Cell surface serine proteases as regulators of epithelial development, repair, and malignancy (33.3% effort) Background: Cell behavior in is regulated by proteases and protease inhibitors that operate in the pericellular environment to provide cytokine/growth factor maturation, matrix remodeling, signaling receptor activation ion channel activity, and more. We aim to understand the molecular functions of membrane-anchored serine proteases and their inhibitors in epithelial development, and homeostasis. The membrane-anchored serine proteases prostasin and matriptase initiate a cell surface proteolytic pathway that is essential for epithelial development and postnatal homeostasis. We have recently reported that mice expressing either catalytically inactive or zymogen-locked prostasin complete development and are viable. In this study, we used knockin mice expressing catalytically inactive endogenous prostasin (Prss8Ki/Ki) to perform a systematic analysis of previously reported in vivo functions of prostasin. We show that these exhibit varying degree of dependence on prostasin proteolytic activity. Thus, while prostasin null (Prss8-/-) mice exhibit partial embryonic and complete perinatal lethality, proteolytically inactive endogenous prostasin is sufficient to fully restore both pre- and post-natal survival. Similarly, both wildtype and catalytically inactive prostasin induced placental defects and embryonic lethality in mice lacking the principal prostasin/matriptase inhibitor HAI-1 and prevented embryonic survival of mice lacking the prostasin/matriptase inhibitor, HAI-2. Nevertheless, inactive prostasin, unlike wildtype prostasin, was insufficient to activate matriptase during placental differentiation. Surprisingly, we also observed that all of the essential functions of prostasin in embryonic and postnatal development are fully compensated for by inactivation of HAI-1, indicating that prostasin is only essential for mouse development and overall viability of mice in the presence of this inhibitor. Endocytic extracellular matrix degradation in physiological and pathophysiological processes (33.3% effort) Background: Research performed within the last five decades led to the identification and extensive characterization of extracellular matrix (ECM)-degrading enzymes. However, the cellular orchestration of ECM degradation and the contribution of endocytic pathways and endocytic receptors to ECM turnover is less studied and understood. We have continued our long-standing research aimed at understanding endocytic ECM turnover. A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo. Extravascular fibrin deposition accompanies many human diseases, including periodontal disease, multiple sclerosis, tissue fibrosis, muscular dystrophy, and arthritis, and causes chronic inflammation and organ damage, unless removed in a timely manner, as illustrated by the inflammation-associated multi-organ pathology and impaired tissue regenerative capacity of humans and mice deficient in the key fibrinolytic protease zymogen, plasminogen. In collaboration with Roberto Weigert, IMTU, Kenn Holmbeck, CSDB, Matt Flick, University of Cincinnati, Daniel Lawrence, University of Michigan, and Francis Castellino, University of Notre Dame, we used intravital microscopy procedures, developed as described above, to investigate how fibrin is removed from extravascular space. We found that fibrin placed into the dermis of mice undergoes cellular endocytosis and lysosomal targeting, revealing an intracellular pathway for extravascular fibrin degradation. A CCR2-positive subpopulation of inflammatory macrophages was found to constitute the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished by diphtheria toxin-mediated selective elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was distinct from the collagen-internalizing M2-like macrophages identified above and displayed a low capacity to internalize collagen. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activation, with urokinase plasminogen activator and tissue plasminogen activator displaying redundant functions in the activation of fibrin during endocytic fibrin uptake. Surprisingly, fibrin endocytosis could take place in the absence of the cellular fibrin receptors, M2 integrin and Intercellular Adhesion Molecule 1, the myeloid cell integrin binding site on fibrin or the MR. The study identifies a novel fibrin endocytic pathway engaged in extravascular fibrin clearance that is mechanistically linked to the well-established pericellular fibrinolytic pathway, and engages fibrin independently of known fibrin receptors and cellular binding sites on fibrin. Furthermore, the study shows that interstitial fibrin and collagen removal is orchestrated by different subsets of macrophages employing distinct molecular pathways. Reengineered bacterial cytotoxins as antitumor agents (33.3% effort) Background: Elevated expression of matrix-degrading proteases is a hallmark of human cancer. We are engaged in a long-standing collaboration with Steve Leppla, NIAID, on the development of reengineered bacterial cytotoxins, activated by proteases expressed in the tumor microenvironment, as novel therapeutic agents for cancer and as tools for the imaging of specific cell surface proteolytic activity. Transient immunosuppression allows sustained tumor treatment with protease-activated toxins. A conceptual problem with systemic application of foreign protein therapeutics is the frequent generation of neutralizing antibodies, which precludes long-term treatment. Our protease-activated anthrax toxins are strong candidates for further clinical development, but would be expected to encounter this problem after sustained usage. Recently, a combination of pentostatin and cyclophosphamide (PC) has been used successfully to prevent neutralizing antibody production against Pseudomonas exotoxin A-based immunotoxins used for treatment of human mesotheliomas, leading to durable remissions of advanced tumors. We are currently investigating whether a PC regimen blocks production of antibodies that neutralize our engineered anthrax toxins. Data generated thus far indicate that tumor-bearing mice, when treated systemically with engineered toxin in combination with a PC regimen, do not develop toxin-neutralizing antibodies. These findings suggest that the engineered toxins can be safely administered in combination with PC to enable sustained and efficacious tumor treatment. Identification of target cells for protease-activated anthrax toxins Solid tumors consist of malignant cells and an assortment of non-malignant cells, termed stromal cells, which may include fibroblasts, macrophages, lymphocytes, neutrophils, mast cells, myoepithelial cells, endothelial cells, lymphendothelial cells, and platelets. In collaboration with Steve Leppla, NIAID, we are using mice with a wholesale or cell type-specific deficiency in the two anthrax toxin receptors, TEM8 and CMG2, to determine which of the cellular constituents that form a solid tumor are targeted by the engineered anthrax toxins. Interestingly, tumor cell lines xenografted or syngrafted into these toxin receptor-deficient mice become refractory to toxin treatment, showing that the toxin suppresses tumor growth by targeting tumor stromal cells. Selective genetic reconstitution of CMG2 expression on endothelial cells of CMG2-deficient mice restores toxin sensitivity to tumors, while, conversely, selective genetic elimination of CMG2 from endothelial cells of wildtype mice makes grafted tumors refractory to treatment. These data tentatively suggest that the broad-spectrum anti-tumor activity of our engineered anthrax toxins is achieved through targeting of the tumor endothelium.