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.

Osteochondromyxoma: Review of a rare carney complex criterion

“…One of them is Runx2 (Runt-related transcription factor-2, MGI:99829), a gene that is claimed to be a major control for osteoblast differentiation [25], [45], [46]. Chromatin immunoprecipitation and luciferase assays also show repression of DNA binding and functioning of Runx2 at its target genes.…”

“…Chromatin immunoprecipitation and luciferase assays also show repression of DNA binding and functioning of Runx2 at its target genes. There are also reductions in Runx2-cooperating transcription factors such as Foxo1 (forkhead box O1, MGI:1890077) and Atf4 (activating transcription factor 4, MGI:88096) [46]. The osteoblasts affected by the Prkar1a mutation also display suppressed bone nodule formation in addition to markers osteocalcin and osteopontin [25], [46].…”

“…There are also reductions in Runx2-cooperating transcription factors such as Foxo1 (forkhead box O1, MGI:1890077) and Atf4 (activating transcription factor 4, MGI:88096) [46]. The osteoblasts affected by the Prkar1a mutation also display suppressed bone nodule formation in addition to markers osteocalcin and osteopontin [25], [46]. Downregulation of other markers includes Il6st (interleukin-6 signal transducer, MGI:96560), a gene that codes for gp130 subunit of the IL-6 receptor [47].…”

Identification of a novel mutation of the PRKAR1A gene in a patient with Carney complex with significant osteoporosis and recurrent fractures

“…16,17 Apart from the secondary osteoporosis due to PPNAD-associated cortisol hypersecretion, the osteogenic potential may also be influenced by PRKAR1A gene ablation that can interfere with signaling pathways which are necessary for osteoblast differentiation, as shown in experimental data from mouse and human cell lines. 18 The osteoporotic bone changes, fractures and secondary hyperparathyroidism that were found in our CNC patient indicated the long duration of the disease.…”

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“…The total RNA was extracted from the tibial bone marrow cells using TRlzol Reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer's instructions. Two μg of total RNA in each group was reverse transcribed into cDNA in a final volume of 50 μl as our previous report (Fu et al, 2014;Zhang et al, 2014). The gene of GAPDH was selected as an endogemous internal control gene and a non-template reaction was included as negative control for each experiment.…”