Abstract
Purpose :
TMEM97, an ER-resident receptor protein, plays a role in cholesterol processing, cancer, and neurodegenerative diseases, impacting various aspects of ocular biology and pathology. The oxysterol 20(S)-OHC serves as a highly selective ligand for TMEM97 (1). Oxysterols, derived from cholesterol via reactive oxygen species or enzymatic oxidation, are involved in numerous biological activities such as lipid metabolism, autophagy, and apoptosis. In this study, we use transcriptome analysis to investigate differential gene expression in human corneal epithelia (HCE) treated with 20(S)-OHC. Significantly altered TMEM97-dependent pathways include cholesterol metabolism and PI3K-Akt signaling, which are both important regulators of corneal epithelial function (2). This data provides key insight regarding the influence of 20(S)-OHC on cholesterol metabolism and signaling in HCE.
Methods :
Human corneal epithelial (HCE) cells were treated with 10 µg/mL of 20(S)-OHC for 8 hours. Cells were harvested and total RNA was prepared with a RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA sequencing and statistical analysis were performed by Novogene (Sacramento, CA) on total RNA from HCE. Signaling pathway findings in RNA seq data were validated using assays for the activity of Akt, where Akt phosphorylation was analyzed by Western blot at several different time points using phospho-Akt (Ser473) antibody according to techniques reported previously (3).
Results :
Transcriptome analysis revealed that 113 genes were significantly differentially expressed following 20(S)-OHC treatment of HCE. Among these, 52 genes were upregulated and 61 genes were downregulated. Enrichment analysis revealed that the differentially expressed genes are associated with cholesterol metabolism and sterol biosynthesis. Additionally, we observed enrichment in the PI3K-Akt signaling pathway. Following treatment with 20(S)-OHC, Akt activation was assayed at several different time points using Western blot analysis, with most prominent activation observed at 2 hours after treatment.
Conclusions :
Our data suggests that 20(S)-OHC stimulates TMEM97-dependent Akt signaling, and that cholesterol metabolism is altered by 20(S)-OHC treatment. These results inform future studies to investigate the applications of oxysterols to treatment of corneal epithelial pathologies.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.