Endocrine Abstracts (2008) 16 OC4.1

Inactivation of the Carney complex (CNC) gene 1 (PKA regulatory subunit 1A, PRKAR1A) by interference RNA alters multiple signaling pathways and decreases apoptosis

Bruno Ragazzon1, Laure Cazabat1, Marthe Rizk-Rabin1, Karine Perlemoine1, Antoine Martinez2 & Jérôme Bertherat1

1INSERM U567, CNRS UMR8104, Institute Cochin, Paris, France; 2CNRS UMR6547, Clermont-Ferrand, France.

The cAMP signaling pathway plays an important role in cell proliferation and differentiation, and can be altered at multiple levels in endocrine tumors. Its central component is the protein kinase A (PKA). Inactivating mutations of PRKAR1A are observed in CNC (a dominant autosomal hereditary disease responsible for primary pigmented nodular adrenocortical disease, cardiac myxoma and lentiginosis). Most PRKAR1A mutations lead to mRNA unstability and protein degradation. To study the consequences of PRKAR1A mutations we have developed an interference RNA approach in the human embryonic kidney (HEK293) and adrenocortical (H295R) cell lines that allow a 50–80% decrease of the R1A protein.

PRKAR1A inactivation in these two cell lines increases by 2-fold the forskolin (FK) stimulation of a PKA-dependent luciferase reporter construct (CRE-Luc). It also increases rapidly endogenous NURR1 (an ubiquitous target of CREB) expression after 1h of FK stimulation (HEK293: x2.2; H295R: x1.2). As expected, the PKA enzymatic activity is stimulated after PRKAR1A inactivation (basal activity +12%, and after FK +20%). But interestingly, PRKAR1A inactivation exerts various effects on others signaling pathways more frequently involved in tumorigenesis. We have observed an activation of MAPK (proliferation) and PI3K/AKT (cell survival) pathways (x2 of P-ERK1/2 and x2 of P-AKT). Moreover, TGFbeta pathway (known to inhibit adrenal steroidogenesis and to induce apoptosis in numerous cell types) is altered by decreased expression of SMAD3 (−40%). This leads to a decreased response to TGFbeta stimulation (−24%) of a SMADs reporter construct (CAGA-Luc). SMAD3 expression is also inhibited by ACTH in vivo in the mice adrenal and by FK in vitro in H295R cells. Finally, PRKAR1A inactivation leads to resistance to apoptosis induction by TNFalpha (−14% of apoptosis cells with 20 ng/ml per 24 h and −26% of dead cells with 100 ng/ml per 24 h).

Tumorigenesis due to PRKAR1A mutations could results from effects on multiple signaling pathways and apoptosis dysregulation.

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