågren et al. 1994, Birkedal-Hansen 1995, Chambers & Matrisian 1997). Increased expression of MMPs is found in various human tumors and cell lines (Liotta et al. 1980, Birkedal-Hansen 1995, MacDougall and Matrisian 1995, Chambers & Matrisian 1997). MMPs are often overexpressed in malignant tumors (Stetler-Stevenson et al. 1993, Mueller 1996).

MMP activity is regulated at various levels, including regulation of the transcription at the mRNA level, activation from the latent form and by inhibition by the inhibitors of matrix metalloproteinases (TIMPs) (Mauviel 1993, Chambers & Matrisian 1997, Pendas et al.1997).

MMP-2 (Gelatinase A, 72 kDa type IV collagenase) is the most widely distributed enzyme of the MMP family and it was originally described and purified as a basement membrane collagen degrading enzyme activity from a metastatic murine tumor (Liotta et al. 1979, Salo et al. 1983). MMP-2 is expressed e.g. by fibroblasts, keratinocytes, epithelial cells, monocytes and osteoblasts (Birkedal-Hansen 1995).

Gelatinases A and B (72 kD /MMP-2 and 92 kD /MMP-9), which also degrade type IV collagen, are members of this proteinase family, and the secretion of type IV collagen degrading activity has been found to be correlated with invasiveness and metastatic capacity in cultured tumor cells (Liotta et al. 1980, Turpeenniemi- Hujanen et al. 1985, Nakajima et al. 1987).

MMP-2 expression is linked to invasiveness in several human neoplasias including breast, colon, ovarian, lung, prostate, urothelial, hepatocellular carcinomas and melanoma. The MMP-2 protein has been localized immunohistochemically to tumor cells, suggesting an interesting role in determining the invasive potential and metastatic capacity of the neoplastic lesions (Liotta et al. 1980, Monteagudo et al. 1990, D"Errico et al. 1991, Campo et al. 1992, Naruo et al. 1993, Montironi et al. 1996, Kodate et al. 1997, Väisänen et al. 1998). mRNA for MMP-2 has been found in stromal cells, the reason for which is not clearly understood, but it seems to be a host response to the presence of invasive malignant cells (Pyke et al. 1992,1993, Autio-Harmainen et al. 1992, 1993, Poulsom et al. 1993).

MMP-2 activation differs from the other MMPs because it is differently regulated at the transcriptional level. It has been demonstrated that MMP-2 activation can take place in the cell surface of fibroblasts (Ward et al. 1994).

Membrane-type MMP-1 (MT-MMP-1, MMP-14) activates 72 kDa progelatinase on the cell surface (Will et al. 1996) and coexpression of the two MMPs is detected in various carcinomas (Nomura et al.1995, Okada et al. 1995, Polette et al. 1996). MT-MMP-1 has also intrinsic proteolytic activity against e.g. gelatin, elastin laminin, fibronectin or types I, II and III collagen (Pei & Weiss 1996, Ohuchi et al. 1997).

MMPs can be regulated by the tissue inhibitor of metalloproteinases (TIMP). TIMPs play a major role in the regulation of MMP activity. The TIMP family members include TIMP-1, -2, -3 and -4 (Silbiger et al. 1994, Greene et al. 1996). TIMPs form binary complexes with active MMPs inhibiting the activity of the enzyme, or they appear in a complex of TIMP and progelatinase regulating the activation of the latent enzyme (Howard et al. 1991a, Goldberg et al. 1992). TIMP-2 is most effective in inhibiting MMP-2 activity (Howard et al. 1991b, Birkedal-Hansen et al. 1993, Corcoran et al. 1996, Imai et al. 1996, Will et al. 1996). By establishing a trimolecular complex, consisting of MT1-MMP/TIMP-2/ progelatinase A, the components are concentrated on the cell surface. This complex acts as a concentration mechanism that is crucial for the efficiency of activation (Atkinson et al. 1995, Cao et al. 1995, Sato et al. 1996). The role of TIMPs in suppressing tumor growth has been demonstrated in malignant tumors overexpressing one of the TIMPs (Khokha 1994, Bian et al. 1996, Imren et al. 1996). TIMPs have also biological activities not related to MMP inhibition: TIMP-1 and -2 are antiangiogenic and possess growth factor-like activity toward a number of cell types. TIMP-2 inhibits microvascular endothelial cell proliferation associated with angiogenesis (Murphy et al. 1993). The balance between the levels of activated MMPs and the free form of TIMPs determines the net activity of MMPs.