Marta L. Corcoran

MMP-2: expression, activation and inhibition.

Remodeling of the extracellular matrix (ECM), which occurs during many physiological and pathological processes, is one of the requisite events of cellular invasion. The matrix metalloproteinases (MMPs) are a family of zinc-dependent proteases that are responsible for proteolytic degradation of specific ECM components. Regulating the activity of the MMPs at both mRNA and/or protein levels modulates the degradation of the ECM components which in turn alter cellular invasion. Although most MMPs are regulated via similar mechanisms at the mRNA and protein levels, the modulation of gelatinase A is unique. Understanding the mechanisms that regulate gelatinase A is important since expression and activation of this particular MMP is consistently correlated with a majority of malignant phenotypes. In this report, we will contrast the mechanisms that regulate the expression, activation and inhibition of gelatinase A with the mechanisms that modulate the rest the MMP family.

Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli COMPARISON OF WILD TYPE AND AMINO-TERMINAL ALANINE APPENDED VARIANT WITH IMPLICATIONS FOR THE MECHANISM OF TIMP FUNCTIONS

Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala+TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala+TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala+TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.

Cell surface binding of TIMP-2 and pro-MMP-2/TIMP-2 complex

Tissue inhibitor of metalloproteinases (TIMP‐2) is a low molecular weight proteinase inhibitor capable of inhibiting activated matrix metalloproteinases (MMPs). TIMP‐2 is found both free and in a 1:1 stoichiometric complex with the pro‐enzyme form of MMP‐2 (pro‐MMP‐2/TIMP‐2 complex). We have measured the binding of recombinant TIMP‐2 to intact HT‐1080 and MCF‐7 cells. HT‐1080 cells in suspension bound 125I‐labeled rTIMP‐2 with a K d of 2.5 nM and 30,000 sites/cell. Monolayers of MCF‐7 cells were similarly found to bind [125I]rTIMP‐2 with a K d of 1.6 nM and 25,000 sites/cell. Specific binding of MMP‐2 alone to HT‐1080 cells was not observed; however, pro‐MMP‐2/TIMP‐2 complex was capable of binding to the surface of HT‐1080 cells in a TIMP‐2‐dependent manner. Binding of rTIMP‐2 was not competed by the presence of TIMP‐1. These results suggest that rTIMP‐2 alone binds directly to the cell surface of HT‐1080 and MCF‐7 cell lines, and TIMP‐2 is capable of localizing MMP‐2 to the surface of HT‐1080 cells via interaction with a specific binding site.

Increased expression of tissue inhibitor of metalloproteinases type 1 (TIMP‐1) in a more tumourigenic colon cancer cell line

Genetic changes occurring in the late stages of colonic tumour progression have received much less attention than those occurring in the early stages. As described in the accompanying paper, SW480 and SW620 cell lines provide a useful model for studying the advanced stages of progression for colon cancer. Comparison of the two cell lines by differential display reveals that SW620 cells express lower levels of the CC3 tumour suppressor gene and also lower levels of the tissue inhibitor of metalloproteinases‐3 (TIMP‐3) gene. Northern blot analysis for TIMP‐3 confirms this finding and shows a similar difference in the expression of TIMP‐2, which seems logical since TIMPs inhibit enzymes that play a role in tumour invasion. For this reason, it was surprising to find that TIMP‐1 messenger RNA expression is markedly increased in SW620 cells. Consistent with this finding, western blot analysis shows a ten‐fold increase in TIMP‐1 protein secretion by SW620 cells. It is noteworthy that high TIMP‐1 expression is associated with poor prognosis in colorectal cancer. This association between TIMP‐1 expression and tumour progression may be related to additional growth factor‐like effects described for TIMP‐1 in some systems. Copyright

Regulation of Matrix Metalloproteinases during Extracellular Matrix Turnover

Extracellular matrix (ECM) turnover is a critical step in the tissue remodeling that accompanies many physiologic as well as pathologic processes. Normal remodeling events, such as wound healing and trophoblast implantation, require coordinated synthesis and removal of ECM components. These physiologic processes are strictly regulated in a spatial and temporal fashion, and are usually limited in extent. They result in a functionally intact matrix and preservation of tissue boundaries. Pathologic events may result in the extensive and uncontrolled ECM destruction that is often seen in the inflammatory collagen vascular diseases. Such processes cause the loss of normal matrix boundaries and matrix functions. Alternately, inactivation of the matrix degradative machinery may result in excessive ECM accumulation that disrupts normal tissue structure and function. Proteases responsible for matrix turnover have been identified in all four classes of proteases (seryl-, cystyl-, aspartyl- and metalloproteinases). However, the matrix metalloproteinase (MMP) family of enzymes appears to be primarily responsible for much of the coordinate or discoordinate degradation of the ECM that occurs during physiological and pathological events, respectively. Comprehending the precise points of regulation for MMP activity may facilitate therapeutic intervention in pathologic situations that characteristically favor either unregulated ECM destruction or excessive deposition of matrix components.

TIMP-2 Mediates Cell Surface Binding of MMP-2

In order to understand the mechanism for neoplastic cell invasion, we utilized binding studies of TIMP-2, gelatinase A and the TIMP-2/gelatinase A complex to neoplastic cells and correlated these results with their capacity to invade a matrix substrate in a modified Boyden chamber assay. Binding studies were performed on malignant human breast cancer cells and fibrosarcoma cells with rTIMP-2, rGelatinase A, and TIMP-2/gelatinase A complex. Competition studies of the binding characteristics of these proteins indicated that gelatinase A and gelatinase A/TIMP-2 complex bound to the surface of cells via TIMP-2. Furthermore, the localization of either latent or active protease to the surface of MDA-MB-435 breast cancer cells facilitated the invasion of these neoplastic cells through a matrigel barrier. This suggests that in addition to binding this complex, these cells can activate this pro-enzyme-inhibitor complex and use this activity to facilitate cellular invasion. Moreover, their enhanced invasion was suppressed by exogenous additions of rTIMP-2. A working hypothesis and model for the role of gelatinase A/TIMP-2 complex in cellular invasion is presented.

Reversal of immune dysfunction in osteopetrotic rats by interferon-γ: Augmentation of macrophage Ia expression and lymphocyte interleukin-2 production and proliferation

Lymphocytes from osteopetrotic (op) rats, compared to their normal (n) littermates, exhibit defective immune functions associated with their inability to resorb bone. Among these immune defects are the failure of their spleen cells to proliferate normally to mitogens and to generate IL-2. Addition of exogenous IL-2 failed to reverse the suppressed proliferation in the op spleen cells, indicating that additional defects were involved in the suppression. Phenotypic analysis of cellular constituents of op and n spleens revealed that the percentages of T cells, macrophages, and IL-2 receptor positive cells were not different. Furthermore, there was no difference in CD4 (W3/25) and CD8 (OX8) cells. However, the Ia+ (OX3) cells in the op spleen represented less than 50% of those found in the n spleen, but the op had higher levels of transferrin receptor (OX26). On the basis of the ability of interferon-gamma (IFN-gamma) to increase Ia expression, this cytokine was added to op spleen cells (10-50 U/ml) and found to increase the number of Ia+ cells to the level found in n spleen cells. Moreover, pretreatment of op spleen cells with IFN-gamma restored their ability to proliferate to mitogens and their responsiveness to IL-2. Not only did IFN-gamma reverse the defective response to IL-2, but it also augmented the defective IL-2 production by op spleen cells. Taken together, these findings demonstrate that IFN-gamma can reverse many of the impaired immune functions characteristic of op spleen cells in vitro. Furthermore, these data suggest that IFN-gamma may provide an important avenue of treatment in these animals that may contribute to restoration of normal bone resorption.