title: 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 Gs alpha 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 Gs alpha. Like skeletal progenitors isolated from natural fibrous dysplasia (FD) lesions, transduced cells could generate bone but not adipocytes or the hematopoietic microenvironment on in vivo transplantation. In vitro osteogenic differentiation was noted for the lack of mineral deposition, a blunted upregulation of osteocalcin, and enhanced upregulation of other osteogenic markers such as alkaline phosphatase (ALP) and bone sialoprotein (BSP) compared with controls. A very potent upregulation of RANKL expression was observed, which correlates with the pronounced osteoclastogenesis observed in FD lesions in vivo. Stable transduction resulted in a marked upregulation of selected phosphodiesterase (PDE) isoform mRNAs and a prominent increase in total PDE activity. This predicts an adaptive response in skeletal progenitors transduced with constitutively active, mutated Gs alpha. 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 Gs alpha and some of its effects on in vitro differentiation of skeletal progenitors.
authors.affiliation: Biomedical Science Park San Raffaele, Rome, Italy.
authors.forename: Stefania
authors.initials: S
authors.lastname: Piersanti
authors.forename: Cristina
authors.initials: C
authors.lastname: Remoli
authors.forename: Isabella
authors.initials: I
authors.lastname: Saggio
authors.forename: Alessia
authors.initials: A
authors.lastname: Funari
authors.forename: Stefano
authors.initials: S
authors.lastname: Michienzi
authors.forename: Benedetto
authors.initials: B
authors.lastname: Sacchetti
authors.forename: Pamela Gehron
authors.initials: PG
authors.lastname: Robey
authors.forename: Mara
authors.initials: M
authors.lastname: Riminucci
authors.forename: Paolo
authors.initials: P
authors.lastname: Bianco
mesh_terms: D001854:Bone Marrow Cells / Q000378:metabolism*; D002454:Cell Differentiation / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D005357:Fibrous Dysplasia of Bone / Q000235:genetics* / Q000473:pathology / Q000503:physiopathology; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D010012:Osteogenesis / Q000235:genetics; D010641:Phenotype; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D013234:Stem Cells / Q000502:physiology*; D017154:Stromal Cells / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D010727:Phosphoric Diester Hydrolases; D019205:GTP-Binding Protein alpha Subunits, Gs