Bone Marrow Stromal Cells Depend on cAMP-dependent Protein Kinase Activity in Adult Mouse Skeleton

Program: Abstracts - Orals, Featured Poster Presentations, and Posters
Session: SUN 303-321-Cancer in Endocrine Tissues
Bench to Bedside
Sunday, June 16, 2013: 1:45 PM-3:45 PM
Expo Halls ABC (Moscone Center)

Poster Board SUN-304
Sisi Liu*1, Costas Petrovas2, Stephan Stanislaw Spath3, Kit Man Tsang4, Emmanouil Saloustros5, Maria V Nesterova5, Maria De La Luz Sierra5 and Constantine A Stratakis5
1Section on Endocrinology and Gen, Bethesda, MD, 2VRC, 3NIH, Bethesda, MD, 4NICHD/NIH, Bethesda, MD, 5National Institutes of Health (NIH), Bethesda, MD
Background: Carney Complex (CNC) is associated with bone lesions (osteochondromyxomas); the most frequent cause for CNC is mutations in PRKAR1A, the gene coding for type-I regulatory subunit of protein kinase A (PKA). To better understand how PKA and/or alternate cAMP signaling lead to bone lesions, mouse models single or double heterozygous for different PKA regulatory and catalytic subunits (Prkar1a+/-, Prkar1a+/-Prkaca+/-, Prkar1a+/-Prkar2a+/- and Prkar1a+/-Prkar2b+/-) were generated. Bone lesions with different time of onset, malignancy and mineralization and organization levels were found in all mouse models; most lesions were found in tail vertebrae. A specific population of adult skeleton-located bone marrow stromal cells (aBSCs) appear to be responsible for the development of these lesions in mice older than 3 months.

Methods:Bone marrow stromal cells were isolated from tail vertebrae before tumor development; cells were cultured with MSC-qualified FBS to maintain their multipotency. FACS was used for identification and isolation of aBSCs from culture based on a four-color selection panel with two positive (CD106 and CD146) and two negative (CD11b and CD45) markers. Colony formation unit (CFU) and differentiation assays were used as evidence for multipotency of the cells after sorting; microarray is used for transcriptomic profiles comparison among these cells from mice with different genotypes.

Results: Cell cultures from tail vertebrae of mice with different genotypes were established. Different percentages of aBSCs were found in mice with different genotypes (non-detectable for WT, 0.777% for Prkar1a+/-, 3.78% for Prkar1a+/-Prkaca+/- and 12.9% for Prkar1a+/-Prkar2a+/-); RNA was extracted from these cells and submitted for transcriptomic analysis. After FACS sorting, cell cultures were established; different colony formation capacities of aBSCs from mice with different genotypes were indicated by CFU, based on the number of colonies formed every 10,000 cells.

 Conclusions: Small changes in PKA types and/or activity caused by deficiencies of different PKA subunits appear to activate different populations of bone lesion-initiating cells; the lesions are consequently different both in number and in histology. These studies shed light in how cAMP-dependent PKA controls multipotency of bone marrow stromal cells in adult skeleton and may lead to tumors if aberrant.

Nothing to Disclose: SL, CP, SSS, KMT, ES, MVN, MDLLS, CAS

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