Copper ions strongly activate the phosphoinositide-3-kinase/Akt pathway independent of the generation of reactive oxygen species.

Copper is implicated in metabolic disorders, such as Wilson's disease or Alzheimer's disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu(2+) resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 microM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu(2+), indicating a requirement of PI3K for Cu(2+)-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu(2+). Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu(2+), however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu(2+) activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu(2+) is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2',7'-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a copper stress.     

Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B

This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis.     

Creatine kinase B is a target molecule of reactive oxygen species in cervical cancer.

Recently, a procedure for detecting ROS-sensitive proteins that contain active cysteine residues was devdoped. The method is based on the fact that biotin-conjugated iodoacetamide (BIAM) and ROS competitively and selectively react with the active cysteine residues in ROS-sensitive proteins. To investigate the role of ROS in cervical cancer, BIAM labeling on cytosolic proteins in normal and cancer tissues was performed, respectively. The BIAM labeling proteins are separated by 2-dimensional electrophoresis, and then identified by MALDI-TOF mass analysis. ROS-sensitive protein is identified as creatine kinase B containing cysteine residue in active center. Activity of creatine kinase B in normal tissue is higher than that of oxidized form in cervical cancer tissues. The result suggests that ROS play an important role in metabolic regulation in cervical cancer cells. However, molecular mechanisms that ROS and creatine kinase B are integrated into a physiological signal leading to the cellular transformation remain to be elucidated.     

Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta

Evidence indicates that both the Rho/Rho kinase signaling pathway and reactive oxygen species (ROS) such as superoxide and H(2)O(2) are involved in the pathogenesis of hypertension. This study aimed to determine whether ROS-induced vascular contraction is mediated through activation of Rho/Rho kinase. Rat aortic rings (endothelium denuded) were isolated and placed in organ chambers for measurement of isometric force development. ROS were generated by a xanthine (X)-xanthine oxidase (XO) mixture. The antioxidants tempol (3 mM) and catalase (1,200 U/ml) or the XO inhibitor allopurinol (400 microM) significantly reduced X/XO-induced contraction. A Rho kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl-N-4-pyridil)cyclohexanecarboxamide dihydrochloride (Y-27632), decreased the contraction in a concentration-dependent manner; however, the Ca(2+)-independent protein kinase C inhibitor rottlerin did not have an effect on X/XO-induced contraction. Phosphorylation of the myosin light chain phosphatase target subunit (MYPT1) was increased by ROS, and preincubation with Y-27632 blocked this increased phosphorylation. Western blotting for cytosolic and membrane-bound fractions of Rho showed that Rho was increased in the membrane fraction by ROS, suggesting activation of Rho. These observations demonstrate that ROS-induced Ca(2+) sensitization is through activation of Rho and a subsequent increase in Rho kinase activity but not Ca(2+)-independent PKC.     

Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.     

Protein synthesis is the primary target of reactive oxygen species in the photoinhibition of photosystem II

Photoinhibition of photosystem II (PSII) occurs when the rate of photodamage to PSII exceeds the rate of the repair of photodamaged PSII. Recent examination of photoinhibition by separate determinations of photodamage and repair has revealed that the rate of photodamage to PSII is directly proportional to the intensity of incident light and that the repair of PSII is particularly sensitive to the inactivation by reactive oxygen species (ROS). The ROS-induced inactivation of repair is attributable to the suppression of the synthesis de novo of proteins, such as the D1 protein, that are required for the repair of PSII at the level of translational elongation. Furthermore, molecular analysis has revealed that the ROS-induced suppression of protein synthesis is associated with the specific inactivation of elongation factor G via the formation of an intramolecular disulfide bond. Impairment of various mechanisms that protect PSII against photoinhibition, including photorespiration, thermal dissipation of excitation energy, and the cyclic transport of electrons, decreases the rate of repair of PSII via the suppression of protein synthesis. In this review, we present a newly established model of the mechanism and the physiological significance of repair in the regulation of the photoinhibition of PSII.     

Decreases in phosphoinositide-3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling activate components of spinal motoneuron death

Motoneuron dependence on target-derived trophic factors during development is well established, with loss of trophic support leading to the death of these cells. A complete understanding of the intracellular signal transduction machinery associated with extracellular survival signals requires the examination of individual pathways in various cellular and environmental contexts. In cells deprived of trophic support, and hence compromised for survival, phosphoinositide-3-kinase (PI3K) is decreased when compared with healthy cells supplied with trophic support. Extracellular signal-regulated kinase 1/2 (ERK1/2) signaling is dramatically decreased in deprived cells. We have examined the role of these two pathways to understand how changes in their activity regulate motoneuron survival and death. Pharmacological inhibition of PI3K attenuated motoneuron survival and was important in the regulation of Bcl-2 serine phosphorylation, limited release of cytochrome c into the cytoplasm and caspase activation. Bax translocation from cytoplasm to mitochondria was not altered when PI3K was inhibited. High levels of ERK1/2 inhibition robustly attenuated motoneuron survival in cells supplied with trophic support, whereas moderate inhibition of ERK1/2 activation had little effect. ERK1/2 inhibition in these cells decreased Bcl-2 phosphorylation and resulted in release of cytochrome c from the mitochondria. Bax translocation and caspase activation were not affected by ERK1/2 inhibition. These data reveal that changes in PI3K and ERK1/2 signaling lead to individual and overlapping effects on the cell-death machinery. Characterizing the role of these pathways is critical for a fundamental understanding of the development and degeneration of specific neuronal populations.     

Phosphoinositide 3-OH kinase/protein kinase B inhibits apoptotic cell death induced by reactive oxygen species in Saccharomyces cerevisiae

Apoptosis is a common mode of programmed cell death in multicellular organisms. However, the recent observation of yeast cell death displaying the morphology of apoptosis has suggested the presence of an ancestral cell death machinery. Here we examined apoptotic features induced by reactive oxygen species (ROS) in yeast. Saccharomyces cerevisiae show typical apoptotic features upon exposure to ROS: membrane staining with annexin V and DNA fragmentation by the TUNEL assay. The detection of apoptotic features in yeast strongly support the existence of molecular machinery performing the basic pathways of apoptosis. The phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in a variety of cells. It is therefore of interest to determine whether the PI3K/PKB signaling pathway is capable of protecting yeast from apoptosis induced by ROS. We determined that PI3K/PKB is capable of significantly inhibiting ROS-evoked apoptosis in yeast. These results suggest that yeast may provide a suitable model system in which to study the apoptotic signaling pathway elicited by a variety of stimuli.     

3,5,3'-triiodothyronine (T3) stimulates cell proliferation through the activation of the PI3K/Akt pathway and reactive oxygen species (ROS) production in chick embryo hepatocytes

Thyroid hormones (THs) have a wide variety of essential roles in vertebrates, ranging from the regulation of key metabolic processes to cell proliferation and apoptosis. The classical mechanism of action of THs is genomic; 3,5,3'-triiodothyronine (T3) binds to specific nuclear receptors (TRs) and modifies the expression of specific genes. Recently, a new category of mechanisms, termed nongenomic, has been discovered for T3. These mechanisms include, among others, the rapid activation of signal transduction pathways, such as PI3K/Akt and MAPK, which eventually lead to cell proliferation. These effects are mediated in some cell types by a plasma membrane receptor, identified as integrin αvβ3, and in other cell types by cytoplasmic TRβ1. The aim of this work was to analyze the effect of T3 on the cell growth of chick embryo hepatocytes at two different stages of development, 14 and 19 days, and to determine the activation of the signal transduction pathways, focusing on the potential involvement of a plasma membrane receptor and the possible participation of PI3K/Akt and reactive oxygen species (ROS). Our results clearly show that T3 stimulates cell proliferation at both stages of development through the activation of the PI3K/Akt pathway and the production of small amounts of ROS, which operate as effective second messengers. Moreover, we prove that these effects are not initiated at the plasma membrane receptor for T3.     

SH2-containing inositol phosphatase 2 predominantly regulates Akt2, and not Akt1, phosphorylation at the plasma membrane in response to insulin in 3T3-L1 adipocytes

SH2-containing inositol phosphatase 2 (SHIP2) is a physiologically important negative regulator of insulin signaling by hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI 3,4,5-trisphosphate in the target tissues of insulin. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity without affecting biological systems other than insulin signaling. Therefore, we investigated the molecular mechanisms by which SHIP2 specifically regulates insulin-induced metabolic signaling in 3T3-L1 adipocytes. Insulin-induced phosphorylation of Akt, one of the molecules downstream of PI3-kinase, was inhibited by expression of wild-type SHIP2, whereas it was increased by expression of 5'-phosphatase-defective (DeltaIP) SHIP2 in whole cell lysates. The regulatory effect of SHIP2 was mainly seen in the plasma membrane (PM) and low density microsomes but not in the cytosol. In this regard, following insulin stimulation, a proportion of Akt2, and not Akt1, appeared to redistribute from the cytosol to the PM. Thus, insulin-induced phosphorylation of Akt2 at the PM was predominantly regulated by SHIP2, whereas the phosphorylation of Akt1 was only minimally affected. Interestingly, insulin also elicited a subcellular redistribution of both wild-type and DeltaIP-SHIP2 from the cytosol to the PM. The degree of this redistribution was inhibited in part by pretreatment with PI3-kinase inhibitor. Although the expression of a constitutively active form of PI3-kinase myr-p110 also elicited a subcellular redistribution of SHIP2 to the PM, expression of SHIP2 appeared to affect the myr-p110-induced phosphorylation, and not the translocation, of Akt2. Furthermore, insulin-induced phosphorylation of Akt was effectively regulated by SHIP2 in embryonic fibroblasts derived from knockout mice lacking either insulin receptor substrate-1 or insulin receptor substrate-2. These results indicate that insulin specifically stimulates the redistribution of SHIP2 from the cytosol to the PM independent of 5'-phosphatase activity, thereby regulating the insulin-induced translocation and phosphorylation of Akt2 at the PM.     

Cholesterol, not polyunsaturated fatty acids, is target molecule in oxidation induced by reactive oxygen species in membrane of human erythrocytes

The oxidation of polyunsaturated fatty acids (PUFAs) by reactive oxygen species (ROS) is linked to aging and to many diseases. We herein employ initiating peroxyl radical (ROO•) derived from the decomposition of 2,2′-azobis(2-amidinopropane dihydrochloride), hydroxyl radical generated by the Fenton reaction and peroxyl radical (ROO•) and alkoxyl radical (LO•) derived from PUFAs by addition of Cu²⁺ as ROS sources to oxidize glycerides under alkaline conditions in the presence of methanol instead of being treated traditionally by diazomethane (CH₂N₂) under acidic conditions (pH=2.0), to obtain corresponding methyl esters for the combination of gas chromatography with mass spectrometry determination. It was found that all the PUFAs in the membrane are perfectly preserved after oxidation by ROS, even though sufficient time is available for the interaction between human erythrocytes and ROS. This indicates that ROS do not damage PUFAs during reaction time. However, three products (cholesta-4,6-dien-3-ol, cholesta-4,6-dien-3-one, and cholesta-3,5-dien-7-one) are produced from the oxidation of cholesterol within this time frame. This qualitative finding, suggests that the cholesterol in the membrane of human erythrocytes is more susceptible to ROS-induced oxidation than are PUFAs, and compels us to re-evaluate the physiological roles of cholesterol and PUFAs in the human erythrocyte membrane.     

Down-regulation of c-FLIP increases reactive oxygen species,induces phosphorylation of serine/threonine kinase Akt,and impairs motility of cancer cells

The role of c-FLIP in cell motility was investigated using RNA interference. Down-regulation of c-FLIP increased amounts of reactive oxygen species (ROS), while over-expression of c-FLIP produced opposite effect. ROS induced phosphorylation of Akt and impaired cell motility.     

MAO-A-induced mitogenic signaling is mediated by reactive oxygen species, MMP-2, and the sphingolipid pathway

The degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2(-/-) fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H(2)O(2)-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall.