Abstract
Purpose :
Purpose: The insulin like growth factor binding protein 3 (IGFBP-3) is a pleiotropic protein with known roles in cell growth and survival. Our prior findings suggest that IGFBP-3 may play a novel role in mitochondrial homeostasis in corneal epithelial cells (CECs). The purpose of this study was to determine expression levels of IGFBP-3 in CECs exposed to hyperosmolar stress and in a mouse dry eye model.
Methods :
Methods: Telomerase-immortalized human corneal epithelial (hTCEpi) cells were cultured in serum-free keratinocyte basal media (330 mOsm, isotonic control). To induce hyperosmolar stress, cells were cultured in 450 mOsm media with or without recombinant human (rh)IGFBP-3 for 2, 6 or 24 hours. Mitochondrial respiration, morphology and polarization were assessed using Seahorse, transmission electron microscopy, MitoTracker and TMRE staining, respectively. Mitophagy was examined by live cell fluorescent imaging, immunofluorescence and western blotting. ROS levels were quantified using Amplex red. Dry eye was induced in C57BL6/N mice by injecting the extraorbital lacrimal gland with 20 milliunits of botulinum toxin. Mice were assessed for dry eye using phenol red thread and corneal staining using fluorescein at baseline, 7, 14 and 28 days. IGFBP-3 expression was quantified in both models at all time points using an IGFBP-3 sandwich ELISA.
Results :
Results: Intra- and extracellular levels of IGFBP-3 were decreased in hTCEpi cells exposed to hyperosmolar stress. Intracellular levels of IGFBP-3 were also decreased in the corneal epithelium of mice with clinical signs of dry eye. hTCEpi cells in hyperosmolar culture further showed a loss in mitochondrial membrane polarization. This was associated with a shift towards a glycolytic phenotype and an increase in mitophagy. Mitochondria in cells exposed to hyperosmolar stress were small and irregular with a balloon-like morphology and loss of cristae. Cells co-treated with rhIGFBP-3 had a metabolic profile similar to control cells. There was a corresponding reduction in mitophagy. Mitochondrial morphology were elongated with intact cristae.
Conclusions :
Conclusions: Taken together, these data confirm an important role for IGFBP-3 in the regulation of mitochondrial homeostasis in CECs exposed to hyperosmolar stress. Further studies are needed to determine the mechanism by which IGFBP-3 regulates these mitochondrial changes and metabolism.
This is a 2021 ARVO Annual Meeting abstract.