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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 attenuates thrombin-induced NF-κB reporter activity.HPAEC were transfected with control siRNA or BiP siRNA by use of DharmaFect1. Twenty-four hours later, cells were again transfected with NF-κBLUC construct by using DEAE-dextran as described in Materials and Methods. Cells were then challenged with (A) thrombin (5 U/ml) for 6 h, and the cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a fold increase in firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ##p<0.01 difference from controls; **p<0.01 difference from thrombin stimulated controls. (B) HPAEC were transfected with NF-κBLUC in combination with BiP WT (pCMV-BiP-Myc-KDEL-WT) or BiP dominant negative (pCMV-BiP-Myc-KDEL-T37G) as described in Materials and Methods. Sixteen hours after transfection cells were treated with thrombin (5 U/ml) for 6 hrs. Cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a ratio of firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.
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pone-0110949-g002: BiP knockdown attenuates thrombin-induced NF-κB reporter activity.HPAEC were transfected with control siRNA or BiP siRNA by use of DharmaFect1. Twenty-four hours later, cells were again transfected with NF-κBLUC construct by using DEAE-dextran as described in Materials and Methods. Cells were then challenged with (A) thrombin (5 U/ml) for 6 h, and the cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a fold increase in firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ##p<0.01 difference from controls; **p<0.01 difference from thrombin stimulated controls. (B) HPAEC were transfected with NF-κBLUC in combination with BiP WT (pCMV-BiP-Myc-KDEL-WT) or BiP dominant negative (pCMV-BiP-Myc-KDEL-T37G) as described in Materials and Methods. Sixteen hours after transfection cells were treated with thrombin (5 U/ml) for 6 hrs. Cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a ratio of firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.

Mentions: Next, to determine the role of BiP-mediated ER stress in EC inflammation, we monitored the effect of BiP knockdown on the activity of NF-κB, the master regulator of inflammation. Cells were transfected with pNF-κB -LUC in combination with either control-siRNA or BiP-siRNA. Results showed that thrombin challenge of cells transfected with control-siRNA resulted in increased NF-κB reporter activity and this response was inhibited in cells transfected with BiP-siRNA (Fig. 2A). In a reciprocal approach we addressed the effect of overexpression of BiP wild-type and a BiP dominant negative mutant on thrombin-induced NF-κB activity. Transfection of cells with a construct encoding wild-type BiP (pCMV-BiP-Myc-KDEL-WT) showed no effect on thrombin-induced NF-κB activity. However, expression of a construct encoding a dominant negative BiP (pCMV-BiP-Myc-KDEL-T37G) significantly inhibited thrombin-induced NF-κB activity (Fig. 2B). In view of the essential role of NF-κB in adhesion molecule expression, we determined the effect of BiP knockdown on thrombin-induced expression of ICAM-1 and VCAM-1. We found that depletion of BiP attenuated thrombin-induced ICAM-1 and VCAM-1 expression consistent with its effect on NF-κB activity (Fig. 3A–C). To further ascertain our observation that ER stress inhibits NF-κB activity, cells were treated with a known inducer of ER stress, tunicamycin. Results (Figure S1) show that tunicamycin significantly inhibited thrombin-induced NF-κB activity and adhesion molecule expression. Additionally, to investigate the specificity of the above response, we determined whether knockdown of BiP affected TNFα activation of NF-κB-induced ICAM-1 and VCAM-1 expression. Results showed that depletion of BiP (Fig. 3D–F) failed to inhibit TNFα-induced ICAM-1 and VCAM-1 expression. Together, these results indicate that in HPAEC, BiP regulates adhesion molecules expression in a stimulus specific manner.


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 attenuates thrombin-induced NF-κB reporter activity.HPAEC were transfected with control siRNA or BiP siRNA by use of DharmaFect1. Twenty-four hours later, cells were again transfected with NF-κBLUC construct by using DEAE-dextran as described in Materials and Methods. Cells were then challenged with (A) thrombin (5 U/ml) for 6 h, and the cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a fold increase in firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ##p<0.01 difference from controls; **p<0.01 difference from thrombin stimulated controls. (B) HPAEC were transfected with NF-κBLUC in combination with BiP WT (pCMV-BiP-Myc-KDEL-WT) or BiP dominant negative (pCMV-BiP-Myc-KDEL-T37G) as described in Materials and Methods. Sixteen hours after transfection cells were treated with thrombin (5 U/ml) for 6 hrs. Cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a ratio of firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.
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pone-0110949-g002: BiP knockdown attenuates thrombin-induced NF-κB reporter activity.HPAEC were transfected with control siRNA or BiP siRNA by use of DharmaFect1. Twenty-four hours later, cells were again transfected with NF-κBLUC construct by using DEAE-dextran as described in Materials and Methods. Cells were then challenged with (A) thrombin (5 U/ml) for 6 h, and the cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a fold increase in firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ##p<0.01 difference from controls; **p<0.01 difference from thrombin stimulated controls. (B) HPAEC were transfected with NF-κBLUC in combination with BiP WT (pCMV-BiP-Myc-KDEL-WT) or BiP dominant negative (pCMV-BiP-Myc-KDEL-T37G) as described in Materials and Methods. Sixteen hours after transfection cells were treated with thrombin (5 U/ml) for 6 hrs. Cell extracts were prepared and assayed for firefly and Renilla luciferase activities. The data were expressed as a ratio of firefly to Renilla luciferase activities. Data are means ± SE (n = 4–6 for each condition). ###p<0.001 difference from controls; **p<0.01 difference from thrombin stimulated controls.
Mentions: Next, to determine the role of BiP-mediated ER stress in EC inflammation, we monitored the effect of BiP knockdown on the activity of NF-κB, the master regulator of inflammation. Cells were transfected with pNF-κB -LUC in combination with either control-siRNA or BiP-siRNA. Results showed that thrombin challenge of cells transfected with control-siRNA resulted in increased NF-κB reporter activity and this response was inhibited in cells transfected with BiP-siRNA (Fig. 2A). In a reciprocal approach we addressed the effect of overexpression of BiP wild-type and a BiP dominant negative mutant on thrombin-induced NF-κB activity. Transfection of cells with a construct encoding wild-type BiP (pCMV-BiP-Myc-KDEL-WT) showed no effect on thrombin-induced NF-κB activity. However, expression of a construct encoding a dominant negative BiP (pCMV-BiP-Myc-KDEL-T37G) significantly inhibited thrombin-induced NF-κB activity (Fig. 2B). In view of the essential role of NF-κB in adhesion molecule expression, we determined the effect of BiP knockdown on thrombin-induced expression of ICAM-1 and VCAM-1. We found that depletion of BiP attenuated thrombin-induced ICAM-1 and VCAM-1 expression consistent with its effect on NF-κB activity (Fig. 3A–C). To further ascertain our observation that ER stress inhibits NF-κB activity, cells were treated with a known inducer of ER stress, tunicamycin. Results (Figure S1) show that tunicamycin significantly inhibited thrombin-induced NF-κB activity and adhesion molecule expression. Additionally, to investigate the specificity of the above response, we determined whether knockdown of BiP affected TNFα activation of NF-κB-induced ICAM-1 and VCAM-1 expression. Results showed that depletion of BiP (Fig. 3D–F) failed to inhibit TNFα-induced ICAM-1 and VCAM-1 expression. Together, these results indicate that in HPAEC, BiP regulates adhesion molecules expression in a stimulus specific manner.

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