Epidermal Growth Factor Receptor Regulates Beclin-1 in Hyperoxia

Abstract

ABSTRACTIntroductionMore than 300,000 patients are placed on mechanical ventilation in the ICU annually in the United States. Many of these patients will require hyperoxia (>70% oxygen) to prevent tissue hypoxia. We have previously shown that the tyrosine kinase receptor epidermal growth factor receptor (EGFR) regulates alveolar epithelial apoptosis in hyperoxia. Mice with reduced total EGFR activity (EGFRWa5/+mice) show improved survival and reduced markers of ALI compared with WT in hyperoxia. How EGFR regulates additional cell death pathways in hyperoxia remains to be explored.MethodsWe tested the hypothesis that EGFR regulates the molecule coiled-coil moesin-like BCL2-interacting protein (Beclin-1 [BCN1]), which is known to induce autophagy, in hyperoxia. Effects of 100% oxygen were studied in EGFRWa5/+mice and wildtype (WT) littermates. Mice were exposed to 100% oxygen for 24, 48, and 72 hours and compared with normoxia controls. Lungs were analyzed for BCN1 via Western blot (WB), RT-qPCR, immunohistochemistry (IHC), and flow cytometry. Whole-lung and cell-specific analyses were completed. The specific BCN1 phosphorylation site (p-BCN1), serine residue 91 and 94 (Ser91, −94) for mouse (Ser93, −96 for human), that regulates autophagy was examined.ResultsIn WT mice, hyperoxia induced increased whole-lung BCN1 compared with normoxia via WB and RT-qPCR. Whole-lung and epithelial-specific p-BCN1 was increased compared with normoxia via IHC and flow cytometry. Hyperoxia led to increased autophagy via WB and RT-qPCR. EGFRWa5/+mice showed increased BCN1 and reduced p-/total BCN1 ratios in hyperoxia compared with WT via WB. EGFRWa5/+mice also showed increased BCN1 via RT-qPCR and decreased epithelial-specific p-/total BCN1 ratios via flow cytometry, although this did not reach statistical significance. EGFRWa5/+mice showed reduced autophagy via WB compared with WT in hyperoxia.ConclusionsHyperoxia induced increased whole-lung and epithelial-specific BCN1 in hyperoxia and resulted in increased autophagy. Decreased EGFR activity, which leads to improved survival in hyperoxia, resulted in decreased whole-lung p-/total BCN1 ratios and decreased epithelial-specific p-/total BCN1 ratios in hyperoxiain vivo. Because BCN1 and autophagy can regulate cell death, EGFR regulation of p-BCN1 and autophagy is a mechanism in hyperoxia with therapeutic potential.