Abstract 974: Aldosterone Decreases Glucose-6-phosphate Dehydrogenase Expression in Vascular Endothelial Cells by Increased Expression of Cyclic AMP Response Element Modulator
Increased levels of aldosterone (ALDO) promote vascular endothelial cell (EC) dysfunction by decreasing expression of glucose-6-phosphate dehydrogenase (G6PD). G6PD, the first enzyme in the pentose phosphate pathway, modulates EC function by limiting oxidant stress. Decreased G6PD expression is associated with activation of protein kinase A (PKA); however, the molecular mechanisms by which PKA decreases G6PD expression remain unknown. We hypothesized that ALDO decreases transcription of G6PD by PKA-dependent upregulation of cyclic AMP response element modulator (CREM). To examine this hypothesis, bovine aortic EC (BAEC) were treated with ALDO [10−9, 10−8, or 10−7 mol/L] or vehicle (V) for 24 h. Compared to V-EC, ALDO-EC demonstrated a dose-dependent increase in cAMP levels (1.5 ± 0.1 vs 2.3 ± 0.2 vs 3.9 ± 0.4 vs 5.8 ± 0.4 pmol/mg protein, p<0.01) and a 411% increase in PKA activity (at 10−7 mol/L). PKA activation increased phosphorylated CREB expression by 150%; however, CREB-CRE binding was decreased in ALDO-EC compared to V-EC (0.21 ± 0.03 vs 0.06 ± 0.01 arb units, p<0.01). In ALDO-EC, this effect occurred due to a 322% increase in CREM mRNA and a 176% increase in CREM protein expression. To demonstrate that CREM mediated the decrease in G6PD transcription, BAEC were transfected with siRNA to decrease CREB or CREM protein expression by 72% or 66%, respectively. A nuclear run-on assay revealed that G6PD protein expression was similar to V-EC in CREM-transfected EC (0.7 ± 0.1 vs 0.7 ± 0.1, p=NS) yet remained downregulated in CREB-transfected cells (0.1 ± 0.04, p<0.01). To confirm that these effects were PKA-dependent, cells were treated with the PKA inhibitor, PKI. In PKI-treated ALDO-EC, there was a decrease in CREM expression (0.3 ± 0.05 vs 1.3 ± 0.01 arb units, p<0.01) and an increase in G6PD mRNA and protein levels compared to ALDO-EC. Furthermore, ALDO did not activate serum-glucocorticoid kinase, MSK, Ca2+-calmodulin kinase, or MAPK to influence CREB-CRE binding. These studies demonstrate that ALDO decreases G6PD expression by increasing CREM mRNA and protein levels in a PKA-dependent manner. ALDO-mediated activation of PKA to decrease G6PD expression may represent one mechanism by which elevated levels of ALDO impair vascular endothelial cell function.