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Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.

Membrane type 1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP-2 is thought to be important in the proteolysis of extracellular matrix in pathological events in which monocytes/macrophages are found. Here we report on the induction and regulation of human monocyte MT1-MMP and its role in MMP-2 activation. Activation of monocytes by lipopolysaccharide resulted in the induction of MT1-MMP mRNA and protein that was suppressed by inhibitors of prostaglandin synthesis (indomethacin), adenylyl cyclase (SQ 22536), and protein kinase A (Rp-cAMPs). Suppression of MT1-MMP by indomethacin and SQ 22536 was reversed by prostaglandin E(2) and dibutyryl cyclic AMP, respectively, demonstrating that induction of monocyte MT1-MMP is regulated through a prostaglandin-cAMP pathway. Functional analysis revealed that pro-MMP-2 in the supernatants from human bone marrow stromal fibroblasts, normal male-derived fibroblasts and melanoma cells (A2058) was converted to active MMP-2 when cultured with activated but not control monocytes. Antibodies against MT1-MMP blocked the activation of MMP-2. Tissue inhibitor of metalloproteinase-2 regulation of MMP-2 activation was shown through the addition of varying amounts of recombinant tissue inhibitor of metalloproteinase-2 with pro-MMP-2 to MT1-MMP-expressing monocytes. These findings demonstrate that activated monocytes express functionally active MT1-MMP that may play a significant role in the activation of MMP-2 produced by other cells and as such influence developmental and pathological conditions.

The extracellular matrix plays an important role in breast remodeling. We have shown that matrix metalloprotease-2 (MMP2) cleaves laminin-5 (Ln-5), a basement membrane component, generating a fragment called gamma2x. Human breast epithelial cells, while constitutively immobile on intact Ln-5, acquire a motile phenotype on MMP2-cleaved Ln-5. We hypothesize that this mechanism may underlie cell mobilization across the basement membrane during branching morphogenesis in breast development regulated by sex steroids. We report that the expression of MMP2 and cleavage of Ln-5 correlate well with tissue remodeling and epithelial rearrangement of the breast both in vivo and in vitro. Thus, the Ln-5 gamma2x fragment was detected by immunoblotting in sexually mature, pregnant, and postweaning, but not in prepubertal or lactating mammary glands. Furthermore, cleaved Ln-5, as well as MMP2, became detectable in remodeling glands from sexually immature rats treated with sex steroids. In rat mammary gland explants, epithelial reorganization and luminal cell morphological changes were induced by the addition of exogenous MMP2, in parallel to the appearance of cleaved Ln-5. Similar effects were observed in epithelial monolayers plated on human Ln-5 and exposed to MMP2. These results suggest that cleavage of Ln-5 by MMP2 might be regulated by sex steroids and that it may contribute to breast remodeling under physiological and possibly pathological conditions.

The fibrous tissue of the articular disc of the dysfunctional temporomandibular joint undergoes deep and variable structural modifications. Here the concurrence of morphological changes and the expression of matrix metalloproteinase-2 (MMP-2) in damaged discs from individuals suffering joint dysfunction was investigated. Microscopic, ultrastructural and immunocytochemical investigations were made on variously damaged articular discs and on one control sample. Disaggregation of collagen fibres, an increase in cellular components and calcification of large areas of tissue were observed in the damaged discs. These modifications were accompanied by a positive immunoreaction pattern for MMP-2. Fibroblast-, chondroblast- and osteoblast-like cells displayed a positive cytoplasmic reaction. In samples displaying evidence of synovial hyperplasia, some cells of the synovial protrusions were MMP-2 immunoreactive. No MMP-2 staining was observed in the control sample. These findings demonstrate that structural modifications of the articular disc could be specific responses to changes in the function of the temporomandibular joint. Variations in extrinsic stimuli may activate intrinsic factors, such as MMPs, that induce structural modifications in the discal tissue.

Metastatic disease is responsible for the majority of cancer-related deaths, either directly due to tumor involvement of critical organs or indirectly due to complications of therapy to control tumor growth and spread. An understanding of the mechanisms of tumor cell invasion and metastasis may be important for devising therapies aimed at preventing tumor cell spread. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endoproteinases whose enzymatic activity is directed against components of the extracellular matrix (ECM). In humans, 16 members of this family have been identified by cloning and sequencing. These proteinases are linked by a core of common domain structures and by their relationship to a family of proteinase inhibitors called the tissue inhibitors of metalloproteinases (TIMPs). Four members of the TIMP family have been cloned and sequenced in humans and they inhibit MMPs by forming tight-binding, noncovalent associations with the active site of the MMPs. MMPs facilitate tumor cell invasion and metastasis by at least three distinct mechanisms. First, proteinase action removes physical barriers to invasion through degradation of ECM macromolecules such as collagens, laminins, and proteoglycans. This has been demonstrated in vitro through the use of chemoinvasion assays and in vivo by the presence of active MMPs at the invasive front of tumors. Second, MMPs have the ability to modulate cell adhesion. For cells to move through the ECM, they must be able to form new cell-matrix and cell-cell attachments and break existing ones. Using a cell transfection system that altered the ratio of MMP-2 to TIMP-2 we have demonstrated significant variation in the adhesive phenotype of tumor cells. Finally, MMPs may act on ECM components or other proteins to uncover hidden biologic activities. For example, the angiogenesis inhibitor angiostatin may be produced from plasminogen by MMP action and laminin-5 is specifically degraded by MMP-2 to produce a soluble chemotactic fragment. Thus MMPs play multiple key roles in facilitating the metastasis of tumor cells. Therapies designed to interfere with specific MMP actions may be useful in the control of metastatic disease.

Background: Thoracic aortic aneurysms (TAAs) and valvular insufficiency, the main cardiovascular lesions in Marfan's syndrome, are associated with destruction of connective tissue; however, their pathogenesis remains unclear., Methods and Results: To test the hypothesis that changes in the activity of the matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are responsible for the damage to connective tissue in these lesions, histochemical studies of the immunoreactivity (IR) for MMPs and their tissue TIMPs (MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2) were made in TAAs (n = 7) and aortic valves (n = 5) from 7 patients with Marfan's syndrome. All TAAs showed cystic medial necrosis (CMN), with loss of elastic fibers and smooth muscle cells. Extensive areas of myxoid change were found in all aortic valves. Areas of CMN showed no IR for any MMPs or TIMPs. The IR of smooth muscle cells at the borders of areas of CMN was stronger for all MMPs, especially MMP-2 and MMP-9, than in other regions. The surfaces of disrupted elastic fibers showed IR for MMP-2 and MMP-9. Areas of myxoid change showed similar but less pronounced alterations., Conclusions: We hypothesize that the defect in fibrillin-1 in Marfan's syndrome leads to (1) formation of elastin that is abnormally aggregated and more easily degraded by MMPs than is normal elastin, (2) upregulation of the synthesis of MMPs, (3) progressive destruction of connective tissue by these enzymes, and (4) development of TAAs and valvular lesions.

Chondroitin sulfate proteoglycans (CSPGs) are implicated in the regulation of axonal growth. We previously reported that the neurite-promoting activity of laminin is inhibited by association with a Schwann cell-derived CSPG and that endoneurial laminin may be inhibited by this CSPG as well [Zuo J, Hernandez YJ, Muir D (1998) Chondroitin sulfate proteoglycan with neurite-inhibiting activity is upregulated after peripheral nerve injury. J Neurobiol 34:41-54]. Mechanisms regulating axonal growth were studied by using an in vitro bioassay in which regenerating embryonic dorsal root ganglionic neurons (DRGn) were grown on sections of normal adult nerve. DRGn achieved slow neuritic growth on sections of normal nerve, which was reduced significantly by treatment with metalloproteinase inhibitors. Similar results were obtained on a synthetic substratum composed of laminin and inhibitory CSPG. DRGn expressed the matrix metalloproteinase, MMP-2, which was transported to the growth cone. Recombinant MMP-2 inactivated the neurite-inhibiting CSPG without hindering the neurite-promoting potential of laminin. Similarly, neuritic growth by DRGn cultured on normal nerve sections was increased markedly by first treating the nerve sections with MMP-2. The proteolytic deinhibition by MMP-2 was equivalent to and nonadditive with that achieved by chondroitinase, suggesting that both enzymes inactivated inhibitory CSPG. Additionally, the increases in neuritic growth resulting from treating nerve sections with MMP-2 or chondroitinase were blocked by anti-laminin antibodies. From these results we conclude that MMP-2 provides a mechanism for the deinhibition of laminin in the endoneurial basal lamina and may play an important role in the regeneration of peripheral nerve.

Background: Extracellular matrix macromolecules regulate morphogenesis of embryonic organs, and are developmentally regulated. Their expression and turnover is regulated by matrix metalloproteinases (MMPs). Recently, an epithelial cell "membrane" associated metalloproteinase (MT-1-MMP) has been identified that acts as an activator of a "secreted" MMP-2, and is produced by mesenchymal fibroblasts. The activity of MMP-2 is inhibited by a "soluble" tissue inhibitor of MMP-2, TIMP-2. The role of MT-1-MMP in renal development is unknown., Methods: MT-1-MMP was cloned from embryonic mouse kidney cDNA library, and its spatio-temporal distribution during development in the context of MMP-2 and tissue inhibitor of metalloproteinase-2 (TIMP-2) was studied., Results: The cloned MT-1-MMP exhibited approximately 86% nucleotide sequence homology with human MT-1-MMP, and had a catalytic domain and a zinc binding site preceded by a RRKR furin recognition motif. A approximately 4.5 Kb MT-1-MMP mRNA transcript was detected, and its expression was developmentally regulated. A parallel developmental regulation of MMP-2 mRNA expression was also observed. TIMP-2 expression was also developmentally regulated, but lagged behind MT-1-MMP and MMP-2. By in situ hybridization, MT-1-MMP mRNA was seen to be confined to ureteric bud epithelia, and was absent in the mesenchyme, while MMP-2 was confined to the mesenchyme. MT-1-MMP protein expression was seen on ureteric bud epithelia, induced mesenchyme and nascent nephrons, and it was highest during mid gestation. Similar spatio-temporal expressions of MMP-2 and TIMP-2 proteins were observed., Conclusions: mRNAs of MT-MMP-1 and MMP-2 are expressed in the respective epithelial and mesenchymal compartments, while their proteins are co-expressed in the epithelia suggest that MT-1-MMP and MMP-2, in conjunction with TIMP-2, may be involved in paracrine/juxtacrine epithelial:mesenchymal interactions during metanephrogenesis.

Background: Migration of arterial smooth muscle cells (SMCs) is regulated by basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and matrix metalloproteinases (MMPs) in the injured rat carotid artery. We have recently shown that migration of SMCs from baboon aortic explants depends on the activity of MMPs, but the identity of the stimulatory MMPs and the role of bFGF and PDGF in this primate system are not known., Methods and Results: These experiments were designed to determine whether MMP2, MMP9, bFGF, or PDGF plays a role in SMC migration from medial explants of baboon aorta. Explants were cultured in serum-free medium with insulin, transferrin, and ovalbumin. Neutralizing antibodies to MMP2 and antibodies that inhibit activation of proMMP9 decreased SMC migration from the aortic explants. Antibodies to bFGF and to the alpha- and beta-subunits of the PDGF receptor also inhibited migration from the explants. Addition of bFGF and PDGF-BB but not PDGF-AA increased migration. The antibodies to bFGF but not the antibodies to the PDGF receptor subunits decreased the levels of MMP9, whereas all the antibodies decreased activated MMP2., Conclusions: These data demonstrate that SMC migration from primate aortic explants is dependent on endogenous MMP2, MMP9, PDGF, and bFGF. The data also suggest that PDGF-induced (PDGF-BB or possibly PDGF-AB) migration is dependent on MMP2, whereas bFGF-induced migration depends on both MMP2 and MMP9.

Objective: To evaluate the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the pathogenesis of the lesions of pulmonary Langerhans' cell granulomatosis., Design: Immunohistochemical and confocal microscopic studies were made of lung biopsy specimens from five patients with pulmonary Langerhans' cell granulomatosis., Results: The reactivity of Langerhans' cells was moderate to intense for MMP-2, weaker for MMP-9, and faint for TIMP-1 and TIMP-2. Type IV collagen colocalized with MMP-2 in areas of damage to epithelial basement membranes, a finding that emphasizes the potential importance of this enzyme in the pathogenesis of the destructive lesions of pulmonary Langerhans' cell granulomatosis. In the more advanced fibrotic lesions, TIMP-2 colocalized with basement membranes and with fibrillar collagen, suggesting that it contributes to the permanence of the fibrosis., Conclusion: These results indicate an important role for MMPs and TIMPs in pulmonary Langerhans' cell granulomatosis.

Matrix metalloproteinases (MMPs), or matrixins, are a family of zinc endopeptidases that play a key role in both physiological and pathological tissue degradation. Normally, there is a careful balance between cell division, matrix synthesis and matrix degradation, which is under the control of cytokines, growth factors and cell matrix interactions. The MMPs are involved in remodelling during tissue morphogenesis and wound healing. Under pathological conditions, this balance is altered: in arthritis, there is uncontrolled destruction of cartilage; in cancer, increased matrix turnover is thought to promote tumour cell invasion. The demonstration of a functional role of MMPs in arthritis and tumour metastasis raises the possibility of therapeutic intervention using synthetic MMP inhibitors with appropriate selectivity and pharmacokinetics. As the process of drug discovery focuses on structure-based design, efforts to resolve the 3-dimensional structures of the MMP family have intensified. Several novel MMP inhibitors have been identified and are currently being investigated in clinical trials. The structural information that is rapidly accumulating will be useful in refining the available inhibitors to selectively target specific MMP family members. In this review, we focus on the role of MMPs and their inhibitors in tumour invasion, metastasis and angiogenesis, and examine how MMPs may be targeted to prevent cancer progression.

To evaluate the role of matrix metalloproteinases (MMPs) and their specific tissue inhibitors (TIMPs) in the pathogenesis of the structural damage and cystic lesions found in pulmonary lymphangioleiomyomatosis (LAM), immunohistochemical studies were made of the localization of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1, TIMP-2, HMB-45, and type IV collagen in sections of lung biopsy specimens from four patients with this disorder. These studies showed increased immunoreactivity compared with that in normal bronchiolar and vascular smooth muscle cells, of MMP-2 and, to a lesser extent, MMP-9 and MMP-1 in the LAM cells. MMP-2 was also localized in some elastic fibers and in the basement membranes of LAM cells and overlying epithelial cells. The basement membranes in both of these sites often showed colocalization of MMP-2 and type IV collagen. Some epithelial basement membranes showing this colocalization were disrupted. These changes were not accompanied by increased immunoreactivity for TIMPs. Taken together with previous observations showing structural damage to elastic fibers and collagen fibrils, and with the absence of demonstrable neutrophil or pancreatic types of elastase, these findings suggest that MMP-2 and MMP-9 (both of which can degrade elastin as well as collagens) are responsible for the connective tissue destruction and cyst formation in LAM.

Objectives: Tumors are thought to metastasize by a process involving tumor cell attachment to extracellular matrix, degradation of matrix components by tumor-associated proteases, and cellular movement into the area modified by protease activity. Type IV collagen comprises the major element tumor cells must degrade to gain access to the rest of the body. Renal cancer cell line progelatinase A (E.C. 3.4.24.24; 72-kDa type IV collagenase; MMP-2) mRNA expression was correlated with patient survival., Methods: Total cellular mRNA was extracted from tumor cell lines derived from patients with metastatic renal cell carcinoma. The results of the densitometric analysis of Northern blots were correlated with patient survival. Formalin-fixed, paraffin-embedded tissue sections of primary renal cancers were examined for immunohistochemical expression of MMP-2., Results: Cell lines established from 23 primary renal tumors and six metastatic sites in 26 patients with metastatic renal carcinoma were studied. Variable expression of progelatinase A, relative to A2058 melanoma cells (mean +/- SEM, 0.60 +/- 0.21; median, 0.082; range, 0 to 4.78), was found. There was a significant inverse association between patient survival and the log of the MMP-2 expression (P = 0.045 by the Cox proportional-hazards model). Using a cutoff value of 0.10, the closest round number to the median expression of MMP-2, a significant difference between survival of patients with lower and higher MMP-2 expression in their primary renal cell line was found (P = 0.0054). Cell lines with low, intermediate, and high expression of MMP-2 mRNA all had primary tumors with high tissue immunohistochemical expression of MMP-2., Conclusions: These studies demonstrate an inverse relationship between renal cancer cell line MMP-2 mRNA expression and patient survival. Immunohistochemical studies of the primary tumors from which the cell lines were derived uniformly showed high MMP-2 expression. Previous work suggests local renal factors upregulate cellular expression of MMP-2 in the primary tumor, and are not active at extrarenal sites.

Structural changes in the extracellular matrix are necessary for cell migration during tissue remodeling and tumor invasion. Specific cleavage of laminin-5 (Ln-5) by matrix metalloprotease-2 (MMP2) was shown to induce migration of breast epithelial cells. MMP2 cleaved the Ln-5 gamma2 subunit at residue 587, exposing a putative cryptic promigratory site on Ln-5 that triggers cell motility. This altered form of Ln-5 is found in tumors and in tissues undergoing remodeling, but not in quiescent tissues. Cleavage of Ln-5 by MMP2 and the resulting activation of the Ln-5 cryptic site may provide new targets for modulation of tumor cell invasion and tissue remodeling.

Gelatinases have been shown to be regulated by many cytokines and growth factors, and have been implicated in the pathogenesis of certain autoimmune diseases via tissue destruction. High levels of several cytokines, including IFN-gamma and TNF-alpha, have been demonstrated in the salivary gland microenvironment of patients with Sjogren's syndrome (SS). How these cytokines may be contributing to the pathogenesis of this disease is not well understood. We hypothesized that IFN-gamma with or without (+/-) TNF-alpha could be playing a role in the pathogenesis of SS via the regulation of matrix metalloproteinase (MMP) levels. This study examined the role of IFN-gamma and (+) TNF-alpha in the regulation of the matrix metalloproteinases, MMP-2 (72 kD gelatinase A) and MMP-9 (92 kD gelatinase B). A human salivary gland cell line (HSG) has been used as a possible in vitro model to study the role of IFN-gamma + TNF-alpha in the pathogenesis of SS. The HSG cell line, in the presence of IFN +/- TNF-alpha, displays increased MMP-2 and MMP-9 gelatinolytic activity, protein and RNA levels. The increase in MMP activity was partially blocked with an antibody against the IFN-gamma receptor, and this was associated with a complete inhibition of the previously described IFN-gamma +/- TNF-alpha antiproliferative effect. However, incubation of IFN-gamma treated HSG cells with the synthetic MMP inhibitor BB94 did not alleviate this antiproliferative effect. In addition, we demonstrate that there are very high levels of MMP-9 in the saliva of patients with SS when compared to healthy control subjects. These data suggest that cytokines could be regulating MMP production by salivary epithelial cells and thus indicate a potential role for these cells in the pathogenesis of SS.

We have studied the expression of gelatinase A, gelatinase B, interstitial collagenase, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in reactive lymphoid cells, as well as in a series of cell lines derived from neoplasms of B- and T-cell lineage. Using both Northern blot analysis and zymography, gelatinase B activity was detected by zymography in two Burkitt cell lines and in a tonsillar cell suspension, while gelatinase A and interstitial collagenase activities were not detected by either method. TIMP-1 expression was demonstrated by Northern blot analysis in the multipotential neoplastic K-562 cell line, the high grade Burkitt's B-cell lymphoma lines, isolated tonsillar B cells and at low levels in peripheral blood T cells, but was not expressed in any of the neoplastic T-cell lines or isolated peripheral blood B cells. In contrast, TIMP-2 expression was restricted to tissues containing cells of T-cell lineage with high levels being observed in the neoplastic T-cell lines and lower levels in normal peripheral blood T cells and hyperplastic tonsil. Expression of TIMP-1 and TIMP-2 was confirmed at the protein level by reverse zymography and immunofluorescence assays using antihuman TIMP polyclonal antibodies. Expression of gelatinase B by the high grade B-cell Burkitt's lymphoma cell lines is consistent with previous findings in large cell immunoblastic lymphomas and indicates that this enzyme may play an important role in high grade non-Hodgkin's lymphomas. TIMP expression correlated with cell lineage in that TIMP-1 was primarily observed in B cells and TIMP-2 was restricted to T cells.

Matrix metalloproteinases are a growing family of neutral pH optima, zinc atom-dependent endopeptidases that collectively degrade all components of the extracellular matrix. This family of related proteases is further defined by their inhibition of protease activity by a class of low-molecular-weight endogenous inhibitors known as tissue inhibitors of metalloproteinases or TIMPs. Reverse zymography is an electrophoretic technique used to identify TIMP inhibitory activity within acrylamide gels. Previous methods have generally used biochemically complex sources of proteolytic activity (such as cell culture conditioned media) copolymerized with a proteinase substrate in the gel to identify the zones of inhibited proteolysis. We describe a novel system for reverse zymography using purified recombinant human gelatinase A or gelatinase B in place of conditioned media. These reverse zymograms using recombinant gelatinase have sensitivities for TIMPs that are favorable in comparison to immunoblotting techniques but have the benefit of visualizing multiple inhibitors simultaneously. We have developed and characterized these methods for the evaluation of inhibitors and have shown them to be highly sensitive, convenient, and reproducible. Both systems detect TIMPs 1, 2, and 3 simultaneously, but with differential sensitivities for TIMPs 1 and 2. Using gelatinase A the system can detect as little as 1 pg of rTIMP-2, but the limit of detection for rTIMP-1 is 40 pg. Gelatinase B shows less differential activity in that the limits of detection are 60 and 40 pg for TIMP-2 and TIMP-1, respectively. We demonstrate how these varied sensitivities of the gelatinases for the TIMPs can contribute to potential pitfalls in systems using uncharacterized reagents (i.e., conditioned media).

The clinical behavior of giant cell tumors (GCTs) is unpredictable. To gain insight into this tumor's biological behavior, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) were studied. These substances play essential roles in wound healing and neoplastic invasion and metastasis. Paraffin-embedded tissue was collected from 18 cases of histologically benign GCT, with 17 treated by curettage and 1 by resection. Eight cases showed no recurrence after a minimum of 2.5 years, and 10 had local recurrence. One showed metastasis. Antibodies to MMP-9, MMP-2, TIMP-1, and TIMP-2 were applied by immunohistochemical methods. In all cases, MMP-9 was strongly expressed in giant cells predominantly in a diffuse pattern and was strong but focal in stromal cells. MMP-2 decorated stromal cells and giant cells heterogeneously. TIMP-1 was variably expressed in giant cells of the nonrecurrent cases and was strongly present in a diffuse or patchy distribution in the stromal cells in 6 of 8 cases. However, in 9 of 10 recurrent cases, TIMP-1 was expressed weakly by both giant and stromal cells. TIMP-2 was variably expressed in the giant cells of the nonrecurrent cases, but 6 of 8 nonrecurrent cases showed strong stromal cell positivity for TIMP-2. Weak staining for TIMP-2 was observed in 7 of 10 recurrent cases in the stromal cells and 9 of 10 recurrent cases in the giant cells. These results indicate that expression of MMPs and TIMPs differs in giant cells and stromal cells in the same tumor. More significantly, in contrast to the nonrecurrent giant cell tumors, there is an imbalance in the MMPs and TIMPs in the recurrent tumors with a net excess of MMPs. This unopposed expression of MMPs in GCTs may play a role in breakdown of extracellular matrix and tissue invasion. Finally, these markers may prove useful in predicting behavior in these tumors.

Immunohistochemical and confocal microscopic studies of the localization of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen were made in lung tissues from patients with normal pulmonary histology (n = 3), diffuse alveolar damage (n = 14), and idiopathic pulmonary fibrosis (n = 12). Pretreatment with pepsin revealed otherwise undetectable MMP- and TIMP-immunoreactive sites. In normal lung, MMP-2, MMP-9, TIMP-1, and TIMP-2 were localized in ciliated cells, endothelial cells, pneumocytes, macrophages, and smooth muscle cells; fibroblasts showed a strong reaction only for MMP-2. Only TIMP-2 showed co-localization with type IV collagen. Myofibroblasts and epithelial cells expressed increased reactivity for MMPs and TIMPs in both disorders. The reactivities for MMPs and TIMPs were stronger in diffuse alveolar damage. MMP-2 showed focal co-localization in capillary endothelial and disrupted epithelial basement membranes, suggesting activation of collagenolysis. A protective effect against this lysis was suggested by the extensive co-localization of TIMP-2 with type IV collagen and fibrillar collagens. Alveolar buds showed increased reactivity for MMPs and TIMPs in their lining epithelial cells, myofibroblasts, and their basement membranes; however, their matrices were mostly unreactive. These findings emphasize the complexity of the roles of MMPs and TIMPs in collagen turnover in diffuse alvcolar damage and idiopathic pulmonary fibrosis.

Human mesangial cells grown in either 5 or 25 mM glucose were cultured on type IV collagen which had been previously control-incubated or in vitro glycated. Northern blot analysis revealed that after 3-7 days in culture mesangial cells on glycated type IV collagen expressed approximately 25-200% more alpha 1 (IV), approximately 20-50% less matrix metalloproteinase 2 (MMP-2), and 65-75% more tissue inhibitor of metalloproteinase 1 (TIMP-1). Decreased immunoreactivity (approximately 30-40%) and collagenolytic activity (approximately 10-40%) corresponding to MMP-2 was also detected in media conditioned during the third day of culture on glycated type IV collagen. These effects on cell function were related to the extent of type IV collagen modification and were similar for cells cultured in 5 or 25mM glucose. Elevated glucose (25 vrs 5 mM) increased expression of alpha 1 (IV) mRNA (approximately 40-70%) and in conjunction with matrix glycation resulted in detectable levels of MMP-9 message by northern blot although collagenolytic activity corresponding to MMP-9 was not detectable by zymography. We conclude that glucose and matrix glycation may each alter mesangial cell function, perhaps leading to an imbalance in mesangial matrix synthesis and degradation which could contribute to mesangial expansion characteristic of diabetic renal disease.

Lichenoid lesions of the skin are characterized by a band-like dermal inflammatory infiltrate and structural alterations of the basement membrane (BM). The etiopathogenesis of these lesions, of which lichen planus (LP) is perhaps the prototypic example, is unknown. Acute cases of LP are accompanied by the destruction of epidermal BM, degeneration of basal keratinocytes with loss of tonofilaments and hemidesmosomes, vesicular alterations, and even blister formation. Chronic LP is characterized by hyperkeratosis and acanthosis in the epidermis, fibrosis, and dense infiltrate in dermis. We found that acute LP lesions are characterized by uneven or absent immunostaining for laminin-5, laminin-1, and collagen type IV. Distribution and activity of gelatinases matrix metalloproteinase (MMP)-9 and MMP-2, and a specific inhibitor of MMP-2, tissue inhibitor of metalloprotein-2, were analyzed. The expression and activity of MMP-2 were increased, whereas tissue inhibitor of metalloprotein-2 expression was weak in the involved areas during the acute phase of LP. Moreover, we demonstrated in vitro that MMP-2 is directly capable of digesting laminin-5 gamma 2 chains to yield a 80-kd fragment. We also observed a weak or absent staining of all examined integrin receptors in the acute LP lesions. In chronic lesions, the staining of BM components was similar to normal controls. In these sections, normal expression of gelatinases and inhibitor was observed. There was, however, up-regulation and altered polarity of integrin receptors. These results indicate a link between overexpression of gelatinases, BM disruption, and altered integrin expression. In LP, the digestion of BM by MMP-2 may contribute to the pathogenesis by inducing altered integrin expression in basal keratinocytes and ultimately blister formation.

Tumor cell invasion is now viewed as dysregulated physiologic invasion. Investigators have started to define the molecular events that are involved in this process. We find that there are many functional similarities with molecular events involved in physiologic process such as angiogenesis and wound healing. Matrix metalloproteinase activity is a common denominator in these pathologic conditions and in normal responses. Studies using endogenous metalloproteinase inhibitors suggest that targeting matrix metalloproteinase activity may prevent tumor cell dissemination. The development and pre-clinical testing of novel, low molecular weight matrix metalloproteinase inhibitors support this concept and suggest that an exciting new era of cancer therapy is on the horizon.

Summary: Cellular invasion depends on cooperation between adhesive and proteolytic mechanisms. Evidence is provided that the matrix metalloproteinase MMP-2 can be localized in a proteolytically active form on the surface of invasive cells, based on its ability to bind directly integrin alpha v beta 3. MMP-2 and alpha v beta 3 were specifically colocalized on angiogenic blood vessels and melanoma cells in vivo. Expression of alpha v beta 3 on cultured melanoma cells enabled their binding to MMP-2 in a proteolytically active form, facilitating cell-mediated collagen degradation. In vitro, these proteins formed an SDS-stable complex that depended on the noncatalytic C-terminus of MMP-2, since a truncation mutant lost the ability to bind alpha v beta 3. These findings define a single cell-surface receptor that regulates both matrix degradation and motility, thereby facilitating directed cellular invasion.

Gelatinase A is secreted as a proenzyme (progelatinase A) which is activated and bound on the surface of tumor and normal cells. We have reported that the expression of a membrane-type-1-matrix metalloproteinase (MT1-MMP) induces activation of progelatinase A. Here we demonstrate that the expression of MT1-MMP in COS-1 cells induces cell-surface binding of progelatinase A which is consequently processed to an intermediate form. Processing from the intermediate to the fully active form is dependent on the gelatinase A concentration. These results suggest that the cell-surface binding concentrates the gelatinase A intermediate form locally to allow autoproteolytic processing to the fully active form.

The immunohistochemical expression of 72-kDa type IV collagenase [matrix-metalloproteinase (MMP)-21], basement membrane component type IV collagen and proliferation-related antigen Ki 67 were investigated in 43 benign, borderline, and malignant serous tumors of the ovary. The results were compared with the histotypes of ovarian serous tumors and with their clinical behavior. Serum evaluation of MMP-2 was performed in 14 patients with cystadenocarcinoma and the data compared with that of a control group. The basement membrane (BM) was continuous in benign cystadenomas and in some borderline tumors, whereas it was discontinuous or absent in other borderline tumors, in borderline tumors with microinvasion, and cystadenocarcinomas. The percentage of MMP-2- and Ki 67-expressing cells increased from cystadenomas to borderline tumors, being the highest in malignant tumors; a frequent basal disposition of the MMP-2 cytoplasmic granules also was observed in cystadenocarcinomas. Statistical analysis demonstrated that MMP-2 expression was inversely related to BM integrity. Serum MMP-2 values did not differ from that of the control group. Cox regression analysis showed that tumor stage and grade were significant prognostic factors, whereas MMP-2 and Ki 67 immunohistochemical expression added no further significant information to the prognosis. The investigators conclude that the correlation between increasing MMP-2 expression and BM alteration gives support to the hypothesis of a direct role of the metalloproteinase in the process of destructive stromal invasion. MMP-2, type IV collagen, and Ki 67 immunodetection varied according to the histologic classification of ovarian serous tumors. However, neither these factors nor the serum evaluation of MMP-2 appear useful as prognostic predictors in this series.

Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

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.

The balance between the activities of matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) is an important control point during the turnover of the extracellular matrix. Previous studies have demonstrated that an uncontrolled rate of production of these proteins leads to various pathological conditions. Overexpression of MMPs by malignant cells can increase the invasive behavior of solid tumors and their metastatic potential. In an increasing number of reports, it has become evident that expression of MMPs and TIMPs is not restricted to solid tumors. Normal and malignant hematological cells also express these proteins and play a pivotal role in the hematological-cell physiology. In addition to modifying the extracellular matrix, MMPs and TIMPs exert other functions in hematological cells, including growth-factor activity, removal of cell-surface receptors, and autoimmunity. The goal of this review is to gather recent studies concerning the properties of MMPs and TIMPs in hematological cells, their regulation of gene expression, their cellular distribution, and their potential role in the pathogenesis of hematological disorders such as inflammation and neoplasia.

MMP-2, a secreted 72-kd metalloproteinase that specifically degrades type IV collagen as well as denatured collagens, has been implicated in smooth muscle cell migration. To evaluate the possible contribution of this enzyme to the formation and progression of the atherosclerotic lesion, the expression of MMP-2 was studied in human aortic tissue. MMP-2 was visualized in frozen sections of the aortic wall by an immunofluorescent technique with a polyclonal antibody. Expression of MMP-2 in the aortic extracts was also studied by zymography and Western blotting. Our results reveal that a greater amount of MMP-2 is present in fatty streaks and atherosclerotic plaques as compared with normal regions of the aorta. Immunoblotting analysis showed that MMP-2 was expressed in atherosclerotic plaque > fatty streak > normal aortic wall in a ratio of approximately 4:2:1. Zymograms show that both forms (activated and latent) of MMP-2 increased in the atherosclerotic plaques. The presence of macrophages, detected by an immunohistochemical technique in some areas of higher MMP-2 expression suggests that these cells are a possible source of MMP-2. We conclude that MMP-2 collagenase may have a role in the formation and progression of the atherosclerotic lesion and may be involved in clinical complications of atherosclerosis, such as fissure and rupture, leading to thrombosis.

The 72 kDa type IV collagenase (gelatinase), a matrix metalloproteinase (MMP-2), has been proposed to potentiate the invasion and metastasis of malignant tumors. To determine the potential role of the MMP-2 in human gliomas and normal brain tissue, we examined the relative amounts of protein, mRNA, and distribution. Using gelatin zymography, densitometry, and an enzyme-linked immunosorbent assay for the quantitative determination of the MMP-2, we found that the enzyme's activity was significantly elevated in malignant astrocytomas, especially in glioblastoma multiforme, compared to low-grade glioma and normal brain tissues. As determined by Northern blot analysis, the amount of MMP-2 mRNA transcript was higher in anaplastic astrocytomas and glioblastoma multiforme tumors than in normal brain tissues or low-grade gliomas, a finding that was consistent with the amounts of MMP-2 protein detected in these tissues. Immunohistochemical studies demonstrated that MMP-2 was localized in tumor cells and vasculature cells of malignant astrocytomas. Staining intensity was clearly lower in low-grade astrocytomas, and immunoreactivity was very low or undetectable in normal brain astrocytes. The results suggest that expression of the MMP-2 is dramatically upregulated in malignant gliomas, correlating with the malignant progression of human gliomas in vivo.

Extracellular matrix (ECM) turnover is an event that is tightly regulated. Much of the coordinate (physiological) or discoordinate (pathological) degradation of the ECM is catalyzed by a class of proteases known as the matrix metalloproteinases (MMPs) or matrixins. Matrixins are a family of homologous Zn atom dependent endopeptidases that are usually secreted from cells as inactive zymogens. Net degradative activity in the extracellular environment is regulated by specific activators and inhibitors. One member of the matrixin family, gelatinase A, is regulated differently from other MMPs, suggesting that it may play a unique role in cell-matrix interactions, including cell invasion. The conversion from the 72 kDa progelatinase A to the active 62 kDa species may be a key event in the acquisition of invasive potential. This discussion reviews some recent findings on the cellular mechanisms involved in progelatinase A activation and, in particular, the role of tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) and transmembrane containing metalloproteinases (MT-MMP) in this process.

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.

Proteolytic damage is a late event in the molecular cascade initiated by brain injury. Earlier, we proposed that matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) are important in secondary brain injury. We have shown that intracerebral injection of activated 72-kDa type IV collagenase (gelatinase A) opens the blood-brain barrier, and that during hemorrhagic brain injury there is endogenous production of 92-kDa type IV collagenase (gelatinase B) and uPA. Therefore, to study the functional link between proteolytic enzymes and blood-brain barrier damage, we induced MMP expression by infusing tumor necrosis factor-alpha (TNF) intracerebrally in rats. Initially, the effect on capillary permeability of increasing doses of TNF, using [14C]sucrose uptake, was measured. Then, the time-course of the capillary permeability change was studied at 4, 16, 24 and 72 h. Expression of MMP and uPA was measured by zymography at 24 h after TNF injection and compared to saline-injected controls. A dose-dependent increase in capillary permeability was seen 24 h after TNF injection. Maximal uptake of [14C]sucrose occurred at 24 h compared to saline-injected controls (P < 0.05). Zymography showed production of gelatinase B, which was significantly greater than in saline-injected controls at 24 h (P < 0.05). Batimastat, a synthetic inhibitor to metalloproteinases, reduced sucrose uptake at 24 h (P < 0.0001), and was effective even when given 6 h after TNF (P < 0.01). Thus, gelatinase B is the intermediate substance linking TNF to modulation of capillary permeability. Agents that interfere with transcription of proteolytic enzymes or block their action may reduce delayed capillary injury, extending the therapeutic window.

Over-production of gelatinase A (MMP-2) or under-production of its inhibitor (TIMP-2) may result in the matrix degradation crucial for metastasis, and early evaluation of their expression in primary tumor would offer important prognostic informations. RT-PCR amplicons of MMP-2 and TIMP-2 mRNA from tissue biopsies of 13 breast carcinomas and one fibrocystic mastopathy were quantitated. In comparison with their normal-tissue counterparts, their expression trends were not uniform: in some cases MMP-2 increased in the tumor without changes in TIMP-2, in others TIMP-2 expression also increased, although to a lesser extent than MMP-2; only in 2 cases was it slightly lower in the tumor tissue. Nevertheless, clearer insights were gained from the comparison of the ratio (R) between MMP-2tumor/normal and TIMP-2tumor/normal: as in the fibrocystic mastopathy, the R in carcinomas without lymph-node involvement (LN-) was usually lower than I in most cases. In contrast, in 5 out of 6 patients with lymph-node metastasis (LN+), the ratio ranged between 2 and 4. While the R magnitude was not related to the frequency of positive lymph nodes out of the total analyzed, nor to relapse status at follow-up (all relapse-free), the clear-cut difference between the LN- and LN+ groups was statistically significant. Results suggest that evaluation of MMP-2/TIMP-2 mRNA balance may constitute an early prognostic approach, which may also be more reliable concerning cancer aggressiveness as compared with the MMP-2 alone, and that boosting TIMP-2 expression may be a therapeutic strategy to prevent metastasis.

Numerous studies have reported a correlation between the production of gelatinases A and B by cancer cells and invasive and metastatic potential. It has been suggested that the expression of gelatinase A (72-kDa type IV collagenase) is associated more closely with the metastatic phenotype of malignant cells in vitro and in vivo than that of gelatinase B (92-kDa type IV collagenase). We have established a rat bladder carcinoma cell line, MYU3L, which is tumorigenic and locally invasive but is not metastatic to the distal organs in nude mice. The MYU3L cell line secretes pro-gelatinase B but not any detectable level of pro-gelatinase A. We undertook the present study to determine whether over-expression of gelatinase A can affect the metastatic potential of MYU3L cells. We transfected MYU3L cells with an expression vector containing human pro-gelatinase A cDNA under the transcriptional control of the SR alpha promoter. Two stable transfectants over-expressing gelatinase A activity were isolated. We assessed the biological behavior of the transfectants by an orthotopic site (urinary bladder) inoculation and an i.v. injection in nude mice. Our results demonstrate that the induced expression of human gelatinase A enzyme markedly accelerates the metastatic phenotype of the rat bladder carcinoma cell line MYU3L. Our results suggest that gelatinase A produced by tumor cells plays a major role in the metastatic process.

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 Kd of 2.5 nM and 30,000 sites/cell. Monolayers of MCF-7 cells were similarly found to bind [125I]rTIMP-2 with a Kd 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.

Interaction of cells with the extracellular matrix (ECM) plays an important role in the regulation of cell behavior. Formation of adhesive contacts leads to transduction of signals into the cell and results in altered gene expression and modulation of the cellular phenotype. Specific adhesive interactions of the fibronectin and vitronectin receptors with their ligands in the matrix modulates expression of ECM-degrading metalloproteases. These proteases are involved in the acquisition of the invasive phenotype by a number of cell types. The activity of matrix metalloproteases (MMPs) is reduced by endogenous inhibitors referred to as tissue inhibitors of metalloproteases (TIMPs). Alterations in the balance between the activity of MMPs and TIMPs alters cellular invasion through effects on matrix degradation. In this study we demonstrate that inhibition of endogenous gelatinase A activity in A2058 human melanoma cells results in enhanced cellular adhesion. To further explore this phenomenon, we have used retroviral infection vectors to control the amount of the MMP inhibitor TIMP-2 in human melanoma A2058 cells. Altering the production of TIMP-2 modulates not only proteolysis of the extracellular matrix, but also the adhesive and spreading properties of the cells and results in altered cell morphology. These effects of TIMP-2 appear to be mediated by inhibition of gelatinase A activity. We conclude that gelatinase A, in addition to contributing to proteolysis of ECM components, also functions to proteolyse cell surface components that mediate attachment of A2058 cells to the ECM. Thus, gelatinase A may function to modulate cell attachment and facilitate cell migration and invasion.

Tissue inhibitors of metalloproteinases (TIMPs) are negative regulators of matrix metalloproteinases (MMPs) which degrade major components of the extracellular matrix. The aberrant expression of TIMPs is believed to represent an important modulating factor in the invasive capacity of human tumors. In the present study we analyzed the expression of TIMPs in human brain tumor tissue samples by an enzyme-linked immunosorbent assay (ELISA) and by Northern blotting analysis. Quantitation of TIMP-1 and TIMP-2 by ELISA demonstrated low levels of TIMP-1 and TIMP-2 proteins in glioblastomas, and moderate levels in anaplastic astrocytomas compared with normal brain tissues low-grade gliomas and metastatic tumors (renal and breast carcinomas and melanomas). Northern blot analysis of TIMP-1 transcripts demonstrated higher expression in meningioma, normal brain tissues and other metastatic tumors than in anaplastic astrocytoma and glioblastoma. Two distinct transcripts of 1.0 and 3.5 kb were observed for TIMP-2 mRNA in normal brain tissue and in tumor extracts. In addition, TIMP-2 mRNA expression was lower in glioblastoma and anaplastic astrocytoma than in meningioma, normal brain tissues and metastatic tumors. These findings suggest that down-regulation of both TIMP-1 and TIMP-2 contributes significantly to the invasive potential of human glioblastoma multiforme and anaplastic astrocytomas.

Tissue development and structure is controlled by dynamic and interactive relationships between cells and the extra-cellular matrix (ECM) which they secrete. We have investigated the occurrence and distribution of metalloproteinase-2 (MMP-2), an enzyme involved in the catabolism of ECM components, in human embryonic tissues by immunocytochemistry. Cells displaying MMP-2 immunoreactivity showed a widespread distribution in human embryonic tissues and organs. Cytoplasmic staining was detected in cells deriving from all three embryonic layers. Although further studies are needed to clarify the possible substrates of MMP-2 in developing tissues, these morphological data lend support to the hypothesis that ECM remodelling and degradation may represent a physiological counterpart of ECM deposition that occur during development.

Gelatinase A (MMP-2) and cathepsin B are proteinases which have been proposed to participate in human tumor invasion and metastasis. Precise quantitation of the activity of these enzymes in invading tumors has not been previously described. We utilized a novel tissue microdissection technique to determine levels of enzyme activity in specific microscopic areas of invasive human colon cancer. Tissue specimens smaller than one high power field can be extracted from the samples and analyzed. Increased levels of pro-enzyme and active enzyme forms of gelatinase A (MMP-2) and increased cathepsin B activity were localized in regions of tumor invasion as compared with a matched number of normal epithelial cells from the same patient. Levels of progelatinase B (MMP-9) were also increased in the tumors; however, we did not observe activation of this enzyme. To investigate the mechanism of gelatinase A activation, we amplified DNA of specific microdissected tumor cell populations using polymerase chain reaction. We did not detect a mutation in the activation locus of the enzyme in any of the tumors studied, which suggests that activation may be due to up-regulation of a tumor-associated gelatinase A activating species. Microdissection of frozen tissue sections may prove valuable in the study of proteinases in human tumor invasion as well as in the detection of genetic alterations in human cancers.

The primary structure of galectin-3, a approximately 30 kDa galactoside-binding protein (aka CBP-35, mL-34, hL-31, L-29, Mac-2, and epsilon BP), reveals two structural domains: an amino-terminal domain consists of a Pro-Gly-rich motif, and a globular carboxyl-terminal domain containing a carbohydrate-binding site. In this study, we report that the amino-terminal domain of galectin-3 contains a cleavage site for two members of the matrix metalloproteinase family of enzymes: the 72 kDa (gelatinase A, MMP-2) and the 92 kDa (gelatinase B, MMP-9) proteinases. The major cleavage site for the gelatinases in galectin-3 is at the Ala62-Tyr63 bond, and its hydrolysis by these enzymes was inhibited by TIMP-2. Cell-surface expression of galectin-3 was reduced following treatment of viable T47D human breast carcinoma cells with gelatinase A. These results suggest that galectin-3 may be a substrate for gelatinases and that its degradation may play a role in modulating the biological activities of galectin-3.

One critical event of tumour invasion that signals the initiation of the metastatic cascade is thought to be interaction of the tumour cell with the basement membrane. Basement membranes may also pose as barriers to tumour cell invasion at multiple points later in the metastatic cascade, including during the processes of vascular infiltration and extravasation. Thus, an important proteolytic event in the metastatic cascade, and also angiogenesis, appears to be degradation of basement membrane components. A specific class of extracellular matrix degrading metalloenzymes, the matrix metalloproteases, and their endogenous inhibitors, the tissue inhibitors of metalloproteases, are thought to have a role in the creation of the proteolytic defect in basement membrane type IV collagen. We will review the evidence which indicates that matrix metalloproteases and tissue inhibitors of metalloproteases are essential for tumour cell invasion and angiogenesis. The regulation of matrix metalloproteases will be discussed, including gene activation and transcription, messenger ribonucleic acid (mRNA) stability, binding of proenzymes to cell membranes and/or matrix components, proenzyme activation, and inactivation by endogenous inhibitors. We will also discuss the mechanism for tissue inhibitor of metalloproteases-mediated inhibition of tumour invasion and angiogenesis. This appears, at least in part, to be through inhibition of protease activity required for cellular invasion, although recent observations suggest that tissue inhibitors of metalloproteases affect other distinct groups of biological activities through mechanisms other than matrix metalloprotease inhibition.

Simultaneous presence of gelatinase A (MMP-2) and MMP-2 messenger RNA (mRNA) in 30 malignant tumors with various degrees of differentiation and biological behavior was evaluated by immunohistochemistry and in situ hybridization. The series consisted of 10 gastric carcinomas, 10 colorectal carcinomas, five squamous skin carcinomas, and five basal cell skin tumors. MMP-2 was detected in all cases. MMP-2 mRNA was expressed in the stromal cells in all cases and was more marked in the less-differentiated gastric and colonic carcinomas; it was also detected in the neoplastic cells of poorly differentiated tumors, particularly in those of the signet-ring cell type, both in the colon and stomach. The study confirmed that stromal cells have a specific role in tumor invasion and suggests a direct relationship between neoplastic epithelium and stromal cells in the most aggressive varieties.

Altered regulation of metalloproteinases may play a role in a variety of pathologic conditions including cancer. Previous studies have demonstrated transforming growth factor-beta 1 (TGF-beta 1)-mediated stimulation of expression and activation, and phorbol ester-mediated inhibition of matrix metalloproteinase (MMP)-2 (72-kDa type IV collagenase/gelatinase A), indicating a role for transmembrane signal transduction in MMP-2 regulation. We now describe a role for calcium mobilization in the regulation of MMP-2 expression. Receptor-operated calcium influx has been shown to be inhibited by a novel synthetic inhibitor, carboxy amido-triazole (CAI). Incubation of A2058 human melanoma, HT-1080 human fibrosarcoma, and OVCAR3 human ovarian cancer cells with CAI (0-10 microM) resulted in a dose-dependent reduction in MMP-2 latent and activated species activity by zymogram analysis of conditioned medium. This reduction is not due to direct inhibition of the enzyme by CAI or CAI-induced MMP-2 degradation. Decreased quantity of secreted MMP-2 protein in CAI-treated cells was shown by immunoblot and pulse-chase analysis of newly synthesized MMP-2. Cell coincubation with CAI (2 microM) and TGF-beta 1 (5 ng/ml) caused a decrease in the overall amount of latent and activated MMP-2 by zymogram and immunoblot analysis and showed that CAI inhibited TGF-beta 1 stimulation of MMP-2 production at the level of RNA expression. This was confirmed by Northern analysis of A2058 cells treated with CAI (2 microM) for 24 and 48 h and demonstrated a 55% reduction in message for MMP-2 and a 61% reduction in message for MMP-1, 54-kDa interstitial collagenase. Specificity for CAI action was demonstrated by equivalent MMP-2 inhibitory activity from analogs of CAI that retained the ability to inhibit calcium influx and by lack of inhibition by exposure to inactive CAI analogs that could not inhibit calcium influx. As an independent verification of specificity, a marked reduction in MMP-2 gelatinase activity by zymogram was shown after treatment of A2058 cells with SK&F 96365, an unrelated inhibitor of receptor-operated calcium influx. These results suggest a role for calcium-mediated signal transduction in the expression of metalloproteinases.

Monocytes/macrophages are associated with chronic inflammatory lesions, such as periodontal disease and rheumatoid arthritis, in which there is extensive connective tissue destruction. Stimulation of human monocytes results in the production of matrix metalloproteinases (MMPs) via a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Modulation of many monocyte functions by interleukin 10 (IL-10) suggested that this cytokine may influence the signal transduction pathway leading to the production of MMPs by monocytes. Pre-incubation of monocytes with IL-10 for 1 h prior to stimulation with ConA resulted in significant inhibition of prostaglandin H synthase-2 (PGHS-2, the inducible form of prostaglandin synthase). In contrast, PGHS-1, the constitutive PGHS, was not affected by IL-10. Suppression of PGHS-2 mRNA and protein levels was detected at 1 ng/ml of IL-10 with maximal inhibition at 20 ng/ml. Nuclear run-on transcription assays performed on monocytes exposed to ConA or the combination of ConA and IL-10 indicated that IL-10 treatment suppressed PGHS-2 expression at the level of transcription. Attenuation of PGHS-2 by IL-10 was accompanied by decreased prostaglandin production, including PGE2. The decrease in prostaglandin production was primarily related to the effect of IL-10 on PGHS-2, since the release of arachidonic acid was unaffected by this cytokine. The inhibition of PGE2 production by IL-10 resulted in the suppression of mRNA and protein for interstitial collagenase and 92-kDa type IV collagenase/gelatinase (gelatinase B). This conclusion is supported by the ability of exogenously added PGE2 or dibutyryl cAMP to restore the production of MMPs in IL-10-treated monocytes. Additionally, PGHS-2 was also restored by PGE2 or dibutyryl cAMP, indicating that PGHS-2 is regulated through a PGE2-cAMP amplification pathway. These data add further support to the anti-inflammatory properties of IL-10.

Activation of human monocytes induces the production of matrix metalloproteinases (MMPs) through a prostaglandin E2 (PGE2)-cAMP-dependent pathway. Since G-proteins have been documented to modulate adenylyl cyclase, we examined the effect of G-protein ADP-ribosylating agents, cholera toxin (CT) and pertussis toxin (PT), on the signal transduction pathway that culminates in the production of monocyte MMPs. Although CT elevated cAMP levels in both unstimulated and concanavalin A (Con A)-stimulated monocytes, it enhanced the production of prostaglandin H synthase-2 (PGH synthase-2, PGHS-2) protein, prostaglandins, interstitial collagenase, and 92-kDa type IV collagenase/gelatinase only in Con A-stimulated monocytes. Additionally, the indomethacin-mediated suppression of Con A-induced monocyte interstitial collagenase and 92-kDa type IV collagenase/gelatinase production could be reversed by CT. In contrast to the actions of CT, PT treatment suppressed the levels of cAMP, PGHS-2, PGE2, interstitial and 92-kDa type IV collagenase/gelatinase in Con A-stimulated monocytes. The regulation of MMP production by these toxins appears to be mediated primarily through their effect on adenylyl cyclase since the release of arachidonic acid was relatively unaffected by these agents. These findings provide evidence that G-proteins may be involved in either the enhancement or suppression of the eicosanoid-cAMP-dependent signal transduction pathway that results in the production of monocyte MMPs.