Smad3-Mediated TGF ß1 Signaling Represses NF-B Transactivation of PRDX6 Gene, a Survival Signaling in Lens Epithelial Cells Facing Oxidative Stress

Abstract

Purpose:: Oxidative stress has been implicated in age-related cataract. We previously reported the prevalence of reactive oxygen species (ROS)-driven Smad3-mediated, TGFß1-induced deleterious signaling in eye lens/lens epithelial cells (LECs) that lack the antioxidant Peroxiredoxin 6 (PRDX6) protein. Herein, using LECs from Prdx6 and Smad3 knockout mice, we investigated the TGFß1-induced Smad3-mediated repression of NF-ΚB transactivation of Prdx6 gene in LECs facing oxidative stress.

Methods:: LECs were generated from Smad3^-/-, Prdx6^-/- and wild type mice and maintained in complete DMEM. Western analysis and real time quantitative-PCR were used to measure expression levels of PRDX6, α-sm-actin, NF-ΚB, and Smad3/pSmad3, αA-crystallin using specific antibodies and primers. Transactivation assay was done with various deletion mutants of Prdx6 gene promoter linked to CAT to define the contribution of Smad3-mediated TGFß1 signaling in repressing NF-ΚB transactivation of Prdx6 gene. SN50, an inhibitor of NF-ΚB, was used to confirm the results. MTS assay was conducted to assess the viability of LECs.

Results:: LECs isolated from Smad3^-/- or Prdx6^-/- and wild type mice expressed α A-crystallin, a specific marker for LECs. A significant increase in the expression of PRDX6 mRNA and protein occurred in Smad3^-/- LECs; these cells showed resistance against TGF ß1- and H₂O₂-induced insult compared to Smad3^+/+ LECs. The Prdx6-CAT promoter (-1139, -839 and -513 to + 109 nts), which contains NF-ΚB sites, was transactivated more efficiently in Smad3^-/- LECs than a mutant promoter that lacked such sites. Addition of SN50, an NF-ΚB inhibitor, diminished the transcription of Prdx6. Repression of Prdx6 promoter in Smad3^+/+ but not in Smad3^-/- following TGFß1 or H₂O₂ treatment, demonstrated the Smad3-mediated repression of NF-ΚB transactivation of Prdx6 gene. Transactivation of HIV-1LTR-CAT constructs, which contain multiple NF-ΚB sites, validated that NF-ΚB stimulates the transcription of Prdx6 gene in Smad3^-/- LECs.

Conclusions:: Smad3-mediated TGFß1 signaling represses the transactivation of Prdx6 by NF-ΚB. NF-ΚB signaling may protect lens cells from damage during oxidative stress or aging. Understanding the regulation and regulatory function of PRDX6 will help to discover new approaches to prevent ROS-driven deleterious signaling in eye lens.

Keywords: oxidation/oxidative or free radical damage • gene/expression • transcription