Thioredoxin reductase regulates the induction of haem oxygenase-1 expression in aortic endothelial cells - PubMed (original) (raw)

Thioredoxin reductase regulates the induction of haem oxygenase-1 expression in aortic endothelial cells

Wendy L Trigona et al. Biochem J. 2006.

Abstract

Certain selenoproteins such as GPX-1 (glutathione peroxidase-1) and TrxR1 (thioredoxin reductase-1) possess important antioxidant defence functions in vascular endothelial cells. Reduced selenoprotein activity during dietary selenium (Se) deficiency can result in a compensatory increase of other non-Se-dependent antioxidants, such as HO-1 (haem oxygenase-1) that may help to counteract the damaging effects of oxidant stress. However, the role of individual selenoproteins in regulating vascular-derived protective gene responses such as HO-1 is less understood. Using an oxidant stress model based on Se deficiency in BAECs (bovine aortic endothelial cells), we sought to determine whether TrxR1 activity may contribute to the differential regulation of HO-1 expression as a function of altered redox environment. Se-sufficient BAECs up-regulated HO-1 expression following stimulation with the pro-oxidant, 15-HPETE (15-hydroperoxyeicosatetraenoic acid), and levels of this antioxidant inversely correlated with EC apoptosis. While Se-deficient BAECs exhibited higher basal levels of HO-1, it was not up-regulated upon 15-HPETE treatment, which resulted in significantly higher levels of pro-apoptotic markers. Subsequent results showed that HO-1 induction depended on the activity of TrxR1, as proved with chemical inhibitor studies and direct inhibition with TrxR1 siRNA. Finally, restoring intracellular levels of the reduced substrate Trx (thioredoxin) in Sedeficient BAECs was sufficient to increase HO-1 activation following 15-HPETE stimulation. These data provide evidence for the involvement of the Trx/TrxR system, in the regulation of HO-1 expression in BAECs during pro-oxidant challenge.

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Figures

Figure 1

Figure 1. Se deficiency increases expression of HO-1 mRNA and protein

ECs were cultured in +Se or −Se medium for two to three passages. (A) Cells were transiently transfected with HO-1 4.0 kb Luc plasmid and 48 h later were analysed for luciferase activity. Luciferase activity is expressed as fold increase of firefly/Renilla over +Se unstimulated BAECs (unstim). Results are means±S.E.M. for three separate experiments. (B) RNA was isolated and QC RT–PCR was performed. PCR products were separated on a 2% agarose gel, and densitometry was performed in order to quantify the amount of HO-1 mRNA. Results are expressed as fold increase of HO-1 mRNA over +Se unstimulated BAECs (unstim) and are means±S.E.M. for five separate experiments. (C) Whole-cell lysates were harvested, equal amounts of protein were resolved by SDS/12% PAGE, and a Western blot was performed. Results are expressed as fold increase of HO-1 protein/actin ratio over +Se unstimulated BAECs (unstim) and are means±S.E.M. for three separate experiments. Values denoted by different lower-case letters are statistically different from each other (P<0.05) as determined using a paired Student's t test.

Figure 2

Figure 2. Differential levels of HO-1 expression in +Se and −Se BAECs following 15-HPETE stimulation

Following stimulation of ECs with dose titration of (A) 15-HPETE or (B) 15-HETE for 2 h, total RNA was isolated, and QC RT–PCR for HO-1 mRNA was then performed. PCR products were separated on a 2% agarose gel, and densitometry of HO-1 mRNA was measured. Results are expressed as fold increase of HO-1 mRNA over +Se unstimulated BAECs (unstim) and are means±S.E.M. for three separate experiments. (C) Following stimulation of ECs with dose titration of 15-HPETE for 2 h, whole-cell lysates were harvested, and equal amounts of protein were resolved by SDS/12% PAGE for Western blot analysis. (D) Densitometry results from three separate experiments in (C) were averaged and means±S.E.M. for three separate experiments are shown. Values denoted by different lower-case letters are statistically different from each other (P<0.05) as determined by ANOVA.

Figure 3

Figure 3. HO-1 activity regulates apoptosis during Se deficiency

+Se and −Se BAECs were pre-treated with the indicated doses of the HO-1 inhibitor, SnPPIX, for 15 h, followed by treatment with 30 μM 15-HETE or 15-HPETE for 6 h. (A) Caspase 3/7 activation was measured, and results are expressed as fold increase in caspase-3/7 activation over +Se unstimulated BAECs (unstim) as means±S.E.M. for four separate experiments. (B) For Hoechst 33342 staining, approx. 200 ECs per well, in triplicate per treatment, were manually scored for apoptosis based on the presence or absence of nucleic acid condensation and results are reported as the percentage of BAECs with condensed chromatin. Values denoted by different lower-case letters are statistically different from each other (P<0.05) as determined by ANOVA.

Figure 4

Figure 4. TrxR1 siRNA decreases TrxR mRNA, protein and activity

BAECs were transfected with siRNA for TrxR1 or control sequence and were grown to confluence under antibiotic selection for stable expression of siRNA construct. (A) Total RNA was isolated, and QC RT–PCR for TrxR1 mRNA was then performed. PCR products were separated on a 2% agarose gel, and densitometry of TrxR1 mRNA and internal standard was measured. (B) Whole-cell lysates were harvested, equal amounts of protein were resolved by SDS/10% PAGE, and a Western blot was performed. A representative blot is shown. (C) Results in (B) are expressed as fold increase TrxR protein over actin and are means±S.E.M. for three separate experiments. (D) Whole-cell lysates were harvested and tested for TrxR1 activity using the insulin-based method. Results are expressed in _A_412 units×1000/min per mg of protein, and are means±S.E.M. for three separate clones. Values denoted by different lower-case letters are statistically different from each other (P<0.05) as determined by ANOVA.

Figure 5

Figure 5. TrxR1 siRNA inhibits HO-1 induction in BAECs following 15-HPETE stimulation

BAECs were transfected with siRNA for TrxR1 or control sequence and were grown to confluence under antibiotic selection for expression of siRNA construct. (A) Whole-cell lysates from control and TrxR1 siRNA-transfected cells were harvested, and equal amounts of protein were resolved by SDS/12% PAGE for measurement of HO-1 protein levels. (B) Densitometry results from three separate experiments in (A) were averaged, and results are means±S.E.M. for three separate experiments are shown. (C) Caspase 3/7 activation was measured in TrxR siRNA and control siRNA BAECs following 2 h of 15-HPETE stimulation, and results are expressed as fold increase in caspase-3/7 activation over unstimulated BAECs (unstim). Results are means±S.E.M. for four separate experiments. Values denoted by different lower-case letters are statistically different from each other (P<0.05) as determined by ANOVA.

Figure 6

Figure 6. Trx localization following 15-HPETE stimulation

+Se and −Se BAECs were stimulated with 30 μM 15-HPETE or 15-HPETE for 2 h. (A) Cytosolic and nuclear extracts were separated, and equal amounts of protein were resolved by SDS/15% PAGE. (B) Densitometry results from three separate experiments performed with nuclear extracts in (A) were averaged, and results are means±S.E.M. for three separate experiments. Values denoted by different lower-case letters are statistically different from each other (P<0.05) as determined by ANOVA.

Figure 7

Figure 7. Recombinant Trx induces levels of HO-1 in −Se BAECs following 15-HPETE stimulation

(A) +Se and −Se BAECs were pre-treated with the indicated doses of rTrx for 3 h, followed by treatment with 30 μM 15-HPETE for 2 h. RNA was isolated, and QC RT–PCR was performed. PCR products were separated on a 2% agarose gel, and densitometry was performed in order to quantify the amount of HO-1 mRNA. Results are expressed as fold increase HO-1 mRNA over +Se unstimulated (unstim), and results are means±S.E.M. for three separate experiments. Values denoted by different lower-case letters are statistically different from each other (P<0.05) as determined by ANOVA. (B) BAECs were stimulated with 1 or 10 μg/ml rTrx for 3 h, whole-cell lysates were harvested, and equal amounts of protein were resolved by SDS/15% PAGE. A Western blot was performed to confirm increased intracellular Trx protein following rTrx treatment. (C) +Se and −Se BAECs were pre-treated with 10 μg/ml rTrx for 3 h followed by treatment with 30 μM 15-HPETE for 2 h. Whole-cell lysates were harvested, equal amounts of protein were resolved by SDS/12.5% PAGE and a Western blot was performed for HO-1 protein levels. A representative blot of three is shown.

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