Autosomal ichthyosis with hypotrichosis syndrome displays low matriptase proteolytic activity and is phenocopied in ST14 hypomorphic mice

Human autosomal recessive ichthyosis with hypotrichosis (ARIH) is an inherited disorder recently linked to homozygosity for a point mutation in the ST14 gene that causes a G827R mutation in the matriptase serine protease domain (G216 in chymotrypsin numbering). Here we show that human G827R matriptase has strongly reduced proteolytic activity toward small molecule substrates, as well as toward its candidate epidermal target, prostasin. To further investigate the possible contribution of low matriptase activity to ARIH, we generated an ST14 hypomorphic mouse strain that displays a 100-fold reduction in epidermal matriptase mRNA levels. Interestingly, unlike ST14 null mice, ST14 hypomorphic mice were viable and fertile but displayed a spectrum of abnormalities that strikingly resembled ARIH. Thus, ST14 hypomorphic mice developed hyperproliferative and retention ichthyosis with impaired desquamation, hypotrichosis with brittle, thin, uneven, and sparse hair, and tooth defects. Biochemical analysis of ST14 hypomorphic epidermis revealed reduced prostasin proteolytic activation and profilaggrin proteolytic processing, compatible with a primary role of matriptase in this process. This work strongly indicates that reduced activity of a matriptase-prostasin proteolytic cascade is the etiological origin of human ARIH and provides an important mouse model for the exploration of matriptase function in ARIH, as well as multiple other physiological and pathological processes.     

Increased matriptase zymogen activation in inflammatory skin disorders

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of 23 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in keratinocytes at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(2)O(2) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.     

ABCA12 maintains the epidermal lipid permeability barrier by facilitating formation of ceramide linoleic esters

Harlequin ichthyosis is a congenital scaling syndrome of the skin in which affected infants have epidermal hyperkeratosis and a defective permeability barrier. Mutations in the gene encoding a member of the ABCA transporter family, ABCA12, have been linked to harlequin ichthyosis, but the molecular function of the protein is unknown. To investigate the activity of ABCA12, we generated Abca12 null mice and analyzed the impact on skin function and lipid content. Abca12-/- mice are born with a thickened epidermis and die shortly after birth, as water rapidly evaporates from their skin. In vivo skin proliferation measurements suggest a lack of desquamation of the skin cells, rather than enhanced proliferation of basal layer keratinocytes, accounts for the 5-fold thickening of the Abca12-/- stratum corneum. Electron microscopy revealed a loss of the lamellar permeability barrier in Abca12-/- skin. This was associated with a profound reduction in skin linoleic esters of long-chain omega-hydroxyceramides and a corresponding increase in their glucosyl ceramide precursors. Because omega-hydroxyceramides are required for the barrier function of the skin, these results establish that ABCA12 activity is required for the generation of long-chain ceramide esters that are essential for the development of normal skin structure and function.     

Mutation G827R in matriptase causing autosomal recessive ichthyosis with hypotrichosis yields an inactive protease

Matriptase is a member of the novel family of type II transmembrane serine proteases. It was recently shown that a rare genetic disorder, autosomal recessive ichthyosis with hypotrichosis, is caused by a mutation in the coding region of matriptase. However, the biochemical and functional consequences of the G827R mutation in the catalytic domain of the enzyme have not been reported. Here we expressed the G827R-matriptase mutant in bacterial cells and found that it did not undergo autocatalytic cleavage from its zymogen to its active form as did the wild-type matriptase. Enzymatic activity measurements showed that the G827R mutant was catalytically inactive. When expressed in HEK293 cells, G827R-matriptase remained inactive but was shed as a soluble form, suggesting that another protease cleaved the full-length mature form of matriptase. Molecular modeling based on the crystal structure of matriptase showed that replacing Gly(827) by Arg blocks access to the binding/catalytic cleft of the enzyme thereby preventing autocatalysis of the zymogen form. Our study, thus, provides direct evidence that the G827R mutation in patients with autosomal recessive ichthyosis with hypotrichosis leads to the expression of an inactive protease.     

A Matriptase-Prostasin Reciprocal Zymogen Activation Complex with Unique Features: PROSTASIN AS A NON-ENZYMATIC CO-FACTOR FOR MATRIPTASE ACTIVATION*

Matriptase and prostasin are part of a cell surface proteolytic pathway critical for epithelial development and homeostasis. Here we have used a reconstituted cell-based system and transgenic mice to investigate the mechanistic interrelationship between the two proteases. We show that matriptase and prostasin form a reciprocal zymogen activation complex with unique features. Prostasin serves as a critical co-factor for matriptase activation. Unexpectedly, however, prostasin-induced matriptase activation requires neither prostasin zymogen conversion nor prostasin catalytic activity. Prostasin zymogen conversion to active prostasin is dependent on matriptase but does not require matriptase zymogen conversion. Consistent with these findings, wild type prostasin, activation cleavage site-mutated prostasin, and catalytically inactive prostasin all were biologically active in vivo when overexpressed in the epidermis of transgenic mice, giving rise to a severe skin phenotype. Our finding of non-enzymatic stimulation of matriptase activation by prostasin and activation of prostasin by the matriptase zymogen provides a tentative mechanistic explanation for several hitherto unaccounted for genetic and biochemical observations regarding these two membrane-anchored serine proteases and their downstream targets.     

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.     

Prometastatic effect of N-acetylglucosaminyltransferase V is due to modification and stabilization of active matriptase by adding beta 1-6 GlcNAc branching

Oligosaccharide moieties of glycoproteins are structurally altered during development, carcinogenesis, and malignant transformations. It is well known that beta1-6 GlcNAc branching, a product of UDP-GlcNAc alpha-mannoside beta1-6-N-acetylglucosaminyltransferase (GnT-V), is associated with malignant transformation as the results of such alterations. However, the mechanism by which beta1-6 GlcNAc branching is linked to metastasis remains unclear, because the identification of specific glycoprotein(s) that are glycosylated by GnT-V and its biological function have not been examined. We herein report that matriptase, which activates both urokinase-type plasminogen activator and hepatocyte growth factor, is a target protein for GnT-V. The overexpression of GnT-V in gastric cancer cells leads to severe peritoneal dissemination in athymic mice, which can be attributed to the increased expression of matriptase. This increase was due to the acquired resistance of matriptase to degradation, since it is glycosylated by GnT-V and a corresponding increase in the active form. These results indicate that this process is a key element in malignant transformation, as the direct result of oligosaccharide modification.     

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.     

Mechanisms for the control of matriptase activity in the absence of sufficient HAI-1

Matriptase proteolytic activity must be tightly controlled for normal placental development, epidermal function, and epithelial integrity. Although hepatocyte growth factor activator inhibitor-1 (HAI-1) represents the predominant endogenous inhibitor for matriptase and the protein molar ratio of HAI-1 to matriptase is determined to be >10 in epithelial cells and the majority of carcinoma cells, an inverse HAI-1-to-matriptase ratio is seen in some ovarian and hematopoietic cancer cells. In the current study, cells with insufficient HAI-1 are investigated for the mechanisms through which the activity of matriptase is regulated. When matriptase activation is robustly induced in these cells, activated matriptase rapidly forms two complexes of 100- and 140-kDa in addition to the canonical 120-kDa matriptase-HAI-1 complex already described. Both 100- and 140-kDa complexes contain two-chain, cleaved matriptase but are devoid of gelatinolytic activity. Further biochemical characterization shows that the 140-kDa complex is a matriptase homodimer and that the 100-kDa complexes appear to contain reversible, tight binding serine protease inhibitor(s). The formation of the 140-kDa matriptase dimer is strongly associated with matriptase activation, and its levels are inversely correlated with the ratio of HAI-1 to matriptase. Given these observations and the likelihood that autoactivation requires the interaction of two matriptase molecules, it seems plausible that this activated matriptase homodimer may represent a matriptase autoactivation intermediate and that its accumulation may serve as a mechanism to control matriptase activity when protease inhibitor levels are limiting. These data suggest that matriptase activity can be rapidly inhibited by HAI-1 and other HAI-1-like protease inhibitors and "locked" in an inactive autoactivation intermediate, all of which places matriptase under very tight control.     

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.     

Regulation of the activity of matriptase on epithelial cell surfaces by a blood-derived factor.

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 acid-sensitive, 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 two-chain 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.     

Addition of beta1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin

beta1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis-related glycoproteins that contain beta1-6 GlcNAc branching have appeared, the precise function of beta1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of beta1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (beta1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes beta1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains beta1-6 GlcNAc branching. The beta1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of beta1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the beta1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between beta1-6 GlcNAc branching and a biological function at the protein level.     

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.     

Simultaneous activation and hepatocyte growth factor activator inhibitor 1-mediated inhibition of matriptase induced at activation foci in human mammary epithelial cells

Activation of single-chain, latent matriptase, a type II transmembrane serine protease, depends on the weak proteolytic activity of its own zymogen as well as its cognate inhibitor, hepatocyte growth factor activator inhibitor 1 (HAI-1). Oligomerization of matriptase zymogens and HAI-1, and probably its interaction with other proteins, has been proposed to occur during matriptase activation. In the present study, we examined the cellular events associated with matriptase activation triggered either by the physiological inducer sphingosine 1-phosphate (S1P) or by a chemical inducer, the polyanionic compound suramin. S1P-induced matriptase translocation to cell-cell contacts, where it is activated, is an F-actin polymerization-dependent process. Conversely, suramin-induced matriptase accumulation and activation at vesicle-like structures is an F-actin polymerization-independent process. While matriptase activation can occur at different subcellular locations, both S1P- and suramin-induced matriptase accumulation form unique subcellular structures, termed activation foci, where oligomerization of matriptase zymogens and HAI-1 may occur, promoting matriptase activation. Furthermore, matriptase activation may be regulated by intracellular signaling, because Ro 31-8220, a bisindolylmaleimide protein kinase C inhibitor, inhibited both S1P- and suramin-induced activation. The requirement of HAI-1 for matriptase activation and the coincidence of HAI-1 and matriptase in activation foci apparently provide rapid access of HAI-1 for the inhibition of matriptase immediately after its activation. Indeed, all activated matriptase was detected in complexes with HAI-1 only 5 min after suramin stimulation. The close temporospatial coupling of matriptase activation with its inhibition suggests that the proteolytic activity of this enzyme must be well controlled and that the proteolysis of matriptase substrates may be tightly regulated by this mechanism. sphingosine 1-phosphate; suramin     

Decreased matriptase/HAI-1 ratio in advanced colorectal adenocarcinoma of Chinese patients

Matriptase is a serine protease expressed by tumors of surface epithelial origin. We tested the expressions of matriptase and hepatocyte growth factor activator inhibitor-1 (HAI-1) maybe associated with the progression of colorectal adenocarcinoma. Immunohistochemical analysis of matriptase and HAI-1 was performed in tissue microarray slides of 91 colorectal adenocarcinomas with various degrees. The matriptase scores in moderately (346.7 +/- 10.6) and poorly differentiated (248.1 +/- 12.9) were significantly lower than those in well differentiated (368.4 +/- 9.6) colorectal adenocarcinomas. The matriptase/HAI-1 ratios in poorly (1.8 +/- 0.4) and moderately differentiated (1.8 +/- 0.3) were significantly lower than in well differentiated (2.2 +/- 0.2) colorectal adenocarcinomas. Otherwise, the matriptase scores and matriptase/HAI-1 ratio showed significant reverse correlation with more advanced TNM stages of colorectal adenocarcinomas in Chinese patients. In conclusion, pharmacological inhibitors of matriptase may not be effective treatment for advanced colorectal adenocarcinomas.     

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.     

Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptase-antithrombin complexes were not detected in all but two of a panel of seven breast, prostate, and ovarian cancer cell lines. Using purified active matriptase, we further characterize the formation of matriptase-antithrombin complex and show that heparin can significantly potentiate the inhibitory potency of antithrombin against matriptase. Second-order rate constants for the inhibition were determined to be 3.9 × 10(3) M(-1)s(-1) in the absence of heparin and 1.2 × 10(5) M(-1)s(-1) in the presence of heparin, a 30-fold increase, consistent with the established role of heparin in activating antithrombin function. Taken together these data suggest that normal epithelial cells employ a dual mechanism involving HAI-1 and antithrombin to control matriptase and that the antithrombin-based mechanism appears lost in the majority of carcinoma cells.     

Inhibition of tumor invasion by genomic down-regulation of matriptase through suppression of activation of receptor-bound pro-urokinase

Urokinase-type plasminogen activator (uPA) degrades the extracellular matrix and plays critical roles in tumor invasion and metastasis. Matriptase, a membrane-bound serine protease, was shown to activate uPA in a uPA receptor-free, solution-based study. We now investigate whether matriptase affects activation of receptor-bound uPA and contributes to the invasiveness of HRA human ovarian cancer cells in vitro and tumor behavior in nude mice. Here we show the following. 1) uPA expression was effectively stimulated by TGF-beta1 in HRA cells. 2) Antisense (AS)-matriptase transfection achieved a marked inhibition of receptor-bound pro-uPA activation without altering expression of uPA and uPA receptor mRNA and proteins, irrespective of whether cells were stimulated with TGF-beta1. 3) Tumor cell receptor-bound pro-uPA could be efficiently cleaved by matriptase to generate enzymatically active two-chain uPA. Thus, matriptase can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active uPA. 4) The AS-matriptase-treated cells had a decreased ability to invade an extracellular matrix layer, as compared with control cells. 5) When the AS-matriptase-treated cells were injected intraperitoneally into nude mice, the mice developed smaller tumors. Our data identify a novel role for matriptase for activation of receptor-bound uPA.     

Expression of filaggrin-2 protein in the epidermis of human skin diseases: A comparative analysis with filaggrin

Filaggrin-2 is a member of the S100 fused-type protein family, and the structural features and expression of filaggrin-2 are similar to those of profilaggrin, a protein essential for keratinization. In the present study, we investigated the expression profile of filaggrin-2 in patients with skin diseases using antibodies against the repetitive region of filaggrin-2. In tissue samples from patients with skin diseases which are associated with a decrease in filaggrin, including ichthyosis vulgaris, atopic dermatitis and psoriasis vulgaris, the expression level of filaggrin-2 was markedly decreased compared to that in normal skin samples. In contrast, the expression of filaggrin-2 increased in parallel with that of filaggrin in samples of tissue from patients with skin diseases associated with hyperkeratosis, such as lichen planus and epidermolytic ichthyosis. Interestingly, filaggrin-2 signals were observed in slightly higher layers of the epidermis in comparison to those of filaggrin. Similarly, the expression of filaggrin-2 proteins was induced slightly later than filaggrin in the cultured keratinocytes. These findings suggest that filaggrin-2 may play an overlapping role with filaggrin in epithelial cornification; however, it may also have a partially distinct role in the molecular processes of cornification.     

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.