Novel AKT Activator SC79 Protects Airway Epithelial Cells Against Cadmium‐Induced Lung Injury

Study Objective Activation of constitutively expressed nitric oxide synthase (NOS) isoforms has beneficial effects via increased ciliary beat frequency and antibacterial nitric oxide (NO) production in the airway and anti‐inflammatory effects in other tissues. It is known that activation of Ca 2+ /Calmodulin can activate endothelial (e) NOS and subsequent NO production. However, other signaling pathways (e.g., AKT, also known as protein kinase B) can activate e/neuronal (n) NOS. These signaling pathways have not been investigated in the airway cells. We hypothesized that activation of the AKT/eNOS pathway, which also activates nuclear factor erythroid 2–related factor 2 (NRF‐2) in many tissues, may have protective effects in lung cells during injury. We tested the responses of airway cells to SC79, a novel AKT activator. To model epithelial injury, we used cadmium (Cd 2+ ), a highly reactive component of cigarette smoke that has been shown to disrupt epithelial barrier integrity and cause oxidative stress in some cell types. Methods To test if SC79 up‐regulates phospho (p)‐AKT, p‐eNOS, and/or NRF‐2 levels, A549 human type II alveolar epithelial cells were stimulated with SC79 for 2 hrs ± pre‐treatment with phosphoinositide‐3‐kinase (PI3K) inhibitor LY294002 for 1hr. Cell lysates were subjected for Western blotting. To determine the nuclear translocation of NRF‐2, cells were subjected to immunofluorescence (IF) with a specific NRF‐2 primary antibody after stimulation with SC79 for 2 hrs. To test if SC79 could protect against disruption of tight junction integrity during lung injury, 16HBE human bronchial epithelial cells were pretreated with Cd 2+ for 1hr and then stimulated with SC79 for an additional 2hrs. Transepithelial electrical resistant (TEER) measurements were taken using an epithelial ohm meter. Results We observed an approximately 2‐fold induction of p‐AKT, p‐eNOS and NRF‐2 with SC79 treatment that lasted >2 hrs. Pre‐incubation with LY294002 blocked p‐AKT, p‐eNOS, and NRF‐2 induction. IF revealed a 2‐fold increase in nuclear‐localized NRF‐2 upon SC79 stimulation. Additionally, exposure of 16HBE cells to Cd 2+ led to an almost complete reduction in TEER, which was blocked by SC79 for up to 24 hrs. Conclusions Our preliminary data suggest that SC79 upregulates pAKT, peNOS and NRF‐2 in lung cells through a mechanism involving PI3K. Furthermore, treatment with SC79 protects the epithelial integrity against cadmium‐induced injury. Future work will be directed toward testing the role(s) of eNOS and Nrf2 in the protective effects of SC79. Support or Funding Information R01DC016309, R21AI137484 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .