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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.

The cranial neural crest (CNC) undergoes complex molecular and morphological changes during embryogenesis in order to form the vertebrate skull, and nearly three quarters of all birth defects result from defects in craniofacial development. The molecular events leading to CNC differentiation have been extensively studied; however, the role of the cAMP-dependent protein kinase [protein kinase A (PKA)] during craniofacial development has only been described in palate formation. Here, we provide evidence that strict PKA regulation in postmigratory CNC cells is essential during craniofacial bone development. Selective inactivation of Prkar1a, a regulatory subunit of the PKA holoenzyme, in the CNC results in perinatal lethality caused by dysmorphic craniofacial development and subsequent asphyxiation. Additionally, aberrant differentiation of CNC mesenchymal cells results in anomalous intramembranous ossification characterized by formation of cartilaginous islands in some areas and osteolysis of bony trabeculae with fibrous connective tissue stabilization in others. Genetic interaction studies revealed that genetic reduction of the PKA catalytic subunit C(alpha) was able to rescue the phenotype, whereas reduction in Cbeta had no effect. Overall, these observations provide evidence of the essential role of proper regulation of PKA during the ossification of the bones of the skull. This knowledge may have implications for the understanding and treatment of craniofacial birth defects.

Carney complex (CNC) is an inherited neoplasia syndrome characterized by spotty skin pigmentation, myxomas, endocrine tumors, and schwannomas. Among the endocrine tumors that comprise the syndrome, GH-producing pituitary tumors are seen in approximately 10% of patients, although biochemical abnormalities of the GH axis are much more common. To explore the role of loss of the CNC gene PRKAR1A on pituitary tumorigenesis, we produced a tissue-specific knockout (KO) of this gene in the mouse. For these studies, we generated a mouse line expressing the cre recombinase in pituitary cells using the rat GHRH receptor promoter. These mice were then crossed with Prkar1a conditional null animals to produce tissue-specific KOs. Although prolactinomas were observed in KO and control mice, the KO mice exhibited a significantly increased frequency of pituitary tumors compared with wild-type or conventional Prkar1a(+/-) mice. Characterization of the tumors demonstrated they were composed of cells of the Pit1 lineage that stained for GH, prolactin, and TSH. At the biochemical level, levels of GH in the serum of KO animals were markedly elevated compared with controls, regardless of the presence of a frank tumor. These data indicate that complete loss of Prkar1a is sufficient to allow the formation of pituitary tumors and abnormalities of the GH axis, in close analogy to human patients with CNC.

Carney complex (CNC) is an autosomal dominant neoplasia syndrome caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A (PKA). This genetic defect induces skin pigmentation, endocrine tumors, myxomas, and schwannomas. Some patients with the complex also develop myxoid bone tumors termed osteochondromyxomas. To study the link between the PRKAR1A mutations and tumor formation, we generated a mouse model of this condition. Prkar1a(+/-) mice develop bone tumors with high frequency, although these lesions have not yet been characterized, either from human patients or from mice. Bone tumors from Prkar1a(+/-) mice were heterogeneous, including elements of myxomatous, cartilaginous, and bony differentiation that effaced the normal bone architecture. Immunohistochemical analysis identified an osteoblastic origin for the abnormal cells associated with islands of bone. To better understand these cells at the biochemical level, we isolated primary cultures of tumoral bone and compared them with cultures of bone from wild-type animals. The tumor cells exhibited the expected decrease in Prkar1a protein and exhibited increased PKA activity. At the phenotypic level, we observed that tumor cells behaved as incompletely differentiated osteoblasts and were able to form tumors in immunocompromised mice. Examination of gene expression revealed down-regulation of markers of bone differentiation and increased expression of locally acting growth factors, including members of the Wnt signaling pathway. Tumor cells exhibited enhanced growth in response to PKA-stimulating agents, suggesting that tumorigenesis in osteoblast precursor cells is driven by effects directly mediated by the dysregulation of PKA.