The Role of ARL13B in Mitochondrial Homeostasis in Cultured Human Corneal Endothelial Cells.

Purpose: The purpose of this study was to investigate the relationship between primary cilia and mitochondrial function in cultured human corneal endothelial cells (HCECs). Methods: HCECs obtained from donor corneas were cultured and categorized into either differentiated cells or cell-state transition (CST) cells based on the expression of CD44, a non-kinase transmembrane glycoprotein. The frequency of primary cilia was then compared between the differentiated and CST cells using immunofluorescence. Expression of ADP-ribosylation factor-like protein 13B (ARL13B) was inhibited with small interfering RNA (siRNA) to assess the effects on key proteins and mitochondrial function following siRNA treatment via reverse-transcription quantitative polymerase chain reaction, immunofluorescence, and Seahorse XFe24 Extracellular Flux Analyzer (Agilent Technologies) findings. Results: Compared with the differentiated cells, the CST cells exhibited a higher frequency of primary cilia, a lower cell density, and larger nuclei. ARL13B knockdown significantly reduced alpha-tubulin messenger RNA (mRNA) expression, yet did not affect the frequency of primary cilia. On the other hand, ARL13B knockdown altered the localization of aquaporin-1 (AQP1) to the cell membrane and significantly increased mitochondrial oxidative phosphorylation (OXPHOS) without affecting glycolysis. Conclusions: The findings in this study highlight the importance of ARL13B in cultured HCECs and its impact on mitochondrial function and AQP1 localization. Although ARL13B knockdown did not affect the frequency of primary cilia, it significantly enhanced mitochondrial OXPHOS and led to a shift in AQP1 localization, thus suggesting a functional modulation of metabolism in cultured HCECs.