Transfer, analysis, and reversion of the fibrous dysplasia cellular phenotype in human skeletal progenitors

Abstract

Human skeletal progenitors were engineered to stably express R201C mutated, constitutively active Gsα using lentiviral vectors. Long-term transduced skeletal progenitors were characterized by an enhanced production of cAMP, indicating the transfer of the fundamental cellular phenotype caused by activating mutations of Gsα. Like skeletal progenitors isolated from natural FD lesions, transduced cells could generate bone, but not adipocytes or the hematopoietic microenvironment, upon in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted up-regulation of osteocalcin, but with enhanced up-regulation of other osteogenic markers, such as ALP and BSP compared to controls. A very potent up-regulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked up-regulation of selected phosphodiesterase (PDE) isoform mRNAs, and in a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gsα. Indeed, like measurable cAMP levels, the differentiative responses of transduced skeletal progenitors were profoundly affected by inhibition of PDEs, or lack thereof. Finally, using lentiviral vectors encoding short hairpin (sh) RNA interfering sequences, we demonstrated that selective silencing of the mutated allele is both feasible and effective in reverting the aberrant cAMP production brought about by the constitutively active Gsα, and some of its effects on in vitro differentiation of skeletal progenitors.

Wnt/β-catenin signaling is differentially regulated by Gα proteins and contributes to fibrous dysplasia

“…Osteoprotegrin (OPG), a target of Wnt/β-catenin signaling (33), was up-regulated in FD cells, and its expression was decreased by reducing β-catenin levels. Others have reported that bone sialoprotein (BSP) was up-regulated in FD-like cells (34). However, we found that BSP expression levels were similar in control and FD BMSCs, although reduction of β-catenin levels did decrease BSP expression.…”

“…Clearly other Gα s -dependent pathways are required to generate the full spectrum of FD phenotypes. For example, the enhanced osteoclastogenesis and osteolysis present in FD is likely attributable to elevated RANKL expression that is regulated by CREB (34,35).…”

Prompt clinical and biochemical response to denosumab in a young adult patient with craniofacial fibrous dysplasia

“…The finding of a successful effect of Dmab even in craniofacial MFD is consistent with previous studies, reporting the efficacy of Dmab in controlling PFD bone pain (7-9). The better biochemical and clinical response with Dmab as compared with bisphosphonates, might be explained by the fact that Dmab not only induces osteoclasts apoptosis but also affects osteoclasts recruitment from the precursor cells (12) and that Dmab directly inhibits RANKL levels, that are particularly elevated in FD (6). However, we cannot exclude that the incomplete reduction of the pain scores during the period of pamidronate treatment was due, at least in part, to the relatively low dose used (13).…”

“…Denosumab (Dmab), a fully-humanized monoclonal antibody to the receptor activator of nuclear factor kappa-B ligand (RANKL) has been approved for the treatment of osteoporosis and bone metastases from solid tumours in adults (5) and its use in bone diseases other than osteoporosis and bone metastases is off-label. However, in FD, Dmab is of great interest since in vitro studies suggested that in FD Gsα mutation dramatically upregulate RANKL expression (6). Up to now, Dmab treatment has been successfully used in few patients with PFD, (7)(8)(9)(10) but no data are available on craniofacial MFD patients.…”

Orthopaedic Research in Italy: State of the Art

“…They overexpress molecular effectors of osteoclastogenesis, thus promoting inappropriate bone resorption (19). A cellular model was developed by engineering human skeletal progenitors to stably express R201C mutated, constitutively active Gs alpha using lentiviral vectors (20).…”