Posttranscriptional regulation of FOXO expression: microRNAs and beyond.

Forkhead box, class O (FOXO) transcription factors are major regulators of diverse cellular processes, including fuel metabolism, oxidative stress response and redox signalling, cell cycle progression and apoptosis. Their activities are controlled by multiple posttranslational modifications and nuclear-cytoplasmic shuttling. Recently, post-transcriptional regulation of FOXO synthesis has emerged as a new regulatory level of their functions. Accumulating evidence suggests that this post-transcriptional mode of regulation of FOXO activity operates in response to stressful stimuli, including oxidative stress. Here, we give a brief overview on post-transcriptional regulation of FOXO synthesis by microRNAs (miRNAs) and by RNA-binding regulatory proteins, human antigen R (HuR) and quaking (QKI). Aberrant post-transcriptional regulation of FOXOs is frequently connected with various disease states. We therefore discuss characteristic examples of FOXO regulation at the post-transcriptional level under various physiological and pathophysiological conditions, including oxidative stress and cancer. The picture emerging from this summary points to a diversity of interactions between miRNAs/miRNA-induced silencing complexes and RNA-binding regulatory proteins. Better insight into these complexities of post-transcriptional regulatory interactions will add to our understanding of the mechanisms of pathological processes and the role of FOXO proteins. LINKED ARTICLES: This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Ceruloplasmin expression in rat liver cells is attenuated by insulin: role of FoxO transcription factors.

The phosphoinositide 3'-kinase (PI3 K)/Akt pathway controls the activity of a number of proteins important in the regulation of apoptosis and cell proliferation. FoxO (forkhead box, class O) transcription factors, substrates of the Ser/Thr kinase Akt, control the expression of several target genes that are crucial to the defense against oxidative stress, the regulation of cell cycle, and apoptosis in mammalian cells. Here, expression of ceruloplasmin (CP), the major copper-containing protein in blood released by the liver, was investigated. We observed a significant downregulation of CP mRNA levels after insulin treatment in H4IIE rat hepatoma cells. The PI3K inhibitor wortmannin counteracted this insulin effect on CP mRNA levels, indicating that the PI3K/Akt cascade is involved in the regulation of CP expression. Stimulation of FoxO1 was induced in H4IIE rat hepatoma cells expressing a conditionally active FoxO1 construct, resulting in significant upregulation of CP mRNA levels. This upregulation was prevented in the presence of insulin. In parallel, mRNAs of established FoxO target genes were analyzed: like CP mRNA, selenoprotein P and glucose 6-phosphatase mRNAs were upregulated by FoxO1, which was prevented by insulin. The same effects of insulin on CP mRNA levels were detected in primary rat hepatocytes. Furthermore, CP release into cell culture media was analyzed with primary hepatocytes and found to be attenuated by insulin. In line with its insulin-mimetic effects on cultured cells, Cu (2+) imitated the effect of insulin on CP expression and caused a downregulation of CP mRNA levels in rat hepatoma cells.

Linking Alzheimer's disease to insulin resistance: the FoxO response to oxidative stress.

Oxidative stress is an important determinant not only in the pathogenesis of Alzheimer's disease (AD), but also in insulin resistance (InsRes) and diabetic complications. Forkhead box class O (FoxO) transcription factors are involved in both insulin action and the cellular response to oxidative stress, thereby providing a potential integrative link between AD and InsRes. For example, the expression of intra- and extracellular antioxidant enzymes, such as manganese-superoxide dismutase and selenoprotein P, is regulated by FoxO proteins, as is the expression of important hepatic enzymes of gluconeogenesis. Here, we review the molecular mechanisms involved in the pathogenesis of AD and InsRes and discuss the function of FoxO proteins in these processes. Both InsRes and oxidative stress may promote the transcriptional activity of FoxO proteins, resulting in hyperglycaemia and a further increased production of reactive oxygen species (ROS). The consecutive activation of c-Jun N-terminal kinases and inhibition of Wingless (Wnt) signalling may result in the formation of ß-amyloid plaques and τ protein phosphorylation. Wnt inhibition may also result in a sustained activation of FoxO proteins with induction of apoptosis and neuronal loss, thereby completing a vicious circle from oxidative stress, InsRes and hyperglycaemia back to the formation of ROS and consecutive neurodegeneration. In view of their central function in this model, FoxO proteins may provide a potential molecular target for the treatment of both InsRes and AD.

Regulation of glucose-6-phosphatase gene expression by insulin and metformin.

The biguanide derivative metformin is a potent anti-diabetic drug widely used in the treatment of type 2 diabetes mellitus. Its major effect on glucose metabolism consists in the inhibition of hepatic glucose production. Since the mechanisms of metformin action are only partially understood at the molecular level, we studied the regulation of the gene promoter activity of glucose-6-phosphatase (G6Pase), the central hepatic gluconeogenic enzyme, by this drug. We have found that both metformin and insulin inhibit the basal and dexamethasone/cAMP-stimulated G6Pase promoter activity in hepatoma cells. Since one of the pharmacological targets of metformin is AMP-activated protein kinase (AMPK) and activation of AMPK is known to inhibit hepatic glucose production by the suppression of G6Pase gene transcription, we studied the effect of AMPK in this context. Under nonstimulated conditions, the inhibitory effect of both insulin and metformin was partially counteracted to a similar extent by treatment with compound C, a specific inhibitor of AMPK. In contrast, under conditions of stimulation with dexamethasone and cAMP, treatment with compound C reversed the inhibitory effect of metformin on G6Pase promoter activity to a similar extent as compared to nonstimulated conditions, whereas the effect of insulin was almost resistant to treatment with the AMPK-antagonist. These data indicate a differential AMPK-dependent regulation of G6Pase gene expression by insulin and metformin under basal and dexamethasone/cAMP-stimulated conditions.

(-)-Epicatechin inhibits nitration and dimerization of tyrosine in hydrophilic as well as hydrophobic environments.

The flavanol (-)-epicatechin is known to protect against peroxynitrite-induced nitration and oxidation reactions. This study investigated the protection afforded by (-)-epicatechin against both these reaction types on one target molecule, the aminoacid tyrosine, in a hydrophilic milieu as well as with a lipophilic tyrosine derivative, N-t-BOC l-tyrosine tert-butyl ester (BTBE), bound to liposomes. The flavanol efficiently attenuated both tyrosine nitration and tyrosine dimerization (which is based on an initial oxidation reaction) and was active in the hydrophilic and hydrophobic systems at similar IC(50) values, approximately 0.02-0.05 mol (-)-epicatechin/mol peroxynitrite. Related procyanidin oligomers of different chain-length (dimer to octamer) were also tested for their protective properties, and exhibited protection that, on a monomer basis, was in the same order of magnitude as those for (-)-epicatechin.

Epicatechin selectively prevents nitration but not oxidation reactions of peroxynitrite.

The flavanol (-)-epicatechin has been found to protect against damage inflicted by peroxynitrite, an inflammatory intermediate. Here, epicatechin was tested in systems of increasing complexity. The compound efficiently protected against nitration of protein tyrosine residues by peroxynitrite (IC(50) approximately 0.02 mol epicatechin/mol peroxynitrite). However, at epicatechin concentrations completely preventing nitration of tyrosine by peroxynitrite, protection against the oxidative inactivation of glyceraldehyde-3-phosphate dehydrogenase or soybean lipoxygenase-1 was marginal (IC(50) > 1 mol epicatechin/mol peroxynitrite), approximately two orders of magnitude less. Likewise, epicatechin was relatively ineffective against oxidation of thiols in cell lysates, and against the oxidation of 2',7'-dichlorodihydrofluorescein in cultured cells. The activation of the kinases Akt/protein kinase B, ERK1/2 and p38-MAPK by peroxynitrite in murine aorta endothelial cells was not altered by epicatechin, suggesting that activation of these kinases is due to processes other than tyrosine nitration.

Protein oxidation and proteolysis by the nonradical oxidants singlet oxygen or peroxynitrite.

Exposure of proteins to oxidants leads to increased oxidation followed by preferential degradation by the proteasomal system. The role of the biologically occurring oxidants singlet oxygen and peroxynitrite in oxidation of proteins in living cells and enhanced degradation of these proteins was examined in this study. Subsequent to treatment of an isolated model protein, ferritin, with singlet oxygen or peroxynitrite, there was enhanced degradation by the isolated 20S proteasome. Treatment of clone 9 liver cells (normal liver epithelia) with two different singlet oxygen-generating systems or peroxynitrite leads to a concentration-dependent increase in cellular protein turnover. At high concentrations of these oxidants, the protein turnover decreases without significant loss of cell viability and proteasome activity. To compare the increase of intracellular protein turnover with that obtained with other oxidants, cells were exposed to hydrogen peroxide or xanthine/xanthine oxidase. The maximal increase in protein turnover was similar with the various oxidants. The oxidized protein moieties were removed by enhanced protein turnover. Removal of singlet oxygen- or peroxynitrite-damaged proteins is dependent on the proteasomal system, as suggested by the sensitivity to lactacystin. Our results provide evidence that the proteasomal system is able to selectively recognize and degrade proteins modified by singlet oxygen or peroxynitrite in vitro as well as in living cells.

High efficiency of 5-aminolevulinate-photodynamic treatment using UVA irradiation.

Photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. One concept of delivering the employed photosensitizer directly to target cells is to stimulate cellular synthesis of sensitizers such as porphyrins. ALA (5-aminolevulinate) is applied as a precursor of porphyrins which then serve as endogenous photosensitizers. Upon irradiation, reactive oxygen species, predominantly singlet oxygen, are generated, leading to cell death. ALA-PDT using red light (550-750 nm) is known to lead to the activation of stress kinases, such as c-Jun-N-terminal kinase and p38. These kinases are also activated by UVA (320-400 nm), whose biological effects are mediated in part by singlet oxygen. In the present study, the efficiency of a combination of both treatment strategies, ALA-PDT and UVA, in cytotoxicity and activation of stress kinases was investigated taking human skin fibroblasts as a model. Compared with the commonly used ALA-PDT with red light (LD(50) = 13.5 J/cm(2)), UVA-ALA-PDT was 40-fold more potent in killing cultured human skin fibroblasts (LD(50) = 0.35 J/cm(2)) and still 10-fold more potent than ALA-PDT with green light (LD(50) = 4.5 J/cm(2)). Its toxicity relied on the formation of singlet oxygen, as was shown employing modulators of singlet oxygen lifetime. In line with these data, strong activation of the stress kinase p38 was obtained in ALA-pretreated cells irradiated with UVA at doses two orders of magnitude lower than necessary for a comparable activation of p38 by UVA in control cells. Taken together, these data suggest UVA-ALA-PDT as a potentially interesting new approach in the photodynamic treatment of skin diseases.

Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state.

gadd153, also known as chop, is a highly stress-inducible gene that is robustly expressed following disruption of homeostasis in the endoplasmic reticulum (ER) (so-called ER stress). Although all reported types of ER stress induce expression of Gadd153, its role in the stress response has remained largely undefined. Several studies have correlated Gadd153 expression with cell death, but a mechanistic link between Gadd153 and apoptosis has never been demonstrated. To address this issue we employed a cell model system in which Gadd153 is constitutively overexpressed, as well as two cell lines in which Gadd153 expression is conditional. In all cell lines, overexpression of Gadd153 sensitized cells to ER stress. Investigation of the mechanisms contributing to this effect revealed that elevated Gadd153 expression results in the down-regulation of Bcl2 expression, depletion of cellular glutathione, and exaggerated production of reactive oxygen species. Restoration of Bcl2 expression in Gadd153-overexpressing cells led to replenishment of glutathione and a reduction in levels of reactive oxygen species, and it protected cells from ER stress-induced cell death. We conclude that Gadd153 sensitizes cells to ER stress through mechanisms that involve down-regulation of Bcl2 and enhanced oxidant injury.

Polyphenols of cocoa: inhibition of mammalian 15-lipoxygenase.

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Peroxynitrite activates the phosphoinositide 3-kinase/Akt pathway in human skin primary fibroblasts.

Peroxynitrite is a potent oxidizing and nitrating species formed in a diffusion-limited reaction between nitrogen monoxide and superoxide. It induces apoptosis through unknown mechanisms and is believed to interfere with receptor tyrosine kinase signalling through nitration of tyrosine residues. One pathway emanating from receptor tyrosine kinases is that leading to activation of the anti-apoptotic kinase Akt. In the present study we provide evidence that peroxynitrite, administered to cells using two different delivery systems, results in the dose- and time-dependent activation of Akt. Akt activation is rapid and followed by phosphorylation of glycogen synthase kinase-3, an established substrate of Akt. Akt activation is inhibited in the presence of the phosphoinositide 3-kinase (PI-3K) inhibitors wortmannin and LY294002, and by treatment with the platelet-derived growth factor (PDGF) receptor (PDGFR) inhibitor AG1295, indicating a requirement for PDGFR and PI-3K in mediating peroxynitrite-induced Akt activation. Accordingly, the PDGFR-A and PDGFR-B isoforms were shown to undergo rapid tyrosine phosphorylation on treatment with peroxynitrite. Prior exposure of cells to peroxynitrite interferes with PDGF-induced Akt phosphorylation. Our findings suggest that Akt activation occurs as an acute response to peroxynitrite treatment and could play an important role in influencing cell survival and/or alter the cellular response to other growth regulatory signals.

Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes.

We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

Activation pattern of mitogen-activated protein kinases elicited by peroxynitrite: attenuation by selenite supplementation.

Peroxynitrite is a mediator of toxicity in pathological processes in vivo and causes damage by oxidation and nitration reactions. Here, we report a differential induction of mitogen-activated protein kinases (MAPKs) in WB-F344 rat liver epithelial cells by peroxynitrite. For the exposure of cultured cells with peroxynitrite, we employed a newly developed infusion method. At 6.5 microM steady-state concentration, the activation of p38 MAPK was immediate, while JNK1/2 and ERK1/2 were activated 60 min and 15 min subsequent to 3 min of exposure to peroxynitrite, respectively. Protein-bound 3-nitrotyrosine was detected. When cells were grown in a medium supplemented with sodium selenite (1 microM) for 48 h, complete protection was afforded against the activation of p38 and against nitration of tyrosine residues. These data suggest a new role for peroxynitrite in activating signal transduction pathways capable of modulating gene expression. Further, the abolition of the effects of peroxynitrite by selenite supplementation suggests a protective role of selenium-containing proteins.

Mitogen-activated protein kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA.

Ultraviolet A (UVA; 320-400 nm) radiation in human skin fibroblasts induces a pattern of mitogen-activated protein kinase (MAPK) activation consisting of a rapid and transient induction of p38 and c-Jun-N-terminal kinase (JNK) activity but not extracellular signal-regulated kinases (ERK). UVA activation of p38 can be inhibited by the singlet oxygen (1O2) quenchers azide and imidazole, but not by the hydroxyl radical scavengers mannitol or dimethylsulfoxide, pointing to the involvement of 1O2. The same effect has been shown for JNK. Like UVA, 1O2 generated intracellularly upon photoexcitation of Rose Bengal activates p38 and JNK but not ERK. p38 and JNK activation was also elicited by chemiexcitation for the intracellular generation of 1O2 by the lipophilic 1,4-endoperoxide of N,N'-di(2,3-dihydroxypropyl)-1, 4-naphthalene dipropionamide. In contrast, extracellular generation of 1O2, by irradiation of Rose Bengal immobilized on agarose beads or by chemiexcitation employing the hydrophilic 1,4-endoperoxide of disodium 3,3'-(1,4-naphthylidene) dipropionate, was ineffective in activating p38 or JNK. These data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.

Activation of JNK and p38 but not ERK MAP kinases in human skin cells by 5-aminolevulinate-photodynamic therapy.

5-Aminolevulinate (ALA) photodynamic therapy (PDT) is being used clinically for the treatment of skin cancers. ALA is applied as a precursor of porphyrins serving as endogenous photosensitizers. Irradiation of HaCaT cells preincubated with 1 mM ALA for 24 h with red light of 570-750 nm at a dose of 4.5 J/cm2 leads to a 6-fold elevation of cellular c-Jun N-terminal kinase activity; phosphorylation of p38 mitogen-activated protein kinase (MAPK) is enhanced to a similar extent. In contrast, neither activation nor increased phosphorylation of the extracellular stimulus-regulated kinase MAPKs is detected. p38 is also phosphorylated by ALA-PDT in the human melanoma cell lines Bro and SkMel-23, applying doses that lead to 80-95% cell death after 24 h. Hence, the effects of ALA-PDT on MAPKs are similar to stresses like UV irradiation or exposure to hydrogen peroxide with respect to activation of JNK and p38 MAPKs. They are different, however, in that extracellular stimulus-regulated kinase activity is not raised by ALA-PDT. Of the 830 pmol porphyrins/mg protein that were present at 24 h in HaCaT cells, 99 pmol/mg were intracellular. When extracellular porphyrins had been removed by washing, p38 responses were retained. Thus, intracellular porphyrins synthesized from ALA are sufficient to elicit activation of p38 on photosensitization.

Protection against peroxynitrite by selenoproteins.

Cellular defense against excessive peroxynitrite generation is required to protect against DNA strand-breaks and mutations and against interference with protein tyrosine-based signaling and other protein functions due to formation of 3-nitrotyrosine. We recently demonstrated a role of selenium-containing enzymes catalyzing peroxynitrite reduction. Glutathione peroxidase (GPx) protected against the oxidation of dihydrorhodamine 123 (DHR) by peroxynitrite more effectively than ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound exhibiting a high second-order rate constant for the reaction with peroxynitrite, 2 x 10(6) M-1s-1. The maintenance of protection by GPx against peroxynitrite requires GSH as reductant. Similarly, selenomethionine but not selenomethionine oxide exhibited inhibition of rhodamine 123 formation from DHR caused by peroxynitrite. In steady-state experiments, in which peroxynitrite was infused to maintain a 0.2 microM concentration, GPx in the presence of GSH, but neither GPx nor GSH alone, effectively inhibited the hydroxylation of benzoate by peroxynitrite. Under these steady-state conditions peroxynitrite did not cause loss of 'classical' GPx activity. GPx, like selenomethionine, protected against protein 3-nitrotyrosine formation in human fibroblast lysates, shown in Western blots. The formation of nitrite rather than nitrate from peroxynitrite was enhanced by GPx, ebselen or selenomethionine. The selenoxides can be effectively reduced by glutathione, establishing a biological line of defense against peroxynitrite. The novel function of GPx as a peroxynitrite reductase may extend to other selenoproteins containing selenocysteine or selenomethionine. Recent work on organotellurium compounds revealed peroxynitrite reductase activity as well. Inhibition of dihydrorhodamine 123 oxidation correlated well with the GPx-like activity of a variety of diaryl tellurides.

Protection by organotellurium compounds against peroxynitrite-mediated oxidation and nitration reactions.

Diaryl tellurides effectively protect against peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR), hydroxylation of benzoate, and nitration of 4-hydroxyphenylacetate (HPA). Bis(4-aminophenyl) telluride offered the most efficient protection against oxidation of DHR induced by peroxynitrite. Protection by this compound was approximately 3 times more effective than that afforded by its selenium analog, bis(4-aminophenyl) selenide, and 11 times more effective than selenomethionine. When peroxynitrite was infused to maintain a steady-state concentration, bis(4-aminophenyl) telluride in the presence of GSH, but neither bis(4-aminophenyl) telluride nor GSH alone, effectively inhibited the peroxynitrite-mediated hydroxylation of benzoate. The inhibition of nitration was most pronounced using bis(4-hydroxyphenyl) telluride, and this compound was ca. 3 times more effective than selenomethionine. Bis(4-aminophenyl) telluride also protected proteins in lysates from human skin fibroblasts from peroxynitrite-mediated nitration of tyrosine residues more effectively than selenomethionine. These data establish a potential biological or pharmacological role of organotellurium compounds in the defense against peroxynitrite.