Abstract 16445: Ca2+/calmodulin-Dependent Protein Kinase Kinase and Akt Pathway-Mediated Activation of P300 Regulates Laminar Shear Stress-Induced Upregulation of Endothelial Ca2+-activated K+ Channels KCa2.3 and KCa3.1
Introduction: The Ca2+-activated K+ channels KCa2.3 and KCa3.1 in endothelial cells (ECs) play a crucial role in the regulation of arterial tone via producing NO and endothelium-derived hyperpolarizing factors. Since a rise in intracellular Ca2+ levels and activation of p300 histone acetyltransferase are early EC responses to laminar shear stress (LS) for the transcriptional activation of genes, we examined the role of Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) and p300 in LS-dependent regulation of these ion channels.
Methods and Results: Exposure to LS (15 dyn/cm2) for 24 hours in a cone-plate apparatus markedly increased KCa2.3 and KCa3.1 mRNA expression in cultured human coronary artery ECs (3.1±0.4 and 74±12 fold increase, respectively, p<0.05 vs. static, n=9-14), while oscillatory shear (OS; ±5 dyn/cm2 x 1Hz) moderately increased KCa3.1 but did not affect KCa2.3. Expression of KCa2.1 and KCa2.2 was suppressed under both LS and OS conditions, while KCa1.1 was slightly elevated in LS and unchanged in OS. Addition of STO-609 (10 μ g/ml), a CaMKK inhibitor, significantly decreased LS-induction of KCa2.3 and KCa3.1 expression (1.6±0.2 and 21±4 fold increase, respectively, p<0.05 vs. LS, n=6-7). Western blotting revealed that STO-609 suppressed LS-induced phosphorylation of Akt (Ser473) and p300 (Ser1834). Inhibition of Akt (Inhibitor IV; 1 μ M) or disruption of the interaction between p300/CREB-binding protein and transcription factors such as cAMP response element binding protein (CREB) (KG-501; 25 μ M) also decreased LS-induction of both KCa2.3 and KCa3.1 expression (0.84±0.32 and 1.14±0.32 fold, respectively, after Akt inhibition, n=7; and 0.8±0.1 and 24.0±7.1 fold, respectively, after KG-501 treatment, n=7-9, p<0.05 vs. LS). Moreover, levels of phosphorylated p300 were diminished after Akt inhibition. None of these inhibitors affected the induction of LS-induced phosphorylated CREB (Ser133).
Conclusions: LS increases KCa2.3 and KCa3.1 expression in ECs via a CaMKK/Akt cascade-mediated activation of p300 allowing the binding of transcriptional factors to the DNA. Thus, LS plays an important role in EC adaptation to hemodynamic changes and in the maintenance of EC function via increasing KCa2.3 and KCa3.1 expression.
- © 2012 by American Heart Association, Inc.