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Activation of peroxisome proliferator-activated receptor γ by rosiglitazone inhibits lipopolysaccharide-induced release of high mobility group box 1.

Hwang JS, Kang ES, Ham SA, Yoo T, Lee H, Paek KS, Park C, Kim JH, Lim DS, Seo HG - Mediators Inflamm. (2012)

Bottom Line: Among the ligands of PPAR, the effect of rosiglitazone, a specific ligand for PPARγ, was superior in the inhibition of HMGB1 release induced by LPS.Notably, the administration of rosiglitazone to mice improved survival rates in an LPS-induced animal model of endotoxemia, where reduced levels of circulating HMGB1 were demonstrated.Taken together, these results suggest that PPARs play an important role in the cellular response to inflammation by inhibiting HMGB1 release.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea.

ABSTRACT
Peroxisome proliferator-activated receptors (PPARs) are shown to modulate the pathological status of sepsis by regulating the release of high mobility group box 1 (HMGB1), a well-known late proinflammatory mediator of sepsis. Ligand-activated PPARs markedly inhibited lipopolysaccharide- (LPS) induced release of HMGB1 in RAW 264.7 cells. Among the ligands of PPAR, the effect of rosiglitazone, a specific ligand for PPARγ, was superior in the inhibition of HMGB1 release induced by LPS. This effect was observed in cells that received rosiglitazone before LPS or after LPS treatment, indicating that rosiglitazone is effective in both treatment and prevention. Ablation of PPARγ with small interfering RNA or GW9662-mediated inhibition of PPARγ abolished the effect of rosiglitazone on HMGB1 release. Furthermore, the overexpression of PPARγ markedly potentiated the inhibitory effect of rosiglitazone on HMGB1 release. In addition, rosiglitazone inhibited LPS-induced expression of Toll-like receptor 4 signal molecules, suggesting a possible mechanism by which rosiglitazone modulates HMGB1 release. Notably, the administration of rosiglitazone to mice improved survival rates in an LPS-induced animal model of endotoxemia, where reduced levels of circulating HMGB1 were demonstrated. Taken together, these results suggest that PPARs play an important role in the cellular response to inflammation by inhibiting HMGB1 release.

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PPARγ regulates LPS-induced release of HMGB1 in RAW 264.7 cells. (a) Cells pretreated for 1 h with GW9662 were stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was collected and subjected to Western blot analysis for determination of HMGB1 levels. (b) Cells transfected with PPARγ siRNA for 38 h were incubated with serum-free medium for 24 h, and then treated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis. (c) Cells were transfected with a vector encoding one hairpin siRNA against PPARγ or encoding a scrambled shRNA control. Stable transfectants were selected with 100 μg/mL hygromycin, and the expression levels of PPARγ were determined by Western blot analysis. (d) Cells expressing PPARγ shRNA or scrambled control shRNA were treated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was subjected to Western blot analysis for the determination of HMGB1 levels. (e) Cells transfected with pcDNA3.1-PPARγ, or pcDNA3.1 vector for 48 h were harvested and subjected to Western blot analysis with indicated antibodies. (f) Cells transfected with pcDNA 3.1 or pcDNA3.1-PPARγ for 48 h were incubated with serum-free medium for 24 h and then stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis for the detection of HMGB1. Ponceau S staining was used as a loading control. The results shown are representative of three independent experiments.
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fig4: PPARγ regulates LPS-induced release of HMGB1 in RAW 264.7 cells. (a) Cells pretreated for 1 h with GW9662 were stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was collected and subjected to Western blot analysis for determination of HMGB1 levels. (b) Cells transfected with PPARγ siRNA for 38 h were incubated with serum-free medium for 24 h, and then treated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis. (c) Cells were transfected with a vector encoding one hairpin siRNA against PPARγ or encoding a scrambled shRNA control. Stable transfectants were selected with 100 μg/mL hygromycin, and the expression levels of PPARγ were determined by Western blot analysis. (d) Cells expressing PPARγ shRNA or scrambled control shRNA were treated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was subjected to Western blot analysis for the determination of HMGB1 levels. (e) Cells transfected with pcDNA3.1-PPARγ, or pcDNA3.1 vector for 48 h were harvested and subjected to Western blot analysis with indicated antibodies. (f) Cells transfected with pcDNA 3.1 or pcDNA3.1-PPARγ for 48 h were incubated with serum-free medium for 24 h and then stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis for the detection of HMGB1. Ponceau S staining was used as a loading control. The results shown are representative of three independent experiments.

Mentions: To examine the role of PPARγ in rosiglitazone-mediated inhibition of HMGB1 release induced by LPS, RAW 264.7 cells were treated with siRNA against PPARγ or GW9662, an irreversible inhibitor of PPARγ [21]. In LPS-treated RAW 264.7 cells, the addition of PPARγ siRNA or GW9662 almost completely abolished the rosiglitazone-mediated inhibition of HMGB1 release (Figures 4(a) and 4(b)). To further ascertain the effect of endogenous PPARγ on LPS-induced HMGB1 release, knockdown or overexpression of PPARγ using a specific shRNA or vector-host systems, respectively, was carried out. RAW 264.7 cells stably expressing PPARγ shRNA were established and shown to exhibit a reduced level of PPARγ expression, whereas PPARγ expression in cells transfected with a vector expressing scrambled shRNA was unaffected (Figure 4(c)). This PPARγ-shRNA-mediated downregulation of PPARγ counteracted the inhibitory effects of rosiglitazone on the HMGB1 release induced by LPS (Figure 4(d)). Furthermore, overexpression of PPARγ had more pronounced effects in HMGB1 release (Figures 4(e) and 4(f)). These data clearly indicate that PPARγ directly regulates LPS-induced HMGB1 release in RAW 264.7 cells.


Activation of peroxisome proliferator-activated receptor γ by rosiglitazone inhibits lipopolysaccharide-induced release of high mobility group box 1.

Hwang JS, Kang ES, Ham SA, Yoo T, Lee H, Paek KS, Park C, Kim JH, Lim DS, Seo HG - Mediators Inflamm. (2012)

PPARγ regulates LPS-induced release of HMGB1 in RAW 264.7 cells. (a) Cells pretreated for 1 h with GW9662 were stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was collected and subjected to Western blot analysis for determination of HMGB1 levels. (b) Cells transfected with PPARγ siRNA for 38 h were incubated with serum-free medium for 24 h, and then treated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis. (c) Cells were transfected with a vector encoding one hairpin siRNA against PPARγ or encoding a scrambled shRNA control. Stable transfectants were selected with 100 μg/mL hygromycin, and the expression levels of PPARγ were determined by Western blot analysis. (d) Cells expressing PPARγ shRNA or scrambled control shRNA were treated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was subjected to Western blot analysis for the determination of HMGB1 levels. (e) Cells transfected with pcDNA3.1-PPARγ, or pcDNA3.1 vector for 48 h were harvested and subjected to Western blot analysis with indicated antibodies. (f) Cells transfected with pcDNA 3.1 or pcDNA3.1-PPARγ for 48 h were incubated with serum-free medium for 24 h and then stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis for the detection of HMGB1. Ponceau S staining was used as a loading control. The results shown are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: PPARγ regulates LPS-induced release of HMGB1 in RAW 264.7 cells. (a) Cells pretreated for 1 h with GW9662 were stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was collected and subjected to Western blot analysis for determination of HMGB1 levels. (b) Cells transfected with PPARγ siRNA for 38 h were incubated with serum-free medium for 24 h, and then treated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis. (c) Cells were transfected with a vector encoding one hairpin siRNA against PPARγ or encoding a scrambled shRNA control. Stable transfectants were selected with 100 μg/mL hygromycin, and the expression levels of PPARγ were determined by Western blot analysis. (d) Cells expressing PPARγ shRNA or scrambled control shRNA were treated with LPS in the presence or absence of rosiglitazone for 24 h. Conditioned medium was subjected to Western blot analysis for the determination of HMGB1 levels. (e) Cells transfected with pcDNA3.1-PPARγ, or pcDNA3.1 vector for 48 h were harvested and subjected to Western blot analysis with indicated antibodies. (f) Cells transfected with pcDNA 3.1 or pcDNA3.1-PPARγ for 48 h were incubated with serum-free medium for 24 h and then stimulated with LPS in the presence or absence of rosiglitazone for 24 h. Equal volumes of conditioned media were subjected to Western blot analysis for the detection of HMGB1. Ponceau S staining was used as a loading control. The results shown are representative of three independent experiments.
Mentions: To examine the role of PPARγ in rosiglitazone-mediated inhibition of HMGB1 release induced by LPS, RAW 264.7 cells were treated with siRNA against PPARγ or GW9662, an irreversible inhibitor of PPARγ [21]. In LPS-treated RAW 264.7 cells, the addition of PPARγ siRNA or GW9662 almost completely abolished the rosiglitazone-mediated inhibition of HMGB1 release (Figures 4(a) and 4(b)). To further ascertain the effect of endogenous PPARγ on LPS-induced HMGB1 release, knockdown or overexpression of PPARγ using a specific shRNA or vector-host systems, respectively, was carried out. RAW 264.7 cells stably expressing PPARγ shRNA were established and shown to exhibit a reduced level of PPARγ expression, whereas PPARγ expression in cells transfected with a vector expressing scrambled shRNA was unaffected (Figure 4(c)). This PPARγ-shRNA-mediated downregulation of PPARγ counteracted the inhibitory effects of rosiglitazone on the HMGB1 release induced by LPS (Figure 4(d)). Furthermore, overexpression of PPARγ had more pronounced effects in HMGB1 release (Figures 4(e) and 4(f)). These data clearly indicate that PPARγ directly regulates LPS-induced HMGB1 release in RAW 264.7 cells.

Bottom Line: Among the ligands of PPAR, the effect of rosiglitazone, a specific ligand for PPARγ, was superior in the inhibition of HMGB1 release induced by LPS.Notably, the administration of rosiglitazone to mice improved survival rates in an LPS-induced animal model of endotoxemia, where reduced levels of circulating HMGB1 were demonstrated.Taken together, these results suggest that PPARs play an important role in the cellular response to inflammation by inhibiting HMGB1 release.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Biotechnology, Konkuk University, Seoul, Republic of Korea.

ABSTRACT
Peroxisome proliferator-activated receptors (PPARs) are shown to modulate the pathological status of sepsis by regulating the release of high mobility group box 1 (HMGB1), a well-known late proinflammatory mediator of sepsis. Ligand-activated PPARs markedly inhibited lipopolysaccharide- (LPS) induced release of HMGB1 in RAW 264.7 cells. Among the ligands of PPAR, the effect of rosiglitazone, a specific ligand for PPARγ, was superior in the inhibition of HMGB1 release induced by LPS. This effect was observed in cells that received rosiglitazone before LPS or after LPS treatment, indicating that rosiglitazone is effective in both treatment and prevention. Ablation of PPARγ with small interfering RNA or GW9662-mediated inhibition of PPARγ abolished the effect of rosiglitazone on HMGB1 release. Furthermore, the overexpression of PPARγ markedly potentiated the inhibitory effect of rosiglitazone on HMGB1 release. In addition, rosiglitazone inhibited LPS-induced expression of Toll-like receptor 4 signal molecules, suggesting a possible mechanism by which rosiglitazone modulates HMGB1 release. Notably, the administration of rosiglitazone to mice improved survival rates in an LPS-induced animal model of endotoxemia, where reduced levels of circulating HMGB1 were demonstrated. Taken together, these results suggest that PPARs play an important role in the cellular response to inflammation by inhibiting HMGB1 release.

Show MeSH
Related in: MedlinePlus