IGFBP-3 Mediates Metabolic Homeostasis During Hyperosmolar Stress in the Corneal Epithelium

Abstract

Purpose The insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional secretory protein with well-known roles in cell growth and survival. Data in our laboratory suggest that IGFBP-3 may be functioning as a stress response protein in the corneal epithelium. The purpose of this study is to determine the role of IGFBP-3 in mediating the corneal epithelial cell stress response to hyperosmolarity, a well-known pathophysiological event in the development of dry eye disease. Methods Telomerase-immortalized human corneal epithelial (hTCEpi) cells were used in this study. Cells were cultured in serum-free media with (growth) or without (basal) supplements. Hyperosmolarity was achieved by increasing salt concentrations to 450 and 500 mOsM. Metabolic and mitochondrial changes were assessed using Seahorse metabolic flux analysis and assays for mitochondrial calcium, polarization and mtDNA. Levels of IGFBP-3 and inflammatory mediators were quantified using ELISA. Cytotoxicity was evaluated using a lactate dehydrogenase assay. In select experiments, cells were cotreated with 500 ng/mL recombinant human (rh)IGFBP-3. Results Hyperosmolar stress altered metabolic activity, shifting cells towards a respiratory phenotype. Hyperosmolar stress further altered mitochondrial calcium levels, depolarized mitochondria, decreased levels of ATP, mtDNA, and expression of IGFBP-3. In contrast, hyperosmolar stress increased production of the proinflammatory cytokines IL-6 and IL-8. Supplementation with rhIGFBP-3 abrogated metabolic and mitochondrial changes with only marginal effects on IL-8. Conclusions These findings indicate that IGFBP-3 is a critical protein involved in hyperosmolar stress responses in the corneal epithelium. These data further support a new role for IGFBP-3 in the control of cellular metabolism.

IGFBP-3 Regulates Mitochondrial Hyperfusion and Metabolic Activity in Ocular Surface Epithelia during Hyperosmolar Stress

“…To determine whether IGFBP-3 impacts cell growth in cells exposed to hyperosmolar stress, hTCEpi cells were cultured in KBM under isotonic or hyperosmolar conditions (450 mOsm), with or without 500 ng/mL rhIGFBP-3 for six or twenty-four hours. Consistent with our prior work, six hours of hyperosmolar culture arrested hTCEpi cells in the G2/M phase ( Figure 2 A, p < 0.001) [ 41 ]. Unlike the beneficial effects on mitochondrial respiration, treatment with rhIGFBP-3 did not affect cell cycle or cell number ( Figure 2 A,B).…”

“…Similar to the effects of hyperosmolar stress on cells in culture, IGFBP-3 expression was decreased in the mouse corneal epithelium compared to control at all time points tested ( Figure 7 D, p < 0.001 compared to baseline). To further examine the effect of IGFBP-3 on corneal health, IGFBP-3 was topically applied to mouse corneas twice a day every other day for seven days [ 41 ]. Treatment was initiated after 21 days of DED.…”

“…We recently reported that IGFBP-3 functions to inhibit mitophagy and promote mitochondrial respiration in corneal and conjunctival epithelial cells [ 42 ]. We have further shown that IGFBP-3 is decreased during hyperosmolar stress, and thus may play a role in stabilizing mitochondria [ 41 ]. The mechanism by which this occurs is unclear.…”

“…IGFBP-3 is also produced locally in many tissues and is present within the human precorneal tear film [ 39 , 40 ]. We have previously shown that IGFBP-3 is downregulated in response to hyperosmolar stress in corneal epithelial cells [ 41 ]. We further identified a novel role for IGFPB-3 in mitochondrial homeostasis and quality control in corneal and conjunctival epithelial cells [ 42 ].…”

Ameliorative Potential of Resveratrol in Dry Eye Disease by Restoring Mitochondrial Function

“…In diabetic mice, Qu et al [ 27 ] demonstrated that hyperglycemia-induced mitochondrial bioenergetic inadequacy of the lacrimal gland ameliorates early onset of dry eye. Bogdan et al [ 28 ] proposed that insulin-like growth factor binding protein-3 (IGFBP-3) is involved in hyperosmolar stress responses in the corneal epithelium by modulating mitochondrial function. Hyperosmolarity can increase ROS and apoptosis of HCEpiCs ( Figure 1 ), and our results confirm this.…”