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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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SubAB failed to block thrombin-induced IκBα degradation.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A) thrombin (5 U/ml) for 1 h or with (C) TNFα (100 U/ml) for 30 min. Total cell lysates were prepared and immunoblotted with anti-IκBα antibody to determine degradation of IκBα. Levels of RelA/p65 was used to monitor loading. The bar graphs represent the effect SubAB and SuBAA272B on (B) thrombin-induced or (D) TNFα-induced IκBα degradation normalized to total RelA/p65 levels. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls.
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pone-0110949-g012: SubAB failed to block thrombin-induced IκBα degradation.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A) thrombin (5 U/ml) for 1 h or with (C) TNFα (100 U/ml) for 30 min. Total cell lysates were prepared and immunoblotted with anti-IκBα antibody to determine degradation of IκBα. Levels of RelA/p65 was used to monitor loading. The bar graphs represent the effect SubAB and SuBAA272B on (B) thrombin-induced or (D) TNFα-induced IκBα degradation normalized to total RelA/p65 levels. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls.

Mentions: Next we determined whether the reduced NF-κB activity and subsequent adhesion molecule and chemokine expression following preconditioning of HPAEC with SubAB is due to reduced degradation of IκBα, the cytoplasmic inhibitor that sequesters NF-κB in the cytosol and thereby blocks its translocation to the nucleus and consequently gene transcription. Interestingly, analysis of the cytoplasmic extracts showed that preconditioning of cells with SubAB did not inhibit thrombin or TNFα-induced IαBα degradation (Fig. 12A–D). Furthermore, our data showed that the nuclear translocation of the liberated NF-κB also remained unaffected upon preconditioning with ER stress (Fig. 13A & B).


Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

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

SubAB failed to block thrombin-induced IκBα degradation.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A) thrombin (5 U/ml) for 1 h or with (C) TNFα (100 U/ml) for 30 min. Total cell lysates were prepared and immunoblotted with anti-IκBα antibody to determine degradation of IκBα. Levels of RelA/p65 was used to monitor loading. The bar graphs represent the effect SubAB and SuBAA272B on (B) thrombin-induced or (D) TNFα-induced IκBα degradation normalized to total RelA/p65 levels. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214695&req=5

pone-0110949-g012: SubAB failed to block thrombin-induced IκBα degradation.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A) thrombin (5 U/ml) for 1 h or with (C) TNFα (100 U/ml) for 30 min. Total cell lysates were prepared and immunoblotted with anti-IκBα antibody to determine degradation of IκBα. Levels of RelA/p65 was used to monitor loading. The bar graphs represent the effect SubAB and SuBAA272B on (B) thrombin-induced or (D) TNFα-induced IκBα degradation normalized to total RelA/p65 levels. The data are the means ± S.E. (n = 3–6 for each condition). #p<0.05 difference from controls.
Mentions: Next we determined whether the reduced NF-κB activity and subsequent adhesion molecule and chemokine expression following preconditioning of HPAEC with SubAB is due to reduced degradation of IκBα, the cytoplasmic inhibitor that sequesters NF-κB in the cytosol and thereby blocks its translocation to the nucleus and consequently gene transcription. Interestingly, analysis of the cytoplasmic extracts showed that preconditioning of cells with SubAB did not inhibit thrombin or TNFα-induced IαBα degradation (Fig. 12A–D). Furthermore, our data showed that the nuclear translocation of the liberated NF-κB also remained unaffected upon preconditioning with ER stress (Fig. 13A & B).

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