Phosphodiestarase-mediatd adaptation to Gsalpha mutations is developmentally regulated in embryonicand post-natal stem cells.

Fibrous dysplasia is a skeletal disorder caused by gain-of-function mutations of Gs-alpha. Although the genetic event occurs in early development, pre-natal skeletal growth is unaffected in FD patients. This suggests that skeletal progenitors display a different sensitivity to aberrant Gs signaling at different developmental ages. To address this issue, we infected mouse embryonic stem cells (ES) and human post-natal skeletal stem cells (Bone Marrow Stromal Cells, BMSCs) with a lentivector expressing the R201C rat Gs-alpha cDNA and analyzed their ability to differentiate into skeletal phenotypes. The expression of the transgene was confirmed at both RNA and protein levels. Surprisingly, neither ES cells nor BMSCs showed excess intracellular cAMP upon infection. However, treatment with IBMX revealed a different response in the two cell types. In transduced BMSCs, IBMX significantly increased intracellular cAMP compared to wild-type cells. Accordingly, high expression of IBMX-sensitive PDE isozymes (i.e. PDE 3, 4 and 7) was detected by q-PCR in basal conditions. IBMX alone did not modify cAMP levels in transgenic ES cells in which increased expression of the IBMX-insensitive PDE 8 was observed. Following incubation in specific inductive media, the expression of cartilage and bone markers was, overall, unaffected in mutated ES cells. Furthermore, mature cartilage undergoing endochondral ossification and normal bone were observed in teratomas generated upon in vivo transplantation. In contrast, differentiation of BMSC was profoundly affected by the mutation. In vitro, abnormal expression of skeletal markers was observed in specific differentiation assays. In vivo, ectopic osteogenic activity was limited to the deposition of a scarce amount of woven bone. In conclusion, these data demonstrate the different behavior of Gs-alpha mutated embryonic vs adult stem cells during skeletal differentiation. Most important, they show that a PDE-mediated adaptive response to Gs-alpha activating mutations operates in both cell types. However, different, and differentially regulated, PDEs isozymes are activated in mutated ES cells compared to postnatal skeletal progenitors, and the resulting adaptive response seems more efficient in ES cells than in postnatal stem cells. This could contribute to explain the normal development and abnormal postnatal growth of FD bone.