Membrane-anchored serine proteases in epithelial biology 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: A model for initiation of epithelial matriptase activity The membrane-anchored serine proteases, matriptase and prostasin, constitute a proteolytic cascade critical for the development and homeostasis of multiple epithelia. In collaboration with Lotte Vogel, University of Copenhagen, we performed a detailed biochemical investigation of the mechanistic relationship between matriptase and prostasin by using a reconstituted cell-based system, employing a battery of matriptase and prostasin mutants. Matriptase and prostasin formed a reciprocal cell surface zymogen activation complex in which both prostasin mutants locked in the zymogen state and catalytically inactive prostasin mutants activated matriptase. Conversely, prostasin activation by matriptase could be mediated by matriptase mutants that were locked in the zymogen state. We propose that matriptase and prostasin form a reciprocal cell surface zymogen activation complex in which prostasin allosterically activates matriptase, and the matriptase zymogen, in turn, can activate prostasin through a low catalytic activity intrinsic to the zymogen state. To challenge this model, we next generated mice engineered to express only catalytically inactive prostasin. These mice, unlike prostasin null mice, were born with Mendelian frequency, developed barrier function, and were healthy when followed for up to 30 weeks. Furthermore, zymogen-locked and catalytically inactive prostasin mutants were biologically active in vivo when overexpressed in the epidermis of transgenic mice. The apparent ability of prostasin to allosterically activate other protease zymogens is a hitherto unique feature of a vertebrate protease, with similar functions previously only described for non-protease components of the coagulation system and for bacterial plasminogen activators, such as streptokinase and staphylokinase. Dual activation of cMet-Akt-mTor and PAR-2-Gai-NFkB signaling accounts for the transforming potential of matriptase In collaboration with Silvio Gutkind, OPCB, we recently identified cMet-Akt-mTor signaling, elicited by proteolytic activation of pro-hepatocyte growth factor on the keratinocyte cell surface, as one essential pathway by which matriptase promotes malignant transformation. Protease activated receptor (PAR)-2 is a key regulator of inflammation that is activated by picomolar concentrations of matriptase in vitro. PAR-2 activation by matriptase in cell-based assays caused robust induction of NFkB through Gai and expression of NFkB target genes. Surprisingly, genetic elimination of PAR-2 from mice completely prevented matriptase-induced premalignant progression. These findings suggest that dual activation of pro-hepatocyte growth factor-cMet-Akt-mTor proliferation/survival signaling and PAR-2-Gai-NFkB inflammatory signaling accounts for the transforming potential of matriptase. A function for TMPRSS13 in squamous epithelial differentiation and epidermal barrier acquisition In a screen for membrane-anchored serine proteases involved in squamous epithelial differentiation we identified transmembrane protease, serine 13, TMPRSS13 as highly expressed in squamous epithelium of the oral cavity, epidermis, and upper digestive tract. Compatible with this expression pattern, Tmprss13-deficient mice displayed abnormal squamous differentiation and compromised epithelial barrier function, as measured by the trans-epidermal fluid loss rate of newborn mice. The study demonstrates a biological function of TMPRSS13 in promoting squamous epithelial barrier acquisition. Cellular collagen receptors in collagen homeostasis in physiology and pathophysiology Background: The urokinase plasminogen activator (uPA) receptor-associated protein (uPARAP) and mannose receptor (MR) are members of the of the mannose receptor family of endocytic transmembrane proteins that mediate collagen uptake and lysosomal degradation. Research accomplished: In vivo imaging of collagen degradation identifies M2-polarized macrophages as critical mediators of intracellular collagen degradation Although paramount to the morbidity and mortality of cancer, tumor-associated interstitial collagen degradation remains poorly understood at the cellular and molecular level, chiefly due to the lack of assays for imaging collagen turnover in vivo. In collaboration with Roberto Weigert, IMTU, OPCB, and Kenn Holmbeck, CSDB, we found that intracellular collagen degradation was predominantly executed by a minor population of M2-polarized macrophages, while more abundant fibroblasts and inflammatory macrophages internalized collagen at lower levels. Systemic inhibition of matrix metalloproteinases or the use of a mutated collagen in which the collagenase cleavage site was removed, reduced intracellular collagen degradation in vivo, suggesting that collagen endocytosis occurs secondary to initial collagen fragmentation by pericellular collagenases. Genetic ablation of MR or uPARAP impaired this intracellular collagen degradation pathway establishing a role of the two receptors in intracellular collagen degradation. The study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-polarized macrophages in this process. Reengineered bacterial cytotoxins as antitumor and protease imaging agents Background: We are engaged in a long-standing collaboration with Steve Leppla, NIAID and Scott Martin, NHGRI, on the development of reengineered bacterial cytotoxins as novel therapeutic agents for cancer and as tools for the imaging of specific cell surface proteolytic activity. Comparative toxicity and efficacy study of protease-activated cytotoxins We have previously generated versions of PrAg requiring activation by matrix metalloproteinases (MMPs, PrAg-L1), urokinase plasminogen activator (uPA, PrAg-U2) or co-localized MMP/uPA activities (intercomplementing PrAg, consisting of two separate proteins, PrAg L1 I210A and PrAg-U2-R200A). While these toxin combinations all exhibit potent anti-tumor activity, simultaneous organ toxicities of varying severity have been reported with their use, and the lack of a systematic comparative study of the toxicity and efficacy of the various toxin variants has hampered clinical development. To alleviate this deficiency, we systematically evaluated their maximum tolerated dose and dose-limiting toxicities. The dual activated cytotoxin proved to display a superior safety profile. At a dose 9-fold below the maximum tolerated dose, this toxin variant achieved a remarkable 58% reduction in tumor burden. Taken together, the data suggest that an engineered version of anthrax lethal toxin can be administered safely, is highly effective, and is a promising candidate for clinical use.
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