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Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

Leonard A, Paton AW, El-Quadi M, Paton JC, Fazal F - PLoS ONE (2014)

Bottom Line: We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation.Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines.In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Lung Biology and Disease Program, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

ABSTRACT
Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

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BiP knockdown blocks thrombin-induced IKKβ phosphorylation and IκBα degradation.HPAEC were transfected with control siRNA or BiP siRNA using DharmaFect1. After 24–36 h, the cells were challenged for 1 h with (A) thrombin (5 U/ml) or 0.5 h with (C) TNFα (100 U/ml). Total cell lysates were prepared and immunoblotted with anti-phospho-IKKβ and anti-IκBα to determine the phosphorylation of IKKβ and degradation of IκBα respectively. Total levels of IKKβ were used to monitor loading. The bar graphs represent the effect of BiP depletion on (B) thrombin-induced or (D) TNFα-induced IKKβ phosphorylation normalized to total IKKβ levels. The data are the means ± S.E. (n = 3–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.
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pone-0110949-g004: BiP knockdown blocks thrombin-induced IKKβ phosphorylation and IκBα degradation.HPAEC were transfected with control siRNA or BiP siRNA using DharmaFect1. After 24–36 h, the cells were challenged for 1 h with (A) thrombin (5 U/ml) or 0.5 h with (C) TNFα (100 U/ml). Total cell lysates were prepared and immunoblotted with anti-phospho-IKKβ and anti-IκBα to determine the phosphorylation of IKKβ and degradation of IκBα respectively. Total levels of IKKβ were used to monitor loading. The bar graphs represent the effect of BiP depletion on (B) thrombin-induced or (D) TNFα-induced IKKβ phosphorylation normalized to total IKKβ levels. The data are the means ± S.E. (n = 3–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.

Mentions: Next we analyzed the mechanism by which BiP depletion inhibits the NF-κB signaling cascade in endothelial cells. Phosphorylation of IκBα and its subsequent degradation is a requirement for the release and translocation of NF-κB to the nucleus. Since phosphorylation of IκBα is mediated by the IKK complex, we first evaluated the role BiP in IKK activation. Depletion of BiP inhibited activation of IKK upon thrombin challenge, as determined by decreased phosphorylation of IKKβ at Ser176/181 (Fig. 4A & B). We next examined whether knockdown of BiP affects IKKβ-mediated degradation of IκBα. Results show that depletion of BiP was effective in inhibiting IκBα degradation. In contrast, BiP depletion had no significant effect on TNFα-induced IKKβ phosphorylation and consequently on IκBα degradation (Fig. 4C & D).


Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

Leonard A, Paton AW, El-Quadi M, Paton JC, Fazal F - PLoS ONE (2014)

BiP knockdown blocks thrombin-induced IKKβ phosphorylation and IκBα degradation.HPAEC were transfected with control siRNA or BiP siRNA using DharmaFect1. After 24–36 h, the cells were challenged for 1 h with (A) thrombin (5 U/ml) or 0.5 h with (C) TNFα (100 U/ml). Total cell lysates were prepared and immunoblotted with anti-phospho-IKKβ and anti-IκBα to determine the phosphorylation of IKKβ and degradation of IκBα respectively. Total levels of IKKβ were used to monitor loading. The bar graphs represent the effect of BiP depletion on (B) thrombin-induced or (D) TNFα-induced IKKβ phosphorylation normalized to total IKKβ levels. The data are the means ± S.E. (n = 3–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.
© Copyright Policy
Related In: Results  -  Collection

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pone-0110949-g004: BiP knockdown blocks thrombin-induced IKKβ phosphorylation and IκBα degradation.HPAEC were transfected with control siRNA or BiP siRNA using DharmaFect1. After 24–36 h, the cells were challenged for 1 h with (A) thrombin (5 U/ml) or 0.5 h with (C) TNFα (100 U/ml). Total cell lysates were prepared and immunoblotted with anti-phospho-IKKβ and anti-IκBα to determine the phosphorylation of IKKβ and degradation of IκBα respectively. Total levels of IKKβ were used to monitor loading. The bar graphs represent the effect of BiP depletion on (B) thrombin-induced or (D) TNFα-induced IKKβ phosphorylation normalized to total IKKβ levels. The data are the means ± S.E. (n = 3–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.
Mentions: Next we analyzed the mechanism by which BiP depletion inhibits the NF-κB signaling cascade in endothelial cells. Phosphorylation of IκBα and its subsequent degradation is a requirement for the release and translocation of NF-κB to the nucleus. Since phosphorylation of IκBα is mediated by the IKK complex, we first evaluated the role BiP in IKK activation. Depletion of BiP inhibited activation of IKK upon thrombin challenge, as determined by decreased phosphorylation of IKKβ at Ser176/181 (Fig. 4A & B). We next examined whether knockdown of BiP affects IKKβ-mediated degradation of IκBα. Results show that depletion of BiP was effective in inhibiting IκBα degradation. In contrast, BiP depletion had no significant effect on TNFα-induced IKKβ phosphorylation and consequently on IκBα degradation (Fig. 4C & D).

Bottom Line: We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation.Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines.In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Lung Biology and Disease Program, University of Rochester School of Medicine and Dentistry, Rochester, New York, United States of America.

ABSTRACT
Endoplasmic Reticulum (ER) stress, caused by disturbance in ER homeostasis, has been implicated in several pathological conditions such as ischemic injury, neurodegenerative disorders, metabolic diseases and more recently in inflammatory conditions. Our present study aims at understanding the role of ER stress in endothelial cell (EC) inflammation, a critical event in the pathogenesis of acute lung injury (ALI). We found that preconditioning human pulmonary artery endothelial cells (HPAEC) to ER stress either by depleting ER chaperone and signaling regulator BiP using siRNA, or specifically cleaving (inactivating) BiP using subtilase cytotoxin (SubAB), alleviates EC inflammation. The two approaches adopted to abrogate BiP function induced ATF4 protein expression and the phosphorylation of eIF2α, both markers of ER stress, which in turn resulted in blunting the activation of NF-κB, and restoring endothelial barrier integrity. Pretreatment of HPAEC with BiP siRNA inhibited thrombin-induced IκBα degradation and its resulting downstream signaling pathway involving NF-κB nuclear translocation, DNA binding, phosphorylation at serine536, transcriptional activation and subsequent expression of adhesion molecules. However, TNFα-mediated NF-κB signaling was unaffected upon BiP knockdown. In an alternative approach, SubAB-mediated inactivation of NF-κB was independent of IκBα degradation. Mechanistic analysis revealed that pretreatment of EC with SubAB interfered with the binding of the liberated NF-κB to the DNA, thereby resulting in reduced expression of adhesion molecules, cytokines and chemokines. In addition, both knockdown and inactivation of BiP stimulated actin cytoskeletal reorganization resulting in restoration of endothelial permeability. Together our studies indicate that BiP plays a central role in EC inflammation and injury via its action on NF-κB activation and regulation of vascular permeability.

Show MeSH
Related in: MedlinePlus