Nox4 redox regulation of PTP1B contributes to the proliferation and migration of glioblastoma cells by modulating tyrosine phosphorylation of coronin-1C

Glioblastoma multiforme is a common primary brain tumor in adults and one of the most devastating human cancers. Reactive oxygen species (ROS) generated by NADPH oxidase (Nox) 4 have recently been a focus of attention in the study of glioblastomas, but the molecular mechanisms underlying the actions of Nox4 remain elusive. In this study, we demonstrated that silencing of Nox4 expression by Nox4-targeted siRNA suppressed cell growth and motility of glioblastoma U87 cells, indicating the involvement of Nox4. Furthermore, Nox4-derived ROS oxidized and inactivated protein tyrosine phosphatase (PTP):1B: PTP1B in its active form downregulates cell proliferation and migration. By affinity purification with the substrate-trapping mutant of PTP1B, tyrosine-phosphorylated coronin-1C was identified as a substrate of PTP1B. Its tyrosine phosphorylation level was suppressed by Nox4 inhibition, suggesting that tyrosine phosphorylation of coronin-1C is regulated by the Nox4–PTP1B pathway. Finally, ablation of coronin-1C attenuated the proliferative and migratory activity of the cells. Collectively, these findings reveal that Nox4-mediated redox regulation of PTP1B serves as a modulator, in part through coronin-1C, of the growth and migration of glioblastoma cells, and provide new insight into the mechanistic aspect of glioblastoma malignancy.     

Reactive oxygen species as mediators of cell adhesion

The intracellular production of reactive oxygen species (ROS) has a fundamental importance in both cell proliferation and apoptosis induction. Moreover, many experimental and epidemiological evidence indicate that ROS contribute to the initiation and promotion of carcinogenesis, and that drugs or treatments aimed to reduce the tissue content of ROS can be chemopreventive and curative against cancer. Recently, important observations on the role of ROS as physiological regulators of intracellular signaling cascades activated by growth factors through their tyrosine-kinase receptors have shed new light on the possible mechanisms that can sustain the promoting activity of ROS. The downstream effect of ROS production is the reversible oxidation of proteins. Redox sensitive proteins include protein tyrosine phosphatases (PTPs) as the active-site cysteine is the target of specific oxidation, and this modification can be reversed by intracellular reducing agents. The reversible oxidation of PTPs family member was demonstrated firstly for PTP1B during EGF signaling and then for LMW-PTP and SHP-2 during PDGF stimulation. The inhibition exerted by ROS on tyrosine-phosphatases helps the propagation of RTK signals mediated by protein tyrosine phosphorylation, generally associated with the proliferative stimulus. Our new data are consistent with a model in which ROS take a role in integrin signaling, and in which synergistic activation of Rac-1 by growth factors and adhesion molecules translates in a critical increase of intracellular oxidants up to a threshold level where inhibition of the tyrosine phosphatase LMW-PTP takes place. In seeking for potential molecular mechanisms for oxidative signaling by integrins, we found that transient oxidation/inactivation of LMW-PTP, a known negative regulator of RTK signaling, occurred during fibroblast adhesion to matrix, with a kinetic which paralleled the generation of ROS. Moreover, overexpression of LMW-PTP in NIH-3T3 fibroblasts delayed cell attachment to the substrate. Finally, constitutively high levels of intracellular ROS, as are observed in cells expressing active Rac, would attenuate anchorage dependence for growth, by substituting for integrin signaling in non adherent cells.     

Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation

Oncogene-induced reactive oxygen species (ROS) have been proposed to be signaling molecules that mediate proliferative cues. However, ROS may also cause DNA damage and proliferative arrest. How these apparently opposite roles can be reconciled, especially in the context of oncogene-induced cellular senescence, which is associated both with aberrant mitogenic signaling and DNA damage response (DDR)-mediated arrest, is unclear. Here, we show that ROS are indeed mitogenic signaling molecules that fuel oncogene-driven aberrant cell proliferation. However, by their very same ability to mediate cell hyperproliferation, ROS eventually cause DDR activation. We also show that oncogenic Ras-induced ROS are produced in a Rac1 and NADPH oxidase (Nox4)-dependent manner. In addition, we show that Ras-induced ROS can be detected and modulated in a living transparent animal: the zebrafish. Finally, in cancer we show that Nox4 is increased in both human tumors and a mouse model of pancreatic cancer and specific Nox4 small-molecule inhibitors act synergistically with existing chemotherapic agents.     

Nox4-Derived H2O2 Mediates Endoplasmic Reticulum Signaling through Local Ras Activation

The unfolded-protein response (UPR) of the endoplasmic reticulum (ER) has been linked to oxidant production, although the molecular details and functional significance of this linkage are poorly understood. Using a ratiometric H₂O₂ sensor targeted to different subcellular compartments, we demonstrate specific production of H₂O₂ by the ER in response to the stressors tunicamycin and HIV-1 Tat, but not to thapsigargin or dithiothreitol. Knockdown of the oxidase Nox4, expressed on ER endomembranes, or expression of ER-targeted catalase blocked ER H₂O₂ production by tunicamycin and Tat and prevented the UPR following exposure to these two agonists, but not to thapsigargin or dithiothreitol. Tat also triggered Nox4-dependent, sustained activation of Ras leading to ERK, but not phosphatidylinositol 3-kinase (PI3K)/mTOR, pathway activation. Cell fractionation studies and green fluorescent protein (GFP) fusions of GTPase effector binding domains confirmed selective activation of endogenous RhoA and Ras on the ER surface, with ER-associated K-Ras acting upstream of the UPR and downstream of Nox4. Notably, the Nox4/Ras/ERK pathway induced autophagy, and suppression of autophagy unmasked cell death and prevented differentiation of endothelial cells in 3-dimensional matrix. We conclude that the ER surface provides a platform to spatially organize agonist-specific Nox4-dependent oxidative signaling events, leading to homeostatic protective mechanisms rather than oxidative stress.Coupled in part to its function as a major site of protein synthesis, the endoplasmic reticulum (ER) has emerged as an important signaling organelle, responding to various cell stresses and controlling cell fate. Much of this signaling is initiated on the ER membrane surface. In response to an overload of misfolded client proteins in the ER lumen, for example, transmembrane ER stress sensors such as IRE1, PERK, and ATF6 initiate signals on the cytosolic face of the ER to reduce global protein synthesis, promote protein folding, and increase the degradation of misfolded proteins (36). Failure of this response to alleviate protein misfolding stress leads to late expression of proteins such as CHOP, culminating in cell death. In addition to factors controlling this unfolded-protein response (UPR), cyclins, pro- and anti-apoptotic BH3 domain proteins, caspases, and signaling adapters associate with the ER surface and control cell cycle entry, cell death, Ca²⁺ flux, amino acid metabolism, oxidative-stress response, and autophagy (15, 17-19, 41). Thus, the ER integrates a variety of stresses (metabolic, protein misfolding, and oxidative) to coordinate cellular stress responses.Another ER transmembrane protein is the NADPH oxidase Nox4. Like other Nox family members, Nox4 produces H₂O₂ and is thought to function primarily in cell signaling. Consistent with its ER localization, Nox4 mediates oxidative inactivation of the ER-resident phosphatase PTP1B and responds to ER stress induced by the LDL oxysterol 7-ketocholesterol (10, 34). The presence of Nox4 on the ER surface is notable, since ER stress is associated with the production of reactive oxygen species (ROS), though the nature of this association is poorly understood. Agents that cause ER stress initiate ATF4-dependent glutathione (GSH) synthesis, which is necessary to diminish ROS production and subsequent cell death (15). The mechanism of ROS production is also unclear, but it is thought to be a consequence of the UPR, possibly downstream of CHOP (25, 26). For the most part, such studies suggest a role for oxidants as late effectors of cell death, downstream from the UPR. The ER itself may be a source of oxidants, as knockdown of the ER oxidase Ero-1 in pek-1-null worms diminishes oxidant production (15). To some extent, the excessive production of ROS by the stressed ER would seem to run counter to recent observations that chemical and physiologic ER stressors converge on the production of a hyperreduced ER interior (28). In addition, [rho⁰] Saccharomyces cerevisiae lacking mitochondria also fails to produce ROS in response to ER stress, suggesting a mitochondrial source (16). In part, such studies linking the ER and oxidative stress have been hampered by reliance on probes, such as dichlorofluorescein, that are untargeted and nonspecific, reacting with a broad variety of oxidants as well as non-ROS compounds, such as cytosolic cytochrome c.In this study, using an H₂O₂-specific probe targeted to the ER, we found that Nox4 participates early in ER stress signaling in an agonist-specific fashion through a novel process involving focal activation of Ras on the ER. This Nox4-dependent pathway leads to activation of autophagy, which prevents the progression of the UPR to cell death, thus distinguishing Nox4 oxidant signaling from oxidative stress.     

PTP1B Targets the Endosomal Sorting Machinery: DEPHOSPHORYLATION OF REGULATORY SITES ON THE ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT COMPONENT STAM2*

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling in a previously unrecognized manner.     

Regulation of insulin signaling through reversible oxidation of the protein-tyrosine phosphatases TC45 and PTP1B

Many studies have illustrated that the production of reactive oxygen species (ROS) is important for optimal tyrosine phosphorylation and signaling in response to diverse stimuli. Protein-tyrosine phosphatases (PTPs), which are important regulators of signal transduction, are exquisitely sensitive to inhibition after generation of ROS, and reversible oxidation is becoming recognized as a general physiological mechanism for regulation of PTP function. Thus, production of ROS facilitates a tyrosine phosphorylation-dependent cellular signaling response by transiently inactivating those PTPs that normally suppress the signal. In this study, we have explored the importance of reversible PTP oxidation in the signaling response to insulin. Using a modified ingel PTP assay, we show that stimulation of cells with insulin resulted in the rapid and transient oxidation and inhibition of two distinct PTPs, which we have identified as PTP1B and TC45, the 45-kDa spliced variant of the T cell protein-tyrosine phosphatase. We investigated further the role of TC45 as a regulator of insulin signaling by combining RNA interference and the use of substrate-trapping mutants. We have shown that TC45 is an inhibitor of insulin signaling, recognizing the beta-subunit of the insulin receptor as a substrate. The data also suggest that this strategy, using ligand-induced oxidation to tag specific PTPs and using interference RNA and substrate-trapping mutants to illustrate their role as regulators of particular signal transduction pathways, may be applied broadly across the PTP family to explore function.     

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.     

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.     

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 PDGFβR phosphorylation in two independent cell systems demonstrated a decrease in PDGFβR 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 PDGFβR-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 PDGFβR phosphorylation, showing that mitochondrial ROS in general promote PDGFβR 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 1B in intact cells by S-nitrosothiols

Protein tyrosine phosphatases (PTPases) contain an active site cysteine which when oxidized leads to loss of phosphatase activity and accumulation of phosphoproteins. For example, oxidants produced following EGF stimulation inhibit PTP1B and enhance EGF receptor phosphorylation. Because NO-derived species also modify reactive thiols, we postulated that NO would reversibly inhibit PTP1B. In our studies we exposed A431 or Jurkat cells to NO donors and measured PTP1B activity or used 3-maleimidylpropionylbiocytin (MPB) to measure thiol redox status. Nitrosothiols led to a rapid inhibition of PTP1B through a mechanism that was greatly enhanced by addition of cysteine to the medium. Analysis of thiol oxidation status using immunoprecipitated PTP1B showed modification consistent with loss of activity. Both enzyme inhibition and modification were reversible in intact cells or after addition of DTT to cell lysates. While DTT reversed oxidation, ascorbate did not, suggesting that formation of a mixed disulfide (possibly glutathionylation) rather than S-nitrosylation accounts for PTP1B inhibition. Importantly, PTP1B inhibition by nitrosothiols led to EGF receptor phosphorylation even in the absence of exogenously added EGF. These findings suggest an important role for NO in modulating signaling pathways since inhibition of PTPases could potentially enhance or prolong activity of phosphoproteins.     

Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor

The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states.     

Pyrroloquinoline quinone stimulates epithelial cell proliferation by activating epidermal growth factor receptor through redox cycling

Pyrroloquinoline quinone (PQQ), a redox cofactor for bacterial dehydrogenases, has been implicated to be an important nutrient in mammals functioning as a potent growth factor. However, the underlying molecular mechanisms have not been elucidated. The present study revealed that PQQ induces the activation (tyrosine autophosphorylation) of epidermal growth factor receptor (EGFR) and its downstream signaling in a ligand-independent manner, leading to increased cellular proliferation in an epithelial cell line A431. PQQ inhibited protein tyrosine phosphatase 1B (PTP1B), which negatively regulates the EGFR signaling by tyrosine dephosphorylation, to oxidatively modify the catalytic cysteine through its redox cycling activity to generate H(2)O(2). PQQ-inducible intracellular ROS production and EGFR activation were significantly suppressed by the pre-treatment with antioxidants. The intracellular redox state regulates the EGFR signaling through the redox-sensitive catalytic cysteine of PTP1B and modulates cell proliferation. Our data suggest that PQQ may stimulate epithelial cell proliferation by activating EGFR by oxidation and subsequent inactivation of PTP1B via its redox cycling. Our results provide novel insight into the mechanisms by which PQQ may function as a growth factor to contribute to mammalian growth.     

Nox4 and redox signaling mediate TGF-β-induced endothelial cell apoptosis and phenotypic switch

Transforming growth factor-β (TGF-β) triggers apoptosis in endothelial cells, while the mechanisms underlying this action are not entirely understood. Using genetic and pharmacological tools, we demonstrated that TGF-β induced a moderate apoptotic response in human cultured endothelial cells, which was dependent upon upregulation of the Nox4 NADPH oxidase and production of reactive oxygen species (ROS). In contrast, we showed that ectopic expression of Nox4 via viral vectors (vNox4) produced an antiapoptotic effect. TGF-β caused ROS-dependent p38 activation, whereas inhibition of p38 blunted TGF-β-induced apoptosis. However, vNox4, but not TGF-β, activated Akt, and inhibition of Akt attenuated the antiapoptotic effect of vNox4. Akt activation induced by vNox4 was accompanied by inactivation of the protein tyrosine phosphatase-1B (PTP1B) function and enhanced vascular endothelial growth factor receptor (VEGFR)-2 phosphorylation. Moreover, we showed that TGF-β enhanced Notch signaling and increased expression of the arterial marker EphrinB2 in a redox-dependent manner. In summary, our results suggest that Nox4 and ROS have pivotal roles in mediating TGF-β-induced endothelial apoptosis and phenotype specification. Redox mechanisms may influence endothelial cell functions by modulating p38, PTP1B/VEGFR/Akt and Notch signaling pathways.     

Molecular interaction of NADPH oxidase 1 with betaPix and Nox Organizer 1

It is well established that growth-factor-induced reactive oxygen species (ROS) act as second messengers in cell signaling. We have previously reported that betaPix, a guanine nucleotide exchange factor for Rac, interacts with NADPH oxidase 1 (Nox1) leading to EGF-induced ROS generation. Here, we report the identification of the domains of Nox1 and betaPix responsible for the interaction between the two proteins. GST pull-down assays show that the PH domain of betaPix binds to the FAD-binding region of Nox1. We also show that overexpression of the PH domain of betaPix results in inhibition of superoxide anion generation in response to EGF. Additionally, NADPH oxidase Organizer 1 (NoxO1) is shown to interact with the NADPH-binding region of Nox1. These results suggest that the formation of the complex consisting of Nox1, betaPix, and NoxO1 is likely to be a critical step in EGF-induced ROS generation.     

Identification of novel Nox4 splice variants with impact on ROS levels in A549 cells

NAD(P)H oxidases (Nox) generate reactive oxygen species (ROS) that function in host defense and cellular signaling. While analyzing the expression of Nox4 at the protein and the mRNA levels, we identified four novel Nox4 splice-variants Nox4B, Nox4C, Nox4D, and Nox4E, which are expressed in human lung A549 cell line and lung tissues. One Nox4 isoform lacks the first NAD(P)H binding site (Nox4B) while another lacks all FADH and NAD(P)H binding sites (Nox4C). Cells over-expressing NoxB or Nox4C exhibited a decrease in ROS levels. Thus, these isoforms have dominant negative characteristics for ROS generation. Two other splice-variants (Nox4D, Nox4E) lack the transmembrane domains, suggesting these as non-membrane associated isoforms. Nox4D contains all FADH and NAD(P)H binding domains and shows the same rate of ROS generation as Nox4 prototype. Taken together, we suggest that Nox4 exists as several isoforms that may have different functions in ROS-related cell signaling.     

Protein Tyrosine Phosphatase 1B and Insulin Resistance: Role of Endoplasmic Reticulum Stress/Reactive Oxygen Species/Nuclear Factor Kappa B Axis

Obesity-induced endoplasmic reticulum (ER) stress has been proposed as an important pathway in the development of insulin resistance. Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and is tethered to the ER-membrane. The aim of the study was to determine the mechanisms involved in the crosstalk between ER-stress and PTP1B. PTP1B whole body knockout and C57BL/6J mice were subjected to a high-fat or normal chow-diet for 20 weeks. High-fat diet feeding induced body weight gain, increased adiposity, systemic glucose intolerance, and hepatic steatosis were attenuated by PTP1B deletion. High-fat diet- fed PTP1B knockout mice also exhibited improved glucose uptake measured using [³H]-2-deoxy-glucose incorporation assay and Akt phosphorylation in the skeletal muscle tissue, compared to their wild-type control mice which received similar diet. High-fat diet-induced upregulation of glucose-regulated protein-78, phosphorylation of eukaryotic initiation factor 2α and c-Jun NH₂-terminal kinase-2 were significantly attenuated in the PTP1B knockout mice. Mice lacking PTP1B showed decreased expression of the autophagy related protein p62 and the unfolded protein response adaptor protein NCK1 (non-catalytic region of tyrosine kinase). Treatment of C2C12 myotubes with the ER-stressor tunicamycin resulted in the accumulation of reactive oxygen species (ROS), leading to the activation of protein expression of PTP1B. Furthermore, tunicamycin-induced ROS production activated nuclear translocation of NFκB p65 and was required for ER stress-mediated expression of PTP1B. Our data suggest that PTP1B is induced by ER stress via the activation of the ROS-NFκB axis which is causes unfolded protein response and mediates insulin resistance in the skeletal muscle under obese condition.     

Identification of phosphocaveolin-1 as a novel protein tyrosine phosphatase 1B substrate

Protein tyrosine phosphatase 1B (PTP1B) is implicated in a number of signaling pathways including those mediated by insulin, epidermal growth factor (EGF), and the Src family kinases. The scaffolding protein caveolin-1 is also a participant in these pathways and is specifically phosphorylated on tyrosine 14, when these pathways are activated. Here, we provide evidence that PTP1B can efficiently catalyze the removal of the phosphoryl group from phosphocaveolin-1. Overexpression of PTP1B decreases tyrosine 14 phosphorylation in caveolin-1, while expression of the substrate-trapping mutant PTP1B/D181A causes the accumulation of phosphocaveolin-1 and prevents its dephosphorylation by endogenous PTPs. We further demonstrate that PTP1B physically associates with caveolin-1. Finally, we show that inhibition of PTP1B activity with a potent and specific small molecule PTP1B inhibitor blocks the PTP1B-catalyzed caveolin-1 dephosphorylation both in vitro and in vivo. Taken together, the results strongly suggest that caveolin-1 is a specific substrate for PTP1B. Identification of caveolin-1 as a PTP1B substrate represents an important new step in further understanding the signaling pathways regulated by PTP1B.     

Phagocyte-like NADPH oxidase generates ROS in INS 832/13 cells and rat islets: role of protein prenylation

Recent evidence suggests that an acute increase in the generation of phagocyte-like NADPH-oxidase (Nox)-mediated reactive oxygen species (ROS) may be necessary for glucose-stimulated insulin secretion. Using rat islets and INS 832/13 cells, we tested the hypothesis that activation of specific G proteins is necessary for nutrient-mediated intracellular generation of ROS. Stimulation of β-cells with glucose or a mixture of mitochondrial fuels (mono-methylsuccinate plus α-ketoisocaproic acid) markedly elevated intracellular accumulation of ROS, which was attenuated by selective inhibitors of Nox (e.g., apocynin or diphenyleneiodonium chloride) or short interfering RNA-mediated knockdown of p47(phox), one of the subunits of Nox. Selective inhibitors of protein prenylation (FTI-277 or GGTI-2147) markedly inhibited nutrient-induced ROS generation, suggesting that activation of one (or more) prenylated small G proteins and/or γ-subunits of trimeric G proteins is involved in this signaling axis. Depletion of endogenous GTP levels with mycophenolic acid significantly reduced glucose-induced activation of Rac1 and ROS generation in these cells. Other immunosuppressants, like cyclosporine A or rapamycin, which do not deplete endogenous GTP levels, failed to affect glucose-induced ROS generation, suggesting that endogenous GTP is necessary for glucose-induced Nox activation and ROS generation. Treatment of INS 832/13 cells or rat islets with pertussis toxin (Ptx), which ADP ribosylates and inhibits inhibitory class of trimeric G proteins (i.e., G(i) or G(o)), significantly attenuated glucose-induced ROS generation in these cells, implicating activation of a Ptx-sensitive G protein in these signaling cascade. Together, our findings suggest a prenylated Ptx-sensitive signaling step couples Rac1 activation in the signaling steps necessary for glucose-mediated generation of ROS in the pancreatic β-cells.     

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells

Aims

Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs).

Main methods

AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry.

Key findings

AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation.

Significance

This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.