Cell surface serine proteases as regulators of epithelial development, repair, and malignancy (50% effort) Background: Cell behavior in higher eukaryotes is regulated by a large number of proteases and cognate protease inhibitors that operate in the pericellular environment to provide focal proteolysis essential for cytokine/growth factor maturation, matrix remodeling, signaling, receptor shedding, and more. Research accomplished: Regulation of membrane serine protease activity: Hepatocyte growth factor activator inhibitor (HAI)-1 is an epithelial transmembrane serine protease inhibitor encoded by the SPINT1 gene that displays potent inhibitory activity (sub-nanomolar IC50) towards a large number of trypsin-like serine proteases in vitro. Despite this promiscuity of HAI-1 in assays with purified components and its proposed role of the regulation of multiple serine proteases, we have previously shown that the membrane serine protease, matriptase, is the only essential target for HAI-1 during development. HAI-1 is essential not only for embryonic development, but also for postnatal homeostasis, as evidenced by the abnormal differentiation of squamous epithelium, severe growth retardation, and early death that follows the conditional ablation of Spint1 from adult mice. In collaboration with Karin List, Wayne State University, we have extended our previous findings to show that matriptase is the essential inhibitory target for HAI-1 not only during embryogenesis, but also in adult tissue homeostasis. Matriptase and HAI-1 co-localize in all tissues affected by postnatal Spint1 disruption. Furthermore, Spint1-deficient mice with low matriptase levels, caused by introducing a hypomorphic mutation in the St14 gene encoding matriptase, not only survive the neonatal period, but also are healthy, fertile, and display normal long-term survival. Analysis of neonatal, young adult, as well as aged mice did not reveal any abnormalities in Spint1-deficent mice with low matriptase. This study identifies matriptase suppression as the only essential function of HAI-1 in postnatal tissue homeostasis in the absence of external challenges or additional genetic deficits. Furthermore, the study underscores that protease inhibitor specificity in vivo is determined primarily by the microenvironmental context, rather than by the kinetic rate constants of individual protease-inhibitor interactions. Hypomorphic mutations in the human SPINT2 gene were recently shown to cause a broad spectrum of developmental abnormalities in humans, including cleft palate, hypertelorism, choanal atresia, organ and digit duplications, fistulas, and hamartoma. Individuals born with SPINT2 deficiency also display abnormalities in epithelial homeostasis, such as persistent diarrhea, corneal erosions, and abnormal hair, and they usually die within the first year of life. As SPINT2 encodes the transmembrane serine protease inhibitor, HAI-2/placental bikunin, the developmental and postnatal effects of human SPINT2 deficiency could be hypothesized to be a consequence of excess pericellular activity of unidentified target proteases. Indeed, in collaboration with Karin List, and Chen-Yong Lin, University of Maryland, we have found that HAI-2 displays potent inhibitory activity towards matriptase in vitro, forms SDS-stable complexes with the serine protease, and blocks its activation of the candidate physiological substrates, pro-prostasin and cell surface-bound pro-uPA in cell culture models. Global high-resolution mapping of HAI-2 and matriptase expression also revealed a striking co-localization of HAI-2 with matriptase in epithelial cells of both developing and adult organs. To definitively determine the functional relationship between HAI-2 and matriptase, we inactivated the Spint2 gene in mice. All Spint2 null mice underwent embryonic demise associated with defects in neural tube closure, abnormal placental labyrinth formation and loss of epithelial cell polarity. Interestingly, labyrinth formation and term development of Spint2-null mice were completely restored by simultaneous genetic inactivation of matriptase. However, neural tube defects were detected with low frequency in Spint2-null mice, even in the absence of matriptase, indicating that inhibition of additional serine protease(s) besides matriptase by HAI-2 is specifically required to complete neural development. Uniquely, the combined heterozygosity for HAI-1 and HAI-2 also caused embryonic lethality (nonallelic noncomplementation), which could be completely rescued by the loss of just a single matriptase allele, revealing complex functional interactions between matriptase and its two inhibitors. Taken together, these studies suggest that: a) vertebrate tissue morphogenesis requires regulation of membrane-associated serine proteases by a network of partially redundant transmembrane Kunitz-type serine protease inhibitors, b) dysregulation of matriptase proteolytic activity disrupts epithelial cell polarity and can cause both teratogenesis and oncogenesis, and c) pharmacological inhibition of matriptase activity could serve as an efficient strategy for the treatment of the postnatal manifestations of human HAI-2 deficiency. Matriptase is constitutively expressed in multiple other epithelia, suggesting a putative role of the protease in general epithelial homeostasis. However, exploration of postnatal functions of matriptase has been hampered by the perinatal lethality of St14 null mice. To circumvent this problem, in a collaboration with Karin List, we have generated mice with conditional deletion of the St14 gene, and have found that matriptase indeed is essential for the maintenance of multiple types of epithelia in the mouse. Thus, tissue-specific embryonic or postnatal ablation of St14 in epithelial tissues of diverse origin and function caused severe organ dysfunction, which was often associated with increased permeability, loss of tight junction function, mislocation of tight junction-associated proteins, and generalized epithelial demise. These studies have provided tentative evidence for the existence of a, hitherto unknown, pericellular proteolytic pathway that regulates either tight junction formation or maintenance. Reengineered bacterial cytotoxins as antitumor and protease imaging agents (50% effort): Background: We are engaged in a long-standing collaboration with Steve Leppla, NIAID, and Art Frankel, Scott &White, Temple, Texas, on the development of cell surface protease-activated bacterial cytotoxins as therapeutic agents for cancer. In an independent effort, we have retooled protease-activated cytotoxins for use in non-invasive imaging of specific cell surface proteolytic activity. Research accomplished: Solid tumor growth is dependent on angiogenesis, the formation of neovasculature from existing vessels. In a collaboration with Art Frankel, Steve Leppla, Silvio Gutkind and Nicolas Duesbury, Van Andel Research Institute, Grand Rapids, we recently demonstrated that anthrax toxin engineered to be activated by MMPs displays broad anti-tumor activity, but limited systemic toxicity, by inactivating mitogen-activated protein kinase signaling pathways. We now have expanded these initial findings by showing that the MMP-activated toxin reduces endothelial pro-angiogenic MMP expression, thus causing a diminished proteolytic capacity for extracellular matrix remodeling and endothelial differentiation into capillary networks. Additionally, the data suggest that inhibition of the c-jun NH(2)-terminal kinase and p38 pathways, but not extracellular signal-regulated kinase-1/2 pathways, is significant in the anti-angiogenic activity of the MMP-activated anthrax toxin. Collectively, our results support the clinical development of the MMP-activated anthrax toxin for the treatment of solid tumors.

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Project End
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Budget End
Support Year
10
Fiscal Year
2009
Total Cost
$1,614,146
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
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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
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Flick, M J; Bugge, T H (2017) Plasminogen-receptor KT : plasminogen activation and beyond. J Thromb Haemost 15:150-154
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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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