Proper regulation of cAMP-dependent protein kinase (PKA) is essential to normal cell proliferation, but PKA dysregulation leads to tumor formation in a range of endocrine tissues. Resulting from mutations in the main PKA regulatory subunit, PRKAR1A, Carney complex is characterized by tumors of various endocrine glands, as well as bone and cartilage tumors, Schwann cell tumors, and skin discoloration. Since PKA dysregulation affects several neural crest-derived tissues, we sought to examine the role of PKA in the neural crest.
The cranial neural crest (CNC) undergoes complex molecular and morphological changes during embryogenesis in order to form the vertebrate skull, and nearly 75% of all birth defects result from defective craniofacial development. The molecular events leading to CNC differentiation have been extensively studied; however the role of PKA during craniofacial development has only been described in palate formation. Selective inactivation of Prkar1a in the CNC results in perinatal lethality caused by dysmorphic craniofacial bone and cartilage development and subsequent asphyxiation. Aberrant differentiation of Prkar1a-null CNC mesenchymal cells resulted in anomalous intramembranous ossification and cartilage dysplasia. These observations provide new evidence for the importance of PKA regulation in craniofacial development, which may be beneficial to understanding and treating craniofacial birth defects.
In addition to the facial structures, proper PKA regulation is required in Schwann cells. Tissue-specific ablation of Prkar1a in a subset of neural crest precursor cells caused schwannomas with high frequency. Previously, signaling events leading to Schwann cell tumor initiation have been characterized in the context of Neurofibromatosis (NF). At the molecular level, Prkar1a-null tumors revealed loss of both NF proteins, despite the fact that transcript levels were increased, implying post-transcriptional regulation. Furthermore, the small G-proteins Ras, Rac1, and RhoA are all regulated by NF signaling. In Prkar1a-null tumors, all three molecules showed modest increases in total protein, but only Rac1 was activated. These data suggest that dysregulated PKA activation causes Schwann cell tumorigenesis via pathways that overlap but are distinct from those described in NF tumorigenesis.
Genetic interaction studies aimed at identifying the role of NF protein loss in Prkar1a-null tumors revealed that loss of Nf2, specifically, is rate-limiting for PKA Schwann cell tumorigenesis. Because Rac1 was highly activated in Prkar1a-null schwannomas, the p21-activated kinases (Paks) were evaluated for their activity. Pak6 was significantly over-expressed in the schwannomas at both the transcript and protein levels, however its activity was not different from normal Schwann cells. Despite no change in transcript levels, Pak2 protein expression was upregulated, and it was highly activated in the tumors. Downstream targets of Pak were also up-regulated in Prkar1a-null tumors suggesting in vivo activation of Pak signaling. Altogether these results suggest that PKA suppression of Nf2 causes Schwannomas via Rac1-Pak signaling.
The results from this work reveal that proper PKA regulation is required in the developing neural crest, as well as in the adult neural crest-derived tissues. Not only do these studies highlight novel roles for PKA in both differentiation and proliferation, they further emphasize the importance of precise PKA regulation from embryogenesis to adulthood.