(Verbatim from the Applicant): Bone remodeling is a sensitively regulated dynamic process in which bone resorption by multinucleated osteoclasts (OCs) is normally closely followed by osteoblast (OB)-mediated bone formation. The multifunctional signal molecule, nitric oxide (NO), serves as an important intercellular autocrine and paracrine signal modulator in normal, as well as in pathological bone modeling and remodeling processes, and NO appears to exert profound effects on OB proliferation/survival, OC development and function. NO is formed from L-arginine in an oxidative reaction catalyzed by NO synthase (NOS) isoenzymes that are either constitutively expressed and calcium-activated (endothelial eNOS and neuronal nNOS isoforms), or transcriptionally induced (inducible NOS isoform) in response to inflammatory stimuli. Elevated NO levels potently inhibit bone resorption, both in vitro and in vivo, and NO actions on OCs, like other cells, involves a cGMP-dependent mechanism. The possible autocrine effect of NO on OCs has also begun to be examined, since it is now well established that OCs express both iNOS and eNOS. Although avian OCs express both iNOS messenger RNA (mRNA) and protein, inflammatory cytokines or LPS do not induce iNOS mRNA or protein in authentic mature Ocs, whereas elevated levels of calcium do. In contrast to Ocs, LPS and inflammatory cytokines induce iNOS and NO in many other cell types, including avian non-resorptive marrow-derived giant cells (MAGC). Therefore, we hypothesize that as OCs differentiate into fully functional resorption competent cells, the regulation of INOS expression is modified and this change in iNOS regulation impacts on OC-function in normal, as well as pathological OC function. The goal of this revised renewal application is to begin to understand the cell and molecular mechanisms involved in this unique OC iNOS regulation. The following Specific Aims are proposed: (1) to determine if the change in cytokine- and LPS-mediated OC iNOS regulation is a consequence of alterations in signaling pathways during the process of OC differentiation. As part of this aim, we will compare chicken, mouse, and human OC developmental models; (2) to identify promoter regulatory sequences and transcription factors that mediate (a) the differential induction of iNOS gene by calcium, PMA and inflammatory agents in MAGCs compared to mature resorbing OC, and (b) the inhibition of inflammatory-mediated iNOS gene induction following attachment of MAGCs to mineralized bone matrices; and (3) to analyze the signaling mechanisms by which NO alters OC activity. Such studies are anticipated to reveal new aspects of normal bone remodeling mechanisms and have potential to lend insight into skeletal pathologies such as implant loosening, osteoarthritis, other inflammatory skeletal disorders, and osteoporosis.
