Wnt/β-catenin signaling is differentially regulated by Gα proteins and contributes to fibrous dysplasia

Abstract

Skeletal dysplasias are common disabling disorders characterized by aberrant growth of bone and cartilage leading to abnormal skeletal structures and functions, often attributable to defects in skeletal progenitor cells. The underlying molecular and cellular mechanisms of most skeletal dysplasias remain elusive. Although the Wnt/β-catenin signaling pathway is required for skeletal progenitor cells to differentiate along the osteoblastic lineage, inappropriately elevated levels of signaling can also inhibit bone formation by suppressing osteoblast maturation. Here, we investigate interactions of the four major Gα protein families (Gα_s, Gα_i/o, Gα_q/11, and Gα_12/13) with the Wnt/β-catenin signaling pathway and identify a causative role of Wnt/β-catenin signaling in fibrous dysplasia (FD) of bone, a disease that exhibits abnormal differentiation of skeletal progenitor cells. The activating Gα_s mutations that cause FD potentiated Wnt/β-catenin signaling, and removal of Gα_s led to reduced Wnt/β-catenin signaling and decreased bone formation. We further show that activation of Wnt/β-catenin signaling in osteoblast progenitors results in an FD-like phenotype and reduction of β-catenin levels rescued differentiation defects of FD patient-derived stromal cells. Gα proteins may act at the level of β-catenin destruction complex assembly by binding Axin. Our results indicate that activated Gα proteins differentially regulate Wnt/β-catenin signaling but, importantly, are not required core components of Wnt/β-catenin signaling. Our data suggest that activated Gα proteins are playing physiologically significant roles during both skeletal development and disease by modulating Wnt/β-catenin signaling strength.

Fibrous dysplasia (FD) of bone [Online Mendelian Inheritance in Man (OMIM) 174800] is a skeletal dysplasia caused by mosaic gain-of-function mutations in GNAS, which results in constitutive activation of Gα_s (1). FD occurs when these mutations arise in skeletal progenitor cells (2); the result is a disorder that presents during childhood and is characterized by bone marrow fibrosis, the presence of intramedullary and immature woven bone, failure of mature lamellar bone to form, and high bone turnover (2). In particular, the bone marrow fibrosis phenotype is characterized by persistent proliferation and lack of differentiation of osteoblastic precursor cells (3). When FD occurs in combination with multiple endocrinopathies and skin pigmentation defects, it is classically called McCune–Albright syndrome (MAS). Although the genetic mutations that cause FD are well characterized, the molecular mechanism by which activated Gα_s leads to the FD phenotype is unclear.

During normal tissue development and homeostasis, tightly regulated progenitor cell proliferation/renewal and differentiation are required to ensure proper organ function. The Wnt/β-catenin (or “canonical” Wnt) signaling pathway plays critical roles regulating the formation and homeostasis of most tissues, and the importance of this pathway in the skeleton is underscored by the number of disorders caused by mutations in Wnt/β-catenin signaling components (4–7). Proper modulation of Wnt/β-catenin signaling levels is critical, and both decreases and increases in signaling activities are associated with abnormal bone formation. During skeletal development, conditional inactivation of β-catenin in skeletal progenitor cells leads to reduced osteoblast differentiation and decreased bone formation (8–11). Considerable evidence also indicates that the effect of Wnt/β-catenin signaling depends on the stage of osteoblast differentiation and that pathway activation in uncommitted progenitor cells can also suppress osteoblast differentiation (9, 12–14) and block the terminal differentiation of committed osteoblasts (10, 15, 16).

During Wnt/β-catenin signaling, Wnt ligands bind to Frizzled (Fzd) and low-density lipoprotein-related protein 5 or 6 (Lrp5/6), initiating a pathway leading to disruption of the Axin-containing β-catenin “destruction complex.” Fzd receptors contain seven transmembrane domains and are classified as a distinct family of G protein-coupled receptors (GPCRs) (17), although the precise roles of G proteins in transducing Wnt/β-catenin signaling remain controversial. GPCRs signal through four relatively small families of Gα proteins (Gα_s, Gα_i/o, Gα_q, and Gα_12/13), and if Fzd receptors are classic GPCRs, they should signal through one of these four Gα families. Indeed, several groups have suggested that Fzd receptors require G proteins for signaling (18–21). Despite these findings, genetic screens in Drosophila and targeted screens in mammalian cells failed to identify G proteins as core pathway components of Wnt/β-catenin signaling (22, 23). Here, we investigate the interaction of these pathways in the context of a disease of skeletal progenitor cells and explore mechanisms by which Gα proteins regulate Wnt/β-catenin signaling.

Results

Wnt/β-Catenin Signaling Is Up-Regulated in FD/MAS Tissues.

Because Wnt/β-catenin signaling has been implicated to interact with Gα_s signaling (24–26) and accumulation of β-catenin protein is a hallmark of Wnt/β-catenin pathway activation, we first examined β-catenin protein levels in tissues isolated from patients with FD/MAS. We found that β-catenin levels were elevated in lesional bone tissue from three patients with FD relative to three control bones by immunohistochemistry (Fig. 1 A and B). In thyroid tissues from two controls, three patients with MAS [1 with thyroid carcinoma (27)], and one patient with Graves disease (OMIM 275000, increased activation of Gα_s through antibody-dependent activation of the thyroid-stimulating hormone receptor), β-catenin levels in patient tissues were also elevated compared with those in the control tissues by immunoblotting (Fig. 1C). These results suggest that Wnt/β-catenin signaling is more highly activated in affected tissues from the patients with FD/MAS that we have examined.

Fig. 1.

Wnt/β-catenin signaling is up-regulated in FD and MAS tissues. (A) Immunohistochemistry for β-catenin in control adult human bone sections counterstained with nuclear fast red. (Magnification: 10×.) (B) Immunohistochemistry for β-catenin on lesional bone sections from three patients with FD demonstrates elevated levels of expression. Arrows point to bone marrow stromal cells. (C) Immunoblotting for β-catenin and β-actin in thyroid gland lysates from two control individuals, three patients with MAS, and one patient with Graves disease. β-catenin levels were elevated in the patient samples relative to control. (D) qPCR data showing elevated expression of Wnt/β-catenin signaling target genes in BMSCs derived from FD bone when cultured under osteogenic conditions for 3 d (n = 3, average ± SD; *P = 0.02 and *P = 0.01 for AXIN2 and TCF1, respectively). (E) Transcription controlled by Wnt/β-catenin signaling as assayed by Supertopflash luciferase activities was up-regulated in FD BMSCs relative to control when cotransfected with a Wnt3a expression construct (*P = 0.047 and *P = 0.003 for 10 and 24 h, respectively). (F) When expressed in MEF cells, the human R201C/H mutant Gα_s proteins do not affect basal activation of Wnt/β-catenin signaling but potentiate signaling induced by exogenous Wnt3a (*P < 0.01). human Gs, hGs.

Because bone marrow stromal cells (BMSCs) from patients with FD demonstrate enhanced proliferation and decreased osteoblastic differentiation (3), we next asked if these cells also showed elevation of Wnt/β-catenin signaling. BMSCs isolated from healthy controls or patients with FD were cultured in vitro, and expression of transcriptional targets of Wnt/β-catenin signaling activity was examined. Under subconfluent conditions, AXIN2 and TCF1 expression was similar in the FD and control BMSCs (Fig. 1D). However, when allowed to reach confluence and placed in osteogenic media to promote differentiation, FD BMSCs showed elevated expression of both AXIN2 and TCF1 relative to control cells (Fig. 1D). This might be caused by more abundant Wnt ligands produced by the confluent cells. BMSCs were then transfected with a luciferase reporter construct of Wnt/β-catenin activity [Supertopflash (28)]. In the absence of exogenous Wnt ligands, control and FD BMSCs showed similar basal Wnt/β-catenin signaling activities (Fig. 1E). When cotransfected with a Wnt3a expression construct, however, FD cells showed higher Wnt/β-catenin signaling activity relative to control BMSCs (Fig. 1E). These results suggest that mutant activated Gα_s in FD cells caused the elevated Wnt/β-catenin activities. To test this more definitively, either WT or mutant Gα_s carrying the common human FD/MAS-associated mutations (R201C and R201H) was transfected into mouse embryonic fibroblast (MEF) cells. Expression of both Gα_s mutants but not the WT Gα_s resulted in potentiated Wnt3a-dependent transcription (Fig. 1F). The relatively rapid kinetics of this interaction (3–4 h) suggest that the interaction of Gα_s and the Wnt/β-catenin signaling pathway may be direct, although we cannot exclude the possibility that secondary effects also exist. These observations indicate that activated Gα_s sensitizes BMSCs to Wnt signaling and activated Gα_s mutations in FD/MAS may exert their effects by up-regulating Wnt/β-catenin signaling.

Loss of Gα_s Leads to Reduced Wnt/β-Catenin Signaling.

Because both Wnt/β-catenin and Gα_s signaling are important for bone formation, we tested whether Gα_s is required to regulate Wnt/β-catenin signaling in vivo during skeletal development. We conditionally removed Gα_s (29) from the developing limb mesenchyme with the Prx1-Cre line (30) and used the Topgal reporter mice (31) to assess endogenous Wnt/β-catenin signaling. At embryonic day 16.5, the Prx1-Cre;Gα_s^flox/−;Topgal mutant mice demonstrated decreased X-gal staining in the bone collar and primary spongiosum (SI Appendix, Figs. S1 and S2), indicating that loss of Gα_s leads to decreased Wnt/β-catenin signaling in vivo in osteoblastic cells. In further support of this, expression of endogenous Wnt/β-catenin target genes in the developing limb of the mutant mice was decreased relative to littermate controls (SI Appendix, Fig. S3). Consistent with Gα_s promoting Wnt/β-catenin signaling during osteoblast differentiation, removal of Gα_s from mouse BMSCs cultured under osteoblastic conditions also led to reduced expression of Wnt/β-catenin target genes (SI Appendix, Fig. S4). Therefore, Gα_s signaling can regulate bone formation by enhancing Wnt/β-catenin signaling.

Activation of β-Catenin in Osteoblast Precursors Phenocopies Key Aspects of FD in Vivo.

Because Wnt/β-catenin signaling is elevated in the FD BMSCs, we tested whether inappropriate activation of Wnt/β-catenin signaling in osteoprogenitor cells is sufficient to cause the FD phenotypes in vivo. This was accomplished by mating mice harboring a conditional allele of β-catenin (Catnb^Ex3) (32) that produces an activated form of β-catenin following Cre-mediated recombination to Osterix (Osx)-Cre mice (10). FD is characterized by extensive marrow fibrosis associated with increased proliferation and reduced differentiation of osteoblastic cells and poorly mineralized bone (3). In the limbs of the Osx-Cre;Catnb^Ex3/+ mice, large portions of the marrow cavity were filled with osteoblastic cells at the expense of hematopoietic cells. In addition, intramedullary bone was immature and woven in the mutant (Fig. 2 A and B). Von Kossa staining of the undecalcified tibia showed the mutant osteoid to be undermineralized (Fig. 2 C and D). Immunohistochemical staining of bone sections with an anti–β-catenin antibody shows elevated expression of β-catenin in the osteoblastic cells in the marrow compartment of Osx-Cre;Catnb^Ex3/+ mutant mice (Fig. 2 E and F). This staining is similar to β-catenin staining in human FD bone (Fig. 1B) and suggests that activation of Wnt/β-catenin signaling in osteoprogenitor cells can lead to their direct expansion in the marrow compartment. These analyses demonstrate that activation of Wnt/β-catenin signaling in early osteoblasts can recapitulate several important phenotypes associated with FD, including the presence of marrow fibrosis, inappropriate presence of intramedullary and woven bone, and failure of lamellar bone to form and mineralize correctly.

Fig. 2.

Activation of β-catenin partially phenocopies FD in vivo, and reduction of β-catenin rescues FD phenotypes. (A) Sections of a control mouse tibia at postnatal day (P) 12 were stained with H&E and demonstrate normal bone marrow cavities with hematopoietic cells (arrows). (B) H&E-stained sections of a tibia from an Osx-Cre;Catnb^Ex3/+ littermate show abnormal intramedullary bone with many mesenchymal cells (arrowheads) in the marrow compartment and only residual hematopoietic cells (arrow). (C) Von Kossa staining of an undecalcified tibia section from a control animal at P12 indicating normal mineralization of cortical bone. The section is counterstained with Giemsa. (D) Von Kossa staining of an undecalcified section from an Osx-Cre;Catnb^Ex3/+ littermate tibia demonstrates the undermineralized, osteomalacic nature of the mutant osteoid (arrows). The section is counterstained with Giemsa. (E) Immunohistochemistry for β-catenin performed on paraffin sections from a control mouse shows basal levels of β-catenin (arrow). The section is counterstained with hematoxylin. (F) Immunohistochemistry for β-catenin performed on paraffin sections from an Osx-Cre;Catnb^Ex3/+ mouse shows extensive accumulation in fibroblastic/mesenchymal cells (arrows) in the marrow compartment. The section is counterstained with hematoxylin. (Magnification: A and B, 10×; C–F, 20×.) (G) Von Kossa staining of WT and FD BMSCs pretreated with the indicated shRNA lentiviruses (LV) and grown under osteogenic conditions for 4 wk. Note that the presence of mineralized nodules (arrow) in the WT samples is largely missing in the FD samples treated with control LV. Decreasing β-catenin levels causes the reappearance of mineral nodules (arrow). (Scale bar = 1 mm.) (H) qPCR results demonstrate that the dysregulated expression of some genes in FD cells can be corrected by reducing β-catenin levels (n = 3–5, average ± SD; *P = 0.01, *P = 0.03, *P = 0.003, and *P = 0.02 for ALK Phos, BSP, OC, and OPG, respectively). b-cat, β-catenin; hBMSCs, human BMSCs.

Reduction of β-Catenin in Patient-Derived BMSCs Rescues FD in Vitro.

To assess whether the up-regulated Wnt/β-catenin signaling contributes to the pathology of FD in humans, we turned to a cell culture model of osteoblast maturation. Human BMSCs grown to confluence were switched to osteogenic media and cultured for 4 wk, allowing osteoprogenitor cells to differentiate into mature osteoblasts. Relative to control BMSCs, BMSCs isolated from patients with FD demonstrated a block in maturation as seen by the elevated expression of the osteoprogenitor marker alkaline phosphatase (ALK PHOS), decreased mRNA expression of the mature osteoblast marker osteocalcin (OC), and decreased formation of calcified matrix nodules by Von Kossa staining (Fig. 2 G and H).

To test whether elevated Wnt/β-catenin signaling contributes to the phenotypes of the human FD BMSCs, we reduced β-catenin levels by infecting cells with lentiviruses that express shRNAs targeting β-catenin (SI Appendix, Fig. S5). We found that reducing β-catenin levels rescued the ability of FD BMSCs to form calcified matrix nodules in vitro (Fig. 2G). This effect was more pronounced when β-catenin levels were decreased more efficiently (20% vs. 80% knockdown). Quantitative PCR (qPCR) analysis demonstrated that the expression of some genes dysregulated in FD was normalized by reducing β-catenin levels. Expression of ALK PHOS was decreased, and expression of OC was increased, suggesting that the blockage of osteoblast maturation of the FD osteoprogenitor cells had been partially corrected by reducing the β-catenin levels (Fig. 2H). Osteoprotegrin (OPG), a target of Wnt/β-catenin signaling (33), was up-regulated in FD cells, and its expression was decreased by reducing β-catenin levels. Others have reported that bone sialoprotein (BSP) was up-regulated in FD-like cells (34). However, we found that BSP expression levels were similar in control and FD BMSCs, although reduction of β-catenin levels did decrease BSP expression. Receptor activator of NF-κB ligand (RANKL) was up-regulated in FD cells, and this elevation was not reversed by decreasing β-catenin levels but, instead, was further increased. This is consistent with the notion that RANKL expression is positively regulated by Gα_s activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB) (35) and inhibited by Wnt/β-catenin signaling (36). These data suggest that the fibrotic and osteolytic phenotypes in FD can be uncoupled. Taken together, our data indicate that enhanced Wnt/β-catenin signaling in osteoprogenitor cells contributes to the pathophysiology of FD and that inhibition of Wnt/β-catenin signaling may represent a novel therapeutic approach.

Gα Proteins Differentially Regulated Wnt/β-Catenin Signaling.

Gα_s can be activated by GPCRs, such as the parathyroid hormone receptor, and our results suggest that GPCR signaling may regulate bone formation by modulating Wnt/β-catenin signaling activities. Because GPCRs play significant roles during both embryonic development and adult homeostasis, and usually activate more than a single G protein family, we explored the mechanism by which Gα_s and the other major G protein families (Gα_q/11, Gα_i/o, and Gα_12/13) regulate Wnt/β-catenin signaling. We first tested whether all major Gα protein families exhibit similar regulatory activities on Wnt/β-catenin signaling by generating dose–response curves using a wide range of Wnt ligand concentrations and the Supertopflash assay (Fig. 3A). MEF cells transfected with either an empty vector or a vector encoding constitutively active Gα_i2 (Gα_i2-QL) gave similar curves, demonstrating a lack of interaction between Gα_i2 and the Wnt/β-catenin signaling pathway. Expression of Gα_s-QL caused the dose–response curve to shift to the left, but it reached a similar maximum as the control curve. By contrast, Gα_q-QL and Gα₁₃-QL shifted the Wnt3a dose–response curve to the right, decreasing the cells’ responsiveness to Wnt3a. Our results demonstrate that Gα proteins by themselves are not sufficient to stimulate Wnt/β-catenin signaling in MEF cells; however, they exhibit differential activities in modulating Wnt/β-catenin signaling in the presence of exogenous Wnt3a. Under the same experimental conditions, Gα_s potentiates, whereas Gα_q and Gα₁₃ inhibit, Wnt/β-catenin signaling and Gα_i has no effect. Activation of different G protein pathways by distinct GPCRs yielded similar results (SI Appendix, Figs. S6 and S7). Expression of constitutively active forms of three distinct Gα_i/o family members (Gα_i2-QL, Gα_o-QL, and Gα_z-QL) failed to activate basal or Wnt3a-induced β-catenin–dependent transcription (SI Appendix, Fig. S8). Because the stimulatory effects of Gα_s were not mimicked by the PKA activators forskolin or 3-isobutyl-1-methylxanthine, Gα_s may act through a PKA-independent mechanism to regulate Wnt/β-catenin signaling (SI Appendix, Fig. S8). Forskolin treatment did, however, lead to phosphorylation of CREB and enhanced CREB transcriptional activities (SI Appendix, Fig. S9). The inhibitory effects of Gα_q-QL and Gα₁₃-QL were specific to Supertopflash, because these same G protein constructs promoted transcriptional activation of Ap1- and serum response factor-luciferase constructs, as expected (SI Appendix, Fig. S10).

Fig. 3.

Activated Gα proteins differentially regulate Wnt/β-catenin signaling. (A) Dose–response curve obtained by treating MEF cells transiently transfected with activated (QL) Gα protein constructs with increasing concentrations of recombinant Wnt3a. Wnt3a stimulation with concomitant G protein activation demonstrates that Gα_s-QL leads to increased Wnt/β-catenin signaling, whereas both Gα_q-QL and Gα₁₃-QL cause decreased Wnt/β-catenin signaling. (B) HEK293T cells were cotransfected with WT or the activated QL form of rat Gα proteins and Axin or GFP. Activated forms of Gα_s, Gα_q, and Gα₁₃ showed enhanced coimmunoprecipitation with Axin relative to WT Gα forms. (C) Coimmunoprecipitation experiments performed with WT or mutant (R201C, R201H) forms of human Gα_s demonstrate enhanced binding of mutant forms to Axin. (D) Axin (green) subcellular localization is affected by the presence of Gα proteins. Under control conditions, transfected Axin is present in cytoplasmic puncta (1 and 2). When cotransfected with Gα_s-QL (red, 3 and 4), Axin distribution is more widespread and present at the plasma membrane. When cotransfected with Gα₁₃-QL (red, 5 and 6), Axin remains largely cytoplasmic and aligns parallel to f-actin (Magnification: 63×).

To investigate the mechanism of Wnt/β-catenin signaling regulation by Gα proteins, we activated the pathway using lithium chloride (LiCl), a Gsk3 inhibitor. Because Gsk3 promotes β-catenin degradation, LiCl treatment abrogated all Gα effects, suggesting that the Gα proteins act upstream of Gsk3 (SI Appendix, Fig. S8). Indeed, activated Gα proteins differentially affected accumulation of cytoplasmic β-catenin, possibly by regulating the Gsk3-containing β-catenin destruction complex (SI Appendix, Fig. S11). In the absence of exogenous Wnt3a, cytoplasmic β-catenin levels were undetectable regardless of the activation status of Gα proteins. Addition of Wnt3a to MEF cells resulted in accumulation of cytoplasmic β-catenin. In the presence of Gα_s-QL, the amount of cytoplasmic β-catenin increased, whereas Gα_q-QL or Gα₁₃-QL expression resulted in decreased β-catenin levels (SI Appendix, Fig. S11). Addition of Gα_i2-QL had no effect on cytoplasmic β-catenin accumulation. Taken together, these results demonstrate that activation of Gα proteins alone is not sufficient to activate the Wnt/β-catenin pathway de novo but can differentially regulate the activity of this pathway by acting upstream of Gsk3 to modulate β-catenin protein stability.

Because Axin contains a regulator of G protein signaling domain, a protein interaction module known to bind Gα subunits, and is required for the assembly of the β-catenin destruction complex, we explored the interaction between all four major Gα subunits and Axin. Consistent with previous observations (24), overexpressed Gα_s-QL could be coimmunoprecipitated with Axin with considerably higher efficiency than WT Gα_s (Fig. 3B). Similar to the constitutively activating Q227L Gα_s mutations, the common human FD/MAS-associated mutations (R201C and R201H) demonstrated increased affinity for Axin (Fig. 3C). Although robustly expressed, neither WT Gα_i2 nor Gα_i2-QL could be coimmunoprecipitated with Axin. Interestingly, activated forms of both Gα₁₃ and Gα_q could be coimmunoprecipitated with Axin, whereas WT forms either failed to coimmunoprecipitate or did so less robustly. Gα_q-QL tended to be coimmunoprecipitated less efficiently than either Gα_s-QL or Gα₁₃-QL. Because the three activated Gα subunits that interacted with Axin in this assay are the same that affected Wnt/β-catenin signaling in MEF cells (Fig. 3A), these Gα subunits may exert their effects by binding to Axin.

Activation of Gα proteins leads to their subcellular redistribution (37, 38) and may modulate Wnt/β-catenin signaling by affecting Axin localization. To test this, we performed immunofluorescent staining to determine the effects of activated Gα proteins on Axin distribution. In the absence of Gα proteins, exogenously expressed Axin was present in cytoplasmic puncta (Fig. 3D, 1 and 2). Consistent with differential localization of distinct activated Gα proteins, coexpression of Axin with Gα_s-QL led to colocalization and redistribution of Axin to the plasma membrane and throughout the cytoplasm in a diffuse pattern (Fig. 3D, 3 and 4). Consistent with Gα_s potentiating Wnt/β-catenin signaling, others have shown that the membrane localization of Axin is associated with pathway activation (39). By contrast, coexpression with Gα₁₃-QL also colocalized and caused Axin puncta to aggregate between actin stress fibers (Fig. 3D, 5 and 6). These data suggest that the opposing effects of Gα_s and Gα₁₃ on Wnt/β-catenin signaling may be attributable to altered subcellular localization of Axin. These results further suggest that different Gα proteins, when activated simultaneously, might exert their effects in modulating Wnt/β-catenin signaling by competing for binding to Axin. To test this, we coexpressed Axin along with either Gα₁₃-QL or Gα_s-QL, or Axin with both Gα₁₃-QL and Gα_s-QL. We found that when both Gα₁₃-QL and Gα_s-QL were coexpressed, they demonstrated decreased binding to Axin relative to when either Gα₁₃-QL or Gα_s-QL was expressed singly, suggesting that these Gα proteins are competing for common binding sites on Axin (SI Appendix, Fig. S12).

Gα Proteins Are Not Required for Wnt/β-Catenin Signaling.

Although it has been reported that Fzds may act as GPCRs and require Gα proteins to activate Wnt/β-catenin signaling (18, 40), the precise role of G proteins in Wnt signaling remains controversial. If Gα proteins are core components of Wnt/β-catenin signaling, they should be required for Wnt/β-catenin signaling in all cell types. To test this, we genetically removed either Gα_s, Gα_q/11, or Gα_12/13 from MEF cells. Importantly, genetic removal of these Gα proteins failed to affect Wnt/β-catenin signaling as assayed by Supertopflash, accumulation of cytoplasmic β-catenin, or phosphorylation of Lrp6 (Fig. 4 A–C and SI Appendix, Figs. S13–15). Inhibition of Gα_i/o, achieved by pertussis toxin pretreatment, alone or in a Gα_q/11-null background, similarly did not affect Wnt/β-catenin signaling (Fig. 4B and SI Appendix, Fig. S14). Our data indicate that Gα proteins are not part of the core Wnt/β-catenin signaling pathway and are not generally required for pathway transduction.

Fig. 4.

Gα proteins are not required for Wnt/β-catenin signaling. MEF cells were isolated from mice carrying indicated floxed Gα alleles and infected with either a GFP- or Cre-adenovirus (Ad). Loss of protein was confirmed by Western blot analysis. Stimulation of the Wnt/β-catenin signaling by increasing amounts of Wnt3a conditioned medium (CM) or 40 mM LiCl demonstrates normal Wnt/β-catenin signaling activation in all cases. (A) MEF cells were isolated from Gα_s^flox/flox mice. (B) MEF cells were isolated from Gα_q^flox/flox; Gα₁₁KO mice. Cells were pretreated with pertussis toxin (PTX; 100 ng/mL) to inhibit Gα_i/o signaling. (C) MEF cells were isolated from Gα₁₂KO; Gα₁₃^flox/flox mice.

Discussion

Here, we provide unique insights into the mechanisms by which aberrant G protein signaling leads to human diseases. Our data show that activated mutant Gα_s protein in osteoprogenitor cells up-regulated Wnt/β-catenin signaling and inhibited osteoblast maturation and that elevated Wnt/β-catenin signaling contributed to the pathophysiological features of FD. FD is a complex bone dysplasia that presents with bone marrow fibrosis, presence of intramedullary and immature woven bone, abnormal bone mineralization, and accelerated osteolysis attributable to increased osteoclasts. Here, we show that activation of β-catenin in osteoprogenitor cells could recapitulate three of these four phenotypes in mice. Furthermore, inhibition of Wnt/β-catenin signaling in patient-derived FD cells could partially rescue differentiation defects in vitro. Consistent with our findings, expression of Dkk1, which encodes an inhibitor of Wnt signaling, in osteoblasts blocks development of bone marrow fibrosis phenotypes in mouse models of hyperparathyroidism (41).

Generating this FD-like mouse model required placing mice on both doxycycline-containing chow and water, largely suppressing Cre expression in the Osx-Cre mouse. This may have produced a mosaic state, like that seen in human FD (42), allowing the mice to survive for the several weeks required for the FD-like phenotype to develop. In the absence of doxycycline treatment, these mice perished at birth with intramedullary bone but a less developed stromal cell phenotype. It is important to note that in other mouse models in which Wnt signaling is elevated in more committed osteoblasts, FD-like phenotypes have not been reported (33, 36, 43–45). Consistent with this, when we activated β-catenin in mature osteoblasts using the OC-Cre line (46), we saw a considerably milder phenotype, although areas of fibrosis and abnormal mineralization were still present. These data support our conclusion that elevated Wnt/β-catenin signaling in osteoprogenitor cells, as opposed to mature osteoblasts, produces the FD phenotype. Clearly other Gα_s-dependent pathways are required to generate the full spectrum of FD phenotypes. For example, the enhanced osteoclastogenesis and osteolysis present in FD is likely attributable to elevated RANKL expression that is regulated by CREB (34, 35).

This work represents a systematic gain- and loss-of-function analysis exploring the role of Gα proteins in Wnt/β-catenin signaling. Our data support a model in which Gα proteins are playing important regulatory roles in this pathway at least in the skeletal system, although they are not required Wnt signaling core components. The differential effects of Gα proteins suggest they may have a “rheostat-like” function within cells, either increasing or decreasing the strength of Wnt/β-catenin signaling, depending on the particular G protein pathway activated. Recently, considerable interest has revolved around targeting the Wnt/β-catenin pathway for therapeutic purposes. Enhancing Wnt/β-catenin signaling is viewed as therapeutically beneficial in conditions like osteoporosis, whereas inhibiting this pathway is desired in treating certain cancers. Because of the pleiotropic nature of the core Wnt/β-catenin signaling complex and its central role in organ homeostasis, directly targeting this complex may lead to unacceptable toxicities. Our data suggest that indirectly targeting this pathway through GPCR signaling represents an attractive alternative in certain tissues. For instance, the well-documented oncoprotective effects of nonsteroidal antiinflammatory drugs may be attributable to decreased production of prostaglandin E2, leading to decreased activation of prostaglandin E2 receptor (EP) receptors/Gα_s and Wnt/β-catenin signaling (24, 47). Intriguingly, it has recently been shown that Gα_s R201C mutations interact with mutations in adenomatous polyposis coli, a Wnt pathway inhibitor, to promote intestinal tumorigenesis (48, 49), suggesting this mechanism may be relevant in multiple progenitor/stem cells and human diseases.

Several groups have proposed models by which Gα_s stimulates β-catenin through both PKA-dependent and -independent mechanisms (24–26). We found that Gα_s, Gα_q, and Gα₁₃ all showed activity-dependent enhanced affinity for the regulatory scaffolding protein Axin. Although the precise mechanisms by which Gα_s enhances, whereas Gα_q and Gα₁₃ inhibit, Wnt/β-catenin signaling will require further experimentation, our data suggest that competition for Axin binding resulting in altered subcellular trafficking is involved. It is likely there are multiple pathways by which Gα proteins can affect Wnt/β-catenin signaling; for example, Gα_q can also inhibit the pathway by promoting calpain-dependent β-catenin degradation (50). The presence and relative importance of these distinct mechanisms are likely cell context-dependent and remain to be determined. Our findings should stimulate the further identification of biologically important GPCR/Wnt/β-catenin interactions.

Materials and Methods

Descriptions of mice, plasmids, antibodies, cell culture methods, immunohistochemistry, immunofluorescence, histology, and statistics are provided in SI Appendix.

Tissue Collection.

Tissues were collected from patients enrolled in ongoing National Institutes of Health Clinical Center Institutional Review Board-approved studies. All subjects or their parents gave informed consent.

qPCR.

Human and mouse primer sequences are provided in SI Appendix, Table S1. RNA was isolated and cDNA was generated as previously described (51). qPCR was performed using sybr green and an ABI 7900 cycler (Applied Biosystems) with 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Relative gene expression was determined using the 2^-ΔΔCt method.

Luciferase Assays.

Human BMSCs were transfected with Supertopflash/Renilla-null constructs and pIRES-control or pIRES-Wnt3a plasmids by nucleofection (Amaxa). Cells were allowed to recover overnight in α-MEM/20% (vol/vol) FBS growth media and then placed in similar media containing 10% FBS for the indicated time points. MEF cells were transfected with Supertopflash/Renilla-null and other constructs and allowed to recover for 5–6 h. Cells were then placed in DMEM, 0.2% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM glutamine overnight. On the following morning, cells were placed in 0.2% FBS media with recombinant or Wnt3a-conditioned media for 4–6 h. Luciferase activity was determined using the Dual Luciferase Reporter Assay (Promega). For luciferase based assays, n = 3–4 and experiments were repeated a minimum of three times.

Lentiviral Production.

Lentivirus was produced, and BMSCs were infected as described (52). Selection was performed for 48 h using puromycin (2 μg/mL).