Limits...
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.

Show MeSH

Related in: MedlinePlus

SubAB induces BiP cleavage and ER stress.(A) HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for the indicated time points. Cell lysates were immunoblotted with an anti-phospho eIF2α antibody and anti-phospho- AKT antibody to determine the induction of ER stress. The levels of total eIF2α were used to monitor loading. BiP antibody was used to monitor the cleavage of BiP by SubAB. (B) HPAECs were treated with 0.1 µg SubAB for 3 hours and cell lysates were immunoblotted for ATF-4 antibody. Actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4214695&req=5

pone-0110949-g009: SubAB induces BiP cleavage and ER stress.(A) HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for the indicated time points. Cell lysates were immunoblotted with an anti-phospho eIF2α antibody and anti-phospho- AKT antibody to determine the induction of ER stress. The levels of total eIF2α were used to monitor loading. BiP antibody was used to monitor the cleavage of BiP by SubAB. (B) HPAECs were treated with 0.1 µg SubAB for 3 hours and cell lysates were immunoblotted for ATF-4 antibody. Actin was used as a loading control.

Mentions: In order to further ascertain the role of BiP in EC inflammation and injury we used an alternative approach. Cells were treated with SubAB, the prototype of a family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli that specifically cleaves and inactivates BiP, resulting in the activation ER stress and therefore UPR [48]. A time course experiment showed that treatment of HPAEC with SubAB induced BiP cleavage at 1 hour, which coincided with the phosphorylation of eIF2α. We also observed a decrease in AKT-phosphorylation at 1 hour of SubAB treatment. In contrast, treatment of cells with SubAA272B, a nontoxic variant of SubAB, failed to cleave BiP, induce eIF2α phosphorylation and decrease AKT-phosphorylation (Fig. 9A). As expected the level of ATF4 was also induced upon SubAB treatment (Fig. 9B). These data indicate that SubAB specifically cleaves BiP and thereby induce ER stress.


Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

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

SubAB induces BiP cleavage and ER stress.(A) HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for the indicated time points. Cell lysates were immunoblotted with an anti-phospho eIF2α antibody and anti-phospho- AKT antibody to determine the induction of ER stress. The levels of total eIF2α were used to monitor loading. BiP antibody was used to monitor the cleavage of BiP by SubAB. (B) HPAECs were treated with 0.1 µg SubAB for 3 hours and cell lysates were immunoblotted for ATF-4 antibody. Actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0110949-g009: SubAB induces BiP cleavage and ER stress.(A) HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for the indicated time points. Cell lysates were immunoblotted with an anti-phospho eIF2α antibody and anti-phospho- AKT antibody to determine the induction of ER stress. The levels of total eIF2α were used to monitor loading. BiP antibody was used to monitor the cleavage of BiP by SubAB. (B) HPAECs were treated with 0.1 µg SubAB for 3 hours and cell lysates were immunoblotted for ATF-4 antibody. Actin was used as a loading control.
Mentions: In order to further ascertain the role of BiP in EC inflammation and injury we used an alternative approach. Cells were treated with SubAB, the prototype of a family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli that specifically cleaves and inactivates BiP, resulting in the activation ER stress and therefore UPR [48]. A time course experiment showed that treatment of HPAEC with SubAB induced BiP cleavage at 1 hour, which coincided with the phosphorylation of eIF2α. We also observed a decrease in AKT-phosphorylation at 1 hour of SubAB treatment. In contrast, treatment of cells with SubAA272B, a nontoxic variant of SubAB, failed to cleave BiP, induce eIF2α phosphorylation and decrease AKT-phosphorylation (Fig. 9A). As expected the level of ATF4 was also induced upon SubAB treatment (Fig. 9B). These data indicate that SubAB specifically cleaves BiP and thereby induce ER stress.

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