Specificity protein, Sp1-mediated increased expression of Prdx6 as a curcumin-induced antioxidant defense in lens epithelial cells against oxidative stress

Abstract

Peroxiredoxin 6 (Prdx6) is a pleiotropic oxidative stress-response protein that defends cells against reactive oxygen species (ROS)-induced damage. Curcumin, a naturally occurring agent, has diversified beneficial roles including cytoprotection. Using human lens epithelial cells (hLECs) and Prdx6-deficient cells, we show the evidence that curcumin protects cells by upregulating Prdx6 transcription via invoking specificity protein 1 (Sp1) activity against proapoptotic stimuli. Curcumin enhanced Sp1 and Prdx6 mRNA and protein expression in a concentration-dependent manner, as evidenced by western and real-time PCR analyses, and thereby negatively regulated ROS-mediated apoptosis by blunting ROS expression and lipid peroxidation. Bioinformatic analysis and DNA–protein binding assays disclosed three active Sp1 sites (−19/27, −61/69 and −82/89) in Prdx6 promoter. Co-transfection experiments with Sp1 and Prdx6 promoter–chloramphenicol acetyltransferase (CAT) constructs showed that CAT activity was dramatically increased in LECs or Sp1-deficient cells (SL2). Curcumin treatment of LECs enhanced Sp1 binding to its sites, consistent with curcumin-dependent stimulation of Prdx6 promoter with Sp1 sites and cytoprotection. Notably, disruption of Sp1 sites by point mutagenesis abolished curcumin transactivation of Prdx6. Also, curcumin failed to activate Prdx6 expression in the presence of Sp1 inhibitors, demonstrating that curcumin-mediated increased expression of Prdx6 was dependent on Sp1 activity. Collectively, the study may provide a foundation for developing transcription-based inductive therapy to reinforce endogenous antioxidant defense by using dietary supplements.

Delivery of a protein transduction domain-mediated Prdx6 protein ameliorates oxidative stress-induced injury in human and mouse neuronal cells

“…Total RNA was isolated using the single-step guanidine thiocyanate/phenol/chloroform extraction method (TRIzol, Invitrogen) and converted to cDNA using Superscript II RNAase H-Reverse Transcriptase. Quantitative real-time PCR was performed with SYBR Green Master Mix (Roche Diagnostic, Indianapolis, IN) in a Roche LC480 Sequence detector System (LightCycler 480II) as described previously (12,61). Primers were used as shown in Table 1 and the comparative crossing point (C p) method was used to calculate relative fold expression levels using LightCycler 480 software (release 1.5.0 SP3).…”

“…To evaluate whether loss of Prdx6 in neuronal cells is associated with increased ROS expression and decreased cell viability during oxidative stress, we exposed primary cell culture of HCN-2 to different concentrations of paraquat, a superoxide generator (12,19). We assessed cell viability and expression levels of ROS and Prdx6, as shown in Fig.…”

“…The antioxidant protein Prdx6 has GSH peroxidase as well as acidic Ca 2ϩ -independent phospholipase activities (aiPLA 2 ) (13,25,43). Prdx6 uses redoxactive Cys47 to reduce peroxides and protect cells from membrane, DNA, and protein damage mediated by lipid peroxidation (12,43). Prdx6, a cytosolic protein, is also present in lysosomes (59), endoplasmic reticulum (ER), plasma membrane, and mitochondria.…”

“…Thus it seems reasonable that modulation of ROS production at certain levels might alter the activity of NF-B, leading to repression of Prdx6 in cells. Prdx6 is known to protect many cell types derived from both mouse and human against various oxidative stresses, such as H 2 O 2 , paraquat, UVB, heat stress, and ER stress (12,25). Furthermore, Prdx6 is a potent cytoprotective enzyme in the epidermis (40) and is required for blood vessel integrity in wounded skin (41).…”

Isolation and characterisation of peroxiredoxin 6 promoter from sheep (Ovis aries)

“…Sp-1 is an oxidative stress-induced transcription factor (11) that can bind Sp1-responsive elements and increase transcription of Prdx6 gene (2). TFBSs for Sp-1 were ubiquitous in 5′-proximal region of Prdx6 gene promoter.…”