Proteolytic modification of the extracellular environment is essential for physiological tissue remodeling, as well as for the progression of a number of diseases, including oral cancer. The overall aim of this project is to understand the biochemistry, biology, and pathology of cell surface-associated serine proteases; in particular, their relation to the development, regeneration, and malignant transformation of epidermal tissues. Plasminogen activation in malignant tissue remodeling Initiation of the urokinase-mediated plasminogen activation cascade Attempts to identify the initiation routes and the sites of plasminogen (Plg) activation during tissue remodeling in vivo are hampered by the absence of reagents capable of detecting the activation state of the minute quantities of Plg activators that are present in living tissues. In collaboration with the MPS, NIAID, we have performed proof of concept studies to demonstrate that engineered urokinase Plg activator (uPA)-activated cytotoxins can serve to detect cell surface uPA activity in vivo. These studies have established that the uPA receptor (uPAR) is critical for generating cell surface uPA activity in vivo, that plasmin is critical in vivo for the efficient conversion of pro-uPA to active uPA, and that Plg activator inhibitor-1 actively restricts the generation of cell surface uPA activity in vivo. We are continuing the development of modified cytotoxins as imaging agents for cell surface protease activity, and we have obtained outside funding for this project. The uPAR-Associated Protein, uPARAP The uPAR-associated protein (uPARAP), is a recently identified transmembrane glycoprotein that binds ligand-occupied uPAR and collagen. uPARAP expression is highly upregulated at sites of active tissue remodeling during development and in tumor progression, including oral cancer. By introducing a deletion in the mouse uPARAP gene, we have identified an unexpected but critical function of uPARAP in the cellular uptake and lysosomal degradation of collagen. These studies have also revealed a specific role of uPARAP in both the adhesion of cells to collagen and in cell migration on fibrillar collagen matrices. Our current studies of uPARAP aim at defining basic properties of this novel collagen internalization pathway. This includes understanding the functional relationship between uPARAP-dependent lysosomal collagen degradation and pericellular matrix metalloproteinase-dependent collagen degradation, developing an understanding of the relationship between uPARAP-dependent collagen endocytosis and a previously described integrin-dependent phagocytic pathway for collagen uptake, and delineating uPARAPs functional contribution to collagen remodeling during tumor progression. Urokinase-activated bacterial cytotoxins for the treatment of tumors The acquisition of cell surface uPA activity is a hallmark of human malignancy. In collaboration with the MPS,NIAID, we generated engineered anthrax toxins that are specifically activated by cell surface uPA, by replacing the furin activation sequence in anthrax toxin protective antigen with artificial peptide sequences efficiently activated by uPA. These mutations confer cell surface uPA-dependent toxin activation in vivo, as determined using a panel of Plg, Plg activator, - receptor, and - inhibitor-deficient mice. The engineered toxin displayed limited toxicity to normal tissue but potent tumor cell cytotoxicity to a spectrum of transplanted tumors of diverse origin, and could eradicate established solid tumors. This tumoricidal activity was strictly dependent on tumor cell surface uPA. The data show that a simple change of protease activation specificity converts anthrax toxin from a highly lethal to a potent tumoricidal agent. We are currently expanding our joint efforts with MPS, NIAID along several lines to develop uPA-activated cytotoxins for cancer treatment. A collaboration has been initiated with a clinical investigator (Dr. Arthur Frankel, Wake Forest University) who has long-standing experience in the use of modified cytotoxins for the treatment of human malignancies. The biotechnology company, OncoTac Pharmaceuticals, has licensed the new technology, and is performing preclinical testing (toxicology, pharmacology, pharmacokinetics) of the engineered cytotoxins. To further accelerate the introduction of uPA-activated cytotoxins for the treatment of cancer, we are reengineering preexisting cytotoxin fusion proteins that are already in clinical use, to confer cell surface uPA activation to these toxins. Results to date suggest that this strategy may improve the therapeutic index of these drugs and reduce critical side effects. Novel cell surface serine proteases in epidermal development, repair, and malignancy Identification and characterization of novel type II transmembrane serine proteases The recent availability of the complete human and mouse genome sequences and expressed sequence tag databases, precipitated the unveiling of an unanticipated large, new family of membrane anchored serine proteases, the type II transmembrane serine proteases (TTSPs). We have worked with Dr. Toni Antalis, American Red Cross, to characterize and expand the new family using public and commercially accessible databases combined with classical molecular biology. These efforts have led to the identification and characterization of three new TTSPs (Matripase-3, Downregulated in Epidermal Squamous Cell Carcinoma (DESC)-2, and DESC-3), to the classification of the TTSP family into four phylogenetically related subfamilies, and to the realization that the TTSPs most likely arose by convergent evolution from more than one ancestral gene. We are currently characterizing the expression and function of a subset of these novel TTSPs. Matriptase/MT-SP1 in epidermal development We have characterized one member of the TTSPs, Matriptase/MT-SP1, in detail and shown that the novel protease has pleiotropic functions in epidermal development and is required for epidermal barrier function, hair follicle development, and thymic homeostasis. Epidermal Matriptase/MT-SP1-deficiency perturbs stratum corneum lipid matrix formation, cornified envelope morphogenesis, and desquamation, with prolonged exposure of Matriptase/MT-SP1-deficient skin to arileading to severe ichthyosis. Interestingly, proteomic analysis of Matriptase/MT-SP1-deficient epidermis revealed the complete and selective loss of the proteolytically processed forms of the major epidermis-specific protein filaggrin. This loss was associated with a profound accumulation of profilaggrin and aberrant profilaggrin processing products in the stratum corneum. The data identifies keratinocyte Matriptase/MT-SP1 as an essential component of the profilaggrin processing pathway and a key regulator of terminal epidermal differentiation. Moreover, the phenotype of Matriptase/MT-SP1-deficient mice bears striking resemblance to the lethal human congenital autosomal recessive disease, Harlequin ichthyosis suggesting that deficiencies in a component of a Matriptase/MT-SP1-dependent epidermal differentiation pathway could be an underlying cause of this disease.