Cell surface proteolysis in development, tissue repair, and malignancy

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications, trials & patents

Abstract

The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated proteolysis, with an emphasis on determining the contribution to the development, homeostasis, regeneration, and malignant transformation of oral tissues. Cell surface serine proteases as regulators of epithelial development, homeostasis regeneration, and malignancy Background: Cell behavior is regulated by a vast number of proteases and protease inhibitors that function in the pericellular environment to provide focal proteolysis essential for cytokine/growth factor maturation, matrix remodeling, signaling receptor activation and shedding, ion channel activity, and more. We are continuing our efforts to understand the molecular functions of membrane-anchored serine proteases and their inhibitors in vertebrate development, epithelial homeostasis, and epithelial carcinogenesis by using a combined biochemical, cell biological, and genetic approach. Iterative, multiplexed CRISPR-mediated gene editing for functional analysis of complex protease gene clusters Functions of species-conserved kallikreins in squamous epithelial biology Kallikreins are a family of secreted trypsin-like serine proteases encoded by the kallikrein gene cluster the largest protease gene cluster in the human genome. This mammalian-specific gene cluster contains an assortment of species-conserved (KLK1, KLK4-15) and species-specific (e.g. human KLK2 and KLK3 and mouse Klk1-b1, Klk1-b3, Klk1-b4, Klk1-b5, Klk1-b8, Klk1-b9, Klk1-b11, Klk1-b16, Klk1-b21, Klk1-b22, Klk1-b24, Klk1-b26, Klk1-b27) genes. We have undertaken an analysis of kallikrein function in squamous epithelium. By performing RNAseq analysis of both intact skin and EpCAM-sorted keratinocytes, we detailed the expression of kallikreins in mouse epidermis. This analysis revealed epidermal expression of the species-conserved kallikrein genes, Klk5, Klk7, Klk8, Klk9, Klk10, Klk11, Klk13, and Klk14, and very low or absent expression of the species-specific kallikreins. Importantly, this pattern of expression of species-conserved mouse kallikreins in the epidermis mirrored the pattern of epidermal expression of human kallikreins, suggesting the mouse as a suitable model for the genetic exploration of kallikrein biology. Accordingly, we currently are using iterative, multiplexed CRISPR-mediated gene editing to engineer subtle null mutations into epidermally-expressed kallikrein genes in congenic mice. Preliminary phenotypic analysis is compatible with our anticipation of functional redundancy between individual kallikreins. Extracellular matrix degradation in physiological and pathophysiological processes 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 aberrant ECM turnover pathways to human disease are still incompletely understood. Molecular mechanisms of fibrin-driven alveolar bone destruction in periodontitis Fibrin, formed by polymerization of fibrinogen following activation of the coagulation system, is proinflammatory and causes tissue damage, unless removed in a timely manner. Importantly in this respect, humans with compromised fibrinolytic function display excess gingival fibrin deposition and severe periodontal disease, which often results in complete tooth loss early in life. We have continued our collaboration with Niki Moutsopoulos, NIDCR, Yasmine Belkaid, NIAID, Matt Flick and Kimon Divaris, University of North Carolina, Francis Castellino, University of Notre Dame, and Christian Kastrup, University of British Columbia on the link between persistent fibrin deposition and periodontal disease. Histological characterization of periodontal tissue in mice with fibrinolytic deficits revealed exuberant gingival fibrin deposition and periodontal bone loss that was completely reversed in mice expressing no fibrinogen or mutant fibrinogen lacking the neutrophil aMb2 integrin binding site. This pathogenic cascade was in large part triggered by the commensal microbiome, as germ-free mice with fibrinolytic deficits displayed little periodontal bone loss. In collaboration with Andrew Doyle, NIC, we developed novel ex vivo assays to study the effect of fibrin on neutrophil effector functions. By applying these assays, we found that neutrophils plated on the mutant fibrin showed decreased reactive oxygen species (ROS) production and neutrophil extracellular trap formation (NETosis) compared to neutrophils plated on wildtype fibrin, indicating that neutrophil aMb2 integrin-engagement of fibrin leads to neutrophil activation. Accordingly, genetic or pharmacologic elimination of NETosis prevented fibrinolysis deficit-associated periodontal bone loss. Concordant with these findings, genetic polymorphisms in the human PLG gene, encoding the key fibrinolytic protease, plasminogen, were found to be associated with common forms of periodontitis. These data demonstrate that commensal microbiota-induced local engagement of gingival fibrin by neutrophils induces periodontal bone loss by triggering key neutrophil effector functions. Collectively, this study suggests a critical role of fibrin-neutrophil engagement in periodontitis and identifies novel pharmacological targets for therapeutic intervention in the disease. 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. A novel assay for direct visualization of anthrax intoxication in animals reveals shared and individual functions of CMG-2 and TEM-8 in cellular toxin entry The virulence of Bacillus anthracis is linked to the secretion of anthrax lethal toxin and anthrax edema toxin. These binary toxins consist of a common cell-binding moiety, protective antigen (PA), and the enzymatic moieties, lethal factor (LF) and edema factor (EF). PA binds either of two specific cell surface receptors, capillary morphogenesis protein-2 (CMG-2) or tumor endothelial marker-8 (TEM-8), which triggers the binding, endocytosis, and cytoplasmic translocation of LF and EF. The cellular distribution of functional TEM-8 and CMG-2 receptors during anthrax toxin intoxication in animals remains unclear, due to low expression and the absence of monospecific antibodies. We developed a novel assay to directly image anthrax toxin intoxication in animals, and we used the assay to visualize TEM-8- and CMG-2-dependent intoxication. 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 red fluorescent protein in the absence of Cre activity and a green fluorescent protein in the presence of Cre activity, anthrax toxin intoxication could be visualized at single-cell resolution by confocal microscopy or flow cytometry. By using the novel assay we found that: a) CMG-2 is critical for intoxication in the liver and heart, b) TEM-8 is required for full intoxication in the kidney and spleen, c) CMG-2 and TEM-8 are redundant for intoxication of some tissues, d) combined loss of CMG-2 and TEM-8 completely abolishes intoxication, and e) CMG-2 is the dominant receptor on spleen leukocytes of myeloid and lymphoid lineages.