A novel host/tumor cell interaction activates matrix metalloproteinase 1 and mediates invasion through type I collagen.

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10(-6) M) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.     

Characterization of a rainbow trout matrix metalloproteinase capable of degrading type I collagen.

Matrix metalloproteinases (MMPs) are widely distributed in vertebrate tissues and form a large family consisting of at least four distinct subfamilies. Higher vertebrate MMP-13 is well-known as collagenase-3, which represents the third member of a collagenase subfamily. In this study, we cloned cDNA coding for a unique fish homologue of human MMP-13 from a rainbow trout fibroblast cDNA library. The cDNA was 2.1 kb long and contained an open reading frame encoding a protein of 475 amino acids. The catalytic domain of the protein was 66% identical to the human counterpart with the greatest degree of identity occurring in the zinc binding site. In addition, it possessed three amino-acid residues (Tyr122, Asp233 and Gly235) characteristic of the collagenase subfamily, together with a six residue insertion which did not occur in the collagenase subfamily. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and skin type I collagen. It is worth noting that rainbow trout type I collagen was more susceptible to proteolysis with the recombinant protein when compared with the calf one. It appeared that the recombinant protein also cleaved the nonhelical regions of rainbow trout muscle type V collagen. These results have revealed that the cDNA encodes a unique MMP-13 of rainbow trout. This is the first report of cDNA coding for fish MMP capable of degrading type I collagen.     

Membrane type 1 matrix metalloproteinase and gelatinase A synergistically degrade type 1 collagen in a cell model

A fibrosarcoma cell line transfected with the matrix metalloproteinase MT1 MMP showed an enhanced ability to degrade 14C-labelled collagen films. As previously shown for proMMP 2 activation, TIMP 1 was an ineffective inhibitor of the process of collagenolysis whereas TIMP 2 was efficient and completely prevented collagen degradation. In the presence of the calcium ionophore, ionomycin, proteolytic processing of MT1 MMP was restricted and collagenolysis did not occur indicating that the 63 kDa form of the enzyme is not a functional collagenase. The collagenolytic activity of MT1 MMP was shown to be enhanced by the addition of proMMP 2, but TIMP 1 inhibition remained poor relative to that of TIMP 2. The study demonstrated that synergy between two non-conventional collagenases effectively degrades insoluble pericellular collagen. Due to the membrane localisation of MT1 MMP, this could potentially occur in a highly localised manner.     

Collagen dissolution by keratinocytes requires cell surface plasminogen activation and matrix metalloproteinase activity

Matrix metalloproteinase-14 is required for degradation of fibrillar collagen by mesenchymal cells. Here we show that keratinocytes use an alternative plasminogen and matrix metalloproteinase-13-dependent pathway for dissolution of collagen fibrils. Primary keratinocytes displayed an absolute requirement for serum to dissolve collagen. Dissolution of collagen was abolished in plasminogen-depleted serum and could be restored by the exogenous addition of plasminogen. Both plasminogen activator inhibitor-1 and tissue inhibitor of metalloproteinase blocked collagen dissolution, demonstrating the requirement of both plasminogen activation and matrix metalloproteinase activity for degradation. Cell surface plasmin activity was critical for the degradation process as aprotinin, but not alpha(2)-antiplasmin, prevented collagen dissolution. Keratinocytes with single deficiencies in either urokinase or tissue plasminogen activator retained the ability to dissolve collagen. However, collagen fibril dissolution was abolished in keratinocytes with a combined deficiency in both urokinase and tissue plasminogen activator. Combined, but not single, urokinase and tissue plasminogen activator deficiency also completely blocked the activation of the fibrillar collagenase, matrix metalloproteinase-13, by keratinocytes. The activation of matrix metalloproteinase-13 in normal keratinocytes was prevented by plasminogen activator inhibitor-1 and aprotinin but not by tissue inhibitor of metalloproteinase-1 and -2, suggesting that plasmin activates matrix metalloproteinase-13 directly. We propose that plasminogen activation facilitates keratinocyte-mediated collagen breakdown via the direct activation of matrix metalloproteinase-13 and possibly other fibrillar collagenases.     

Cause and effect relationship between myocardial mast cell number and matrix metalloproteinase activity

The objectives of this study were to investigate the temporal response of left ventricular (LV) matrix metalloproteinase (MMP) activity and collagen volume fraction (CVF) induced by an aortocaval fistula and the role of cardiac mast cells in regulating MMP activity. LV tissue was analyzed for MMP activity, CVF, and mast cell number in rats euthanized at 0.5, 1, 2, 3, 5, 14, 21, 35, and 56 days. Additional rats treated with the mast cell membrane-stabilizing drug cromolyn sodium were euthanized 1, 2, and 3 days postfistula. Marked increases in MMP activity occurred rapidly and remained significantly elevated for 5 days before returning toward normal. A significant decrease in CVF occurred by day 5, but thereafter CVF rebounded to normal or above normal values. The number of myocardial mast cells also significantly increased postfistula, and there was a close association between mast cell density and MMP activity. Cromolyn treatment prevented the increase in mast cell number and MMP activity. Thus it is concluded that cardiac mast cells play a major role in the regulation of MMP activity.     

Membrane type 1 matrix metalloproteinase (MT1-MMP) ubiquitination at Lys581 increases cellular invasion through type I collagen

Membrane type 1 matrix metalloproteinase (MT1-MMP/MMP14) is a zinc-dependent type I transmembrane metalloproteinase playing pivotal roles in the regulation of pericellular proteolysis and cellular migration. Elevated expression levels of MT1-MMP have been demonstrated to correlate with a poor prognosis in cancer. MT1-MMP has a short intracellular domain (ICD) that has been shown to play important roles in cellular migration and invasion, although these ICD-mediated mechanisms remain poorly understood. In this study, we report that MT1-MMP is mono-ubiquitinated at its unique lysine residue (Lys(581)) within the ICD. Our data suggest that this post-translational modification is involved in MT1-MMP trafficking as well as in modulating cellular invasion through type I collagen matrices. By using an MT1-MMP Y573A mutant or the Src family inhibitor PP2, we observed that the previously described Src-dependent MT1-MMP phosphorylation is a prerequisite for ubiquitination. Taken together, these findings show for the first time an additional post-translational modification of MT1-MMP that regulates its trafficking and cellular invasion, which further emphasizes the key role of the MT1-MMP ICD.     

Exosite interactions impact matrix metalloproteinase collagen specificities

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. However, the substrate structural determinants that facilitate interaction with specific MMPs are not well defined. We hypothesized that type I-III collagen sequences located N- or C-terminal to the physiological cleavage site mediate substrate selectivity among MMP-1, MMP-2, MMP-8, MMP-13, and MMP-14/membrane-type 1 (MT1)-MMP. The enzyme kinetics for hydrolysis of three fluorogenic triple-helical peptides (fTHPs) was evaluated herein. The first fTHP contained consensus residues 769-783 from type I-III collagens, the second inserted α1(II) collagen residues 763-768 N-terminal to the consensus sequence, and the third inserted α1(II) collagen residues 784-792 C-terminal to the consensus sequence. Our analyses showed that insertion of the C-terminal residues significantly increased k(cat)/K(m) and k(cat) for MMP-1. MMP-13 showed the opposite behavior with a decreased k(cat)/K(m) and k(cat) and a greatly improved K(m) in response to the C-terminal residues. Insertion of the N-terminal residues enhanced k(cat)/K(m) and k(cat) for MMP-8 and MT1-MMP. For MMP-2, the C-terminal residues enhanced K(m) and dramatically decreased k(cat), resulting in a decrease in the overall activity. These changes in activities and kinetic parameters represented the collagen preferences of MMP-8, MMP-13, and MT1-MMP well. Thus, interactions with secondary binding sites (exosites) helped direct the specificity of these enzymes. However, MMP-1 collagen preferences were not recapitulated by the fTHP studies. The preference of MMP-1 for type III collagen appears to be primarily based on the flexibility of the hydrolysis site of type III collagen compared with types I and II. Further characterization of exosite determinants that govern interactions of MMPs with collagenous substrates should aid the development of pharmacotherapeutics that target individual MMPs.     

Thrombospondin type 1 repeats interact with matrix metalloproteinase 2. Regulation of metalloproteinase activity

Thrombospondins are thought to function as inhibitors of angiogenesis. However, the mechanism(s) of this activity is not well understood. In this study, we have used the yeast two-hybrid system to identify proteins that interact with the thrombospondins 1 (TSP1) and 2 (TSP2) properdin-like type 1 repeats (TSR). One of the proteins identified that interacted with both TSR was matrix metalloproteinase 2 (MMP2). The isolated MMP2 cDNA clone encoded amino acid residues 237-633, which include the fibronectin-like gelatin binding region flanking the catalytic center and the carboxyl hemopexin-like region. Further testing of this clone demonstrated that the TSR interacted with the NH(2)-terminal region of the MMP2 that contains the catalytic domain. The protein interaction observed in yeast was further demonstrated by immunoprecipitation and Western blotting using purified intact TSP1, TSP2, MMP2, and MMP9. Although MMP2 interacted with TSP1 and TSP2 via its gelatin-binding domain or a closely mapping site, neither TSP1 nor TSP2 was degraded by MMP2 in vitro. Tissue culture and in vitro assays demonstrated that the presence of purified TSR and intact TSP1 resulted in inhibition of MMP activity. The ability of TSP1 to inhibit MMP3-dependent activation of pro-MMP9 and thrombin-induced activation of pro-MMP2 suggests that the TSPs may inhibit MMP activity by preventing activation of the MMP2 and MMP9 zymogens.     

Distinct modes of collagen type I proteolysis by matrix metalloproteinase (MMP) 2 and membrane type I MMP during the migration of a tip endothelial cell: insights from a computational model

Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the proteolytic processing of extracellular matrix (ECM) structural proteins under physiological and pathological conditions. During sprouting angiogenesis, the MMPs expressed by a single "tip" endothelial cell exhibit proteolytic activity that allows the cells of the sprouting vessel bud to migrate into the ECM. Membrane type I matrix metalloproteinase (MT1-MMP) and the diffusible matrix metalloproteinase MMP2, in the presence of the tissue inhibitor of metalloproteinases TIMP2, constitute a system of proteins that play an important role during the proteolysis of collagen type I matrices. Here, we have formulated a computational model to investigate the proteolytic potential of such a tip endothelial cell. The cell expresses MMP2 in its proenzyme form, pro-MMP2, as well as MT1-MMP and TIMP2. The interactions of the proteins are described by a biochemically detailed reaction network. Assuming that the rate-limiting step of the migration is the ability of the tip cell to carry out proteolysis, we have estimated cell velocities for matrices of different collagen content. The estimated velocities of a few microns per hour are in agreement with experimental data. At high collagen content, proteolysis was carried out primarily by MT1-MMP and localized to the cell leading edge, whereas at lower concentrations, MT1-MMP and MMP2 were found to act in parallel, causing proteolysis in the vicinity of the leading edge. TIMP2 is a regulator of the proteolysis localization because it can shift the activity of MT1-MMP from its enzymatic toward its activatory mode, suggesting a tight mechanosensitive regulation of the enzymes and inhibitor expression. The model described here provides a foundation for quantitative studies of angiogenesis in extracellular matrices of different compositions, both in vitro and in vivo. It also identifies critical parameters whose values are not presently available and which should be determined in future experiments.     

Membrane type I matrix metalloproteinase usurps tumor growth control imposed by the three-dimensional extracellular matrix

Cancer cells are able to proliferate at accelerated rates within the confines of a three-dimensional (3D) extracellular matrix (ECM) that is rich in type I collagen. The mechanisms used by tumor cells to circumvent endogenous antigrowth signals have yet to be clearly defined. We find that the matrix metalloproteinase, MT1-MMP, confers tumor cells with a distinct 3D growth advantage in vitro and in vivo. The replicative advantage conferred by MT1-MMP requires pericellular proteolysis of the ECM, as proliferation is fully suppressed when tumor cells are suspended in 3D gels of protease-resistant collagen. In the absence of proteolysis, tumor cells embedded in physiologically relevant ECM matrices are trapped in a compact, spherical configuration and unable to undergo changes in cell shape or cytoskeletal reorganization required for 3D growth. These observations identify MT1-MMP as a tumor-derived growth factor that regulates proliferation by controlling cell geometry within the confines of the 3D ECM.     

A cell surface receptor complex for collagen type I recognizes the Arg- Gly-Asp sequence

To isolate collagen-binding cell surface proteins, detergent extracts of surface-iodinated MG-63 human osteosarcoma cells were chromatographed on affinity matrices of either type I collagen-Sepharose or Sepharose carrying a collagen-like triple-helical peptide. The peptide was designed to be triple helical and to contain the sequence Arg-Gly-Asp, which has been implicated as the cell attachment site of fibronectin, vitronectin, fibrinogen, and von Willebrand factor, and is also present in type I collagen. Three radioactive polypeptides having apparent molecular masses of 250 kD, 70 kD, and 30 kD were distinguishable in that they showed affinity toward the collagen and collagen-like peptide affinity columns, and could be specifically eluted from these columns with a solution of an Arg-Gly-Asp-containing peptide, Gly-Arg-Gly-Asp-Thr-Pro. These collagen-binding polypeptides associated with phosphatidylcholine liposomes, and the resulting liposomes bound specifically to type I collagen or the collagen-like peptide but not to fibronectin or vitronectin or heat-denatured collagen. The binding of these liposomes to type I collagen could be inhibited with the peptide Gly-Arg-Gly-Asp-Thr-Pro and with EDTA, but not with a variant peptide Gly-Arg-Gly-Glu-Ser-Pro. We conclude from these data that these three polypeptides are membrane molecules that behave as a cell surface receptor (or receptor complex) for type I collagen by interacting with it through the Arg-Gly-Asp tripeptide adhesion signal. The lack of binding to denatured collagen suggests that the conformation of the Arg-Gly-Asp sequence is important in the recognition of collagen by the receptor complex.     

Integrin-mediated cell adhesion to type I collagen fibrils

In the integrin family, the collagen receptors form a structurally and functionally distinct subgroup. Two members of this subgroup, alpha(1)beta(1) and alpha(2)beta(1) integrins, are known to bind to monomeric form of type I collagen. However, in tissues type I collagen monomers are organized into large fibrils immediately after they are released from cells. Here, we studied collagen fibril recognition by integrins. By an immunoelectron microscopy method we showed that integrin alpha(2)I domain is able to bind to classical D-banded type I collagen fibrils. However, according to the solid phase binding assay, the collagen fibril formation appeared to reduce integrin alpha(1)I and alpha(2)I domain avidity to collagen and to lower the number of putative alphaI domain binding sites on it. Respectively, cellular alpha(1)beta(1) integrin was able to mediate cell spreading significantly better on monomeric than on fibrillar type I collagen matrix, whereas alpha(2)beta(1) integrin appeared still to facilitate both cell spreading on fibrillar type I collagen matrix and also the contraction of fibrillar type I collagen gel. Additionally, alpha(2)beta(1) integrin promoted the integrin-mediated formation of long cellular projections typically induced by fibrillar collagen. Thus, these findings suggest that alpha(2)beta(1) integrin is a functional cellular receptor for type I collagen fibrils, whereas alpha(1)beta(1) integrin may only effectively bind type I collagen monomers. Furthermore, when the effect of soluble alphaI domains on type I collagen fibril formation was tested in vitro, the observations suggest that integrin type collagen receptors might guide or even promote pericellular collagen fibrillogenesis.     

The cell surface: the stage for matrix metalloproteinase regulation of migration

Matrix metalloproteinases are important for the turnover of extracellular matrix in tissue. Recent studies have expanded their roles well beyond extracellular matrix degradation - they also cleave many growth factors, cytokines and cell adhesion molecules in the extracellular milieu, modulating their functions irreversibly. In particular, some matrix metalloproteinases that associate with the cell surface have arisen as intriguing regulators of cellular functions, including migration.     

Control of matrix metalloproteinase catalytic activity

As their name implies, MMPs were first described as proteases that degrade extracellular matrix proteins, such as collagens, elastin, proteoglycans, and laminins. However, studies of MMP function in vivo have revealed that these proteinases act on a variety of extracellular protein substrates, often to activate latent forms of effector proteins, such as antimicrobial peptides and cytokines, or to alter protein function, such as shedding of cell-surface proteins. Because their substrates are diverse, MMPs are involved in variety of homeostatic functions, such as bone remodeling, wound healing, and several aspects of immunity. However, MMPs are also involved in a number of pathological processes, such as tumor progression, fibrosis, chronic inflammation, tissue destruction, and more. A key step in regulating MMP proteolysis is the conversion of the zymogen into an active proteinase. Several proMMPs are activated in the secretion pathway by furin proprotein convertases, but for most the activation mechanisms are largely not known. In this review, we discuss both authentic and potential mechanisms of proMMP activation.     

Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr(573), we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.     

Hyaluronan cell surface binding is induced by type I collagen and regulated by caveolae in glioma cells

Hyaluronan (HA) is a component of the brain extracellular matrix environment that is synthesized and secreted by glioma cells. The primary cell surface receptor for HA is CD44, a membrane glycoprotein that is functionally regulated by a membrane type 1 matrix metalloproteinase (MT1-MMP). Both CD44 and MT1-MMP are partially located in Triton X-100-insoluble domains, but no functional link has yet been established between them. In the present study, we studied the regulation of HA cell surface binding in U-87 glioma cells. We show that an MMP-dependent mechanism regulates the intrinsic cell surface binding of HA as ilomastat, a broad MMP inhibitor, increased HA binding to glioma cells. HA binding was also rapidly and specifically up-regulated by 3-fold by type I collagen in U-87 cells, which also induced a significant morphological reorganization associated with the activation of a latent form of MMP-2 through a MT1-MMP-mediated mechanism. Interestingly, caveolae depletion with a cell surface cholesterol-depleting agent beta-cyclodextrin triggered an additional increase (9-fold) in the binding of HA, in synergy with type I collagen. On the other hand, HA cell surface binding was diminished by the MEK inhibitor PD98059 and by the overexpression of a recombinant, wild type MT1-MMP, whereas its cytoplasmic-deleted form had no effect. Taken together, our results suggest that MT1-MMP regulates, through its cytoplasmic domain, the cell surface functions of CD44 in a collagen-rich pericellular environment. Additionally, we describe a new molecular mechanism regulating the invasive potential of glioma cells involving a MT1-MMP/CD44/caveolin interaction, which could represent a potential target for anti-cancer therapies.     

Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis

The extracellular matrix (ECM) plays a prominent role in ovarian function by participating in processes such as cell migration, proliferation, growth, and development. Although some of these signaling processes have been characterized in the mouse, the relative quantity and distribution of ECM proteins within developing follicles of the ovary have not been characterized. This study uses immunohistochemistry and real-time PCR to characterize the ECM components type I collagen, type IV collagen, fibronectin, and laminin in the mouse ovary according to follicle stage and cellular compartment. Collagen I was present throughout the ovary, with higher concentrations in the ovarian surface epithelium and follicular compartments. Collagen IV was abundant in the theca cell compartment with low-level expression in the stroma and granulosa cells. The distribution of collagen was consistent throughout follicle maturation. Fibronectin staining in the stroma and theca cell compartment increased throughout follicle development, while staining in the granulosa cell compartment decreased. Heavy staining was also observed in the follicular fluid of antral follicles. Laminin was localized primarily to the theca cell compartment, with a defined ring at the exterior of the follicular granulosa cells marking the basement membrane. Low levels of laminin were also apparent in the stroma and granulosa cell compartment. Taken together, the ECM content of the mouse ovary changes during follicular development and reveals a distinct spatial and temporal pattern. This understanding of ECM composition and distribution can be used in the basic studies of ECM function during follicle development, and could aid in the development of in vitro systems for follicle growth.