Bloch and Richard T. Lee P. Christian Schulze, Heling Liu, Elizabeth ...
Bloch and Richard T. Lee P. Christian Schulze, Heling Liu, Elizabeth ... Bloch and Richard T. Lee P. Christian Schulze, Heling Liu, Elizabeth ...
2668 Arterioscler Thromb Vasc Biol. December 2006 versus unstimulated cells), 1.70.2-fold at 2 hours (P0.01 versus unstimulated cells), and 1.90.5-fold after 4 hours of stimulation (P0.05 versus unstimulated cells) (Figure 1G). Further, GSNO increased thioredoxin activity 1.50.5-fold at 10 mol/L (pNS), 1.90.5-fold at 100 mol/L (P0.05 versus unstimulated cells), and 1.90.2-fold at 500 mol/L (P0.001 versus unstimulated cells) of GSNO stimulation (Figure 1H). The increase of thioredoxin activity after 4 hours of GSNO stimulation was reflected in a decrease by 5315% Figure 1. Regulation of Txnip and thioredoxin reductase expression. A, GSNO stimulation resulted in a time-dependent reduction of Txnip mRNA and induction of thioredoxin reductase mRNA. B, Incubation with increasing levels of GSNO resulted in a concentration-dependent reduction of Txnip mRNA and an increase in thioredoxin reductase mRNA. C, Stimulation with GSNO resulted in time-dependent reduction of Txnip protein levels while thioredoxin protein levels remain unchanged. D, Stimulation of RPaSMC with GSNO induces protein levels of thioredoxin reductase. E, Stimulation with the NO donors GSNO, NOC- 15, and SNAP resulted in comparable reduction of Txnip protein levels. F, Preincubation with the pharmacological inhibitor ODQ did not inhibit the GSNOinduced effects on Txnip and thioredoxin reductase expression (representative blots from 3 independent experiments). G, Incubation of RPaSMC with GSNO resulted in a time-dependent increase of thioredoxin activity by 1.6-fold at 1 hour, 1.7-fold at 2 hours, and 1.8-fold after 4 hours (n3 per data point). H, GSNO increased thioredoxin activity 1.5-fold at 10 m, 1.8-fold at 100 m, and 1.8-fold at 500 m GSNO (stimulation for 2 hours; n3 per data point). in levels of hydrogen peroxide as measured by DCFDA fluorescence (P0.05 versus unstimulated cells). NO Suppresses Txnip mRNA Expression Without Changing Its Stability To determine whether GSNO reduces Txnip mRNA accumulation by decreasing the rate of synthesis or by increasing the rate of degradation, RPaSMCs were pretreated with actinomycin D (5 g/mL) to inhibit transcriptional activity and then Downloaded from http://atvb.ahajournals.org/ by guest on July 30, 2013
exposed to 100 mol/L GSNO for several time intervals. The half-life of Txnip mRNA was not affected by GSNO stimulation (1.00.2 hour versus 0.80.2 hour; PNS) (Figure 2A). Using nuclear run-off experiments, we observed that de novo synthesis of Txnip mRNA was reduced in cells exposed to GSNO (Figure 2B). Assessment of specific radioactive count activity revealed a reduction of de novo Txnip mRNA Schulze et al NO Regulates Thioredoxin Through Txnip 2669 Figure 2. GSNO does not alter Txnip mRNA stability. A, RPaSMCs were pretreated with actinomycin D to inhibit transcriptional activity and then exposed to GSNO for 2, 4 and 6 hours. The halflife of Txnip mRNA was not affected by GSNO. B, De novo Txnip mRNA synthesis decreased from 0.280.03 to 0.190.05 (P0.05) under GSNO stimulation while levels of thioredoxin mRNA remained stable (0.40.02 vs 0.390.15; all n3 per data point). levels from 0.280.03 to 0.190.05 (n3; P0.05), whereas levels of de novo thioredoxin mRNA remained stable (0.40.02 versus 0.390.15; n3; PNS). NO Effects on Txnip Promoter Activity While several studies have previously investigated the transcriptional regulation of thioredoxin reductase, 22,23 little is Figure 3. Deletion analysis of the human Txnip promoter. A, Schematic representation of the human Txnip promoter and deletion constructs. Numbers refer to base pairs upstream of the ATG codon. B, Transfection of Txnip promoter constructs reveals activation of the Txnip promoter in RPaSMCs. Luciferase activities are expressed as percentages of full-length promoter activity. Empty vector transfected cells served as control. C, GSNO stimulation of RPaSMC’s transfected with the Txnip promoter constructs showed strong suppression of luciferase activity only in cells transfected with the 1777 Txnip promoter (*P0.05 vs nonstimulated cells; n5 per data point). D, Transfection of RPaSMC with full-length Txnip promoter constructs followed by incubation in high glucose (22.4 mmol/L) or low glucose (5.6 mmol/L) medium revealed a strong induction of Txnip promoter activity in RPaSMCs under hyperglycemic conditions. Stimulation of transfected cells with GSNO reduced Txnip promoter activity at 5.6 mmol/L glucose but had no effect at 22.4 mmol/L glucose (*P0.05 vs nonstimulated cells; #P0.001 vs low glucose; n5 per data point). Downloaded from http://atvb.ahajournals.org/ by guest on July 30, 2013
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2668 Arterioscler Thromb Vasc Biol. December 2006<br />
versus unstimulated cells), 1.70.2-fold at 2 hours (P0.01<br />
versus unstimulated cells), <strong>and</strong> 1.90.5-fold after 4 hours of<br />
stimulation (P0.05 versus unstimulated cells) (Figure 1G).<br />
Further, GSNO increased thioredoxin activity 1.50.5-fold at<br />
10 mol/L (pNS), 1.90.5-fold at 100 mol/L (P0.05<br />
versus unstimulated cells), <strong>and</strong> 1.90.2-fold at 500 mol/L<br />
(P0.001 versus unstimulated cells) of GSNO stimulation<br />
(Figure 1H). The increase of thioredoxin activity after 4 hours<br />
of GSNO stimulation was reflected in a decrease by 5315%<br />
Figure 1. Regulation of Txnip <strong>and</strong> thioredoxin<br />
reductase expression. A, GSNO<br />
stimulation resulted in a time-dependent<br />
reduction of Txnip mRNA <strong>and</strong> induction<br />
of thioredoxin reductase mRNA. B, Incubation<br />
with increasing levels of GSNO<br />
resulted in a concentration-dependent<br />
reduction of Txnip mRNA <strong>and</strong> an<br />
increase in thioredoxin reductase mRNA.<br />
C, Stimulation with GSNO resulted in<br />
time-dependent reduction of Txnip protein<br />
levels while thioredoxin protein levels<br />
remain unchanged. D, Stimulation of<br />
RPaSMC with GSNO induces protein<br />
levels of thioredoxin reductase. E, Stimulation<br />
with the NO donors GSNO, NOC-<br />
15, <strong>and</strong> SNAP resulted in comparable<br />
reduction of Txnip protein levels. F, Preincubation<br />
with the pharmacological inhibitor<br />
ODQ did not inhibit the GSNOinduced<br />
effects on Txnip <strong>and</strong> thioredoxin<br />
reductase expression (representative<br />
blots from 3 independent experiments).<br />
G, Incubation of RPaSMC with GSNO<br />
resulted in a time-dependent increase of<br />
thioredoxin activity by 1.6-fold at 1 hour,<br />
1.7-fold at 2 hours, <strong>and</strong> 1.8-fold after 4<br />
hours (n3 per data point). H, GSNO<br />
increased thioredoxin activity 1.5-fold at<br />
10 m, 1.8-fold at 100 m, <strong>and</strong> 1.8-fold<br />
at 500 m GSNO (stimulation for 2<br />
hours; n3 per data point).<br />
in levels of hydrogen peroxide as measured by DCFDA<br />
fluorescence (P0.05 versus unstimulated cells).<br />
NO Suppresses Txnip mRNA Expression Without<br />
Changing Its Stability<br />
To determine whether GSNO reduces Txnip mRNA accumulation<br />
by decreasing the rate of synthesis or by increasing the<br />
rate of degradation, RPaSMCs were pretreated with actinomycin<br />
D (5 g/mL) to inhibit transcriptional activity <strong>and</strong> then<br />
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