Cell surface proteolysis in development, tissue repair, and malignancy

National Institute Of Dental & Craniofacial Research

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Abstract

The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated proteolysis, with an emphasis on understanding the contribution of proteolytic pathways to the development, homeostasis, regeneration, and pathology of oral tissues. Cell surface serine proteases as regulators of epithelial development, homeostasis, and regeneration EpCAM as a candidate physiological substrate for matriptase Matriptase is a membrane-anchored serine protease expressed in most vertebrate epithelia. Previous studies of matriptase-deficient mice, by us and by others, and clinical studies of matriptase-deficient individuals have uncovered a plethora of functions of the protease in the development and/or homeostasis of epithelial tissues. While impaired epithelial tight junction function appears to be the common denominator of these matriptase deficiency-associated pathologies, no definitive substrate, whose cleavage by matriptase executes the physiological functions of the protease, has been identified, despite considerable effort. EpCAM (Epithelial Cell Adhesion Molecule) is a type I transmembrane glycoprotein expressed on the surface of most epithelial cells. EpCAM was recently reported to maintain tight junction homeostasis through sequestration and proteolysis-regulated release of claudin-7 and possibly other members of the claudin family of epithelial tight junction proteins. Congenital tufting enteropathy (CTE) and syndromic CTE (sCTE) are life-threatening recessive human genetic disorders characterized by severe intestinal dysfunction, resulting from loss-of-function mutations in EPCAM and SPINT2, respectively. We recently reported that mice deficient in Spint2, encoding the matriptase inhibitor, HAI-2, spontaneously develop CTE-like early-onset intestinal failure associated with a progressive loss of EpCAM protein, and that these pathologies are largely caused by the unchecked activity of matriptase. The identification of EpCAM as a potential pathogenic substrate for dysregulated matriptase in sCTE naturally raised the question as to whether EpCAM also constitutes a physiologic target for matriptase in intestinal homeostasis. In the normal intestine, EpCAM is found in both a full-length and in a cleaved form (cleavage after Arg80). EpCAM has been proposed to function as a homodimer, and, intriguingly, in the crystal structure of EpCAM, the matriptase cleavage site forms part of the EpCAM dimerization interface. In collaboration with Michael Ploug, University of Copenhagen, Copenhagen, Denmark, we identified EpCAM mutants that are resistant to protease cleavage, but retain their ability to dimerize in cell-based assays. Informed by these studies, we proceeded to generate three mouse strains carrying different point mutations (Arg80Ala, Arg80Leu, Arg80Gln) in endogenous EpCAM by using CRISPR-mediated gene editing. Elimination of the matriptase cleavage site strongly suppressed proteolytic processing of EpCAM in vitro and in vivo. Unexpectedly, expression of either of the three cleavage-resistant EpCAM variants failed to prevent early-onset intestinal failure and postnatal lethality in Spint2-deficient mice. In addition, genetic inactivation of intestinal matriptase largely counteracted the effect of Spint2 deficiency in mice expressing cleavage-resistant EpCAM. These data demonstrate that excessive proteolysis of EpCAM, through unrestricted matriptase activity, is not sufficient to drive intestinal dysfunction in syndromic CTE. Interestingly, however, mice expressing the cleavage-resistant EpCAM variants developed late-onset intestinal pathologies that closely mimicked those observed after intestinal ablation of matriptase itself. This suggests that EpCAM cleavage by matriptase is indispensable for EpCAM physiological function, and thereby identifies EPCAM as a candidate physiological substrate for matriptase. Cellular orchestration of extracellular matrix degradation Background: Our Section has a long-standing interest in pathways of extracellular matrix (ECM) degradation and the pathological consequences of insufficient or excessive ECM turnover. Working with Niki Moutsopoulos, OIIS, and a range of intramural and extramural investigators, we are studying the role of fibrin deposition in the pathogenesis of periodontal disease. Molecular mechanisms that drive fibrin-mediated periodontal disease Fibrin is a provisional ECM protein that is formed by the polymerization of thrombin-cleaved fibrinogen followed by cross-linking of the fibrin polymer by coagulation factor XIII. Fibrin is deposited into the extravascular space in response to tissue injury, where it serves to stem the loss of blood, immobilize bacteria, and provide a provisional matrix for tissue regeneration. Fibrin, however, is highly proinflammatory and causes chronic inflammation and tissue damage, unless removed in a timely manner. In this respect, we recently demonstrated that fibrin is a key contributor to periodontal disease progression under both normal and compromised fibrinolytic conditions, thereby identifying a new target for therapeutic intervention in this prevalent disease. Furthermore, we were able to demonstrate that fibrin promoted periodontal disease progression through the local engagement and activation of gingival neutrophils, leading to ROS production and neutrophil extracellular trap formation (NETosis). Our current efforts are directed at uncovering the upstream mechanisms leading to the pathogenic deposition of fibrin in gingiva and preclinical studies aimed at targeting gingival fibrin deposition in periodontal disease. c-Myc signaling in host-derived tumor endothelial cells is essential for solid tumor growth We have previously generated several tumor-selective variants of anthrax toxin and demonstrated their potential use in solid tumor treatment in experimental animals and in veterinary oncology settings. These include variants of PrAg requiring activation by urokinase plasminogen activator, matrix metalloproteinases or both. Previous studies indicate that our reengineered anthrax toxins likely suppress the growth of solid tumors by targeting tumor endothelial cells via CMG2. LF, the effector moiety of the tumor-targeting toxin, possesses potent proteolytic activity towards MEK kinases, thereby inactivating the RAS-RAF-MEK-ERK pathway required for endothelial cell proliferation and tumor angiogenesis. Because cellular intoxication requires the surface expression of CMG2, genetic manipulation of CMG2 expression using cell-type-specific CMG2 transgenic mice allowed for specific identification of the contribution of individual tumor stromal cell types to tumor development. We found that disruption of ERK signaling in tumor endothelial cells suffices to block the growth of transplanted tumors in mice. Furthermore, we identified c-Myc as a downstream effector of ERK signaling, and that a MEK-ERK-c-Myc metabolic axis in tumor endothelial cells is essential for tumor progression. Taken together, these data show that disruption of ERK-c-Myc signaling in host-derived tumor endothelial cells by our tumor-selective anthrax toxins explains their high efficacy in solid tumor treatment.