Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification

Abstract

Heterotopic ossification, the pathologic formation of extraskeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gαs restricts bone formation to the skeleton by inhibiting Hedgehog signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia, a human disease caused by null mutations in GNAS, which encodes Gαs, Hedgehog signaling is upregulated in ectopic osteoblasts and progenitor cells. In animal models, we show that genetically-mediated ectopic Hedgehog signaling is sufficient to induce heterotopic ossification, whereas inhibition of this signaling pathway by genetic or pharmacological means strongly reduces the severity of this condition. As our previous work has shown that GNAS gain-of-function mutations upregulate WNT-β-catenin signaling in osteoblast progenitor cells, resulting in their defective differentiation and fibrous dysplasia, we identify Gαs as a key regulator of proper osteoblast differentiation through its maintenance of a balance between the Wnt-β-catenin and Hedgehog pathways. Also, given the results here of the pharmacological studies in our mouse model, we propose that Hedgehog inhibitors currently used in the clinic for other conditions, such as cancer, may possibly be repurposed for treating heterotopic ossification and other diseases caused by GNAS inactivation.

Figure 1. Loss of Gnas in limb mesenchyme leads to HO

(a, b) Representative alizarin red and alcian blue staining of forelimbs from wild-type littermate control (WT) and Prx1-Cre;Gnas^f/− mutant mice at E17.5 (a) and P4 (b). Regions of initiating HO (a) and overt HO (b) are indicated (arrows). Scale bar, 1 mm. (c) Representative computed tomography (CT) scans of forelimbs from P20 WT littermate and Prx1-Cre;Gnas^f/− mutant mice. (d) Representative alizarin red and alcian blue staining of hindlimbs from P20 WT littermate and Prx1-Cre;Gnas^f/− mutant mice. A region of unmineralized Achilles tendon (left) and of ossified Achilles tendon (right) are indicated (arrowheads). Regions of HO are also indicated (arrows). Scale bar, 0.5 mm. (e, f) Longitudinal sections of the autopod of a P4 Prx1-Cre;Gnas^f/f mouse counterstained with alcian blue and Sirius red and processed by Von Kossa staining (e) or by Osx immunohistochemistry (DAB, brown) (f). Regions of ectopic mineralization (black arrows) and chondrocyte hypertrophy and joint fusion (yellow arrows) are indicated (e), as are the brown nuclear staining of Osx-positive cells (black arrows) in interdigital regions of surrounding light-blued stained ossicles (f). The boxed interdigital regions in each panel are shown in higher magnification on the right. Scale bars, 0.2 mm (left), 0.05 mm (right).

Figure 2. Loss of Gnas in adult subcutaneous tissue leads to HO

(a) Representative alizarin red and alcian blue staining of the Gnas^f/f mice injected with either Ad-GFP or Ad-Cre virus in the subcutaneous regions (shown in supplemental Fig. 3). N=18. Extensive ectopic bone formation is indicated (arrow). Scale bar, 1 mm. (b) Histological analyses of ectopic bone formation shown in (a) by Osx and Oc immunohistochemistry (arrows) and Von Kossa staining (dark brown). Scale bar, 0.05 mm. (c, d) Representative Von Kossa staining of the ectopic bone (green arrows) in the subcutaneous (c) and the deep muscular regions (d) in the hindlimbs of Gnas^f/f mice 12 weeks after Ad-Cre virus injection. H: hair follicle; M: muscle. Scale bars, 0.2 mm (c), 0.05 mm (d).

Figure 3. Removal of Gnas from mesenchymal progenitor cells upregulates Hh signaling in vitro and in vivo

(a, b) Representative Von Kossa (a) or alizarin red staining (b) of Ad-Cre or Ad-GFP infected BMSCs from Gnas^f/f mice after 10 days in osteogenic medium. (c) qRT-PCR analysis of osteoblast markers at day 7 following adenovirus infection (mean±SD; n=3; * p=0.017 for Osx; 0.022 for Col1a1; 0.005 for Alpl; 0.003 for BSP; 0.003 for Oc). (d) Representative in situ hybridization performed on E14.5 forelimbs from the WT littermate control (left) and the Prx1-Cre; Gnas^f/− embryos (right). Scale bar, 0.5 mm. (e) qRT-PCR analysis of RNA isolated from E14.5 forelimbs. Expression of Gnas and transcription targets of Hh pathway is shown (mean±SD; n=4; * p=2.8×10⁻⁵ for Gnas; 0.012 for Ptch1; 0.036 for Gli1; 0.01 for Hhip). (f) qRT-PCR analysis of Hh pathway target genes in the BMSCs 2-3 days following Ad-Cre or Ad-GFP infection (mean±SD; n=3; * p=1.1×10⁻⁴ for Ptch1; 0.001 for Gli1; 0.007 for Hhip).

Figure 4. Gα_s acts through cAMP and PKA to suppress Hh signaling

(a) cAMP levels in SMPs from Gnas^f/f mice as measured 3 days after adenovirus infection (mean±SD; n=3; *p=8.5×10⁻⁵). (b) PKA activity indicated by Phospho-Creb (P-Creb) protein levels in SMPs from Gnas^f/f mice 5 days after adenovirus infection. (c) qRT-PCR assay of Hh target gene expression in SMPs from Gnas^f/f mice 5 days after adenovirus infection (mean±SD; n=3; *p=4.4×10⁻⁴ for Ptch1; 2.4×10⁻⁵ for Gli1; 2.6×10⁻⁶ for Hhip). (d) Alizarin red staining of the SMPs from the Gnas^f/f mice 14 days after the indicated treatment. (e) PKA activity indicated by P-Creb levels in SMPs from the Gnas^f/f mice 5 days after the indicated treatment. (f) q-RT-PCR assay of Hh target expression in SMPs from the Gnas^f/f mice that had been infected with adenovirus for 7 days and treated as indicated for 5 days (mean±SD; n=3; *p=2.1×10⁻⁴ for Ptch1; 8.7×10⁻⁴ for Gli1; 2.6×10⁻⁴ for Hhip). (g) PKA activity indicated P-Creb protein levels in WT SMPs infected by dnPKA adenovirus for 5 days. (h) qRT-PCR analysis of Hh target gene expression in WT SMPs infected by dnPKA adenovirus for 5 days (mean±SD; n=3; *p=0.001 for Ptch1; 2.1×10⁻⁴ for Gli1; 3.9×10⁻⁴ for Hhip). (i) qRT-PCR analysis of osteoblast differentiation marker expression in WT SMPs infected by dnPKA adenovirus for 5 days (mean±SD; n=3; *p=0.003 for Osx; 4.8×10⁻⁴ for Alpl). (j) Alizarin red staining of WT SMP cells infected by dnPKA adenovirus for 14 days.

Figure 5. Reducing Hh signaling inhibits HO in vivo and in vitro

(a, b) Representative alizarin red and alcian blue staining of the limbs from E18.5 embryos with the indicated genotypes. Less severe HO, particularly in the hindlimb, is indicated (arrow) . In (b), the E18.5 embryos were from the pregnant female mice that had been injected with vehicles or the indicated Hh antagonists three times (E13.5, E15.5 and E17.5). Scale bars, 0.5 mm. (c) Alizarin red staining of the differentiating BMSCs from the Gnas^f/f mice with the indicated treatment.

Figure 6. Hh signaling is activated in ectopic bone from POH individuals and activation of Hh signaling is sufficient to cause HO

(a-c) GLI1 immunohistochemical staining of human samples. (a, b) Samples from a normal human subject. Scale Bars: 1 μm. (c) GLI1 expression in ectopic osteoblasts of the POH samples (arrows). Scale Bar: 1 μm. (d) Representative alizarin red and alcian blue staining of the limbs from the R26SmoM2 mice injected with the Ad-GFP (left limb) or the Ad-Cre (right limb). Ectopic bone formation is indicated (arrows). N=8. (e) Alizarin red staining of the cultured SMP cells. (f) Schematic illustration showing the fundamental roles of Gα_s in bone formation and the mechanisms of GNAS mutations in bone disease (see text for more details). L: low, H: high, GOF: gain of function, LOF: loss of function.