Our long-term goal is to define the molecular mechanisms by which Ca2+ oscillations in osteoclasts (OCs) contribute to bone remodeling and provide the basis for guiding more effective therapies to promote skeleton health. Recent breakthrough studies have shown RANKL-evoked Ca2+ oscillations play a switch-on role in inducing NFATc1 activation and OC differentiation. However, the factors induced by RANKL that initiate and maintain Ca2+ oscillations for OC differentiation and how Ca2+ oscillations induc osteoclastogenesis at the molecular level are still largely unknown. Our recent investigations suggest that Regulator of G-protein Signaling 12 (RGS12), a multi-domain and the largest protein in the RGS family, plays essential roles in Ca2+ oscillations and OC differentiation. We deleted RGS12 in hematopoietic lineage cells using inducible Mx1-cre and found that the mutant mice (RGS12?/?/cre) are osteopetrotic with significantly reduced OC numbers and bone resorption. Additionally, we found that RGS12 directly interacts with N-type Ca2+ channels and calcium- sensing receptors (CaRs), likely through phosphotyrosine-binding (PTB) and RGS domains, and that ectopic expression of RGS12 and its PTB domain respectively increases RANKL sensitivity during OC differentiation. It is known that the G protein-coupled CaRs play a pivotal role in controlling signaling pathways involved in OC differentiation and survival and maintaining extracellular Ca2+ concentrations, and that RGS proteins play essential roles in modulating CaRs and stimulating PLC? activity by inhibiting G?i signaling. Furthermore, cytosolic Ca2+ oscillations are generated mainly by influx of extracellular Ca2+ through L- and N-type calcium channels, and that RGS12 is capable of direct interaction with N-type Ca2+ channel through its PTB domain and modulates channel activity. Based on these findings, our central hypothesis is that RGS12 is required for Ca2+ oscillations in OC differentiation and function, and that RGS12 interacts with G?i protein, CaRs, Ca2+ channels, and other heterodimerization partners to regulate the RANKL-induced Ca2+ oscillation-NFATc1 pathway during OC differentiation. We will test this hypothesis through two specific aims:
Aim 1, we will determine where in the OC lineage RGS12-induced Ca2+ oscillations are required for OC differentiation and function in bone development and remodeling by analyzing the bone phenotype of RGS12 mutant mice with deletion of RGS12 at early and late stages of OC differentiation using RGS12flox/flox/Mx1-Cre, RGS12flox/flox/LysM-Cre and RGS12flox/flox/CathepsinK-Cre. We will further etermine the role of RGS12 in the enhanced bone resorption and formation that occurs after OVX. We will then characterize the role of RGS12 in regulating Ca2+ oscillations, OC differentiation and function at early and late stages of OC differentiation in vitro.
Aim 2, we will investigate the signaling pathways that impair OC differentiation in RGS12?/?/cre mice. We will further elucidate the mechanism by identifying and characterizing the functional domains of RGS12 and their heterodimerization partners involved in RGS12-induced Ca2+ oscillation in OCs.

Public Health Relevance

Osteoporosis is a major public health threat affecting approximately 44 million women and men aged 50 and older in the US which results in 1.5 million fractures annually with the cost of $12 billion to $18 billion for caring of these fracture. Moreover, the current treatment options such as bisphosphonates, selective estrogen receptor modulators and parathyroid hormone have been constrained with lower response rates or side effects and ineffectively tackle the quantitative burden and heterogeneity of osteoporosis. Thus, identifying new proteins such as RGS12 which target bone resorbing osteoclasts and bone remodeling has potential to provide a significant impact on patients.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
1R01AR066101-01
Application #
8673136
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Chen, Faye H
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
State University of New York at Buffalo
Department
Dentistry
Type
Schools of Dentistry/Oral Hygn
DUNS #
City
Buffalo
State
NY
Country
United States
Zip Code
14260