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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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Related in: MedlinePlus

BiP knockdown blocks thrombin-induced RelA/p65 binding to DNA.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 or 0.5 h with (B) TNFα. Nuclear extracts were prepared and assayed for DNA binding of RelA/p65 by EMSA as described in the Materials and Methods. Results are representatives of two experiments.
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pone-0110949-g006: BiP knockdown blocks thrombin-induced RelA/p65 binding to DNA.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 or 0.5 h with (B) TNFα. Nuclear extracts were prepared and assayed for DNA binding of RelA/p65 by EMSA as described in the Materials and Methods. Results are representatives of two experiments.

Mentions: Since IκBα degradation is a prerequisite for the release of NF-κB for its nuclear translocation, we next assessed the effect of BiP depletion on NF-κB nuclear translocation and subsequent DNA binding. Nuclear extracts from control and treated cells were analyzed by immunoblotting and electrophoretic mobility shift assay. Results showed a marked decrease in nuclear translocation (Fig. 5A & B) and DNA binding of RelA/p65 (Fig. 6A) in thrombin-treated cells transfected with BiP-siRNA, as compared to cells transfected with control-siRNA. Together these results are consistent with our previous data showing a block in IκBα degradation upon BiP depletion. Interestingly, results show that TNFα-induced RelA/p65 nuclear translocation (Fig. 5C & D) and subsequent DNA binding (Fig. 6B) were unchanged upon knockdown of BiP.


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 RelA/p65 binding to DNA.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 or 0.5 h with (B) TNFα. Nuclear extracts were prepared and assayed for DNA binding of RelA/p65 by EMSA as described in the Materials and Methods. Results are representatives of two experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110949-g006: BiP knockdown blocks thrombin-induced RelA/p65 binding to DNA.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 or 0.5 h with (B) TNFα. Nuclear extracts were prepared and assayed for DNA binding of RelA/p65 by EMSA as described in the Materials and Methods. Results are representatives of two experiments.
Mentions: Since IκBα degradation is a prerequisite for the release of NF-κB for its nuclear translocation, we next assessed the effect of BiP depletion on NF-κB nuclear translocation and subsequent DNA binding. Nuclear extracts from control and treated cells were analyzed by immunoblotting and electrophoretic mobility shift assay. Results showed a marked decrease in nuclear translocation (Fig. 5A & B) and DNA binding of RelA/p65 (Fig. 6A) in thrombin-treated cells transfected with BiP-siRNA, as compared to cells transfected with control-siRNA. Together these results are consistent with our previous data showing a block in IκBα degradation upon BiP depletion. Interestingly, results show that TNFα-induced RelA/p65 nuclear translocation (Fig. 5C & D) and subsequent DNA binding (Fig. 6B) were unchanged upon knockdown of BiP.

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