Akt-dependent Regulation of Nitric Oxide and Inflammation in Airway Cells

Abstract

Activation of endothelial nitric oxide synthase (eNOS) increases ciliary beating, kills bacteria, facilitates mucus clearance, and maybe anti-inflammatory in airway cells. Protein kinase B/Akt activation of eNOS is understudied in airway cells. We hypothesized that Akt plays a role in the activation of eNOS/ nitric oxide (NO) and protects against inflammation in airway cells. To test if SC79, a direct Akt activator induces phospho (p)-eNOS, A549 type-II alveolar epithelial cells were treated with phosphoinositide-3-kinase (PI3K) inhibitor LY294002 (10μg/ml) for 1hr before stimulating with SC79 (10μg/ml) for 2 hrs. Next, cells were lysed and were run by Western blotting. To measure NO production, A549 cells were loaded with a fluorescent probe (DAF-FM) for 60 min and then stimulated with SC79. To investigate whether SC79 could protect against tumor necrosis factor (TNF-α) induced cytokine production, A549 cells were treated with TNF-α (0.1μg/ml) ± SC79 for 24 hrs and interleukin (IL-6 and IL-8) levels were measured by quantitative (q)-PCR. We observed a 2-fold induction of p-eNOS after the treatment with SC79, which was blocked by LY294002. Live-cell imaging data show a significant increase in NO production with SC79 (p<0.05). TNF-α induced interleukins were reversed by SC79 in airway cells (p<0.05). Our preliminary data suggest Akt plays a role in PI3K-dependent NO production in airway cells. Additionally, treatment with SC79 reversed TNF-α induced IL-6 and IL-8 in airway cells. Future work will be directed toward testing the downstream mechanisms of eNOS/NO in response to SC79 in patient-derived primary cells.