Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathway

Wu, Mengrui; Chen, Wei; Lu, Yun; Zhu, Guochun; Hao, Liang; Li, Yi-Ping

doi:10.1038/ncomms13700

Download PDF

Article
Open access
Published: 19 January 2017

Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathway

Mengrui Wu¹,
Wei Chen¹,
Yun Lu ORCID: orcid.org/0000-0002-4785-3401¹,
Guochun Zhu¹,
Liang Hao¹ &
…
Yi-Ping Li¹

Nature Communications volume 8, Article number: 13700 (2017) Cite this article

6478 Accesses
92 Citations
2 Altmetric
Metrics details

Subjects

An Author Correction to this article was published on 25 November 2019

Abstract

Many positive signalling pathways of osteoclastogenesis have been characterized, but negative signalling pathways are less well studied. Here we show by microarray and RNAi that guanine nucleotide-binding protein subunit α13 (Gα13) is a negative regulator of osteoclastogenesis. Osteoclast-lineage-specific Gna13 conditional knockout mice have a severe osteoporosis phenotype. Gna13-deficiency triggers a drastic increase in both osteoclast number and activity (hyper-activation), mechanistically through decreased RhoA activity and enhanced Akt/GSK3β/NFATc1 signalling. Consistently, Akt inhibition or RhoA activation rescues hyper-activation of Gna13-deficient osteoclasts, and RhoA inhibition mimics the osteoclast hyperactivation resulting from Gna13-deficiency. Notably, Gα13 gain-of-function inhibits Akt activation and osteoclastogenesis, and protects mice from pathological bone loss in disease models. Collectively, we reveal that Gα13 is a master endogenous negative switch for osteoclastogenesis through regulation of the RhoA/Akt/GSK3β/NFATc1 signalling pathway, and that manipulating Gα13 activity might be a therapeutic strategy for bone diseases.

Sex differences orchestrated by androgens at single-cell resolution

Article 10 April 2024

Human skeletal muscle aging atlas

Article Open access 15 April 2024

Single-cell multiplex chromatin and RNA interactions in ageing human brain

Article Open access 27 March 2024

Introduction

Osteoclasts are the principal, if not exclusive, bone-resorbing cells^1,2,3. A balance between bone formation by osteoblasts and bone resorption by osteoclasts is critical to maintain normal bone density and mineral homeostasis^1,2,3. Osteoclastogenesis is initiated by signals transmitted by the receptor activator of nuclear factor-κB (RANK) ligand (RANKL) and macrophage colony-stimulating factor (M-CSF)^1,2,3,4. RANKL and M-CSF, binding to their receptors, RANK and cFms, respectively, on the surface of osteoclast precursors, activates many key transcription factors including nuclear factor of activated T-cells, c1 (NFATc1) and CCAAT/enhancer-binding protein alpha (C/EBPα)^1,2,3,4. Our previous work on cathepsin K^5,6, Atp6i (refs 7, 8), C/EBPα⁹ and RGS10 (ref. 10) has contributed to the discovery of positive regulatory machinery controlling osteoclast differentiation and function. Notably, excessive osteoclast activity is responsible for various bone diseases including osteoporosis, rheumatoid arthritis and Paget’s disease^1,2,3. Thus, we anticipate that hyperactivation of osteoclasts should be antagonized by intrinsic negative regulators, which have received limited attention, to maintain normal bone homeostasis. A decrease in the expression of negative osteoclast regulators might trigger the bone loss associated with many pathological bone disorders.

Guanine nucleotide-binding protein subunit α13 (Gα13; encoded by Gna13) belongs to the G12 subfamily of the G protein superfamily. Gα13 regulates cell cytoskeleton organization by regulating the RhoGEF-RhoGTPase signalling pathway^11,12. Here we utilize a genome-wide screening strategy and characterize Gα13 as an intrinsic negative regulator of osteoclast formation and activity. Absence of Gα13 favours osteoclast formation and enlarges osteoclast size, leading to an osteopenia phenotype in mice. On the other hand, constitutively active Gα13 blocks the formation of multinucleated osteoclasts and their bone resorptive activity. We reveal that Akt-mediated signalling, regulated by RhoA, is critical for the function of Gα13 in osteoclasts.

Results

Gna13 silencing promotes osteoclastogenesis

Given the limited attention attributed to the investigation of negative regulators of osteoclastogenesis, we performed a genome-wide screening to identify factors that negatively regulate osteoclast formation (Fig. 1). Towards this end, we compared gene expression in human blood monocyte-derived osteoclasts with that in their precursors, and found that Gna13 was not only drastically induced by RANKL, but also its expression pattern was comparable to various osteoclast genes (Fig. 1a), and much higher than the genes encoding several other G proteins (Fig. 1b). Moreover, gene expression analysis confirmed that Gna13 was highly expressed in osteoclasts and osteoclast-like cells derived from MOCP-5, an osteoclast precursor cell line that was generated in our lab¹³, as compared with several tissues, such as heart, kidney, lung and intestine (Fig. 1c). Furthermore, using murine bone marrow monocytes (BMMs), which are widely used as primary osteoclast precursors, we demonstrated that whereas Gα13 expression was only mildly induced by M-CSF (∼2-fold at both mRNA and protein level), its expression was strongly induced by combined stimulation with M-CSF and RANKL (∼6-fold at mRNA level and 8-fold at protein level) (Fig. 1d,e). These results indicated that Gα13 might have important roles in osteoclasts. Hence, we then silenced Gα13 expression by lentiviral-mediated expression of short hairpin RNA against Gna13 in BMMs. The knockdown efficiency (∼90%) was confirmed by immunobloting as compared with control cells (Fig. 1f). Interestingly, our data showed that Gα13 silencing strongly increased osteoclast formation and also drastically enhanced osteoclast size (Fig. 1g,h). These results suggested that Gα13 might be a negative regulator of bone resorption.

**Figure 1: Gα13 which is induced by RANKL and M-CSF inhibits osteoclastogenesis.**

Depletion of Gna13 causes osteopenia in mice

To further investigate the role of Gα13 in osteoclast formation and activity, we generated knock-out mice through specific deletion of Gna13 in the osteoclast lineage (Fig. 2). Mice bearing loxP sites encompassing the Gna13 exon2 (Gna13^f/f mice)¹⁴ were crossed with those expressing Cre recombinase driven by the lysozyme M promoter (LysM-Cre mice) or the Cathepsin K promoter (Ctsk-Cre mice). The offspring were intercrossed to get Gna13^f/fLysM-Cre mice or Gna13^f/fCtsk-Cre mice and wild-type (WT) mice. Mouse genotypes were confirmed by PCR (Supplementary Fig. 1a–c). The deletion of Gα13 in Gna13^f/fLysM-Cre BMMs and Gna13^f/fCtsk-Cre pre-osteoclasts was confirmed by quantitative PCR and immunoblotting (Fig. 2a,b). While LysM-Cre deletes Gna13 in the osteoclast precursors (Fig. 2a,b), Ctsk-Cre works at a late stage of osteoclast differentiation (Supplementary Fig. 1d).

**Figure 2: Osteoclast-specific *Gna13*-CKO mice displayed osteoporosis.**

Two-month-old WT and Gna13^f/fLysM-Cre or Gna13^f/fCtsk-Cre mice have no obvious gross morphological changes. As assessed by X-ray analysis, skeletal mass was decreased in distal femurs of both male and female Gna13^f/fLysM-Cre mice as compared with those of WT littermates (Fig. 2c). Quantitative microtomography (μ-CT) analysis showed that Gna13^f/fLysM-Cre mice exhibited ∼30% reduction in bone volume/tissue volume (BV/TV) and trabecular number (Tb.N) as well as ∼40% increase in trabecular space (Tb.Sp), ∼50% decreased in bone mineral density (BMD) as compared with WT littermates (Fig. 2d,e). Tartrate-resistant acid phosphatase (TRAP) activity was markedly increased in the primary spongiosa of 2-month old Gna13^f/fLysM-Cre mouse femur as compared with that of WT littermates (Fig. 2f–h). Histomorphometric parameters of distal femurs showed that, in the trabecular bone area, Gna13^f/fLysM-Cre mice had ∼3-fold increased in osteoclast numbers per bone surface (N.Oc/BS) as compared with WT littermates (Fig. 2g). Interestingly, osteoclast number was even more drastically increased in the cortical bone area in the mutant mice (Fig. 2f,g). Similarly, X-ray and μ-CT analyses showed that skeletal mass was decreased in distal femurs of 2-month-old Gna13^f/fCtsk-Cre mice (Fig. 2i,j, Supplementary Fig. 2c). TRAP staining showed ∼6-fold increase in osteoclast numbers in femoral sections of Gna13^f/fCtsk-Cre mice as compared with that of WT controls (Fig. 2k, Supplementary Fig. 1d–f). Histomorphometry based on Trichrome staining showed that trabecular bone number was reduced without much change of osteoblast number and osteoblast surface per bone surface in the Gna13^f/fCtsk-Cre mice, confirming that the reduced bone density was not caused by decreased bone formation (Supplementary Fig. 2a,b). In fact, as measured by calcein labelling (Supplementary Fig. 2d), mineral apposition rate was increased by about 35% in Gna13^f/fCtsk-Cre mice. Consistently, serum alkaline phosphatase (ALP) was also increased by about 35% in Gna13^f/fCtsk-Cre mice (Supplementary Fig. 2e). The accelerated bone formation and remodelling might be caused by robust osteoclast formation. These results show that osteoclast-lineage-specific Gna13 deletion promotes osteoclastic bone resorption leading to lower bone density in vivo.

Gna13-depletion hyper-activates osteoclast

Given our data that Gna13 deficiency in vivo led to an increase in osteoclast development (Fig. 2), we examined the effects of using lower doses of RANKL in osteoclastogenesis under Gna13 deficiency. Unlike WT BMMs, Gna13-deficient cells formed osteoclasts when attended by permissive RANKL dose (one tenth of the optimum 10 ng ml⁻¹ RANKL) (Fig. 3a) and formed 25% more osteoclasts at 10 ng ml⁻¹ RANKL (Fig. 3a,b). Further, BMMs deficient in Gna13 generated osteoclast earlier (∼day 3) than WT BMMs (∼day 5) (Supplementary Fig. 3a,b). Gna13 deficiency not only increased the sensitivity of BMMs to RANKL, but also increased the size of osteoclasts (Fig. 3a,b). More osteoclasts with 3–8 nuclei or >13 nuclei were observed in the Gna13-deficient group (increased by 50% and 100%, respectively) (Fig. 3b). Consistently, gene expression analysis revealed that the expression of the osteoclast genes encoding NFATc1, Ctsk, Acp5, Atp6v0d2 and dc-stamp was higher in Gna13^f/fLysM-Cre, but not in WT, BMMs cultured with M-CSF and RANKL for 3 days (Fig. 3c). Importantly, among the aforementioned genes, dc-stamp and Atp6v0d2 are known to have roles in osteoclast fusion^15,16, indicating that Gα13 negatively regulates osteoclast formation by also promoting the fusion of mononucleated osteoclasts into multinucleated osteoclasts.

**Figure 3: Loss of Gα13 promotes osteoclast formation and function.**

To further analyse the role of Gα13 in osteoclastic bone resorption, we performed in vitro bone resorption assays and analysed bone resorption pits by wheat germ agglutinin (WGA) staining and scanning electron microscope (Fig. 3d). Data showed that the total resorption area by Gna13^f/fLysM-Cre osteoclasts was significantly higher than that of WT osteoclasts (Fig. 3e). Collagen I C-terminal telopeptide (CTX-I), released during bone resorption¹⁷, were drastically increased in Gna13^f/fLysM-Cre cell culture medium (Fig. 3f). In addition, WGA-fluorescein isothiocyanate (FITC) staining showed that pits resorbed by Gna13^f/fLysM-Cre osteoclasts are also larger (∼14-fold) and deeper (∼3-fold) than those by WT cells (Fig. 3g,h, Supplementary Fig. 4). Collectively, the results showed that Gα13 negatively regulated osteoclastic bone resorption.

Next, we co-stained the osteoclasts on bone slides with Rhodamine-conjugated-Phalloidin (red), to analyse F-actin ring formation, a critical structure of mature osteoclasts¹⁸, anti-Ctsk antibody (green) and Hoechst (blue) (Fig.3i,j). Data showed that Gna13-deficient osteoclasts exhibited 1.5-fold increased F-actin ring formation (Fig. 3i,k), and the average size of F-actin rings was also about threefold larger in these cells than control cells (Fig. 3j,l).

Furthermore, immunostaining showed a more polarized location of Ctsk in Gna13^f/fLysM-Cre cells (Fig. 3j), indicating a change in Ctsk transportation and secretion. We thus analysed intracellular and medium Ctsk by western blot (Fig. 3m,n). Data showed a slight increase (∼1.5-fold) in intracellular Ctsk level in Gna13^f/fLysM-Cre osteoclasts (Fig. 3m). However, secreted Ctsk concentration in the culture medium by Gna13^f/fLysM-Cre osteoclasts was drastically elevated (∼4-fold) (Fig. 3n). These results demonstrate that Gna13-deficient osteoclasts had enlarged actin ring with increased Ctsk secretion, both of which would contribute to enhanced bone resorption besides enhanced osteoclast formation.

We performed TUNEL staining on day-6 osteoclasts (Supplementary Fig. 3c,e) and cytokine/serum-starved day-5 osteoclasts (Supplementary Fig. 3d,f), and similar apoptosis nuclei ratio are observed in the WT and mutant cells. Similar survival rate was observed in the WT and Gna13^f/fLysM-Cre cells when the osteoclasts were extensively cultured for 7 days (Supplementary Fig. 3h,i). Data indicated that Gna13-deficiency might not influence osteoclast apoptosis. Further, acridine Orange staining showed that acidification is similar between WT and Gna13^f/fLysM-Cre osteoclasts (Supplementary Fig. 3g).

Gα13-RhoA brakes Akt-GSK3β-NFATc1 signalling

RANKL and M-CSF orchestrate multiple signalling pathways to promote osteoclast formation^1,2,3. Expression of RANKL receptor (RANK) and M-CSF receptor (cFms) were similar in WT and Gna13-deficient cells (Supplementary Fig. 3j,k). To analyse signal transduction in response to RANKL or M-CSF stimulation (including Akt, p38, JNK and Erk activation), BMMs were starved for 5 h and then stimulated with RANKL or M-CSF for 5–60 min (Fig. 4a,b). Akt phosphorylation was enhanced in Gna13^f/fLysM-Cre cells by either RANKL or M-CSF as compared with WT BMMs (Fig. 4a,b). Nonetheless, activation of the p38, JNK and Erk signalling pathways were similar between WT and Gna13^f/fLysM-Cre cells (Supplementary Fig. 5).

**Figure 4: Knockout of *Gna13* enhances Akt-GSK3β-Nfatc1 signalling through RhoA.**

Given the drastic increase in Akt activation, we then applied the Akt inhibitor MK2206 2HCl to attenuate Akt activity, which inhibited osteoclastogenesis in a dose-dependent manner (Fig. 4c–e). Notably, Gna13-deficient cells were more resistant to the Akt inhibitory effect on osteoclastogenesis. In the presence of 0.1∼0.2 μM MK2206 2HCl (Fig. 4d,e), Gna13-deficient cells formed osteoclasts at normal level comparatively to WT cells. The results indicate that Gna13 deficiency promotes osteoclastogenesis by increasing Akt activity.

Studies have demonstrated that Gα13 activates RhoA through RhoGEF (p115RhoGEF, LARG et al.)^11,12, and RhoA can inhibit Akt activity¹⁹. Consistently, RhoA activity was dramatically downregulated in Gna13-deficient cells (Fig. 4f). In all, 250 ng ml⁻¹ Rho activator II corrected the hyper-activation of Gna13-deficient osteoclasts (Fig. 4g–i). In addition, Rho activator II could attenuate Akt activation in osteoclasts (Fig. 4m). On the other hand, in the presence of 6–12 ng ml⁻¹ Rho inhibitor, cell-permeable C3 toxin, osteoclast differentiation was enhanced (Fig. 4j–l) and Akt activation was also increased (Fig. 4n). Thus, Gα13 regulated Akt activity through RhoA so as to promote osteoclastogenesis.

It has been established that the PI3K/Akt-GSK3β-NFATc1 signalling cascade is critical for osteoclast differentiation^20,21. Specifically, Akt can inhibit GSK3β, which can promote NFATc1 translocation from the nucleus into the cytoplasm and thereby abrogate osteoclast differentiation. Thus, we characterized GSK3β phosphorylation level and NFATc1 nuclei translocation in WT and Gna13^f/fLysM-Cre cells. We found that GSK3α/β phosphorylation was hyper-activated in Gna13^f/fLysM-Cre monocyte/macrophage in response to RANKL and M-CSF (Fig. 4o). NFATc1 expression increased during osteoclastogenesis in WT and Gna13^f/fLysM-Cre cells, and more quickly in Gna13^f/fLysM-Cre cells (Fig. 4p). Consistently, while similar amounts of NFATc1 were observed in the cytoplasm, much more NFATc1 was observed in the Gna13^f/fLysM-Cre nucleus than WT (Fig. 4q). The results demonstrated that Gα13 mediates Akt-GSK3β-NFATc1 signalling to promote osteoclast gene expression.

Gα13CA inhibits osteoclastogenesis

To confirm the inhibitory effect of Gα13 on osteoclastogenesis, we overexpressed a constitutively active form of Gα13 (Gα13CA) in BMMs and RAW264.7 cells (Fig. 5). Moreover, we overexpressed Gα13CA in WT and Gna13^f/fLysM-Cre pre-osteoclasts using lentivirus²². A successful infection (>90% infection) was assessed by green fluorescent protein expression in control groups (Supplementary Fig. 6a,c), and Gα13CA overexpression was confirmed by western blot (Supplementary Fig. 6b,d,e). Importantly, Gα13CA overexpression drastically blocked osteoclast fusion, inhibited bone resorption and impaired F-actin ring formation in WT and Gna13f/f;LysM-Cre cells (Fig. 5a–f). Additionally, the secretion of Ctsk was decreased after Gα13CA overexpression in WT and Gna13-deficient osteoclasts (Fig. 5g,h). Ctsk and NFATc1 protein levels were also decreased after Gα13CA overexpression (Fig. 5i,j). Furthermore, Gα13CA overexpression attenuated Akt activation in Gna13^f/fLysM-Cre cells, but did not affect the activation of p38 and ERK (Fig. 5k,l; Supplementary Fig. 7a). Gα13CA overexpression in WT BMMs also drastically inhibited osteoclastogenesis (Fig. 5m left panels; 5n). Interestingly, overexpression of a constitutively active form of Akt (AktCA) could partially rescue the inhibitory effects of Gα13CA (Fig. 5m right panels; 5n). These results demonstrate that Gα13CA inhibited osteoclastogenesis by inhibiting Akt activation in BMMs.

**Figure 5: Gα13 gain-of-function inhibits osteoclast formation and function *in vitro*.**

Furthermore, we infected RAW264.7 cells with the same retrovirus we used in BMMs in Fig. 5m,o. Similarly, Gα13CA overexpression inhibited osteoclast-like cell formation (Fig. 5o left panels; 5p), which could be rescued by AktCA overexpression (Fig. 5o right panels; 5p). Akt activation was greatly decreased in RAW264.7 cells overexpressing Gα13CA (Fig. 5q,r), while the activation of p38 and Erk remains unchanged (Supplementary Fig. 7b). The overexpression of Gα13CA and AktCA was confirmed by western blot (Fig. 5s,t). The results indicated that Gα13CA also inhibited osteoclast-like cells formation from the RAW264.7 cells by inhibiting Akt phosphorylation.

Gα13CA protects mice from pathological bone loss

To investigate Gα13 clinical implication potential, we overexpressed Gα13CA locally in mice using adeno-associated virus (AAV) gene expression to investigate its effects in protecting against bone degradation. AAV vectors have advantages compared with many other viral and non-viral based gene delivery platforms in both function and safety for correcting genetic-based diseases²³. To test the use of AAV-Gα13CA, we utilized three disease models of bone loss: the human tumour-necrosis factorα (hTNFα)-transgene expressing mouse model of autoimmune arthritis²⁴ (named TNFα-RA) (Fig. 6), ovariectomized (OVX) animal model of osteoporosis, (Fig. 7a–d) and calvaria-adjacent lipopolysaccharide (LPS)-injection mouse model of osteolysis (Fig. 7e,f)²⁵. Successful infection of AAV in vivo was monitored by yellow fluorescent protein (YFP) expression (Supplementary Figs 8 and 9a). Successful overexpression of G13CA in vivo was confirmed by immunofluorescence staining (Supplementary Fig. 9b).

**Figure 6: Gα13 protects Rhematoid arthritis mice from inflammatory bone loss.**

**Figure 7: Gα13 protects mice from OVX- and LPS-induced bone loss.**

TNFα-RA mice were accompanied by excessive osteoclast formation and bone destruction (Fig. 6). Local administration of AAV-Gα13CA to ankles greatly reduced bone loss, as assessed by X-ray in the TNFα-RA mice as compared with those injected with AAV-YFP (Fig. 6c,d). Not surprisingly, osteoclast number was dramatically decreased in TNFα-RA mice with local injection of AAV-Gα13CA to ankles (Fig. 6g,h). Unexpectedly, local administration of AAV-Gα13CA also relieved ankle swelling (Fig. 6a,b), cartilage destruction (Fig. 6e lower panels, Fig. 6f) and inflammation cell infiltration (Fig. 6e upper and middle panels, Fig. 6f) in TNFα-RA mice. Hence, reduced bone loss and decreased osteoclast number might be a combined effect of Gα13CA and inhibited inflammation.

OVX-induced bone loss and osteoclast number increased due to estrogen depletion (Fig. 7a–d, sham+PBS group compared with OVX+YFP group). OVX mice were subjected to calvaria-adjacent subcutaneous injection of AAV-YFP or AAV-Gα13CA. As assessed by X-ray and u-CT, application of AAV-Gα13CA reduced OVX-induced bone loss (Fig. 7a,b). As assessed by TRAP staining of whole calvarial and calvarial sections, AAV-Gα13CA dramatically reduced OVX-induced osteoclast number increase (Fig. 7c,d) as compared with YFP group.

In the LPS-injection model, both WT and Gna13^f/fLysM-Cre mice were subjected to calvaria-adjacent subcutaneous injection of LPS or PBS control (Fig. 7e,f). Compared with local injection of PBS, LPS induced a dramatic increase in osteoclast number and bone resorption (Fig. 7e,f). The absence of Gα13 further promoted the induction of osteoclastogenesis and bone loss in both PBS and LPS treated mice (Fig. 7e,f). Local administration of AAV-Gα13CA to calvaria had a marked therapeutic effect on osteoclast formation and bone destruction by LPS, compared with local injection of AAV-YFP control (Fig. 7e,f). These results confirm that it is possible to overexpress Gα13CA in vivo to target pathologic bone loss.

Full images of western blots are presented in Supplementary Fig. 10.

Discussion

We proposed a model that Gα13-RhoA antagonizes osteoclast formation and activity by attenuating the Akt-GSK3β-NFATc1 signalling axis (Fig. 8). Briefly, as a downstream of RANK and c-Fms, Akt phosphorylates and inactivates GSK3β, which has a role in NFATc1 nuclei exportation, so as to promote osteoclast differentiation; Meanwhile, Gα13 activates RhoA which in turn inhibits Akt phosphorylation and activity, so as to antagonize the over-activation of osteoclast (Fig. 8). The critical role of PI3K-Akt activity in osteoclast differentiation and activation is well-documented^{20,21,26,27,28,29}. Although several endogenous factors that oppose osteoclast differentiation were described, most are downregulated under osteoclastogenic condition. In contrast, Gα13 is induced by the combined stimulation of RANKL and M-CSF, and in turn inhibit osteoclast differentiation so as to avoid over-activated osteoclast and excessive bone loss. Hence, our data reveal that Gα13 is an intrinsic control mechanism during osteoclastogenesis. In addition, we comprehensively study and report a signalling cascade (Gα13-RhoA-Akt-GSK3β-NFATc1) that negatively regulates osteoclastogenesis and osteoclast function. Most importantly, our results from both loss-of-function and gain-of-function strategies proved that Gα13 is a key negative regulator and main switch in osteoclastogenesis.

**Figure 8: Working model of Gα13 as a negative regulator in osteoclast.**

It was reported that RhoA activity is essential for podosome formation and osteoclastic bone resorption³⁰. On the other hand, it was also reported that increased RhoA activity destabilizes the sealing zone in osteoclasts, and inhibition of RhoA stabilizes the sealing zone correlating with acetylation and stabilization of the microtubule network in these cells^31,32,33,34. Our study showed that inhibition of RhoA activity through Gα13 knockout, RhoA inhibitor and RNA interference favours osteoclastogenesis, while RhoA activator inhibits osteoclastogenesis. Hence, these studies suggest that RhoA is the downstream effector of Gα13 to negatively regulate osteoclastogenesis. Our finding is underscored by genetic evidences that have suggested an important role for the RhoGEF-RhoGTPase (for example, Arhgef3-RhoA) pathway in osteoporosis^35,36,37,38. Genome-wide linkage studies have identified chromosome region 3p14-p21, in which RHOA gene was located, as a quantitative trait locus for BMD^35,36. One RHOA single-nucleotide polymorphism (SNP) rs17595772, and one ARHGEF3 SNP were reported to be significantly associated with decreased BMD) in postmenopausal women^37,38. However, the mechanisms underlying the correlation between RhoGEF-RhoGTPase pathway and BMD remain to be explored.

Current anti-resorptive agents (for example, bisphosphonates and denosumab) are effective but far from ideal. The major problem of bisphosphonates is their permanent deleterious affect on normal bone remodelling. Agents preventing bone resorption by inhibiting late differentiation of osteoclasts without affecting normal bone remodelling are more desired, which are unlikely to interfere with the coupling of osteoblastic bone formation to osteoclastic bone resorption, critical for maintaining normal bone homeostasis³⁹. Here we showed that overexpression of Gα13CA markely inhibits osteoclast formation and bone resorption both in vitro and in vivo. Importantly, although multi-nuclear cell formation was blocked, Gα13CA over-expressing monocytes were TRAP⁺ stained (Fig. 5a), indicating that Gα13CA regulated osteoclast differentiation at a late stage and could be a good target for bone loss without affecting normal bone remodelling. Interestingly, Gα13 might have dual functions in inflammatory disease models (TNFα-RA and LPS-injection models), by concurrently inhibiting osteoclast differentiation and inflammation so as to achieve the ultimate outcome of bone loss protection. Hence, activating Gα13, an endogenous osteoclast inhibitor, may serve as a novel therapeutic approach targeting bone loss in various bone pathological states.

Methods

Mice

Gna13^flox/flox (Gna13^f/f) mice were provided by Dr S. Offermanns (Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany)¹⁴. LysM-Cre transgenic mice were purchased from the Jackson laboratory. Ctsk-Cre knock-in mice were provided by Dr S. Kato (University of Tokyo, Tokyo, Japan)⁴⁰. To obtain mice with Gna13-deficient macrophages, Gna13^f/+LysM-Cre males were bred with Gna13^f/+LysM-Cre females. To obtain mice with Gna13-deficient osteoclasts, Gna13^f/+Ctsk-Cre males were bred with Gna13^f/f females. In all experiments, Gna13^f/+LysM-Cre or Gna13^f/+Ctsk-Cre mice were compared with their WT littermates (Gna13^f/+, Gna13^f/f, Gna13^+/+LysM-Cre or Ctsk-Cre mice). All mice were on a pure C57BL/6 background. All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at University of Alabama at Birmingham (UAB), Birmingham, USA.

GeneChip analysis

Human peripheral blood mononuclear cells were cultured for 7 days with human RANKL and human M-CSF. GeneChip data were analysed using Affymetrics scanner and accompanying gene expression software.

Radiographic analysis.

For X-ray analysis, excised 2-month-old mouse femurs were scanned using a high-resolution soft X-ray system (Faxitron Model MX-20) at 30 kV. For Micro-Computed Tomography (μ-CT) analysis, excised 2-month-old mouse femurs were scanned using the Scanco CT40 desktop cone-beam μ-CT scanner (Scanco Medical AG, Brüttisellen, Switzerland). The scan of the trabecular bone was performed from the growth plate, and consisted of 310 slices (12 μm per slice) and 200 slices were analysed using the CT Evaluation Program (v5.0A, Scanco Medical). The scan and analysis of the cortical bone was performed at the midshaft of the femur and consisted of 25 slices (12 μm per slice).

Histology

Histology was performed as previously described^40,41. Briefly, the femurs of 2-month-old mice were fixed with 70% ethanol followed by plastic embedding and Golden trichrome staining, or with 10% neutral buffered formalin followed by decalcification in 10% EDTA for 2 weeks, paraffin embedding and TRAP staining using Phosphatase, Leukocyte Acid (TRAP) kit (Sigma-Aldrich, 387A-1KT). Osteoclastic and osteoblastic perimeters were measured and analysed using Osteomeasure in a blinded fashion.

In-vivo calcein labelling

Calcein labelling was previously described⁴¹. Briefly, 2-month-old mice were intraperitoneally injected with 20 mg kg⁻¹ of calcein in a 2% sodium bicarbonate solution, 8 days and 2 days before killing of mice. Calvarias were fixed in 4% PFA, soaked in 30% glucose in PB, embedded in OCT and frozen sectioned. Mineral apposition rate is the distance between the midpoints of the two labels divided by the time between the midpoints of the interval.

Serum ALP assay

Two-month old mouse serum was collected after 6-hour fasting, and the serum ALP activity was detected and quantified using Alkaline Phosphatase Assay Kit (Colorimetric) (ab83369) according to the manufacturer’s instructions.

In vitro osteoclast differentiation assay

Mature osteoclasts were generated as described¹⁰. Briefly, isolated BMMs from C57BL/6 mice were cultured in MEM α (pH 6.9) containing 10% FBS, 10 ng ml⁻¹ recombinant RANKL and 10 ng ml⁻¹ recombinant M-CSF for 5 days. Recombinant murine M-CSF and RANKL were obtained from R&D systems, Inc. Cell culture medium was obtained from Gibco, Life Technologies Corporation. Mature osteoclasts were characterized by staining for TRAP activity using a commercial kit (Sigma-Aldrich, 387A-1KT) and TRAP⁺ MNCs were enumerated per well in a 24-well plate.

In vitro bone resorption assay

For bone resorption assay, osteoclasts were seeded on bovine bone slides. Concentration of bovine cross-linked C-telopeptide of type I collagen (CTX-1) in the medium was measured using CrossLaps for Culture ELISA (CTX-I) kit (Immunodiagnostic Systems Limited) following the manufacturer’s instructions. Bone resorption pits were sonicated in PBS, stained with 2 ug ml⁻¹ WGA-lectin (Sigma-Aldrich, L-3892) for two hours and then using DAB peroxidase (horseradish peroxidase) Substrate Kit (Vector laboratories, SK-4100) (ref. 42). To analyse bone resorption pits depth and volume, bone slides were sonicated in PBS, stained with 2 ug ml⁻¹ FITC-WGA (Sigma-Aldrich, L-4895) staining for 2 h and visualized by confocal microscopy⁴³.

Cellular immunofluorescent staining

For F-actin ring staining, osteoclasts on bovine bone slides were incubated with 2 U ml⁻¹ Texas Red-X Phalloidin (Life Technologies, T7471). For anti-Ctsk immunofluorescence stain, osteoclasts on bovine bone slides were fixed by 4% formaldehyde, permeabilized with 0.2% Triton X-100, blocked with 5% goat serum and 100 μg ml⁻¹ unconjugated AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs), incubated with mouse anti-mouse Ctsk primary antibody (1:100, Santa Cruz sc-48353) and then with FITC goat anti-mouse IgG (H+L) secondary antibody. Nuclei were visualized with 1 μg ml⁻¹ DAPI (4’,6-diamidino-2-phenylindole; Sigma-Aldrich, D9542). Three-dimensional images were taken by confocal microscopy and constructed by Imaris software⁴⁴.

Inhibitors and activators treatment.

MK-2206 2HCl was purchased from Selleckchem Inc. (Cat# S1078). Rho Inhibitor I (cell-permeable exoenzyme C3 transferase, or cell-permeable C3 toxin) was purchased from Cytoskeleton Inc. (Cat# CT04). Rho activator II was purchased from Cytoskeleton Inc. (Cat# CN03). Stock solutions were prepared according to the manufacturer’s instructions. During differentiation from BMMs to mature osteoclasts, the cells were treated continuously by those inhibitors or activators at indicated doses.

Quantitative reverse transcription–PCR (qPCR) assay

Total RNA was isolated from cultured cells with TRIzol reagent (Life Technologies, 15596018). Mouse complementary DNA (cDNA) was reverse-transcribed from 0.5 μg total RNA with SuperScript VILO Master Mix (Life Technologies, 11755050). Quantitative reverse transcription–PCR performed on StepOne Real-Time PCR System (Life Technologies, Applied Biosystems) using the TaqMan Gene Expression assays (Life Technologies, Applied Biosystems) or SYBR Green reagents (Life Technologies, 4385610). Taqman probe numbers and Primer sequences are as followings: Ctsk (Mm00484039_m1); Acp5 (Mm00475698_m1), Atp6i (Mm00469394_m1), Nfatc1 (Mm00479445_m1), Hprt1 (Mm00446968_m1), Hprt1 (5′-GGTGGAGATGATCTCTCAACTTTAA-3′, 5′-AGGAAAGCAAAGTCTGCATTGTT-3′), Atp6v0d2 (5′-GAAGCTGTCAACATTGCAGA-3′, 5′-TCACCGTGATCCTTGCAGAAT-3′), DC-stamp (5′-GGGGACTTATGTGTTTCCACG-3′, 5′-ACAAAGCAACAGACTCCCAAAT-3′), Gna13 (5′-TGCTGGTAGATGCCCGAGA-3′, 5′-GATCGTAGGCATTCTGTATACCA-3′), β-actin (5′-TGACGTTGACATCCGTAAAGAC-3′, 5′-TGCTAGGAGCCAGAGCAGTAA-3′).

Western blotting and phosphorylation assay

Cell lysates preparation and SDS–polyacrylamide gel electrophoresis /western blotting analysis were carried out according to a standard protocol^10,45. For the phosphorylation assay, BMMs and pre-osteoclasts (derived from BMMs with 2-day treatment of RANKL and M-CSF) were starved for 5 h and stimulated with RANKL or M-CSF for different times. Proteins were harvested in cell lysis buffer supplemented with proteinase inhibitor cocktail (Sigma-Aldrich Co. LLC, 1:100) and phosphatase inhibitor cocktail 2 (P-5726, Sigma-Aldrich Co. LLC, 1:100). To extract nuclear and cytoplasmic proteins, NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo Fisher Scientific Inc) was used. Western blot was performed using following antibodies:, anti-p84 mouse IgG (GeneTex GTX70220, 1:1,000), anti-IκBα rabbit IgG (Cell Signaling Technology 4812, 1:1,000), anti-phospho-IκBα (Ser32) rabbit IgG (Cell Signaling Technology 2859, 1:1,000), anti-p38 rabbit IgG (Cell Signaling Technology 9212, 1:1,000), anti-phospho-p38 (Thr180/Tyr182) rabbit IgG (Cell Signaling Technology 4631, 1:1,000), anti-Erk rabbit IgG (Cell Signaling Technology 4370, 1:1,000), anti-phospho-Erk (Thr202/Tyr204) rabbit IgG (Cell Signaling Technology 4695, 1:1,000), anti-Akt rabbit IgG (Cell Signaling Technology 4685, 1:1,000), anti-phospho-Akt (Thr308) rabbit IgG (Cell Signaling Technology 2965, 1:1,000), anti-JNK rabbit IgG (Cell Signaling Technology 9251, 1:1,000), anti-phospho-JNK (Thr183/Tyr185) rabbit IgG (Cell Signaling Technology 9258, 1:1,000), anti-c-Jun rabbit IgG (Cell Signaling Technology 9165), anti-phospho-c-Jun (Ser73) rabbit IgG (Cell Signaling Technology 9164, 1:1,000), anti- GSK3β rabbit IgG (Cell Signaling Technology 9315, 1:1,000), anti-phospho-GSK3α/β (Ser21/9) rabbit IgG (Cell Signaling Technology 9331, 1:1,000), anti-Gα13 rabbit IgG(Santa Cruz sc-410, 1:500), anti-Ctsk mouse IgG (Santa Cruz sc-48353, 1:500), anti-NFATc1 mouse IgG (Santa Cruz sc-7294, 1:500), anti-GAPDH mouse IgG (Santa Cruz sc-166574, 1:500), anti-β-Actin mouse monoclonal IgG (Santa Cruz sc-81178, 1:500), Goat anti-rabbit IgG-HRP (Santa Cruz sc-200416, 1:2,500), Rabbit anti-mouse IgG-HRP (Santa Cruz sc358917, 1:2,500). Full images of western blots are presented in Supplementary Fig. 10.

RhoA activation assay

Pre-osteoclasts (derived from BMMs with 2-day treatment of RANKL and M-CSF) were starved for 4 h and stimulated by RANKL and M-CSF for different times. Cell lysates was harvested and RhoA-GTP activity was analysed using commercial kits (Rho Activation Assay Biochem Kit, Cytoskeleton, Inc.) according to the manufacturer’s instructions.

Constructs and virus production and infection

TRC lentiviral mouse Gna13 targeted short hairpin RNA (Clone ID: TRCN0000098146, target sequence 5′-CGCGATCAACACAGAGAACAT-3′), TRC Lentiviral pLKO.1 Empty Vector and mouse Gna13 full-length cDNA were purchased from Open Biosystems, Thermo Fisher Scientific Inc. Lentiviral pLB vector and packaging plasmids (pCMV-VSV-G and pCMV-Dr8.92) were purchased from Addgene. pHR-EF-IRES-Bla (referred to as pHR in the following text) was a gift from Dr Ling Tian in Dr W. Timothy Garvey’s lab and used for lentivirus-mediated overexpression²². Gα13 constitutively active form (Gα13 Q226L, referred to as Gα13CA in the text) full-length cDNA was purchased from Missouri cDNA Resource Center. Akt constitutively active form cDNA was purchased from addgene. Gα13CA was ligated into pHR-EF-IRES-Bla at the BamHI and XhoI sites and substituted the green fluorescent protein fragments, to construct pHR.Gα13CA vector. Retrovirus vector pMXs-ires-puro was a gift from Dr Xu Feng. A 3xFLAG sequence was inserted into pMXs-ires-puro to construct pMXs-3xFLAG-ires-puro vector. Gα13CA was ligated into pMXs-3xFLAG-ires-puro at the EcoRI and NotI sites to construct pMXs-3xFLAG-G13CA-ires-puro vector (referred to as pMXs-Gα13CA). AktCA was ligated into pMXs-ires-puro vector to construct pMXs-AktCA vector. For lentivirus production, lentivirus vectors (with 10% pLB vector or 10% pHR vector) and packaging plasmids were co-transfected into HEK-293T cells using a calcium phosphate co-precipitation method. The lentiviral supernatant was harvested 48–72 h post-transfection. For retrovirus production, pMXs vectors was transfected into 293GPG cells (gift from Dr Xu Feng) using a calcium phosphate co-precipitation method and retrovirus supernatant were harvested between 48–96 h (ref. 46). Gα13CA was ligated into pAAV-CMV vector from Invitrogen at EcoRI and SalI sites to produce pAAV-CMV-G13CA vector (referred to as pAAV-G13CA). Lentivirus and retrovirus titers were determined by transfecting HEK293T cells with serial dilutions of virus supernatant. AAV was produced and tittered according to the manufacturer’s instructions (Life Technology, Inc). BMMs or RAW264.7 cells were transduced with virus supernatant in the presence of 8 μg ml⁻¹ polybrene (Sigma) for 24 h before induced with RANKL.

OVX-induced bone destruction and AAV-G13CA treatment

OVX or sham was performed on two-month-old female mice. One week later and two weeks later those mice were administered with a local calvarial injection of 30 ul AAV (titer=10^9-10 ml⁻¹) expressing YFP or G13CA. Mice were harvested 5 weeks after OVX operation and fixed in 4% PFA. Calvaria bone were analysed by X-ray, u-CT and whole-mount TRAP staining. Calvarial were also decalcified for 3 days, immersed in 30% sucrose overnight and then submitted to frozen section and TRAP staining and immunofluorescent staining.

Immunofluorescent staining

Frozen sections were fixed in 4% PFA for half an hour and heat-induced antigen retrieval was performed (abcam, ab973). Sections were incubated with mouse IgG blocking solution and universal blocking solution, and then overnight with primary antibodies: anti-YFP rabbit IgG (Santa Cruz sc-410, 1:100) or anti-Gα13 rabbit IgG (Santa Cruz sc-410, 1:100) and anti-Ctsk mouse IgG (Santa Cruz sc-48353, 1:200). Ctsk was then labelled by biotin-anti-mouse IgG (Vector laboratories, BMK-2202) and streptavidin-FITC (sigma). YFP and Gα13 were labelled by TR-goat-anti-rabbit IgG.

LPS-induced bone destruction and AAV-G13CA treatment

Seven-week-old female mice were administered with a local calvarial injection of AAV expressing YFP or G13CA. Six days later those mice were administered with a local calvarial injection of LPS (sigma) at 25 mg kg⁻¹ body weight or PBS and analysed after 5 days following a previously described model²⁵. For analysis, calvarial with skin tissue were fixed in 4% PFA for 6 h at 4 °C, decalcified for 3 days, immersed in 30% sucrose overnight and then submitted to frozen section and TRAP staining.

TNFα-induced rheumatoid arthritis and AAV-Gα13CA treatment

13-week old WT and human-TNFα-transgenic (hTNFtg) mice was injected with AAV (titer=10^9-10 ml⁻¹) expressing YFP or G13CA over ankles. The injection was performed four times every week successively. Photographic and radiographic analysis were performed before and 35 days after the first injection. Samples were harvested 35 days after the first injection, and Safranin O (SO) stain, H&E stain and TRAP stain were performed. Hind paw volume was quantified by water displacement method⁴⁷. Bone destruction in X-ray was quantified by Larsen method in RA lesion area⁴⁸. Cartilage destruction was assessed using safranin O staining and was measured as OARSI grade in ankle joint area⁴⁹.

Statistics

Data represent mean±s.d. Analysis was performed using GraphPad Prism, version 5. Statistical significance was assessed using a two-tailed Student’s t-test or ANOVA analysis, as indicated in the figure legend, considering a P value≤0.05 as significant.

Data availability

The authors declare that the data supporting the findings of this study are available within the article and its Supplementary Information files.

Additional information

How to cite this article: Wu, M. et al. Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathway. Nat. Commun. 8, 13700 doi: 10.1038/ncomms13700 (2017).

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

Takayanagi, H. New immune connections in osteoclast formation. Ann. N. Y. Acad. Sci. 1192, 117–123 (2010).
Article ADS CAS Google Scholar
Boyle, W. J., Simonet, W. S. & Lacey, D. L. Osteoclast differentiation and activation. Nature 423, 337–342 (2003).
Article ADS CAS Google Scholar
Teitelbaum, S. L. & Ross, F. P. Genetic regulation of osteoclast development and function. Nat. Rev. 4, 638–649 (2003).
Article CAS Google Scholar
Chen, W. et al. C/EBPalpha regulates osteoclast lineage commitment. Proc. Natl Acad. Sci. USA 110, 7294–7299 (2013).
Article ADS CAS Google Scholar
Chen, W. et al. Novel pycnodysostosis mouse model uncovers cathepsin K function as a potential regulator of osteoclast apoptosis and senescence. Hum. Mol. Genet. 16, 410–423 (2007).
Article CAS Google Scholar
Li, Y. P. et al. Cloning and complete coding sequence of a novel human cathepsin expressed in giant cells of osteoclastomas. J. Bone Miner. Res. 10, 1197–1202 (1995).
Article CAS Google Scholar
Li, Y. P., Chen, W. & Stashenko, P. Molecular cloning and characterization of a putative novel human osteoclast-specific 116-kDa vacuolar proton pump subunit. Biochem. Biophys. Res. Commun. 218, 813–821 (1996).
Article CAS Google Scholar
Li, Y. P., Chen, W., Liang, Y., Li, E. & Stashenko, P. Atp6i-deficient mice exhibit severe osteopetrosis due to loss of osteoclast-mediated extracellular acidification. Nat. Genet. 23, 447–451 (1999).
Article CAS Google Scholar
Chen, W. et al. C/EBPα regulates osteoclast lineage commitment. Proc. Natl Acad. Sci. 110, 7294–7299 (2013).
Article ADS CAS Google Scholar
Yang, S. & Li, Y. P. RGS10-null mutation impairs osteoclast differentiation resulting from the loss of [Ca2+]i oscillation regulation. Genes Dev. 21, 1803–1816 (2007).
Article CAS Google Scholar
Siehler, S. Regulation of RhoGEF proteins by G12/13-coupled receptors. Br. J. Pharmacol. 158, 41–49 (2009).
Article CAS Google Scholar
Worzfeld, T., Wettschureck, N. & Offermanns, S. G(12)/G(13)-mediated signalling in mammalian physiology and disease. Trends Pharmacol. Sci. 29, 582–589 (2008).
Article CAS Google Scholar
Chen, W. & Li, Y. P. Generation of mouse osteoclastogenic cell lines immortalized with SV40 large T antigen. J. Bone Miner. Res. 13, 1112–1123 (1998).
Article CAS Google Scholar
Moers, A. et al. G13 is an essential mediator of platelet activation in hemostasis and thrombosis. Nat. Med. 9, 1418–1422 (2003).
Article CAS Google Scholar
Yagi, M. et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J. Exp. Med. 202, 345–351 (2005).
Article CAS Google Scholar
Lee, S. H. et al. v-ATPase V0 subunit d2-deficient mice exhibit impaired osteoclast fusion and increased bone formation. Nat. Med. 12, 1403–1409 (2006).
Article CAS Google Scholar
Zhuo, Y., Gauthier, J. Y., Black, W. C., Percival, M. D. & Duong, L. T. Inhibition of bone resorption by the cathepsin K inhibitor odanacatib is fully reversible. Bone 67, 269–280 (2014).
Article CAS Google Scholar
Kanehisa, J. et al. A band of F-actin containing podosomes is involved in bone resorption by osteoclasts. Bone 11, 287–293 (1990).
Article CAS Google Scholar
Muppidi, J. R. et al. Loss of signalling via G[agr]13 in germinal centre B-cell-derived lymphoma. Nature 516, 254–258 (2014).
Article ADS CAS Google Scholar
Moon, J. B. et al. Akt induces osteoclast differentiation through regulating the GSK3beta/NFATc1 signaling cascade. J. Immunol. 188, 163–169 (2012).
Article CAS Google Scholar
Jang, H. D., Noh, J. Y., Shin, J. H., Lin, J. J. & Lee, S. Y. PTEN regulation by the Akt/GSK-3beta axis during RANKL signaling. Bone 55, 126–131 (2013).
Article CAS Google Scholar
Zhang, W. et al. TRIB3 mediates glucose-induced insulin resistance via a mechanism that requires the hexosamine biosynthetic pathway. Diabetes 62, 4192–4200 (2013).
Article CAS Google Scholar
Daya, S. & Berns, K. I. Gene therapy using adeno-associated virus vectors. Clin. Microbiol. Rev. 21, 583–593 (2008).
Article CAS Google Scholar
Keffer, J. et al. Transgenic mice expressing human tumour necrosis factor: a predictive genetic model of arthritis. EMBO J. 10, 4025–4031 (1991).
Article CAS Google Scholar
Chiang, C. Y., Kyritsis, G., Graves, D. T. & Amar, S. Interleukin-1 and tumor necrosis factor activities partially account for calvarial bone resorption induced by local injection of lipopolysaccharide. Infect. Immun. 67, 4231–4236 (1999).
CAS PubMed PubMed Central Google Scholar
Pilkington, M. F., Sims, S. M. & Dixon, S. J. Wortmannin inhibits spreading and chemotaxis of rat osteoclasts in vitro. J. Bone Miner. Res. 13, 688–694 (1998).
Article CAS Google Scholar
Nakamura, I. et al. Phosphatidylinositol-3 kinase is involved in ruffled border formation in osteoclasts. J. Cell. Physiol. 172, 230–239 (1997).
Article CAS Google Scholar
Shinohara, M. et al. Class IA phosphatidylinositol 3-kinase regulates osteoclastic bone resorption through protein kinase B-mediated vesicle transport. J. Bone Miner. Res. 27, 2464–2475 (2012).
Article CAS Google Scholar
Takeshita, S. et al. SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts. Nat. Med. 8, 943–949 (2002).
Article CAS Google Scholar
Chellaiah, M. A. et al. Rho-A is critical for osteoclast podosome organization, motility, and bone resorption. J. Biol. Chem. 275, 11993–12002 (2000).
Article CAS Google Scholar
Destaing, O. et al. A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts. J. Cell Sci. 118, 2901–2911 (2005).
Article CAS Google Scholar
Ory, S., Brazier, H., Pawlak, G. & Blangy, A. Rho GTPases in osteoclasts: orchestrators of podosome arrangement. Eur. J. Cell Biol. 87, 469–477 (2008).
Article CAS Google Scholar
Ory, S., Munari-Silem, Y., Fort, P. & Jurdic, P. Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation. J. Cell Sci. 113, (Pt 7): 1177–1188 (2000).
CAS PubMed Google Scholar
Granot-Attas, S., Luxenburg, C., Finkelshtein, E. & Elson, A. Protein tyrosine phosphatase epsilon regulates integrin-mediated podosome stability in osteoclasts by activating Src. Mol. Biol. Cell 20, 4324–4334 (2009).
Article CAS Google Scholar
Ioannidis, J. P. et al. Meta-analysis of genome-wide scans provides evidence for sex- and site-specific regulation of bone mass. J. Bone Miner. Res. 22, 173–183 (2007).
Article CAS Google Scholar
Wilson, S. G. et al. Comparison of genome screens for two independent cohorts provides replication of suggestive linkage of bone mineral density to 3p21 and 1p36. Am. J. Hum. Genet. 72, 144–155 (2003).
Article CAS Google Scholar
Mullin, B. H. et al. Further genetic evidence suggesting a role for the RhoGTPase-RhoGEF pathway in osteoporosis. Bone 45, 387–391 (2009).
Article CAS Google Scholar
Mullin, B. H. et al. Identification of a role for the ARHGEF3 gene in postmenopausal osteoporosis. Am. J. Hum. Genet. 82, 1262–1269 (2008).
Article CAS Google Scholar
Martin, T. J. & Sims, N. A. Osteoclast-derived activity in the coupling of bone formation to resorption. Trends Mol. Med. 11, 76–81 (2005).
Article CAS Google Scholar
Nakamura, T. et al. Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell 130, 811–823 (2007).
Article CAS Google Scholar
Su, X. et al. The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling. J. Clin. Invest. 122, 3579–3592 (2012).
Article CAS Google Scholar
Zhao, H. et al. Synaptotagmin VII regulates bone remodeling by modulating osteoclast and osteoblast secretion. Dev. Cell 14, 914–925 (2008).
Article CAS Google Scholar
DeSelm, C. J. et al. Autophagy proteins regulate the secretory component of osteoclastic bone resorption. Dev. Cell 21, 966–974 (2011).
Article CAS Google Scholar
Yang, D. Q. et al. V-ATPase subunit ATP6AP1 (Ac45) regulates osteoclast differentiation, extracellular acidification, lysosomal trafficking, and protease exocytosis in osteoclast-mediated bone resorption. J. Bone Miner. Res. 27, 1695–1707 (2012).
Article CAS Google Scholar
Feng, S. et al. Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts. Biochem. J. 417, 195–203 (2009).
Article CAS Google Scholar
Ory, D. S., Neugeboren, B. A. & Mulligan, R. C. A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl Acad. Sci. USA 93, 11400–11406 (1996).
Article ADS CAS Google Scholar
Asagiri, M. et al. Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science 319, 624–627 (2008).
Article CAS Google Scholar
Boini, S. & Guillemin, F. Radiographic scoring methods as outcome measures in rheumatoid arthritis: properties and advantages. Ann. Rheum. Dis. 60, 817–827 (2001).
CAS PubMed PubMed Central Google Scholar
Glasson, S. S., Chambers, M. G., Van Den Berg, W. B. & Little, C. B. The OARSI histopathology initiative—recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthr. Cartil. 18, S17–S23 (2010).
Article Google Scholar

Download references

Acknowledgements

We would like to thank Dr S. Offermanns from Max-Planck-Institute for kindly giving us the Gna13^f/f mice. We would like to thank Dr Garvey from UAB for kindly giving us the pHR-EF-IRES-Bla vector, and Dr Xu Feng from UAB for kindly giving us the pMXs-3xFLAG-ires-puro retroviral vector and 293GPG retroviral packaging cell line. We also would like to thank Dr Joel Jules, Matthew McConnell and Dr Jay M. McDonald for their extensive reading, discussion and excellent assistance with this manuscript. We appreciate the assistance of the Center for Metabolic Bone Disease at the University of Alabama at Birmingham (UAB) (P30 AR046031). We are also grateful for the assistance of the UAB Small Animal Phenotyping Core, the UAB Metabolism Core, the UAB Vector Production Facility and the UAB Neuroscience Molecular Detection Core Laboratory (P30 NS0474666). This work was supported by NIH grants R01-AR-044741 (Y-.P.L.) and R01-DE-023813 (Y-.P.L.).

Author information

Authors and Affiliations

Department of Pathology, University of Alabama at Birmingham, SHEL 810, 1825 University Boulevard, Birmingham, 35294-2182, Alabama, USA
Mengrui Wu, Wei Chen, Yun Lu, Guochun Zhu, Liang Hao & Yi-Ping Li

Authors

Mengrui Wu
View author publications
You can also search for this author in PubMed Google Scholar
Wei Chen
View author publications
You can also search for this author in PubMed Google Scholar
Yun Lu
View author publications
You can also search for this author in PubMed Google Scholar
Guochun Zhu
View author publications
You can also search for this author in PubMed Google Scholar
Liang Hao
View author publications
You can also search for this author in PubMed Google Scholar
Yi-Ping Li
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Y-.P.L., M.W. and W.C. designed the experiments. M.W., W.C., Y.L., G.Z. and L.H. conducted the studies. M.W., W.C., Y.L., G.Z., L.H. and Y-.P.L. collected the data and analysed the data. M.W., W.C. and Y-.P.L. drafted the manuscript. All authors approved the final version of the manuscript for submission.

Corresponding authors

Correspondence to Wei Chen or Yi-Ping Li.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-10. (PDF 1741 kb)

Rights and permissions

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Reprints and permissions

About this article

Cite this article

Wu, M., Chen, W., Lu, Y. et al. Gα13 negatively controls osteoclastogenesis through inhibition of the Akt-GSK3β-NFATc1 signalling pathway. Nat Commun 8, 13700 (2017). https://doi.org/10.1038/ncomms13700

Download citation

Received: 25 November 2015
Accepted: 25 October 2016
Published: 19 January 2017
DOI: https://doi.org/10.1038/ncomms13700

This article is cited by

RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signaling
- Jirong Wang
- Chengyun Xu
- Genxiang Mao
Molecular Medicine (2023)
Dlk2 interacts with Syap1 to activate Akt signaling pathway during osteoclast formation
- Xinwei Chen
- Xuzhuo Chen
- Shanyong Zhang
Cell Death & Disease (2023)
Macrophage-specific RhoA knockout delays Wallerian degeneration after peripheral nerve injury in mice
- Jiawei Xu
- Jinkun Wen
- Jiasong Guo
Journal of Neuroinflammation (2021)
GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner
- Zhizhou Xia
- Xiuli Zhang
- Ruibao Ren
Cell Death & Disease (2021)
Site-1 protease controls osteoclastogenesis by mediating LC3 transcription
- Zeyu Zheng
- Xuyang Zhang
- Jian Chen
Cell Death & Differentiation (2021)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.