G-protein coupled receptor (GPCR) signaling pathways mediate a wide spectrum of biological activities in humans. McCune-Albright Syndrome (MAS) is a mosaic disease caused by a somatic activating mutation in the GNAS gene (c.602G>A, p.R201H). The GNAS complex locus encodes the stimulatory alpha subunit of the guanine nucleotide binding protein (Gs?) and regulates production of cAMP. MAS is characterized by the classic triad of polyostotic fibrous dysplasia, caf-au-lait skin lesions, and precocious puberty. It can also cause hyperthyroidism, Cushing?s disease, acromegaly, and malignancies of the thyroid, pituitary and pancreas. The R201H mutation is thought to occur post-zygotically since tissues from all 3 germ layers can be affected. There is no known vertical transmission of MAS in humans; therefore, germline mutations are thought to be embryonically lethal; however, the precise mechanism leading to lethality is not fully understood. There is emerging literature suggesting that GPCR signaling pathways play a role in early development and stem cell fate, and we hypothesize that the early lethality seen in MAS may be a consequence of over-activation of the Gs-signaling pathway which may create a critical block in the development of certain cell lineages. Unfortunately, our ability to study this mechanism further is hampered by our lack of animal models carrying the GNAS R201H mutation in the endogenous locus. We propose developing a novel, robust human model of MAS using induced pluripotent stem cells (iPSCs) to explore how activated GNAS and elevated cAMP levels affect stem cell fate and lineage commitment in MAS. First, we will test how increased cAMP levels, pharmacologically induced by forskolin, affect pluripotency and lineage commitment in control human iPSCs. Next, we will create a human model of MAS by introducing the R201H mutation into control iPSCs at the endogenous locus using CRISPR/Cas9 gene-editing techniques. We will then compare these two models and examine whether pharmacologically-induced and R201H mutation-induced cAMP activation have similar effects on iPSC pluripotency and lineage commitment. We will then differentiate our engineered R201H iPSCs into osteogenic precursors and examine the effect of the mutation on osteoblast commitment and maturation. Our results will provide insight into the potential mechanisms contributing to early cell fate changes and embryonic lethality in MAS. This new knowledge will guide future studies in mature cell types that can be generated from iPSCs, including adrenal cortical cells, pituitary cells, and pancreatic ductal cells. As GPCR signaling pathways mediate many critical biological activities in the human body, this model will support the study of Gs-signaling in other tissues and diseases. The results of our current study will also be critical for developing screening tools and identifying key endpoints for GNAS-specific high-throughput drug screens. Finally, this training plan will help the candidate gain the necessary skills to apply for a NIH K08 award and develop into an independent translational clinician-scientist in the field of endocrinology.

Public Health Relevance

G-protein coupled receptor (GPCR) signaling pathways mediate a wide-spectrum of biological activities in humans, and we are interested in exploring their effect on early cell fate by creating a novel human pluripotent stem cell model of McCune-Albright Syndrome (MAS). MAS is a mosaic disease that occurs as a consequence of abnormal activation of the stimulatory G-protein signaling pathway and produces the classic triad of polyostotic fibrous dysplasia, caf-au-lait skin hyperpigmentation and precocious puberty, as well as other endocrinopathies (hyperthyroidism, Cushing's disease and acromegaly) and solid organ malignancies of the thyroid, pancreas and pituitary. This proposal also supports a coordinated postdoctoral training program that will position the candidate to develop into a strong, independent translational physician-scientist.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Postdoctoral Individual National Research Service Award (F32)
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Special Emphasis Panel (ZRG1)
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Mukhopadhyay, Mahua
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University of California San Francisco
Internal Medicine/Medicine
Schools of Medicine
San Francisco
United States
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Barruet, Emilie; Morales, Blanca M; Cain, Corey J et al. (2018) NF-?B/MAPK activation underlies ACVR1-mediated inflammation in human heterotopic ossification. JCI Insight 3: