Delineation of Matriptase Protein Expression by Enzymatic Gene Trapping Suggests Diverging Roles in Barrier Function, Hair Formation, and Squamous Cell Carcinogenesis
2006; Elsevier BV; Volume: 168; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2006.051071
ISSN1525-2191
AutoresKarin List, Roman Szabo, Alfredo Molinolo, Boye Schnack Nielsen, Thomas Bugge,
Tópico(s)Skin and Cellular Biology Research
ResumoThe membrane serine protease matriptase is required for epidermal barrier function, hair formation, and thymocyte development in mice, and dysregulated matriptase expression causes epidermal squamous cell carcinoma. To elucidate the specific functions of matriptase in normal and aberrant epidermal differentiation, we used enzymatic gene trapping combined with immunohistochemical, ultrastructural, and barrier function assays to delineate the spatio-temporal expression and function of matriptase in mouse keratinized tissue development, homeostasis, and malignant transformation. In the interfollicular epidermis, matriptase expression was restricted to postmitotic transitional layer keratinocytes undergoing terminal differentiation. Matriptase was also expressed in keratinizing oral epithelium, where it was required for oral barrier function, and in thymic epithelium. In all three tissues, matriptase colocalized with profilaggrin. In staged embryos, the onset of epidermal matriptase expression coincided with that of profilaggrin expression and acquisition of the epidermal barrier. In marked contrast to stratifying keritinized epithelium, matripase expression commenced already in undifferentiated and rapidly proliferating profilaggrin-negative matrix cells and displayed hair growth cycle-dependent expression. Exposure of the epidermis to carcinogens led to the gradual appearance of matriptase in a keratin-5-positive proliferative cell compartment during malignant progression. Combined with previous studies, these data suggest that matriptase has diverging functions in the genesis of stratified keratinized epithelium, hair follicles, and squamous cell carcinoma. The membrane serine protease matriptase is required for epidermal barrier function, hair formation, and thymocyte development in mice, and dysregulated matriptase expression causes epidermal squamous cell carcinoma. To elucidate the specific functions of matriptase in normal and aberrant epidermal differentiation, we used enzymatic gene trapping combined with immunohistochemical, ultrastructural, and barrier function assays to delineate the spatio-temporal expression and function of matriptase in mouse keratinized tissue development, homeostasis, and malignant transformation. In the interfollicular epidermis, matriptase expression was restricted to postmitotic transitional layer keratinocytes undergoing terminal differentiation. Matriptase was also expressed in keratinizing oral epithelium, where it was required for oral barrier function, and in thymic epithelium. In all three tissues, matriptase colocalized with profilaggrin. In staged embryos, the onset of epidermal matriptase expression coincided with that of profilaggrin expression and acquisition of the epidermal barrier. In marked contrast to stratifying keritinized epithelium, matripase expression commenced already in undifferentiated and rapidly proliferating profilaggrin-negative matrix cells and displayed hair growth cycle-dependent expression. Exposure of the epidermis to carcinogens led to the gradual appearance of matriptase in a keratin-5-positive proliferative cell compartment during malignant progression. Combined with previous studies, these data suggest that matriptase has diverging functions in the genesis of stratified keratinized epithelium, hair follicles, and squamous cell carcinoma. The epithelial compartment of the skin is composed of a multilayered interfollicular epidermis and a follicular epidermis consisting of hair follicles with associated sebaceous glands.1Fuchs E Raghavan S Getting under the skin of epidermal morphogenesis.Nat Rev Genet. 2002; 3: 199-209Crossref PubMed Scopus (569) Google Scholar During epidermal development and homeostasis, cell proliferation and differentiation are compartmentalized and tightly regulated processes. The interfollicular epidermis undergoes continuous renewal when proliferative cells residing in the basal layer commit to differentiation and move outwards to give rise to the spinous layer, the granular layer, the transitional layer, and the terminally differentiated cornified layer, the stratum corneum.2Presland RB Dale BA Epithelial structural proteins of the skin and oral cavity: function in health and disease.Crit Rev Oral Biol Med. 2000; 11: 383-408Crossref PubMed Scopus (308) Google Scholar The interfollicular epidermis serves a critical function as a first line of defense against the external environment by providing a protective barrier against mechanical, chemical, and biological insults.3Elias PM Choi EH Interactions among stratum corneum defensive functions.Exp Dermatol. 2005; 14: 719-726Crossref PubMed Scopus (162) Google Scholar The epidermis also provides a water-impermeable barrier that prevents excessive loss of body fluids, a function that is critical for the survival of all terrestrial vertebrates. The epidermal barrier function resides in the stratum corneum and consists of an interlocking meshwork of flattened terminally differentiated keratinocytes in which the plasma membrane is replaced by a highly cross-linked, insoluble cornified envelope of the corneocytes. The corneocytes are connected by desmosomes and are embedded in a specialized intercorneocyte lipid matrix. 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Rapidly proliferating transient amplifying cells located in the follicular matrix, a zone located at the proximal end of the hair surrounding the dermal papilla, give rise to the cortex, medulla, and cuticle of the hair shaft and to the inner root sheath cells, which continuously move upwards. The outer root sheath of the hair follicle is continuous with the basal layer of the interfollicular epidermis. The matrix transient amplifying cells are relatively undifferentiated and periodically withdraw from the cell cycle and commit to terminal differentiation. As the matrix cells lose their proliferative capacity, the hair follicle growth subsides, and the follicular regression phase progresses. 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The membrane protease was required for completion of a terminal differentiation program in the interfollicular epidermis that included cornified envelope and lipid lamellar granule formation. The defects caused by matriptase deficiency included severely impaired formation of extracellular skin lipids, abnormal cornified envelope formation, and loss of epidermal barrier function. They correlated at the molecular level with an impaired proteolytic processing of profilaggrin, leading to a complete loss of both filaggrin monomer and filaggrin S-100 protein in the absence of alterations in the expression of other major differentiation markers.16List K Haudenschild CC Szabo R Chen W Wahl SM Swaim W Engelholm LH Behrendt N Bugge TH Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis.Oncogene. 2002; 21: 3765-3779Crossref PubMed Scopus (282) Google Scholar, 17List K Szabo R Wertz PW Segre J Haudenschild CC Kim SY Bugge TH Loss of proteolytically processed filaggrin caused by epidermal deletion of Matriptase/MT-SP1.J Cell Biol. 2003; 163: 901-910Crossref PubMed Scopus (178) Google Scholar Loss of matriptase also seriously affected hair follicle development and resulted in generalized follicular hypoplasia, absence of erupted vibrissae, and lack of vibrissal hair canal formation.16List K Haudenschild CC Szabo R Chen W Wahl SM Swaim W Engelholm LH Behrendt N Bugge TH Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis.Oncogene. 2002; 21: 3765-3779Crossref PubMed Scopus (282) Google Scholar Furthermore, the thymus, an organ rich in keratinized epithelium,7Laster AJ Itoh T Palker TJ Haynes BF The human thymic microenvironment: thymic epithelium contains specific keratins associated with early and late stages of epidermal keratinocyte maturation.Differentiation. 1986; 31: 67-77Crossref PubMed Scopus (81) Google Scholar, 8Hale LP Markert ML Corticosteroids regulate epithelial cell differentiation and Hassall body formation in the human thymus.J Immunol. 2004; 172: 617-624PubMed Google Scholar displayed pronounced hypoplasia caused by dramatically increased thymocyte apoptosis, leading to depletion of thymocytes.16List K Haudenschild CC Szabo R Chen W Wahl SM Swaim W Engelholm LH Behrendt N Bugge TH Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis.Oncogene. 2002; 21: 3765-3779Crossref PubMed Scopus (282) Google Scholar Matriptase was identified in part through its consistent overexpression in human carcinoma and has been causally linked to epithelial carcinogenesis in a large number of studies.18Kang JY Dolled-Filhart M Ocal IT Singh B Lin CY Dickson RB Rimm DL Camp RL Tissue microarray analysis of hepatocyte growth factor/Met pathway components reveals a role for Met, matriptase, and hepatocyte growth factor activator inhibitor 1 in the progression of node-negative breast cancer.Cancer Res. 2003; 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61: 228Crossref PubMed Scopus (78) Google Scholar, 28Santin AD Zhan F Bellone S Palmieri M Cane S Bignotti E Anfossi S Gokden M Dunn D Roman JJ O'Brien TJ Tian E Cannon MJ Shaughnessy Jr, J Pecorelli S Gene expression profiles in primary ovarian serous papillary tumors and normal ovarian epithelium: identification of candidate molecular markers for ovarian cancer diagnosis and therapy.Int J Cancer. 2004; 112: 14-25Crossref PubMed Scopus (253) Google Scholar, 29List K Szabo R Molinolo A Sriuranpong V Redeye V Murdock T Burke B Nielsen BS Gutkind JS Bugge TH Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation.Genes Dev. 2005; 19: 1934-1950Crossref PubMed Scopus (211) Google Scholar Most recently, modest matriptase overexpression in transgenic mice was found to suffice to initiate multistage squamous cell carcinogenesis and to promote strongly carcinogen-induced epidermal tumor formation.29List K Szabo R Molinolo A Sriuranpong V Redeye V Murdock T Burke B Nielsen BS Gutkind JS Bugge TH Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation.Genes Dev. 2005; 19: 1934-1950Crossref PubMed Scopus (211) Google Scholar Expression studies of normal human tissues have shown that matriptase is widely expressed in epithelia including the epidermis.30Oberst MD Singh B Ozdemirli M Dickson RB Johnson MD Lin CY Characterization of matriptase expression in normal human tissues.J Histochem Cytochem. 2003; 51: 1017-1025Crossref PubMed Scopus (140) Google Scholar To provide a sensitive and specific method for studying matriptase expression in the mouse, we have now generated two transgenic mouse strains in which the endogenous matriptase gene is fused to a bacterial β-galactosidase marker gene under transcriptional control of the endogenous matriptase locus. We used these transgenic mice to delineate matriptase expression and function in normal and aberrant keratinized epithelia by combining the enzymatic detection of β-galactosidase with immunohistochemistry, electron microscopy, and epidermal barrier function assays. We show that matriptase is expressed in the keratinized part of the oral and thymic epithelium and that the requirement of matriptase expression for acquisition of barrier formation extends to the oral cavity. Moreover, we show that matriptase is expressed in three functionally very different keratinocyte populations: 1) keratinized stratified epithelium (terminally differentiating profilaggrin-processing cells), 2) hair follicles (rapidly proliferating, profilaggrin-negative, transit amplifying matrix cells), and 3) epi-dermal carcinomas (well-differentiated and keratin-5-expressing poorly differentiated proliferating cells). Together with previous studies of the effects of epidermal ablation and overexpression of matriptase, these data suggest divergent roles of the membrane protease in the three tissues and provide a platform for further exploration of matriptase molecular functions in the development and malignant transformation of keratinized epithelium. ES cell lines XM184 and RST485 with a gene trap in-sertion into introns 1 and 16, respectively, of the mouse matriptase gene were obtained from Bay Genomics (baxgenomics.ucsf.edu, San Francisco, CA). The embryonic stem cells (ES) cell lines were generated using a gene trap protocol with the trapping construct vectors pGT0pfs and pGt1TM, respectively, containing the intron from the engrailed 2 gene upstream of the gene encoding the β-galactosidase/neomycin-phosphotransferase fusion protein (Bay Genomics). The ES cells were injected into the blastocoel cavity of C57Bl/6J-derived blastocysts and implanted into pseudopregnant females. Chimeric male offspring were bred to NIH Black Swiss females (Taconic Farms, Germantown, NY) to generate offspring carrying one β-galactosidase-targeted ma-triptase allele. Conventional matriptase gene-targeted mice were generated as described previously.16List K Haudenschild CC Szabo R Chen W Wahl SM Swaim W Engelholm LH Behrendt N Bugge TH Matriptase/MT-SP1 is required for postnatal survival, epidermal barrier function, hair follicle development, and thymic homeostasis.Oncogene. 2002; 21: 3765-3779Crossref PubMed Scopus (282) Google Scholar The gene trap insertion sites in the mouse matriptase gene were confirmed by the identification of truncated matriptase-β-galactosidase fusion mRNA species in ES cell clones XM184 and RST485 by RT-PCR and Northern blot. Total RNA from ES cells was prepared by extraction in Trizol reagent (Gibco-BRL), as recommended by the manufacturer. RNA from ES cells was amplified by reverse transcription followed by PCR amplification using Ready-to-go RT-PCR beads (Amersham Pharmacia Biotech Inc., Piscataway, NJ), as recommended by the manufacturer. First-strand cDNA synthesis was performed using gene-trap-specific primers (clone XM184, 5′- TGGGTAACGCCAGGGTTT-3′, and clone RST485, 5′- GGTTGCTAGTAGACTTCTGCAC-3′). The subsequent PCR amplification (annealing temperature, 55°C; denaturation temperature, 92°C; extension temperature, 72°C; 40 cycles) was performed with the first-strand primer in combination with a matriptase exon 1-specific primer (5′-AGCAATCGGGGCCGCAAGGCCG-3′) or a matri-ptase exon 16-specific primer (5′-CCAGGGCCACTTGTGTGGGGCCT-3′). The RT-PCR products were excised from agarose gels after electrophoresis, purified, and subsequently analyzed by DNA sequencing. For Northern blot analysis, total RNA (1 μg) from ES clones XM184 and RST485 were fractionated electrophoretically on formaldehyde agarose gels, blotted onto Nytran SuperCharge nylon membranes (Schleicher and Schuell, Keene, NH), and hybridized to a 32P-labeled 3.5-kb matriptase expressed sequence tag probe (I.M.A.G.E. ID 2609399) that contains the complete full-length murine matriptase cDNA or a trap-specific probe amplified by PCR using primers 5′- GCTGGCTGGAGTGCGATCTT-3′ and 5′-ACTGTCCTGGCCGTAACCGA-3′. The membranes were subjected to PhosphorImage analysis using ImageQuant software from Molecular Dynamics (Sunnyvale, CA). In situ hybridization on tissues from newborn mice was performed as previously described.29List K Szabo R Molinolo A Sriuranpong V Redeye V Murdock T Burke B Nielsen BS Gutkind JS Bugge TH Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation.Genes Dev. 2005; 19: 1934-1950Crossref PubMed Scopus (211) Google Scholar In brief, an expressed sequence tag containing the full-length murine matriptase cDNA (I.M.A.G.E. ID 2609399) was used as a template to generate two nonoverlapping PCR fragments for transcription of antisense and sense probes. Riboprobes were labeled with [35S]UTP (NEN, Boston, MA) by in vitro transcription using T7 and T3 RNA polymerases (Roche, Basel, Switzerland) and approximately 1 μg of template. Paraffin sections were deparaffinized in xylene and hydrated with graded ethanol solutions. Sections were incubated at 99°C for 2 minutes in Tris EGTA buffer (10 mmol/L Tris, pH 9.0, 0.5 mmol/L EGTA) using a T/T Micromed microwave processor (Milestone, Sorisol, Italy). After an additional 20 minutes at room temperature, the sections were dehydrated with graded ethanol, and the 35S-labeled probes (2 × 106 cpm in 20 μl of hybridization mixture per slide) were incubated overnight at 55°C in a humidified chamber. Sections were washed with standard saline citrate (SSC) buffers containing 0.1% sodium dodecyl sulfate and 10 mmol/L dithiothreitol at 150 rpm at 55°C for 10 minutes in 2× SSC, 10 minutes in 0.5× SSC, and 10 minutes in 0.2× SSC. Sections were then RNase A treated for 10 minutes to remove nonspecifically bound riboprobe. Subsequent washes were performed in 0.2× SSC as specified above. Sections were dehydrated and soaked in an autoradiographic emulsion, exposed for 10 to 14 days, developed, and counterstained with hematoxylin and eosin. Skin samples were fixed for 1 hour in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS), rinsed in PBS, and stained with a β-galactosidase staining kit (Roche, Indianapolis, IN) overnight at 37°C. The tissues were post-fixed for 1 hour in 4% PFA, embedded in paraffin, and sectioned. Sections were counterstained with nuclear fast red or hematoxylin. Tissues were fixed with 4% PFA, X-gal-stained, processed into paraffin, and sectioned as described above. Antigens were retrieved by incubation with 5 μg/ml proteinase K, blocked with 2% bovine serum albumin, and incubated overnight at 4°C with rabbit antibodies to mouse keratin 5, keratin 6, or filaggrin (Covance, Richmond, CA). The polyclonal antibody mIRSa used to visualize type I inner root sheath keratin mIRSa3.1 was a kind gift from Dr. Rebecca M. Porter.38Porter RM Gandhi M Wilson NJ Wood P McLean WH Lane EB Functional analysis of keratin components in the mouse hair follicle inner root sheath.Br J Dermatol. 2004; 150: 195-204Crossref PubMed Scopus (21) Google Scholar The antibody AE13 (Abcam, Cambridge MA) was used to detect hair cortex keratins. Bound antibodies were visualized using Envision system HPRT secondary antibodies (DAKO, Carpentaria, CA) and a NovaRED substrate kit (Vector Laboratories, Burlingame, CA). Cell proliferation was visualized by intraperitoneal injection of 100 μg/g bromodeoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) 2 hours before euthanasia. BrdU incorporation was detected with a mouse anti-BrdU antibody (Accurate Chemical & Scientific Corporation, Westbury, NY), and bound antibodies were visualized with a Vectastain ABC peroxidase kit (Vector Laboratories). Cell proliferation was also visualized using a rabbit polyclonal Ki67 antibody (NovoCastra, Newcastle, United Kingdom) as described above. The procedure described by Aoyama et al32Aoyama N Molin DG Mentink MM Koerten HK De Ruiter MC Gittenberger-De Groot AC Poelmann RE Changing intracellular compartmentalization of beta-galactosidase in the ROSA26 reporter mouse during embryonic development: a light- and electron-microscopic study.Anat Rec A Discov Mol Cell Evol Biol. 2004; 279: 740-748Crossref PubMed Scopus (5) Google Scholar was followed. Skin samples were trimmed into 1-mm3 pieces and fixed in 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.4, for 1 hour at 4°C. The tissues were then incubated in 0.1% 5-bromo-3-indolyl-d-galactoside (Bluo-gal) (Sigma-Aldrich, St. Louis, MO) with 5 mmol/L potassium ferricyanide, 5 mmol/L potassium ferrocyanide, and 2 mmol/L MgCl2 overnight at 37°C. At the end of incubation, the tissues were briefly rinsed in PBS and postfixed in 2% paraformaldehyde and 2% glutaraldehyde in sodium cacodylate buffer for 12 hours. The samples were further fixed in 1% OsO4 followed by ethanol dehydration, embedding, and staining with uranyl acetate and lead citrate. The samples were examined with a JEOL-1010 transmission electron microscope operated at 80 kV. Newborn mice were euthanized and subjected to methanol dehydration and subsequent rehydration as described previously.33Hardman MJ Sisi P Banbury DN Byrne C Patterned acquisition of skin barrier function during development.Development. 1998; 125: 1541-1552Crossref PubMed Google Scholar Whole pups or dissected tongues and hard palates were then stained for 1 hour at room temperature in 0.1% toluidine blue/PBS (Fisher Scientific, Pittsburgh, PA), destained for 15 minutes in PBS at room temperature, and examined with a dissection microscope for epidermal dye penetration. The dorsal skin of 6- to 8-week-old matriptase+/β-gal and wild-type littermate mice was shaved and treated 2 days later with a single topical application of 25 μg of 7,12-dimethylbenzanthracene (DMBA) (Sigma) in 200 μl of acetone, followed 2 weeks later by weekly applications of 12 μg of phorbol 12-myristate 13-acetate (PMA) (Sigma) for up to 30 weeks. Previous in situ hybridization studies of mouse tissues did not enable a detailed high-resolution analysis of matriptase expression,29List K Szabo R Molinolo A Sriuranpong V Redeye V Murdock T Burke B Nielsen BS Gutkind JS Bugge TH Deregulated matriptase causes ras-independent multistage carcinogenesis and promotes ras-mediated malignant transformation.Genes Dev. 2005; 19: 1934-1950Crossref PubMed Scopus (211) Google Scholar and immunohistochemical screening of a series of commercial and in-house-generated matriptase antibodies using matriptase-deficient mouse tissue sections as specificity control revealed prohibitively high cross-reactivity in keratinized tissues (K. List and T.H. Bugge, unpublished data). We therefore used the technology of enzymatic gene trapping to study matriptase expression in the mouse. A search of the BayGenomics Web site revealed two ES cell clones in which a promoterless β-galactosidase-neomycin gene trap was inserted between exons 1 and 2 (ES clone XM184) and 16 and 17 (ES clone RST485) of matriptase. These insertions would give rise to a soluble intracellular matriptase-β-galactosidase fusion protein (gene trap insertion between exons 1 and 2) or a membrane-anchored fusion protein (gene trap insertion between exons 16 and 17) anchored via the CD4 transmembrane segment provided by the gene trap and the matriptase signal anchor. Both fusion proteins would be expressed under the control of the endogenous matriptase promoter. Northern blot (data not shown) and RT-PCR analysis combined with DNA sequencing (Figure 1) confirmed the expression of the predicted matriptase-β-galactosidase fusion mRNA in both ES cell clones. The two ES cell lines were microinjected into blastocysts, and breeding of the ensuing chimeras for germline transmission gave rise to two mouse strains carrying a gene trapped matriptase allele (hereafter referred to as matriptase+/E1β-gal and matriptase+/E16β-gal). Expression analysis of tissues from both matriptase+/E1β-gal and matriptase+/E16β-gal mice showed X-gal staining in the same cell populations in all epithelia studied, including vibrissae, follicular epidermis, interfollicular epidermis, thymic and oral epithelium (Figure 2, A and B; data not shown). The only difference detected was that the staining intensity tended to be higher in the matriptase+/E16β-gal strain. The analysis in all cases also included matriptase+/+ littermate controls, which were negative for X-gal staining in the tissues that were examined in this study (Figure 2, Figure 6, data not shown). Furthermore, to confirm that the expression pattern of both the matriptase E1-β-galactosidase and E16-β-galactosidase fusion proteins properly reflected that of the wild-type matriptase transcript, parallel in situ hybridizations of vibrissal hair follicles were performed (Figure 2, D and E). Vibrissal hair follicles were suitable for this comparative analysis because of their large size and high matriptase expression level, which enabled relatively high resolution by in situ hybridization. The expression of matriptase as detected by X-gal staining and by in situ
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