Although, we know much about the molecular signals that regulate OC function, we know relatively little about the lineage development and mechanisms that OC use to develop from progenitors. The goal of this application is to better define OC progenitor (OCP) development and trafficking and the mechanisms regulating this process in health and disease so that we can identify potential drug targets to develop superior therapies for bone diseases. The central hypotheses are: 1) During homeostasis marrow-resident cells are the principal OCP source, while during inflammation or fracture repair, circulating cells become a significant source of OCP. 2) The mechanisms regulating OCP migration, engraftment and maturation to OC in bone differ between healthy and disease states. To test these hypotheses, we propose the following aims: 1. Define the role that CX3CR1+ OCP have in OC development during homeostasis and identify mechanisms regulating their homing and engraftment. 1A) Perform time course studies in CX3CR1-CreERT2-Ai14 mice at various ages to examine the kinetics of labeled OC. We will also monitor the kinetics of labeled OCP in the bone marrow, blood and spleen. 1B) The cell receptors EBI2 and CX3CR1 are expressed on OCP and have previously been implicated to influence OCP homing, engraftment and maturation. We will determine their role in OCP lineage development and trafficking in vivo under homeostatic conditions using CX3CR1-CreERT2-Ai14 mice and gene deletion. 2. Examine OCP homing from the circulation during bone inflammation and fracture repair. These studies will examine two disease models in which we previously demonstrated that circulating OCP are recruited to engraft in bone: TNFa-induced bone inflammation and a repairing fracture. 2A) Study a TNFa-induced inflammatory bone model (a WT parabiont has TNF? injected over its calvaria; the other parabiont is a CX3CR1-EGFP; TRAP-tdTomato mouse) and determine the rate that circulating labeled cells home to the inflammatory site, form OC and disappear. OCP kinetics will be measured as in 1A. 2B) Study a parabiosis fracture model (a WT parabiont receives a femur fracture; the other parabiont is a CX3CR1-EGFP; TRAP-tdTomato mouse) and determine the rate that circulating labeled cells home to the repairing callus, form OC and disappear. OCP kinetics will be measured as in 1A. 2C) Determine the phenotype and kinetics of circulating OCP that home to bone with TNFa-induced inflammation or fracture repair by injecting various populations of OCP from CX3CR1-EGFP; TRAP-tdTomato mice and monitoring the rates that labeled OC appear and disappear in bone. 2D) Determine if gene deletion of EBI2 or CX3CR1 CXCR4 alters the ability of circulating OCP to home and mature into OC in the models of TNFa-induced bone inflammation and fracture repair studied in aim 2C.
This application will test the hypotheses that: 1) during homeostasis marrow-resident cells are the principal source of osteoclast precursors (OCP) while during states of inflammation or fracture repair, circulating cells become a significant OCP source and 2) there are different mechanisms regulating OCP homing, engraftment and differentiation between healthy and disease states. Experiments are proposed that will define the role and identify mechanisms that regulate OCP development into osteoclasts in homeostasis and examine OCP homing engraftment and differentiation from the circulation during bone inflammation and fracture repair.
