Tong-Shin Chang

Cellular Regulation by Hydrogen Peroxide

Substantial evidence suggests that the transient production of H(2)O(2) is an important signaling event triggered by the activation of various cell surface receptors. Understanding the intracellular messenger function of H(2)O(2) calls for studies of how receptor occupation elicits the production of H(2)O(2), what kinds of molecules are targeted by the produced H(2)O(2), and how H(2)O(2) is eliminated after the completion of its mission. Recent studies suggest that growth factor-induced H(2)O(2) production requires the activation of PtdIns 3-kinase. The essential role of PtdIns 3-kinase is likely to provide PI(3,4,5)P(3) that recruits and activates a guanine nucleotide exchange factor of Rac, which is required for the activation of NADPH oxidase. The targets of H(2)O(2) action include proteins that contain a reactive Cys residue. Thus, H(2)O(2) produced in response to growth factor causes inactivation of protein tyrosine phosphatases in various cells by oxidizing specifically the catalytic Cys. These results, together with other observations, indicate that the activation of a receptor tyrosine kinase per se by binding of the corresponding growth factor might not be sufficient to increase the steady-state level of protein tyrosine phosphorylation in cells. Rather, the concurrent inhibition of protein tyrosine phosphatases by H(2)O(2) might also be required. Peroxiredoxins, members of a newly discovered family of peroxidases, efficiently reduced the intracellular level of H(2)O(2) produced in the cells stimulated with various cell surface ligands. Furthermore, the activity of peroxiredoxin enzymes seems to be regulated via protein phosphorylation as in the case of many other intracellular messenger metabolizing enzymes.

2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications

n n H2O2 is a reactive oxygen species that has drawn much interest because of its role as a second messenger in receptor-mediated signaling. Mammalian 2-Cys peroxiredoxins have been shown to eliminate efficiently the H2O2 generated in response to receptor stimulation. 2-Cys peroxiredoxins are members of a novel peroxidase family that catalyze the H2O2 reduction reaction in the presence of thioredoxin, thioredoxin reductase and NADPH. Several lines of evidence suggest that 2-Cys peroxiredoxins have dual roles as regulators of the H2O2 signal and as defenders of oxidative stress. In particular, 2-Cys peroxiredoxin appears to provide selective, specific and localized control of receptor-mediated signal transduction. Thus, the therapeutic potential of 2-Cys peroxiredoxins is clear for diseases, such as cancer and cardiovascular diseases, that involve reactive oxygen species.n n

Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Hydrogen peroxide (H2O2) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H2O2 requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H2O2 in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H2O2, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H2O2. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H2O2-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H2O2 in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H2O2 can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H2O2, they cannot be used to monitor transient changes in H2O2 concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H2O2 and can be targeted to various compartments of the cell, but they are not selective for H2O2 because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H2O2-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.

Molecular Mechanism of the Reduction of Cysteine Sulfinic Acid of Peroxiredoxin to Cysteine by Mammalian Sulfiredoxin

Among many proteins with cysteine sulfinic acid (Cys-SO2H) residues, the sulfinic forms of certain peroxiredoxins (Prxs) are selectively reduced by sulfiredoxin (Srx) in the presence of ATP. All Srx enzymes contain a conserved cysteine residue. To elucidate the mechanism of the Srx-catalyzed reaction, we generated various mutants of Srx and examined their interaction with PrxI, their ATPase activity, and their ability to reduce sulfinic PrxI. Our results suggest that three surface-exposed amino acid residues, corresponding to Arg50, Asp57, and Asp79 of rat Srx, are critical for substrate recognition. The presence of the sulfinic form (but not the reduced form) of PrxI induces the conserved cysteine of Srx to take the γ-phosphate of ATP and then immediately transfers the phosphate to the sulfinic moiety of PrxI to generate a sulfinic acid phosphoryl ester (Prx-Cys-S(=O)batchmode documentclass[fleqn,10pt,legalpaper]{article} usepackage{amssymb} usepackage{amsfonts} usepackage{amsmath} pagestyle{empty} begin{document} (mathrm{OPO}_{3}^{2-}) end{document}). This ester is reductively cleaved by a thiol molecule (RSH) such as GSH, thioredoxin, and dithiothreitol to produce a disulfide-S-monoxide (Prx-Cys-S(=O)-S-R). The disulfide-S-monoxide is further reduced through the oxidation of three thiol equivalents to complete the catalytic cycle and regenerate Prx-Cys-SH.

Sulfiredoxin Translocation into Mitochondria Plays a Crucial Role in Reducing Hyperoxidized Peroxiredoxin III

The mitochondria are the major intracellular source of reactive oxygen species (ROS), which are generated during cellular respiration. The role of peroxiredoxin (Prx) III, a 2-Cys Prx family member, in the scavenging of mitochondrial H2O2 has recently been emphasized. While eliminating H2O2, Prx can become overoxidized and inactivated by modifying the active cysteine into cysteine sulfinic acid (Cys-SO2H). When 2-Cys Prxs are inactivated in vitro, sulfiredoxin (Srx) reduces the cysteine sulfinic acid to cysteines. However, whereas Srx is localized in the cytoplasm, Prx III is present exclusively in the mitochondria. Although Srx reduces sulfinic Prx III in vitro, it remains unclear whether the reduction of Prx III in cells is actually mediated by Srx. Our gain- and loss-of-function experiments show that Srx is responsible for reducing not only sulfinic cytosolic Prxs (I and II) but also sulfinic mitochondrial Prx III. We further demonstrate that Srx translocates from the cytosol to mitochondria in response to oxidative stress. Overexpression of mitochondrion-targeted Srx promotes the regeneration of sulfinic Prx III and results in cellular resistance to apoptosis, with enhanced elimination of mitochondrial H2O2 and decreased rates of mitochondrial membrane potential collapse. These results indicate that Srx plays a crucial role in the reactivation of sulfinic mitochondrial Prx III and that its mitochondrial translocation is critical in maintaining the balance between mitochondrial H2O2 production and elimination.

Sulfiredoxin Protein Is Critical for Redox Balance and Survival of Cells Exposed to Low Steady-state Levels of H2O2

Background: Sulfiredoxin catalyzes the reactivation of hyperoxidized peroxiredoxins, which are peroxidases. Results: Under low steady-state levels of H2O2 ([H2O2]ss), loss of function of sulfiredoxin leads to redox imbalance and sensitizes cells to apoptosis. Conclusion: Sulfiredoxin functions as a critical antioxidant for redox balance and survival of cells exposed to low [H2O2]ss. Significance: Sulfiredoxin may serve as a cell survival factor even in the presence of low [H2O2]ss. Sulfiredoxin (Srx) is an enzyme that catalyzes the reduction of cysteine sulfinic acid of hyperoxidized peroxiredoxins (Prxs). Having high affinity toward H2O2, 2-Cys Prxs can efficiently reduce H2O2 at low concentration. We previously showed that Prx I is hyperoxidized at a rate of 0.072% per turnover even in the presence of low steady-state levels of H2O2. Here we examine the novel role of Srx in cells exposed to low steady-state levels of H2O2, which can be achieved by using glucose oxidase. Exposure of low steady-state levels of H2O2 (10–20 μm) to A549 or wild-type mouse embryonic fibroblast (MEF) cells does not lead to any significant change in oxidative injury because of the maintenance of balance between H2O2 production and elimination. In contrast, loss-of-function studies using Srx-depleted A549 and Srx−/− MEF cells demonstrate a dramatic increase in extra- and intracellular H2O2, sulfinic 2-Cys Prxs, and apoptosis. Concomitant with hyperoxidation of mitochondrial Prx III, Srx-depleted cells show an activation of mitochondria-mediated apoptotic pathways including mitochondria membrane potential collapse, cytochrome c release, and caspase activation. Furthermore, adenoviral re-expression of Srx in Srx-depleted A549 or Srx−/− MEF cells promotes the reactivation of sulfinic 2-Cys Prxs and results in cellular resistance to apoptosis, with enhanced removal of H2O2. These results indicate that Srx functions as a novel component to maintain the balance between H2O2 production and elimination and then protects cells from apoptosis even in the presence of low steady-state levels of H2O2.

Reactive oxygen species play a critical role in collagen-induced platelet activation via SHP-2 oxidation.

AIMSnThe collagen-stimulated generation of reactive oxygen species (ROS) regulates signal transduction in platelets, although the mechanism is unclear. The major targets of ROS include protein tyrosine phosphatases (PTPs). ROS-mediated oxidation of the active cysteine site in PTPs abrogates the PTP catalytic activity. The aim of this study was to elucidate whether collagen-induced ROS generation leads to PTP oxidation, which promotes platelet stimulation.nnnRESULTSnSH2 domain-containing PTP-2 (SHP-2) is oxidized in platelets by ROS produced upon collagen stimulation. The oxidative inactivation of SHP-2 leads to the enhanced tyrosine phosphorylation of spleen tyrosine kinase (Syk), Vav1, and Brutons tyrosine kinase (Btk) in the linker for the activation of T cells signaling complex, which promotes the tyrosine phosphorylation-mediated activation of phospholipase Cγ2 (PLCγ2). Moreover, we found that, relative to wild-type platelets, platelets derived from glutathione peroxidase 1 (GPx1)/catalase double-deficient mice showed enhanced cellular ROS levels, oxidative inactivation of SHP-2, and tyrosine phosphorylation of Syk, Vav1, Btk, and PLCγ2 in response to collagen, which subsequently led to increased intracellular calcium levels, degranulation, and integrin αIIbβ3 activation. Consistent with these findings, GPx1/catalase double-deficiency accelerated the thrombotic response in FeCl3-injured carotid arteries.nnnINNOVATIONnThe present study is the first to demonstrate that SHP-2 is targeted by ROS produced in collagen-stimulated platelets and suggests that a novel mechanism for the regulation of platelet activation by ROS is due to oxidative inactivation of SHP-2.nnnCONCLUSIONnWe conclude that collagen-induced ROS production leads to SHP-2 oxidation, which promotes platelet activation by upregulating tyrosine phosphorylation-based signal transduction.

Processed Panax ginseng, Sun Ginseng Increases Type I Collagen by Regulating MMP-1 and TIMP-1 Expression in Human Dermal Fibroblasts

In the present study, effects of sun ginseng (SG) on the collagen synthesis and the proliferation of dermal fibroblast were investigated. Collagen synthesis was measured by assaying procollagen type I C-peptide production. In addition, the level of matrix metalloproteinase (MMP)-1 was assessed by western blot analysis. SG suppressed the MMP-1 protein level in a dose-dependent manner. In contrast, SG dose-dependently increased tissue inhibitors of MMP (TIMP)-1 production in fibroblasts. SG increased type I collagen production directly and/or indirectly by reducing MMP-1 and stimulating TIMP-1 production in human dermal fibroblasts. SG dose-dependently induced fibroblast proliferation and this, in turn, can trigger more collagen production. These results suggest that SG may be a potential pharmacological agent with anti-aging properties in cultured human skin fibroblast.

Kaempferol suppresses collagen-induced platelet activation by inhibiting NADPH oxidase and protecting SHP-2 from oxidative inactivation.

Reactive oxygen species (ROS) generated upon collagen stimulation act as second messengers to propagate various platelet-activating events. Among the ROS-generating enzymes, NADPH oxidase (NOX) plays a prominent role in platelet activation. Thus, NOX has been suggested as a novel target for anti-platelet drug development. Although kaempferol has been identified as a NOX inhibitor, the influence of kaempferol on the activation of platelets and the underlying mechanism have never been investigated. Here, we studied the effects of kaempferol on NOX activation, ROS-dependent signaling pathways, and functional responses in collagen-stimulated platelets. Superoxide anion generation stimulated by collagen was significantly inhibited by kaempferol in a concentration-dependent manner. More importantly, kaempferol directly bound p47(phox), a major regulatory subunit of NOX, and significantly inhibited collagen-induced phosphorylation of p47(phox) and NOX activation. In accordance with the inhibition of NOX, ROS-dependent inactivation of SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) was potently protected by kaempferol. Subsequently, the specific tyrosine phosphorylation of key components (Syk, Vav1, Btk, and PLCγ2) of collagen receptor signaling pathways was suppressed by kaempferol. Kaempferol also attenuated downstream responses, including cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Ultimately, kaempferol inhibited platelet aggregation and adhesion in response to collagen in vitro and prolonged in vivo thrombotic response in carotid arteries of mice. This study shows that kaempferol impairs collagen-induced platelet activation through inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2. This effect suggests that kaempferol has therapeutic potential for the prevention and treatment of thrombovascular diseases.

Resveratrol inhibits collagen-induced platelet stimulation through suppressing NADPH oxidase and oxidative inactivation of SH2 domain-containing protein tyrosine phosphatase-2

Reactive oxygen species (ROS) produced upon collagen stimulation are implicated in propagating various platelet-activating pathways. Among ROS-producing enzymes, NADPH oxidase (NOX) is largely responsible for collagen receptor-dependent ROS production. Therefore, NOX has been proposed as a novel target for the development of antiplatelet agent. We here investigate whether resveratrol inhibits collagen-induced NOX activation and further examine the effects of resveratrol on ROS-dependent signaling pathways in collagen-stimulated platelets. Collagen-induced superoxide anion production in platelets was inhibited by resveratrol. Resveratrol suppressed collagen-induced phosphorylation of p47(phox), a major regulatory subunit of NOX. Correlated with the inhibitory effects on NOX, resveratrol protected SH2 domain-containing protein tyrosine phosphatase-2 (SHP-2) from ROS-mediated inactivation and subsequently attenuated the specific tyrosine phosphorylation of key components (spleen tyrosine kinase, Vav1, Brutons tyrosine kinase, and phospholipase Cγ2) for collagen receptor signaling cascades. Resveratrol also inhibited downstream responses such as cytosolic calcium elevation, P-selectin surface exposure, and integrin-αIIbβ3 activation. Furthermore, resveratrol inhibited platelet aggregation and adhesion in response to collagen. The antiplatelet effects of resveratrol through the inhibition of NOX-derived ROS production and subsequent oxidative inactivation of SHP-2 suggest that resveratrol is a potential compound for prevention and treatment of thrombovascular diseases.