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Background: PEPT1 is a rheogenic transport protein in the apical membrane of intestinal epithelial cells capable of transporting essentially all possible di- and tripeptides that are generated from the luminal protein breakdown. In addition, several anticancer, antimicrobial and antiviral drugs are taken up from the intestinal lumen via PEPT1 and therefore PEPT1 is a target for efficient drug delivery via prodrug approaches. Thus, understanding PEPT1 gene regulation is not only of importance for dietary adaptation but also for drug treatment., Methods: In silico analysis of the Pept1 promoter was performed using MatInspector. Pept1 promoter constructs were generated and cotransfected with an Nrf2 expression plasmid. Caco-2 cells were stimulated with Nrf2 inducers followed by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP). Biological relevance was investigated using western blot analysis and transport activity assays., Results: Reporter gene assays showed transcriptional activation of the Pept1 promoter in response to Nrf2 overexpression. EMSA as well as ChIP analysis validated Nrf2 binding to the ARE located closest to the start codon (Pept1-ARE1). Induction of the Nrf2 pathway resulted in increased endogenous PEPT1 protein abundance as well as transport activity. Moreover, we demonstrate that also the induction of autophagy by MG132 resulted in elevated Nrf2 binding to Pept1-ARE1 and increased PEPT1 protein expression., Conclusion: In summary, we identified a biologically active Nrf2 binding site within the Pept1 promoter which links Pept1 to the cellular defense program activated by Nrf2., General Significance: This study identifies Pept1 as an inducible target gene of the Nrf2 pathway., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Saturated fatty acids (SFAs) induce hepatocyte cell death, wherein oxidative stress is mechanistically involved. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a master transcriptional regulator of cellular antioxidant defense enzymes. Therefore, Nrf2 activation is regarded as an effective strategy against oxidative stress-triggered cellular damage. In this study, tert-butylhydroquinone (tBHQ), a widely used Nrf2 activator, was initially employed to investigate the potential protective role of Nrf2 activation in SFA-induced hepatoxicity. As expected, SFA-induced hepatocyte cell death was prevented by tBHQ in both AML-12 mouse hepatocytes and HepG2 human hepatoma cells. However, the protective effect of tBHQ is Nrf2-independent, because the siRNA-mediated Nrf2 silencing did not abrogate tBHQ-conferred protection. Alternatively, our results revealed that autophagy activation was critically involved in the protective effect of tBHQ on lipotoxicity. tBHQ induced autophagy activation and autophagy inhibitors abolished tBHQ's protection. The induction of autophagy by tBHQ exposure was demonstrated by the increased accumulation of LC3 puncta, LC3-II conversion, and autophagic flux (LC3-II conversion in the presence of proteolysis inhibitors). Subsequent mechanistic investigation discovered that tBHQ exposure activated AMP-activated protein kinase (AMPK) and siRNA-mediated AMPK gene silencing abolished tBHQ-induced autophagy activation, indicating that AMPK is critically involved in tBHQ-triggered autophagy induction. Furthermore, our study provided evidence that tBHQ-induced autophagy activation is required for its Nrf2-activating property. Collectively, our data uncover a novel mechanism for tBHQ in protecting hepatocytes against SFA-induced lipotoxicity. tBHQ-triggered autophagy induction contributes not only to its hepatoprotective effect, but also to its Nrf2-activating property., (© 2013.)

Background: The thioredoxin system maintains redox balance through the action of thioredoxin and thioredoxin reductase. Thioredoxin regulates the activity of various substrates, including those that function to counteract cellular oxidative stress. These include the peroxiredoxins, methionine sulfoxide reductase A and specific transcription factors. Of particular relevance is Redox Factor-1, which in turn activates other redox-regulated transcription factors., Scope of Review: Experimentally defined transcription factor binding sites in the human thioredoxin and thioredoxin reductase gene promoters together with promoters of the major thioredoxin system substrates involved in regulating cellular redox status are discussed. An in silico approach was used to identify potential putative binding sites for these transcription factors in all of these promoters., Major Conclusions: Our analysis reveals that many redox gene promoters contain the same transcription factor binding sites. Several of these transcription factors are in turn redox regulated. The ARE is present in several of these promoters and is bound by Nrf2 during various oxidative stress stimuli to upregulate gene expression. Other transcription factors also bind to these promoters during the same oxidative stress stimuli, with this redundancy supporting the importance of the antioxidant response. Putative transcription factor sites were identified in silico, which in combination with specific regulatory knowledge for that gene promoter may inform future experiments., General Significance: Redox proteins are involved in many cellular signalling pathways and aberrant expression can lead to disease or other pathological conditions. Therefore understanding how their expression is regulated is relevant for developing therapeutic agents that target these pathways., (© 2013.)

Background: Ochratoxin A (OTA), a mycotoxin, causes extensive cell damage, affecting liver and kidney cells. OTA toxicity is fairly well characterized where oxidative stress is believed to play a role, however, the sequence of molecular events after OTA-exposure, have not been characterized in literature. Further, antidotes for alleviating the toxicity are sparsely reported. The aim of this study was to understand the sequence of some molecular mechanisms for OTA-induced toxicity and the cytoprotective effect of quercetin on OTA-induced toxicity., Methods: Time course studies to evaluate the time of intracellular calcium release and ROS induction were carried out. The time of activation and induction of two key redox- sensitive transcription factors, NF-κB and Nrf-2 were determined by nuclear localization and expression respectively. The time of expression of inflammatory marker COX-2 was determined. Oxidative DNA damage by comet assay and micronucleus formation was studied. The ameliorative effect of quercetin on OTA-induced toxicity was also determined on all the above-mentioned parameters., Results: OTA-induced calcium release, ROS generation and activated NF-κB nuclear translocation and expression. Pre-treatment with quercetin ameliorated ROS and calcium release as well as NF-κB induction and expression. Quercetin induced Nrf-2 nuclear translocation and expression. Quercetin's anti-inflammatory property was exhibited as it down regulated COX-2. Anti-genotoxic effect of quercetin was evident in prevention of DNA damage and micronucleus formation., Conclusion: Quercetin modulated OTA-induced oxidative stress and redox-signaling in HepG2 cells., General Significance: The results of the study demonstrate for the first time that quercetin prevents OTA-induced toxicity in HepG2 cells., (© 2013.)

The endoplasmic-reticulum (ER) stress response constitutes a cellular process that is triggered by a variety of conditions that disturb folding of proteins in the ER. Eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. In cases where ER stress cannot be reversed, cellular functions deteriorate, often leading to cell death. Accumulating evidence implicates ER stress-induced cellular dysfunction and cell death as major contributors to many diseases, making modulators of ER stress pathways potentially attractive targets for therapeutics discovery. Here, we summarize recent advances in understanding the diversity of molecular mechanisms that govern ER stress signaling in health and disease. This article is part of a Special Section entitled: Cell Death Pathways., (© 2013.)

In various human malignancies, widespread dysregulation of microRNA (miRNA) expression is reported to occur and affects various cell growth programs. Recent studies suggest that the expression levels of miRNAs that act as tumor suppressors are frequently reduced in cancers because of chromosome deletions, epigenetical changes, aberrant transcription, and disturbances in miRNA processing. MiR-143 and -145 are well-recognized miRNAs that are highly expressed in several tissues, but down-regulated in most types of cancers. However, the mechanism of this down-regulation has not been investigated in detail. Here, we show that DEAD-box RNA helicase 6, DDX6 (p54/RCK), post-transcriptionally down-regulated miR-143/145 expression by prompting the degradation of its host gene product, NCR143/145 RNA. In human gastric cancer cell line MKN45, DDX6 protein was abundantly expressed and accumulated in processing bodies (P-bodies). DDX6 preferentially increased the instability of non-coding RNA, NCR143/145, which encompasses the miR-143/145 cluster, and down-regulated the expression of mature miR-143/145. In human monocytic cell line THP-1, lipopolysaccharide treatment promoted the assembly of P-bodies and down-regulated the expression of NCR143/145 and its miR-143/145 rapidly. In these cells, cycloheximide treatment led to a loss of P-bodies and to an increase in NCR143/145 RNA stability, thus resulting in up-regulation of miR-143/145 expression. These data demonstrate that DDX6 contributed to the control of NCR143/145 RNA stability in P-bodies and post-transcriptionally regulated miR-143/145 expression in cancer cells., (© 2013.)