RGS12 is essential for RANKL-evoked signaling for terminal differentiation of osteoclasts in vitro.
How RANKL evokes [Ca(2+)](i) oscillations and leads to osteoclast differentiation is unclear. We identified a new signaling protein, RGS12, and found that RGS12 is essential for [Ca(2+)](i) oscillations and osteoclast differentiation induced by RANKL. RGS12 may play a critical role in the RANKL-evoked PLCgamma-calcium channels-[Ca(2+)](i) oscillation-NFAT2 pathway.


BACKGROUND
RANKL-induced [Ca(2+)](i) oscillations play a switch-on role in NFAT2 expression and osteoclast differentiation. However, RANKL evokes [Ca(2+)](i) oscillations and leads to osteoclast differentiation by an unknown mechanism. In this study, we identified a new RANKL-induced signaling protein, regulator of G signaling protein 12 (RGS12), and investigated its effect on osteoclast differentiation in vitro.


METHODS
We used a genome-wide screening approach to identify genes that are specifically or prominently expressed in osteoclasts. To study the role of the RGS12 in osteoclast differentiation, we used vector and lentivirus-based RNAi gene silencing technology to silence the RGS12 gene in the monocyte progenitor cell lines and primary bone marrow-derived monocytes (BMMs). The interaction between RGS12 and N-type calcium channels was elucidated using co-immunoprecipitation and immunoblotting.


RESULTS
We found that RGS12 was prominently expressed in osteoclast-like cells (OLCs) induced by RANKL. This result was further confirmed at both the mRNA and protein level in human osteoclasts and mouse OLCs. Silence of RGS12 expression using vector and lentivirus based RNA interference (RNAi) impaired phosphorylation of phospholipase C (PLC)gamma and blocked [Ca(2+)](i) oscillations, NFAT2 expression, and osteoclast differentiation in RANKL-induced RAW264.7 cells and BMMs. We further found that N-type calcium channels were expressed in OLCs after RANKL stimulation and that RGS12 directly interacted with the N-type calcium channels.


CONCLUSIONS
These results reveal that RGS12 is essential for the terminal differentiation of osteoclasts induced by RANKL. It is possible that RGS12 regulates osteoclast differentiation through a PLC gamma-calcium channel-[Ca(2+)](i) oscillation-NFAT2 pathway.
Yang
Shuying
S
Department of Cytokine Biology, The Forsyth Institute, Boston, MA 02115, USA.
Li
Yi-Ping
YP
D000818:Animals; D017209:Apoptosis; D001862:Bone Resorption; D002118:Calcium / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010010:Osteoclasts / Q000166:cytology* / Q000502:physiology*; D053245:RANK Ligand / Q000502:physiology*; D020710:RGS Proteins / Q000235:genetics / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*
D053245:RANK Ligand; D020710:RGS Proteins; C106375:RGS12 protein, human; C508173:RGS12 protein, mouse; C505688:TNFSF11 protein, human; C505689:Tnfsf11 protein, mouse; D002118:Calcium