Insulin-like growth factor 2 (IGF2) expression in adrenocortical disease due to PRKAR1A mutations compared to other benign adrenal tumors.

PURPOSE: Insulin-like growth factor-II (IGF2), a key regulator of cell growth and development, is tightly regulated in its expression by epigenetic control that maintains its monoallelic expression in most tissues. Biallelic expression of IGF2 resulting from loss of imprinting (LOI) has been reported in adrenocortical tumors. In this study, we wanted to check whether adrenocortical lesions due to PRKAR1A mutations lead to increased IGF2 expression from LOI and compare these findings to those in other benign adrenal lesions. METHODS: We compared the expression of IGF2 by RNA and protein studies in primary pigmented nodular adrenocortical disease (PPNAD) caused by PRKAR1A gene mutations to that in primary macronodular adrenocortical hyperplasia (PMAH) and cortisol-producing adenomas (CPA) that did not have any mutations in known genes. We also checked LOI in all lesions by DNA allelic studies and the expression of other components of IGF2 signaling at the RNA and protein level. RESULTS: We identified cell clusters overexpressing IGF2 in PPNAD; although immunostaining was patchy, overall, by RNA and immunoblotting PPNAD expressed high IGF2 message and protein. However, this was not due to LOI, as there was no correlation between IGF2 expression and the presence of LOI. CONCLUSIONS: Our data pointed to over-expression of IGF2 protein in PPNAD compared to other benign adrenocortical lesions, such as PMAH and CPA. However, there was no correlation of IGF2 mRNA levels with LOI of IGF2/H19. The discrepancy between mRNA and protein levels with regards to LOI points, perhaps, to different control of IGF2 gene expression in PPNAD.

Regulation of actin function by protein kinase A-mediated phosphorylation of Limk1.

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a(-/-) MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.

Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: implications for transformation.

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1); however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM) that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated. RESULTS: We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R) in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1alpha (MIP-1alpha), a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was enhanced due to activation of lymphocytes by HTLV-1 infection. CONCLUSION: These data demonstrate that PTHrP and its receptor are up-regulated specifically during immortalization of T-lymphocytes by HTLV-1 infection and may facilitate the transformation process.

Targeted deletion of Prkar1a reveals a role for protein kinase A in mesenchymal-to-epithelial transition.

Dysregulation of protein kinase A (PKA) activity, caused by loss of function mutations in PRKAR1A, is known to induce tumor formation in the inherited tumor syndrome Carney complex (CNC) and is also associated with sporadic tumors of the thyroid and adrenal. We have previously shown that Prkar1a(+/-) mice develop schwannomas reminiscent of those seen in CNC and that similar tumors are observed in tissue-specific knockouts (KO) of Prkar1a targeted to the neural crest. Within these tumors, we have previously described the presence of epithelial islands, although the nature of these structures was unclear. In this article, we report that these epithelial structures are derived from KO cells originating in the neural crest. Analysis of the mesenchymal marker vimentin revealed that this protein was markedly down-regulated not only from the epithelial islands, but also from the tumor as a whole, consistent with mesenchymal-to-epithelial transition (MET). In vitro, Prkar1a null primary mouse embryonic fibroblasts, which display constitutive PKA signaling, also showed evidence for MET, with a loss of vimentin and up-regulation of the epithelial marker E-cadherin. Reduction of vimentin protein occurred at the posttranslational level and was rescued by proteasomal inhibition. Finally, this down-regulation of vimentin was recapitulated in the adrenal nodules of CNC patients, confirming an unexpected and previously unrecognized role for PKA in MET.

Disruption of protein kinase a regulation causes immortalization and dysregulation of D-type cyclins.

Phosphorylation is a key event in cell cycle control, and dysregulation of this process is observed in many tumors, including those associated with specific inherited neoplasia syndromes. We have shown previously that patients with the autosomal dominant tumor predisposition Carney complex carry inactivating mutations in the PRKAR1A gene, which encodes the type 1A regulatory subunit of protein kinase A (PKA), the cyclic AMP-dependent protein kinase. This defect was associated with dysregulation of PKA signaling, and genetic analysis has suggested that complete loss of the gene may be required for tumorigenesis. To determine the mechanism by which dysregulation of PKA causes tumor formation, we generated in vitro primary mouse cells lacking the Prkar1a protein. We report that this genetic disruption of PKA regulation causes constitutive PKA activation and immortalization of primary mouse embryonic fibroblasts (MEFs). At the molecular level, knockout of Prkar1a leads to up-regulation of D-type cyclins, and this increase occurs independently of other pathways known to increase cyclin D levels. Despite the immortalized phenotype, known mediators of cellular senescence (e.g., p53 and p19ARF) seem to remain intact in Prkar1a-/- MEFs. Mechanistically, cyclin D1 mRNA levels are not altered in the knockout cells, but protein half-life is markedly increased. Using this model, we provide the first direct genetic evidence that dysregulation of PKA promotes important steps in tumorigenesis, and that cyclin D1 is an essential target of PKA.