Jaeyul Kwon

Reversible Inactivation of the Tumor Suppressor PTEN by H2O2

The tumor suppressor PTEN regulates cell migration, growth, and survival by removing the 3′-phosphate of phosphoinositides. Exposure of purified PTEN or of cells to H2O2 resulted in inactivation of PTEN in a time- and H2O2concentration-dependent manner. Analysis of various cysteine mutants, including mass spectrometry of tryptic peptides, indicated that the essential Cys124 residue in the active site of PTEN specifically forms a disulfide with Cys71during oxidation by H2O2. The reduction of H2O2-oxidized PTEN in cells appears to be mediated predominantly by thioredoxin. Thus, thioredoxin was more efficient than glutaredoxin, glutathione, or a 14-kDa thioredoxin-like protein with regard to the reduction of oxidized PTEN in vitro. Thioredoxin co-immunoprecipitated with PTEN from cell lysates; and incubation of cells with 2,4-dinitro-1-chlorobenzene (an inhibitor of thioredoxin reductase) delayed the reduction of oxidized PTEN, whereas incubation with buthioninesulfoximine (an inhibitor of glutathione biosynthesis) did not. These results suggest that the reversible inactivation of PTEN by H2O2might be important for the accumulation of 3′-phosphorylated phosphoinositides and that the uncontrolled generation of H2O2 associated with certain pathological conditions might contribute to cell proliferation by inhibiting PTEN function.

Hydrogen Peroxide: A Key Messenger That Modulates Protein Phosphorylation Through Cysteine Oxidation

Ligand-receptor interactions can generate the production of hydrogen peroxide (H2O2) in cells, the implications of which are becoming appreciated. Fluctuations in H2O2 levels can affect the intracellular activity of key signaling components including protein kinases and protein phosphatases. Rhee et al. discuss recent findings on the role of H2O2 in signal transduction. Specifically, H2O2 appears to oxidize active site cysteines in phosphatases, thereby inactivating them. H2O2 also can activate protein kinases; however, although the mechanism of activation for some kinases appears to be similar to that of phosphatase inactivation (cysteine oxidation), it is unclear how H2O2 promotes increased activation of other kinases. Thus, the higher levels of intracellular phosphoproteins observed in cells most likely occur because of the concomitant inhibition of protein phosphatases and activation of protein kinases.

The Nonphagocytic NADPH Oxidase Duox1 Mediates a Positive Feedback Loop During T Cell Receptor Signaling

Reactive oxygen species enhance T cell receptor signaling by promoting the phosphorylation of a proximal kinase. Oxidative Feedback Reactive oxygen species (ROS) are formed as unwanted by-products of cellular respiration and have deleterious effects, such as causing tissue damage. However, ROS also have beneficial uses—for example, in the destruction of pathogens by phagocytic cells. ROS also enhance T cell receptor (TCR) responses; however, the source of ROS and the mechanism by which ROS modulate signaling in T cells are unclear. Kwon et al. show that stimulation of the TCR in human CD4+ T cells activates the Ca2+-dependent, nonphagocytic NADPH oxidase Duox1 to generate ROS in a manner that depends on TCR-proximal kinases. ROS enhanced TCR signaling in a positive feedback loop by inhibiting the inactivation of a key TCR-dependent kinase called ZAP-70. Knockdown of Duox1 inhibited signaling downstream of the TCR and the production of cytokines. Together with other studies of the effects of ROS on B cells, these data suggest that ROS are functionally important in nonphagocytic cells of the immune system. Production of reactive oxygen species, often by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidases, plays a role in the signaling responses of cells to many receptor stimuli. Here, we describe the function of the calcium-dependent, nonphagocytic NADPH oxidase Duox1 in primary human CD4+ T cells and cultured T cell lines. Duox1 bound to inositol 1,4,5-trisphosphate receptor 1 and was required for early T cell receptor (TCR)–stimulated production of hydrogen peroxide (H2O2) through a pathway that was dependent on TCR-proximal kinases. Transient or stable knockdown of Duox1 inhibited TCR signaling, especially phosphorylation of tyrosine-319 of ζ chain–associated protein kinase of 70 kilodaltons (ZAP-70), store-operated entry of calcium ions (Ca2+), and activation of extracellular signal–regulated kinase. The production of cytokines was also inhibited by knockdown of Duox1. Duox1-mediated inactivation of Src homology 2 domain–containing protein tyrosine phosphatase 2 promoted the phosphorylation of ZAP-70 and its association with the Src family tyrosine kinase Lck and the CD3ζ chain of the TCR complex. Thus, we suggest that activation of Duox1, downstream of proximal TCR signals, generates H2O2 that acts in a positive feedback loop to enhance and sustain further TCR signaling.

T cell receptor ligation triggers novel nonapoptotic cell death pathways that are Fas-independent or Fas-dependent.

Short-term culture of activated T cells with IL-2 renders them highly susceptible to apoptotic death triggered by TCR cross-linking. Activation-induced apoptosis is contingent upon caspase activation and this is mediated primarily by Fas/Fas ligand (FasL) interactions that, in turn, are optimized by p38 mitogen-activated protein kinase (MAPK)-regulated signals. Although T cells from mice bearing mutations in Fas (lpr) or FasL (gld) are more resistant to activation-induced cell death (AICD) than normal T cells, a significant proportion of CD8+ T cells and to a lesser extent CD4+ T cells from mutant mice die after TCR religation. Little is known about this Fas-independent death process. In this study, we demonstrate that AICD in lpr and gld CD4+ and CD8+ T cells occurs predominantly by a novel mechanism that is TNF-α-, caspase-, and p38 MAPK-independent and has morphologic features more consistent with oncosis/primary necrosis than apoptosis. A related Fas- and caspase-independent, nonapoptotic death process is revealed in wild-type (WT) CD8+ T cell blasts following TCR ligation and treatment with caspase inhibitors, the p38 MAPK inhibitor, SB203580, or neutralizing anti-FasL mAb. In parallel studies with WT CD4+ T cells, two minor pathways leading to nonapoptotic, caspase-independent AICD were identified, one contingent upon Fas ligation and p38 MAPK activation and the other Fas- and p38 MAPK-independent. These data indicate that TCR ligation can activate nonapoptotic death programs in WT CD8+ and CD8+ T blasts that normally are masked by Fas-mediated caspase activation. Selective use of potentially proinflammatory oncotic death programs by activated lpr and gld T cells may be an etiologic factor in autosensitization.

Oxidation of thioredoxin reductase in HeLa cells stimulated with tumor necrosis factor‐α

Stimulation of cells with tumor necrosis factor‐α (TNF‐α) results in the increase in generation of H2O2 in mitochondria that leads to apoptosis. The effect of H2O2 produced by TNF‐α on the redox status of selenocysteine (SeCys) residue essential for mitochondrial thioredoxin reductase (TrxR2) was investigated in HeLa cells. TNF‐α caused accumulation of oxidized TrxR2 with a thioselenide bond. The conditional induction of SeCys‐deficient TrxR2 resulted in the increased production of H2O2 and apoptosis. These results suggest that the SeCys residue of TrxR2 plays a critical role in cell survival by serving as an electron donor for Trx‐II and subsequent peroxiredoxin‐III, which is a primary line of defense against H2O2 in mitochondria.

Zinc Finger Protein Tristetraprolin Interacts with CCL3 mRNA and Regulates Tissue Inflammation

Zinc finger protein tristetraprolin (TTP) modulates macrophage inflammatory activity by destabilizing cytokine mRNAs. In this study, through a screen of TTP-bound mRNAs in activated human macrophages, we have identified CCL3 mRNA as the most abundantly bound TTP target mRNA and have characterized this interaction via conserved AU-rich elements. Compared to the wild-type cells, TTP−/− macrophages produced higher levels of LPS-induced CCL3. In addition, the plasma level of CCL3 in TTP−/− mice was markedly higher than that in wild-type mice. To determine the in vivo significance of TTP-regulated CCL3, we generated CCL3−/−TTP−/− double-knockout mice. Along with decreased proinflammatory cytokines in their paw joints, there were significant functional and histologic improvements in the inflammatory arthritis of TTP−/− mice when CCL3 was absent, although cachexia, reflecting systemic inflammation, was notably unaffected. Furthermore, the marked exacerbation of aortic plaque formation caused by TTP deficiency in the APOE−/− mouse model of atherosclerosis was also rescued by disrupting CCL3. Taken together, our data indicate that the interaction between TTP and CCL3 mRNA plays an important role in modulating localized inflammatory processes in tissues that are dissociated from the systemic manifestations of chronic inflammation.

Decreased STAT5 phosphorylation and GATA-3 expression in NOX2 deficient T cells: Role in T helper development

Absence of phagocyte NADPH oxidase (NOX2) activity causes chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent bacterial infections. In contrast to this innate immune deficit, CGD patients and animal models display a predisposition toward autoimmune disease and enhanced response to Helicobacter pylori and influenza virus infection. These data imply an altered, perhaps augmented, adaptive immune response in CGD. As previous data demonstrated functional NOX2 expression in T cells, our goal here was to determine if NOX2‐deficient T cells are inherently altered in their responses. Activation of purified naive CD4+ T cells from NOX2‐deficient mice led to augmented IFN‐γ and diminished IL‐4 production and an increased ratio of expression of the TH1‐specific transcription factor T‐bet versus the TH2‐specfic transcription factor GATA‐3, consistent with a TH1 skewing of naïve T cells. Selective inhibition of TCR‐induced STAT5 phosphorylation was identified as a potential mechanism for skewed T helper differentiation. Exposure to antioxidants inhibited, while pro‐oxidants augmented TH2 cytokine secretion and STAT5 phosphorylation, supporting the redox dependence of these signaling changes. These data suggest that TCR‐induced ROS generation from NOX2 activation can regulate the adaptive immune response in a T‐cell‐inherent fashion, and propose a possible role for redox signaling in T helper differentiation.

Peroxiredoxin 6 (Prdx6) supports NADPH oxidase1 (Nox1)-based superoxide generation and cell migration.

Nox1 is an abundant source of reactive oxygen species (ROS) in colon epithelium recently shown to function in wound healing and epithelial homeostasis. We identified Peroxiredoxin 6 (Prdx6) as a novel binding partner of Nox activator 1 (Noxa1) in yeast two-hybrid screening experiments using the Noxa1 SH3 domain as bait. Prdx6 is a unique member of the Prdx antioxidant enzyme family exhibiting both glutathione peroxidase and phospholipase A2 activities. We confirmed this interaction in cells overexpressing both proteins, showing Prdx6 binds to and stabilizes wild type Noxa1, but not the SH3 domain mutant form, Noxa1 W436R. We demonstrated in several cell models that Prdx6 knockdown suppresses Nox1 activity, whereas enhanced Prdx6 expression supports higher Nox1-derived superoxide production. Both peroxidase- and lipase-deficient mutant forms of Prdx6 (Prdx6 C47S and S32A, respectively) failed to bind to or stabilize Nox1 components or support Nox1-mediated superoxide generation. Furthermore, the transition-state substrate analogue inhibitor of Prdx6 phospholipase A2 activity (MJ-33) was shown to suppress Nox1 activity, suggesting Nox1 activity is regulated by the phospholipase activity of Prdx6. Finally, wild type Prdx6, but not lipase or peroxidase mutant forms, supports Nox1-mediated cell migration in the HCT-116 colon epithelial cell model of wound closure. These findings highlight a novel pathway in which this antioxidant enzyme positively regulates an oxidant-generating system to support cell migration and wound healing.

Hydrogen Peroxide as Intracellular Messenger: Identification of Protein Tyrosine Phosphatases and Pten as H2O2 Target

The proposed role of H2O2 in growth factor signaling is depicted in Fig. 5. In the model, H2O2 induces reversible inactivation of PTPs and PTEN through oxidation of their essential Cys residues. This model suggests that the receptor-mediated activation of RTK and PI 3-kinase may not be sufficient for the accumulation of tyrosine phosphorylated proteins and 3′-phosphorylated PIs because of the opposing activity of PTPs and PTEN, respectively. The concomitant inactivation of PTPs and PTEN by H2O2 produced in response to receptor stimulation might also be necessary for these effects. This model is consistent with the previous observations that H2O2 generation and accumulation are necessary for downstream actions of PDGF and EGF that are mediated by RTK and PI 3-kinase. In the absence of the proposed function of H2O2 the activation of RTK and PI 3-kinase would result in futile cycles of phosphorylation / dephosphorylation of proteins and phosphoinositide.