The Role of RANKL and RANKL Inhibition in Fibrous Dysplasia

Objectives:
Fibrous dysplasia of bone (FD) is a benign skeletal disease caused by activating mutations in the GNAS gene, resulting in Gs-alpha dysregulation and elevated cAMP. In an in vitro model of FD, this results in upregulation of receptor activator of nuclear factor kappa-B ligand (RANKL) expression. This study seeks to characterize the role of RANKL expression in FD and its inhibition with the anti-RANKL antibody/drug denosumab.

Methods:
Immunohistochemistry for RANKL expression was performed on archived FD specimens. Primary cultures of patient-derived bone marrow stromal cells (BMSCs), naturally occurring and engineered, were used to create model FD cell lines. Immortalized BMSCs were transfected with Gs-alpha constructs: R201R (WT), R201C (C), or R201H (H), representing wildtype and FD Gs-alpha mutants. Expression of Gs-alpha constructs was confirmed by western blot. Cell cultures were exposed to denosumab or vehicle and the effects evaluated by measuring cAMP using ELISA and osteogenic markers by RT-PCR and western blotting after immunoprecipitation.

Results:
Stained sections showed robust RANKL expression. Cells transfected with C and H mutations produced higher cAMP versus WT. Treatment with denosumab resulted in decreased cAMP. Expression of osteogenic markers alkaline phosphatase, bone sialoprotein, and RANKL were higher in C and H, while the RANKL decoy receptor osteoprotegerin was similarly expressed in all cells. Western blots of whole cell extracts showed elevated production of RANKL in the C and H cells. Immunoprecipitation of RANKL using denosumab demonstrated increased production of shed/soluble RANKL.

Conclusions:
These data confirm the role of RANKL in FD and suggest that RANKL inhibition with denosumab may have a direct effect on FD cells, independent of the established role of RANKL in osteoclastogenesis, suggesting potential utility in treating FD.