Loss of NF-κB Control and Repression of Prdx6 Gene Transcription by Reactive Oxygen Species-driven SMAD3-mediated Transforming Growth Factor β Signaling

Abstract

Activation of transforming growth factor ␤ (TGF␤) in response to increased reactive oxygen species (ROS) leads to pathophysiology of cells/tissues by overmodulation of gene transcription. PRDX6 plays a rheostat role in regulating gene transcription by controlling cellular ROS in maintaining homeostasis; thus, fine tuning of Prdx6 expression is required to optimize ROS levels. Ϫ/Ϫ or Prdx6 Ϫ/Ϫ cells was moderately increased by disruption of NF-B sites, suggesting the role of NF-B in tuning of Prdx6 expression. Findings revealed a mechanism of repression and regulation of PRDX6 expression in cells facing stress or aging and provided clues for antioxidant(s)-based new approaches in preventing ROS-driven deleterious signaling. PRDX6 (peroxiredoxin 6) is a member of an emerging peroxiredoxin family that has GSH peroxidase as well as acidic Ca 2ϩ -independent phospholipase A2 activities (1-7). The peroxiredoxins function in concert to detoxify reactive oxygen species (ROS) 2 and play a cytoprotective role by removing ROS and by limiting ROS levels regulating cell survival signaling (1, 3, 4, 6, 8, 9 -11). The unique ability to regulate signaling and to maintain phospholipid turnover distinguishes PRDX6 from the other five peroxiredoxins (PRDX1 to -5). This molecule is widely expressed, occurring in high levels in the liver, lung, eye lens, and keratinocytes (1, 2, 10, 12-18), whereas reduced expression of PRDX6 can lead to cell death and tissue degeneration (10, 11, 15, 19 -22). PRDX6 has been implicated in maintenance of blood vessel integrity in wounded skin (23, 24) and in development and progression of several diseases, including oxidative-induced cataractogenesis (25), psoriasis (12, 26) atherosclerosis (22), and Parkinsonian dementia (27). Accumulating evidence indicates that expression levels of PRDX6 contribute to pathophysiology of cells and tissues. Several studies have reported decreases in antioxidant levels and increases in ROS levels, leading to a decline in a number of physiological functions because of overmodulation of ROS-mediated gene expression and activation of such factors as TGF␤ and NF-B, hallmarks of the aging process and age-associated degenerative diseases (28 -30). However, given the role of ROS as a mediator of normal or redox signaling, we believe that optimal regulation of Prdx6 expression may require fine tuning to avoid overshooting the desired beneficial effects to the point of perturbing the delicate redox balance essential to maintenance of normal cellular function. Recently, using targeted inactivation of the Prdx6 gene, we reported activation and expression of TGF␤ and TGF␤-mediated suppression of Prdx6 mRNA and protein (1,4,5,31), suggesting that this molecule may have a role in repression of Prdx6 transcription. Quantification by staining with H2DCF-DA established a higher prevalence of ROS in these cells. Also, the activation of TGF␤ in Prdx6-deficient cells was associated with ROS and inhibited by PRDX6 overexpression or MnTBAP, a superoxide dismutase mimetic and a known ROS...

Curcumin abates hypoxia-induced oxidative stress based-ER stress-mediated cell death in mouse hippocampal cells (HT22) by controlling Prdx6 and NF-κB regulation

“…However, alterations in cellular environment have been shown to be associated with the modulation in the activity of several transcription factors including NF-B (78,83,88). On the basis of our previous work as well as other published reports (26,31) showing that NF-B is regulator of Prdx6 transcription, it seems reasonable that ROS induced by hypoxic stress at certain levels could work as a biosignal and thereby alter the activity of NF-B and influence Prdx6 expression in cell. Furthermore, in normal physiological condition, NF-B is inactive (85,86).…”

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

“…To learn whether NF-B is activated in H22 neuronal cells facing H 2O2-induced oxidative stress, and the activation of the NF-B signaling is functional, HIV-1 LTR-CAT constructs and its mutant at NF-B site (a kind gift from Dr. Carole Kretz-Remy) were used to transfect HT22 cells. The HIV-1 promoter contains binding sites for many transcriptional factor NF-B, and can be upregulated 12-to 150-fold following various stresses, including oxidative stress (23). Thus, we used pLTR-CAT wild type and pLTR-CAT PstI (where NF-B sites are disrupted) to monitor the activation of NF-B.…”

“…Because NF-B can function as a repressor of Prdx6 in situations of cellular background (13,23,27), we examined the expression levels of Prdx6 protein and cellular activity of NF-B in HT22 cells during acute oxidative stress. The HT22 cells were pretreated with SN50, a cell-permeable inhibitor peptide that blocks translocation of the NF-B active complex into nucleus.…”

“…Initially, we tested whether NF-B is activated in HT22 cells under oxidative stress, whether its activation is a cause of Prdx6 repression, and whether the process can be blocked by addition of TAT-HA-Prdx6. To this end, we performed transactivation experiments utilizing pHIV-1 linked to CAT vector (13,23). HT22 cells were transfected with pLTR-CAT construct consisting of binding sites for NF-B (Fig.…”

“…Absorbance was measured at 405 nm using a microtiter plate ELISA reader. Transactivation activities were adjusted for transfection efficiencies using GFP/SEAP values (13,23).…”