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Dimethylfumarate protects against TNF-α-induced secretion of inflammatory cytokines in human endothelial cells.

Gerhardt S, König V, Doll M, Hailemariam-Jahn T, Hrgovic I, Zöller N, Kaufmann R, Kippenberger S, Meissner M - J Inflamm (Lond) (2015)

Bottom Line: Furthermore, we found that DMF slightly inhibited the early degradation of IκBα.This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536.These effects were independent of the p38, PI3K and p42/44 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany ; Department of Cardiology, Gutenberg-University, Mainz, Germany.

ABSTRACT

Background: Inflammation, angiogenesis and oxidative stress have been implicated in the pathogenesis of various vascular diseases. Recent evidence suggests that dimethylfumarate (DMF), an antiposriatic and anti-multiple sclerosis agent, possesses anti-inflammatory, anti-oxidative and anti-angiogenic properties. Here, we analyze the influence of DMF on TNF-α-induced expression of the important pro-inflammatory and pro-atherogenic chemokine MCP-1 and investigate the underlying mechanisms of this expression.

Findings: We analyzed constitutive and TNF-α-induced expression of MCP-1 in human umbilical vascular endothelial cells (HUVEC) +/- DMF treatment via enzyme-linkes immunosorbent assay (ELISA). DMF significantly inhibited the protein expression levels in a time- and concentration-dependent manner. Furthermore, MCP-1 mRNA expression was also reduced in response to DMF, as demonstrated by RT-PCR. Thus, the regulation occurs at the transcriptional level. Interestingly, DMF prolonged the TNF-α-induced p38 and JNK phosphorylation in HUVEC, as demonstrated by Western blot analysis; however, the p38 and JNK inhibitor SB203580 did not affect the DMF-conveyed suppression of TNF-α-induced MCP-1 expression. DMF suppressed the TNF-α-induced nuclear translocation and phosphorylation (Serine 536) of p65 in these cells. These results were additionally approved by p65 luciferase promoter assays. Furthermore, we found that DMF slightly inhibited the early degradation of IκBα. In addition, we verified our results using other important inflammatory cytokines such as CCL-5, PDGF-BB, GM-CSF and IL-6.

Conclusion: DMF suppresses various TNF-α-induced pro-inflammatory and pro-atherogenic cytokines/chemokines in human endothelial cells. This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536. These effects were independent of the p38, PI3K and p42/44 signaling pathways. As a result, DMF might be suitable for treating patients with vascular diseases.

No MeSH data available.


Related in: MedlinePlus

Analysis of TNF-α-induced p65 nuclear entry, phosphorylation (Ser 536), promoter activity and IκBα degradation during DMF treatment. a Nuclear p65 translocation and phosphorylation: Western blot analysis of nuclear proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3-h pre-treatment) + TNF-α for 60 min. The phosphorylated p65 (Serine 536) band is marked by an arrow head. b Representative immunofluorescent analysis of p65 in HUVECs that were treated with vehicle, TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 1 h. c IkB degradation: Western blot analysis cytosolic proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. d Analyses of the NfκB luciferase (Luc) reporter constructs in HUVECs treated with vehicle (solvent only), DMF (80 μM), TNF-α (20 ng/ml) or DMF (80 μM) + TNF-α for 24 h, respectively. The Luc activities are expressed as relative luciferase activity as a percent (mean ± SEM of at least five independent triplicate assays). *p < 0.05 versus TNF-α; **p < 0.05 versus control
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Fig4: Analysis of TNF-α-induced p65 nuclear entry, phosphorylation (Ser 536), promoter activity and IκBα degradation during DMF treatment. a Nuclear p65 translocation and phosphorylation: Western blot analysis of nuclear proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3-h pre-treatment) + TNF-α for 60 min. The phosphorylated p65 (Serine 536) band is marked by an arrow head. b Representative immunofluorescent analysis of p65 in HUVECs that were treated with vehicle, TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 1 h. c IkB degradation: Western blot analysis cytosolic proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. d Analyses of the NfκB luciferase (Luc) reporter constructs in HUVECs treated with vehicle (solvent only), DMF (80 μM), TNF-α (20 ng/ml) or DMF (80 μM) + TNF-α for 24 h, respectively. The Luc activities are expressed as relative luciferase activity as a percent (mean ± SEM of at least five independent triplicate assays). *p < 0.05 versus TNF-α; **p < 0.05 versus control

Mentions: It is well known that TNF-α mediates its effects, such as increased MCP-1 expression, mainly by nuclear translocation and phosphorylation of p65 after the degradation of IκBα. Recently, Loewe et al. demonstrated the reduced nuclear translocation of p65 in response to DMF treatment in HUVEC, partially reversing the TNF-α effects. In our experiments, we observed reduced p65 translocation but additionally recorded reduced nuclear p65 phosphorylation at Serine 536 during DMF treatment (Fig. 4a). Furthermore, we found a significant reduction in nuclear translocation of p65 by immunofluorescence staining of NFκB p65 in HUVECs treated with a combination of DMF and TNF-α compared with a treatment of TNF-α alone (Fig. 4b). To exclude a reduced overall expression of p65 during the treatment with DMF, we analyzed p65 expression by Western blot analysis (Additional file 1); no reduction of p65 expression was observed.Fig. 4


Dimethylfumarate protects against TNF-α-induced secretion of inflammatory cytokines in human endothelial cells.

Gerhardt S, König V, Doll M, Hailemariam-Jahn T, Hrgovic I, Zöller N, Kaufmann R, Kippenberger S, Meissner M - J Inflamm (Lond) (2015)

Analysis of TNF-α-induced p65 nuclear entry, phosphorylation (Ser 536), promoter activity and IκBα degradation during DMF treatment. a Nuclear p65 translocation and phosphorylation: Western blot analysis of nuclear proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3-h pre-treatment) + TNF-α for 60 min. The phosphorylated p65 (Serine 536) band is marked by an arrow head. b Representative immunofluorescent analysis of p65 in HUVECs that were treated with vehicle, TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 1 h. c IkB degradation: Western blot analysis cytosolic proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. d Analyses of the NfκB luciferase (Luc) reporter constructs in HUVECs treated with vehicle (solvent only), DMF (80 μM), TNF-α (20 ng/ml) or DMF (80 μM) + TNF-α for 24 h, respectively. The Luc activities are expressed as relative luciferase activity as a percent (mean ± SEM of at least five independent triplicate assays). *p < 0.05 versus TNF-α; **p < 0.05 versus control
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Related In: Results  -  Collection

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Fig4: Analysis of TNF-α-induced p65 nuclear entry, phosphorylation (Ser 536), promoter activity and IκBα degradation during DMF treatment. a Nuclear p65 translocation and phosphorylation: Western blot analysis of nuclear proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3-h pre-treatment) + TNF-α for 60 min. The phosphorylated p65 (Serine 536) band is marked by an arrow head. b Representative immunofluorescent analysis of p65 in HUVECs that were treated with vehicle, TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 1 h. c IkB degradation: Western blot analysis cytosolic proteins of HUVECs treated with vehicle (solvent only), TNF-α (20 ng/ml) or DMF (80 μM, 3 h pre-treatment) + TNF-α for 60 min. d Analyses of the NfκB luciferase (Luc) reporter constructs in HUVECs treated with vehicle (solvent only), DMF (80 μM), TNF-α (20 ng/ml) or DMF (80 μM) + TNF-α for 24 h, respectively. The Luc activities are expressed as relative luciferase activity as a percent (mean ± SEM of at least five independent triplicate assays). *p < 0.05 versus TNF-α; **p < 0.05 versus control
Mentions: It is well known that TNF-α mediates its effects, such as increased MCP-1 expression, mainly by nuclear translocation and phosphorylation of p65 after the degradation of IκBα. Recently, Loewe et al. demonstrated the reduced nuclear translocation of p65 in response to DMF treatment in HUVEC, partially reversing the TNF-α effects. In our experiments, we observed reduced p65 translocation but additionally recorded reduced nuclear p65 phosphorylation at Serine 536 during DMF treatment (Fig. 4a). Furthermore, we found a significant reduction in nuclear translocation of p65 by immunofluorescence staining of NFκB p65 in HUVECs treated with a combination of DMF and TNF-α compared with a treatment of TNF-α alone (Fig. 4b). To exclude a reduced overall expression of p65 during the treatment with DMF, we analyzed p65 expression by Western blot analysis (Additional file 1); no reduction of p65 expression was observed.Fig. 4

Bottom Line: Furthermore, we found that DMF slightly inhibited the early degradation of IκBα.This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536.These effects were independent of the p38, PI3K and p42/44 signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany ; Department of Cardiology, Gutenberg-University, Mainz, Germany.

ABSTRACT

Background: Inflammation, angiogenesis and oxidative stress have been implicated in the pathogenesis of various vascular diseases. Recent evidence suggests that dimethylfumarate (DMF), an antiposriatic and anti-multiple sclerosis agent, possesses anti-inflammatory, anti-oxidative and anti-angiogenic properties. Here, we analyze the influence of DMF on TNF-α-induced expression of the important pro-inflammatory and pro-atherogenic chemokine MCP-1 and investigate the underlying mechanisms of this expression.

Findings: We analyzed constitutive and TNF-α-induced expression of MCP-1 in human umbilical vascular endothelial cells (HUVEC) +/- DMF treatment via enzyme-linkes immunosorbent assay (ELISA). DMF significantly inhibited the protein expression levels in a time- and concentration-dependent manner. Furthermore, MCP-1 mRNA expression was also reduced in response to DMF, as demonstrated by RT-PCR. Thus, the regulation occurs at the transcriptional level. Interestingly, DMF prolonged the TNF-α-induced p38 and JNK phosphorylation in HUVEC, as demonstrated by Western blot analysis; however, the p38 and JNK inhibitor SB203580 did not affect the DMF-conveyed suppression of TNF-α-induced MCP-1 expression. DMF suppressed the TNF-α-induced nuclear translocation and phosphorylation (Serine 536) of p65 in these cells. These results were additionally approved by p65 luciferase promoter assays. Furthermore, we found that DMF slightly inhibited the early degradation of IκBα. In addition, we verified our results using other important inflammatory cytokines such as CCL-5, PDGF-BB, GM-CSF and IL-6.

Conclusion: DMF suppresses various TNF-α-induced pro-inflammatory and pro-atherogenic cytokines/chemokines in human endothelial cells. This action is regulated by reduced p65 activity and nuclear translocation, which can be explained in part by the reduced early degradation of IκBα and more important the reduced phosphorylation of p65 at Serine 536. These effects were independent of the p38, PI3K and p42/44 signaling pathways. As a result, DMF might be suitable for treating patients with vascular diseases.

No MeSH data available.


Related in: MedlinePlus