Abstract
Purpose :
Purpose: Epithelial mesenchymal transition (EMT) of lens epithelial cells (LECs) is one of the most important pathogenic mechanisms in lens fibrotic disorders such as anterior subcapsular cataract (ASC) and posterior capsule opacification (PCO). We recently showed that the transcriptional factor CREB regulates the stress response and aging process of LECs in human cataractogenesis. In the present study, we demonstrated that CREB plays an essential role in lens EMT.
Methods :
Methods: All experiments were based on injury induced ASC mouse model and TGF β-induced EMT of mouse lens epithelial cells. Quantitative real-time PCR, Western Blot, immunofluorescence, and whole-mount immunofluorescence were used to examine the differential EMT gene expression levels.
Results :
Results: Knockout or gene silencing of CREB significantly attenuated EMT progression of mouse LECs, and CREB phosphorylation level at S133 was upregulated in EMT of mouse LECs. The overexpression of wild-type CREB enhanced the expression of mesenchymal marker genes. However, the overexpression of S133A-CREB, a mutant mimicking constant dephosphorylation at S133, further enhanced mesenchymal gene expression of mouse LECs. Meanwhile, the lens capsule of S133A-CREB mutant mice demonstrated enhanced mesenchymal gene expression. ChIP assay and CREB mutant functional assay showed that S133A-CREB serves as a novel transcription factor to activate mesenchymal gene expression. Moreover, the introduction of R314A mutation in WT-CREB, which abolish the interaction of CREB and CRTC family co-activators, has no effect on EMT gene expression. While R314A mutation in S133A-CREB led to suppressed expression of mesenchymal genes in mouse LECs.
Conclusions :
Conclusions: CREB plays important roles in EMT of mouse LECs partially through the S133 phosphorylation-dependent manner. It appears that S133A-CREB serves as a novel transcription factor regulating mesenchymal genes in mouse LECs. Mechanistically, S133A-CREB interacts with CRTC family co-activators, and thereby resides on the promoter regions of the mesenchymal genes to activate transcription. [Supported by NSFC#82271071, #81970787, #81970784, #82000876, #81700821, #81770910, NSF-Guangdong Province and Guangzhou City Joint Program#20191515120014, ZOC-SKLO#3030901010110 & 030901010111].
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.