PRSS8) supports epidermal development and postnatal homeostasis independent of its enzymatic activity.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 21, pp. 14740 –14749, May 23, 2014 Published in the U.S.A.

The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity* Received for publication, December 6, 2013, and in revised form, March 24, 2014 Published, JBC Papers in Press, April 4, 2014, DOI 10.1074/jbc.M113.541318

Background: Prostasin is a membrane-anchored serine protease with essential functions in epithelial development and homeostasis. Results: Mice expressing enzymatically inactive endogenous prostasin, unlike prostasin null mice, display normal tissue development and homeostasis. Conclusion: Essential in vivo functions of prostasin are independent of the catalytic activity of prostasin. Significance: Prostasin may have a unique role as an allosteric regulator of other membrane-anchored proteases. 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 (Prss8ⴚ/ⴚ) 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 (Prss8Catⴚ/Catⴚ 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 Prss8ⴚ/ⴚ and Prss8Catⴚ/Catⴚ mice born within the same litter. Furthermore, Prss8Catⴚ/Catⴚ 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 membraneanchored serine protease.

Prostasin (also known as channel-activating protease 1, CAP1, and PRSS8) is a phylogenetically conserved membrane-

* This work was supported by the NIDCR Intramural Research Program (to K. H. and T. H. B.), by The Harboe Foundation, The Lundbeck Foundation, and the Foundation of 17.12.1981 (to S. F.). 1 To whom correspondence should be addressed: Proteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, 30 Convent Drive, Rm. 211, Bethesda, MD 20892. Tel.: 301-435-1840; Fax: 301402-0823; E-mail: [email protected].

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anchored serine protease, encoded by the PRSS8 gene, that is widely expressed in epithelial tissues. Loss-of-function studies in mice have revealed an essential role of prostasin in terminal epidermal differentiation and postnatal survival. Prostasin null (Prss8⫺/⫺) mice, lacking any prostasin protein, display placental insufficiency that leads to complete or partial embryonic lethality, while mice with epidermal deletion of Prss8 die shortly after birth due to lack of epidermal barrier formation and fatal dehydration (1, 2). Prostasin is also an essential regulator of the epithelial sodium channel in the context of alveolar fluid clearance, lung fluid balance, and intestinal sodium and water absorption (3, 4). It is now generally recognized that prostasin and the type II transmembrane serine protease, matriptase, form part of a single epithelial proteolytic cascade in the context of placental development, terminal epidermal differentiation, and epithelial tight junction formation (5–10). The specific mechanistic interrelationship between the two proteases, however, has remained unclear, with different studies placing prostasin either upstream or downstream from matriptase depending on the specific context (5–7, 9). Using a reconstituted cell-based assay, we recently found that prostasin can form complexes with matriptase and can stimulate matriptase autoactivation independent of prostasin’s own catalytic activity. In this regard, a catalytically inactive prostasin mutant was able to both activate matriptase and to stimulate the activity of matriptase toward a physiological target substrate, proteinase-activated receptor-2 (11). To determine the biological relevance of these observations, we herein generated and characterized knock-in mice with a point mutation engineered into the endogenous Prss8 gene, VOLUME 289 • NUMBER 21 • MAY 23, 2014

Downloaded from http://www.jbc.org/ at Australian Natl Univ (CAUL) on March 15, 2015

Diane E. Peters‡§, Roman Szabo‡, Stine Friis‡¶, Natalia A. Shylo‡, Katiuchia Uzzun Sales‡储, Kenn Holmbeck**, and Thomas H. Bugge‡1 From the ‡Proteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, the §Program of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts 02111, the ¶Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen, Denmark, the **Connective Tissue Remodeling Section, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892, and the 储 Clinical Research Core, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, Maryland 20892

Non-enzymatic in Vivo Functions of Prostasin which resulted in the synthesis of a catalytically inactive protease. Analysis of these mice unexpectedly revealed that prostasin supports both epidermal development and long term survival independent of its enzymatic activity.

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EXPERIMENTAL PROCEDURES Generation of Catalytically Inactive Prostasin Knock-in Mice— All experiments were performed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited vivarium following institutional guidelines and standard operating procedures. Prss8Cat⫺/Cat⫺ mice were generated by homologous recombination in embryonic stem (ES) cells using a replacement targeting vector. A 2.5-kb fragment containing exon 6 of the mouse Prss8 gene was amplified from feeder-free W4129 S6 ES cell DNA using high-fidelity long range PCR. A NotI site was incorporated at the 5⬘ end during amplification and an EcoRI site existed at the 3⬘ end. This fragment was inserted between the EcoRI and NotI sites of pBluescript II KS. A T3 G substitution was introduced into exon 6 (corresponding to nucleotide 943 of the mouse Prss8 cDNA, NM_133351.3) by site-directed mutagenesis using a QuikChange kit (Stratagene, La Jolla, CA). Mutagenesis primers used were as follows: 5⬘-TTTCTCCTTAGGGTGACGCTGGGGGCCC-3⬘ and 5⬘-GGGCCCCCAGCGTCACCCTAAGGAG AAA-3⬘ (mutation underlined). Following introduction of the desired point mutation, an adjacent 2.5-kb DNA fragment was amplified containing a native EcoRI site at its 5⬘ end and with both NotI and SalI sites added at the 3⬘ end. This was inserted into the vector via EcoRI/SalI sites effectively reassembling a homologous 5-kb DNA fragment, which included the introduced T3 G substitution in exon 6 of Prss8 and also contained NotI sites at both 5⬘ and 3⬘ ends. The final targeting vector was generated in the vector PL452 (12), which contains a neomycin cassette under the control of the 3-phosphoglycerate kinase promoter flanked by LoxP sites, by ligating the 5-kb NotI/NotI fragment from pBluescript into the targeting vector downstream from the neomycin cassette to form a 5⬘ homology region. A contiguous 1.5-kb DNA fragment containing Prss8 exons 3 through 5 was then inserted upstream of the neo cassette to form the 3⬘ homology region. The integrity of the targeting vector was confirmed by DNA sequencing. The targeting vector was linearized by SacII digestion and was electroporated into 129-strain-derived W4129 S6 ES cells (Taconic, Germantown, NY). Culture and selection of recombinant ES cell clones was performed as described previously (13). 506 neomycin-resistant ES cell clones were screened by PCR using a primer set spanning from within the PL452 targeting vector to endogenous genomic sequences located externally to the targeting construct (forward, 5⬘-CTTAGCTCTCCTGTCCTTGGGATG-3⬘; reverse, 5⬘-ATTGGGCTGCAGGAATTCGATAGC-3⬘). A positive clone was identified, and correct targeting was verified by extensive sequencing using PCR-amplified DNA sequences spanning Prss8 exons 1– 6 (forward, 5⬘-AGCTGTGACCATTCTGCTCCTTCT-3⬘; reverse, 5⬘-CCAGTTTCTAGGATGGCAGCCTA-3⬘). This ES cell clone was injected into C57BL/6J blastocysts. Male progeny with a high percentage of coat color chimerism were bred to Black Swiss females (Taconic, Germantown, NY) to establish

germ line transmission. Heterozygous mice from this breeding were mated with FVB/N-Tg(EIIa-cre)C5379Lmgd/J mice (The Jackson Laboratory, Bar Harbor, ME) to produce a Cre-recombined F2 generation, Prss8 ⫹/Cat⫺ offspring with the neomycin cassette excised. Mouse genotypes were identified by PCR using primers flanking the neo cassette (forward, 5⬘-GCAGCTCGAGGTACCACTCATCAGC-3⬘; reverse, 5⬘-AACTCACAATGCCTGCCAAGTACC-3⬘). F2 Prss8⫹/Cat⫺ generation offspring were interbred to yield an F3 generation, allowing for enrollment of Prss8⫹/⫹ and Prss8Cat⫺/Cat⫺ littermate cohorts. In parallel, F2 generation Prss8⫹/Cat⫺ offspring were bred to Prss8⫹/⫺ females (see below). The F3 generation offspring from these matings (Prss8Cat⫺/⫺, Prss8⫹/⫺, Prss8⫹/Cat⫺) were then crossed to produce an F4 generation allowing for a direct comparison of the Prss8Cat⫺/Cat⫺ and Prss8⫺/⫺ phenotypes on identical genetic backgrounds. Long term cohorts of Prss8⫹/⫹ and Prss8Cat⫺/Cat⫺ littermates were co-housed at 3–5 mice per cage and monitored daily. Generation of Prostasin Knock-out Mice—Prss8⫺/⫺ mice were generated from mouse C57BL/6N embryonic stem cells carrying a retroviral genetrap vector inserted into intron 2 of the mouse Prss8 gene (Texas A&M Institute for Genomic Medicine, College Station, TX, clone no. IST10122F12). The Prss8targeted ES cells were injected into the blastocoel cavity of C57BL/6-cBrd/cBrd-derived blastocysts and implanted into pseudopregnant females. The presence of the targeted allele in the offspring from ensuing chimeras bred to C57BL/6J females (The Jackson Laboratory) was identified by PCR genotyping using V76R (5⬘-CCAATAAACCCTCTTGCAGTTGC-3⬘) and Prss8_52 (5⬘-ACTTAGCCACACTAAGTGTCCC-3⬘) primers. RNA Preparation and RT-PCR—Tissues were collected from newborn mice, snap-frozen in liquid nitrogen, and ground to a fine powder with mortar and pestle. Total RNA was prepared by extraction in TRIzol reagent (Invitrogen) as recommended by the manufacturer. Reverse transcription and PCR amplification were performed using a High Capacity cDNA reverse transcription kit (Invitrogen), per the manufacturer’s instructions. First strand cDNA synthesis was performed using an oligo(dT) primer. The primer pair utilized for Prss8 RT-PCR was as follows: 5⬘-TTGCTGTAGGAGTCTAGC-3⬘ and 5⬘-AAGCTGTGACCATTCTGC-3⬘. Annealing temperature for this primer set was 55 °C. Expression levels were normalized to S15 mRNA levels in each sample. Western Blot Analysis—Tissues from newborn mice were homogenized in ice-cold PBS containing 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitors (Sigma) and incubated on ice for 10 min. The lysates were centrifuged at 20,000 ⫻ g for 30 min at 4 °C to remove tissue debris, and the supernatant was used for further analysis as described below. For profilaggrin processing analysis, epidermis was homogenized in 50 mM Tris/HCl, pH 8.0, 10 mM EDTA, and 8 M urea. Protein concentrations were determined using a BCA assay to allow loading of equivalent total protein. Samples were mixed with 4⫻ SDS sample buffer (Invitrogen) containing 7% ␤-mercaptoethanol, boiled for 5 min, separated on 4 –12% Bis-Tris NuPage gels (Invitrogen), and transferred to 0.2-␮m pore size PVDF membranes (Invitrogen). Membranes were blocked with 5% BSA in

Non-enzymatic in Vivo Functions of Prostasin

RESULTS Characterization of Mice Expressing Catalytically Inactive Endogenous Prostasin—To generate mice expressing catalytically inactive prostasin, we introduced a c.943T3 G nucleotide substitution into exon 6 of the Prss8 gene by homologous recombination in embryonic stem cells (Fig. 1A). This resulted in the substitution of serine 238 of the catalytic histidine-aspartate-serine triad with alanine. Genomic analysis confirmed the introduction of the point mutation in the Prss8 gene (Fig. 1, B


and C, the targeted allele hereafter referred to as Prss8Cat⫺). Analysis of mRNA transcripts showed that the introduction of the point mutation and a neighboring LoxP site into intron 5 did not affect Prss8 expression (Fig. 1D). Western blot analysis of protein extracts from skin, kidney, and lungs of newborn Prss8Cat⫺/Cat⫺ pups and wild-type (Prss8⫹/⫹) littermates demonstrated that the mutant prostasin was expressed at levels similar to wild-type prostasin (Fig. 1, E and F). An additional faster migrating prostasin species was detected in kidney and lung of Prss8Cat⫺/Cat⫺ mice (red triangle in Fig. 1E, kidney and lung panels, compare lanes 1–3 with 4 – 6), which may represent an activation site-cleaved mutant prostasin unable to be cleared through serpin or Kunitz-type inhibitor complex formation. Immunohistochemistry of skin and other major organ systems (Fig. 2) showed normal spatial localization of the catalytically inactive mutant prostasin. Expression was predominantly restricted to epithelia, and no differences were identified relative to wild-type prostasin, as published previously (10). The Enzymatic Activity of Prostasin Is Dispensable for Mouse Development and Long Term Survival—We interbred Prss8⫹/Cat⫺ mice and genotyped 111 offspring from a total of 13 litters and found that Prss8Cat⫺/Cat⫺ pups were born in Mendelian frequency (Fig. 3A, black bars). Surprisingly, no Prss8Cat⫺/Cat⫺ pups died within the 21-day preweaning period (Fig. 3A, gray bars). Furthermore, no deaths were observed in a prospective cohort of 20 Prss8Cat⫺/Cat⫺ mice and 24 Prss8⫹/⫹ littermates followed for 56 to 140 days postweaning (Fig. 3B). One possible explanation for the survival discrepancy between Prss8Cat⫺/Cat⫺ and Prss8⫺/⫺ mice relates to the targeting strategy employed to generate prostasin null mice. Prss8⫺/⫺ mice were originally made by the insertion of LoxP sites into introns 2 and 5, followed by Cre-mediated recombination (1, 2). Thus, inadvertent removal of an essential non-protein coding gene or a critical regulatory element may have contributed to the observed postnatal lethality. To investigate this possibility, we generated Prss8⫺/⫺ mice via an alternate strategy, using ES cells with a retroviral insertion in intron 2. This insertion resulted in a null mutation by placing a strong splice acceptor site in intron 2, leading to formation of a truncated Prss8 mRNA fused to a ␤-geo reporter gene (Fig. 4). Importantly, this targeting strategy did not delete any native DNA sequences. Nevertheless, Prss8⫺/⫺ mice generated by this strategy also displayed uniform postnatal lethality (data not shown and Fig. 3D). It is known that subtle genetic background differences and maternal effects can strongly influence the phenotypic expression of induced mouse mutations (17). We, therefore, next interbred mice carrying Prss8Cat⫺ and Prss8⫺ alleles to generate breeding pairs capable of producing Prss8Cat⫺/Cat⫺ and Prss8⫺/⫺ offspring within the same litter. Whereas Prss8⫺/⫺ offspring from these crosses died within 48 h after birth, Prss8Cat⫺/Cat⫺ littermates, again, were born in the expected frequency and displayed normal preweaning survival (Fig. 3C). Prss8Cat⫺/⫺ offspring were also born in the expected ratio and 73% survived the preweaning period (Fig. 3C). Similar results were obtained when using a breeding scheme that allowed VOLUME 289 • NUMBER 21 • MAY 23, 2014


Tris-buffered saline containing 0.05% Tween 20 for 1 h at room temperature. Individual PVDF membranes were then probed with primary antibodies diluted in 1% BSA in TBS-T overnight at 4 °C. The antibodies used included mouse anti-human prostasin (BD Transduction Laboratories, catalog no. 612173), rabbit anti-human GAPDH (Cell Signaling, Danvers, MA, catalog no. 2118), and a polyclonal rabbit anti-mouse profilaggrin/ filaggrin (Covance, Inc., Chantilly, VA, catalog no. PRB-417P). The next day, membranes were washed 3 ⫻ 5 min with TBS-T and incubated for 1 h with alkaline phosphatase-conjugated secondary antibodies (Dako, Carpinteria, CA). After 3 ⫻ 5 min washes with TBS-T, the signal was developed using nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Pierce). Densitometric scans were quantified using ImageJ software (14). Histological Analysis—Newborn to 24-h-old pups were euthanized by decapitation and prepared for histology as described previously (15). Hair follicle density and diameter were determined on a region-matched area of dorsal skin; all hair follicles within a 1-cm segment were measured. Epidermal thickness (excluding stratum corneum) and stratum corneum thickness were measured in this same 1-cm segment of dorsal skin and were calculated by averaging 100 independent measurements per skin specimen. Immunohistochemistry—Antigens from 5 ␮m paraffin sections were retrieved by incubation for 20 min at 100 °C in 0.01 M sodium citrate buffer, pH 6.0. The sections were blocked with 2.5% BSA in PBS and incubated overnight at 4 °C with mouse anti-human prostasin (1:125, BD Transduction Laboratories, catalog no. 612173). Bound antibodies were visualized using a biotin-conjugated anti-mouse secondary antibody (1:400, Vector Laboratories, Burlingame, CA) and a Vectastain ABC kit (Vector Laboratories) using 3,3⬘-diaminobenzidine as the substrate (Sigma-Aldrich). Transepidermal Fluid Loss Assay—Transepidermal fluid loss assay was performed exactly as described (15). Cutaneous Wound Repair—Full thickness incisional skin wounds (15 mm) were made in the interscapular dorsum, and healing was assessed by daily inspection of wounds by an investigator blinded as to genotype, as described previously (16). Specifically, macroscopic closure of the incisional interface was evaluated both visually and by palpation. At the time of gross inspection, maximal longitudinal wound length was measured, and each wound was photographed. Wound areas were then determined using ImageJ software (14). Wounds were collected for histological examination at days 5, 10, 14, and 21. Histological and morphometric analysis of wounds was performed as described (16).


for the generation of Prss8⫹/⫹, Prss8Cat⫺/Cat⫺, Prss8Cat⫺/⫺, and Prss8⫺/⫺ mice on the same genetic background (Fig. 3D). Prostasin Supports Terminal Epidermal Differentiation through a Non-catalytic Mechanism—The normal survival of Prss8Cat⫺/Cat⫺ mice indicated that prostasin could support terminal epidermal differentiation and epidermal barrier formation by a mechanism that was independent of its enzymatic activity. In agreement with this, the outward appearance of newborn Prss8Cat⫺/Cat⫺ pups was indistinguishable from Prss8⫹/⫹ littermates (Fig. 5A). At the histological level, the epidermis of Prss8⫹/⫹ (Fig. 5B) and Prss8Cat⫺/Cat⫺ (Fig. 5C) mice MAY 23, 2014 • VOLUME 289 • NUMBER 21

were also strikingly similar with both exhibiting normal stratum corneum featuring a characteristic “basket weave” pattern with intercorneocyte lacunae formed by a meshwork of interlocking flattened layers of corneocytes connected by desmosomes (18). In contrast, Prss8⫺/⫺ stratum corneum was completely compacted (Fig. 5D), presenting with few intercorneocyte lacunae (2). Histomorphometric analysis, revealed a small (18%), but significant, reduction in the thickness of Prss8Cat⫺/Cat⫺ stratum corneum (Fig. 5, E and F). In accordance with the normal stratum corneum structure, transepidermal water loss of Prss8Cat⫺/Cat⫺ and Prss8Cat⫺/⫺ pups was only marginally increased relative to Prss8⫹/⫹ littermates (Fig. 5G), whereas JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 1. Generation of mice expressing only catalytically inactive endogenous prostasin. A, structure of targeting vector (top), wild-type Prss8 allele (middle), and targeted Prss8 allele with the neomycin cassette removed (bottom). Exons are indicated as blue boxes and intron sequences as black lines. The locations of primers used for PCR screening of ES cell clones and genotyping of mice are indicated by green and black arrowheads, respectively. Homologous recombination in ES cells introduced a c.943T3 G substitution into exon 6 and introduced a neomycin selection cassette (orange) flanked by LoxP sites (red triangles) into intron 5. G indicates the position of the serine to alanine codon change in exon 6. B, PCR analysis of DNA from Prss8⫹/⫹ (lane 1), Prss8⫹/Cat⫺ (lane 2), Prss8Cat⫺/Cat⫺ (lane 3) offspring from Prss8⫹/Cat⫺ intercrosses. Positions of amplicons from the wild-type and targeted alleles are shown on the right. Positions of molecular weight markers (bp) are indicated on the left. C, sequence analysis of exon 6 from Prss8⫹/⫹ (left) and Prss8Cat⫺/Cat⫺ (right) mice confirms the introduction of the c.943T3 G substitution causing the serine 238 to alanine substitution in prostasin (red letters in nucleotide and amino acid sequence). D, RT-PCR analysis of Prss8 mRNA (upper panel) and ribosomal protein S15 mRNA (lower panel) in skin (lanes 1, 5, and 9), large and small intestine (lanes 2, 6, and 10), kidney (lanes 3, 7, and 11), and lung (lanes 4, 8, and 12) of Prss8⫹/⫹ (lanes 1– 4), Prss8Cat⫺/Cat⫺ (lanes 5– 8), and Prss8⫺/⫺ (lanes 9 –12) mice. A no reverse transcriptase control is included in lane 13. E, prostasin (upper) and GAPDH (lower) Western blots of skin (left panels), kidney, (middle panels), and lung (right panels) from Prss8⫹/⫹ (lanes 1–3), Prss8Cat⫺/Cat⫺ (lanes 4 – 6), and Prss8⫺/⫺ (lane 7) mice. Positions of prostasin are indicated with black and green arrowheads at the right. A prostasin species specific for Prss8Cat⫺/Cat⫺ is indicated with a red arrowhead. The positions of molecular mass markers (kDa) are indicated at the left. F, densitometric quantification of prostasin protein levels normalized to GAPDH. AU, arbitrary units. p ⫽ N.S., not significant; Student’s t test, two-tailed.


Prss8⫺/⫺ littermates dehydrated at a rapid pace (Fig. 5G), as described previously (2). Prostasin is known to be required for the proteolytic processing of the key epidermal polyprotein, profilaggrin, into filaggrin monomers during stratum corneum formation (2). Analysis of epidermal extracts from newborn Prss8Cat⫺/Cat⫺ pups by SDSPAGE (Fig. 5H) and by Western blot (Fig. 5I) showed that prostasin-mediated profilaggrin processing did not require the enzymatic activity of prostasin, although the level of processed filaggrin monomer was reduced in Prss8Cat⫺/Cat⫺ epidermis (Figs. 5, H and I, compare lanes 3–5 with lanes 6 – 8). Impaired Hair Follicle Development in Mice Expressing Catalytically Inactive Prostasin—Subtle differences in whiskers and pelage hair were apparent in Prss8Cat⫺/Cat⫺ mice. Prss8Cat⫺/Cat⫺ pups were born either without whiskers or with shorter, kinky, and curly whiskers (Fig. 6A). The whiskers remained abnormal throughout the observation period (Fig. 6B). The time of pelage hair eruption of Prss8Cat⫺/Cat⫺ mice was normal (data not shown), as were hair follicle density (Fig. 6C) and hair follicle diameter (Fig. 6D). The coat of newly weaned Prss8Cat⫺/Cat⫺ mice, however, varied from indistinguishable from Prss8⫹/⫹ littermates (⬃86% of mice, example in Fig. 6E, middle) to markedly thinner and sparser (⬃14% of mice, example in Fig. 6E, right). The latter phenotype persisted throughout the observation period in ⬃5% of Prss8Cat⫺/Cat⫺ mice.


Reduced Body Weights of Mice Expressing Catalytically Inactive Prostasin—The body weights of newborn Prss8Cat⫺/Cat⫺ pups were indistinguishable from Prss8⫹/⫹ littermates. However, Prss8Cat⫺/Cat⫺ pups displayed reduced body weight at day 14, which became significant at 3 weeks for females and at 2 weeks for males, and persisted throughout the 11-week observation period (Fig. 6, F and G). The body weights of Prss8Cat⫺/Cat⫺ mice were 10–26% reduced within the 11-week period, with the greatest weight differences manifesting the weeks prior to and after weaning. Prss8Cat⫺/Cat⫺ mice presented with no obvious outward phenotype that could explain the lower body weight. Likewise, non-epidermal tissues of Prss8Cat⫺/Cat⫺ mice were histologically unremarkable when compared with wild-type littermates (Fig. 6H). Epidermal Barrier Restoration after Incisional Wounding— To determine whether catalytically inactive prostasin could support the restoration of the epidermal barrier after disruption, we generated 1.5-cm full-thickness incisional skin wounds in the mid-scapular dorsal region of Prss8Cat⫺/Cat⫺ mice and their wild-type littermates. The wounds were left unsutured and undressed and were observed daily by an investigator blinded as to mouse genotype. The wounds were scored as healed based on the macroscopic closure of the incision interface and restoration of epithelial covering (Fig. 7A, representative photographs in Fig. 7D). Interestingly, mice expressing catalytically inactive prostasin were capable of healing their VOLUME 289 • NUMBER 21 • MAY 23, 2014


FIGURE 2. Spatial localization of catalytically inactive prostasin is normal in skin and non-epidermal tissues. Prostasin immunohistochemistry of representative sections of skin (A–C), tongue (D–F), lung (G–I), and kidney (J–L) from newborn Prss8⫹/⫹ (A, D, G, J), Prss8Cat⫺/Cat⫺ (B, E, H, and K), and Prss8⫺/⫺ (C, F, I, and L) mice. Insets are high magnification images demonstrating prostasin staining in epithelial cells of Prss8⫹/⫹ and Prss8Cat⫺/Cat⫺, but not Prss8⫺/⫺ mice. Scale bars are 50 ␮m for large panels and 10 ␮m for insets. More specifically, expression of both wild-type and mutant prostasin is observed in suprabasal layers of the interfollicular epidermis and in the hair follicle (A and B), in suprabasal layers of the tongue (D and E), in bronchial epithelium of lungs (G and H), and in distal and collecting duct epithelia of the kidney (J and K).

Non-enzymatic in Vivo Functions of Prostasin wounding, 4 of 4 Prss8Cat⫺/Cat⫺ and 4 of 4 control wounds had restored epidermal covering. Taken together, these data demonstrate that mice expressing only catalytically inactive prostasin can restore epidermal covering after wounding.

wounds, although healing was delayed by 26% (mean healing time (days) ⫾ S.D. Prss8⫹/⫹ ⫽ 12.3 ⫾ 1.7, Prss8Cat⫺/Cat⫺ ⫽ 15.5 ⫾ 0.8, p ⫽ 0.003 log-rank test, two-tailed). Histologic examination of the wounds at day 21 showed complete restoration of epidermal covering, including a well developed stratum corneum in the regenerated epidermis of both Prss8⫹/⫹ and Prss8Cat⫺/Cat⫺ mice (Fig. 7, B and C). We next performed a histological analysis of the kinetics of wound healing by analyzing wounds at days 5, 10, and 14 after incisional wounding (Fig. 7E). At day 5 after wounding, 0 of 5 Prss8Cat⫺/Cat⫺ and 1 of 5 Prss8⫹/⫹ wounds had restored epidermal covering. The length of the newly formed epidermal wedges migrating into the wounds in the five Prss8Cat⫺/Cat⫺ wounds was 663 ⫾ 152 ␮m and was 765 ⫾ 277 ␮m in the four non-reepithelialized Prss8⫹/⫹ wounds (p ⫽ N.S.,2 Student’s t test, two-tailed). At day 10 after wounding, 2 of 4 Prss8Cat⫺/Cat⫺ and 3 of 4 control wounds had restored epidermal covering, and at day 14 after 2

The abbreviation used is: N.S., not significant.



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FIGURE 3. Mice expressing catalytically inactive prostasin develop to term and exhibit normal postnatal survival in a genetic background where complete absence of prostasin leads to uniform lethality. A, genotype distribution of offspring from Prss8⫹/Cat⫺ intercrosses at 0 –24 h after birth (black bars) and at weaning (gray bars). 111 offspring from a total of 13 litters were analyzed. B, postweaning survival of a prospective cohort of 24 Prss8⫹/⫹ (black) and 20 Prss8Cat⫺/Cat⫺ (green) littermates that were followed for up to 20 weeks. C, genotype analysis of newborn offspring from intercrossed Prss8Cat⫺/⫺ mice. Thirty offspring from a total of seven litters were genotyped shortly after birth and tracked until weaning. Note that Prss8Cat⫺/Cat⫺ and Prss8⫺/⫺ mice could be born within the same litter but that all Prss8⫺/⫺ mice died, whereas littermate Prss8Cat⫺/Cat⫺ mice survived normally. D, postnatal survival of a prospective cohort of 21 Prss8⫹/⫹ (black circles), 11 Prss8Cat⫺/Cat⫺ (green squares), 17 Prss8Cat⫺/⫺ (yellow triangles), and 14 Prss8⫺/⫺ (red diamonds) mice on the same genetic background that were followed for 21 days. Prss8⫹/⫹ versus Prss8Cat⫺/Cat⫺, p ⫽ N.S.; Prss8⫹/⫹ versus Prss8Cat⫺/⫺, p ⬍ 0.05; Prss8⫹/⫹ versus Prss8⫺/⫺, p ⬍ 0.0001; Prss8Cat⫺/Cat⫺ versus Prss8Cat⫺/⫺, p ⫽ N.S; Prss8Cat⫺/Cat⫺ versus Prss8⫺/⫺, p ⬍ 0.0001 (logrank test, two-tailed).

DISCUSSION The PRSS8 gene is conserved in all vertebrate species examined, and it encodes a single-chain protease zymogen that can be proteolytically converted to an active two-chain trypsin-like serine protease. Aligned with this high conservation, prostasin is essential for mouse survival and promotes key proteolytic processes in epithelial tissues (reviewed in Ref. 19). The results of the current study, which show that prostasin can support both epidermal development and long term mouse survival independent of its catalytic activity, are therefore unexpected. Particularly striking are the normal interfollicular epidermal differentiation and the formation of a functional epidermal barrier in mice engineered to express only catalytically inactive endogenous prostasin, Prss8Cat⫺/Cat⫺ mice, as compared with the lack of terminal epidermal differentiation and uniform lethality observed in mice lacking prostasin protein, Prss8⫺/⫺ mice. Furthermore, Prss8Cat⫺/Cat⫺ mice were able to restore their epidermal covering after wounding, albeit with increased healing times. At present, it is not possible to determine whether the reduction in profilaggrin processing, the marginal impairment of epidermal barrier function, and the increased skin wound healing times observed for Prss8Cat⫺/Cat⫺ mice are due to the loss of catalytic activity or instead due to subtle changes in the trafficking, subcellular localization or turnover of the mutant prostasin. What is clear, however, is that prostasin can support these activities independent of its enzymatic activity. The specific mechanism by which catalytically inactive prostasin supports interfollicular epidermal development remains to be established and is beyond the scope of the current study. However, in a cell-based assay, we recently found that catalytically inactive prostasin, was capable of stimulating both matriptase auto-activation and cleavage of a physiological matriptase substrate (11). Furthermore, in a reconstituted Xenopus oocyte system, catalytically inactive prostasin has been reported to stimulate the activation of the epithelial sodium channel by inducing proteolytic cleavage of the epithelial sodium channel ␥-chain (20, 21). These findings, in conjunction with the in vivo observations presented herein, lend support to the hypothesis that an allosteric interaction of prostasin with another membrane-bound serine protease, matriptase, may be required for normal epidermal barrier formation. Proteolytic cleavage of single-chain prostasin to two-chain prostasin has been proposed to be the key function of matriptase in epidermal development (5). It follows, logically, that if this assertion is correct, then the non-catalytic function by which prostasin supports terminal epidermal differentiation will be expressed only after conversion of single-chain prostasin to two-chain prostasin. In this respect, prostasin would be similar to hepatocyte growth factor and macrophage stimulating protein, both of which are trypsin-like serine protease-like proteins that are competent to execute their biological functions only after canonical activation site cleavage (22). Importantly,


FIGURE 5. Terminal epidermal differentiation and epidermal barrier formation require prostasin, but not prostasin’s enzymatic activity. (A) Representative photographs of newborn Prss8⫹/⫹ (left), and Prss8Cat⫺/Cat⫺ (right) littermates demonstrating similarity in size and outward appearance. B–D, H&E staining of dorsal skin from newborn Prss8⫹/⫹ (B), Prss8Cat⫺/Cat⫺ (C), and Prss8⫺/⫺ (D) mice. The position of the stratum corneum is indicated to the right of each panel. Note the abnormally compacted Prss8⫺/⫺ stratum corneum, and the normal morphology of Prss8Cat⫺/Cat⫺ stratum corneum. Scale bars are 50 ␮m. E and F, thickness of the epidermis (excluding the stratum corneum) (E) and the stratum corneum (F) of Prss8⫹/⫹ (black circles, n ⫽ 11) and Prss8Cat⫺/Cat⫺ (purple squares, n ⫽ 14) littermates. Horizontal bars indicate medians (*, p ⬍ 0.05, Student’s t test, two tailed). G, rate of epidermal fluid loss from newborn mice was estimated by measuring reduction of body weight as a function of time. The data are expressed as the average % of initial body weight for Prss8⫹/⫹ (black circles; n ⫽ 10), Prss8Cat⫺/Cat⫺ (green squares; n ⫽ 9), Prss8Cat⫺/⫺ (yellow triangles; n ⫽ 5), and Prss8⫺/⫺ (red diamonds; n ⫽ 3) pups in the same genetic background. Error bars indicate S.D. *, p ⬍ 0.001 and **, p ⬍ 0.0001, relative to Prss8⫹/⫹ (Student’s t test, two-tailed). Note that Prss8⫺/⫺ littermates have impaired barrier function and dehydrate rapidly, whereas the epidermal barrier of Prss8Cat⫺/Cat⫺ mice is functional. H and I, profilaggrin processing was assessed by performing SDS-PAGE with Coomassie Brilliant Blue staining (H) and profilaggrin/filaggrin Western blotting (I) on epidermal protein extracts from Prss8⫺/⫺ (lanes 1 and 2), Prss8⫹/⫹ (lanes 3–5), and Prss8Cat⫺/Cat⫺ (lanes 6 – 8) mice. Position of the processed filaggrin monomer is indicated at the right, and the positions of molecular mass markers (kDa) are indicated on the left.


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FIGURE 4. Generation of Prss8ⴚ/ⴚ mice by retroviral insertion. A, structure of targeting vector (top), wild-type Prss8 allele (middle), and targeted Prss8 allele (bottom). Exons are indicated as blue boxes, and intron sequences are shown as black lines. The ␤-geo cassette is indicated as an orange box and retroviral long terminal repeats (LTR) as filled green arrows. SA indicates the position of the engrailed splice acceptor site, and pA indicates the position of the polyadenylation site. The positions of the primers used for genotyping of mice by PCR are indicated by black arrowheads. The locations of primers used for Prss8 transcript analysis are indicated by green arrowheads. Insertion of the retroviral targeting construct into intron 2 of Prss8 leads to fusion of Prss8 exon 2 to the ␤-geo gene, resulting in a null mutation. B, RT-PCR of Prss8 (top panel) and ribosomal protein S15 (bottom panel) mRNA isolated from skin (lanes 1 and 5), intestine (lanes 2 and 6), kidneys (lanes 3 and 7), and lungs (lanes 4 and 8) of newborn Prss8⫹/⫹ (lanes 1– 4) and Prss8⫺/⫺ (lanes 5– 8) littermates using primer pairs amplifying mRNA sequences derived from exons 1 through 4 (see position of green primers in A). No reverse transcriptase was added to the reaction in lane 9. C, Western blot of proteins extracted from the placenta of two Prss8⫺/⫺ embryos (lanes 1 and 2) and two Prss8⫹/⫹ littermates (lanes 3 and 4). Position of prostasin is indicated at the right, and the positions of molecular mass markers (kDa) are indicated at the left. fwd, forward; rev, reverse.


prostasin differs from these two growth factors, which are devoid of proteolytic activity, by having non-enzymatic functions while at the same time being an enzymatically active serine protease. Mice expressing catalytically inactive prostasin presented with a distinct whisker and pelage hair phenotype. It is known that prostasin expression in the mouse hair follicle is restricted to Henle’s layer of the inner root sheath of the non-proliferative compartment where it co-localizes with matriptase (10), and the observed phenotype for Prss8Cat⫺/Cat⫺ mice MAY 23, 2014 • VOLUME 289 • NUMBER 21

is quite similar to the phenotype observed in both matriptase hypomorphic mice (23) and in the spontaneous mutant mouse strain, frizzy (24). The latter mouse strain carries a point mutation in the Prss8 gene (Prss8fr/fr) that results in a non-conservative V170D amino acid substitution in the prostasin protein. We have reported previously that recombinant soluble V170D prostasin has a low residual enzymatic activity, and we attributed the frizzy pelage hair and whisker phenotype to this reduced proteolytic activity (6). The V170D mutation in prostasin, however, is predicted by in silico modeling to result in a JOURNAL OF BIOLOGICAL CHEMISTRY

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FIGURE 6. Prss8Catⴚ/Catⴚ have a visible phenotype including whisker and pelage hair defects and reduced body weights; however, there are no differences in the histologic appearance of tissues. A, representative example of the appearance of whiskers in newborn Prss8⫹/⫹ (left) and Prss8Cat⫺/Cat⫺ (right) littermates. B, appearance of whiskers of 21-day-old Prss8⫹/⫹ (left) and Prss8Cat⫺/Cat⫺ (middle and right) littermates. C and D, hair follicle density (C) and hair follicle diameter (D) of newborn Prss8⫹/⫹ (black circles, n ⫽ 11) and Prss8Cat⫺/Cat⫺ (purple squares, n ⫽ 14) littermates. For each mouse, all hair follicles in a region-matched 1-cm segment of dorsal skin were counted, and the diameter was measured. Horizontal bars indicate medians (p ⫽ N.S. Student’s t test, two tailed). E, representative examples of pelage hair of 21-day-old Prss8⫹/⫹ (left) and Prss8Cat⫺/Cat⫺ (middle and right) littermates. The coat of Prss8Cat⫺/Cat⫺ mice varies from indistinguishable from Prss8⫹/⫹ littermates (middle) to markedly sparser (right). F and G, plot of age versus weight of female (F) Prss8⫹/⫹ (black circles) and littermate Prss8Cat⫺/Cat⫺ (red squares) mice and of male (G) Prss8⫹/⫹ (black circles) and littermate Prss8Cat⫺/Cat⫺ (blue squares) mice. The weights were determined at 1- to 2-week intervals. Bars indicate S.D. (*, p ⬍ 0.05, Student’s t test, two-tailed). H, H&E staining of sections of lung (left), liver (middle left), kidney (middle right), and small intestine (right) from newborn Prss8⫹/⫹ (top) and Prss8Cat⫺/Cat⫺ (bottom) mice. Scale bars are 100 ␮m.


2.4-kcal energy loss and was shown experimentally to display abnormal glycosylation (3). V170D prostasin, therefore, may also display non-enzymatic activity-associated deficiencies, which could cause the pelage hair and whisker phenotype. The S238A prostasin that is expressed by Prss8Cat⫺/Cat⫺ mice, however, can be presumed to be deficient solely in its enzymatic activity, which provides additional support for prostasin proteolysis, possibly dependent on matriptase, being important for hair follicle development. Nevertheless, we cannot completely exclude that the pelage hair and whisker phenotype of Prss8Cat⫺/Cat⫺ mice may be due to a “gain of function” acquired by the S238A mutation. Arguing strongly against this, however, the pelage hair phenotype displayed greater penetrance in Prss8Cat⫺/⫺ mice, when compared with Prss8Cat⫺/Cat⫺ mice.3 In conclusion, this study reveals that critical biologic functions of prostasin in epithelial development and homeostasis 3

D. E. Peters, R. Szabo, S. Friis, N. A. Shylo, K. Uzzun Sales, K. Holmbeck, and T. H. Bugge, unpublished observations.


are independent of the enzymatic activity of this protease and adds further support to the hypothesis that an allosteric interaction of prostasin with matriptase is a mechanistic requirement for normal epidermal barrier formation. Acknowledgments—We thank Drs. Silvio Gutkind and Mary Jo Danton for critically reviewing this manuscript and Advait Limaye and Glenn Longenecker from the NIDCR Gene Targeting Facility for mouse generation.

REFERENCES 1. Hummler, E., Dousse, A., Rieder, A., Stehle, J. C., Rubera, I., Osterheld, M. C., Beermann, F., Frateschi, S., and Charles, R. P. (2013) The channelactivating protease CAP1/Prss8 is required for placental labyrinth maturation. PLoS One 8, e55796 2. Leyvraz, C., Charles, R. P., Rubera, I., Guitard, M., Rotman, S., Breiden, B., Sandhoff, K., and Hummler, E. (2005) The epidermal barrier function is dependent on the serine protease CAP1/Prss8. J. Cell Biol. 170, 487– 496 3. Frateschi, S., Keppner, A., Malsure, S., Iwaszkiewicz, J., Sergi, C., Merillat,

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FIGURE 7. The enzymatic activity of prostasin is not required for restoration of the epidermis following cutaneous wounding. A, rate of healing of 1.5-cm incisional skin wounds in Prss8⫹/⫹ (black, n ⫽ 6) mice and Prss8Cat⫺/Cat⫺ (green, n ⫽ 7) littermates. Note that wound healing was significantly delayed in Prss8Cat⫺/Cat⫺ mice relative to Prss8⫹/⫹ littermate controls (log-rank test, two-tailed). B and C, H&E sections of healed wounds collected on day 21 from Prss8⫹/⫹ (B) and Prss8Cat⫺/Cat⫺ (C) littermates. Note the well developed stratum corneum (SC, dashed lines) of the regenerated epidermis in both cases. Arrows indicate wound margins, and asterisks indicate granulation tissue. Scale bars for low magnification images were 500 ␮m, and scale bars for high magnification were 50 ␮m. D, Photographic time course of wound healing for a Prss8⫹/⫹ mouse scored as healed on day 11 (upper panels) and a Prss8Cat⫺/Cat⫺ littermate scored as healed on day 16 (lower panels). All images are shown at the same magnification. Scale bar is 1 cm. E, histologic time course of wound healing in representative Prss8⫹/⫹ (upper panels) and Prss8Cat⫺/Cat⫺ (lower panels) littermates. Arrows indicate wound margins, arrowheads indicate epithelial tongues in un-healed wounds, and asterisks indicate granulation tissue. Scale bars, 500 ␮m.


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

J. Biol. Chem. 2014, 289:14740-14749. doi: 10.1074/jbc.M113.541318 originally published online April 4, 2014

Access the most updated version of this article at doi: 10.1074/jbc.M113.541318 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 24 references, 9 of which can be accessed free at http://www.jbc.org/content/289/21/14740.full.html#ref-list-1


Diane E. Peters, Roman Szabo, Stine Friis, Natalia A. Shylo, Katiuchia Uzzun Sales, Kenn Holmbeck and Thomas H. Bugge

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