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 NF?B, 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.
Project description:Regulators of G protein signaling (Rgs) have pivotal roles in controlling various cellular processes, such as cell differentiation. How Rgs proteins regulate osteoclast (OC) differentiation, function and bone homeostasis is poorly understood. It was previously demonstrated that Rgs12, the largest protein in the Rgs family, is predominantly expressed in OCs and regulates OC differentiation in vitro. To further understand the role and mechanism of Rgs12 in OC differentiation and bone diseases in vivo, we created OC-targeted Rgs12 knockout mice by using inducible Mx1-Cre and CD11b-Cre. Deletion of Rgs12 in hematopoietic cells or specifically in OC precursors resulted in increased bone mass with decreased OC numbers. Loss of Rgs12 impaired OC differentiation and function with impaired Ca(2+) oscillations and reduced nuclear factor of activated T cells (NFAT) 2 expression. The introduction of wild-type osteoblasts did not rescue the defective osteoclastogenesis. Ectopic expression of NFAT2 rescued defective OC differentiation in CD11b;Rgs12(fl/fl) cells and promoted normal OC differentiation. Moreover, deletion of Rgs12 significantly inhibited pathological osteoclastogenesis and bone destruction in Rgs12-deficient mice that were subjected to ovariectomy and lipodysaccharide for bone loss. Thus our findings demonstrate that Rgs12 is an important regulator in OC differentiation and function and identify Rgs12 as a potential therapeutic target for osteoporosis and inflammation-induced bone loss.
Project description:Osteoclasts are the sole bone-resorbing cells that play an essential role in homeostatic bone remodeling and pathogenic bone destruction such as inflammatory arthritis. Pharmacologically targeting osteoclasts has been a promising approach to alleviating bone disease, but there remains room for improvement in mitigating drug side effects and enhancing cell specificity. Recently, we demonstrated the crucial role of MYC and its downstream effectors in driving osteoclast differentiation. Despite these advances, upstream regulators of MYC have not been well defined. In this study, we identify nuclear factor erythroid 2-related factor 2 (NRF2), a transcription factor known to regulate the expression of phase II antioxidant enzymes, as a novel upstream regulator of MYC. NRF2 negatively regulates receptor activator of nuclear factor-?B ligand (RANKL)-induced osteoclastogenesis through the ERK and p38 signaling-mediated suppression of MYC transcription. Furthermore, the ablation of MYC in osteoclasts reverses the enhanced osteoclast differentiation and activity in NRF2 deficiency in vivo and in vitro in addition to protecting NRF2-deficient mice from pathological bone loss in a murine model of inflammatory arthritis. Our findings indicate that this novel NRF2-MYC axis could be instrumental for the fine-tuning of osteoclast formation and provides additional ways in which osteoclasts could be therapeutically targeted to prevent pathological bone erosion.
Project description:The G12 family of G protein alpha subunits has been shown to participate in the regulation of various physiological processes. However, the role of G?12 in bone physiology has not been well described. Here, by micro-CT analysis, we discovered that G?12-knockout mice have an osteopetrotic phenotype. Histological examination showed lower osteoclast number in femoral tissue of G?12-knockout mice compared to wild-type mice. Additionally, in vitro osteoclastic differentiation of precursor cells with receptor activator of nuclear factor-?B ligand (RANKL) showed that G?12 deficiency decreased the number of osteoclast generated and the bone resorption activity. The induction of nuclear factor of activated T-cell c1 (NFATc1), the key transcription factor of osteoclastogenesis, and the activation of RhoA by RANKL was also significantly suppressed by G?12 deficiency. We further found that the RANKL induction of NFATc1 was not dependent on RhoA signalling, while osteoclast precursor migration and bone resorption required RhoA in the G?12-mediated regulation of osteoclasts. Therefore, G?12 plays a role in differentiation through NFATc1 and in cell migration and resorption activity through RhoA during osteoclastogenesis.
Project description:Regulators of G-protein Signaling (Rgs) proteins are the members of a multigene family of GTPase-accelerating proteins (GAP) for the Galpha subunit of heterotrimeric G-proteins. Rgs proteins play critical roles in the regulation of G protein couple receptor (GPCR) signaling in normal physiology and human diseases such as cancer, heart diseases, and inflammation. Rgs12 is the largest protein of the Rgs protein family. Some in vitro studies have demonstrated that Rgs12 plays a critical role in regulating cell differentiation and migration; however its function and mechanism in vivo is largely unknown. Here, we generated a floxed Rgs12 allele (Rgs12(flox/flox) ) in which the exon 2, containing both PDZ and PTB_PID domains of Rgs12, was flanked with two loxp sites. By using the inducible Mx1-cre and Poly I:C system to specifically delete Rgs12 at postnatal 10 days in interferon-responsive cells including monocyte and macrophage cells, we found that Rgs12 mutant mice had growth retardation with the phenotype of increased bone mass. We further found that deletion of Rgs12 reduced osteoclast numbers and had no significant effect on osteoblast formation. Thus, Rgs12(flox/flox) conditional mice provide a valuable tool for in vivo analysis of Rgs12 function and mechanism through time- and cell-specific deletion of Rgs12.
Project description:ASXL2 is an ETP family protein that interacts with PPAR?. We find that ASXL2-/- mice are insulin resistant, lipodystrophic, and fail to respond to a high-fat diet. Consistent with genetic variation at the ASXL2 locus and human bone mineral density, ASXL2-/- mice are also severely osteopetrotic because of failed osteoclast differentiation attended by normal bone formation. ASXL2 regulates the osteoclast via two distinct signaling pathways. It induces osteoclast formation in a PPAR?/c-Fos-dependent manner and is required for RANK ligand- and thiazolidinedione-induced bone resorption independent of PGC-1?. ASXL2 also promotes osteoclast mitochondrial biogenesis in a process mediated by PGC-1? but independent of c-Fos. Thus, ASXL2 is a master regulator of skeletal, lipid, and glucose homeostasis.
Project description:Bone resorption by osteoclasts is necessary to maintain bone homeostasis. Osteoclast differentiation from hematopoietic progenitors and their activation depend on M-CSF and RANKL, but also requires co-stimulatory signals acting through receptors associated with DAP12 and FcRgamma adaptors. Dap12 mutant mice (KDelta75) are osteopetrotic due to inactive osteoclasts but, surprisingly, these mice are more sensitive than WT mice to bone loss following an ovariectomy. Because estrogen withdrawal is known to disturb bone mass, at least in part, through lymphocyte interaction, we looked at the role of mature lymphocytes on osteoclastogenesis and bone mass in the absence of functional DAP12. Lymphocytes were found to stimulate an early osteoclast differentiation response from Dap12-deficient progenitors in vitro. In vivo, Rag1-/- mice lacking mature lymphocytes did not exhibit any bone phenotype, but lost their bone mass after ovariectomy like KDelta75 mice. KDelta75;Rag1-/- double mutant female mice exhibited a more severe osteopetrosis than Dap12-deficient animals but lost their bone mass after ovariectomy, like single mutants. These results suggest that both DAP12 and mature lymphocytes act synergistically to maintain bone mass under physiological conditions, while playing similar but not synergistic co-stimulatory roles in protecting bone loss after gonadal failure. Thus, our data support a role for lymphocytes during osteoclast differentiation and suggest that they may function as accessory cells when regular osteoclast function is compromised.
Project description:Cdc42 mediates bone resorption principally by stimulating osteoclastogenesis. Whether its sister GTPase, Rac, meaningfully impacts upon the osteoclast and, if so, by what means, is unclear. We find that whereas deletion of Rac1 or Rac2 alone has no effect, variable reduction of Rac1 in osteoclastic cells of Rac2(-/-) mice causes severe osteopetrosis. Osteoclasts lacking Rac1 and Rac2 in combination (Rac double-knockout, RacDKO), fail to effectively resorb bone. By contrast, osteoclasts are abundant in RacDKO osteopetrotic mice and, unlike those deficient in Cdc42, express the maturation markers of the cells normally. Hence, the osteopetrotic lesion of RacDKO mice largely reflects impaired function, and not arrested differentiation, of the resorptive polykaryon. The dysfunction of RacDKO osteoclasts represents failed cytoskeleton organization as evidenced by reduced motility of the cells and their inability to spread or generate the key resorptive organelles (i.e. actin rings and ruffled borders), which is accompanied by abnormal Arp3 distribution. The cytoskeleton-organizing capacity of Rac1 is mediated through its 20-amino-acid effector domain. Thus, Rac1 and Rac2 are mutually compensatory. Unlike Cdc42 deficiency, their combined absence does not impact upon differentiation but promotes severe osteopetrosis by dysregulating the osteoclast cytoskeleton.
Project description:Glycogen synthase kinase-3? (GSK-3?) is a serine/threonine kinase originally identified as a regulator of glycogen deposition. Although the role of GSK-3? in osteoblasts is well characterized as a negative regulator of ?-catenin, its effect on osteoclast formation remains largely unidentified. Here, we show that the GSK-3? inactivation upon receptor activator of NF-?B ligand (RANKL) stimulation is crucial for osteoclast differentiation. Regulation of GSK-3? activity in bone marrow macrophages by retroviral expression of the constitutively active GSK-3? (GSK3?-S9A) mutant inhibits RANKL-induced osteoclastogenesis, whereas expression of the catalytically inactive GSK-3? (GSK3?-K85R) or small interfering RNA (siRNA)-mediated GSK-3? silencing enhances osteoclast formation. Pharmacological inhibition of GSK-3? further confirmed the negative role of GSK-3? in osteoclast formation. We also show that overexpression of the GSK3?-S9A mutant in bone marrow macrophages inhibits RANKL-mediated NFATc1 induction and Ca(2+) oscillations. Remarkably, transgenic mice expressing the GSK3?-S9A mutant show an osteopetrotic phenotype due to impaired osteoclast differentiation. Further, osteoclast precursor cells from the transgenic mice show defects in expression and nuclear localization of NFATc1. These findings demonstrate a novel role for GSK-3? in the regulation of bone remodeling through modulation of NFATc1 in RANKL signaling.
Project description:Osteoclasts are multinuclear cells that resorb bone. Osteoclast differentiation is regulated by multiple transcription factors which may be acting in a single or multiple factor complex to regulate gene expression. Myocyte enhancer factor 2 (MEF2) is a family of transcription factors whose role during osteoclast differentiation has not been well characterized. Because MEF2A and MEF2D are the family members most highly expressed during osteoclast differentiation, we created conditional knockout mice models for MEF2A and/or MEF2D. In vitro cultures of A- and D-KO osteoclasts were smaller and less numerous than wild type cultures, while AD-KO osteoclasts were almost completely devoid of TRAP positive mononuclear cells. Female A-KO mice are osteopetrotic while male A- and D-KO mice of either sex had no significant in vivo skeletal phenotype, suggesting a sex-specific regulation of osteoclasts by MEF2A. Lastly, in vivo male AD-KO mice are osteopenic, indicating while MEF2 is required for M-CSF and RANKL-stimulated osteoclastogenesis in vitro, osteoclasts can form in the absence of MEF2 in vivo via a RANKL-alternative pathway.