Sumoylation At K16 Of Specificity Protein 1 Represses Prdx6 Transcription Leading To Lens Epithelial Cell Death During Oxidative Stress And Aging

Abstract

Purpose : A decline in antioxidant defense in aging or oxidative stress is a major culprit in age-related blinding diseases. Specificity protein (Sp)1 transactivates antioxidant protein Peroxiredoxin (Prdx)6 by directly binding to its Sp1-response elements. Using Prdx6-deficient (Prdx6^-/-) lens epithelial cells (LECs) and LECs facing oxidative stress, we showed that aberrant Sp1 Sumoylation at K (lysine) 16 during oxidative stress and aging dysregulates its DNA binding activity and represses Prdx6 expression.

Methods : Sumoylation status of Sp1 in Prdx6^-/- mouse (m) LECs, aging human (h) LECs and LECs facing oxidative stress (H₂O₂ and/or UVB ) for variable times was examined, along with its correlation with expression of Prdx6, Sumo1 and Senp1 by using Sandwich-Sumo1/Prdx6 or Sp1 Specific ELISA, immunoprecipitation, and Western and qPCR analyses with specific probes. Sumo1, Senp1 and Sp1 and its mutant at Sumo1 site, K16 to Arginine (R) created by Site-Directed-Mutagenesis were cloned into pGEX-2T and pCMV expression vectors, respectively. Sp1 and Sp1^K16R transfected cells were treated with cycloheximide or actinomycinD to examine their relative protein stability. MTS and H2DCF-DA dye assays were done to measure cell viability and ROS levels, respectively. ChIP or gel-shift assay and transactivation assay with Prdx6 promoter containing Sp1 sites ( -19/27, -61/69 and -82/89) monitored the effects of Sumoylation on Sp1’s DNA binding activity and transactivation potential.

Results : Sumoylation of Sp1 was increased in aging LECs and LECs facing oxidative stress, and was well correlated with increased Sumo1 and decreased Senp1, Prdx6 and Sp1 abundance. Increased Sumoylation patterns of Sp1 in Prdx6^-/- cells were similar to those in aging lenses/LECs. Sumoylation assays revealed that Sumo1 conjugation occurred at K16, and Sumoylation of Sp1 reduced its cellular stability and decreased Prdx6 expression and cell viability. Downregulation of Prdx6 was due to inhibition of DNA binding activity of Sumoylated Sp1. Sp1-siRNA and cotransfection with Sumo1 or Senp1 and/or Sp1^K16R showed that Prdx6 was regulated by oxidative stress in an Sp1 Sumoylation-dependent manner.

Conclusions : This study may provide a foundation for a strategy to repair the effects of aberrant Sumoylation signaling in aging or oxidative stress by controlling the deleterious process of Sumoylation.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.