PROJECT SUMMARY: Craniofacial fibrous dysplasia (CFD) is a disabling skeletal disease with no curative medical therapies. It is characterized by fibrotic, expansile bone lesions that compress surrounding craniofacial structures and cause significant morbidity, including vision loss, hearing impairment, dysmorphic facies, fractures, pain, and dental and jaw abnormalities. Fibrous dysplasia (FD) can affect one or more bones throughout the skeleton, but the femur, ribs and craniofacial skeleton are the most commonly affected sites. FD is caused by a somatic, mosaic activating mutation in GNAS, the gene encoding the alpha subunit of the stimulatory guanine nucleotide binding protein, Gsa, leading to constitutive activation of Gsa and elevated intracellular cAMP levels in affected tissues. CFD can occur in isolation or in association with McCune-Albright Syndrome (MAS), which is characterized by polyostotic FD (FD affecting more than one bone), café-au-lait-skin hyperpigmentation and precocious puberty. There are no approved medical treatment options for FD, and there is a critical need to understand the pathways that are activated downstream of Gsa to identify potential therapeutic targets for this disabling disease. Our single cell RNA sequencing (scRNAseq) data on cells collected from human craniofacial and non- craniofacial FD bone lesions has identified critical signaling pathways that are up-regulated in FD, including TGFβ signaling. TGFb signaling is critical for healthy skeletal development and repair, but when inappropriately activated, it is a significant driver of tissue fibrosis, including pulmonary, renal and systemic fibrosis. Despite its established role in driving fibrosis of non-skeletal tissues, the contributory role of TGFb signaling in FD bone lesions remains unknown. The goal of this proposal is to elucidate the role of TGFβ signaling in driving the fibrotic bone phenotype seen in FD/MAS. Our central hypothesis is that TGFβ signaling is activated in response to Gs-GPCR signaling and contributes to the fibrosis and aberrant osteogenesis seen in FD lesions. To test our hypothesis, we will pursue 2 aims. The first aim will elucidate the mechanisms driving TGFβ signaling in response to Gs-GPCR overactivation at a cellular level. We will use our iPSC model of FD/MAS to generate neural crest-derived mesenchymal osteogenic lineage cells and test the effect of TGFb stimulation and inhibition on the fibrotic profile of these cells at different developmental stages. The second aim will use an in vivo murine model of FD in which GNASR201H is expressed in early osteoblastic lineages to test the effect of TGFβ signaling inhibition on the development of fibrosis and impaired osteogenesis using a pan-TGFβ antibody. Upon successful completion of this project, we aim to shed light on the interplay between Gs-GPCR and TGFβ signaling in the skeleton and apply this knowledge to the treatment of CFD as well as more common diseases of impaired osteogen...