MITOCHONDRIAL CALCIUM SIGNALING IN AIRWAY INFLAMMATION: MECHANISTIC INSIGHTS AND REGULATORY DYNAMICS

Mitochondria act as central signaling hubs, coordinating metabolic and immune pathways under physiological and stress conditions. Among these, mitochondrial DNA (mtDNA) extrusion serves as a crucial link between mitochondrial dysfunction and innate immune activation. Here, novel split-GFP based biosensors, TFAMs11 and CpGs11 (FLAME probes), were developed to visualize and quantify mtDNA release in living cells. Using these probes, we show that mitochondrial depolarization triggered by CCCP treatment or MITOK overexpression induces transient openings in the outer membrane that permit the escape of TFAM-bound mtDNA. Similarly, exposure of BEAS-2B airway epithelial cells to diesel exhaust particles or the viral mimic poly I:C elicited mtDNA release and pro-inflammatory signaling. Mechanistically, mitochondrial Ca²⁺ uptake via the MCU complex was essential for this process, as pharmacological inhibition (MCU-i11) markedly reduced mtDNA extrusion and cytokine release. Furthermore, miR-16-5p was identified as a post-transcriptional regulator of MCU and MICU1, modulating mitochondrial Ca²⁺ homeostasis in a cell type dependent manner through altered organelle contact dynamics. Collectively, these findings reveal that mitochondrial Ca²⁺ signaling governs organelle integrity and mtDNA stability, providing a mechanistic framework that links microRNA regulation, inter-organelle communication, and immune activation. Targeting MCU or miR-16-5p may thus offer therapeutic strategies against mitochondrial stress driven inflammation.