The cyclic AMP/PKA signalling cascade and PKA subunits are involved in the pathogenesis of a subset of cortisol-secreting adrenocortical tumors (ACT). In addition, steroid excess causes morbidity of all types of ACT.
The PKA regulatory subunits PRKARIA, PRKARIIb control proliferation/apoptosis in the H295R adrenocortical cell line. Their inactivation enhances the accumulation of cells in the G2 phase, increases steroidogenesis and activates the PKA and MAPkinases pathways.
The goal is to study the correlation between the cell cycle phases and the steroid secretion, and its control by PKA in H295R cells.
Methods: Using pharmacologic drugs, cells were synchronized at specific cell cycle check point (G1 phase), (S phase) and (G2 phase). We analysed the cell cycle distribution (Cytometry), the expression, of cyclins and cdks, the PKA subunits and cell signalling pathways, the expression of StAR and steroidogenic enzymes. We studied the effect of PKA activation by PRKARIA inactivation and PRKACAa overexpression along the cell cycle synchronization.
Results: The synchronization of H295R cells at G2 phase increased the expression of the steroidogenic enzymes and steroid secretion. Arresting H295R in G1 phase decreased the steroidogenic enzymes expression resulting in a decrease of cortisol secretion. PKA subunits distribution and PKA activity were modulated during the cell cycle progression.
Moreover the PRKARIA inactivation counteracted the decrease of steroidogenesis by enhancing StAR/luc reporter gene activity only in cells arrest in G1 phase, and enforced their progression in G2 phase. While PRKACAa overexpression increased the StAR/luc reporter gene activity, independently of the cell cycle check point arrest.
In Conclusion; we have demonstrated a tight link between the cell cycle check points and the regulation of steroidogenesis, where the PRKARIA subunit had a role as a key regulator of cell cycle and steroidogenesis in low steroidogenic G1 phase arrested cells.