Rgs12 enhances osteoclastogenesis by suppressing Nrf2 activity and promoting the formation of reactive oxygen species

The Regulator of G-protein Signaling 12 (Rgs12) is important for osteoclast (OC) differentiation, and its deletion in vivo protected mice against pathological bone loss. To characterize its mechanism in osteoclastogenesis, we selectively deleted Rgs12 in OC precursors using the LysM-Cre transgenic line or overexpressed the gene in RAW264.7 cells. Rgs12 deletion led to increased bone mass with decreased OC numbers, whereas its overexpression increased OC number and size. Proteomics analysis of Rgs12-deficient OCs 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 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. We therefore identified a novel role of Rgs12 in regulating Nrf2, thereby controlling cellular redox state and OC differentiation.


INTRODUCTION 29
Based on our proteomics analysis, we hypothesized that Rgs12 is needed to suppress Nrf2 1 activity and facilitate the formation of ROS, which has been previously shown to play a critical 2 role in OC differentiation (10, 21). To test this hypothesis, we assessed Nrf2 activity and the 3 expression of Nrf2 and Keap1 in Rgs12 fl/fl and LysM;Rgs12 fl/fl BMMs (Fig. 4). Western blotting of 4 Nrf2 in day 3 OCs also showed increased levels of Nrf2 in LysM;Rgs12 fl/fl cells ( Fig. 4A and B). 5 Keap1, however, which is known to suppresses Nrf2 activity by facilitating its degradation via 6 the proteasome pathway, was unexpectedly elevated in Rgs12-deficient cells.  Under basal conditions (i.e. absence of cellular stress), Nrf2 remains inactive through its 23 interaction with Keap1, which causes its continual ubiquitination and degradation via the 24 proteasome pathway (22, 23). A variety of stress conditions can induce conformational changes 25 in Keap1, thereby releasing Nrf2 from the ubiquitin-proteasome pathway, allowing it to 26 accumulate and translocate into the nucleus (24, 25). To better understand the mechanism by 1 which Rgs12 suppresses Nrf2 activity, we therefore first determined whether the ability of Rgs12 2 to suppress Nrf2 activity relies on this canonical mechanism (Fig. 5A). Given that Rgs12 3 deletion resulted in elevated Nrf2 expression and nuclear translocation, we first determined 4 whether Rgs12 overexpression could exert the opposite effect ( Fig. 5A and B). We measured 5 Nrf2 protein levels in RAW264.7 cells stably transfected with the Rgs12-His or empty vector and 6 found no difference when cells are at their basal state (i.e. uninduced). Stimulation of 7 RAW264.7 cells with tert-buthylhydroquinone (tBHQ), which is known to directly bind Keap1 and 8 attenuate its inhibitory effect on Nrf2 (26), caused a robust induction of Nrf2 protein levels in a 9 dose-dependent manner ( Fig. 5A and B). More importantly, RAW264.7 cells overexpressing 10 Rgs12 showed a significant reduction of Nrf2 protein levels that resulted from Keap1 inhibition 11 compared to those in the control cells. Moreover, the ability of Rgs12 to facilitate Nrf2 12 degradation despite the inhibition of Keap1 suggests that Rgs12 functions downstream of 13 Keap1, either by controlling the ubiquitination or proteasomal degradation of Nrf2. 14 15 Given the possibility that the reduction of Nrf2 levels in Rgs12 overexpression cells may be a 16 result of increased Nrf2 degradation, we further tested whether inhibiting the proteasome, a step 17 downstream of Keap1, could attenuate the ability of Rgs12 to facilitate Nrf2 degradation ( Fig. 5D  18 and E). Similar to tBHQ, preventing Nrf2 degradation using the proteasome inhibitor MG-132 19 caused Nrf2 protein to substantially accumulate (Fig. 5D, left panel). Interestingly, when Nrf2 20 protein levels were artificially induced, we observed the presence of a lower molecular weight 21 band, which could correspond to a different post-translational modification state (e.g. 22 unphosphorylated or non-ubiquitinated). Furthermore, we did not observe any changes in 23 Keap1 protein levels. In the previous scenario wherein Rgs12 overexpression could still 24 promote Nrf2 degradation in spite of tBHQ treatment, this was not the case when using MG-25 132. In fact, inhibiting the proteasome was able to reverse the ability of Rgs12 to promote Nrf2 26 degradation, indicating its requirement for the proteasome's function. Repeating this experiment 1 in RAW264.7 cells differentiated for 3 days with RANKL showed that Nrf2 levels were 2 suppressed in OCs (Fig. 5D, right panel); likely due to reduced transcriptional activity, which 3 corroborates with findings from previous studies (Fig. 5F) (21, 27). More importantly, Rgs12 4 overexpression could suppress Nrf2 protein levels, but inhibition of the proteasome using MG-5 132 reversed this effect (Fig. 5D, right panel). To confirm that Rgs12 inhibits Nrf2 through a 6 post-translational mechanism, we measured Nrf2 transcript levels by qPCR and found no 7 difference between wild-type or Rgs12-overexpressing cells (Fig. 5F). Overall, our data 8 collectively indicate that Rgs12 suppresses Nrf2 activity by facilitating its degradation through 9 the proteasome-dependent pathway. 10 11

Rgs12-mediated activation of osteoclast MAPK and NFκB signaling is dependent on 12 intracellular ROS. 13
It was previously demonstrated that ROS could act as an intracellular signal mediator OC 14 differentiation, and is required for the RANKL-dependent activation of p38 mitogen-activated 15 protein kinase (MAPK), extracellular signal-regulated kinase (ERK), and NFκB (10, 28). Given 16 our findings that Rgs12 could suppress the activity of Nrf2 and thereby promoting intracellular 17 ROS, we hypothesized that Rgs12 could promote RANKL-dependent signaling, and that this 18 effect would be abrogated by the addition of an antioxidant ( Fig. 6A  The importance of ROS in osteoclasts has been underlined by the growing body of evidence 1 that ROS increased with aging or during inflammation can stimulate bone resorption and 2 exacerbate bone loss (9). Targeting ROS in diseases of excess bone resorption such as 3 osteoporosis could therefore be an important therapeutic strategy. Additionally, an important 4 mechanism of cellular ROS clearance relies on the Keap1-Nrf2 pathway which is very well 5 characterized, especially in the context of cancer biology (29). However, the upstream signaling 6 molecules that could regulate the Keap1-Nrf2 axis in OCs remains unknown. Targeting this gap 7 in knowledge, our study uncovered a novel role of the signaling protein Rgs12 in regulating 8 Nrf2, thereby controlling cellular redox state and OC differentiation (Fig. 6C). 9

10
In this study, we demonstrated the essential role of Rgs12 in OC differentiation such that Rgs12 11 knockout mice exhibited increased bone mass (Fig. 1) and OC precursors isolated from these 12 mice showed reduced OC differentiation (Fig. 2). On the contrary, overexpressing Rgs12 in 13 RAW264.7 cells significantly promoted OC formation and increased the size of resultant OCs. 14 However, the mechanism by which Rgs12 regulates OC differentiation remains unclear. 15

16
Proteomics is a powerful tool that has led to numerous discoveries of proteins and biological 17 processes that drive OC differentiation (30). Notably, this technique was recently used to map 18 the podosome proteome which helped to advance our understanding of determinants in the 19 macrophage multinucleation process (31, 32), and how metabolism and energy is redirected 20 towards bone resorption in OCs (33). Proteomics can therefore provide a broad yet informative 21 overview of the systemic changes in the differentiating OC. To discover the cellular function of 22 Rgs12 in OCs, we employed a robust and high-throughput quantitative proteomics approach to 23 characterize the global protein changes of OCs derived from Rgs12 fl/fl and LysM;Rgs12 fl/fl BMMs 24 (Fig. 3). Interestingly, the analysis identified the upregulation of a collection of antioxidant 25 enzymes that are transcriptionally regulated by the antioxidant response element (ARE) in the 26 promoter region, which is activated by the transcription factor Nrf2 (34). Based on this evidence, 1 we further investigated the role of Rgs12 in Nrf2 signaling. We found that Nrf2 protein levels and 2 nuclear translocation were increased in OC precursors in which Rgs12 was deleted (Fig. 4). 3 Because our data showed that Rgs12 deficiency upregulated Nrf2, we expected that Keap1 4 levels should be reduced in order to facilitate the increased Nrf2 activity. On the contrary, Keap1 5 levels were upregulated in Rgs12-deficient cells, suggesting that the Nrf2 upregulation may be 6 independent of the Keap1. We speculate that Rgs12-deficient cells may be overcompensating 7 Keap1 expression in order to rein back the increased Nrf2 activity. Nevertheless, consistent with 8 the upregulation of Nrf2 and its corresponding antioxidant enzymes, the RANKL-dependent 9 induction of ROS was attenuated in Rgs12-deficient cells. Taking an opposite approach, we 10 demonstrated that Rgs12 overexpression in OC precursors could also enhance RANKL-11 mediated activation of ERK and NFκB, which is known to be dependent on ROS (Fig. 7). 12 Inhibition of intracellular ROS blocked the effect of Rgs12 overexpression, indicating that Rgs12 13 promotes RANKL-dependent signaling by facilitating ROS production. Overall, the data 14 collectively demonstrate that Rgs12 promotes osteoclastogenesis by facilitating ROS generation 15 through the suppression of Nrf2 and its target antioxidant genes. 16 17 Nrf2 is constitutively expressed but its activity is inhibited through its interaction with Keap1. 18 Under basal conditions, Nrf2 is restricted to the cytoplasm where it is continually depleted 19 through the proteosomal degradation pathway. When bound to Nrf2, Keap1 recruits the Cul3-20 dependent E3 ubiquitin ligase complex, which ubiquitinates and targets Nrf2 for degradation by 21 the 26S proteasome (23, 24, 35, 36). The Keap1 protein containing multiple reactive cysteine 22 residues that serve as redox sensors (26) Stressor conditions including oxidative stress causes 23 the electrophilic modification of Keap1, inducing conformational changes which cause the 24 protein to dissociate from Nrf2 and allow the transcription factor to translocate into the nucleus 25 (22). We therefore determined how Rgs12 regulates this well-defined mechanism (Fig. 5). tBHQ is a selective inhibitor of Keap1 activity by covalently binding the protein's reactive thiols and 1 could activate Nrf2 and its downstream proteins in RAW264.7 cells (26). Furthermore, tBHQ 2 inhibited OC differentiation via the upregulation of heme oxygenase-1, a Nrf2-dependent 3 antioxidant enzyme (Yamaguchi et al., 2014). In our study, we determined whether Rgs12 could 4 suppress the tBHQ-dependent upregulation of Nrf2 (Fig. 5B). We reasoned that if Rgs12 relies 5 on a Keap1-dependent mechanism, then the inhibition of Keap1 by tBHQ should prevent the 6 ability of Rgs12 to suppress Nrf2. However, we observed that Rgs12 was still able to suppress 7 Nrf2 despite blocking Keap1 activity, indicating that Rgs12 functions downstream of Keap1. 8 Following Keap1-mediated ubiquitination of Nrf2, the targeted protein is degraded by the 9 proteasome. Again, we reasoned that if Rgs12 is dependent on the proteasomal degradation 10 pathway, then inhibition of this pathway using MG-132 should prevent Rgs12-mediated 11 suppression of Nrf2. Indeed, we found that Inhibiting the proteasome was able to reverse the 12 Rgs12-mediated degradation of Nrf2, which places Rgs12 in between Keap1 and the 13 proteasome in the Nrf2 degradation pathway (Fig. 5D and E). Thus, Rgs12 could either regulate 14 the Cul3-dependent E3 ubiquitin ligase complex to facilitate the ubiquitination of Nrf2, or directly 15 control proteasome activity. It is interesting to note that NFκB activation is also dependent on 16 the proteasomal degradation of inhibitor of κB (IκB), which otherwise sequesters NFκB to the 17 cytoplasm (37). If Rgs12 could modulate proteasome activity, it is possible that Rgs12 could 18 directly promote NFκB by facilitating the degradation of IκB. However, the fact that antioxidant 19 treatment to suppress ROS could almost completely block the phosphorylation of NFκB points 20 to an important role of ROS, and not just the proteasomal degradation of IκB in NFκB activation 21 (Fig. 6). This potential crosstalk between the NFκB and Nrf2 pathways will need to be evaluated 22 in future studies. 23

24
In conclusion, our study points to a novel role of Rgs12 in OC redox biology, thus forming the 25 molecular basis for developing therapies for osteoporosis and other diseases of bone loss. 26 Additionally, we found a new factor that could modulate the Nrf2-Keap1 pathway, which is 1 important within the context of ROS biology. BMMs were obtained from the tibia and femur of 8-weeks-old C57BL/6J mice as described 24 previously (13). BMMs were seeded at 2 × 10 6 cells per 24-well and stimulated with 100 ng/mL 25 RANKL and 20 ng/mL M-CSF for 5 days to generate mature OCs. RAW264.7 cells were seeded 26 at 1.35 × 10 4 cells per 24-well and stimulated with 100 ng/mL RANKL for 5 days. Prior to fixing 1 and staining, RAW264.7-derived OCs were rinsed thoroughly with PBS to remove mononuclear 2 cells that tend to obscure OCs during imaging. TRAP staining was performed using the acid   Figure 7A. Sections 1 shown in Figure 6C are highlighted with dashed boxes. 2