Thioredoxin reductase 1 and NADPH directly protect protein tyrosine phosphatase 1B from inactivation during H2O2 exposure.

Regulation of growth factor signaling involves reversible inactivation of protein tyrosine phosphatases (PTPs) through the oxidation and reduction of their active site cysteine. However, there is limited mechanistic understanding of these redox events and their co-ordination in the presence of cellular antioxidant networks. Here we investigated interactions between PTP1B and the peroxiredoxin 2 (Prx2)/thioredoxin 1 (Trx1)/thioredoxin reductase 1 (TrxR1) network. We found that Prx2 becomes oxidized in PDGF-treated fibroblasts, but only when TrxR1 has first been inhibited. Using purified proteins, we also found that PTP1B is relatively insensitive to inactivation by H2O2 but found no evidence for a relay mechanism in which Prx2 or Trx1 facilitates PTP1B oxidation. Instead, these proteins prevented PTP1B inactivation by H2O2 Intriguingly, we discovered that TrxR1/NADPH directly protects PTP1B from inactivation when present during the H2O2 exposure. This protection was dependent on the concentration of TrxR1 and independent of Trx1 and Prx2. The protection was blocked by auranofin and required an intact selenocysteine residue in TrxR1. This activity likely involves reduction of the sulfenic acid intermediate form of PTP1B by TrxR1 and is therefore distinct from the previously described reactivation of end-point oxidized PTP1B, which requires both Trx1 and TrxR1. The ability of TrxR1 to directly reduce an oxidized phosphatase is a novel activity that can help explain previously observed increases in PTP1B oxidation and PDGF receptor phosphorylation in TrxR1 knockout cells. The activity of TrxR1 is therefore of potential relevance for understanding the mechanisms of redox regulation of growth factor signaling pathways.

Glioma-induced inhibition of caspase-3 in microglia promotes a tumor-supportive phenotype.

Glioma cells recruit and exploit microglia (the resident immune cells of the brain) for their proliferation and invasion ability. The underlying molecular mechanism used by glioma cells to transform microglia into a tumor-supporting phenotype has remained elusive. We found that glioma-induced microglia conversion was coupled to a reduction in the basal activity of microglial caspase-3 and increased S-nitrosylation of mitochondria-associated caspase-3 through inhibition of thioredoxin-2 activity, and that inhibition of caspase-3 regulated microglial tumor-supporting function. Furthermore, we identified the activity of nitric oxide synthase 2 (NOS2, also known as iNOS) originating from the glioma cells as a driving stimulus in the control of microglial caspase-3 activity. Repression of glioma NOS2 expression in vivo led to a reduction in both microglia recruitment and tumor expansion, whereas depletion of microglial caspase-3 gene promoted tumor growth. Our results provide evidence that inhibition of the denitrosylation of S-nitrosylated procaspase-3 mediated by the redox protein Trx2 is a part of the microglial pro-tumoral activation pathway initiated by glioma cancer cells.

Clinical Relevance of Biomarkers of Oxidative Stress.

SIGNIFICANCE: Oxidative stress is considered to be an important component of various diseases. A vast number of methods have been developed and used in virtually all diseases to measure the extent and nature of oxidative stress, ranging from oxidation of DNA to proteins, lipids, and free amino acids. RECENT ADVANCES: An increased understanding of the biology behind diseases and redox biology has led to more specific and sensitive tools to measure oxidative stress markers, which are very diverse and sometimes very low in abundance. CRITICAL ISSUES: The literature is very heterogeneous. It is often difficult to draw general conclusions on the significance of oxidative stress biomarkers, as only in a limited proportion of diseases have a range of different biomarkers been used, and different biomarkers have been used to study different diseases. In addition, biomarkers are often measured using nonspecific methods, while specific methodologies are often too sophisticated or laborious for routine clinical use. FUTURE DIRECTIONS: Several markers of oxidative stress still represent a viable biomarker opportunity for clinical use. However, positive findings with currently used biomarkers still need to be validated in larger sample sizes and compared with current clinical standards to establish them as clinical diagnostics. It is important to realize that oxidative stress is a nuanced phenomenon that is difficult to characterize, and one biomarker is not necessarily better than others. The vast diversity in oxidative stress between diseases and conditions has to be taken into account when selecting the most appropriate biomarker.

Knockout of mitochondrial thioredoxin reductase stabilizes prolyl hydroxylase 2 and inhibits tumor growth and tumor-derived angiogenesis.

AIMS: Mitochondrial thioredoxin reductase (Txnrd2) is a central player in the control of mitochondrial hydrogen peroxide (H2O2) abundance by serving as a direct electron donor to the thioredoxin-peroxiredoxin axis. In this study, we investigated the impact of targeted disruption of Txnrd2 on tumor growth. RESULTS: Tumor cells with a Txnrd2 deficiency failed to activate hypoxia-inducible factor-1α (Hif-1α) signaling; it rather caused PHD2 accumulation, Hif-1α degradation and decreased vascular endothelial growth factor (VEGF) levels, ultimately leading to reduced tumor growth and tumor vascularization. Increased c-Jun NH2-terminal Kinase (JNK) activation proved to be the molecular link between the loss of Txnrd2, an altered mitochondrial redox balance with compensatory upregulation of glutaredoxin-2, and elevated PHD2 expression. INNOVATION: Our data provide compelling evidence for a yet-unrecognized mitochondrial Txnrd-driven, regulatory mechanism that ultimately prevents cellular Hif-1α accumulation. In addition, simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems was used as an efficient therapeutic approach in hindering tumor growth. CONCLUSION: This work demonstrates an unexpected regulatory link between mitochondrial Txnrd and the JNK-PHD2-Hif-1α axis, which highlights how the loss of Txnrd2 and the resulting altered mitochondrial redox balance impairs tumor growth as well as tumor-related angiogenesis. Furthermore, it opens a new avenue for a therapeutic approach to hinder tumor growth by the simultaneous targeting of both the mitochondrial thioredoxin and glutathione systems.

Effects of epigenetic modificators in combination with small molecule inhibitors of receptor tyrosine kinases on medulloblastoma growth.

Epigenetic alterations and aberrant expression of genes controlling epigenetic mechanisms have been identified in several cancers, including medulloblastoma, the most common brain tumor in children. Here we show that combining drugs that inhibit two of the most important epigenetic factors, gene methylation and post-translational modifications of protein histone-associated DNA, with small molecule inhibitors of receptor tyrosine kinases induces apoptosis. The histone deacetylation inhibitor, 4-phenylbutyrate (4-PB) and the demethylation agent, 5-Aza-2'deoxycytidine (5-Aza-dC) had minor effects on medulloblastoma cell cytotoxity in single agent treatment whereas a significant enhancement in cell cytotoxity was seen when these drugs were combined with Gleevec. Triple treatment of medulloblastoma cells with 4-PB, 5-Aza and Gleevec were associated with reduced DNA methyltransferase activity, reduced global methylation and induction of apoptosis. Taken together these results suggest that a combination of these drugs may be beneficial in the treatment of medulloblastoma.

Cancer-associated fibroblasts expressing CXCL14 rely upon NOS1-derived nitric oxide signaling for their tumor-supporting properties.

Cancer-associated fibroblasts (CAF) stimulate tumor growth and metastasis. Signals supporting CAF function are thus emerging as candidate therapeutic targets in the tumor microenvironment. The chemokine CXCL14 is a potent inducer of CAF protumorigenic functions. This study is aimed at learning how the protumoral functions of CXCL14-expressing CAF are maintained. We found that the nitric oxide synthase NOS1 is upregulated in CXCL14-expressing CAF and in fibroblasts stimulated with CXCL14. Induction of Nos1 was associated with oxidative stress and occurred together with activation of NRF2 and HIF1α signaling in CXCL14-expressing CAF. Genetic or pharmacologic inhibition of NOS1 reduced the growth of CXCL14-expressing fibroblasts along with their ability to promote tumor formation following coinjection with prostate or breast cancer cells. Tumor analysis revealed reduced macrophage infiltration, with NOS1 downregulation in CXCL14-expressing CAF and lymphangiogenesis as a novel component of CXCL14-promoted tumor growth. Collectively, our findings defined key components of a signaling network that maintains the protumoral functions of CXCL14-stimulated CAF, and they identified NOS1 as intervention target for CAF-directed cancer therapy.

The mitochondrial reactive oxygen species regulator p66Shc controls PDGF-induced signaling and migration through protein tyrosine phosphatase oxidation.

Growth factor receptors induce a transient increase in reactive oxygen species (ROS) levels upon receptor binding to promote signaling through oxidation of protein tyrosine phosphatases (PTPs). Most studies have focused on NADPH oxidases as the dominant source of ROS to induce PTP oxidation. A potential additional regulator of growth factor-induced PTP oxidation is p66Shc, which stimulates mitochondrial ROS production. This study explores the contribution of p66Shc-induced ROS to PTP oxidation and growth factor receptor-induced signaling and migration through analyses of p66Shc-KO fibroblasts and cells with siRNA-mediated p66Shc downregulation. Analyses of PDGFßR phosphorylation in two independent cell systems demonstrated a decrease in PDGFßR phosphorylation after p66Shc deletion or downregulation, which occurred in a partially site-selective and antioxidant-sensitive manner. Deletion of p66Shc also reduced PDGF-induced activation of downstream signaling of Erk, Akt, PLCγ-1, and FAK. Importantly, reduced levels of p66Shc led to decreased oxidation of DEP1, PTP1B, and SHP2 after PDGF stimulation. The cell biological relevance of these findings was indicated by demonstration of a significantly reduced migratory response in PDGF-stimulated p66Shc-KO fibroblasts, consistent with reduced PDGFßR-Y1021 and PLCγ-1 phosphorylation. Downregulation of p66Shc also reduced EGFR phosphorylation and signaling, indicating that the positive role of p66Shc in receptor tyrosine kinase signaling is potentially general. Moreover, downregulation of the mitochondrial hydrogen peroxide scavenger peroxiredoxin 3 increased PDGFßR phosphorylation, showing that mitochondrial ROS in general promote PDGFßR signaling. This study thus identifies a previously unrecognized role for p66Shc in the regulation of PTP oxidation controlling growth factor-induced signaling and migration. In more general terms, the study indicates a regulatory role for mitochondrial-derived ROS in the control of PTP oxidation influencing growth factor signaling.

Regulation of protein tyrosine phosphatase oxidation in cell adhesion and migration.

SIGNIFICANCE: Redox-regulated control of protein tyrosine phosphatases (PTPs) through inhibitory reversible oxidation of their active site is emerging as a novel and general mechanism for control of cell surface receptor-activated signaling. This mechanism allows for a previously unrecognized crosstalk between redox regulators and signaling pathways, governed by, for example, receptor tyrosine kinases and integrins, which control cell proliferation and migration. RECENT ADVANCES: A large number of different molecules, in addition to hydrogen peroxide, have been found to induce PTP inactivation, including lipid peroxides, reactive nitrogen species, and hydrogen sulfide. Characterization of oxidized PTPs has identified different types of oxidative modifications that are likely to display differential sensitivity to various reducing systems. Accumulating evidence demonstrates that PTP oxidation occurs in a temporally and spatially restricted manner. Studies in cell and animal models indicate altered PTP oxidation in models of common diseases, such as cancer and metabolic/cardiovascular disease. Novel methods have appeared that allow characterization of global PTP oxidation. CRITICAL ISSUES: As the understanding of the molecular and cellular biology of PTP oxidation is developing, it will be important to establish experimental procedures that allow analyses of PTP oxidation, and its regulation, in physiological and pathophysiological settings. Future studies should also aim to establish specific connections between various oxidants, specific PTPs, and defined signaling contexts. FUTURE DIRECTIONS: Modulation of PTP activity still appears as a valid strategy for correction or inhibition of dys-regulated cell signaling. Continued studies on PTP oxidation might present yet unrecognized means to exploit this regulatory mechanism for pharmacological purposes.

Selective activation of oxidized PTP1B by the thioredoxin system modulates PDGF-ß receptor tyrosine kinase signaling.

The inhibitory reversible oxidation of protein tyrosine phosphatases (PTPs) is an important regulatory mechanism in growth factor signaling. Studies on PTP oxidation have focused on pathways that increase or decrease reactive oxygen species levels and thereby affect PTP oxidation. The processes involved in reactivation of oxidized PTPs remain largely unknown. Here the role of the thioredoxin (Trx) system in reactivation of oxidized PTPs was analyzed using a combination of in vitro and cell-based assays. Cells lacking the major Trx reductase TrxR1 (Txnrd1(-/-)) displayed increased oxidation of PTP1B, whereas SHP2 oxidation was unchanged. Furthermore, in vivo-oxidized PTP1B was reduced by exogenously added Trx system components, whereas SHP2 oxidation remained unchanged. Trx1 reduced oxidized PTP1B in vitro but failed to reactivate oxidized SHP2. Interestingly, the alternative TrxR1 substrate TRP14 also reactivated oxidized PTP1B, but not SHP2. Txnrd1-depleted cells displayed increased phosphorylation of PDGF-ß receptor, and an enhanced mitogenic response, after PDGF-BB stimulation. The TrxR inhibitor auranofin also increased PDGF-ß receptor phosphorylation. This effect was not observed in cells specifically lacking PTP1B. Together these results demonstrate that the Trx system, including both Trx1 and TRP14, impacts differentially on the oxidation of individual PTPs, with a preference of PTP1B over SHP2 activation. The studies demonstrate a previously unrecognized pathway for selective redox-regulated control of receptor tyrosine kinase signaling.

Regulation of protein tyrosine phosphatases by reversible oxidation.

Oxidation of the catalytic cysteine of protein-tyrosine phosphatases (PTP), which leads to their reversible inactivation, has emerged as an important regulatory mechanism linking cellular tyrosine phosphorylation and signalling by reactive-oxygen or -nitrogen species (ROS, RNS). This review focuses on recent findings about the involved pathways, enzymes and biochemical mechanisms. Both the general cellular redox state and extracellular ligand-stimulated ROS production can cause PTP oxidation. Members of the PTP family differ in their intrinsic susceptibility to oxidation, and different types of oxidative modification of the PTP catalytic cysteine can occur. The role of PTP oxidation for physiological signalling processes as well as in different pathologies is described on the basis of well-investigated examples. Criteria to establish the causal involvement of PTP oxidation in a given process are proposed. A better understanding of mechanisms leading to selective PTP oxidation in a cellular context, and finding ways to pharmacologically modulate these pathways are important topics for future research.

12/15-lipoxygenase-derived lipid peroxides control receptor tyrosine kinase signaling through oxidation of protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like H(2)O(2), as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF beta-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX-derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than H(2)O(2) in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation.

Differential up-regulation of MAP kinase phosphatases MKP3/DUSP6 and DUSP5 by Ets2 and c-Jun converge in the control of the growth arrest versus proliferation response of MCF-7 breast cancer cells to phorbol ester.

Different levels of regulation account for the inactivation of MAP kinases by MAPK phosphatases (MKPs), in a cell type- and stimuli-dependent manner. MCF-7 human breast carcinoma cells treated with the phorbol 12-myristate 13-acetate (PMA) suffer growth arrest and show morphological alterations, which depend on the activation of the ERK1/2 MAP kinases. MKP3/DUSP6 and DUSP5 MAP kinase phosphatases, two negative regulators of ERK1/2, were specifically up-regulated in MCF-7 and SKBR3 cells in response to PMA. MKP3 and DUSP5 up-regulation required the prolonged activation of the ERK1/2 pathway, and correlated with the shutdown of this route. MKP3 induction relied on the activation of the Ets2 transcription factor, whereas DUSP5 induction depended on the activation of c-Jun. Diminishing the expression of MKP3 and DUSP5 raised the activation of ERK1/2, and accelerated growth arrest of PMA-treated MCF-7 cells. Conversely, MCF-7 cell lines expressing high levels of MKP3 or DUSP5 did not undergo PMA-triggered growth arrest, displayed a migratory phenotype, and formed colonies in soft agar. We propose that the differential up-regulation of MKP3 by Ets2 and of DUSP5 by c-Jun may converge in similar functional roles for these MAP kinase phosphatases in the growth arrest versus proliferation decisions of breast cancer cells.