Comparison of the effects of PRKAR1A and PRKAR2B depletion on signaling pathways, cell growth, and cell cycle control of adrenocortical cells

Abstract

The cyclic AMP/protein kinase A signaling cascade is one of the main pathways involved in the pathogenesis of adrenocortical tumors. The PKA R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. Inactivating mutations of PRKAR1A are associated with Carney complex and a subset of sporadic tumors and the abundance of R2B protein is low in a subset of secreting adrenocortical adenomas. We previously showed that PRKAR1A and PRKAR2B inactivation have anti-apoptotic effects on the adrenocortical carcinoma cell line H295R. The aim of this study was to compare the effects of PRKAR1A and PRKAR2B depletion on cell proliferation, apoptosis, cell signaling pathways, and cell cycle regulation. We found that PRKAR2B depletion is compensated by an upregulation of R1A protein, whereas PRKAR1A depletion has no effect on the production of R2B. The depletion of either PRKAR1A or PRKAR2B promotes the expression of Bcl-xL and resistance to apoptosis; and is associated with a high percentage of cells in S and G2 phase, activates PKA and MEK/ERK pathways, and impairs the expression of IkB leading to activate the NF-κB pathway. However, we observed differences in the regulation of cyclins. The depletion of PRKAR1A leads to the accumulation of cyclin D1 and p27kip, whereas the depletion of PRKAR2B promotes the accumulation of cyclin A, B, cdk1, cdc2, and p21Cip. In conclusion, although the depletion of PRKAR1A and PRKAR2B in adrenocortical cells has similar effects on cell proliferation and apoptosis; loss of these PKA subunits differentially affects cyclin expression.

Fig. 1

Effect of PRKAR1A or PRKAR2B depletion on the abundance of other PKA subunits, cell signaling pathways, apoptosis and cell proliferation. a Histogram showing the abundance of PKA R1A, R2B and Cα mRNA and protein. b PKA activity of cells incubated with FSK for 5 or 10 min, 48 h after siRNA transfection. DEAE Chromatography indicates the activity of Type I and Type II PKA in the absence or presence of cAMP in siR1A and siR2B cells. c Apoptosis was assessed by annexin V-FITC/IP staining in transfected cells with or without TNFα. Results are expressed as the percentage of viable cells (hatched bars) and cells undergoing apoptosis (annexin +/IP, plus annexin +/IP +, black bars). Western blot of Bcl-xL shows the accumulation of the protein in both siR1A and siR2B cells. No differences were observed in the abundance of Bax. BrdU incorporation reveals that the proportion of cells in S phase is higher in both siR1A and siR2B cells than in control cells. d NF-κB cell signaling; the abundance of IkBα expression is low in siR1A and siR2B cells. Analysis of NF-κB p50 and p65 in cytosolic and nuclear fractions reveal different pattern of NF-κB proteins in siR1A and siR2B cells.

Fig. 2

Effects of PRKAR1A or PRKAR2B depletion on cell cycle regulators. a The abundance of cyclin mRNA and the distribution of cyclin proteins between the cytosol and the nucleus. Cyclin D accumulates in siR1A cells whereas cyclins A and B accumulate in siR2B cells. b The distribution of cdks between the cytosol and nucleus is consistent with the distribution of their partner cyclins. c The abundance of the cdk inhibitors p21Cip and p27 Kip is affected by the depletion of PKA subunits.