GCN5-TFEB Pathway Regulates Inflammation by Tuning Autophagic Degradation of Retinoic Acid-Induced Gene-I in Human Conjunctival Epithelial Cells.

Abstract

Purpose: To evaluate the pathological role of the GCN5-TFEB axis in dry eye diseases (DEDs) by detecting the autophagic degradation of retinoic acid-inducible gene I (RIG-I), a master RNA-sensing receptor in cells. Methods: RNA-sequencing analysis and quantitative PCR (qPCR) analysis of the expression level of genes related to the interferon (IFN)-I signaling pathway was used to evaluate the inflammatory level of cells overexpressed with General control non-repressed protein 5 (GCN5) or empty vector, which were further confirmed by Western Blot (WB) analysis. siRNA and chemical treatment were used to inhibit GCN5. The acetylation level of transcription factor EB (TFEB) was detected by WB, and qPCR analysis confirmed the transcriptional activity of TFEB. Immunofluorescence and WB were used to measure autophagy and lysosomal biogenesis. When the GCN5 activity was regulated, the autophagic degradation of RIG-I and its pathological role in DEDs were determined by detecting the protein level of RIG-I and the level of cell inflammation. Results: Cells that overexpressed GCN5 showed increased expression of genes involved in the IFN-I signaling pathway compared with cells transfected with an empty vector. Inhibition of GCN5 decreased the acetylation level of TFEB and increased the transcriptional activity of TFEB, combined with the elevated autophagy and lysosomal biogenesis in human corneal epithelial cells (HCECs). Promoting the autophagic degradation of RIG-I by GCN5 inhibition could alleviate the IFN-I signaling pathway. Conclusions: GCN5 could aggravate the activity of the IFN-I signaling pathway in HCECs by acetylating TFEB and inhibiting autophagy, which caused the accumulation of RIG-I. This process could be used to control the overactivation of inflammation in the pathological development of DED.