Expression of an active Gαs mutant in skeletal stem cells is sufficient and necessary for fibrous dysplasia initiation and maintenance

Abstract

Fibrous dysplasia (FD) is a disease caused by postzygotic activating mutations of GNAS (R201C and R201H) that encode the α-subunit of the G_s stimulatory protein. FD is characterized by the development of areas of abnormal fibroosseous tissue in the bones, resulting in skeletal deformities, fractures, and pain. Despite the well-defined genetic alterations underlying FD, whether GNAS activation is sufficient for FD initiation and the molecular and cellular consequences of GNAS mutations remains largely unresolved, and there are no currently available targeted therapeutic options for FD. Here, we have developed a conditional tetracycline (Tet)-inducible animal model expressing the Gα_s^R201C in the skeletal stem cell (SSC) lineage (Tet-Gα_s^R201C/Prrx1-Cre/LSL-rtTA-IRES-GFP mice), which develops typical FD bone lesions in both embryos and adult mice in less than 2 weeks following doxycycline (Dox) administration. Conditional Gα_s^R201C expression promoted PKA activation and proliferation of SSCs along the osteogenic lineage but halted their differentiation to mature osteoblasts. Rather, as is seen clinically, areas of woven bone admixed with fibrous tissue were formed. Gα_s^R201C caused the concomitant expression of receptor activator of nuclear factor kappa-B ligand (Rankl) that led to marked osteoclastogenesis and bone resorption. Gα_s^R201C expression ablation by Dox withdrawal resulted in FD-like lesion regression, supporting the rationale for Gα_s-targeted drugs to attempt FD cure. This model, which develops FD-like lesions that can form rapidly and revert on cessation of mutant Gα_s expression, provides an opportunity to identify the molecular mechanism underlying FD initiation and progression and accelerate the development of new treatment options.

Keywords: GNAS; PKA; fibrous dysplasia; mouse models; skeletal stem cell.

Fig. 1.

Generation of the transgenic mouse model of fibrous dysplasia (FD). (A) Schematic representation of the transgenic mouse model to express Gα_s-activating mutations in a tissue-specific manner. (B) Quantitative PCR (qPCR) analysis (n = 6) and (C) immunoblotting showing elevated expression of Gα_s in limb bone from Gα_s^R201C mice relative to control after doxycycline (Dox) administration. Both intercellular cyclic adenosine monophosphate (cAMP) level (D) and protein kinase A (PKA) phosphorylation (E) are dramatically elevated in Gα_s^R201C BMSCs in response of Dox treatment. (F) Fluorescence imaging from Rosa26 mT/mG reporter mice (mT/mG) shows the expression of Tet-Gα_s/linker/Prrx1-Cre cassette and the initiation of FD (white arrows). White dashed lines outline ossification zone (oz) of growth plate and bone marrow (bm) in control (Prrx1-Cre/mT/mG) tissue or unaffected bone marrow (bm) in mutant (Tet-Gα_s/linker/Prrx1-Cre/mT/mG) tissue. The genotype of the mice corresponds to control-A = linker, control-B = linker/Prrx1-Cre, control-C = Tet-Gα_s/linker, Gα_s^R201C = Tet-Gα_s/linker/Prrx1-Cre. Note: control mice, if not indicated, were selected randomly from mice which did not include the three Tet-Gα_s/linker/Prrx1-Cre transgenes. These terms are also applied for the rest of figure legends. Data are presented as mean ± SD, and significance was calculated by Student’s t test (P > 0.05; *P < 0.05; and ***P < 0.001).

Fig. 2.

Gα_s^R201C expression during embryogenesis results in FD-like lesions. (A) Representative image of the expended limbs of Gα_s^R201C mice (red arrows). (B) Whole-mount staining showing the deformity and cortical lytic changes of the limb bone (red arrows) and large nonmineralized areas of the skull in Gα_s^R201C mice (red arrows). (C) H&E sections present limb bone deformity of Gα_s^R201C mice with predominance of chondroid matrix (arrowheads) and reduced endochondral ossification. In the higher magnification of the blue square of Gα_s^R201C mice, chondroblasts (black arrows) can be clearly observed, and irregularly shaped woven bone (white arrows) embedded in a fibrocellular matrix (stars) and not rimmed by osteoblasts.

Fig. 3.

FD lesions in limbs and skulls of postnatal Gα_s^R201C mice. (A) Representative image of the expanded limbs of adult Gα_s^R201C mice. (B) Kaplan–Meier curve of mice free of visible disease symptoms (limb expansion and limping behavior). Gα_s^R201C (n = 14) mice developed bone lesions within 14 d after Dox administration. Control (n = 15) mice remained lesion-free. (C) X-ray examination shows the expansive deformity of long bones. A mixture of lucent and sclerotic images, representing cortical lytic-sclerotic changes, described as ground glass appearance found in human FD patients. (D) Whole-mount staining showing the expansive deformity and lytic changes of the limb bone from Gα_s^R201C mice. (E) μCT examination of limbs showing the lytic-sclerotic lesions with subtle spontaneous fracture (red arrows). (F) μCT examination of skulls, showing the lytic defect (red arrow). (G) Bone volume (BV) fraction (BV/TV) in the regions of limb with FD lesions (total volume, TV) and the corresponding regions in control mice (n = 4). Data are presented as mean ± SD, and significance was calculated by Student’s t test (***P < 0.001).

Fig. 4.

Histopathological analyses of the FD lesions. (A) Normal forelimb bone structure of a control mouse. (B) FD bone lesions of a Gα_s^R201C mouse after Dox administration. Left shows a low magnification image of a histological section (H&E), showing a mixture of lighter and dense areas, representing the cystic and fibrotic areas (circled in red) seen in the X-ray, compared with the structure of the unaffected humerus (circled in black). Right represents higher magnification of the blue squares at Left, to show irregularly shaped trabeculae of immature, undermineralized woven bone (white arrows), embedded in a fibrocellular matrix of soft collagen (stars). The trabeculae are not rimmed by osteoblasts, one of the distinctive features of fibrous dysplasia. The presence of multinucleated giant cells (standard arrows) is evident at this magnification. All these findings highlight similarities with a case of human FD (Fig. S3A) with the same legends. (C and D) Transmitted (Left) and polarized light (Right) views Sirius Red staining show a widespread disorganized pattern of woven bone in Gα_s^R201C mice. Right represents higher magnification of the blue squares at Left, to show green birefringent collagen-rich fibrosis as seen under polarized light. Red color represents bone. Toluidine Blue (E) and von Kossa (F) staining of undecalcified limb sections show irregular shaped undermineralized woven bone (white arrows), which are surrounded by excess osteoid (black arrows) in Gα_s^R201C mice. (G) Histomorphometric analyses of the regions of limb with FD lesions and the corresponding regions in controls. Bone surface (B.Pm), osteoblast number (N.Ob), osteoblast surface (Ob.Pm), osteoclast number (N.Oc), osteoclast surface (Oc.Pm), osteoid thickness (O.Wi), osteoid surface (O.Pm). n = 8 normal and 8 FD tissues sections. Data are presented as mean ± SD, and significance was calculated by Student’s t test (**P < 0.01 and ***P < 0.001).

Fig. 5.

The preosteogenic feature of Gα_s^R201C mutant cells. (A) Immunohistochemistry of osteogenic markers in control and FD limb bone sections. Runt-related transcription factor 2 (Runx2), osterix (Osx), and alkaline phosphatase (Alp) are enriched in the fibroblastic-like cells within fibrous tissue (ft); Ocn is only found positive in the cells in contact with lesional trabeculae. (B) Double-labeling immunofluorescence staining show elevated expression of early osteogenic markers in Glu-Glu (EE) tag-positive Gα_s^R201C mutant cells along the development of FD. In tissues of day 0, white dashed lines outline boundaries between the hypertrophic zone (hz) and ossification zone (oz) of growth plate or boundaries between cortical bone (cb) and bone marrow (bm); in tissues of day 2 and day 7, white dashed lines outline boundaries between FD lesions (fibrous tissue, ft) and unaffected cortical bone (cb) or bone marrow (bm). Col1; Collagen I. 5-ethynyl-2′-deoxyuridine (EdU) click staining (C) and cleaved caspase-3 immunofluorescence staining (D) showed high proliferation and relatively low apoptosis rate of Gα_s^R201C mutant cells.

Fig. 6.

Gα_s^R201C leads to increased bone resorption via the Rankl pathway. (A) Tartrate-resistant acid phosphatase (TRAP) staining of limb bone indicates the presence of numerous osteoclasts in Gα_s^R201C mice (n = 4). (B) Serum level of tartrate-resistant acid phosphatase 5b (TracP 5b) was elevated in Gα_s^R201C mice (n = 4). Scatterplots are plus mean (Middle line). (C) qPCR analysis shows significantly increased Rankl and Rankl/Opg ratio (n = 6). (D) Serum level of Rankl was significantly increased in Gα_s^R201C mice (n = 4). Scatterplots are plus mean (Middle line). (E) Immunofluorescence staining shows colocalization of Rankl with EE tag in fibroblastic-like cells expressing Gα_s^R201C. (F) Immunofluorescence staining shows binding of Rankl with osteoclasts, which are marked by cathepsin K (Ctsk). Opg, osteoprotegerin; Rankl, receptor activator of nuclear factor kappa-B ligand. Data are presented as mean ± SD, and significance was calculated by Student’s t test (**P < 0.01 and ***P < 0.001).

Fig. 7.

Reducing GNAS expression following Dox withdrawal results in clinical improvement of the preexisting FD bone lesions. (A) Schematic representation of the Dox treatment of control and Gα_s^R201C mice. (B) μCT examination showed the slightly thickened limb bone of the Gα_s^R201C mice without porous-like lytic phenotype or fracture. (C) No significant difference of bone volume fraction between control and Gα_s^R201C mice (n = 4). (D) H&E section of both control and Gα_s^R201C mouse. The preexisting FD lesions circled in red exhibit a dramatic recovery, dense cortical bone with disordered trabecular processes. The diaphyseal marrow cavity exhibits clear boundaries, although slightly narrower than in littermate control, and are composed of hematopoietic cells and adipocytes (black arrows). Details can be better viewed in the Middle and Right which represent higher magnification of the blue squares and stars area in Left, respectively. Note the collagen-rich fibrocellular matrix and multinucleated giant cells are not present at this stage. (E) Von Kossa staining of undecalcified limb tissue shows normal mineralization of cortical bone without lesional osteoid in Gα_s^R201C mice. (F) TRAP staining shows dramatically reduced number of osteoclasts in remineralized bone 16 wk after discontinuing Dox (compare with Fig. 6A). (G) μCT examination of the recovery skull of Gα_s^R201C mice compared with its littermate. Data are presented as mean ± SD, and significance was calculated by Student’s t test. No significant differences were observed.