Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1–deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2–deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.
Vertebrate embryogenesis is dependent upon a series of precisely coordinated cell proliferation, migration, and differentiation events. Recently, the execution of these events was shown to be guided in part by extracellular cues provided by focal pericellular proteolysis by a newly identified family of membrane-anchored serine proteases. We now show that two of these membrane-anchored serine proteases, prostasin and matriptase, constitute a single proteolytic signaling cascade that is active at multiple stages of development. Furthermore, we show that failure to precisely regulate the enzymatic activity of both prostasin and matriptase by two developmentally co-expressed transmembrane serine protease inhibitors, hepatocyte growth factor activator inhibitor-1 and -2, causes an array of developmental defects, including clefting of the embryonic ectoderm, lack of placental labyrinth formation, and inability to close the neural tube. Our study also provides evidence that the failure to regulate the prostasin–matriptase cascade may derail morphogenesis independent of the activation of known protease-regulated developmental signaling pathways. Because hepatocyte growth factor activator inhibitor–deficiency in humans is known to cause an assortment of common and rare developmental abnormalities, the aberrant activity of the prostasin–matriptase cascade identified in our study may contribute importantly to genetic as well as sporadic birth defects in humans.
Matriptase, a membrane-associated serine protease, plays an essential role in epidermal barrier function through activation of the glycosylphosphatidylinositol (GPI)-anchored serine protease prostasin. The matriptase-prostasin proteolytic cascade is tightly regulated by hepatocyte growth factor activator inhibitor (HAI)-1 such that matriptase autoactivation and prostasin activation occur simultaneously and are followed immediately by the inhibition of both enzymes by HAI-1. However, the mechanisms whereby matriptase acts on extracellular substrates remain elusive. Here we report that some active matriptase can escape HAI-1 inhibition by being rapidly shed from the cell surface. In the pericellular environment, shed active matriptase is able to activate hepatocyte growth factor (HGF), accelerate plasminogen activation, and shed syndecan 1. The amount of active matriptase shed is inversely correlated with the amount of antithrombin (AT) bound to the surface of the keratinocytes. Binding of AT to the surface of keratinocytes is dependent on a functional heparin binding site, Lys-125, and that the N-glycosylation site Asn-135 be unglycosylated. This suggests that β-AT, and not α-AT, is responsible for regulation of pericellular matriptase activity in keratinocytes. Keratinocytes appear to rely on AT to regulate the level of pericellular active matriptase much more than breast and prostate epithelial cells in which AT regulation of matriptase activity occurs at much lower levels than keratinocytes. These results suggest that keratinocytes employ two distinct serine protease inhibitors to control the activation and processing of two different sets of matriptase substrates leading to different biological events: 1) HAI-1 for prostasin activation/inhibition, and 2) AT for the pericellular proteolysis involved in HGF activation, accelerating plasminogen activation, and shedding of syndecans.
The type 2 transmembrane serine protease matriptase is broadly expressed in human carcinomas and hematological cancers. The proteolytic activity of matriptase is a potential target of drugs and imaging probes. We assessed the fate of active matriptase following the induction of matriptase zymogen activation. Exposing eight human carcinoma cells to pH 6.0 buffer induced robust matriptase zymogen activation followed by rapid inhibition of the nascent active matriptase by hepatocyte growth factor activator inhibitor (HAI)-1. Consequently, no enzymatically active matriptase was detected in these cells. Some active matriptase is, however, rapidly shed to the extracellular milieu by these carcinoma cells. The lack of cell-associated active matriptase and the shedding of active matriptase were also observed in two hematological cancer lines. Matriptase shedding is correlated closely with the induction of matriptase activation, suggesting that matriptase activation and shedding are kinetically coupled. The coupling allows a proportion of active matriptase to survive HAI-1 inhibition by rapid shedding from cell surface. Our study suggests that cellular free, active matriptase is scarce and might not be an effective target for in vivo imaging and drug development.
Matriptase is a member of the family of type II transmembrane serine proteases that is essential for development and maintenance of several epithelial tissues. Matriptase is synthesized as a single-chain zymogen precursor that is processed into a two-chain disulfide-linked form dependent on its own catalytic activity leading to the hypothesis that matriptase functions at the pinnacle of several protease induced signal cascades. Matriptase is usually found in either its zymogen form or in a complex with its cognate inhibitor hepatocyte growth factor activator inhibitor 1 (HAI-1), whereas the active non-inhibited form has been difficult to detect. In this study, we have developed an assay to detect enzymatically active non-inhibitor-complexed matriptase by using a biotinylated peptide substrate-based chloromethyl ketone (CMK) inhibitor. Covalently CMK peptide-bound matriptase is detected by streptavidin pull-down and subsequent analysis by Western blotting. This study presents a novel assay for detection of enzymatically active matriptase in living human and murine cells. The assay can be applied to a variety of cell systems and species.
Matriptase is a type II transmembrane serine protease containing two complement proteases C1r/C1s–urchin embryonic growth factor–bone morphogenetic protein domains (CUB repeat) and four low-density lipoprotein receptor class A domains (LDLRA repeat). The single-chain zymogen of matriptase has been found to exhibit substantial protease activity, possibly causing its own activation (i.e. conversion to a disulfide-linked two-chain fully active form), although the activation seems to be mediated predominantly by two-chain molecules. Our aim was to assess the roles of CUB and LDLRA repeats in zymogen activation. Transient expression studies of soluble truncated constructs of recombinant matriptase in COS-1 cells showed that the CUB repeat had an inhibitory effect on zymogen activation, possibly because it facilitated the interaction of two-chain molecules with a matriptase inhibitor, hepatocyte growth factor activator inhibitor type-1. By contrast, the LDLRA repeat had a promoting effect on zymogen activation. The effect of the LDLRA repeat seems to reflect its ability to increase zymogen activity. The proteolytic activities were higher in pseudozymogen forms of recombinant matriptase containing the LDLRA repeat than in a pseudozymogen without the repeat. Our findings provide new insights into the roles of these non-catalytic domains in the generation of active matriptase.
CUB repeat; hepatocyte growth factor activator inhibitor type-1; LDLRA repeat; matriptase; zymogen activation
The type II transmembrane serine protease (TTSP) family consists of eighteen closely related serine proteases that are implicated in multiple functions. To identify selective, inhibitory antibodies against one particular TTSP, matriptase (MT-SP1), a phage display library with a natural repertoire of Fabs from human naïve B cells was created. Fab A11 was identified with a 720 picomolar inhibition constant and high specificity for matriptase over other trypsin-fold serine proteases. A Trichoderma reesei system expressed A11 with ~200 mg/L yield. The crystal structure of A11 in complex with matriptase has been determined and compared to the crystal structure of another antibody inhibitor (S4) in complex with matriptase. Previously discovered from a synthetic scFv library, S4 is also a highly selective and potent matriptase inhibitor. The crystal structures of the A11/matriptase and S4/matriptase complexes were solved to 2.1 Å and 1.5 Å respectively. Although these antibodies, discovered from separate libraries, interact differently with the protease surface loops for their specificity, the structures reveal a similar novel mechanism of protease inhibition. Through the insertion of the H3 variable loop in a reverse orientation at the substrate-binding pocket, these antibodies bury a large surface area for potent inhibition and avoid proteolytic inactivation. This discovery highlights the critical role the antibody scaffold plays in positioning loops to bind and inhibit protease function in a highly selective manner. Additionally, Fab A11 is a fully human antibody that specifically inhibits matriptase over other closely related proteases, suggesting this approach could be useful for clinical applications.
antibody; specificity; matriptase; structure; protease inhibitor
Matriptase is a type II transmembrane protease that is characterized by an N-terminal transmembrane and multiple extracellular domains, in addition to the conserved extracellular serine protease catalytic domain. The expression pattern of matriptase suggests that this protease may play broad roles in the biology of surface lining epithelial cells. In this study we report that α1-antitrypsin (AAT), an endogenous inhibitor of serine proteases, inhibits the catalytic domain of human recombinant matriptase in vitro. Co-incubation of AAT with matriptase (at a molar ratio 1:2) resulted in the formation of heat stable complexes, clearly seen in sodium dodecyl sulfate electrophoresis and Western blots. AAT was found to be a slow, tight-binding inhibitor of the catalytic domain of matriptase with a second order reaction rate constant of 0.31 × 103 M−1s−1. Notably, the oxidized form of AAT, which lacks serine protease inhibitor activity, failed to generate matriptase complexes and to inhibit matriptase activity. Since matriptase is involved in a number of physiologic processes, including activation of epithelial sodium channels, our findings offer considerable new insights into new regulatory function of AAT in vivo.
serine proteases; α1-antitrypsin; matriptase; complex formation; kinetics
Matriptase, a type II transmembrane serine protease, has been linked to initiation and promotion of epidermal carcinogenesis in a murine model, suggesting that deregulation of its role in epithelia contributes to transformation. In human prostate cancer, matriptase expression correlates with progression. It is therefore of interest to determine how matriptase may contribute to epithelial neoplastic progression. One approach for studying this is to identify potential matriptase substrates involved in epithelial integrity and/or transformation like the extracellular matrix macromolecule, laminin-332 (Ln-332), which is found in the basement membrane of many epithelia, including prostate. Proteolytic processing of Ln-332 regulates cell motility of both normal and transformed cells, which has implications in cancer progression.
In vitro cleavage experiments were performed with purified Ln-332 protein and matriptase. Western blotting, enzyme inhibition assays, and mass spectrometry were used to confirm cleavage events. Matriptase overexpressing LNCaP prostate cancer cells were generated and included in Transwell migration assays and single cell motility assays, along with other prostate cells.
We report that matriptase proteolytically cleaves Ln-332 in the β3 chain. Substrate specificity was confirmed by blocking cleavage with the matriptase inhibitor, Kunitz domain-1. Transwell migration assays showed that DU145 cell motility was significantly enhanced when plated on matriptase-cleaved Ln-332. Similarly, Transwell migration of matriptase-overexpressing LNCaP cells was significantly increased on Ln-332 and, as determined by live single-cell microscopy, two motility parameters of this cell line, speed and directional persistence, were also higher.
Proteolytic processing of Ln-332 by matriptase enhances speed and directional persistence of prostate cancer cells.
laminin-332; matriptase; type II transmembrane serine protease; proteolysis; prostate cancer; cell migration
Hepatocyte growth factor/scatter factor (HGF/SF) plays critical roles in cancer progression through its specific receptor, MET. HGF/SF is usually synthesized and secreted as an inactive proform (pro-HGF/SF) by stromal cells, such as fibroblasts. Several serine proteases are reported to convert pro-HGF/SF to mature HGF/SF and among these, HGF activator (HGFA) and matriptase are the most potent activators. Increased activities of both proteases have been observed in various cancers. HGFA is synthesized mainly by the liver and secreted as an inactive pro-form. In cancer tissues, pro-HGFA is likely activated by thrombin and/or human kallikrein 1-related peptidase (KLK)-4 and KLK-5. Matriptase is a type II transmembrane serine protease that is expressed by most epithelial cells and is also synthesized as an inactive zymogen. Matriptase activation is likely to be mediated by autoactivation or by other trypsin-like proteases. Recent studies revealed that matriptase autoactivation is promoted by an acidic environment. Given the mildly acidic extracellular environment of solid tumors, matriptase activation may, thus, be accelerated in the tumor microenvironment. HGFA and matriptase activities are regulated by HGFA inhibitor (HAI)-1 (HAI-1) and/or HAI-2 in the pericellular microenvironment. HAIs may have an important role in cancer cell biology by regulating HGF/SF-activating proteases.
hepatocyte growth factor; hepatocyte growth factor activator; matriptase; TTSP; HAI-1; HAI-2
belongs to trypsin-like serine proteases involved in matrix remodeling/degradation,
growth regulation, survival, motility, and cell morphogenesis. Herein,
we report a structure-based approach, which led to the discovery of
sulfonamide and amide derivatives of pyridyl bis(oxy)benzamidine as
potent and selective matriptase inhibitors. Co-crystal structures
of selected compounds in complex with matriptase supported compound
designing. Additionally, WaterMap analyses indicated the possibility
of occupying a distinct pocket within the catalytic domain, exploration
of which resulted in >100-fold improvement in potency. Co-crystal
structure of 10 with matriptase revealed critical interactions
leading to potent target inhibition and selectivity against other
Matriptase; pyridyl dibenzimidamide; SAR; crystal structure; cancer
The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival.
Development of mammalian embryos is dependent on an efficient exchange of nutrients, oxygen, and waste products between the mother and the embryo. The interface between the two systems is provided by the placenta in a form of a specialized epithelium that both facilitates the transport of molecules between the mother and the embryo and screens the substances that can pass between the maternal and fetal tissues. We now show that two independent signaling pathways that include the serine proteases, matriptase and prostasin, and a G protein-coupled receptor PAR-2, are critical for the establishment of a functional feto-maternal interface by specifically regulating the barrier properties of the placental epithelium. Because aberrant formation of epithelial barriers is an underlying feature of a great variety of human developmental abnormalities, the identification of the two protease-dependent signaling pathways critical for the barrier formation in embryonic tissues may help pinpoint molecular mechanisms involved in the etiology of these conditions.
Cleavage activation of the hemagglutinin (HA) precursor is an essential step in the influenza virus replication cycle that is driven by host cell proteases. HA cleavage activation is required for virus-endosome membrane fusion and the subsequent release of the influenza virus genome into the cytoplasm. Previous studies have determined that HA cleavage is most likely driven by either membrane-bound or extracellular trypsin-like proteases that reside in the respiratory tract. However, there is still uncertainty regarding which proteases are critical for HA cleavage in vivo. Therefore, further investigation of HA cleavage activation is needed in order to gain insight into the critical proteases involved. Matriptase is a member of the type II transmembrane serine protease family that is highly expressed in a membrane-bound form throughout the respiratory tract. One feature of matriptase is that, once activated, the catalytic domain is secreted into the extracellular space and so serves as a functional extracellular protease. In this study, we have determined that the secreted, catalytic domain of matriptase has the ability to cleave and activate HA from the influenza virus H1 subtype but not the H2 and H3 subtypes. Furthermore, matriptase selectively cleaved the HA of particular strains within the H1 subtype, revealing both subtype and H1 strain specificity. Matriptase was also found to activate thrombolytic zymogens that have been shown to cleave and activate the influenza virus HA. Our data demonstrate that matriptase has the ability to cleave HA directly or indirectly by activating HA-cleaving zymogens.
Matriptase is a type II transmembrane serine protease that is found on the surfaces of epithelial cells and certain cancer cells. Matriptase has been implicated in the degradation of certain extracellular matrix components as well as the activation of various cellular proteins and proteases, including hepatocyte growth factor and urokinase. Sunflower trypsin inhibitor-1 (SFTI-1), a cyclic peptide inhibitor originally isolated from sunflower seeds, exhibits potent inhibitory activity toward matriptase.
We have engineered and produced recombinant proteins of the matriptase protease domain, and have determined the crystal structures of the protease:SFTI-1 complex at 2.0 Å as well as the protease:benzamidine complex at 1.2 Å. These structures elaborate the structural basis of substrate selectivity of matriptase, and show that the matriptase S1 substrate specificity pocket is larger enough to allow movement of benzamidine inside the S1 pocket. Our study also reveals that SFTI-1 binds to matriptase in a way similar to its binding to trypsin despite the significantly different isoelectric points of the two proteins (5.6 vs. 8.2).
This work helps to define the structural basis of substrate specificity of matriptase and the interactions between the inhibitor and protease. The complex structure also provides a structural template for designing new SFTI-1 derivatives with better potency and selectivity against matriptase and other proteases.
Influenza A viruses of the subtype H9N2 circulate worldwide and have become highly prevalent in poultry in many countries. Moreover, they are occasionally transmitted to humans, raising concern about their pandemic potential. Influenza virus infectivity requires cleavage of the surface glycoprotein hemagglutinin (HA) at a distinct cleavage site by host cell proteases. H9N2 viruses vary remarkably in the amino acid sequence at the cleavage site, and many isolates from Asia and the Middle East possess the multibasic motifs R-S-S-R and R-S-R-R, but are not activated by furin. Here, we investigated proteolytic activation of the early H9N2 isolate A/turkey/Wisconsin/1/66 (H9-Wisc) and two recent Asian isolates, A/quail/Shantou/782/00 (H9-782) and A/quail/Shantou/2061/00 (H9-2061), containing mono-, di-, and tribasic HA cleavage sites, respectively. All H9N2 isolates were activated by human proteases TMPRSS2 (transmembrane protease, serine S1 member 2) and HAT (human airway trypsin-like protease). Interestingly, H9-782 and H9-2061 were also activated by matriptase, a protease widely expressed in most epithelia with high expression levels in the kidney. Nephrotropism of H9N2 viruses has been observed in chickens, and here we found that H9-782 and H9-2061 were proteolytically activated in canine kidney (MDCK-II) and chicken embryo kidney (CEK) cells, whereas H9-Wisc was not. Virus activation was inhibited by peptide-mimetic inhibitors of matriptase, strongly suggesting that matriptase is responsible for HA cleavage in these kidney cells. Our data demonstrate that H9N2 viruses with R-S-S-R or R-S-R-R cleavage sites are activated by matriptase in addition to HAT and TMPRSS2 and, therefore, can be activated in a wide range of tissues what may affect virus spread, tissue tropism and pathogenicity.
Influenza viruses do not encode any proteases and must rely on host proteases for the proteolytic activation of their surface hemagglutinin proteins in order to fuse with the infected host cells. Recent progress in the understanding of human proteases responsible for influenza virus hemagglutinin activation has led to the identification of members of the type II transmembrane serine proteases TMPRSS2 and TMPRSS4 and human airway trypsin-like protease; however, none has proved to be the sole enzyme responsible for hemagglutinin cleavage. In this study, we identify and characterize matriptase as an influenza virus-activating protease capable of supporting multicycle viral replication in the human respiratory epithelium. Using confocal microscopy, we found matriptase to colocalize with hemagglutinin at the apical surface of human epithelial cells and within endosomes, and we showed that the soluble form of the protease was able to specifically cleave hemagglutinins from H1 virus, but not from H2 and H3 viruses, in a broad pH range. We showed that small interfering RNA (siRNA) knockdown of matriptase in human bronchial epithelial cells significantly blocked influenza virus replication in these cells. Lastly, we provide a selective, slow, tight-binding inhibitor of matriptase that significantly reduces viral replication (by 1.5 log) of H1N1 influenza virus, including the 2009 pandemic virus. Our study establishes a three-pronged model for the action of matriptase: activation of incoming viruses in the extracellular space in its shed form, upon viral attachment or exit in its membrane-bound and/or shed forms at the apical surface of epithelial cells, and within endosomes by its membrane-bound form where viral fusion takes place.
Recent investigations point to an important role for peptidases in regulating transcellular ion transport by the epithelial Na+ channel, ENaC. Several peptidases, including furins and proteasomal hydrolases, modulate ENaC maturation and disposal. More idiosyncratically, apical Na+ transport by ENaC in polarized epithelia of kidney, airway, and gut is stimulated constitutively by one or more trypsin-family serine peptidases, as revealed by inhibition of amiloride-sensitive Na+ transport by broad-spectrum antipeptidases, including aprotinin and bikunin/SPINT2. In vitro, the transporting activity of aprotinin-suppressed ENaC can be restored by exposure to trypsin. The prototypical channel-activating peptidase (CAP) is a type 1 membrane-anchored tryptic peptidase first identified in Xenopus kidney cells. Frog CAP1 strongly upregulates Na+ transport when coexpressed with ENaC in oocytes. The amphibian enzyme's apparent mammalian orthologue is prostasin, otherwise known as CAP1, which is coexpressed with ENaC in a variety of epithelia. In airway cells, prostasin is the major basal regulator of ENaC activity, as suggested by inhibition and knockdown experiments. Other candidate regulators of mature ENaC include CAP2/TMPRSS4 and CAP3/matriptase (also known as membrane-type serine protease 1/ST14). Mammalian CAPs are potential targets for treatment of ENaC-mediated Na+ hyperabsorption by the airway in cystic fibrosis (CF) and by the kidney in hypertension. CAPs can be important for mammalian development, as indicated by embryonic lethality in mice with null mutations of CAP1/prostasin. Mice with selectively knocked out expression of CAP1/prostasin in the epidermis and mice with globally knocked out expression of CAP3/matriptase exhibit phenotypically similar defects in skin barrier function and neonatal death from dehydration. In rats, transgenic overexpression of human prostasin disturbs salt balance and causes hypertension. Thus, several converging lines of evidence indicate that ENaC function is regulated by peptidases, and that such regulation is critical for embryonic development and adult function of organs such as skin, kidney, and lung.
The cell surface protease membrane-type serine protease 1 [MT-SP1]/matriptase is often upregulated in epithelial cancers. A dysregulation in MT-SP1/matriptase levels with respect to its cognate inhibitor hepatocyte growth factor activator inhibitor-1 [HAI-1] suggests that it is an increase in proteolytic activity that significantly differentiates malignant from normal tissue. Here we use antibodies to demonstrate that MT-SP1 is active on cancer cells and that this activity may be targeted for tumor detection in vivo. A proteolytic activity assay with the MT-SP1-positive human cancer cell lines MCF-7, HT29, LNCaP, and MDA-MB-468 showed that the antibodies, which inhibit recombinant catalytic MT-SP1, are able to bind and inhibit the full-length enzyme. The same experiment with the MT-SP1-negative breast cancer cell lines MDA-MB-231, COLO 320DM and HT1080 showed no inhibition of proteolysis. Fluorescent microscopy then confirmed localization of labeled antibodies to the surface of MT-SP1-positive cells. To evaluate these antibodies as probes for targeting MT-SP1 activity in vivo, 0.7-2 nanomoles of fluorescently labeled antibodies were administered to xenograft mouse cancer models. The antibodies localized to the MT-SP1-positive MCF-7 and MCF-7/Luc+ tumors (n=3), permitting visualization of MT-SP1 activity. Fluorescence was not observed in MT-SP1-negative MDA-MD-231/Luc+ tumors (n=2), suggesting that MT-SP1 activity is a novel biomarker for epithelial cancer and these antibodies provide a non-invasive method for detecting this activity in vivo.
Cancer; MT-SP1; Matriptase; Protease Activity
The liver peptide hepcidin regulates body iron, is upregulated in iron overload and inflammation and downregulated in iron deficiency/hypoxia. The transmembrane serine protease matriptase-2 (TMPRSS6) inhibits the hepcidin response and its mutational inactivation causes iron-deficient anemia in mice and humans. Here we confirm the inhibitory effect of matriptase-2 on hepcidin promoter; we show that matriptase-2 lacking the serine protease domain, identified in the anemic Mask mouse (matriptase-2MASK), is fully inactive and that mutant R774C found in patients with genetic iron deficiency has decreased inhibitory activity. Matriptase-2 cleaves hemojuvelin (HJV), a regulator of hepcidin, on plasma membrane; matriptase-2MASK shows no and the human mutant only partial cleavage capacity. Matriptase-2 interacts with HJV through the ectodomain since the interaction is conserved in matriptase-2MASK. The expression of matriptase-2 mutants in zebrafish results in anemia, confirming the matriptase-2 role in iron metabolism and its interaction with HJV.
Matriptase is a member of the type II transmembrane serine
family. Several studies have reported deregulated matriptase expression
in several types of epithelial cancers, suggesting that matriptase
constitutes a potential target for cancer therapy. We report herein
a new series of slow, tight-binding inhibitors of matriptase, which
mimic the P1–P4 substrate recognition sequence of the enzyme.
Preliminary structure–activity relationships indicate that
this benzothiazole-containing RQAR-peptidomimetic is a very potent
inhibitor and possesses a good selectivity for matriptase versus other
serine proteases. A molecular model was generated to elucidate the
key contacts between inhibitor 1 and matriptase.
matriptase; type II transmembrane serine protease; slow tight-binding inhibitor
Clinical trials where cancer patients were treated with protease inhibitors have suggested that the serine protease, prostasin, may act as a tumour suppressor. Prostasin is proteolytically activated by the serine protease, matriptase, which has a very high oncogenic potential. Prostasin is inhibited by protease nexin-1 (PN-1) and the two isoforms encoded by the mRNA splice variants of hepatocyte growth factor activator inhibitor-1 (HAI-1), HAI-1A, and HAI-1B.
Using quantitative RT-PCR, we have determined the mRNA levels for prostasin and PN-1 in colorectal cancer tissue (n = 116), severe dysplasia (n = 13), mild/moderate dysplasia (n = 93), and in normal tissue from the same individuals. In addition, corresponding tissues were examined from healthy volunteers (n = 23). A part of the cohort was further analysed for the mRNA levels of the two variants of HAI-1, here denoted HAI-1A and HAI-1B. mRNA levels were normalised to β-actin. Immunohistochemical analysis of prostasin and HAI-1 was performed on normal and cancer tissue.
The mRNA level of prostasin was slightly but significantly decreased in both mild/moderate dysplasia (p < 0.001) and severe dysplasia (p < 0.01) and in carcinomas (p < 0.05) compared to normal tissue from the same individual. The mRNA level of PN-1 was more that two-fold elevated in colorectal cancer tissue as compared to healthy individuals (p < 0.001) and elevated in both mild/moderate dysplasia (p < 0.01), severe dysplasia (p < 0.05) and in colorectal cancer tissue (p < 0.001) as compared to normal tissue from the same individual. The mRNA levels of HAI-1A and HAI-1B mRNAs showed the same patterns of expression. Immunohistochemistry showed that prostasin is located mainly on the apical plasma membrane in normal colorectal tissue. A large variation was found in the degree of polarization of prostasin in colorectal cancer tissue.
These results show that the mRNA level of PN-1 is significantly elevated in colorectal cancer tissue. Future studies are required to clarify whether down-regulation of prostasin activity via up regulation of PN-1 is causing the malignant progression or if it is a consequence of it.
Dysregulation of cell surface proteolysis has been strongly implicated in tumorigenicity and metastasis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer (PCa) cell migration, invasion, tumorigenicity and metastasis using a human PCa progression model (103E, N1, and N2 cells) and xenograft models. N1 and N2 cells were established through serial intraprostatic propagation of 103E human PCa cells and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was revealed to gradually increase throughout the serial isolations (103E
prostate cancer; hepatocyte growth factor activator inhibitor-2; cancer cell invasion; tumorigenicity and metastasis
Background & Aims
Matriptase is a membrane-anchored serine protease encoded by Suppression of Tumorigenicity-14 (ST14) that is required for epithelial barrier homeostasis. However its functional role in inflammatory bowel disease (IBD) is unexplored.
Matriptase expression in control, Crohn's disease and ulcerative colitis tissue specimens was studied by qPCR and immunostaining. Matriptase function was investigated by subjecting St14 hypomorphic and control littermates to dextran sodium sulfate (DSS)-induced colitis and by siRNA silencing in cultured monolayers. Mice were analyzed for clinical, histological, molecular and cellular effects.
Matriptase protein and ST14 mRNA levels are significantly down-regulated in inflamed colonic tissues from Crohn's disease and ulcerative colitis patients. Matriptase deficient St14 hypomorphic mice administered DSS for 7 days followed by water without DSS for 3 days develop a severe colitis with only 30% of the St14 hypomorphic mice surviving to day 14, compared with 100% of control littermates. Persistent colitis in surviving St14 hypomorphic mice was associated with sustained cytokine production, an inability to recover barrier integrity, and enhanced claudin-2 expression. Cytokines implicated in barrier disruption during IBD suppress matriptase expression in T84 epithelial monolayers and restoration of matriptase improves barrier integrity in the cytokine-perturbed monolayers.
These data demonstrate a critical role for matriptase in restoring barrier function to injured intestinal mucosa during colitis, which is suppressed by excessive activation of the immune system. Strategies to enhance matriptase-mediated barrier recovery could be important for intervening in the cycle of inflammation associated with IBD.
serine protease; TTSP; DSS; colitis; IBD; matriptase
Increasing evidence indicates the significance of platelet-derived growth factor receptor-β (β-PDGFR) signaling in prostate cancer (PCa). Accordingly, preclinical studies suggest the potential of β-PDGFR as a therapeutic target in metastatic PCa. However, a ligand responsible for β-PDGFR activation in PCa was unknown, and recent clinical trials with imatinib mesylate showed limited success due to normal tissue toxicity. Similarly, in spite of mounting evidence indicating the significance of matriptase in PCa, little is known about its substrates or molecular actions during PCa progression. Here, we identified PDGF-D as a ligand for β-PDGFR in PCa and discovered matriptase as its regulator. Matriptase activates PDGF-D by proteolytic removal of the CUB domain in a two-step process, creating a hemidimer (HD) followed by growth factor domain dimer (GFD-D) generation. Matriptase can deactivate PDGF-D by further proteolytic cleavage within the GFD, revealing its biphasic regulation. Importantly, PDGF-D/matriptase co-localization is accompanied with β-PDGFR phosphorylation in human PCa tissues. This study unveiled a novel signaling axis of matriptase/PDGF-D/β-PDGFR in PCa, providing new insights into functional interplay between serine protease and growth factor signaling networks.
Genitourinary cancers; prostate; Protease-inhibitor systems; Growth factors and receptors
Hepatocyte growth factor activator inhibitor type I (HAI-1) is a membrane-bound, serine protease inhibitor with two protease-inhibitory domains (Kunitz domain I and II). HAI-1 is known as a physiological inhibitor of a membrane-bound serine protease, matriptase. Paradoxically, however, HAI-1 has been found to be required for the extracellular appearance of the protease in an expression system using a monkey kidney COS-1 cell line. In the present study, we show using COS-1 cells that co-expression of recombinant variants of HAI-1 with the inhibition activity toward matriptase, including a variant consisting only of Kunitz domain I (the domain responsible for inhibition of matriptase), allowed for the appearance of this protease in the conditioned medium, whereas that of the variants without the activity did not. These findings suggest that the inhibition activity toward matriptase is critical for the extracellular appearance of protease in COS-1 cells.
Extracellular occurrence of matriptase; Hepatocyte growth factor activator inhibitor type 1; Intracellular environments; Kunitz domain; Matriptase-inhibitory activity
Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.
To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P’) sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1′ position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.
Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1′ positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.
Results 1-25 (1116608)