uPARAP/Endo180 is essential for cellular uptake of collagen and promotes fibroblast collagen adhesion

The uptake and lysosomal degradation of collagen by fibroblasts constitute a major pathway in the turnover of connective tissue. However, the molecular mechanisms governing this pathway are poorly understood. Here, we show that the urokinase plasminogen activator receptor–associated protein (uPARAP)/Endo180, a novel mesenchymally expressed member of the macrophage mannose receptor family of endocytic receptors, is a key player in this process. Fibroblasts from mice with a targeted deletion in the uPARAP/Endo180 gene displayed a near to complete abrogation of collagen endocytosis. Furthermore, these cells had diminished initial adhesion to a range of different collagens, as well as impaired migration on fibrillar collagen. These studies identify a central function of uPARAP/Endo180 in cellular collagen interactions

Evidence for a matriptase-prostasin proteolytic cascade regulating terminal epidermal differentiation

Recent gene ablation studies in mice have shown that matriptase, a type II transmembrane serine protease, and prostasin, a glycosylphosphatidylinositol-anchored membrane serine protease, are both required for processing of the epidermis-specific polyprotein, profilaggrin, stratum corneum formation, and acquisition of epidermal barrier function. Here we present evidence that matriptase acts upstream of prostasin in a zymogen activation cascade that regulates terminal epidermal differentiation and is required for prostasin zymogen activation. Enzymatic gene trapping of matriptase combined with prostasin immunohistochemistry revealed that matriptase was co-localized with prostasin in transitional layer cells of the epidermis and that the developmental onset of expression of the two membrane proteases was coordinated and correlated with acquisition of epidermal barrier function. Purified soluble matriptase efficiently converted soluble prostasin zymogen to an active two-chain form that formed SDS-stable complexes with the serpin protease nexin-1. Whereas two forms of prostasin with molecular weights corresponding to the prostasin zymogen and active prostasin were present in wild type epidermis, prostasin was exclusively found in the zymogen form in matriptase-deficient epidermis. These data suggest that matriptase, an autoactivating protease, acts upstream from prostasin to initiate a zymogen cascade that is essential for epidermal differentiation

The cutting edge: Membrane-anchored serine protease activities in the pericellular microenvironment

The serine proteases of the trypsin-like (S1) family play critical roles in many key biological processes including digestion, blood coagulation, and immunity. Members of this family contain N- or C-terminal domains that serve to tether the serine protease catalytic domain directly to the plasma membrane. These membraneanchored serine proteases are proving to be key components of the cell machinery for activation of precursor molecules in the pericellular microenvironment, playing vital functions in the maintenance of homoeostasis. Substrates activated by membraneanchored serine proteases include peptide hormones, growth and differentiation factors, receptors, enzymes, adhesion molecules and viral coat proteins. In addition, new insights into our understanding of the physiological functions of these proteases and their involvement in human pathology have come from animal models and patient studies.The present reviewdiscusses emerging evidence for the diversity of this fascinating group of membrane serine proteases as potent modifiers of the pericellular microenvironment through proteolytic processing of diverse substrates. We also discuss the functional consequences of the activities of these proteases on mammalian physiology and disease

Abstract 9: Two Distinct Roles of Plasminogen Activator Inhibitor-2 (PAI) and PAI-1 in Deep Vein Thrombosis

Objectives: Urokinase-plasminogen activator (uPA) has a vital role in deep vein thrombosis (DVT). While PAI-1 is the main inhibitor of uPA in vivo , PAI-2, found mostly intracellularly, has proven to have more anti-inflammatory and anti-apoptotic than anti-fibrinolytic activities. We explored the hypothesis that PAI-1 and PAI-2 play roles in DVT through either fibrinolytic or inflammatory pathways. Methods: Wild type C57Bl/6 (WT), PAI-2 -/- , and PAI-1 -/- mice underwent surgical caval ligation. Subsequent thrombus was harvested at various time points and measured for thrombus weight (TW) normalized to body weight. Tissue was analyzed by western blot, ELISAs, zymography and flow cytometry. Results: PAI-2 deficiency resulted in unchanged thrombus formation (TW day 4: WT = 1.037 ± .043 mg/g n=17; PAI-2 -/- = 1.028 ± .067 mg/g n=17, p=.91), while it enhanced thrombus resolution (TW day 12: WT = .43 ± .03 mg/g n=13; PAI-2 -/- = .319 ± .021 mg/g, n=16, p=.004). PAI-1 deficiency caused decreased formation (TW day 12: PAI-1 -/- = .901 ± .043 mg/g n=11, p=.045) and enhanced resolution (TW day 12: PAI-1 -/- = .248 ± .011 mg/g n=10, p=.00004). Though total uPA levels in thrombi were similar between groups, active uPA by ELISA was increased by 5 fold in PAI-1 -/- mice and 40 fold in PAI-2 -/- mice compared to WT mice at day 12, indicating increased uPA-mediated fibrinolysis. Exploring other mediators of resolution, we found that PAI-1 and PAI-2 deficiencies lead to contrasting results. By zymography, PAI-1 deficiency caused an early increase in matrix metalloproteinase (MMP) activity that decreased over time. Conversely, PAI-2 deficiency trended towards less early MMP activity that then increased. Using flow cytometry to assess clot cell populations, on day 4, PAI-2 -/- mice had an increase in neutrophils with a decrease in macrophages, whereas PAI-1 -/- mice had an increase in macrophages. Conclusions: This identifies PAI-2 as a novel regulator of venous thrombus resolution and suggests the first context in vivo in which PAI-2 has an anti-fibrinolytic role. Both PAI-1 and PAI-2 deficiencies enhance thrombus resolution, but through diverging mechanisms. Further examination of these pathways may lead to potential therapeutic prospects in accelerating thrombus resolution. </jats:p

Novel signaling interactions between proteinase-activated receptor 2 and Toll-like receptors <i>in vitro</i> and <i>in vivo</i> (136.36)

Abstract Toll-like receptors (TLRs) and proteinase-activated receptors (PARs) function as innate immune biosensors in mucosal epithelial cells (ECs). We previously reported the functional and physical interactions between TLR4 and PAR2, and more recently, between PAR2 and the TLR adapter, TRIF, and established a novel mechanism of “receptor cooperativity” between PAR2 and TLR4. We hypothesized that intracellular signaling pathways utilized by TLRs and PAR2 would converge either cooperatively or non-cooperatively when co-engaged. We demonstrate herein the cooperation between PAR2 and TLR2, TLR3, or TLR4 for activation of NF-κB-dependent signaling in mucosal EC lines. In contrast, activation of PAR2 negatively regulated TLR3-dependent antiviral pathway, blunting the expression of TLR3/IRF-3-driven genes, as well as activation of IRF-3 and STAT1. Similarly, TLR4-TRIF-activated, IRF-3-driven genes were also attenuated by PAR2 co-activation. Consistent with these in vitro observations, PAR2−/− and TLR4−/− mice, which were refractory to footpad edema induced by PAR2 agonist peptide, were protected from mouse-adapted H1N1 influenza A virus-induced lethality when compared to wild-type mice. These data support and extend our recently described, novel model of PAR2-TLR4 “receptor cooperativity” and highlight the complexity of signaling integration between heterologous innate immune biosensors.</jats:p