Novel anti-oxidative role of calreticulin in protecting A549 human type II alveolar epithelial cells against hypoxic injury.

Short-term hypoxic pretreatment is an effective approach to protect the lung from subsequent prolonged hypoxic injury under conditions such as lung transplantation, shock, and trauma. However, the signaling pathways are not well understood. By use of high-throughput, two-dimensional electrophoresis combined with mass spectrometry, we found that short-term hypoxic treatment upregulated calreticulin (CRT), an endoplasmic-reticulum stress protein, in A549 human type II alveolar epithelial cells. Genetic manipulation of CRT expression in A549 cells through small interferring RNA inhibition or overexpression demonstrated a positive correlation between CRT expression level and cell viability in subsequent prolonged hypoxia, which indicates that CRT is a key mediator of short-term hypoxia-induced cell protection. Importantly, CRT overexpression prevented reactive oxygen species (ROS) accumulation during prolonged hypoxia by inducing the expression of thioredoxin (TRX), an antioxidant, in A549 cells. Furthermore, CRT promoted the nuclear translocation of nuclear factor-E2-related factor 2, the transcription factor of TRX. Finally, overexpressing an inactive TRX mutant reversed the effects of CRT on ROS accumulation and cell protection. Our results demonstrate that CRT stimulates the anti-oxidant pathway and contributes to short-term hypoxia-induced protection in A549 type II alveolar epithelial cells, which may have potential therapeutic ramifications for hypoxic pulmonary diseases.