Regulator of G protein signaling 12 enhances osteoclastogenesis by suppressing Nrf2-dependent antioxidant proteins to promote the generation of reactive oxygen species

Abstract

Regulators of G-protein Signaling are a conserved family of proteins required in various biological processes including cell differentiation. We previously demonstrated that Rgs12 is essential for osteoclast differentiation and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted Rgs12 in C57BL/6J mice targeting osteoclast precursors using LyzM-driven Cre mice or overexpressed Rgs12 in RAW264.7 cells. Rgs12 deletion in vivo led to an osteopetrotic phenotype evidenced by increased trabecular bone, decreased osteoclast number and activity but no change in osteoblast number and bone formation. Rgs12 overexpression increased osteoclast number and size, and bone resorption activity. Proteomics analysis of Rgs12-depleted osteoclasts identified an upregulation of antioxidant enzymes under the transcriptional regulation of Nrf2, the master regulator of oxidative stress. We confirmed an increase of Nrf2 activity and impaired reactive oxygen species production in Rgs12-deficient cells. Conversely, Rgs12 overexpression suppressed Nrf2 through a mechanism dependent on the 26S proteasome, and promoted RANKL-induced phosphorylation of ERK1/2 and NFκB, which was abrogated by antioxidant treatment. Our study therefore identified a novel role of Rgs12 in regulating Nrf2, thereby controlling cellular redox state and osteoclast differentiation.

Bone marrow adipogenic lineage precursors promote osteoclastogenesis in bone remodeling and pathologic bone loss

“…Red O staining or osteogenic medium (αMEM with 10% FBS, 10 nM dexamethasone, 10 mM βglycerophosphate, 50 µg/mL ascorbic acid, 100 IU/mL penicillin, and 100 μg/mL streptomycin) For in vitro osteoclastogenesis, bone marrow cells flushed from long bones were seeded to dishes for obtaining bone marrow macrophages (BMMs) as described previously (64,65).…”

RGS12 is required for the maintenance of mitochondrial function during skeletal development

“…RGS12 is expressed in chondrocyte mitochondria, cytoplasm, and nucleus RGS12 has been identified as a protein related to cell proliferation, differentiation, oxidative phosphorylation, ROS production, and apoptosis 19,25 . These observations indicate that RGS12 may play a key role in mitochondria.…”

“…In addition to a central RGS domain, RGS12 contains a PDZ (PSD-95/discslarge/ZO-1 homology) domain, a PTB (phosphotyrosine-binding) domain, a tandem repeat of Ras-binding domain and a GoLoco domain 24 . Our previous studies found that RGS12 is highly expressed in bone cells and regulates the function of osteoblasts and osteoclasts 19,25 . Endochondral bone growth is critically driven by the transformation of cartilage into bone tissue as a result of cell proliferation, differentiation, and apoptosis within the growth plate.…”

“…Regulator of G-protein signaling (RGS) proteins are a family of proteins that are involved in apoptosis and ROS regulation [18][19][20] . To date, several members of the RGS family of proteins have been isolated 21 , and specific RGS proteins have been reported to regulate specific signaling pathways 22 .…”

The Roles of ROS Generation in RANKL-Induced Osteoclastogenesis: Suppressive Effects of Febuxostat

“…Moreover, OC formation and activity (resorption areas) were also potently suppressed with febuxostat at 60 µM ( Figure 1F). Because ROS has been recognized as an intracellular signal mediator for RANKL-induced OC differentiation [11,21,22], these results suggest that febuxostat can act as an inhibitor for RANKL-induced osteoclastogenesis through the suppression of ROS production.…”