Endocrine Abstracts (2010) 22 P37

Inactivation of PRKARIA or PRKAR2B increases cell proliferation and decreases apoptosis, delineating distinct molecular mechanisms in adrenocortical human H295R cell line

Bruno Ragazzon1, Jerôme Bertherat1,2 & Marthe Rizk-Rabin1

1Institut Cochin, Université Paris Descartes, CNRS UMR 8104, Inserm U1016, Paris, France; 2Service d’Endocrinologie, Groupe Hospitatlier Cochin Saint Vincent de Paul, Paris, France.

The cAMP signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors (ACT). Protein kinase A (PKA) is a key element of this pathway. The R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are observed in Carney complex and a subset of ACT. We have recently reported that inactivation of PRKAR1A dysregulates cAMP pathway and reduces TGFβ-induced apoptosis in the human adrenocortical cell line (H295R) (Ragazzon et al. Cancer Res 2009). A dramatic decreased in R2B protein levels are observed in a subset of adrenocortical adenomas. However, no information is available on the role of PRKAR2B in H295R. This study aims to compare the potential role of R1A and R2B subunits in cell proliferation, control of cell cycle and apoptosis.

We have inactivated PRKAR1A and PRKAR2B in HEK293 and H295R cell lines by RNA interference.

Both inactivation lead to an increased PKA enzymatic activity, disturbs the cell cycle progression by increasing the G2 phase. This increases proliferating cells (BrdU incorporation) and confers resistance to TGFβ- and TNFα-induced apoptosis (Annexine V). However, these apparently similar global cellular responses are mediated by different mechanisms. Differential regulations of cyclins are observed. PRKAR1A inactivation acts on G1/S phase, decreasing the expression and the transcriptional activity of cyclin E and A. PRKAR2B inactivation acts on S/G2 phase increasing the expression and transcriptional activity of cyclin A and B. The resistance to apoptosis displays distinct regulation. The expression of Bax is decreased and Bcl2 is increased under TNFα stimulation in PRKAR2B inactivated cells only.

The effects of PRKAR1A and PRKAR2B inactivation are not antagonist in H295R. However these similar effects of PKA type I or type II on the cell cycles are in fact mediated by different mechanisms.

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