Site-specific regulation of osteoblast activity.

摘要

In normal bone remodeling, bone resorption is coupled to bone formation at all sites in the adult skeleton. This coupling occurs in both a temporally and spatially coordinated fashion. That is, a wave of bone forming activity by osteoblasts follows a bone resorption episode by osteoclasts. Moreover, the formation occurs at the immediate site of bone resorption. In order to investigate the site specificity of this process, we used a phage-display library to probe for specific molecules that could bind to a resorbing surface. In this thesis, our efforts have utilized a random and cDNA phage-display library as a ligand source and type V osteoclast tartrate resistant acid phosphatase (TRAP) as the target.; Our phage-display library utilizes M13 filamentous phage, and T7 cDNA phage to express peptides in fusion with the coat protein (gene III) and protein X respectively. The phage are then used to detect specific protein-protein interactions with a biopanning technique. The target material is purified TRAP immobilized on plastic culture dishes. After 3 rounds of selection/amplification, individual clones are characterized by DNA sequencing.; We sequenced clones that demonstrated high affinity binding. The DNA and peptide sequences as well as their consensus frequency have been determined. The clones that appeared with the highest frequency: (i) recognize TRAP with high specificity and affinity; (ii) localize to osteoclast lacunae created on cortical bone wafers; (iii) code for genes (GPC-4 and TRIP-1) that were present in the osteoblast; (iv) led us to uncover another physiological function of TRAP in bone remodeling.; Identification and characterization of GPC-4 has provided at least one mechanism by which osteoblasts can recognize the skeletal sites where bone formation is required. The discovery of TRIP-1 has provided a mechanism for the stimulation of osteoblast differentiation once the cell is at the formation site. The intracellular pathways stimulated by activation of TRIP-1 include the Smad and MEKK pathways.; In summary, osteoblasts are spatially coordinated with osteoclasts during skeletal remodeling. The research in this thesis has discovered two novel mechanisms by which this coordination can be accomplished. It also has uncovered new and potential targets for the intervention and control of bone cell function, a finding that may have clinical implications.