Role of matrix metalloproteinases in melanoma cell invasion

Cutaneous melanomas are notorious for their tendency to metastasize. Essential steps in this process are the degradation of basement membranes and remodeling of the extracellular matrix (ECM) by proteolytic enzymes such as matrix metalloproteinases (MMPs), which are regulated by their tissue inhibitors (TIMPs). An MMP expression is not restricted to tumor cells but is also found in stromal cells, indicating that stroma-derived proteases may contribute to melanoma progression. The MMPs have been shown to interact with a broad range of non-matrix proteins including adhesion molecules, growth factors and mediators of angiogenesis and apoptosis. In this review, we evaluate new insights into the interplay of MMPs and their molecular partners in melanoma progression.     

Matrix metalloproteinases and their expression in mammary gland

The matrix metalloproteinases (MMPs) are a family of zine-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events.The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation,and is controlled by growth factors,cytokines,hormones,as well as interactions with the ECM macromolecules.Furthermore,the activity of the MMPs is regulated by their natural endogenous inhibitors,which are members of the tissue inhibitor of metalloproteinases (TIMP) family.In the normal mammary gland,MMPs are expressed during ductal development,lobulo-alveolar development in pregnancy and involution after lactation.Under pathological conditions,such as tumorigenesis,the dysregulated expression of MMPs play a role in tumor initiation,progression and malignant conversion as well as facilitating invasion and metastasis of malignant cells through degradation of the ECM and basement membranes.     

Extracellular matrix degradation by cultured mesangial cells: mediators and modulators

Decreased degradation of the glomerular extracellular matrix (ECM) is thought to contribute to the accumulation of glomerular ECM that occurs in diabetic nephropathy and other chronic renal diseases. Several lines of evidence indicate a key role for the plasminogen activator/plasminogen/plasmin system in glomerular ECM degradation. However, which of the two plasminogen activators (PAs) present in renal tissue, tissue plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA), is responsible for plasmin generation and those factors that modulate the activity of this system remain unclear. This study utilized mesangial cells isolated from mice with gene deletions for tPA, uPA, and plasminogen activator inhibitor 1 (PAI-1) to further delineate the role of the PA/plasminogen/plasmin system in ECM accumulation. ECM degradation by uPA-null mesangial cells was not significantly different from controls (92% +/- 1%, n = 12). In contrast, ECM degradation by tPA-null mesangial cells was markedly reduced (-78 +/- 1%, n = 12, P < 0.05) compared with controls, whereas tPA/uPA double-null mesangial cells degraded virtually no ECM. Previous studies from this laboratory have established that transforming growth factor-beta1 (TGFbeta1) inhibits ECM degradation by cultured mesangial cells by increasing the production of PAI-1, the major physiological PA inhibitor. In keeping with this observation, TGFbeta1 (1 ng/ml) had no effect on ECM degradation by PAI-1-null MC. High glucose levels (30 mM) in the presence or absence of insulin (0.1 mM) caused a moderate increase in ECM degradation by normal human mesangial cells. In contrast, glycated albumin, whose concentration is known to increase in diabetes, produced a dose-dependent (0.2-0.5 mg/ml) inhibition of ECM degradation by normal human mesangial cells. Taken together, these results document the importance of tPA versus uPA in renal plasmin production and indicate that in contrast to elevated glucose, glycated albumin may contribute to ECM accumulation in diabetic nephropathy.     

Matrix metalloproteinases and their function in myocardium

A significant number of myocardial diseases are accompanied by increased synthesis and degradation of the extracellular matrix (ECM) as well as by changed maturation and incorporation of ECM components. Important groups of enzymes responsible for both normal and pathological processes in ECM remodeling are matrix metaloproteinases (MMPs). These enzymes share a relatively conserved structure with a number of identifiable modules linked to their specific functions. The most important function of MMPs is the ability to cleave various ECM components; including such rigid molecules as fibrillar collagen molecules. The amount and activity of MMPs in cardiac tissue are regulated by a range of activating and inhibiting processes. Although MMPs play multifarious roles in many myocardial diseases, here we have focused on their function in ischemic cardiac tissue, dilated cardiomyopathy and hypertrophied cardiac tissue. The inhibition of MMPs by means of synthetic inhibitors seems to be a promising strategy in cardiac disease treatment. Their effects on diseased cardiac tissue have been successfully tested in several experimental studies.     

A matrix metalloproteinase gene is expressed at the boundary of senescence and programmed cell death in cucumber

Cell-cell and extracellular cell matrix (ECM) interactions provide cells with information essential for controlling morphogenesis, cell-fate specification, and cell death. In animals, one of the major groups of enzymes that degrade the ECM is the matrix metalloproteinases (MMPs). Here, we report the characterization of the cucumber (Cucumis sativus L. cv Marketmore) Cs1-MMP gene encoding such an enzyme likely to play a role in plant ECM degradation. Cs1-MMP has all the hallmark motif characteristics of animal MMPs and is a pre-pro-enzyme having a signal peptide, propeptide, and zinc-binding catalytic domains. Cs1-MMP also displays functional similarities with animal MMPs. For example, it has a collagenase-like activity that can cleave synthetic peptides and type-I collagen, a major component of animal ECM. Cs1-MMP activity is completely inhibited by a hydroxamate-based inhibitor that binds at the active site of MMPs in a stereospecific manner. The Cs1-MMP gene is expressed de novo at the end stage of developmental senescence, prior to the appearance of DNA laddering in cucumber cotyledons leaf discs and male flowers. As the steady-state level of Cs1-MMP mRNA peaks late in senescence and the pro-enzyme must undergo maturation and activation, the protease is probably not involved in nutrient remobilization during senescence but may have another function. The physiological substrates for Cs1-MMP remain to be determined, but the enzyme represents a good candidate for plant ECM degradation and may be involved in programmed cell death (PCD). Our results suggest that PCD occurs only at the culmination of the senescence program or that the processes are distinct with PCD being triggered at the end of senescence.     

Oxidation of extracellular matrix modulates susceptibility to degradation by the mesangial matrix metalloproteinase-2.

Protein oxidation occurs in aging and in various inflammatory conditions. Glomerulosclerosis is characterized by an accumulation of extracellular matrix (ECM) proteins and a paucity of glomerular mesangial cells and can be seen as an end-result of glomerular injury and in aging. ECM accumulation is the net result of the balance between synthesis and degradation. ECM may become oxidized as a part of inflammatory renal injury and with aging. We evaluated the hypothesis that oxidation of mesangial ECM could alter its susceptibility to the action of ECM degrading enzymes. Radiolabeled mesangial ECM was generated by growing cells on tissue culture plastic and incubating with [3H]proline. After removal of cells, leaving behind ECM, selected wells were oxidized using a FeCl3/EDTA/ascorbate system or treated under control conditions. The control and oxidized matrices were then incubated with concentrated supernatants from mesangial cells containing the major mesangial ECM degrading enzyme, the matrix metalloproteinase-2, whose activity was confirmed by gelatin substrate zymography. Counts released corresponding with ECM degraded were measured. ECM oxidized with this system was significantly less susceptible to degradation compared to control ECM. To confirm that this effect was specifically due to oxidative modification of the ECM rather than changes unrelated to oxidation we coincubated ECM with the oxidizing system plus the radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN). PBN treatment was able to prevent the impaired susceptibility to degradation induced by exposure to the oxidizing system. Exposure of ECM to milder oxidative stress, however, modestly enhanced susceptibility to degradation. These data suggest that oxidation of mesangial ECM can modulate its susceptibility to degradation. This may account for the development of ECM accumulation and glomerulosclerosis in inflammatory renal injury and in aging.     

Diabetic Nephropathy and Extracellular Matrix

Diabetic nephropathy (DN) is a serious complication in diabetes. Major typical morphological changes are the result of changes in the extracellular matrix (ECM). Thus, basement membranes are thickened and the glomerular mesangial matrix and the tubulointerstitial space are expanded, due to increased amounts of ECM. One important ECM component, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs in basement membranes are decreased but increased in the mesangium and the tubulointerstitial space. The amounts and structures of heparan sulfate chains are changed, and such changes affect levels of growth factors regulating cell proliferation and ECM synthesis, with cell attachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfate structures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classes also modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serine proteases, such as plasminogen activator, as well as their corresponding inhibitors. The levels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN. Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesis and turnover in DN. Whether ECM components can be used as markers for early kidney changes is an important research topic, whereas at present, the clinical use remains to be established.     

Ontogeny and regulation of matrix metalloproteinase activity in the zebrafish embryo by in vitro and in vivo zymography

Remodeling of the extracellular matrix (ECM) during development, angiogenesis, wound healing, tumor metastasis, and other morphogenetic processes depends on the exquisitely regulated activities of matrix metalloproteinases (MMPs). Yet very little is known about the activity patterns of these proteases in vivo. We have employed fluorescent MMP-substrates, both in vitro and in vivo, to characterize patterns of MMP activity in the zebrafish embryo. Qualitatively similar patterns of degradation are detected using native Type I or Type IV collagen substrates, suggesting that multiple MMPs are being regulated concomitantly. MMP activity is observed primarily in ECM-rich structures predicted to be undergoing active remodeling, such as the perichordal sheath and somite boundaries. Patterns of Type I and Type IV collagen hydrolysis are similar, but not identical in embryos of any given stage. Conventional gelatin zymography shows MMPs present in embryos as early as 3-somites (11 h) and our in vivo assays detect Type IV collagen degradation at somite boundaries as early as 4-somites (11.5 h). However, we are unable to detect significant in vitro activity using homogenates made from embryos prior to Prim-16 (31 h). Mixed lysate assays demonstrate that this is the result of endogenous inhibitors present in early embryos, suggesting a model of matrix remodeling regulated by spatially heterogeneous MMP inhibition.     

Following matrix metalloproteinases activity near the cell boundary by infrared micro-spectroscopy.

Matrix Metalloproteinases (MMPs) are cell-secreted soluble and membrane-tethered enzymes that degrade extracellular matrix (ECM) proteins. These proteases play a key role in diverse physiological and pathological processes, including embryonic development, wound repair, inflammatory diseases and cancer. Yet, there is insufficient knowledge on the mode by which cell-produced MMPs conduct their action on the ECM. Specifically, the localization and the mode of the degradation within the pericellular space are of great interest. To provide new insights to these questions we utilized Fourier transform infrared (FTIR) micro-spectroscopy to follow proteolytic processes, induced by invasive cancer cells, on insoluble collagen-based matrices. Here we show that FTIR micro-spectroscopy have a great potential for monitoring degradation events near cells. Using this tool we demonstrate that the net proteolysis is unevenly distributed around the cell boundary. The degradation patterns show different levels of proteolytic activity by MMPs within the pericellular space. In addition, our spectral analysis suggests that the enzymatic proteolysis of the collagen-based matrices induces unwinding of the triple helical structures of the macromolecules within the collagen network.     

Matrix metalloproteinases and epidermal wound repair

Epidermal wound healing is a complex and highly coordinated process where several different cell types and molecules, such as growth factors and extracellular matrix (ECM) components, play an important role. Among the many proteins that are essential for the restoration of tissue integrity is the metalloproteinase (MMP) family. MMPs can act on ECM and non-ECM components affecting degradation and modulation of the ECM, growth-factor activation and cell–cell and cell–matrix signalling. MMPs are secreted by different cell types such as keratinocytes, fibroblasts and inflammatory cells at different stages and locations during wound healing, thereby regulating this process in a very coordinated and controlled way. In this article, we review the role of MMPs and their inhibitors (TIMPs), as well as the disintegrin and metalloproteinase with the thrombospondin motifs (ADAMs) family, in epithelial wound repair.     

Matrix metalloproteinases and angiogenesis

Matrix metalloproteinases (MMPs) are a family of enzymes that proteolytically degrade various components of the extracellular matrix (ECM). Angiogenesis is the process of forming new blood vessels from existing ones and requires degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissue. MMPs participate in this remodeling of basement membranes and ECM. However, it has become clear that MMPs contribute more to angiogenesis than just degrading ECM components. Specific MMPs have been shown to enhance angiogenesis by helping to detach pericytes from vessels undergoing angiogenesis, by releasing ECM-bound angiogenic growth factors, by exposing cryptic proangiogenic integrin binding sites in the ECM, by generating promigratory ECM component fragments, and by cleaving endothelial cell-cell adhesions. MMPs can also contribute negatively to angiogenesis through the generation of endogenous angiogenesis inhibitors by proteolytic cleavage of certain collagen chains and plasminogen and by modulating cell receptor signaling by cleaving off their ligand-binding domains. A number of inhibitors of MMPs that show antiangiogenic activity are already in early stages of clinical trials, primarily to treat cancer and cancer-associated angiogenesis. However, because of the multiple effects of MMPs on angiogenesis, careful testing of these MMP inhibitors is necessary to show that these compounds do not actually enhance angiogenesis.     

Matrix metalloproteinase-14 is a mechanically regulated activator of secreted MMPs and invasion

Matrix metalloproteinases (MMPs) are extracellular matrix (ECM) degrading enzymes and have complex and specific regulation networks. This includes activation interactions, where one MMP family member activates another. ECM degradation and MMP activation can be initiated by several different stimuli including changes in ECM mechanical properties or intracellular contractility. These mechanical stimuli are known enhancers of metastatic potential. MMP-14 facilitates local ECM degradation and is well known as a major mediator of cell migration, angiogenesis and invasion. Recently, function blocking antibodies have been developed to specifically block MMP-14, providing a useful tool for research as well as therapeutic applications. Here we utilize a selective MMP-14 function blocking antibody to delineate the role of MMP-14 as an activator of other MMPs in response to changes in cellular contractility and ECM stiffness. Inhibition using function blocking antibodies reveals that MMP-14 activates soluble MMPs like MMP-2 and -9 under various mechanical stimuli in the pancreatic cancer cell line, Panc-1. In addition, inhibition of MMP-14 abates Panc-1 cell extension into 3D gels to levels seen with non-specific pan-MMP inhibitors at higher concentrations. This strengthens the case for MMP function blocking antibodies as more potent and specific MMP inhibition therapeutics.     

MMP Secretion Rate and Inter-invadopodia Spacing Collectively Govern Cancer Invasiveness

Invadopodia are micron-sized invasive structures that mediate extracellular matrix (ECM) degradation through a combination of membrane-bound and soluble matrix metalloproteinases (MMPs). However, how such localized degradation is converted into pores big enough for cancer cells to invade, and the relative contributions of membrane-bound versus soluble MMPs to this process remain unclear. In this article, we address these questions by combining experiments and simulations. We show that in MDA-MB-231 cells, an increase in ECM density enhances invadopodia-mediated ECM degradation and decreases inter-invadopodia spacing. ECM degradation is mostly mediated by soluble MMPs, which are activated by membrane-bound MT1-MMP. We present a computational model of invadopodia-mediated ECM degradation, which recapitulates the above observations and identifies MMP secretion rate as an important regulator of invadopodia stability. Simulations with multiple invadopodia suggest that inter-invadopodia spacing and MMP secretion rate collectively dictate the size of the degraded zones. Taken together, our results suggest that for creating pores conducive for cancer invasion, cells must tune inter-invadopodia spacing and MMP secretion rate in an ECM density-dependent manner, thereby striking a balance between invadopodia penetration and ECM degradation.     

Localizing matrix metalloproteinase activities in the pericellular environment

Matrix metalloproteinases (MMPs) are a group of structurally related proteolytic enzymes containing a zinc ion in the active site. They are secreted from cells or bound to the plasma membrane and hydrolyze extracellular matrix (ECM) and cell surface-bound molecules. They therefore play key roles in morphogenesis, wound healing, tissue repair and remodeling in diseases such as cancer and arthritis. Although the cell anchored membrane-type MMPs (MT-MMPs) function pericellularly, the secreted MMPs have been considered to act within the ECM, away from the cells from which they are synthesized. However, recent studies have shown that secreted MMPs bind to specific cell surface receptors, membrane-anchored proteins or cell-associated ECM molecules and function pericellularly at focussed locations. This minireview describes examples of cell surface and pericellular partners of MMPs, as well as how they alter enzyme function and cellular behaviour.     

Matrix metalloproteinases and gastrointestinal cancers: Impacts of dietary antioxidants

The process of carcinogenesis is tightly regulated by antioxidant enzymes and matrix degrading enzymes, namely, matrix metalloproteinases(MMPs). Degradation of extracellular matrix(ECM) proteins like collagen, proteoglycan, laminin, elastin and fibronectin is considered to be the prerequisite for tumor invasion and metastasis. MMPs can degrade essentially all of the ECM components and, most MMPs also substantially contribute to angiogenesis, differentiation, proliferation and apoptosis. Hence, MMPs are important regulators of tumor growth both at the primary site and in distant metastases; thus the enzymes are considered as important targets for cancer therapy. The implications of MMPs in cancers are no longer mysterious; however, the mechanism of action is yet to be explained. Herein, our major interest is to clarify how MMPs are tied up with gastrointestinal cancers. Gastrointestinal cancer is a variety of cancer types, including the cancers of gastrointestinal tract and organs, i.e., esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum and anus. The activity of MMPs is regulated by its endogenous inhibitor tissue inhibitor of metallopro-teinase(TIMP) which bind MMPs with a 1:1 stoichiometry. In addition, RECK(reversion including cysteinerich protein with kazal motifs) is a membrane bound glycoprotein that inhibits MMP-2,-9 and-14. Moreover, α2-macroglobulin mediates the uptake of several MMPs thereby inhibit their activity. Cancerous conditions increase intrinsic reactive oxygen species(ROS) through mitochondrial dysfunction leading to altered protease/anti-protease balance. ROS, an index of oxidative stress is also involved in tumorigenesis by activation of different MAP kinase pathways including MMP induction. Oxidative stress is involved in cancer by changing the activity and expression of regulatory proteins especially MMPs. Epidemiological studies have shown that high intake of fruits that rich in antioxidants is associated with a lower cancer incidence. Evidence indicates that some antioxidants inhibit the growth of malignant cells by inducing apoptosis and inhibiting the activity of MMPs. This review is discussed in six subchapters, as follows.     

Occurrence and temporal variation in matrix metalloproteinases and their inhibitors during murine secondary palatal morphogenesis

Extracellular matrix (ECM) molecules are known to play a pivotal role in morphogenesis of the secondary palate, and changes in their composition and distribution, not attributable to changes in synthesis, are known to occur during palatogenesis. The present study was undertaken to determine if the enzymes responsible for mediating their degradation, the matrix metalloproteinases (MMP), and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMP), are temporospatially regulated during murine palatal shelf morphogenesis. Palatal shelves were harvested at gestational days (gd) 12, 13 and 14. MMPs were identified by gelatin zymography, with and without inhibitors, and the identity of specific bands confirmed by Western blot analysis. TIMPs were identified by reverse zymography. MMP and TIMP messages were detected using RT-PCR with specific primers to MMPs 2, 3, 7, 9 and 13 and TIMPs 1 and 2. Zymography revealed bands of molecular weights corresponding to MMPs 2, 7, 9 and 13 at all ages examined; the intensity of these bands increased with developmental age. Western blot analysis established the presence of MMP-3 and its developmental variation in expression. RT-PCR demonstrated the presence of mRNA for all MMPs and TIMP at all sampling times and all but MMP-2 showed developmental variation. Whereas increases in mRNA were detected for MMPs 3, 9, and 13, MMP-7 mRNA decreased between gd 12 and 14. The results of this study demonstrate that MMPs 2, 3, 7, 9 and 13 and TIMPs 1 and 2 and their messages are present during the course of palatal shelf remodelling and that their expression is temporally regulated.     

A unifying hypothesis for explaining the mechanism of amyloid formation under conditions of increased oxidative stress

Amyloid related organ dysfunction is a common feature of conditions associated with chronic oxidative injury such as diabetes, inflammation, neurodegenerative disorders, renal failure, and natural aging. Matrix metalloproteinases (MMPs) are a family of calcium and zinc-dependent endopeptidases comprised of 23 enzymes in the human. Among these, MMPs 2 and 9 are known as secretable forms, present in all body fluids and susceptible to activation by oxidants. Although MMPs are generally accepted and named for their effect on extracellular matrix turnover, their non-extracellular-matrix targets have emerged recently. Cystatin C (CysC) is a very potent inhibitor of cysteine proteinases, present in all body fluids. Its solubility is determined by its N-terminal sequence. CysC is known to polimerize and form fibrils and has been isolated from amyloids. The CysC isolated from amyloids is in the N-terminal truncated form. My hypothesis regarding amyloid formation is that CysC could be a substrate for MMPs 2 and 9, which upon cleaving the N-terminal off the CysC protein will render it insoluble and promote amyloid formation. Several in vitro studies have demonstrated degradation of CysC by MMPs. The implications of such a degradation in kidney glomerules (where the clearance of CysC occurs) could be of importance for understanding the mechanism of kidney failure e.g. in diabetes. This proposed mechanism for amyloid formation through degradation of CysC by MMPs, can be proposed for all cases of CysC related amyloid formation, such as those seen in cerebrovascular, cardiac and rheumatoid disorders.     

Role of matrix metalloproteinases in skeletal muscle

Matrix metalloproteases (MMPs) are key regulatory molecules in the formation, remodeling, and degradation of extracellular matrix (ECM) components in both physiological and pathological processes in many tissues. In skeletal muscle, MMPs play an important role in the homeostasis and maintenance of myofiber functional integrity by breaking down ECM and regulating skeletal muscle cell migration, differentiation and regeneration. Skeletal muscle satellite cells, a group of quiescent stem cells located between the basement membrane and the plasmalemma of myofibers, are responsible for lifelong maintenance and repairing, which can be activated and as a result migrate underneath the basement membrane to promote regeneration at the injured site. MMPs are able to degrade ECM components, thereby facilitating satellite cell migration and differentiation. This current review will focus on the critical roles of MMPs in skeletal muscle injury and repair, which include satellite cell activation with migration and differentiation. The effect of MMPs on muscle regeneration and fibrous scar tissue formation, as well as therapeutic insights for the future will be explored.