Human bodies change with age, and the skeleton is among the parts of the body most visibly affected. This is because bone tissue tends to decrease as the skeleton gets older. For example, people often get shorter as they get older, mostly because they lose bone mass in areas of the skeleton that support posture. Severe bone loss can also lead to osteoporosis, a debilitating condition where bones become brittle and fracture easily.

Human skeletons contain cells, called osteoclasts, which break down bone tissue. Osteoclasts normally cooperate with other cells that add new bone, which helps maintain the balance between ‘bone-eating’ and ‘bone-building’ responsible for sculpting a healthy skeleton. This balance is disrupted during old age when the body starts producing too many ‘hyperactive’ osteoclasts, and bone formation cannot keep up with bone loss.

Reactive oxygen species (ROS) are unstable, potentially toxic molecules that have been linked with diseases of aging. Recent research has shown that low amounts of ROS can also drive the formation of new osteoclasts. Ng, Li et al. therefore wanted to determine how exactly ROS did this – specifically, whether ROS works together with the cell signaling mechanisms involved in bone loss controlled by a gene called Rgs12.

Initial experiments, using genetically altered mice, showed that removing Rgs12 from immature osteoclasts was enough to stop them from maturing. The bones of these mice were also stronger and thicker than usual. In contrast, forcing osteoclasts to produce large amounts of the protein encoded by Rgs12 heightened their bone-eating ability.

Analysis of the proteins made by cells without Rgs12 revealed that the cells had turned on the Nrf2 gene, a molecular ‘master switch’ that helps produce the enzymes capable of counteracting ROS (termed antioxidants). These cells therefore contained abnormally high amounts of antioxidants and low levels of ROS. However, osteoclasts where the Rgs12 gene was present were able to generate ROS by switching off the Nrf2 gene, and were thus able to reach maturity.

These results shed new light on the molecular signals that direct the development and activity of osteoclasts. In the future, a better understanding of these mechanisms could help us prevent them going wrong during aging, or even lead to better therapies for osteoporosis and other skeletal disorders.

Osteoporosis is a pervasive disorder characterized by skeletal fragility and microarchitectural deterioration that predisposes individuals to bone fractures. The disease has a significant global impact, affecting an estimated 200 million people worldwide and exerts a heavy economic burden. Moreover, disease prevalence is projected to rise by approximately 50% within the next ten years (Amin et al., 2014). Therefore, understanding the pathogenesis of osteoporosis is an urgent matter to develop better treatments for this debilitating disease.

Bone remodeling is carried out by the coordinated actions of the bone-forming osteoblasts (OBs) and the bone-resorbing osteoclasts (OCs). Disorders of skeletal deficiency such as osteoporosis are typically characterized by enhanced osteoclastic bone resorption relative to bone formation, thereby resulting in net bone loss. Although significant progress has been made in understanding the pathological role of OCs, the molecular pathways that drive OC differentiation remains an area needing further investigation.

Regulators of G-protein Signaling (RGS) are a family comprised of more than thirty proteins that share the eponymous and functionally conserved RGS domain; these proteins play a classical role in attenuating G protein-coupled receptor signaling through its GTPase-accelerating protein activity to inactivate the Gα subunit (Neubig and Siderovski, 2002; Keinan, 2014). RGS proteins are multifunctional proteins that can contribute to various cellular processes including cell differentiation. Rgs12 is unique in that it is the largest protein in its family. In addition to the RGS domain, Rgs12 contains a PSD-95/Dlg/ZO1 (PDZ) domain, a phosphotyrosine-binding (PTB) domain, a tandem Ras-binding domain (RBD1/2), and a GoLoco interaction motif. The multi-domain architecture of Rgs12 is thought to facilitate its role as a scaffolding protein in complexes in which multiple signaling pathways might converge (Snow et al., 2002; Sambi et al., 2006; Snow et al., 1998; Willard et al., 2007; Schiff et al., 2000).

Reactive oxygen species (ROS) are produced as a normal byproduct of cellular metabolism (Callaway and Jiang, 2015) and forms the basis Denham Harman’s free radical theory of aging, which perhaps is the most enduring model of aging. The theory of aging postulates that the gradual accumulation of damage inflicted by ROS eventually manifests as degenerative diseases associated with aging (Harman, 1956; Krause, 2007). In addition to longevity, ROS have been implicated in the management and prevention of cancers, cardiovascular diseases, macular degeneration, Alzheimer’s disease, arthritis, and many other tissues—to which the bone is no exception (Naka et al., 2008; Domazetovic et al., 2017).

More recent studies have shown that RANKL-induced ROS are indispensable for OC differentiation (Callaway and Jiang, 2015; Lee et al., 2005; Kim et al., 2010; Bartell et al., 2014). ROS at high levels induce oxidative stress, which if left unchecked becomes deleterious to cell. At low concentrations, however, ROS have been shown to participate in signaling events in OCs, including the RANKL-dependent activation of mitogen-activated protein kinases (MAPKs), phospholipase C gamma (PLCγ), nuclear factor kappa B (NFκB), and [Ca²⁺] oscillations; all of which contribute to the activation of nuclear factor of T-cells (NFAT), the master regulator of OC differentiation. Multiple lines of evidence have consistently shown that suppression of ROS by various means could inhibit OC differentiation (Lee et al., 2005; Kim et al., 2010; Bartell et al., 2014). In particular, RANKL-dependent activation of PLCγ, [Ca²⁺] oscillations, and NFAT were abrogated when OC precursors were treated with the antioxidant N-acetylcysteine (NAC) (Kim et al., 2010). Furthermore, our previous studies demonstrated that Rgs12 silencing could inhibit PLCγ activation, [Ca²⁺] oscillations, and the expression of NFATc1 and its downstream factors (Yang and Li, 2007). Hence, these findings led us to hypothesize that Rgs12 may play a role in regulating the cellular redox state, thereby controlling OC differentiation.

The importance of ROS in osteoclasts has been underlined by the growing corpus of evidence demonstrating that ROS increased with aging or during inflammation can stimulate bone resorption and exacerbate bone loss (Callaway and Jiang, 2015). Targeting ROS in diseases of excess bone resorption such as osteoporosis could therefore represent a novel therapeutic strategy. An important mechanism of cellular ROS clearance relies on the Keap1-Nrf2 pathway, which has been well characterized especially in the context of cancer biology (Kansanen et al., 2013); however, the upstream signaling molecules that could regulate the Keap1-Nrf2 axis in OCs remains unknown. Targeting this gap in knowledge, our study uncovered a novel role of the signaling protein Rgs12 in regulating Nrf2, thereby controlling cellular redox state and OC differentiation.

Within this study, we first demonstrated the essential role of Rgs12 in OC differentiation such that myeloid cell-targeted Rgs12 knockout mice exhibited an osteopetrotic phenotype, evidenced by reduced OC numbers but no changes to OB numbers nor bone formation (Figure 1). Furthermore, OC precursors isolated from these mice showed reduced in vitro OC differentiation and bone resorptive activity (Figure 2). On the contrary, forced overexpression of Rgs12 in RAW264.7 cells significantly promoted OC formation and increased the size of the resultant OCs, which was also associated with increased bone resorptive activity. However, the mechanism by which Rgs12 regulates OC differentiation remains unclear, to which we investigated more deeply using a leading-edge and high-throughput proteomics technique.

Proteomics is a powerful tool that has led to numerous discoveries of proteins and biological processes that drive OC differentiation (Segeletz and Hoflack, 2016). Notably, this technique was recently used to map the podosome proteome which helped to advance our understanding of determinants in the macrophage multinucleation process (Rotival et al., 2015; Cervero et al., 2012), and how metabolism and energy is redirected towards bone resorption in OCs (An et al., 2014). Proteomics can therefore provide a broad yet informative overview of the systemic changes in the differentiating OC. To discover the cellular function of Rgs12 in OCs, we employed a robust and high-throughput quantitative proteomics approach to characterize the global protein changes of OCs derived from Rgs12 cKO and cKO BMMs (Figure 3). The analysis revealed that most perturbations to the proteome was related to day 3 and 5 of OC differentiation, which coincides with the timing of Rgs12 upregulation in wild-type OCs (Figure 2A–B). This overlap suggests that Rgs12 may be more important to later phases of OC differentiation, including cell-cell fusion and bone resorption. The analysis also identified the upregulation of a collection of antioxidant enzymes that are all transcriptionally regulated by the antioxidant response element (ARE) within the promoter region, which is activated by the transcription factor Nrf2 (Nguyen et al., 2009). While the upregulation of these genes would lead one to surmise that Nrf2 should be similarly increased, the transcription factor was not detected in the proteomics analysis, which is likely result of technical limitations inherent to current LC-MS capabilities. It was previously reported that transcription factor protein levels are notoriously difficult to obtain due to many having relatively low expression levels that are generally below current mass spectrometer detection limits (Simicevic and Deplancke, 2017). The quantitation of transcription factors typically entails an upstream enrichment/separation step before LC-MS analysis. Therefore, the absence of Nrf2 in our proteomics analysis is not an unusual happenstance from a technical standpoint. Alternatively, we confirmed our proteomics findings by showing increased Nrf2 protein levels and nuclear translocation activity in Rgs12-deficient osteoclasts (Figure 4A–C).

Based on our preliminary evidence, we further investigated the role of Rgs12 in Nrf2 signaling and found that Nrf2 protein levels and nuclear translocation were increased in OC precursors in which Rgs12 was depleted (Figure 4). Because our data showed that Rgs12 deficiency upregulated Nrf2, we expected that Keap1 levels should be reduced in order to facilitate the increased Nrf2 activity. On the contrary, Keap1 levels were upregulated in Rgs12-deficient cells, to which we speculate that Rgs12-deficient cells may be overcompensating Keap1 expression in order to rein back the increased Nrf2 activity. Nevertheless, consistent with the upregulation of Nrf2 and its corresponding antioxidant enzymes, the RANKL-dependent induction of ROS was attenuated in Rgs12-deficient cells. Through an orthogonal approach, we demonstrated that Rgs12 overexpression in OC precursors could also enhance RANKL-mediated activation of ERK1/2 and NFκB (Figure 6), which are established to be dependent on ROS (Hyeon et al., 2013). Furthermore, inhibition of intracellular ROS blocked the effect of Rgs12 overexpression, indicating that Rgs12 promotes RANKL-dependent signaling by facilitating ROS production. Overall, the data collectively demonstrate that Rgs12 promotes osteoclastogenesis by facilitating ROS generation through the suppression of Nrf2 and its target antioxidant genes.

The transcription factor Nrf2 is constitutively expressed but its activity is inhibited through its interaction with Keap1. Under basal conditions, Nrf2 is restricted to the cytoplasm where it is continually depleted through the proteosomal degradation pathway. When bound to Nrf2, Keap1 recruits the cullin 3 (Cul3)-dependent E3 ubiquitin ligase complex, which ubiquitinates and targets Nrf2 for degradation by the 26S proteasome (Zhang and Hannink, 2003; Itoh et al., 2003; Nguyen et al., 2005; McMahon et al., 2003). Keap1 protein contains multiple reactive cysteine residues that serve as redox sensors (Abiko et al., 2011) that are sensitive to stressor conditions including oxidative stress causes the electrophilic modification of Keap1, inducing conformational changes, causing the protein to dissociate from Nrf2 and thereby allowing the transcription factor to enter the nucleus (Stewart et al., 2003). We therefore determined how Rgs12 regulates this well-defined mechanism (Figure 5). tBHQ is a selective inhibitor of Keap1 activity by covalently binding the protein’s reactive thiols and as a result activate Nrf2 and its downstream proteins in RAW264.7 cells (Abiko et al., 2011). tBHQ was also previously shown to inhibit OC differentiation via the upregulation of heme oxygenase-1, a Nrf2-dependent antioxidant enzyme (Yamaguchi et al., 2014). In this study, we determined whether Rgs12 could suppress the tBHQ-dependent upregulation of Nrf2 (Figure 5B). We reasoned that if Rgs12 relies on a Keap1-dependent mechanism, then the inhibition of Keap1 by tBHQ should prevent the ability of Rgs12 to suppress Nrf2. However, we observed that Rgs12 was still able to suppress Nrf2 despite the pharmacological blockade of Keap1 activity, thereby indicating that Rgs12 functions downstream of Keap1.

Following Keap1-mediated ubiquitination of Nrf2, the targeted protein should be degraded by the proteasome. Again, we reasoned that if Rgs12 is dependent on the proteasomal degradation pathway, then inhibition of this pathway using the proteasome inhibitor MG-132 should prevent Rgs12-mediated suppression of Nrf2. Indeed, we found that Inhibiting the proteasome was able to reverse the Rgs12-mediated degradation of Nrf2, which places Rgs12 in between Keap1 and the proteasome in the Nrf2 degradation pathway (Figure 5D–E). Thus, Rgs12 could either regulate the Cul3-dependent E3 ubiquitin ligase complex to facilitate the ubiquitination of Nrf2, or directly control proteasome activity. While Keap1 certainly plays a canonical role in Nrf2 suppression, the evidence herein suggest that Keap1 is not involved in the Rgs12 function.

It is also interesting to note that NFκB activation is also dependent on the proteasomal degradation of inhibitor of κB (IκB), which otherwise sequesters NFκB to the cytoplasm (Boyce et al., 2015). If Rgs12 could modulate proteasome activity, it is possible that Rgs12 could directly promote NFκB by facilitating the degradation of IκB. However, the fact that antioxidant treatment to suppress ROS could almost completely block the phosphorylation of NFκB points to an important involvement of ROS, and not just simply the proteasomal degradation of IκB in NFκB activation (Figure 6). Nonetheless, this potential crosstalk between the NFκB and Nrf2 pathways will need to be evaluated in future studies.

ROS is an inevitable byproduct of the mitochondrial electron transport chain (oxidative phosphorylation, OXPHOS) during ATP synthesis. Microscopy analyses have noted the high abundance of mitochondria in osteoclasts as early as 1961 (Gonzales, 1961), but interest in the metabolic profile that supports OC differentiation has made a resurgence in recent years. As examples, we and others through the proteomic study of OCs have reported that proteins involved in ATP synthesis were elevated in mature osteoclasts as compared to precursors (An et al., 2014; Ng et al., 2018). Additionally, it was demonstrated that mature OCs exhibit larger mitochondria, higher levels of enzymes of the electron transport chain, and higher oxygen consumption rates (Lemma et al., 2016). The evidence collectively point towards OXPHOS as a main bioenergetic source for osteoclast differentiation, and can therefore be considered an integral aspect of the process. As a consequence of increased mitochondrial biogenesis, ROS would similarly be elevated due to the ‘leakiness’ of the electron transport chain, which in turn contributes to the ROS that promotes OC differentiation. As such, an increase in ROS has been associated with mitochondria biogenesis in osteoclasts (Ishii et al., 2009), and mitochondrial ROS is essential for osteoclast formation (Bartell et al., 2014). Therefore, the analysis of OXPHOS and glycolytic patterns should not be discounted in a study that focuses on redox biology. To address this concern, we evaluated the molecular subsets in the proteome involved in the energy metabolism pathways including glycolysis, tricarboxylic acid (TCA) cycle, and OXPHOS pathways (Figure 3—figure supplement 1 and Figure 3—source data 2).

The loss of Rgs12 in BMMs led to the inhibition of RANKL-dependent ROS, which we demonstrated was a function of increased Nrf2 activity and elevated levels of its target antioxidant enzymes. At the same time, the proteomics analysis identified a reduction of proteins involved in the TCA cycle and OXPHOS (Figure 3—source data 2). Interestingly, cytochrome b5 type A (CYB5A) was against the general trend and was significantly increased by ~30% in Rgs12-deficient BMMs. The TCA cycle is responsible for the consumption of acetyl-CoA to generate NADH which is fed into the OXPHOS pathway to power ATP synthase and catalyze the formation of ATP. The impediment of OXPHOS, which requires molecular oxygen as co-factor, can shift ATP production towards anaerobic glycolysis under hypoxic and other conditions. However, the proteomic analysis revealed that protein levels of phosphofructokinase (PFK), the rate-limiting enzyme of glycolysis, were unaffected by Rgs12 deletion (Figure 3—figure supplement 1 and Figure 3—source data 2). In fact, all enzymes we detected that are involved in the glycolytic pathway with the exception of enolase 3 (a.k.a. phosphopyruvate hydratase) were unaffected by the loss of Rgs12.

On one hand, the loss of Rgs12 was associated with reduced OXPHOS, which would theoretically contribute to lower ROS levels. Conversely, the inhibition of OC differentiation could lead to reduced mitochondrial biogenesis, which could also explain lowered ROS levels in Rgs12-deficient BMMs. Therefore, mitochondrial ROS could be a contributing factor in Rgs12 function—albeit likely as a secondary effect. Although OXPHOS enzymes were reduced in Rgs12-deficient osteoclasts, only 19/83 proteins identified (22.9%) were altered, as compared to 26/26 proteins (100%) identified as Nrf2 targets (Figure 3—figure supplement 1 and Figure 3—source data 2). Additionally, the magnitude of protein expression fold-changes was markedly higher in Nrf2 response genes as compared to OXPHOS genes. Therefore, the higher degree of intra-group concordance and magnitude of expression of Nrf2 genes both indicate a more targeted response, whereas the weak pattern of expression of OXPHOS genes suggests an equivocal effect. Secondly, Nrf2 antioxidant proteins act downstream of mitochondrial ROS formation by acting directly on ROS. This effect is best exemplified by the fact that the ectopic expression of mitochondria-targeted catalase was able to suppress ROS and inhibit OC differentiation (Bartell et al., 2014). In the case of Rgs12 overexpression, it could be expected that increased mitochondrial ROS production would also need the concomitant suppression of Nrf2 to raise the overall intracellular ROS level. In the current work, we demonstrate that Rgs12 could promote Nrf2 degradation. Therefore, the inhibition of RANKL-dependent ROS associated with loss of Rgs12 was unlikely to be the direct result of glycolytic shift or reduced OXPHOS. While changes in mitochondrial ROS could be an minor aspect of Rgs12 function, but Nrf2 remains an essential component in the overall scheme.

In conclusion, we identified a new gene that could modulate the Nrf2-proteasome axis, which forms the crux of redox homeostasis in many biological contexts. Our study also points to a novel role of Rgs12 in OC redox biology, thus forming the molecular basis for developing therapies to modulate ROS for osteoporosis and other diseases of bone loss. Outside of its role in bone homeostasis, Rgs12 has also been shown to hold diverse and significant roles in other clinical contexts including pathological cardiac hypertrophy (Huang et al., 2016), tumor suppression in African American prostate cancer (Wang et al., 2017), and psychostimulant-induced increases in dopamine levels in the brain (Gross et al., 2018). We speculate that the ability of Rgs12 to regulate Nrf2 could be an important clue to better understand the role of Rgs12 in the context of cardiac disease and tumorigenesis, in which ROS is known to have significant implications. Therefore, our research would have widespread appeal to audiences of different research backgrounds.

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 and 3.

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Author details

1. Andrew Ying Hui Ng

   1. Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, United States
   2. Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, United States
   3. New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, United States

   Present address

   Department of Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Writing—original draft, Writing—review and editing

   Contributed equally with

   Ziqing Li

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7771-6574

2. Ziqing Li

   Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Data curation, Investigation

   Contributed equally with

   Andrew Ying Hui Ng

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3596-3520

3. Megan M Jones

   Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, United States

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

4. Shuting Yang

   Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Data curation, Investigation, Methodology

   Competing interests

   No competing interests declared

5. Chunyi Li

   Department of Oral Biology, School of Dental Medicine, University at Buffalo, Buffalo, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Chuanyun Fu

   Department of Stomatology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China

   Contribution

   Data curation, Methodology

   Competing interests

   No competing interests declared

7. Chengjian Tu

   1. New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, United States
   2. Department of Pharmaceutical Science, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, United States

   Contribution

   Software, Supervision, Validation, Methodology

   Competing interests

   No competing interests declared

8. Merry Jo Oursler

   Division of Endocrinology, Metabolism, Nutrition & Diabetes, Mayo Clinic, Rochester, United States

   Contribution

   Resources, Software, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

9. Jun Qu

   1. New York State Center of Excellence in Bioinformatics and Life Sciences, Buffalo, United States
   2. Department of Pharmaceutical Science, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, Buffalo, United States

   Contribution

   Resources, Software, Supervision, Funding acquisition, Methodology

   Competing interests

   No competing interests declared

10. Shuying Yang

    1. Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, United States
    2. The Penn Center for Musculoskeletal Disorders, School of Medicine, University of Pennsylvania, Philadelphia, United States

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Investigation, Project administration, Writing—review and editing

    For correspondence

    shuyingy@upenn.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-7126-6901

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