Abstract Rationale β-ionone is a volatile apocarotenoid metabolite derived from β-carotene. Our recent work demonstrated that β-ionone directly activates OPN3-expressing airway smooth muscle, reducing contractility through cAMP-dependent mechanisms. However, the endogenous origin of β-ionone in the airway remained unknown. β-Carotene oxygenase-2 (BCO2) is a mitochondrial carotenoid-cleaving enzyme expressed in metabolically active tissues such as liver, retina, and adipose tissue, where it cleaves β-carotene metabolites into b-Ionone molecules. The respiratory epithelium, constantly exposed to dietary and environmental metabolites, represents a plausible but unexplored site of local β-ionone source. To explore this hypothesis, we examined BCO2 expression and localization in immortalized human airway epithelial cells (Beas2B) and murine airway tissue. Methods BCO2 expression was assessed in immortalized human airway epithelial (Beas2B) cells and murine precision-cut lung slices (PCLS). Beas2B cells were fixed in 4% paraformaldehyde, permeabilized, and immunostained with rabbit polyclonal anti-BCO2 (Catalog #14324-1-AP, Thermo Fisher Scientific, 1:200), with a DAPI nuclear counterstain. A no-primary control was included to assess background fluorescence. Mouse precision-cut lung slices were prepared as previously described. Slices were sectioned at 130 µm, fixed in acetone (10 min, 4C), blocked (1% BSA + glycine in PBST), and stained with the same primary/secondary antibody protocol. All samples were imaged using a Nikon A2 confocal microscope under identical laser and exposure settings. RT-PCR was confirmed to measure mRNA transcript presence. Results BCO2 protein was detected in Beas2B cells with punctate cytoplasmic staining consistent with mitochondrial localization. Negligible fluorescence was observed in no-primary controls. In murine airway epithelium, BCO2 signal localized along the luminal epithelial border, confirming epithelial expression in vivo. RT-PCR confirmed transcript presence in Beas2B (BCO2 Ct = 25.0; GAPDH Ct = 15.07). Conclusion Our findings demonstrate that BCO2 is expressed in airway epithelium. This epithelial driven metabolic process represents a possible unrecognized regulatory axis integrating diet, cellular metabolism, and airway physiology. This abstract is funded by: NIH
Uribe et al. (Fri,) studied this question.