Abstract
Purpose :
We previously reported that Per2m/m mice that are lacking circadian rhythms recapitulate the retinal vascular alterations associated with diabetic retinopathy (DR). The vascular dysfunction in Per2m/m mice was largely attributed to dysregulated expression of connective tissue growth factor (CTGF/CCN2), a downstream target and core component of the TGF-β pathway. At the molecular levels, CTGF gene expression is dependent on the canonical Wnt/β-catenin pathway. Interestingly, signaling through PER2 leads to increased casein kinase 1 (CK1)-ε activity, a signaling events also associated with β-catenin phosphorylation and degradation. Therefore, we hypothesized that silencing of Per2 increases β-catenin stability and nuclear translocation and subsequent transactivation of the CTGF gene.
Methods :
Eyes from wild type and Per2m/m were enucleated, sectioned and stained for CTGF. Human retinal endothelial cells (HRECs) were transfected with siRNA for Per2 and the protein expression of CTGF and β-catenin was determined using qRT-PCR and western blot analysis. The nuclear localization of β-catenin was examined by immuonofluorescence. ATCF/LEF luciferase reporter assay (TOPFLASH) assay was used to validate the involvement of β-catenin in the activation of CTGF.
Results :
Per2m/m retinas showed increased CTGF immunostaining in the ganglion cell layer and retinal endothelium. Loss of Per2 function resulted in increased expression of CTGF and β-catenin. siRNA-mediated silencing of Per2 in HRECs increased nuclear accumulation of β-catenin as compared to the control siRNA. The TOPFALSH assay showed an increase in luminescence for HRECs transfected with Per2 siRNA.
Conclusions :
Our studies show that loss of Per2 results in activation of CTGF via nuclear entry of β-catenin. The future therapies aimed at targeting the β-catenin-CTGF pathway may provide a novel tool for correction of vascular dysfunction in DR.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.