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, 9 (7), e100677
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Carbon Monoxide Induces Heme oxygenase-1 to Modulate STAT3 Activation in Endothelial Cells via S-glutathionylation

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Carbon Monoxide Induces Heme oxygenase-1 to Modulate STAT3 Activation in Endothelial Cells via S-glutathionylation

Yan-Chang Yang et al. PLoS One.

Abstract

IL-6/STAT3 pathway is involved in a variety of biological responses, including cell proliferation, differentiation, apoptosis, and inflammation. In our present study, we found that CO releasing molecules (CORMs) suppress IL-6-induced STAT3 phosphorylation, nuclear translocation and transactivity in endothelial cells (ECs). CO is a byproduct of heme degradation mediated by heme oxygenase (HO-1). However, CORMs can induce HO-1 expression and then inhibit STAT3 phosphorylation. CO has been found to increase a low level ROS and which may induce protein glutathionylation. We hypothesized that CORMs increases protein glutathionylation and inhibits STAT3 activation. We found that CORMs increase the intracellular GSSG level and induce the glutathionylation of multiple proteins including STAT3. GSSG can inhibit STAT3 phosphorylation and increase STAT3 glutathionylation whereas the antioxidant enzyme catalase can suppress the glutathionylation. Furthermore, catalase blocks the inhibition of STAT3 phosphorylation by CORMs treatment. The inhibition of glutathione synthesis by BSO was also found to attenuate STAT3 glutathionylation and its inhibition of STAT3 phosphorylation. We further found that HO-1 increases STAT3 glutathionylation and that HO-1 siRNA attenuates CORM-induced STAT3 glutathionylation. Hence, the inhibition of STAT3 activation is likely to occur via a CO-mediated increase in the GSSG level, which augments protein glutathionylation, and CO-induced HO-1 expression, which may enhance and maintain its effects in IL-6-treated ECs.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. CO inhibition of IL-6-induced STAT3 activation.
A. and B. EC cultures were induced by IL-6 treatment (10ng/ml) for 10 min and a portion of these cells were then pretreated with 25 µM TCDC or 5mM MC for the indicated times. Cell lysates were prepared and subjected to western blot analysis with antibodies against pTyr-STAT3 or STAT3 as indicated. The actin band intensities indicate equal loading of each well. C. and D. ECs were pretreated with TCDC or MC for the indicated times and then stimulated with IL-6. Nuclear (N) and cytosolic (C) extracts were then prepared and subjected to western blot analysis using STAT3 antibodies. The tubulin and lamin band intensities indicate equal loading, respectively. E. ECs were co-transfected with the STAT3 luciferase reporter construct and β-galactosidase for 16 hours. Cells were then exposed to TCDC or MC for 12 hours and to IL-6 for another 6 hours. Luciferase activity was normalized against β-galactosidase activity; the untreated value was taken as 1. *P<0.05 compared with untreated ECs, # P<0.05 compared with IL-6 alone (mean ± SEM).
Figure 2
Figure 2. CO induces HO-1 expression upon STAT3 phosphorylation.
A. and B. EC cultures were incubated with 25 µM TCDC or 5mM MC for the indicated periods. Western blotting analysis was then performed with antibodies against HO-1. C. ECs were transfected with control or HO-1 siRNA vectors for 36 hours and exposed to TCDC or MC for 12 hours. Cell lysates were subjected to western blot analysis with antibodies against pTyr-STAT3 or STAT3. D. ECs were transiently transfected with pcDNA vector or HO-1 plasmid. ECs transfected with pcDNA vector were exposed to IL-6 as a positive control. Cell lysates were subjected to western blot analysis with antibodies against pTyr-STAT3 or STAT3 as indicated. The quantification of the band intensities from three independent experiments was normalized to the control values (if the control was at near background, the background was set to 1). The resulting data are the mean ± SEM. *P<0.05 compared with untreated ECs.
Figure 3
Figure 3. CO increases oxidative stress in ECs.
A. and B. ECs were exposed to 25 µM TCDC or 5mM MC for 1, 3, 6 and 12 hours with or without pretreated 500U catalase for 30 min and the intracellular ROS levels were then measured. The results shown are the mean ± SEM. *P<0.05 compared with untreated ECs. C. and D. ECs were subjected to similar treatments prior to the measurement of intracellular ROS levels. The folds of lucigenin-amplified chemiluminescence are shown as the mean ± SEM. Results are presented as the mean ± SEM (n = 3). *P<0.05 compared with untreated ECs.
Figure 4
Figure 4. CO increases the GSSG level in ECs.
A. and B. ECs were exposed to TCDC or MC for 1, 3, 6 and 12 hours and the intracellular SGGS levels were then measured. The results shown are the mean ± SEM. *P<0.05 compared with untreated ECs. C. and D. The intracellular GSH levels of ECs incubated with TCDC or MC for 1, 3, 6 and 12 hours. Results are presented as the mean ± SEM (n = 3). *P<0.05 compared with untreated ECs.
Figure 5
Figure 5. The effects of CO on STAT3 glutathionylation.
A. and B. ECs were treated with 100 µM BioGEE for one hour and loaded with TCDC or MC for the indicated times to induce protein glutathionylation. C. and D. ECs were exposed to TCDC or MC for the indicated periods. S-glutathionylated STAT3 was immunoprecipitated and detected using an antibody against protein-SSG. The quantification of the band intensities was normalized to the control values. Results are presented as the mean ± SEM (n = 3). *P<0.05 compared with untreated ECs.
Figure 6
Figure 6. The STAT3 phosphorylation and glutathionylation levels are dependent on the ROS levels in CO-treated ECs.
A. ECs subjected to IL-6 (10ng/ml) treatment for 10 min were pretreated with or without 0.5 or 1 mM GSSG for 30 minutes. Cell lysates were then subjected to western blotting with antibodies against pTyr-STAT3 or STAT3. B. ECs were pretreated with catalase at the indicated units for 30 min and then cultured without further treatment or exposed to TCDC or MC for 12 hours. S-glutathionylated STAT3 was then detected as above. The quantification of the band intensities was normalized to the control values. Results are presented as the mean ± SEM (n = 3). *P<0.05 compared with untreated ECs. # P<0.05 compared with TCDC or MC alone. C. ECs subjected to IL-6 treatment for 10 min were pretreated with 500 U catalase for 30 min and then cultured without further treatment or exposed to TCDC or MC for 12 hours. Cell lysates were then subjected to western blotting.
Figure 7
Figure 7. The effects of CO are dependent on the intracellular GSH level.
A. ECs were pretreated with 0.5-glutathionylated STAT3 was then detected as described in Figure 5. *P<0.05 compared with untreated ECs. # P<0.05 compared with TCDC or MC alone. B. ECs subjected to IL-6 treatment for 10 min were pretreated with 0.5 mM BSO for 30 min and then cultured without further treatment or exposed to TCDC or MC for 12 hours. Cell lysates were then subjected to western blotting.
Figure 8
Figure 8. HO-1 modulates CO-induced STAT3 glutathionylation.
A. ECs were transiently transfected with pcDNA vector or HO-1 plasmid. S-glutathionylated STAT3 was immunoprecipitated and detected using a protein-SSG antibody. *P<0.05 compared with untreated ECs. B. ECs were transfected with control or HO-1 siRNA vectors for 36 hours and then exposed to TCDC or MC for 12 hours. S-glutathionylated STAT3 was then detected as above. *P<0.05 compared with untreated ECs. # P<0.05 compared with TCDC or MC alone.
Figure 9
Figure 9. Proposed model of the inhibitory effects of CO upon STAT3 activation.
NO and CO have long been known to increase ROS via the inhibition of cytochrome c oxidase function by competing with oxygen binding. The increased intracellular low levels oxidative stress induces protein S-glutathionylation to prevent STAT3 nuclear translocation under inflammatory cytokine treatment. See the “Discussion" section for details.

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Grant support

This work was supported in part by grants (102-2320-B-415-002-MY3) from the National Science Council, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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