The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated proteolysis, with an emphasis on determining its contribution to the development, regeneration, and malignant transformation of oral tissues. Cell surface serine proteases as regulators of epithelial development, homeostasis regeneration, and malignancy Background: Cell behavior in higher eukaryotes is regulated by a large number of proteases and protease inhibitors that operate in the pericellular environment to provide focal proteolysis that is essential for cytokine/growth factor maturation, matrix remodeling, signaling receptor activation and shedding, ion channel activity, and more. Research accomplished: Matriptase drives early-onset intestinal failure in a mouse model of congenital tufting enteropathy. Congenital tufting enteropathy (CTE) is a human genetic disorder with a recessive Mendelian inheritance. CTE is characterized by epithelial dysplasia, villous atrophy and a compromised intestinal epithelial barrier. Mutations in the SPINT2 gene, encoding the transmembrane serine protease inhibitor, hepatocyte growth factor activator inhibitor (HAI)-2, have been identified as a cause of CTE. We recently generated a Spint2-deficient mouse model of CTE that displays histological and biochemical hallmarks of CTE, including widespread villous atrophy, tufted villi, loss of mucin-producing goblet cells, loss of colonic crypt structure, bleeding into the intestinal lumen, and decreased expression of the epithelial junctional proteins, EpCAM, E-cadherin, occludin, and claudin-1 and -7. To directly address the role of the membrane-anchored protease, matriptase in CTE, the protease was ablated from the intestine of Spint2-deficient mice. Interestingly, macroscopic and histological defects observed in the absence of HAI-2 were prevented by the intestinal epithelial cell-specific ablation of matriptase. Furthermore, the expression levels of CTE-associated junctional proteins were normalized. As a result, loss of intestinal matriptase allowed Spint2-deficient mice mice to gain weight and dramatically increased their life span. These data implicate matriptase in CTE and may provide a new target for the treatment of CTE in patients carrying SPINT2 mutations. Extracellular matrix degradation in physiological and pathophysiological processes Background: The cellular orchestration of ECM degradation and the contribution of endocytic pathways and endocytic receptors to ECM turnover is incompletely understood. We have continued our long-standing collaborative research with Niels Behrendt and Lars Engelholm, University of Copenhagen, Denmark, aimed at understanding ECM turnover. Research accomplished: High resolution phenotyping of ECM-degrading macrophage subsets Tissue remodeling and regeneration require the orchestrated deposition and removal of ECM within the pericellular environment. Perturbations in ECM homeostasis may cause delayed or aberrant tissue regeneration and is linked to a wide variety of human degenerative and inflammatory diseases. Two key ECM constituents in this regard are the interstitial collagens and fibrin both of which are primarily degraded by macrophages. To enable the extensive characterization of ECM-degrading macrophage subtypes, we developed a flow cytometry-based assay that allowed for the detailed phenotyping of macrophages engaged in dermal ECM degradation. By using this assay, we found that at least five distinct subpopulations of macrophages are present in the mouse dermis during ECM remodeling. Within these, the dominant macrophage subpopulations engaged in collagen turnover were resident Ly6C-negative, C-C chemokine receptor type 2 (CCR2) high-expressing cells, and resident Ly6C-negative, CCR2 low-expressing cells, while recruited Ly6C-positive, CCR2-positive macrophages account for the large majority of fibrin-degrading cells. Furthermore, we were able to show that Ly6C-negative resident dermal macrophages and Ly6C-positive recruited macrophages utilized distinct pathways for collagen internalization dependent on, respectively, the mannose receptor (MR) and the urokinase plasminogen activator receptor-associated protein (uPARAP). Surprisingly, genetic loss-of-function analysis revealed that not only fibrin degradation, but also collagen degradation was dependent on the integrity of the chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemoattractant protein 1 (MCP1)-CCR2 axis. Moreover, macrophage-mediated endocytic collagen degradation was stimulated by Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF), and Interleukin (IL)-13, while IL4-Ra-dependent signaling was dispensable for this process. Taken together, this study reveals distinct subsets of macrophages engaged in ECM turnover and identifies novel wound healing-associated functions for CCL2 and GM-CSF inflammatory cytokines. Reengineered bacterial cytotoxins as anti-tumor and protease imaging agents Background: Elevated expression of matrix-degrading proteases is a hallmark of malignancy. 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. Modified anthrax toxins as imaging agents for cell surface proteolytic activity Anthrax toxins are binary toxins consisting of a common cell binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds to cell surface receptors, and, following cleavage by furin, mediates the cytoplasmic translocation of LF and EF. The absolute requirement for proteolytic cleavage of PA for cytoplasmic translocation of EF and LF, combined with the ability to reengineer PA to be cleaved by other proteases, including urokinase plasminogen activator (uPA), matrix metalloproteinases (MMPs), and matriptase/testisin, makes modified anthrax toxins attractive as agents for imaging specific cell surface proteolytic activity. We took advantage of the potential of genetic read-out systems to provide near-unlimited signal amplification to image cell surface protease activity using modified anthrax toxins. Specifically, we generated a chimeric protein consisting of the N-terminal domain of LF fused to a nuclear localization signal-tagged Cre recombinase (LFn-NLS-Cre). When PA and LFn-NLS-Cre were co-administered to transgenic mice that ubiquitously express a membrane-tagged red fluorescent protein in the absence of Cre activity and a membrane-tagged green fluorescent protein in the presence of Cre activity, the presence of furin activity could be directly visualized by confocal microscopy at single cell resolution. Furthermore, the plasma membrane illumination provided by the membrane-targeted red and green fluorescent proteins, when combined with the staining of nuclei by an in vivo Hoechst labelling procedure, provided sufficient cellular and tissue morphology information to allow for identification of intoxicated cells in unfixed and unprocessed organ slices. Current efforts are directed at cataloging cell surface uPA, and MMP activity in neoplastic tissues, aimed at resolving long-standing uncertainties as to the cellular sources of these matrix-degrading proteases.
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