Proteases and Tissue Remodeling Unit ? Z01DE0699-05? Thomas H. Bugge? ? Novel cell surface serine proteases in epidermal development, repair, and malignancy (33 % effort)? Prostasin is a GPI-anchored membrane serine protease that is proposed to be critical for the regulation of epithelial sodium channel activity. Prostasin is synthesized as an inactive zymogen that requires a site-specific cleavage by for activation. Epidermal ablation of matriptase and prostasin in mice resulted in identical effects on terminal epidermal differentiation. Enzymatic gene trapping of matriptase and prostasin immunohistochemistry revealed that matriptase is co-localized with prostasin in the oral epithelium and epidermis, and that the onset of expression of the two membrane proteases correlates with the acquisition of epidermal barrier function. Soluble matriptase converted prostasin zymogen to an active two-chain form that formed SDS-stable complexes with serpins. Two forms of prostasin corresponding to the prostasin zymogen and active prostasin were present in wildtype mouse epidermis, but only the zymogen form in matriptase-deficient epidermis. This shows that matriptase acts upstream from prostasin to initiate a proteolytic cascade promoting differentiation of oral epithelium and epidermis. Matriptase and prostasin co-localize in simple, stratified, and pseudo-stratified epithelia suggesting that the matriptase-prostasin proteolytic cascade may have other roles in epithelial biology in addition to regulating terminal oral epithelial and epidermal differentiation. ? Matriptase is consistently expressed in human carcinomas, including oral squamous cell carcinoma, and the ratio of matriptase to its endogenous inhibitor, the transmembrane glycoprotein, hepatocyte growth factor activator inhibitor-1 (HAI-1) is a strong prognostic marker. In collaboration with MCU, OPCB, we have previously shown that matriptase becomes expressed de novo in the undifferentiated proliferative basal epidermal compartment during squamous cell carcinogenesis, and that upregulation of matriptase relative to HAI-1 suffices to cause squamous cell carcinogenesis in mice. To determine the biological functions of HAI-1 and identify physiological HAI-1 targets, we generated HAI-1-deficient mice. HAI-1-deficient mice died at mid-gestation due to disruption of the epithelial integrity of placental chorionic trophoblast stem cells. The loss of HAI-1 in chorionic stem cells induced many stereotypic changes associated with malignant epithelial transformation, including chorionic basement membrane dissolution, loss of E-cadherin, and loss of membrane-associated β-catenin. Matriptase was specifically expressed in these chorionic stem cells, and matriptase gene deletion in HAI-1-deficient embryos restored the integrity of chorionic trophoblast stem cells, and enabled normal placentation and development to term. This establishes HAI-1 as an essential inhibitor of matriptase activity during embryonic development. ? ? Urokinase receptor-associated protein (uPARAP) and intracellular collagen turnover (33 % effort)? uPARAP contributes to collagen turnover and progression of metastatic mammary adenocarcinoma. To determine the possible association between uPARAP and oral cancer progression, with MCU, OPCB we used oral cancer tissue arrays and uPARAP monoclonal antibodies to determine the expression of the collagen internalization receptor in human squamous cell carcinomas of the oral cavity. The specificity of detection was verified by the staining of serial sections with two different monoclonal antibodies. uPARAP is expressed in fibroblast-like, vimentin-positive cells. The expression of the collagen internalization receptor was increased in tumor stroma as compared to tumor-adjacent connective tissue or normal submucosal connective tissue, and uPARAP was most prominent in poorly-differentiated tumors. This suggests that uPARAP participates in connective tissue destruction during oral cancer progression.? uPARAP expression during homeostasis is restricted to tooth and bone forming tissues. uPARAP deletion impaired calvarial and long bone formation, resulting in reduced bone length, cortical thickness, and bone mineral density. This was exacerbated by superimposing MT1-MMP deficiency, suggesting a complementary relationship between intracellular and pericellular collagen degradation. Studies of primary chondrocytes and osteoblasts performed in collaboration with CGRS, OPCB revealed that uPARAP is required for internalization and lysosomal degradation of the major bone collagens by these cells. In situ analysis revealed that uPARAP-mediated intracellular collagen degradation stimulated chondrocyte and osteoblast proliferation and survival within their respective collagen type I and II-rich osteogenic and chondrogenic niches. Taken together, these studies have identified an in vivo function of uPARAP-dependent intracellular collagen degradation in chondrogenesis and osteogenesis.? To gain mechanistic insights into the uPARAP-dependent intracellular pathway of collagen degradation we have used cell culture-based assays of fibrillar collagen turnover. This revealed that cleaved collagen is taken up faster by uPARAP than intact, native collagen due to the gelatin-like structure of collagen acquired after collagenase-mediated cleavage. The growth of uPARAP-deficient fibroblasts on a native fibrillar collagen matrix leads to accumulation of large soluble collagen fragments. In contrast, wildtype fibroblasts possess the ability to direct a complete collagen breakdown sequence, including extracellular cleavage, endocytic (uPARAP-dependent) uptake of large, defined fragments, and a final lysosomal degradation step. These data show that fibrillar collagen degradation is an integrated multi-step process that involves extracellular collagenases, receptor-mediated endocytosis, and lysosomal trafficking. ? ? Bacterial cytotoxins as antitumor and protease imaging agents (33 % effort)? Preclinical studies of uPA-activated anthrax toxins have been completed in collaboration with Stephen Leppla, MPS, NIAID and Art Frankel, Scott & White, Temple, Texas. The studies have shown that uPA-activated anthrax toxin can be safely administered systemically, displays potent antitumor activity against a spectrum of xenografted human and mouse syngeneic tumors, and can cause complete remission of tumors that are refractory to conventional treatment. The potency of the uPA-activated anthrax toxin towards individual tumor cell lines can be predicted by toxin receptor, uPA, and uPAR levels, but is independent of plasminogen activator inhibitor-1 abundance.? We have explored a number of modifications of our newly developed protease imaging assay, aimed at extending the capacity of the assay to include the visualization of specific proteolytic activity in intact tissues and animals. Assay modifications that have been surveyed include the use of near-infrared wavelength excitation and emission fluorogenic β-lactamase substrates, the use of lethal factor (LF)-near-infrared excitation and emission wavelength fluorochrome conjugates, the use of LF-Cre fusion proteins to activate floxed luciferase and β-galactosidase genes, and the use intra-vital multi-photon fluorescence microscopy in combination with conventional fluorogenic β-lactamase substrates. The last approach has emerged as holding the greatest promise. ? We have adapted our protease imaging assay for automated quantitative high throughput screening for protease inhibitors in collaboration with Christopher Austin and James Inglese, NIH Chemical Genomics Center, NHGRI. A proof of principle screen of 70,000 compounds has been completed, resulting in the initial identification of 27 active compounds, of which six were subsequently verified to inhibit toxin internalization
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