ER stress-associated transcription factor CREB3 is essential for normal Ca²⁺, ATP, and ROS homeostasis

In mammalian cells, mitochondrial respiration produces reactive oxygen species (ROS) such as superoxide (O₂^-), which is then converted by the SOD1 enzyme into hydrogen peroxide (H₂O₂), the predominant form of cytosolic ROS. ROS at high levels can be toxic, but below this threshold are important for physiological processes acting as a second messenger similar to Ca²⁺. Mitochondrial Ca²⁺ influx from the ER increases ATP and ROS production, while ATP and ROS can regulate Ca²⁺ homeostasis, leading to an intricate interplay between Ca²⁺, ROS, and ATP synthesis. The Unfolded Protein Response (UPR) proteins ATF6α and XBP1 contribute to protection from oxidative stress through upregulation of Sod1 and Catalase genes. Here, UPR-associated protein CREB3 is shown to play a role in balancing Ca²⁺, ROS, and ATP homeostasis. Creb3-deficient mouse embryonic fibroblast cells (MEF^-/-) were susceptible to H₂O₂-induced oxidative stress while having a functioning antioxidant gene expression response compared to MEF^+/+. MEF^-/- cells also contained elevated basal cytosolic ROS levels, which was attributed to drastically increased basal mitochondrial respiration and spare respiratory capacity relative to MEF^+/+. MEF^-/- cells also showed an increase in endoplasmic reticulum Ca²⁺ release and mitochondrial Ca²⁺ levels hinting at a potential cause for MEF^-/- cell mitochondrial dysfunction. These results suggest that CREB3 is essential for maintaining proper Ca²⁺, ATP, and ROS homeostasis in mammalian cells.