Browsing School, Graduate by Subject "PAR2"
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The Biochemistry and Pathophysiological Function of TestisinTestisin is a unique trypsin-like serine protease that is localized to the extracellular membrane of cells through a glycophosphatidylinositol (GPI)-anchor. Testisin is expressed in sperm, eosinophils, and microvascular endothelial cells, and is overexpressed in ovarian cancers. Little is known about the regulation of testisin activity, proteolytic substrate specificity, or the physiological role of testisin. We generated recombinant human testisin using two expression systems to study its biochemical properties. We found that human testisin may be activated by recombinant hepsin, a transmembrane serine protease. Active human recombinant testisin forms an SDS-stable inhibitory complex with the serpin, protein C inhibitor (PCI). Examination of testisin substrate specificity using commercially available recombinant mouse testisin serine protease domain revealed that testisin prefers to cleave peptide substrates after Arg amino acid residues, and efficiently cleaves a peptide substrate derived from the N-terminal activation domain of the G-protein coupled receptor, Protease-Activated Receptor-2 (PAR2). When PAR2 was expressed in cell lines, we found that recombinant testisin cleaved and activated PAR2 to induce transient release of intracellular calcium and the phosphorylation of ERK1/2. When full-length human testisin and PAR2 are co-expressed on the cell surface, testisin is able to cleave PAR2, causing constitutive activation of several intracellular signaling pathways, the induction of cytokine expression, and internalization and loss of PAR2 from the cell surface. These data provide new insight into the biochemical properties of testisin, and its ability to activate PAR2, a critical signaling receptor important in inflammation, wound healing, and cancer. While testisin is not found in mature vasculature, it is expressed by microvascular endothelial cells in vascular beds associated with active angiogenesis. Testisin-deficient mice display delayed and aberrant corpus luteal angiogenesis. We found that capillary outgrowth from aortic rings isolated from testisin-deficient mice is reduced substantially. Silencing of testisin expression by siRNA knockdown in primary microvascular endothelial cells plated on Matrigel basement membranes suggests that capillary-like tubule formation is also decreased. These data demonstrate a functional role for testisin in processes required by microvascular endothelial cells for capillary formation, and may suggest a potential pathway involving testisin activation of PAR2 on the surface of microvascular endothelial cells.
The role of protease activated receptor 2 (PAR2) in the initiation and maintenance of type 2 immunityMore than one third of the world's population is infected with helminths with the highest prevalence in under developed countries. It is well established that helminth infection induces a highly polarized type 2 immune response in the host that is critical for helminth expulsion; however, the mechanisms by which host immunity is initiated are poorly understood. Helminth infection up-regulation of type 2 cytokines, IL–4 and IL–13 and induces alterations in gut function including an increase in intestinal permeability and a hyper-responsiveness of smooth muscle. Helminths secrete a number of proteases that may be used as molecular mimics to interact with the host. We identified a 26 kDa “trypsin-like protease” generated by helminths that interacts with host protease activated receptor 2 (PAR2). PAR2 is expressed on structural and immune cells throughout the gut and is activated by proteolytic cleavage. Proteolytic pathways are important to immune responses, but the contribution of PAR2 to the Th2 immune response against helminth infection is poorly understood. The central hypothesis of this project is that helminth generated serine proteases activate PAR2 on host cells and play a pivotal role in the induction of host type 2 immunity. To investigate this hypothesis, we propose the following aims; (1) to determine if helminths use molecular mimicry to interact with host proteolytic pathways and (2) to elucidate the role of PAR2 in the development of the type 2-mediated protective immune response in vivo. Our results indicate that nematodes secrete a trypsin–like serine protease that activates PAR2. Sequence and sequence comparisons results demonstrate that this protease is similar to other common human trypsin–like proteases. Finally, we identified a role for PAR2 in the both early and late stages of enteric nematode infection. At early time points, PAR2 plays a role in the increased in intestinal epithelial permeability that acts to facilitate the passage of worm products across the mucosal barrier and contributes to the initial up–regulation of type 2 cytokines. At later time points, PAR2 mediates the smooth muscle response to neural stimulation that is part of the immune–mediated alterations in gut function that promote worm expulsion.