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.     

The Channel-Activating Protease CAP1/Prss8 Is Required for Placental Labyrinth Maturation

The serine protease CAP1/Prss8 is crucial for skin barrier function, lung alveolar fluid clearance and has been unveiled as diagnostic marker for specific cancer types. Here, we show that a constitutive knockout of CAP1/Prss8 leads to embryonic lethality. These embryos presented no specific defects, but it is during this period, and in particular at E13.5, that wildtype placentas show an increased expression of CAP1/Prss8, thus suggesting a placental defect in the knockout situation. The placentas of knockout embryos exhibited significantly reduced vascular development and incomplete cellular maturation. In contrary, epiblast-specific deletion of CAP1/Prss8 allowed development until birth. These CAP1/Prss8-deficient newborns presented abnormal epidermis, and died soon after birth due to impaired skin function. We thus conclude that a late placental insufficiency might be the primary cause of embryonic lethality in CAP1/Prss8 knockouts. This study highlights a novel and crucial role for CAP1/Prss8 in placental development and function.     

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.     

Identification of a subgroup of glycosylphosphatidylinositol-anchored tryptases

The tryptase locus on mouse chromosome 17A3.3 contains 13 genes that encode enzymatically active serine proteases with different tissue expression profiles and substrate specificities. Mouse mast cell protease (mMCP) 6, mMCP-7, mMCP-11/protease serine member S (Prss) 34, tryptase 6/Prss33, tryptase ε/Prss22, implantation serine protease (Isp) 1/Prss28, and Isp-2 are constitutively exocytosed enzymes. We now demonstrate that tryptase 5/Prss32, pancreasin/Prss27, and testis serine protease-1 are inserted into plasma membranes via glycosylphosphatidylinositol (GPI) anchors analogous to Prss21, and that these serine proteases can be released from the cell’s surface by a phosphatidylinositol-specific phospholipase C. These data suggest that the C-terminal residues play key roles in determining where tryptases compartmentalize in cells. GPI-anchored proteins are targeted to lipid rafts. Thus, our identification of a number of GPI-anchored tryptases whose genes reside at mouse chromosome 17A3.3 also implicates important biological functions for this new family of serine proteases on the surfaces of cells.     

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.     

Impaired stratum corneum hydration in mice lacking epidermal water channel aquaporin-3

The water and solute transporting properties of the epidermis have been proposed to be important determinants of skin moisture content and barrier properties. The water/small solute-transporting protein aquaporin-3 (AQP3) was found by immunofluorescence and immunogold electron microscopy to be expressed at the plasma membrane of epidermal keratinocytes in mouse skin. We studied the role of AQP3 in stratum corneum (SC) hydration by comparative measurements in wild-type and AQP3 null mice generated in a hairless SKH1 genetic background. The hairless AQP3 null mice had normal perinatal survival, growth, and serum chemistries but were polyuric because of defective urinary concentrating ability. AQP3 deletion resulted in a > 4-fold reduced osmotic water permeability and > 2-fold reduced glycerol permeability in epidermis. Epidermal, dermal, and SC thickness and morphology were not grossly affected by AQP3 deletion. Surface conductance measurements showed remarkably reduced SC water content in AQP3 null mice in the hairless genetic background (165 +/- 10 versus 269 +/- 12 microsiemens (microS), p < 0.001), as well as in a CD1 genetic background (209 +/- 21 versus 469 +/- 11 microS). Reduced SC hydration was seen from 3 days after birth. SC hydration in hairless wild-type and AQP3 null mice was reduced to comparable levels (90-100 microS) after a 24-h exposure to a dry atmosphere, but the difference was increased when surface evaporation was prevented by occlusion or exposure to a humidified atmosphere (179 +/- 13 versus 441 +/- 34 microS). Conductance measurements after serial tape stripping suggested reduced water content throughout the SC in AQP3 null mice. Water sorption-desorption experiments indicated reduced water holding capacity in the SC of AQP3 null mice. The impaired skin hydration in AQP3 null mice provides the first functional evidence for the involvement of AQP3 in skin physiology. Modulation of AQP3 expression or function may thus alter epidermal moisture content and water loss in skin diseases.     

The important role of epidermal triacylglycerol metabolism for maintenance of the skin permeability barrier function

Survival in a terrestrial, dry environment necessitates a permeability barrier for regulated permeation of water and electrolytes in the cornified layer of the skin (the stratum corneum) to minimize desiccation of the body. This barrier is formed during cornification and involves a cross-linking of corneocyte proteins as well as an extensive remodeling of lipids. The cleavage of precursor lipids from lamellar bodies by various hydrolytic enzymes generates ceramides, cholesterol, and non-esterified fatty acids for the extracellular lipid lamellae in the stratum corneum. However, the important role of epidermal triacylglycerol (TAG) metabolism during formation of a functional permeability barrier in the skin was only recently discovered. Humans with mutations in the ABHD5/CGI-58 (α/β hydrolase domain containing protein 5, also known as comparative gene identification-58, CGI-58) gene suffer from a defect in TAG catabolism that causes neutral lipid storage disease with ichthyosis. In addition, mice with deficiencies in genes involved in TAG catabolism (Abhd5/Cgi-58 knock-out mice) or TAG synthesis (acyl-CoA:diacylglycerol acyltransferase-2, Dgat2 knock-out mice) also develop severe skin permeability barrier dysfunctions and die soon after birth due to increased dehydration. As a result of these defects in epidermal TAG metabolism, humans and mice lack ω-(O)-acylceramides, which leads to malformation of the cornified lipid envelope of the skin. In healthy skin, this epidermal structure provides an interface for the linkage of lamellar membranes with corneocyte proteins to maintain permeability barrier homeostasis. This review focuses on recent advances in the understanding of biochemical mechanisms involved in epidermal neutral lipid metabolism and the generation of a functional skin permeability barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

The identification of novel ovarian proteases through the use of genomic and bioinformatic methodologies

Proteolytic activities are essential for follicular growth, ovulation, as well as for luteal formation and regression. Using suppression subtractive hybridization (SSH), a novel mouse ovary-selective gene (termed protease serine 35, Prss35) was identified. Analysis of the mouse genome database using the Prss35 sequence led to the identification of a homologous protease (protease serine 23, Prss23). PRSS35 possesses general features that are characteristic of serine (Ser) proteases, but is unique in that the canonical Ser that defines this enzyme family is replaced by a threonine (Thr). In contrast, PRSS23 possesses the standard catalytic Ser typical for this family of proteases. As determined by real-time polymerase chain reaction (PCR), the Prss35 mRNA levels increased around the time of ovulation and remained elevated in the developing corpus luteum. Steroid ablation/replacement studies demonstrated progesterone-dependent regulation of Prss35 gene expression prior to follicle rupture. Prss35 gene expression was localized to the theca cells of pre-antral follicles, the theca and granulosa cells of pre-ovulatory and ovulatory follicles, as well as to the developing corpus luteum. In contrast, Prss23 mRNA levels decreased transiently after ovulation induction and again in the postovulatory period. Prss23 gene expression was noted primarily in the granulosa cells of the secondary/early antral follicles. PRSS35 and PRSS23 orthologs in the rat, human, rhesus macaque, chimpanzee, cattle, dog, and chicken were identified and found to be highly homologous to one another (75-99% homology). Collectively, these results suggest that the PRSS35 and PRSS23 genes have been conserved as critical ovarian proteases throughout the course of vertebrate evolution.     

Prss16 is not required for T-cell development

PRSS16 is a serine protease expressed exclusively in cortical thymic epithelial cells (cTEC) of the thymus, suggesting that it plays a role in the processing of peptide antigens during the positive selection of T cells. Moreover, the human PRSS16 gene is encoded in a region near the class I major histocompatibility complex (MHC) that has been linked to type 1 diabetes mellitus susceptibility. The mouse orthologue Prss16 is conserved in genetic structure, sequence, and pattern of expression. To study the role of Prss16 in thymic development, we generated a deletion mutant of Prss16 and characterized T-lymphocyte populations and MHC class II expression on cortical thymic epithelial cells. Prss16-deficient mice develop normally, are fertile, and show normal thymic morphology, cellularity, and anatomy. The total numbers and frequencies of thymocytes and splenic T-cell populations did not differ from those of wild-type controls. Surface expression of MHC class II on cTEC was also similar in homozygous mutant and wild-type animals, and invariant chain degradation was not impaired by deletion of Prss16. These findings suggest that Prss16 is not required for quantitatively normal T-cell development.     

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.     

Positive Promoting Effects of Smilax China Flower Absolute on the Wound Healing/Skin Barrier Repair-Related Responses of HaCaT Human Skin Keratinocytes

Smilax china (SC) has pharmacological effects including anti-inflammatory activity, but its effects on skin wound healing and skin barrier function have not been investigated. Here, we investigated the effects of absolute extracted from SC flowers (SCF) on skin wound healing-linked responses and functional skin barrier proteins using human epidermal keratinocytes (HaCaT cells). SCF absolute contained 20 components and was non-toxic to HaCaT cells. The absolute increased the proliferation, migration, and sprout outgrowth of HaCaT cells, and enhanced the activations of serine/threonine-specific protein kinase and extracellular signal-regulated kinase1/2. In addition, it increased the syntheses of type I and IV collagens and the expressions of skin barrier proteins (filaggrin and loricrin). These results indicate SCF absolute may has positive effects on skin wound healing by accelerating keratinocyte migration and proliferation activities and collagen synthesis, and on skin barrier function by upregulating barrier proteins in keratinocytes. We suggest SCF absolute to be considered as a potential means of promoting skin wound and barrier repair.     

Importance of Mast Cell Prss31/Transmembrane Tryptase/Tryptase-γ in Lung Function and Experimental Chronic Obstructive Pulmonary Disease and Colitis

Protease serine member S31 (Prss31)/transmembrane tryptase/tryptase-γ is a mast cell (MC)-restricted protease of unknown function that is retained on the outer leaflet of the plasma membrane when MCs are activated. We determined the nucleotide sequences of the Prss31 gene in different mouse strains and then used a Cre/loxP homologous recombination approach to create a novel Prss31^−/− C57BL/6 mouse line. The resulting animals exhibited no obvious developmental abnormality, contained normal numbers of granulated MCs in their tissues, and did not compensate for their loss of the membrane tryptase by increasing their expression of other granule proteases. When Prss31-null MCs were activated with a calcium ionophore or by their high affinity IgE receptors, they degranulated in a pattern similar to that of WT MCs. Prss31-null mice had increased baseline airway reactivity to methacholine but markedly reduced experimental chronic obstructive pulmonary disease and colitis, thereby indicating both beneficial and adverse functional roles for the tryptase. In a cigarette smoke-induced model of chronic obstructive pulmonary disease, WT mice had more pulmonary macrophages, higher histopathology scores, and more fibrosis in their small airways than similarly treated Prss31-null mice. In a dextran sodium sulfate-induced acute colitis model, WT mice lost more weight, had higher histopathology scores, and contained more Cxcl-2 and IL-6 mRNA in their colons than similarly treated Prss31-null mice. The accumulated data raise the possibility that inhibitors of this membrane tryptase may provide additional therapeutic benefit in the treatment of humans with these MC-dependent inflammatory diseases.     

Distinct roles of JNK-1 and ERK-2 isoforms in permeability barrier repair and wound healing

c-Jun N-terminal kinases (JNKs, also called stress activated protein kinases) and the extra-cellular signal responsive kinases (ERKs) exert different functions in mitogenesis, maturation and differentiation of immune and epithelial cells. We investigated specific functions of individual JNK and ERK isoforms in skin permeability barrier repair and in wound healing. JNK1, but not JNK2 or JNK3, deficient mice revealed a delay in the permeability barrier repair after superficial injury to the skin (tape-stripping) as well as a delay in the healing of full skin thickness wounds. Skin barrier injury induced an increase in epidermal JNK1 enzyme activity in mouse skin in vivo, and JNK1 activity correlated with the degree of differentiation in organotypic keratinocyte cultures. Skin injury activated epidermal ERK2 enzyme activity with biphasic maxima after 30 min and 3h, and the activity was independent from the differentiation state in keratinocyte culture. In summary, superficial and deep wound healing depends on the differential activity of MAP kinases such as JNK1 in epidermal differentiation and ERK2 in proliferation.     

A testis-specific serine protease,Prss41/Tessp-1, is necessary for the progression of meiosis during murine in vitro spermatogenesis

The function of protease during male meiosis has not been well studied. We previously cloned and characterized four testis-specific serine proteases in the mouse testis. One of the proteases, Prss41/Tessp-1, was expressed in the germ and Sertoli cell. This time, to examine the involvement of Prss41/Tessp-1 in spermatogenesis, we conducted the organ culture of testis fragments in the presence of the anti-Prss41/Tessp-1 antibody. Because in the Sertoli cell, the Prss41/Tessp-1 protein was mostly associated with the membrane of intracellular organelles by glycosylphosphatidylinositol, the antibody was expected to affect Prss41/Tessp-1 at the plasma membrane of spermatogonia. By adding the antibody, the number of germ cells was decreased in some seminiferous tubules. The marker genes expression strongly suggested that meiosis was arrested at spermatogonia, and the number of apoptotic germ cells increased by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. These data indicated that Prss41/Tessp-1 was necessary for the progression of meiosis at the stage of spermatogonia during in vitro spermatogenesis. Together with our previous study, the current results suggest that the Prss/Tessp proteases are important for the progression of meiosis at each stage.     

Selectively reduced glycerol in skin of aquaporin-3-deficient mice may account for impaired skin hydration,elasticity, and barrier recovery

Deletion of the epidermal water/glycerol transporter aquaporin-3 (AQP3) in mice reduced superficial skin conductance by approximately 2-fold (Ma, T., Hara, M., Sougrat, R., Verbavatz, J. M., and Verkman, A. S. (2002) J. Biol. Chem. 277, 17147-17153), suggesting defective stratum corneum (SC) hydration. Here, we demonstrate significant impairment of skin hydration, elasticity, barrier recovery, and wound healing in AQP3 null mice in a hairless (SKH1) genetic background and investigate the cause of the functional defects by analysis of SC morphology and composition. Utilizing a novel (3)H(2)O distribution method, SC water content was reduced by approximately 50% in AQP3 null mice. Skin elasticity measured by cutometry was significantly reduced in AQP3 null mice with approximately 50% reductions in elasticity parameters Uf, Ue, and Ur. Although basal skin barrier function was not impaired, AQP3 deletion produced an approximately 2-fold delay in recovery of barrier function as measured by transepidermal water loss after tape stripping. Another biosynthetic skin function, wound healing, was also approximately 2-fold delayed by AQP3 deletion. By electron microscopy AQP3 deletion did not affect the structure of the unperturbed SC. The SC content of ions (Na(+), K(+), Ca(2+), Mg(2+)) and small solutes (urea, lactic acid, glucose) was not affected by AQP3 deletion nor was the absolute amount or profile of lipids and free amino acids. However, AQP3 deletion produced significant reductions in glycerol content in SC and epidermis (in nmol/microg protein: 5.5 +/- 0.4 versus 2.3 +/- 0.7 in SC; 0.037 +/- 0.007 versus 0.022 +/- 0.005 in epidermis) but not in dermis or blood. These results establish hydration, mechanical, and biosynthetic defects in skin of AQP3-deficient mice. The selective reduction in epidermal and SC glycerol content in AQP3 null mice may account for these defects, providing the first functional evidence for physiologically important glycerol transport by an aquaporin.     

Inflammatory cytokine‐mediated induction of serine racemase in atopic dermatitis

Serine racemase (SR) is an enzyme that catalyses the synthesis of d ‐serine, an endogenous coagonist for N‐methyl‐D‐aspartate (NMDA)‐type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild‐type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune‐reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d ‐serine in stratum corneum in AD patients and healthy controls using a tape‐stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d ‐serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor ‐α or macrophage migration inhibitory factor. Taken together, these findings suggest that d ‐serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.     

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.