title: Knockdown of PRKAR1A, the gene responsible for Carney complex, interferes with differentiation in osteoblastic cells.
abstract: PRKAR1A is the gene encoding the type 1A regulatory subunit of protein kinase A, and it is the cause of the inherited human tumor syndrome Carney complex. Data from our laboratory has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through the failure of terminal differentiation. In the present study, we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. These observations imply that the antiosteogenic effect of Prkar1a ablation is not species or cell line specific. Furthermore, because Runt-related transcription factor-2 (Runx2) is a key mediator of osteoblast differentiation, we reasoned that the function of this transcription factor may be inhibited by Prkar1a knockdown. Chromatin immunoprecipitation and luciferase assays demonstrated that Prkar1a ablation repressed DNA binding and function of Runx2 at its target genes. Additionally, we determined that this effect is likely due to reductions in the Runx2-cooperating transcription factors forkhead box O1 and activating transcription factor 4. Taken together, this study provides direct evidence that ablation of Prkar1a interferes with signaling pathways necessary for osteoblast differentiation.
authors: Departments of Molecular, Virology, Immunology, and Medical Genetics (M.Z., P.K.M., L.S.K.) and Molecular and Cellular Biochemistry (D.W., Q.W.) and Division of Endocrinology, Diabetes, and Metabolism (L.S.K.), The Ohio State University, Columbus, Ohio 43210.
authors: Mei
authors: M
authors: Zhang
authors: Parmeet K
authors: PK
authors: Manchanda
authors: Dayong
authors: D
authors: Wu
authors: Qianben
authors: Q
authors: Wang
authors: Lawrence S
authors: LS
authors: Kirschner
mesh_terms: D016475:3T3 Cells; D000818:Animals; D056733:Carney Complex / Q000235:genetics; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D050659:Core Binding Factor Alpha 1 Subunit / Q000502:physiology; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000502:physiology; D051379:Mice; D010006:Osteoblasts / Q000502:physiology*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D014158:Transcription, Genetic
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; C517835:Prkar1a protein, mouse; D034741:RNA, Small Interfering; D015675:Osteocalcin; D053495:Osteopontin