Abstract
Purpose :
Keratoconus (KC), a leading cause for cornea transplantation, is a progressive corneal thinning disease where the underlying molecular events are poorly understood. Our ongoing RNA seq and proteomics studies of keratoconus corneas and cultured stromal cells suggest changes in AKT/mTOR signaling. Here we tested this signaling mechanism in cultured KC and donor (DN) stromal cells.
Methods :
DN (n = 5) and KC (n = 5) stromal fibroblast cultures were subjected to the following stresses, 1) ER stress with tunicamycin (2ug), 2) calcium ion overload using calcium ionophore A23187 (2uM) and 3) oxidative stress using H2O2 (20 uM), and cell viability measured by the MTT assay. The above concentrations were used after determining the LC50 for each drug by monitoring cell death at increasing concentrations of the drugs. Phosphorylated intermediates of AKT and mTOR signals were analyzed in cell lysates by immunoblotting.
Results :
The expression of mTOR pathway associated proteins were significantly altered in untreated and treated KC cells as compared to DN cells. Basal levels of pmTOR (active form) was 5-fold higher in KC cells, and after subjecting the cells to cellular stresses, pmTOR decreased by 3-fold in KC cells as compare to DN. Consistent with this finding mTOR pathway regulators PI3K (2.6 fold) and P62 (3.4 fold) were increased in KC. cells as compare to DN. At stressed condition, there was 2.5-fold increase in PI3K expression in KC cells whereas there was no change in P62 expression. We examined the expression of downstream effectors of both mTORC1 and mTORC2 complexes and only the downstream effector of the mTORC1 complex, 4EBP1 was 3-fold in KC cells at basal level and 6-fold after subjecting to cellular stress in KC compared to DN cells.
Conclusions :
MTORC1 signaling allows cells to regulate protein synthesis, proliferate, grow or undergo autophagy or apoptosis in response to nutrient levels, energy, growth factors and stress. Therefore, MTORC1 signaling may be central to stromal cell homeostasis in the avascular nutrient-restricted cornea. Our keratoconus cell culture model suggests major changes in this signaling mechanism in keratoconus pathogenesis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.