Matriptase is an epithelial-derived, integral membrane serine protease. The enzyme was initially isolated from human breast cancer cells and has been implicated in breast cancer invasion and metastasis. In the current study, using active matriptase isolated from human milk, we demonstrate that matriptase is able to cleave various synthetic substrates with arginine or lysine as their P1 sites and prefers small side chain amino acids, such as Ala and Gly, at P2 sites. For the most reactive substrates, N-tert-butoxycarbonyl (N-t-Boc)-␥-benzylGlu-Ala-Arg-7-amino-4-methylcoumarin (AMC) and N-tBoc-Gln-Ala-Arg-AMC, the K m values were determined to be 3.81 and 4.89 M, respectively. We further demonstrated that matriptase can convert hepatocyte growth factor/scattering factor to its active form, which can induce scatter of Madin-Darby canine kidney epithelial cells and can activate c-Met tyrosine phosphorylation in A549 human lung carcinoma cells. In addition, we noted that matriptase can activate urokinase plasminogen activator but has no affect on plasminogen. These results suggest that matriptase could act as an epithelial, upstream membrane activator to recruit and activate stromal-derived downstream effectors important for extracellular matrix degradation and epithelial migration, two major events of tissue remodeling, cancer invasion, and metastasis.
Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P 1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P 1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P 1 . We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P 1 , VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P 1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and -arrestins abolished FTY720-P-induced S1P 1 receptor degradation. These data suggest that agonistinduced phosphorylation of S1P 1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P 1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P 1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P 1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.Sphingosine 1-phosphate (S1P) 2 is recognized as a multifunctional bioactive lipid mediator involved in immune cell trafficking, regulation of vascular permeability, and angiogenesis (1, 2). It acts via a family of G protein-coupled receptors referred to as S1P n receptors (3). The prototypical receptor, S1P 1 was originally isolated as an inducible gene from vascular endothelial cells (4). Knock out of S1P 1 resulted in embryonic lethality due to a vascular maturation defect (5). We recently showed that S1P 1 function in endothelial cells is needed for proper endothelial-pericyte interaction, a critical event in vascular maturation (6). In addition, we demonstrated previously that S1P 1 is needed for the assembly of vascular endothelialcadherin-based adherens junctions on vascular endothelial cells (7). This event is needed for regulation of paracellular permeability, a model system vascular leak syndrome (8). In the immune system, selective deletion of S1P 1 in T-cells led to inhibition of lymphocyte egress from lymph nodes and the thymus (9, 10). However, function of S1P 1 in efferent lymphatics may also be important for the regulation of lymphocyte egress, as activation of this receptor may lead to "gate clos...
Matriptase, a trypsin-like serine protease with two potential regulatory modules (low density lipoprotein receptor and complement C1r/s domains), was initially purified from T-47D breast cancer cells. Given its plasma membrane localization, extracellular matrix-degrading activity, and expression by breast cancer cells, this protease may be involved in multiple aspects of breast tumor progression, including cancer invasion. In breast cancer cells, matriptase was detected mainly as an uncomplexed form; however, low levels of matriptase were detected in complexes. In striking contrast, only the complexed matriptase was detected in human milk. The complexed matriptase has now been purified. Amino acid sequences obtained from the matriptaseassociated proteins reveal that they are fragments of a Kunitz-type serine protease inhibitor that was previously reported to be an inhibitor of the hepatocyte growth factor activator. In addition, matriptase and its complexes were detected in milk-derived, SV40 T-antigen-immortalized mammary luminal epithelial cell lines, but not in human foreskin fibroblasts or in HT-1080 fibrosarcoma cells. These results suggest that the milk-derived matriptase complexes are likely to be produced by the epithelial components of the lactating mammary gland in vivo and that the activity and function of matriptase may be differentially regulated by its cognate inhibitor, comparing breast cancer with the lactating mammary gland.Matriptase is a trypsin-like serine protease with two regulatory modules: two tandem repeats of the complement subcomponent C1r/s domain and four tandem repeats of the low density lipoprotein receptor domain (1). Matriptase was initially identified from T-47D human breast cancer cells as a major gelatinolytic activity on a gelatin zymogram, with a migration rate between those of gelatinase A (72 kDa; MMP-2) and gelatinase B (92 kDa; MMP-9) (2); it has been proposed to play a role in breast cancer invasion (3). The primary cleavage specificity of matriptase was identified to be arginine and lysine residues, similar to the majority of serine proteases, including trypsin and plasmin. In addition, matriptase, as does trypsin, exhibits broad spectrum cleavage activity, and such activity is likely to contribute to its gelatinolytic activity on a gelatin zymogram.HAI-1 (hepatocyte growth factor activator inhibitor-1) (4) is a Kunitz-type serine protease inhibitor that is able to inhibit the hepatocyte growth factor (HGF) 1 activator, a blood coagulation factor XII-like serine protease (5). The mature form of this protease inhibitor has 478 amino acid residues, with a calculated molecular mass of 53,319 Da. A putative transmembrane domain is located at its carboxyl terminus. HAI-1 contains two Kunitz domains (domain I spans residues 246 -306, and domain II spans residues 371-431) separated by a low density lipoprotein receptor domain (residues 315-360). The presumed P1 residue of the active-site cleft is likely to be arginine 260 in Kunitz domain I and lysine 385 in domain II by alig...
A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease.Elevated proteolytic activity has been implicated in neoplastic progression. Although the exact role(s) of proteolytic enzymes in the progression of tumor remains unclear, it seems that proteases may be involved in almost every step of the development and spread of cancer. A widely proposed view is that proteases contribute to the degradation of extracellular matrix and to tissue remodeling and are necessary for cancer invasion and metastasis. A wide array of extracellular matrixdegrading proteases have been discovered, the expression of some of which correlates with tumor progression, as reviewed by Magnatti and Rifkin (1). The plasmin/urokinase-type plasminogen activator system and the 72-kDa gelatinase (MMP-2)/ membrane-type MMP system have received the most attention for their potential roles in the process of invasion of breast cancer and other carcinomas. However, both systems appear to be largely synthesized by stromal cells in vivo (2-5) and require indirect mechanisms for their recruitment and activation on the surfaces of cancer cells. The stromal origins of these well characterized extracellular matrix-degrading proteases may suggest that cancer invasion is an event that either depends entirely upon stromal-epithelial cooperation or is controlled by some other unknown epithelium-derived protease(s). A search for these epithelium-derived proteolytic systems that may interact with the plasmin/urokinase-type plasminogen activator system and/or with the MMP family could provide a missing link in our understanding of malignant invasion.We have pursued studies of a novel protease with the hypothesis that a tumor itself may be a major source of proteases important for multiple aspects of malignant behavior, including invasion and ...
Matriptase and its cognate, Kunitz-type serine protease inhibitor, HAI-1, comprise a newly characterized extracellular matrix-degrading protease system that may function as an epithelial membrane activator for other proteases and latent growth factors. Both enzyme and inhibitor have been detected in breast cancer cells, immortalized mammary epithelial cells, and human milk, but not in cultured fibroblasts nor in fibrosarcoma cells. To test the hypothesis that this system is expressed by normal breast epithelium, invasive breast cancers, and other cancers of an epithelial origin (carcinomas) but not in cancers of a mesenchymal origin, we have expanded our expression analysis of matriptase and HAI-1 in vitro and in vivo. Matriptase and HAI-1 were detected at the protein and mRNA levels both in hormone-dependent and hormone-independent cultured breast cancer cells, and this expression correlated with the expression of the epithelial markers E-cadherin or ZO-1. However, none of the breast cancer cell lines tested that express the mesenchymal marker vimentin express matriptase or HAI-1, consistent with an epithelial-selective expression of this system. Expression of matriptase, as determined by Western blot analysis, was observed in primary human breast, gynecological, and colon carcinomas, but not in stromal-derived ovarian tumors and human sarcomas of various origins and histological grades. It has long been proposed that metastasis is a multistep process. This includes the breakdown of the basement membrane, detachment of cancer cells from the primary tumor, invasion into the stroma, intravasation into blood vessels, survival in the blood stream, extravasation through target organ blood vessels, and the establishment and proliferation of cancer cells in remote tissues. To accomplish these events, cancer cells must acquire an enhanced ability to migrate through and degrade extracellular matrix components. An array of extracellular matrix-degrading proteases and cell motility factors have been characterized and implicated in cancer invasion and metastasis.1 Among the protease systems, the plasmin/urokinase-type plasminogen activator (uPA) system, 2-6 and the matrix metalloproteases 7-12 have received the most attention. Although these extracellular matrix-degrading proteases have been implicated in breast cancer invasion and metastasis, they are mainly expressed by stromal components of human breast tumors. 9,11,[13][14][15][16] The stromal origins of these extracellular matrix-degrading proteases in breast cancer suggests that malignant invasion is an event that depends at least in part on a stromal-epithelial interaction. 17 Furthermore, growth and motility factors secreted by stromal cells may also contribute to the ability of cancer cells to migrate through the extracellular matrix. Hepatocyte growth factor (HGF)/scattering factor (SF) is one of these mesenchymal cell-derived proteins. On binding to the c-Met receptor on the surfaces of epithelial cells, HGF can dissociate epithelial colonies and scatter cells. ...
Matriptase is an epithelial-derived, integral membrane, trypsin-like serine protease. We have shown previously that matriptase exists both in complexed and noncomplexed forms. We now show that the complexed matriptase is an activated, two-chain form, which is inhibited in an acidsensitive, reversible manner through binding to its cognate, Kunitz-type inhibitor, HAI-1 (hepatocyte growth factor activator inhibitor-1). Conversely, the majority of the noncomplexed matriptase is a single-chain zymogen, which lacks binding affinity to HAI-1, suggesting that matriptase, similar to most other serine proteases, is activated by proteolytic cleavage at a canonical activation motif. We have now generated mAbs specific for the conformational changes associated with the proteolytic activation of matriptase. Using these mAbs, which specifically recognize the twochain form of matriptase, we demonstrate that matriptase is transiently activated on 184A1N4 human mammary epithelial cell surfaces following their exposure to serum. The ability of serum to activate matriptase is highly conserved across reptilian, avian, and mammalian species. This serum-dependent activation of matriptase on epithelial cell surfaces is followed by ectodomain shedding of both matriptase and its Kunitz-type inhibitor.
.-In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1).In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from cell homogenates by Triton X-100 or sonication of cell homogenates completely inhibited the effect, suggesting that matriptase activation requires proper lipid bilayer microenvironments, potentially allowing appropriate interactions of matriptase zymogens with HAI-1 and other components. Matriptase activation occurred in a narrow pH range (from pH 5.2 to 7.2), with a sharp increase in activation at the transition from pH 5.2 to 5.4, and could be completely suppressed by moderately increased ionic strength. Protease inhibitors only modestly affected activation, whereas 30 nM (5 g/ml) of anti-matriptase LDL receptor domain 3 monoclonal antibodies completely blocked activation. These atypical biochemical features are consistent with a mechanism for autoactivation of matriptase that requires protein-protein interactions but not active proteases.hepatocyte growth factor activator inhibitor 1; protease activation; low-density lipoprotein MATRIPTASE AND HEPATOCYTE growth factor activator inhibitor 1 (HAI-1) are a pair of epithelium-derived, membrane-associated proteins: a proteolytic enzyme and its cognate inhibitor, respectively (22, 49). Matriptase, a member of the type II transmembrane serine protease (8,35,50), contains a transmembrane domain at the amino terminus, followed by a sperm protein, enterokinase, and agrin (SEA) domain, two tandem C1r/s, Uegf, and bone morphogenic protein-1 (CUB) domains, four tandem LDL receptor class A domains, and a trypsin-like serine protease domain (15,22,23,52,54). HAI-1, a type 1 transmembrane protein, contains two Kunitz-type serine protease inhibitor domains and an LDL receptor class A domain (46). Matriptase and HAI-1 are broadly expressed and may have diverging functions in the epithelial cells of most epithelium-containing tissues (15,22,26,36,39,52,54). For example, matriptase was shown to be required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis in matriptase knockout mice (25). The roles of matriptase in epidermal differentiation apparent...
Matriptase, a membrane-tethered serine protease, plays essential roles in epidermal differentiation and barrier function, largely mediated via its activation of prostasin, a glycosylphosphatidylinositol-anchored serine protease. Matriptase activity is tightly regulated by its inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) such that free active matriptase is only briefly available to act on its substrates. In the current study we provide evidence for how matriptase activates prostasin under this tight control by HAI-1. When primary human keratinocytes are induced to differentiate in a skin organotypic culture model, both matriptase and prostasin are constitutively activated and then inhibited by HAI-1. These processes also occur in HaCaT human keratinocytes when matriptase activation is induced by exposure of the cells to a pH 6.0 buffer. Using this acid-inducible activation system we demonstrate that prostatin activation is suppressed by matriptase knockdown and by blocking matriptase activation with sodium chloride, suggesting that prostatin activation is dependent on matriptase in this system. Kinetics studies further reveal that the timing of autoactivation of matriptase, prostasin activation, and inhibition of both enzymes by HAI-1 binding are closely correlated. These data suggest that, during epidermal differentiation, the matriptase-prostasin proteolytic cascade is tightly regulated by two mechanisms: 1) prostasin activation temporally coupled to matriptase autoactivation and 2) HAI-1 rapidly inhibiting not only active matriptase but also active prostasin, resulting in an extremely brief window of opportunity for both active matriptase and active prostasin to act on their substrates.Matriptase, a type II transmembrane serine protease, plays essential roles in epidermal differentiation and barrier function (1, 2). Mutations of matriptase are associated with autosomal recessive ichthyosis and hypotrichosis (3). Four mutations have been identified in ST14, the gene that encodes for matriptase (3-5). Targeted deletion of matriptase in mice also causes severe epidermal defects with impairment of desquamation, lipid matrix formation, and profilaggrin processing. These defects lead to compromised epidermal barrier function and postnatal death (2). The severe epidermal defects appear to result from the lack of activation of the glycosylphosphatidylinositolanchored serine protease prostasin by matriptase, because both matriptase-deficient and prostasin-deficient mice share an almost identical pattern of epidermal defects. Furthermore, lack of prostasin activation was observed in matriptase-deficient mouse skin (6, 7), suggesting that much of the function of matriptase in the epidermis is manifested through the activation of prostasin.Hepatocyte growth factor activator inhibitor-1 (HAI-1) 2 is a transmembrane Kunitz-type serine protease inhibitor (8). HAI-1 can inhibit matriptase in a competitive and reversible manner, consistent with the characteristics of Kunitz-type serine protease inhibitors (...
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