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. 2016 Aug 1;311(2):L517-24.
doi: 10.1152/ajplung.00069.2016. Epub 2016 Jul 1.

Phospholipase C-ε Signaling Mediates Endothelial Cell Inflammation and Barrier Disruption in Acute Lung Injury

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Free PMC article

Phospholipase C-ε Signaling Mediates Endothelial Cell Inflammation and Barrier Disruption in Acute Lung Injury

Kaiser M Bijli et al. Am J Physiol Lung Cell Mol Physiol. .
Free PMC article

Abstract

Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε(-/-) mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-ε(-/-) mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1β, IL-6, macrophage inflammatory protein 2, keratinocyte-derived cytokine, monocyte chemoattractant protein 1, and granulocyte-macrophage colony-stimulating factor), lung neutrophil infiltration and microvascular leakage, and loss of VE-cadherin compared with PLC-ε(+/+) mice. These data identify PLC-ε as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC-ε activity in endothelial cells (EC) could contribute to ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC-ε inhibited NF-κB activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNF-α, and the nonreceptor agonist phorbol 13-myristate 12-acetate (phorbol esters) in EC. Depletion of PLC-ε also inhibited thrombin-induced expression of NF-κB target gene, VCAM-1. Importantly, PLC-ε knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC-ε as a novel regulator of EC inflammation and permeability and show a hitherto unknown role of PLC-ε in the pathogenesis of ALI.

Keywords: adhesion molecules; endothelial cells; lung inflammation; signal transduction; transcription factors.

Figures

Fig. 1.
Fig. 1.
Effect of phospholipase C-ε (PLC-ε) deficiency on LPS-induced proinflammatory mediators in the lung. Age-matched C57BL/6L wild-type (WT) and PLC-ε−/− (knockout, KO) mice were aerosolized with saline alone or saline containing Escherichia coli LPS as described in materials and methods. At 18 h after LPS inhalation, lungs and bronchoalveolar lavage (BAL) fluids from these mice were collected, and levels of proinflammatory mediators were measured. A: lung homogenates were analyzed for ICAM-1 and VCAM-1 (a and b) levels by ELISA and TNF-α, monocyte chemoattractant protein 1 (MCP-1), keratinocyte-derived cytokine (KC), macrophage inflammatory protein 2 (MIP2), and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels (cg) by multiplex immune assay system. B: BAL fluids were analyzed for IL-1β, IL-6, MCP-1, and KC levels (ad) by ELISA. Data are means ± SE (n = 3–5 for each condition). *P <0.05 vs. saline WT; #P vs. LPS WT. nd, not detected.
Fig. 2.
Fig. 2.
Effect of PLC-ε deficiency on LPS-induced lung polymorphonuclear leukocyte (PMN) infiltration and injury. Lung homogenates and BAL fluids were collected from WT and KO mice exposed to LPS as in Fig. 1. Lung homogenates were analyzed for tissue myeloperoxidase (MPO) activity (A), and BAL fluids were analyzed for PMN counts (B) and total protein (C). Data are means ± SE (n = 3–5 for each condition). *P <0.05 vs. saline WT; #P vs. LPS WT.
Fig. 3.
Fig. 3.
Effect of PLC-ε deficiency on LPS-induced decrease in VE-cadherin levels. Lung homogenates from WT and KO mice exposed to LPS as in Fig. 1 were analyzed for VE-cadherin levels by immunoblotting. Actin levels were used to monitor loading. The bar graph represents the effect of PLC-ε deficiency on LPS-induced decrease in VE-cadherin levels normalized to actin levels. Data are means ± SE (n = 3 for each condition). *P <0.05 vs. saline WT; #P vs. LPS WT.
Fig. 4.
Fig. 4.
PLC-ε regulates NF-κB signaling in endothelial cells (EC). A: human pulmonary artery EC (HPAEC) were transfected with control siRNA (si-Con) or PLC-ε-specific siRNA (si-PLC-ε). After 36 h, total cell lysates were immunoblotted with an antibody to PLC-ε. RelA/p65 levels were used to monitor loading. The bar graph represents the effect of siRNA on PLC-ε level normalized to RelA/p65 level. Data are means ± SE (n = 4 for each condition). *P < 0.05 vs. si-Con control. B: HPAEC were transfected with si-Con or si-PLC-ε. After 36 h, cells were challenged with thrombin (5 U/ml) for 6 h, and the cell lysates were immunoblotted with an antibody to VCAM-1. RelA/p65 levels were used to monitor loading. The bar graph represents the effect of PLC-ε knockdown on thrombin-induced VCAM-1 expression normalized to actin level. Data are means ± SE (n = 3 for each condition). *P < 0.05 vs. si-Con untreated controls; #P < 0.05 vs. si-Con thrombin-treated controls. CF: HPAEC were transfected with si-PLC-ε or si-Con using DharmaFect1. After 24 h, cells were again transfected with NF-κBLUC construct using diethylaminoethyl-dextran as described in materials and methods. Eighteen hours later, cells were challenged with thrombin (5 U/ml) (C), LPS (0.1 mg/ml) (D), TNF-α (100 U/ml) (E), or phorbol 13-myristate 12-acetate (PMA) (50 nm) (F) for 6 h. Cell extracts were assayed for firefly and Renilla luciferase activities as a measure of NF-κB activity. Data are means ± SE (n = 3–6 for each condition). *P < 0.05 vs. si-Con untreated controls; #P < 0.05 vs. si-Con thrombin-treated controls.
Fig. 5.
Fig. 5.
PLC-ε regulates NF-κB activity by promoting degradation of Iκ-Bα, nuclear DNA biding, and phosphorylation of RelA/p65. HPAEC were transfected with si-Con or si-PLC-ε. After 36 h, cells were treated with thrombin (5 U/ml) for 1.5 h. A: total cell lysates were prepared and immunoblotted with an anti-Iκ-Bα antibody. RelA/p65 levels were used to monitor loading. B: nuclear extracts were prepared and analyzed for RelA/p65 nuclear DNA binding using Cayman's NF-κB transcription factor assay kit as described in materials and methods. C: total cell lysates were immunoblotted with an anti-phospho-RelA/p65 (Ser536) antibody. RelA/p65 levels were used to monitor loading. Data are means ± SE (n = 6–8 for each condition). *P < 0.05 vs. si-Con untreated controls; #P < 0.05 vs. si-Con thrombin-treated controls.
Fig. 6.
Fig. 6.
PLC-ε regulates endothelial barrier disruption by decreasing cell surface VE-cadherin and increasing actin stress fiber formation. HPAEC were transfected with si-Con or si-PLC-ε. A: after 24 h, cells were reseeded on gold electrode plates and cultured for an additional 48 h. The confluent monolayers were then treated with thrombin, and endothelial barrier disruption was determined by measuring transendothelial electrical resistance (TER). Data are means ± SE (n = 3–5 for each condition). *Difference between si-Con thrombin-treated vs. si-PLC-ε thrombin-treated controls (P < 0.05). B: after 36 h, cells were challenged with thrombin for 5 min, and immunofluorescence was performed using VE-cadherin antibody to visualize adherens junctions. Arrows indicate the sites of disruption of VE-cadherin staining. C: after 36 h, cells were challenged with thrombin for 15 min and labeled with Alexa 488-labeled phalloidin to visualize the actin stress fibers by fluorescence microscopy. Results are representative of 2–3 experiments.

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