Activation of RANK/RANKL/OPG Pathway Is Involved in the Pathophysiology of Fibrous Dysplasia and Associated With Disease Burden

Abstract

Fibrous dysplasia of bone (FD) is a mosaic disease caused by mutations in GNAS. Constitutive activation of the α-subunit of the G_s stimulatory protein (Gαs) leads to dysregulated proliferation of bone marrow stromal cells (BMSCs), generating expansile lesions of fibrotic tissue and abnormal bone. Local bone remodeling regulation by BMSCs is also altered, and FD tissue is characterized by abundant osteoclast-like cells that may be essential for lesion expansion. Animal models show local expression of RANKL in bone lesions, and treatment with the RANKL neutralizing antibody denosumab decreased lesion expansion rate in a patient with aggressive FD. However, the role of RANKL/osteoprotegerin (OPG) in FD pathophysiology is not yet understood. We measured serum levels of RANKL, OPG, and inactive RANKL-OPG complexes in FD patients of known disease burden and in healthy volunteers (HVs). RANK, RANKL, and Ki67 immunohistochemistry were assessed in FD tissue. Cultured FD and HV BMSCs were stimulated with prostaglandin E₂ (PGE₂ ) and 1,25 vitamin D₃ to increase RANKL expression, and media levels of RANKL and OPG were measured. Osteoclastogenic induction by FD or HV BMSCs was assessed in co-cultures with HV peripheral monocytes. FD patients showed a 16-fold increase in serum RANKL compared to HVs. OPG was moderately increased (24%), although RANKL/OPG ratio was 12-fold higher in FD patients than in HVs. These measurements were positively correlated with the skeletal burden score (SBS), a validated marker of overall FD burden. No differences in serum inactive RANKL-OPG complexes were observed. In FD tissue, RANKL+ and Ki67+ fibroblastic cells were observed near RANK+ osteoclasts. High levels of RANKL were released by FD BMSCs cultures, but were undetectable in HV cultures. FD BMSC released less OPG than HV BMSCs. FD, but not HV BMSCs, induced osteoclastogenesis in monocyte co-cultures, which was prevented by denosumab addition. These data are consistent with the role of RANKL as a driver in FD-induced osteoclastogenesis. © 2018 American Society for Bone and Mineral Research.

Trial registration: ClinicalTrials.gov NCT00001727.

Keywords: FIBROUS DYSPLASIA; MCCUNE ALBRIGHT SYNDROME; OPG; RANK; RANKL.

Fig. 1.

Serum RANKL and OPG, but not inactive RANKL-OPG complex, are altered in FD. Levels of serum RANKL (A), OPG (C), RANKL/OPG ratio (E), and RANKL-OPG complex (G) in FD patients compared to HVs. Spearman correlation and linear regression of RANKL (B), OPG (D), RANKL/OPG ratio (F), and RANKL-OPG complex (H) with FD burden as measured by SBS. **p < 0.01, ****p < 0.0001. FD = fibrous dysplasia; HV = healthy volunteer; SBS = skeletal burden score.

Fig. 2.

FD BMSCs promote osteoclastogenesis through RANKL overexpression. (A–D) Serial sections of a representative FD specimen showing closely localized Ki67⁺ (B) and RANKL⁺ (C) cells with RANK⁺ (D) osteoclasts; black arrows = osteoclasts. (E, F) RANKL (E) and OPG (F) release by unstimulated (unst) and 1,25 vitamin D₃-stimulated + PGE₂-stimulated (stim) BMSCs derived from HVs or FD patients. (G) TRAP staining of HV PBMCs co-cultured with HV or FD BMSCs in the presence of DMAB or isotype control antibody (IgG) for 2 weeks. (H) HV and FD BMSCs population doublings with DMAB or IgG2. *p < 0.05 versus unst FD BMSCs. BMSC = bone marrow stromal cell; DMAB = denosumab; FD = fibrous dysplasia; ft = fibrous tissue; HV = healthy volunteer; PBMC = peripheral blood mononuclear cell; stim = stimulated; tb = trabecular bone; unst = unstimulated.