Decoding the Osteoclast Proteome and the Novel RGS12 Function in Redox Biology
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Osteoporosis is a disease of skeletal insufficiency that predisposes elderly individuals to bone fractures, affecting more than 200 million people worldwide. Heightened activity of osteoclasts, the cells uniquely capable of bone resorption, is a principal component in pathological bone loss. Therefore, understanding why and how osteoclasts are hyperactivated with increased age is important towards developing new therapeutics targeting osteoporosis. Moreover, aging was previously thought to manifest from the accumulation of reactive oxygen species (ROS) and oxidative damage, but it is now known that ROS play a physiological role in cell signaling, and notably in promoting osteoclast differentiation. Contributing to this emergent research, we herein describe the novel function of Regulator of G protein Signaling 12 (RGS12) in regulating the osteoclast redox state.Based on its namesake, RGS12 plays a canonical role in attenuating G protein-coupled receptor (GPCR) signaling by inactivating the Gα subunit. Through this action, the RGS family proteins have been demonstrated to control various cellular processes, including cell differentiation. It was previously shown that RGS12 is essential for osteoclast differentiation and function, and that its deletion in mice could substantially protect against pathological bone destruction. However, the molecular mechanism by which RGS12 promotes osteoclasts remains unknown. To investigate its function, we generated a conditional knockout mouse model wherein the RGS12 gene was selectively deleted in cells of the myeloid lineage (including osteoclasts) by means of the LysM promoter-driven Cre recombinase. Quantitative analysis of the bone architecture in RGS12-deficient mice revealed an osteopetrotic phenotype, in which trabecular bone—but not cortical bone—was significantly increased. Osteoclast precursors isolated from these mice show reduced differentiation and resorptive activity. Conversely, the overexpression of RGS12 led to increased osteoclast numbers and size, which were dependent on its RGS and RBD (Ras-binding domain) protein domains. Further assessment found that RGS12-deficient cells could not form filopodia (or microspikes), which are important for the fusion of precursor cells to form the mature, multinucleated osteoclast. RGS12 overexpression on the contrary increased the number and length of filopodia. To better understand RGS12 function on the molecular level, we deployed a robust and high-throughput proteomics platform to analyze the global perturbations in protein levels corresponding to the deletion of RGS12 in osteoclasts. By analyzing 3,700+ proteins we identified that a subset of antioxidant enzymes associated with Nrf2 (nuclear factor (erythroid-derived 2)-like 2), the master regulator of stress response, was significantly upregulated in Rgs12-deficient osteoclasts. Subsequent investigations confirmed that Nrf2 protein levels and transcriptional activity were increased, corresponding with reduced ROS levels in osteoclast precursors. Additionally, we showed that RGS12 post-translationally suppresses Nrf2 by facilitating its degradation in a proteasome-dependent manner. Through its inhibitory action on Nrf2, RGS12 promotes ROS that is required for the activation of ERK1/2 and NFκB, which are important signaling molecules during osteoclast differentiation. Overall, we identified RGS12 as a novel upstream regulator of Nrf2, which expands our understanding of osteoclast redox biology and is a step towards targeting ROS in the clinical context of osteoporosis.