Distinct Developmental Functions of Prostasin (CAP1/PRSS8) Zymogen and Activated Prostasin

The membrane-anchored serine prostasin (CAP1/PRSS8) is essential for barrier acquisition of the interfollicular epidermis and for normal hair follicle development. Consequently, prostasin null mice die shortly after birth. Prostasin is found in two forms in the epidermis: a one-chain zymogen and a two-chain proteolytically active form, generated by matriptase-dependent activation site cleavage. Here we used gene editing to generate mice expressing only activation site cleavage-resistant (zymogen-locked) endogenous prostasin. Interestingly, these mutant mice displayed normal interfollicular epidermal development and postnatal survival, but had defects in whisker and pelage hair formation. These findings identify two distinct in vivo functions of epidermal prostasin: a function in the interfollicular epidermis, not requiring activation site cleavage, that can be mediated by the zymogen-locked version of prostasin and a proteolysis-dependent function of activated prostasin in hair follicles, dependent on zymogen conversion by matriptase.     

The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.     

Evidence for a matriptase-prostasin proteolytic cascade regulating terminal epidermal differentiation

Recent gene ablation studies in mice have shown that matriptase, a type II transmembrane serine protease, and prostasin, a glycosylphosphatidylinositol-anchored membrane serine protease, are both required for processing of the epidermis-specific polyprotein, profilaggrin, stratum corneum formation, and acquisition of epidermal barrier function. Here we present evidence that matriptase acts upstream of prostasin in a zymogen activation cascade that regulates terminal epidermal differentiation and is required for prostasin zymogen activation. Enzymatic gene trapping of matriptase combined with prostasin immunohistochemistry revealed that matriptase was co-localized with prostasin in transitional layer cells of the epidermis and that the developmental onset of expression of the two membrane proteases was coordinated and correlated with acquisition of epidermal barrier function. Purified soluble matriptase efficiently converted soluble prostasin zymogen to an active two-chain form that formed SDS-stable complexes with the serpin protease nexin-1. Whereas two forms of prostasin with molecular weights corresponding to the prostasin zymogen and active prostasin were present in wild type epidermis, prostasin was exclusively found in the zymogen form in matriptase-deficient epidermis. These data suggest that matriptase, an autoactivating protease, acts upstream from prostasin to initiate a zymogen cascade that is essential for epidermal differentiation.     

Regulation of the Matriptase-Prostasin Cell Surface Proteolytic Cascade by Hepatocyte Growth Factor Activator Inhibitor-1 during Epidermal Differentiation

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.     

Transport via the transcytotic pathway makes prostasin available as a substrate for matriptase

The matriptase-prostasin proteolytic cascade is essential for epidermal tight junction formation and terminal epidermal differentiation. This proteolytic pathway may also be operative in a variety of other epithelia, as both matriptase and prostasin are involved in tight junction formation in epithelial monolayers. However, in polarized epithelial cells matriptase is mainly located on the basolateral plasma membrane whereas prostasin is mainly located on the apical plasma membrane. To determine how matriptase and prostasin interact, we mapped the subcellular itinerary of matriptase and prostasin in polarized colonic epithelial cells. We show that zymogen matriptase is activated on the basolateral plasma membrane where it is able to cleave relevant substrates. After activation, matriptase forms a complex with the cognate matriptase inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1 and is efficiently endocytosed. The majority of prostasin is located on the apical plasma membrane albeit a minor fraction of prostasin is present on the basolateral plasma membrane. Basolateral prostasin is endocytosed and transcytosed to the apical plasma membrane where a long retention time causes an accumulation of prostasin. Furthermore, we show that prostasin on the basolateral membrane is activated before it is transcytosed. This study shows that matriptase and prostasin co-localize for a brief period of time at the basolateral plasma membrane after which prostasin is transported to the apical membrane as an active protease. This study suggests a possible explanation for how matriptase or other basolateral serine proteases activate prostasin on its way to its apical destination.     

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency-Associated Developmental Defects by Preventing Matriptase Activation

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.     

Activity and inhibition of prostasin and matriptase on apical and basolateral surfaces of human airway epithelial cells

Prostasin is a membrane-anchored protease expressed in airway epithelium, where it stimulates salt and water uptake by cleaving the epithelial Na(+) channel (ENaC). Prostasin is activated by another transmembrane tryptic protease, matriptase. Because ENaC-mediated dehydration contributes to cystic fibrosis (CF), prostasin and matriptase are potential therapeutic targets, but their catalytic competence on airway epithelial surfaces has been unclear. Seeking tools for exploring sites and modulation of activity, we used recombinant prostasin and matriptase to identify substrate t-butyloxycarbonyl-l-Gln-Ala-Arg-4-nitroanilide (QAR-4NA), which allowed direct assay of proteases in living cells. Comparisons of bronchial epithelial cells (CFBE41o-) with and without functioning cystic fibrosis transmembrane conductance regulator (CFTR) revealed similar levels of apical and basolateral aprotinin-inhibitable activity. Although recombinant matriptase was more active than prostasin in hydrolyzing QAR-4NA, cell surface activity resisted matriptase-selective inhibition, suggesting that prostasin dominates. Surface biotinylation revealed similar expression of matriptase and prostasin in epithelial cells expressing wild-type vs. ΔF508-mutated CFTR. However, the ratio of mature to inactive proprostasin suggested surface enrichment of active enzyme. Although small amounts of matriptase and prostasin were shed spontaneously, prostasin anchored to the cell surface by glycosylphosphatidylinositol was the major contributor to observed QAR-4NA-hydrolyzing activity. For example, the apical surface of wild-type CFBE41o- epithelial cells express 22% of total, extractable, aprotinin-inhibitable, QAR-4NA-hydrolyzing activity and 16% of prostasin immunoreactivity. In conclusion, prostasin is present, mature and active on the apical surface of wild-type and CF bronchial epithelial cells, where it can be targeted for inhibition via the airway lumen.     

Strong expression association between matriptase and its substrate prostasin in breast cancer

Breast cancer tumorigenesis is accompanied by increased levels of extracellular proteases that are capable of remodeling the extracellular matrix as well as cleaving and activating growth factors and signaling receptors that are critically involved in neoplastic progression. Multiple studies implicate the membrane anchored serine protease matriptase (also known as MT-SP1 and epithin) in breast cancer. The pro-form of the GPI-anchored serine protease prostasin has recently been identified as a physiological substrate of matriptase and the two proteases are co-expressed in multiple healthy tissues. In this study, the inter-relationship between the two membrane-anchored serine proteases in breast cancer was investigated using breast cancer cell lines and breast cancer patient samples to delineate the association between matriptase and prostasin. We used Western blotting to determine the expression of matriptase and prostasin proteins in a panel of breast cancer cell lines and immunohistochemistry to assess the expression in serial sections from breast cancer tissue arrays. We demonstrate that the expression of matriptase and prostasin is closely correlated in breast cancer cell lines as well as in breast cancer tissue samples. Furthermore, matriptase and prostasin display a near identical spatial expression pattern in the epithelial compartment of breast cancer tissue. These data suggest that the matriptase-prostasin cascade might play a critical role in breast cancer.     

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. Prostasin null (Prss8^−/−) mice lack barrier formation and display fatal postnatal dehydration. In sharp contrast, mice homozygous for a point mutation in the Prss8 gene, which causes the substitution of the active site serine within the catalytic histidine-aspartate-serine triad with alanine and renders prostasin catalytically inactive (Prss8^{Cat−/Cat−} mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8^−/− and Prss8^{Cat−/Cat−} mice born within the same litter. Furthermore, Prss8^{Cat−/Cat−} mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane-anchored serine protease.     

Matriptase Activation, an Early Cellular Response to Acidosis

Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activities, and activates G protein-coupled receptors. In this report, we demonstrated a novel cellular response to acidosis: induction of the zymogen activation of matriptase. Acid-induced matriptase activation is ubiquitous among epithelial and carcinoma cells and is characterized by rapid onset, fast kinetics, and the magnitude of activation seen. Trace amounts of activated matriptase can be detected 1 min after cells are exposed to pH 6.0 buffer, and the vast majority of latent matriptase within the cells is converted to activated matriptase within 20 min. Matriptase activation may be a direct response to proton exposure because acid-induced matriptase activation also occurs in an in vitro, cell-free setting in which intracellular signaling molecules and ion channel activities are largely absent. Acid-induced matriptase activation takes place both on the cell surface and inside the cells, likely due to the parallel intracellular acidification that activates intracellular matriptase. Following matriptase activation, the active enzyme is immediately inhibited by binding to hepatocyte growth factor activator inhibitor 1, resulting in stable matriptase-hepatocyte growth factor activator inhibitor 1 complexes that are rapidly secreted. As an early response to acidosis, matriptase activation can also be induced by perturbation of intracellular pH homeostasis by 5-(N-methyl-N-isobutyl)-amiloride and 5-(N-ethyl-N-isopropyl)-amiloride, both of which inhibit Na⁺/H⁺ exchangers, and diisothiocyanostilbene-2,2′-disulfonic acid, which can inhibit other acid-base ion channels. This study uncovers a novel mechanism regulating proteolysis in epithelial and carcinoma cells, and also demonstrates that a likely function of matriptase is as an early response to acidosis.     

Regulation of Feto-Maternal Barrier by Matriptase- and PAR-2-Mediated Signaling Is Required for Placental Morphogenesis and Mouse Embryonic Survival

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.     

Hepsin activates prostasin and cleaves the extracellular domain of the epidermal growth factor receptor

The epithelial extracellular serine protease activation cascade involves matriptase (PRSS14) and prostasin (PRSS8), capable of modulating epidermal growth factor receptor (EGFR) signaling. Matriptase activates prostasin by cleaving in the amino-terminal pro-peptide region of prostasin, presumably at the Arg residue of position 44 (R44) of the full-length human prostasin. Using an Arg-to-Ala mutant (R44A) human prostasin, we showed in this report that the cleavage of prostasin by matriptase is at Arg44. This prostasin proteolytic activation site is also cleaved by hepsin (TMPRSS1) to produce active prostasin capable of forming a covalent complex with protease nexin 1 (PN-1). An amino-terminal truncation of EGFR in the extracellular domain (ECD) was observed when the receptor was co-expressed with hepsin. Hepsin and matriptase appear to cleave the EGFR ECD at different sites, while the hepsin cleavage is not affected by active prostasin, which enhances the matriptase cleavage of EGFR. Using hepsin as the prostasin-activating protease in cells co-transfected with EGFR, we showed that active prostasin does not cleave the EGFR ECD directly in the cellular context. Purified active prostasin also does not cleave purified EGFR. Hepsin cleavage of EGFR is not dependent on receptor tyrosine phosphorylation, while the hepsin-cleaved EGFR is phosphorylated at Tyr1068 and no longer responsive to EGF stimulation. The cleavage of EGFR by hepsin does not result in increased phosphorylation of the downstream extracellular signal-regulated kinases (Erk1/2), an event inducible by the matriptase–prostasin cleavage of EGFR. The role of hepsin serine protease should be considered in future studies of epithelial biology concerning matriptase, prostasin, and EGFR.     

Expression of full-length human pro-urokinase in mammary glands of transgenic mice

Human pro-urokinase expressed in the mammary glands of transgenic animals is quickly activated and converted to urokinase by proteases that are present in the milk. Thus, it is nearly impossible to isolate full-sized pro-urokinase from the milk of transgenic animals. To solve this problem, we constructed transgenic mice that express human pro-urokinase and modified ecotin, which is a potent serine protease inhibitor from E. coli, in their mammary glands. The gene encoding ecotin was modified so as to enhance its specificity for the human urokinase-type plasminogen activator. Co-expression of modified ecotin and human pro-urokinase in the mammary glands allows for purification of full-length human pro-urokinase from these transgenic mice. The results described here suggest a general way of preventing the activation of zymogens that are expressed in the mammary glands of transgenic animals by co-expression of a zymogen along with a protease inhibitor.     

Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.     

Divergent Inhibitor Susceptibility among Airway Lumen-Accessible Tryptic Proteases

Tryptic serine proteases of bronchial epithelium regulate ion flux, barrier integrity, and allergic inflammation. Inhibition of some of these proteases is a strategy to improve mucociliary function in cystic fibrosis and asthmatic inflammation. Several inhibitors have been tested in pre-clinical animal models and humans. We hypothesized that these inhibitors inactivate a variety of airway protease targets, potentially with bystander effects. To establish relative potencies and modes of action, we compared inactivation of human prostasin, matriptase, airway trypsin-like protease (HAT), and β-tryptase by nafamostat, camostat, bis(5-amidino-2-benzimidazolyl)methane (BABIM), aprotinin, and benzamidine. Nafamostat achieved complete, nearly stoichiometric and very slowly reversible inhibition of matriptase and tryptase, but inhibited prostasin less potently and was weakest versus HAT. The IC₅₀ of nafamostat’s leaving group, 6-amidino-2-naphthol, was >10⁴-fold higher than that of nafamostat itself, consistent with suicide rather than product inhibition as mechanisms of prolonged inactivation. Stoichiometric release of 6-amidino-2-naphthol allowed highly sensitive fluorometric estimation of active-site concentration in preparations of matriptase and tryptase. Camostat inactivated all enzymes but was less potent overall and weakest towards matriptase, which, however was strongly inhibited by BABIM. Aprotinin exhibited nearly stoichiometric inhibition of prostasin and matriptase, but was much weaker towards HAT and was completely ineffective versus tryptase. Benzamidine was universally weak. Thus, each inhibitor profile was distinct. Nafamostat, camostat and aprotinin markedly reduced tryptic activity on the apical surface of cystic fibrosis airway epithelial monolayers, suggesting prostasin as the major source of such activity and supporting strategies targeting prostasin for inactivation.     

Activation of matriptase zymogen

Matriptase is a type II transmembrane serine protease expressed abundantly in the epithelial cells and keratinocyte. It plays a key role in the establishment and maintenance of epithelial integrity. Matriptase is considered to be at the most upstream in cellular protease cascade. Activation of its zymogen is the most critical step in regulation of downstream proteases activities and physiological functions. It has recently found that the exposure of matriptase-expressing epithelial cells and its homogenate to mildly acidic pH induces the rapid activation of matriptase zymogen. On the other hand, high ionic strength prevents this activation. The activation of the zymogen is thought to be triggered by the acidification and the lowering of ionic strength in cell-surface microenvironments.     

The role of asparagine-linked glycosylation site on the catalytic domain of matriptase in its zymogen activation

Matriptase is a type II transmembrane serine protease containing one potential site for asparagine-linked glycosylation (N-glycosylation) on the catalytic domain (Asn772). It has been found that the activation of matriptase zymogen occurs via a mechanism requiring its own activity and that the N-glycosylation site is critical for the activation. The present study aimed to determine the underlying reasons for the site requirement using Madin–Darby canine kidney cells stably expressing recombinant variants of rat matriptase. A full-length variant with glutamine substitution at Asn772 appeared to be unable to undergo activation because of its catalytic incompetence (i.e., decreased availability of the soluble catalytic domain and/or of the correctly folded domain). This was evidenced by the observations that (i) a recombinant catalytic domain of matriptase with glutamine substitution at the site corresponding to matriptase Asn772 [N772Q-CD-Myc(His)₆] was not detected in the medium conditioned by transfected cells but was on the cell surface and (ii) purified N772Q-CD-Myc(His)₆ exhibited markedly reduced activity toward a peptide substrate. It is concluded that N-glycosylation site at Asn772 of matriptase is required for the zymogen activation because it plays an important role in rendering this protease catalytically competent in the cellular environment.     

Prostasin expression is regulated by airway surface liquid volume and is increased in cystic fibrosis

Airway surface liquid (ASL) absorption is initiated by Na+ entry via epithelial Na+ channels (ENaC), which establishes an osmotic gradient that drives fluid from the luminal to serosal airway surface. We and others have recently reported that a protease/anti-protease balance regulates ENaC in human airway epithelial cells (HAEC) and provides a mechanism for autoregulation of ASL volume. In cystic fibrosis (CF), this balance is disturbed, leading to constitutive proteolytic activation of ENaC and the pathological Na+ hyperabsorption characteristic of this airway disease. Prostasin is a glycosylphosphatidylinositol-anchored serine protease that activates ENaC and is expressed on the surface epithelium lining the airway. In this report we present evidence that prostasin expression is regulated by the ASL volume, allowing for increased proteolytic activation of ENaC when the ASL volume is high. Prostasin activity is further regulated by the cognate serpin protease nexin-1 (PN-1), which is expressed in HAEC and inhibits Na+ absorption by forming an inactive complex with prostasin and preventing the proteolytic processing of prostasin. Whereas these mechanisms regulate prostasin expression in response to ASL volume in non-CF epithelia, HAEC cultured from CF patients express >50% more prostasin on the epithelial surface. These findings suggest that a proteolytic cascade involving prostasin, an upstream prostasin-activating protease, and PN-1 regulate Na+ absorption in the airway and that abnormal prostasin expression contributes to excessive proteolytic activation of ENaC in CF patients.     

Detection of Active Matriptase Using a Biotinylated Chloromethyl Ketone Peptide

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.     

The matriptase-prostasin proteolytic cascade in epithelial development and pathology

The type II transmembrane serine protease matriptase has an essential role in the integrity and function of multiple epithelial tissues. In the epidermis, matriptase activates the glycosylphosphatidylinositol (GPI) anchored membrane serine protease prostasin to initiate a proteolytic cascade that is required for the development of the stratum corneum barrier function. Accordingly, mice deficient for matriptase phenocopy mice deficient for epidermal prostasin and present with impaired corneocyte differentiation, imparied lipid matrix formation, loss of profilaggrin processing and loss of tight junction formation and function. Together, these defects lead to a compromised epidermal barrier and result in fatal dehydration during the neonatal period. Proteolytic activity of the matriptase-prostasin cascade is regulated in the epidermis via inhibition by the Kunitz-type serine protease inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). Importantly, targeted post-natal ablation of matriptase in mice perturbs the function of multiple adult tissues, indicating an ongoing requirement for matriptase proteolysis in the maintenance of diverse types of epithelia. Impaired matriptase proteolytic activity has been linked to human Autosomal Recessive Icthyosis with Hypotrichosis (ARIH), whereas aberrant matriptase activity has been implicated in Netherton’s Syndrome. This review will summarize information pertaining to the role of matriptase in epithelial biology and will discuss recent advancements in our understanding of how matriptase activity is regulated and the down-stream effectors of matriptase proteolysis.