14 research outputs found
Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization
<div><p>Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H<sub>2</sub>O<sub>2</sub>, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases. </p> </div
Schematic representation of TRX-1 and the ERK1/2 MAP kinases nuclear translocation stimulated by nitrosative/oxidative stress conditions.
<p>(A) The Ras-Raf-MEK-ERK 1/2 signaling pathway is activated in HeLa wild-type cells after exposure to 0.5 mM SNAP or 0.5 mM H<sub>2</sub>O<sub>2</sub> (1). Under these conditions expression of TXNIP is down-regulated at mRNA and protein levels (2) and TRX-1 and the ERK 1/2 MAP kinases, independently of each other, migrate to the nuclear compartment (3). (B) Nuclear migration of TRX-1 and the ERK 1/2 MAP kinases is prevented in three situations: HeLa cells pre-incubated with the MEK inhibitor PD98059 (4), HeLa cells over-expressing the cytoplasmic anchor of ERK 1/2 MAP kinases – PEA-15 (5), HeLa cells over-expressing the physiological inhibitor of TRX-1, TXNIP (6).</p
Confocal microscopy based analysis to determine a time-dependent sub-cellular localization of TRX-1 in HeLa cells.
<p>(<b>A</b>) Wild-type HeLa cells, HeLa cells expressing PEA-15, and HeLa cells expressing TXNIP were plated two days prior to experiment. Transient transfections with pEGFP-C1_TRX-1 were performed on day one. On day two, cells were assayed for TRX-1 sub-cellular localization. Cells were kept in a heated stage chamber (37°C) of the confocal microscope, and a first scanning was recorded to serve as control. After that, media was replaced and H<sub>2</sub>O<sub>2</sub> was added to a final concentration of 0.5 mM. Subsequent scanning were performed at every 15 min up to 2 h. Bar = 20µm. (<b>B</b>) Cells were transfected with pEGFP-C1-TRX-1, treated with SNAP (0.5 mM) for 2 h at 37°C and submitted to indirect immunofluorescence for GFP, to show the localization of TRX-1 in HeLa cells. Red arrows indicate TRX-1 nuclear accumulation.</p
TXNIP promoter construction and regulation of its expression by oxidative and nitrosative stress.
<p>(<b>A</b>) Scheme representing the molecular cloning of TXNIP’s promoter region replacing the pEGFP-C1 CMV’s promoter region. (<b>B</b>) HeLa wild-type cells were transiently transfected with the TXNIP promoter construction and relative levels of GFP mRNA were determined by real-time PCR. (<b>C</b>) HeLa cells permanently transfected with the different TXNIP promoter constructs were stimulated with H<sub>2</sub>O<sub>2</sub> (0.5 mM) or SNAP (0.5 mM). Treatments stimulated strong suppression of GFP expression only in cells transfected with the –2056 base pairs TXNIP promoter construct. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *<i>p</i><0.05).</p
HeLa cells submitted to oxidative and nitrosative stress conditions.
<p>(<b>A</b>) HeLa cells were treated with increasing concentrations of SNAP (0 - 1.0 mM) during 2 h. After this period medium was replaced for complete medium (MEM supplemented with 10% FBS). Cell viability was determined 24 h later using the MTT assay as described in Materials and Methods. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *<i>p</i> < 0.05). (<b>B</b>) Intracellular NO detection in HeLa cells after incubation with 0.5 mM SNAP for 2 h at 37°C. DAF fluorescence is directly related to NO generation by intracellular metabolism of SNAP and was determined by flow cytometry. (<b>C</b>) HeLa cells were treated with increasing concentrations of H<sub>2</sub>O<sub>2</sub> (0 - 1.0 mM) during 2 h. After this period medium was replaced for complete medium (MEM supplemented with 10% FBS). Cell viability was determined 24 h later using the MTT assay as described in Materials and Methods. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *<i>p</i> < 0.05). (<b>D</b>) Catalase and Glutathione Peroxidase enzymatic activity assays (described in Materials and Methods) were performed after exposure of HeLa cells to 0.5 mM H<sub>2</sub>O<sub>2</sub> for 2 h at 37°C. (<b>E</b>) HeLa cells were exposed to 0.5 mM SNAP or to 0.5 mM H<sub>2</sub>O<sub>2</sub> for 2 h at 37°C. After cell lysis, 50 μg total protein were subjected to Western blotting with a rabbit polyclonal anti-phospho-Akt antibody and with a mouse monoclonal anti-Akt antibody. Western blot results are representative of three independent experiments. Histogram represents the ratio between the densitometric values of the protein bands corresponding to the phosphorylated form of Akt and of Akt protein of one representative experiment (Lower panel). </p
TXNIP silencing and TRX-1 intracellular localization.
<p>(<b>A</b>) Western blot analysis to determine the effects of mRNA TXNIP knockdown on TXNIP protein levels in HeLa cells permanently transfected with a shRNA for TXNIP. The blot presented is representative of three independent experiments. (<b>B</b>) Indirect immunofluorescence to determine TRX-1 sub-cellular localization in HeLa cells permanently transfected with a shRNA for TXNIP. Images shown are representative from three independent experiments. Blue arrows indicate TRX-1 nuclear accumulation. Bar = 20µm.</p
Determination of sub-cellular compartmentalization of TRX-1 in HeLa cells transiently over-expressing PEA-15; TXNIP; TRX-1 and exposed to 0.5 mM H<sub>2</sub>O<sub>2</sub>.
<p>HeLa wild-type cells were seeded in complete medium and 24 h later were transiently transfected with the following plasmids pcDNA3_PEA-15, pcDNA3_TXNIP, and pEGFP-C1_TRX-1. After 16 h, transiently transfected cultures were divided to perform either western blot analysis, to confirm over expression of designated inserts, or to perform immunofluorescence-based analysis to determine TRX-1 sub cellular localization after exposure to 0.5 mM H<sub>2</sub>O<sub>2</sub>. (<b>A</b>) HeLa cells transfected with pcDNA3_PEA-15. Left panel: western blot analysis of PEA-15 expression. Right panel: Immunofluorescence image of TRX-1 cellular location. (<b>B</b>) HeLa cells transfected with pcDNA3_TXNIP. Left panel: western blot analysis of TXNIP expression. Right panel: Immunofluorescence image of TRX-1 cellular location (<b>C</b>) HeLa cells transfected with pEGFP-C1_TRX-. Left panel: western blot analysis of TRX-1 expression. Right panel: Immunofluorescence image of TRX-1 cellular location. Red arrows indicate TRX-1 nuclear accumulation.</p
SNAP and H<sub>2</sub>O<sub>2</sub> -modulated TXNIP mRNA and protein levels in wild-type Hela cells and in HeLa cells expressing PEA-15.
<p>(<b>A</b>) Relative levels of TXNIP mRNA determined by quantitative real-time PCR in HeLa cells wild-type, in HeLa cells expressing PEA-15, and in HeLa cells wild-type pre-treated with 50μM of PD98059 for 30 min as indicated (see Materials and Methods for details). All cell lines were treated with 0.5mM SNAP or 0.5mM H<sub>2</sub>O<sub>2</sub> for 2 h at 37°C. Values are reported in the bar graphs and expressed as means ± S.D. (n = 3, *<i>p</i> < 0.05; **<i>p</i> < 0.01). (<b>B</b>) wild-type HeLa cells and HeLa cells expressing PEA-15 were incubated with SNAP (0.5 mM) or H<sub>2</sub>O<sub>2</sub> (0.5 mM) for 2 h at 37°C. Cells were lysed and expression levels of TXNIP were examined by western blotting. Western blot results are representative of four independent experiments. Histogram represents the ratio between the densitometric values of the protein bands corresponding to TXNIP and of β-actin (Lower panel). </p
Confocal microscopy and western blot analysis to determine TRX-1 and phospho ERK 1/2 MAP Kinases sub-cellular location in wild-type HeLa cells.
<p>(<b>A</b>) Cells were treated with SNAP (0.5 mM), or H<sub>2</sub>O<sub>2</sub> (0.5 mM) for 2 h at 37°C. Detection of TRX-1 and phospho-ERK 1/2 MAP Kinases was performed as described in Materials and Methods. Bar = 20µm. (<b>B</b>) Cells were treated with SNAP (0.5mM) or H<sub>2</sub>O<sub>2</sub> (0.5 mM) for 2 h at 37°C. HeLa cells were pre-treated with 50μM PD98059, a MEK inhibitor, for 30 min and incubated with 0.5mM SNAP or 0.5 mM H<sub>2</sub>O<sub>2</sub> for 2 h at 37°C. Detection of TRX-1 was performed on a fluorescence microscope as described in Materials and Methods. Images shown are representative from three independent experiments. Bar = 20µm. (<b>C</b>) HeLa cells were incubated with SNAP and H<sub>2</sub>O<sub>2</sub> at the indicated concentrations for 2 h and nuclear extracts were subjected to Western blotting with the anti-human TRX-1 mouse monoclonal antibody and with the anti-human phospho-ERK1/2 MAP Kinases rabbit polyclonal antibody. Western blot results are representative of three independent experiments. Purity of nuclear fractions was estimated by LDH detection as described in Materials and Methods. Values are means ± S.D. of triplicate cultures.</p
Low-temperature EPR spectra of ferricyt <i>c</i> treated with AA.
<p>(A) EPR spectra of (300 µM) ferricyt <i>c</i> after a 12-h incubation with (1.0–5.0 mM) AA. (B) Time-response ERP spectra of ferricyt <i>c</i> treated with (1.0 mM) AA for 30 min. Incubation conditions: 50 mM phosphate buffer, pH 7.4, at 37°C. (C) MCD spectra of ferricyt <i>c</i> in the presence of AA. The conditions are ferricyt <i>c</i> (40 µM) MCD spectrum before treatment with AA (thick line) and MCD ferricyt <i>c</i> spectrum after 12 h of (5.0 mM) AA treatment (thin line). (D) CD spectrum of ferricyt <i>c</i> treated with 5.0 mM AA. Incubation conditions: 50 mM phosphate buffer, pH 7.4, at 37°C.</p