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 a-subunit of the G s stimulatory protein (Gas) 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 E 2 (PGE 2 ) and 1,25 vitamin D 3 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 16fold 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, RANKLþ and Ki67þ fibroblastic cells were observed near RANKþ 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.

RANKL Inhibition in Fibrous Dysplasia of Bone: A Preclinical Study in a Mouse Model of the Human Disease

“…To clarify the effect of RANKL‐inhibition on the radiographic and histological expression of FD, we took advantage of our transgenic murine model of the disease that allowed us to investigate the effects of an anti‐mouse RANKL‐mAb in a sizable number of individuals, homogenous for age and type of lesion. The use of our model was further validated by the demonstration that, as in FD patients, in EF1α‐G s α R201C mice the fibro‐dysplastic tissue was a source of RANKL. In addition, as in primates and humans under treatment with denosumab, a sustained decrease of both CTX‐1 and P1NP occurred during the administration of the anti‐RANKL mAb.…”

“…(23)(24)(25) In addition, serum levels of RANKL have been reported to be increased in mouse models that replicate human FD, (22,26) as well as in FD patients, in which they strongly correlated with the burden of the disease. (25) Accordingly, a limited number of studies have already tested the effect of the humanized anti-RANKL antibody, denosumab, in FD patients and have reported a positive effect on bone turnover, on the growth rate of lesions, as assessed by CT analysis, and on bone pain. (23,(27)(28)(29) However, the role of RANKL in the histopathology of FD, as well as the effect of RANKL inhibition on the natural history of the disease, need to be investigated further.…”

“…Recently, a role for receptor activator of nuclear factor kappa‐B ligand (RANKL; a member of the tumour necrosis factor superfamily and a potent stimulator of osteoclastogenesis), in the disease has emerged from studies performed on human skeletal progenitors transduced with G s α R201C , transgenic mice expressing G s α R201C in skeletal cells, and human FD tissue and cells . In addition, serum levels of RANKL have been reported to be increased in mouse models that replicate human FD, as well as in FD patients, in which they strongly correlated with the burden of the disease . Accordingly, a limited number of studies have already tested the effect of the humanized anti‐RANKL antibody, denosumab, in FD patients and have reported a positive effect on bone turnover, on the growth rate of lesions, as assessed by CT analysis, and on bone pain .…”

Changes in gene expression in human skeletal stem cells transduced with constitutively active Gsα correlates with hallmark histopathological changes seen in fibrous dysplastic bone

“…Thus far, the quest for therapeutic approaches, like attempts to interpret the genesis and mechanisms of the disease, have been dominated by almost exclusive consideration of the effect of constitutively active Gsα, with the idea that reverting the effects of mutant Gsα activity in relevant cells would necessarily lead to a cure. However, multiple lines of evidence point to a more complex scenario, in which tissue changes that have a direct bearing on morbidity at the tissue and organ level may well emanate from a complicated and prolonged in vivo history-of the mutated gene, cell, tissue, and organ [11,[17][18][19][20].…”

First in Mice: RANKL Neutralization in Fibrous Dysplasia

“…This produces a rapidly remodeling fibro‐osseous tissue, reflected by elevated bone formation and resorption markers. Additionally, and possibly quite importantly, osteoclast‐like giant cells not associated with bone surfaces are often found in the areas of most active lesion formation, as evidenced by hypercellularity and Ki67 positivity, suggesting an etiological relationship between RANKL‐driven giant cell formation and disease activity . Clinically, the result is skeletal deformity, fragility, fractures, and bone pain, often independent of associated fractures.…”