Abstract
Purpose :
Many blinding diseases are associated with dysfunction of the antioxidant pathway due to loss of transcription factors, brain and muscle arnt-like protein 1 (Bmal1) and NFE2-related factor 2 (Nrf2) in aging. We previously showed that Metformin (Met)-mediated preservation of Bmal1/E-box and Nrf2/ARE (antioxidant response element) cooperativity was key for Peroxiredoxin (Prdx) 6 expression and cytoprotection. Here we test whether Bmal1-Nrf2 interacts and Met promotes enrichment of the Bmal1-Nrf2 heterodimers at E-Box and ARE sites for peak Prdx6 transcription.
Methods :
Human (h) lens epithelial cells (LECs) overexpressing pGFP/Bmal1 or pGFP/Nrf2 and Bmal1 or Nrf2 depleted (using shRNA) LECs were treated with Met (0-1mM) for variable periods. Immunoprecipitation(IP)-Western blot (WB) analysis was performed to examine Bmal1-Nrf2 interaction by cross probing with Bmal1 or Nrf2 antibody. MTS and H2-DCF-DA dye measured levels of cell viability and ROS against H2O2 (0-150µM). ChIP-qPCR-WB with specific probes was used to examine Bmal1-Nrf2 enrichment at E-box/ARE of Prdx6 gene. Wildtype/mutants biotinylated oligos mutated at E-box (CA(T)CGT(A)G) or ARE (T(G)G(T)AnnnnnGC) was incubated with nuclear extract of LECs, the complex was pulled out with streptavidin and immunoblotted with Bmal1or Nrf2 antibody to validate the results. Prdx6 promoter (-918/+30nt)-LUC or its mutants at E-Box/ARE was used for the promoter assay. Two-tailed Student’s t-test and one-way ANOVA were used for statistical analysis.
Results :
Pulldown assay showed that Bmal1-Nrf2 formed heterodimers, and in response to Met, the complex was increased (p<001). Met treatment led to enhanced Nrf2 and Bmal1and their target Prdx6 gene with increased cell survival. Mechanistically, ChIP-qPCR-WB and the promoter assays coupled with loss-and-gain of function and mutation of E-box/ARE identified that the E-box or ARE was significantly enriched with Bmal1-Nrf2 heterodimers with increased Prdx6 promoter activity in response to Met (p<0.001). Oligos pulldown assay with Met-treated LECs nuclear extracts confirmed that E-Box or ARE was enriched with the heterodimers (P<0.001), and upregulated Prdx6 transcription to protect LECs.
Conclusions :
Our data reveal for the first time that Bmal1-Nrf2 are complexes that regulate Prdx6 gene involved in various aspects of cytoprotective functions through associated E-box or ARE.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.