The Role of Protease Activated Receptors in Osteoblast Differentiation

Abstract

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2010.Protease activated receptors (PARs) are G-protein coupled receptors (GPCRs) that are activated by serine proteases and initiate a variety of cellular responses including proliferation, migration, differentiation, and mitogenesis. In vivo, PARs have been found to be predominately involved with tissue repair and wound healing. Two protease activated receptors, PAR-1 and PAR-2 have been shown to be expressed on the cell membranes of bone making cells, osteoblasts. Little is known regarding their role in osteoblast maturation and bone remodeling overall. In this thesis, our efforts have addressed the goal of discovering how PARs affect osteoblast differentiation and maturation and what function PARs play in vivo in bone development. Our research aims to examine the temporal expression of the receptors during osteoblast maturation, decipher the G-protein signaling pathways involved in osteoblasts, and study the functional response of the receptors in bone remodeling environments. Localization of PAR-1 and PAR-2 expression in active sites of bone formation in addition to temporal expression of both PAR-1 and PAR-2 throughout the development and growth states of the maturing osteoblasts was examined in this work. Using specific G-protein coupled receptor (GPCR) signaling activation, we were able to demonstrate PAR-1 and PAR-2 are active during osteoblast maturation. By examining the functional responses of PAR activation in osteoblasts, we were able to connect temporal PAR expression to osteoblast differentiation and maturation processes. It is known in the literature that PARs signal through different G-protein signaling pathways based on the cell or tissue in which they are active. The G-protein signaling mechanisms by which activated PARs on osteoblast use have not been completely characterized and were therefore examined in this thesis work using inhibitor/reporter and calcium signaling assays. Using luciferase reporters specific for several GPCR signaling endpoints, including serum response element (SRE) and Nuclear factor of activated T-cells (NFAT), we were able to identify specific target pathways that PARs may be signaling through in osteoblasts. Understanding the signaling pathways in which PAR-1 and PAR-2 may signal through in bone can help us to better understand the roles in which PARs play in osteoblast expression and function. In vivo expression and function of both PAR-1 and PAR-2 were examined using PAR-1-/- and PAR-2-/- mice. Characterization of mice absent of the receptor gave insight into the function of PARs in vivo in the musculoskeletal system. Knockout mice for both PAR-1 and PAR-2, when compared to wildtype littermates, showed a significant decrease in long bone trabecular density and decreased maturation of osteoblasts grown in culture. Injections of synthetic peptide agonists for PAR-1 and PAR-2 over the calvaria showed increase in bone deposition in wildtype mice, suggesting that PARs play an important role in bone remodeling and growth. In summary, this work shows how PAR-1 and PAR-2 are important in osteoblast maturation and function. Expression of PARs on the surface of osteoblasts and the active signaling mechanisms that these receptors signal through suggest a primary role in osteoblast function. Functional studies, both in vitro and in vivo have shown evidence that PARs may play an important role in key musculoskeletal processes, including normal and abnormal remodeling of the bone surface, wound repair within the bone environment, and several inflammatory bone diseases including osteoarthritis. The simplified amino-peptide signaling mechanism in which these receptors are activated may present many therapeutic and pharmaceutical opportunities to target several known osteoblast-related bone diseases, including osteoporosis