Abstract
Purpose :
Hyperosmolarity of the precorneal tear film plays a key role in the underlying pathobiology of dry eye disease (DED). The insulin-like growth factor binding protein-3 (IGFBP-3) is a pleiotropic protein present in the precorneal tear film and is downregulated in response to hyperosmolar stress (H-stress) in corneal epithelial cells (CECs). The purpose of this study was to evaluate the role of IGFBP-3 in mitochondrial morphology and metabolic activity in CECs and corneal epithelium exposed to H-stress.
Methods :
Telomerase-immortalized human corneal epithelial (hTCEpi) cells were cultured in isotonic keratinocyte basal media (KBM, 330 mOsm) or KBM with osmolarity values up to 450 mOsm, with or without recombinant human (rh)IGFBP-3 for 2 or 24 hours. Mitochondrial respiration, morphology, and dynamics were analyzed using a seahorse metabolic flux analyzer, real-time PCR, and transmission electron microscopy. Expression and localization of IGFBP-3 and type I IGF-receptor (IGF-1R) were analyzed using subcellular fractionation/immunoblotting. Protein translation was quantified with a SUnSET assay. Injection of 20 mU of botulinum toxin B into the exorbital lacrimal gland of C57BL6/N mice was used to induce DED. DED was confirmed using a phenol red thread test and by corneal staining using fluorescein. After 21 days of DED, mice were treated topically with balanced salt solution with or without rhIGFBP-3. IGFBP-3 expression in the corneal epithelium was assessed using an IGFBP-3 ELISA and immunofluorescence.
Results :
H-stress decreased IGFBP-3 expression in vitro and in vivo. Intra- and extracellular levels of IGFBP-3 parallel mitochondrial respiration in CECs under H-stress. At 2 hours of H-stress, IGFBP-3 induced mitochondrial translocation of IGF-1R. At 24 hours, there was a stress-mediated decrease in oxidative phosphorylation that was blocked by the addition of exogenous IGFBP-3. Treatment with IGFBP-3 also promoted IGFBP-3 nuclear localization, mitochondrial hyperfusion, and protein translation. In vivo, exogenous IGFBP-3 restored CEC health.
Conclusions :
These data show that IGFBP-3 mediates mitochondrial structure and function through an early and late biphasic response. These findings could pave the way for novel therapeutics for diseases in ocular tissues, such as DED, with underlying mitochondrial and metabolic dysfunction.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.