The immediate goal of this application is to understand how osteoclast (OC) differentiation is regulated through negative endogenous regulators, which may provide novel therapeutic targets for bone diseases, such as osteoporosis and rheumatoid arthritis (RA). Although positive regulators of OC differentiation through the receptor activator of nuclear factor kB (RANK) ligand (RANKL)-RANK signaling axis have been extensively studied, understanding of negative regulators of OC differentiation is elusive. To identify key negative regulators of OC differentiation, we utilized microarray, gene downregulation, and osteoclastogenesis assays, leading to the isolation of guanine protein alpha subunit 13 (G?13, encoded by the Gna13 gene) as a potential OC negative regulator. To enable in vivo investigation of the role of G?13 in OC differentiation, we generated an OC-lineage specific Gna13 conditional knockout (CKO) mouse model by crossing Gna13f/f mice with LysM-Cre (OC precursor specific) mice. Our preliminary data showed that Gna13f/fLysM-Cre mice exhibited a severe osteoporosis from a drastic increase in OC differentiation. We also noted that G?13 deficiency attenuates RhoA activity and promotes Akt activity in OCs. Consistently, we showed that, as a proof of principle strategy, local constitutively active G?13 overexpression can protect against bone and cartilage loss while also attenuating inflammation in a mouse model of RA. Based on our preliminary data, we hypothesize that G?13 is a key negative regulator of OC that controls OC cell lineage commitment and differentiation for bone homeostasis under physiological and pathological conditions through activating RhoA and attenuating AKT signaling pathway.
Three specific aims are proposed to test our hypothesis.
In Aim 1, we will characterize the bone phenotypes of Gn?13f/fLysM-Cre mice and elucidate the mechanism underlying how G?13 activates RhoA signaling and attenuates Akt signaling to negatively regulate OC cell lineage commitment and differentiation.
In Aim 2 we will determine the upstream G?13 signaling cascade in OCs through characterization of G protein coupled receptors (GPCRs) coupled with G?13. We will characterize the mechanism underlying the roles of G?13 in bone remodeling and pathological bone loss by using dendritic cell (DC)-, monocyte- and OC-specific Gna13 CKO and overexpression transgenic mice in the loss-of-function and gain-of-function analysis, respectively. This study may provide important insights into the roles of negative regulators of OC differentiation in bone homeostasis and osteolytic bone diseases of excessive OC differentiation. Knowledge gained from this study may generate potential therapeutic targets, through characterization of G?13 signaling in OCs that may be targeted in treating osteolytic bone diseases by mimicking normal OC inhibitory signaling pathway to control OC formation. A multidisciplinary research team, including a bone biologist with expertise in OC biology and animal models of bone diseases; a bone biologist with expertise in OC biology and cell signaling; and an immunologist with expertise in autoimmunity diseases including RA has been established to achieve the research goal.
The goal of this proposal is to understand how osteoclast (OC) differentiation is negatively regulated and to characterize the mechanism by which endogenous negative regulators of osteoclasts control bone homeostasis under physiological and pathological conditions, such as osteoporosis and rheumatoid arthritis (RA). The proposed study will provide important insights into negative regulation of OCs by G?13 signaling pathway. This study will not only improve our understanding of the role of negative regulators in normal OC differentiation and in OC-related bone diseases (e.g., RA and osteoporosis), but it will also facilitate the design of novel therapeutic approaches for osteolytic diseases.
