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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 inhibits NF-κB mediated proinflammatory gene expression.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A & C & E) thrombin (5 U/ml) or (B & D & F) TNFα (100 U/ml) for 6 h. Total cell lysates were immunoblotted with (A & B) anti-ICAM-1 antibody and anti -VCAM-1 antibody. Actin was used to monitor loading. The conditioned media were subjected to ELISA to determine the levels of (C & D) IL-8 or (E & F) MCP-1. Data are means ± S.E. (n = 6–9 for each condition). ###p<0.001 difference from control; ***p<0.001 difference from thrombin and TNFα-stimulated controls.
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pone-0110949-g011: SubAB inhibits NF-κB mediated proinflammatory gene expression.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A & C & E) thrombin (5 U/ml) or (B & D & F) TNFα (100 U/ml) for 6 h. Total cell lysates were immunoblotted with (A & B) anti-ICAM-1 antibody and anti -VCAM-1 antibody. Actin was used to monitor loading. The conditioned media were subjected to ELISA to determine the levels of (C & D) IL-8 or (E & F) MCP-1. Data are means ± S.E. (n = 6–9 for each condition). ###p<0.001 difference from control; ***p<0.001 difference from thrombin and TNFα-stimulated controls.

Mentions: Expression of adhesion molecules, cytokines and chemokines in endothelial cells by inflammatory mediators such as thrombin and TNFα is a central step in the pathogenesis of ALI and is under the tight regulation of the transcription factor NF-κB. We determined whether SubAB-mediated ER stress preconditioning affects thrombin and TNFα-induced NF-κB activation and subsequent expression of adhesion molecules ICAM-1, VCAM-1, and chemo-attractant proteins IL-8 and MCP-1. Cells were transfected with pNF-κB-LUC for 24 hours, followed by treatment with SubAB for 6 hours. Results showed that thrombin or TNFα challenge of cells resulted in increased NF-κB reporter activity and this response was inhibited in cells pretreated with 0.1 µg/ml of SubAB (Fig. 10A & B). Next we determined the effect of SubAB on NF-κB target genes, ICAM-1 and VCAM-1. HPAEC were exposed to 0.1 µg/ml SubAB and SubAA272B for 6 hours and then treated with 5 U/ml thrombin and 100 U/ml of TNFα for 6 hours. Western blot analysis using total cell lysates showed a reduced expression of both thrombin- and TNFα-induced ICAM-1 and VCAM-1 expression, in cells treated with SubAB (Fig. 11A & B). ELISA using culture supernatants showed a marked reduction in the levels of thrombin- and TNFα-induced for IL-8 and MCP-1, in cells preconditioned with SubAB (Fig. 11C–F). In contrast, preconditioning of HPAECs to SubAA272B, did not affect any of the above responses (Fig. 11C–F).


Preconditioning with endoplasmic reticulum stress ameliorates endothelial cell inflammation.

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

SubAB inhibits NF-κB mediated proinflammatory gene expression.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A & C & E) thrombin (5 U/ml) or (B & D & F) TNFα (100 U/ml) for 6 h. Total cell lysates were immunoblotted with (A & B) anti-ICAM-1 antibody and anti -VCAM-1 antibody. Actin was used to monitor loading. The conditioned media were subjected to ELISA to determine the levels of (C & D) IL-8 or (E & F) MCP-1. Data are means ± S.E. (n = 6–9 for each condition). ###p<0.001 difference from control; ***p<0.001 difference from thrombin and TNFα-stimulated controls.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214695&req=5

pone-0110949-g011: SubAB inhibits NF-κB mediated proinflammatory gene expression.HPAEC were treated with 0.1 µg/ml of SubAB or mutant SubAA272B for 6 h, followed by treatment with (A & C & E) thrombin (5 U/ml) or (B & D & F) TNFα (100 U/ml) for 6 h. Total cell lysates were immunoblotted with (A & B) anti-ICAM-1 antibody and anti -VCAM-1 antibody. Actin was used to monitor loading. The conditioned media were subjected to ELISA to determine the levels of (C & D) IL-8 or (E & F) MCP-1. Data are means ± S.E. (n = 6–9 for each condition). ###p<0.001 difference from control; ***p<0.001 difference from thrombin and TNFα-stimulated controls.
Mentions: Expression of adhesion molecules, cytokines and chemokines in endothelial cells by inflammatory mediators such as thrombin and TNFα is a central step in the pathogenesis of ALI and is under the tight regulation of the transcription factor NF-κB. We determined whether SubAB-mediated ER stress preconditioning affects thrombin and TNFα-induced NF-κB activation and subsequent expression of adhesion molecules ICAM-1, VCAM-1, and chemo-attractant proteins IL-8 and MCP-1. Cells were transfected with pNF-κB-LUC for 24 hours, followed by treatment with SubAB for 6 hours. Results showed that thrombin or TNFα challenge of cells resulted in increased NF-κB reporter activity and this response was inhibited in cells pretreated with 0.1 µg/ml of SubAB (Fig. 10A & B). Next we determined the effect of SubAB on NF-κB target genes, ICAM-1 and VCAM-1. HPAEC were exposed to 0.1 µg/ml SubAB and SubAA272B for 6 hours and then treated with 5 U/ml thrombin and 100 U/ml of TNFα for 6 hours. Western blot analysis using total cell lysates showed a reduced expression of both thrombin- and TNFα-induced ICAM-1 and VCAM-1 expression, in cells treated with SubAB (Fig. 11A & B). ELISA using culture supernatants showed a marked reduction in the levels of thrombin- and TNFα-induced for IL-8 and MCP-1, in cells preconditioned with SubAB (Fig. 11C–F). In contrast, preconditioning of HPAECs to SubAA272B, did not affect any of the above responses (Fig. 11C–F).

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