Cell surface serine proteases as regulators of epithelial development, repair, and malignancy (33% effort) Identification of matriptase as a global regulator of vertebrate epithelial homeostasis. Matriptase is a serine protease encoded by the ST14 gene. We determined the effect of tissue-specific embryonic and postnatal ablation of St14 in a variety of epithelial tissues in mice. Matriptase loss caused epithelial dysfunction with two distinct phenotypes emerging: a) loss of epithelial function, but preservation of epithelial anatomy (salivary glands, tear glands, stomach, small intestine), and, b) loss of epithelial function followed by loss of anatomical integrity (orofacial and colonic epithelium). Absence of matriptase was associated with the loss of tight junction function. In collaboration with Toni Antalis, University of Maryland, we studied the effect of reducing matriptase on transepithelial electrical resistance (TEER) in epithelial monolayers. Inhibition of matriptase reduced TEER and increased paracellular permeability to macromolecular FITC-dextran. Matriptase loss was associated with enhanced incorporation of the permeability-associated tight junction protein, claudin-2, into cell-cell junctions. Decrease of claudin-2 enhanced TEER in matriptase-silenced monolayers, indicating that reduced barrier integrity was caused by an inability to regulate claudin-2 expression. This suggests a relationship between matriptase and the composition of tight junction proteins. A role of matriptase in the pathogenesis of Netherton syndrome. Netherton syndrome is a congenital human disorder that features excessive proteolytic degradation of corneodesmosomes, which leads to premature stratum corneum loss, exposure of living layers of the epidermis to the environment, dehydration, severe inflammation, infections, failure to gain weight, and high postnatal mortality. Systemic allergic manifestations, such as asthma, atopic dermatitis, gingivitis, and food allergies are other hallmarks of the syndrome. Netherton syndrome is caused by mutations in the SPINK5 gene that encodes the lympho-epithelial Kazal-type-related inhibitor (LEKTI). In collaboration with Paul A. Overbeek, Baylor College of Medicine, Houston, TX and Roberto Weigert, IMTU, we found that matriptase, which is expressed and active at the granular-transitional cell layer boundary, was a potent activator of epidermal pro-kallikreins. Furthermore, in a LEKTI-deficient mouse model of Netherton syndrome, the ablation of matriptase eliminated aberrant kallikrein activity, restored corneodesmosome integrity, prevented stratum corneum detachment, improved epidermal barrier function, and dampened inflammation. The study uncovers a pathogenic matriptase-pro-kallikrein pathway that plays a key role in Netherton syndrome and could be involved in other epithelial and inflammatory diseases. Complex interactions between matriptase and HAI-1 and HAI-2 enable vertebrate epithelial development and postnatal homeostasis. Hepatocyte growth factor activator inhibitor (HAI)-1 and -2 are transmembrane serine protease inhibitors encoded by, respectively, the SPINT1 and -2 genes. By using a mouse genetic approach, we have previously shown that matriptase is the only essential developmental target for HAI-1, and that HAI-1/matriptase double-deficient, but not HAI-1 single-deficient, mice complete embryonic development. Recent studies by others revealed that HAI-1 is essential not only for embryonic development, but also for postnatal epithelial homeostasis, as evidenced by fatal epithelial dysfunction in mice with conditional ablation of Spint1. We have extended this finding by showing that HAI-1 maintains epithelial homeostasis in the adult mouse exclusively through the inhibition of matriptase. Spint1-null mice in which the level of matriptase had been genetically reduced, were healthy, displayed normal long term survival, and histologically unremarkable epithelia. Mapping of HAI-2 and matriptase expression also revealed a striking co-localization of HAI-2 with matriptase in both developing and adult epithelia. We therefore performed an epistasis analysis in mice, which identified three developmental processes in which HAI-2 inhibition of matriptase was essential: a) Early (before E8.5) embryonic development, in which the ubiquitous demise of Spint2 null embryos could be prevented by the simultaneous loss of either one or two St14 alleles. b) Placental development, where the failure of the placental labyrinth to undergo branching morphogenesis in Spint2 null embryos could be prevented by the introduction of two St14 null alleles. c) Neural tube closure, in which the development of exencephaly and spina bifida could be partially rescued by the loss of both St14 alleles. We also found that the functional interactions between matriptase, HAI-1 and HAI-2 are uniquely complex. Thus, the combined haploinsufficiency for Spint-1 and -2 caused embryonic lethality, which could be prevented by loss of just one St14 allele, suggesting a functional cooperation between HAI-1 and -2 in regulating matriptase. Identification of a novel role of the uPA-uPAR interaction in suppression of fibrin-associated inflammation. The urokinase plasminogen activator (uPA) receptor (uPAR) is a cell surface receptor that has been proposed to play a role in cell adhesion, cell migration, proliferation, differentiation, and cell survival in multiple physiological and pathological contexts. To define the specific function of uPA binding to uPAR in vivo, in collaboration with Michael Ploug, University of Copenhagen and Steve Leppla, NIAID, we developed a new mouse strain (PlauGFDhu/GFDhu), in which the interaction between endogenous uPA and uPAR was selectively abrogated, while all other functions of both the protease and its receptor were retained. Specifically, we introduced four amino acid substitutions into uPA that abrogated uPAR binding, while preserving the overall structure of the domain. Analysis of PlauGFDhu/GFDhu mice revealed an unanticipated role of the uPA-uPAR interaction in suppressing chronic inflammation secondary to fibrin deposition. In contrast, inflammatory cell recruitment and tissue regeneration were unaffected by the selective loss of uPA binding to uPAR. This study identifies a principal in vivo function of the uPA-uPAR interaction in cell-associated fibrinolysis critical for suppression of fibrin-associated inflammation. It also provides a valuable model for the scientific community to further explore this enigmatic protease receptor. uPAR-associated protein (uPARAP) and intracellular collagen turnover (33% effort) Our previous studies revealed that uPARAP is critical for the uptake and lysosomal degradation of collagen by mesenchymal cells. In a collaborative effort with Roberto Weigert, IMTU, OPCB, and Niels Behrendt, University of Copenhagen, we now have succeeded in establishing an assay that images cellular collagen uptake and lysosomal collagen degradation in vivo. Reengineered bacterial cytotoxins as antitumor agents (33% effort) Efficient targeting of tumor stroma with MMP-activated toxins. Our prior research has shown that melanomas with activating B-Raf mutations are sensitive to a matrix metalloproteinase (MMP) activated anthrax lethal toxin (PA-L1 and LF) in ex vivo assays. We now found that PA-L1 and LF inhibits orthotopic anaplastic thyroid xenograft progression in both toxin-sensitive and toxin-resistant tumor cell lines via reduced endothelial cell recruitment and subsequent impaired tumor vascularization. This in turn translates into an improved long-term survival that is comparable with that produced by the multikinase inhibitor, sorafenib. These data indicate that therapy with PA-L1 and LF may be highly effective against advanced tumors with well-established vascular networks.

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National Institute of Dental & Craniofacial Research
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Szabo, Roman; Bugge, Thomas H (2018) Loss of HAI-2 in mice with decreased prostasin activity leads to an early-onset intestinal failure resembling congenital tufting enteropathy. PLoS One 13:e0194660
Liu, Shihui; Ma, Qian; Fattah, Rasem et al. (2017) Anti-tumor activity of anthrax toxin variants that form a functional translocation pore by intermolecular complementation. Oncotarget 8:65123-65131
Madsen, Daniel Hargbøl; Jürgensen, Henrik Jessen; Siersbæk, Majken Storm et al. (2017) Tumor-Associated Macrophages Derived from Circulating Inflammatory Monocytes Degrade Collagen through Cellular Uptake. Cell Rep 21:3662-3671
Nonboe, Annika W; Krigslund, Oliver; Soendergaard, Christoffer et al. (2017) HAI-2 stabilizes, inhibits and regulates SEA-cleavage-dependent secretory transport of matriptase. Traffic 18:378-391
Flick, M J; Bugge, T H (2017) Plasminogen-receptor KT : plasminogen activation and beyond. J Thromb Haemost 15:150-154
Friis, Stine; Tadeo, Daniel; Le-Gall, Sylvain M et al. (2017) Matriptase zymogen supports epithelial development, homeostasis and regeneration. BMC Biol 15:46
Friis, Stine; Madsen, Daniel H; Bugge, Thomas H (2016) Distinct Developmental Functions of Prostasin (CAP1/PRSS8) Zymogen and Activated Prostasin. J Biol Chem 291:2577-82
Liu, Shihui; Liu, Jie; Ma, Qian et al. (2016) Solid tumor therapy by selectively targeting stromal endothelial cells. Proc Natl Acad Sci U S A 113:E4079-87
Bardou, Olivier; Menou, Awen; François, Charlène et al. (2016) Membrane-anchored Serine Protease Matriptase Is a Trigger of Pulmonary Fibrogenesis. Am J Respir Crit Care Med 193:847-60
Engelholm, Lars H; Melander, Maria C; Hald, Andreas et al. (2016) Targeting a novel bone degradation pathway in primary bone cancer by inactivation of the collagen receptor uPARAP/Endo180. J Pathol 238:120-33

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