The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated serine proteases, with emphasis on determining their contribution to the development, regeneration, and malignant transformation of oral tissues.? ? Novel cell surface serine proteases in epithelial development, repair, and malignancy:? The type II transmembrane serine proteases (TTSPs) constitute a recently recognized family of membrane-bound serine proteases with largely unknown functions. We have continued our efforts to identify novel TTSPs and determine their contribution to development and malignancy.? We have previously shown that the TTSP, matriptase, is required for the formation of the epidermal barrier. To determine the potential function of matriptase in oral epithelial barrier development, we used enzymatic gene trapping of matriptase combined with immunohistochemistry, ultrastructural analysis, and barrier function assays. The study revealed that matriptase is expressed in post-mitotic oral keratinocytes undergoing terminal differentiation. Furthermore, matriptase co-localizes with profilaggrin, a critical downstream target for the membrane serine protease. Importantly, mice deficient in matriptase displayed a loss of barrier function within the keratinized portion of the oral epithelium, thus revealing a critical role of the TTSP in oral epithelial development. ? Recent gene ablation studies in mice have shown that matriptase and the GPI-anchored serine protease, prostasin (CAP1/PRSS8), are both required for the processing of the polyprotein, profilaggrin, stratum corneum formation, and acquisition of epidermal barrier function. By enzymatic gene trapping of matriptase combined with prostasin immunohistochemistry, we found that matriptase co-localizes with prostasin in transitional layer keratinocytes of the oral epithelium and the epidermis, and that the developmental onset of expression of the two membrane serine proteases is coordinated and correlate with the 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 serine protease inhibitor, protease nexin-1. Importantly, whereas both prostasin zymogen and active prostasin were present in wildtype epidermis, prostasin was exclusively found in the zymogen form in matriptase-deficient epidermis. These data suggest that matriptase acts upstream of prostasin in a zymogen cascade that is essential for terminal oral epithelial and epidermal differentiation. Since matriptase is an auto-activating protease, it may serve as the initiator of an epidermal proteolytic cascade system, similar to the closely related TTSP, enteropeptidase, in the digestive tract.? Matriptase is overexpressed in most carcinomas, including oral squamous cell carcinoma. We have previously shown that matriptase causes spontaneous squamous cell carcinoma and dramatically potentiates carcinogen-induced tumor formation, when unopposed by its endogenous transmembrane serine protease inhibitor, hepatocyte growth factor activator inhibitor-1 (HAI-1). In collaboration with MCU, OPCB, we generated HAI-1-deficient mice to specifically determine the physiological function of the transmembrane serine protease inhibitor. Mice deficient for HAI-1 displayed embryonic lethality at embryonic day 9.5 due to placental failure. Interestingly, however, mice deficient in both HAI-1 and matriptase developed to term and were indistinguishable from matriptase single-deficient mice. Matriptase and HAI-1 were co-expressed in a distinct population of chorionic trophoblast stem cells of the placenta. Expression of matriptase in this cell population, when unopposed by HAI-1, caused phenotypic changes that mimicked epithelial malignant transformation. These included basement membrane dissolution, loss of E-cadherin, and loss of membrane-associated beta-catenin. This study identified a critical physiological role of endogenous HAI-1 in preserving epithelial integrity through the inhibition of matriptase.? ? ? Urokinase plasminogen activator receptor-associated protein and intracellular collagen turnover:? Local growth, invasion, and metastasis of oral malignancies are associated with extensive degradation and remodeling of the underlying, collagen-rich connective tissue. We previously reported that the urokinase plasminogen activator receptor-associated protein (uPARAP), a recently identified member of the macrophage mannose receptor family, is critical for the cellular uptake and lysosomal degradation of collagen during tumor progression. As a first step towards determining the putative function of uPARAP in oral cancer progression in humans, in a collaboration with MCU/OPCB we used immunohistochemistry to determine the expression of this collagen internalization receptor in 112 squamous cell carcinomas, and 19 normal or tumor-adjacent oral tissue samples resected from the tongue, gingiva, cheek, tonsils, palate, floor of mouth, larynx, maxillary sinus, upper jaw, nasopharynx/nasal cavity, and lymph nodes. The specificity of detection was verified by the staining of serial sections with two different monoclonal antibodies directed against two non-overlapping epitopes on uPARAP and by the use of isotype-matched non-immune antibodies. uPARAP expression was observed frequently in fibroblast-like, vimentin-positive cells within the tumor stroma. Furthermore, the expression of the collagen internalization receptor was increased in tumor stroma compared to tumor-adjacent connective tissue or normal submucosal connective tissue, and expression was most prominent in poorly-differentiated tumors. Taken together, these data suggest that uPARAP participates in the connective tissue destruction associated with oral squamous cell carcinoma progression.? ? Reengineered bacterial cytotoxins as antitumor and protease imaging agents: ? We have continued our long-standing collaboration with Stephen Leppla, MPS, NIAID, on the development of cell surface protease-activated bacterial cytotoxins as therapeutic agents for cancer. Our previous work provided ?proof of concept, by showing that cell surface uPA-activated reengineered anthrax toxin displays limited toxicity to normal tissues, but broad and potent uPA-dependent tumorcidal activity in mice. We have worked via a CRADA with the biopharmaceutical company OncoTac and Arthur Frankel, Scott & White, Temple, Texas on the clinical development of uPA activated anthrax toxins. Specifically, a systematic series of studies have been conducted that have determined the pharmacological properties, optimal route of administration, optimal dosing regiment, toxicity, efficacy against a spectrum of human and murine tumors, and benefit of adjuvant therapy.? The paucity of agents to visualize the activity of individual proteases in situ and monitor their pharmacological inhibition has been an impediment to basic research and to therapeutic targeting of proteases in cancer. To alleviate this deficiency, we have worked collaboratively with Stephen Leppla, MPS, NIAID, on the use of reengineered cytotoxins as protease activity imaging agents. In this respect, we recently developed a simple, sensitive, and non-invasive assay that uses non-toxic, reengineered anthrax toxin??-lactamase fusion proteins with altered protease cleavage specificity to visualize specific cell surface proteolytic activity in single living cells. The assay could specifically and quantitatively image endogenous cell surface furin, urokinase plasminogen activator, and metalloprotease activity, and was adapted for flow cytometry and fluorescent plate reader formats.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Intramural Research (Z01)
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Dental & Craniofacial Research
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Szabo, Roman; Kosa, Peter; List, Karin et al. (2009) Loss of matriptase suppression underlies spint1 mutation-associated ichthyosis and postnatal lethality. Am J Pathol 174:2015-22
Szabo, Roman; Hobson, John P; Christoph, Kristina et al. (2009) Regulation of cell surface protease matriptase by HAI2 is essential for placental development, neural tube closure and embryonic survival in mice. Development 136:2653-63
Chaipan, Chawaree; Kobasa, Darwyn; Bertram, Stephanie et al. (2009) Proteolytic activation of the 1918 influenza virus hemagglutinin. J Virol 83:3200-11
Alfano, Randall W; Leppla, Stephen H; Liu, Shihui et al. (2008) Cytotoxicity of the matrix metalloproteinase-activated anthrax lethal toxin is dependent on gelatinase expression and B-RAF status in human melanoma cells. Mol Cancer Ther 7:1218-26
Alfano, Randall W; Leppla, Stephen H; Liu, Shihui et al. (2008) Potent inhibition of tumor angiogenesis by the matrix metalloproteinase-activated anthrax lethal toxin: implications for broad anti-tumor efficacy. Cell Cycle 7:745-9
Szabo, Roman; Bugge, Thomas H (2008) Type II transmembrane serine proteases in development and disease. Int J Biochem Cell Biol 40:1297-316
Liu, Shihui; Wang, Hailun; Currie, Brooke M et al. (2008) Matrix metalloproteinase-activated anthrax lethal toxin demonstrates high potency in targeting tumor vasculature. J Biol Chem 283:529-40
Zhang, Yahong; Zhou, Zhao-Hua; Bugge, Thomas H et al. (2007) Urokinase-type plasminogen activator stimulation of monocyte matrix metalloproteinase-1 production is mediated by plasmin-dependent signaling through annexin A2 and inhibited by inactive plasmin. J Immunol 179:3297-304
Madsen, Daniel H; Engelholm, Lars H; Ingvarsen, Signe et al. (2007) Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/Endo180, cooperate in fibroblast-mediated collagen degradation. J Biol Chem 282:27037-45
Bugge, Thomas H; List, Karin; Szabo, Roman (2007) Matriptase-dependent cell surface proteolysis in epithelial development and pathogenesis. Front Biosci 12:5060-70

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