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Bhavana K. Chhunchha, Nigar Fatma, Eri Kubo, Biju Bhargavan, Dhirendra P. Singh; Sp1-Dependent Enhanced Prdx6 Transcription: A Novel Action of Curcumin in Protecting Lens Epithelial Cells Against Oxidative Stress. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1549.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress and a decline in antioxidant defense are major factors in the progression of age-related diseases, including cataractogenesis. Curcumin protects cells against stressors by activating expression of antioxidant proteins. Herein, we investigated the mechanism by which curcumin induces expression of peroxiredoxin 6 (Prdx6) and protects human lens epithelial cells (hLECs).
To evaluate the protective potential of curcumin, hLECs were treated with different concentrations of curcumin for variable periods, and were exposed to predefined concentrations of H2O2, Paraquat or UVB. MTS and TUNEL assays were done to evaluate cell viability and define cell death, respectively. H2DCFH-DA dyes measured ROS levels. The Web-based MatInspector program was used to spot out putative transcription factor(s) (TFs) binding sites in Prdx6 promoter, and deletion mutant(s) -839, -513, -339 and -131 constructs were prepared with common 3’ end (+109bps) in CAT-basic vector. Gel-shift and ChIP assays, Site-Directed Mutagenesis and CAT-ELISA were used to characterize functional TFs. pCMV-Sp1expression plasmid and Sp1 inhibitor Artemisinin (ART) was used in cotransfection experiments to validate results. Western and real-time PCR analysis monitored Sp1-dependent Prdx6 expression.
Cell viability assay showed that curcumin (5µM) protected LECs against toxicity induced by stressors. Cells displayed curcumin concentration-dependent elevated expression of Sp1 and Prdx6, and reduced expression of ROS. Transfection assays revealed Sp1-dependent upregulation of Prdx6 mRNA and protein. Bioinformatic analysis predicted three Sp1 sites in Prdx6 promoter; Gel-shift and ChIP assays showed that Sp1 bound to Sp1 site(s) (-17/27, -61/69 and -78/90) in Prdx6 promoter, and that curcumin enhanced binding affinity. Transactivation experiments with Prdx6 promoters with Sp1 sites showed increased promoter activity in cells treated with curcumin and cells overexpressing Sp1 or with disruption of all three Sp1 sites, cells did not respond to curcumin. ART abolished Prdx6 promoter activity, validating the curcumin-mediated Sp1-dependent regulation of Prdx6.
Curcumin protects LECs by enhancing endogenous antioxidant potential. Increased production of Prdx6 depends on activation of Sp1. The study may be the basis of curcumin-based approach to counteracting oxidative injury by reinforcing endogenous defenses.
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