IL-1β suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury
Shiqin Xiong, Zhigang Hong, Long Shuang Huang, Yoshikazu Tsukasaki, Saroj Nepal, Anke Di, Ming Zhong, Wei Wu, Zhiming Ye, Xiaopei Gao, Gadiparthi N. Rao, Dolly Mehta, Jalees Rehman, Asrar B. Malik
Shiqin Xiong, Zhigang Hong, Long Shuang Huang, Yoshikazu Tsukasaki, Saroj Nepal, Anke Di, Ming Zhong, Wei Wu, Zhiming Ye, Xiaopei Gao, Gadiparthi N. Rao, Dolly Mehta, Jalees Rehman, Asrar B. Malik
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Research Article Inflammation Vascular biology

IL-1β suppression of VE-cadherin transcription underlies sepsis-induced inflammatory lung injury

Abstract

Unchecked inflammation is a hallmark of inflammatory tissue injury in diseases such as acute respiratory distress syndrome (ARDS). Yet the mechanisms of inflammatory lung injury remain largely unknown. Here we showed that bacterial endotoxin lipopolysaccharide (LPS) and cecal ligation and puncture–induced (CLP-induced) polymicrobial sepsis decreased the expression of transcription factor cAMP response element binding (CREB) in lung endothelial cells. We demonstrated that endothelial CREB was crucial for VE-cadherin transcription and the formation of the normal restrictive endothelial adherens junctions. The inflammatory cytokine IL-1β reduced cAMP generation and CREB-mediated transcription of VE-cadherin. Furthermore, endothelial cell–specific deletion of CREB induced lung vascular injury whereas ectopic expression of CREB in the endothelium prevented the injury. We also observed that rolipram, which inhibits type 4 cyclic nucleotide phosphodiesterase–mediated (PDE4-mediated) hydrolysis of cAMP, prevented endotoxemia-induced lung vascular injury since it preserved CREB-mediated VE-cadherin expression. These data demonstrate the fundamental role of the endothelial cAMP-CREB axis in promoting lung vascular integrity and suppressing inflammatory injury. Therefore, strategies aimed at enhancing endothelial CREB-mediated VE-cadherin transcription are potentially useful in preventing sepsis-induced lung vascular injury in ARDS.

Authors

Shiqin Xiong, Zhigang Hong, Long Shuang Huang, Yoshikazu Tsukasaki, Saroj Nepal, Anke Di, Ming Zhong, Wei Wu, Zhiming Ye, Xiaopei Gao, Gadiparthi N. Rao, Dolly Mehta, Jalees Rehman, Asrar B. Malik

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Figure 3

IL-1β transcriptionally downregulates CREB-mediated VE-cadherin expression to increase endothelial permeability.

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IL-1β transcriptionally downregulates CREB-mediated VE-cadherin expressi...
(A–C) mLMVECs were transfected with siRNA of sAC or control scrambled siRNA for 3 days. Cells were then stimulated with IL-1β (5 ng/mL) for 12 hours. Intracellular cAMP levels were measured (A) (n = 4). Protein expression by Western blot (B) and permeability to HRP (C) were determined (n = 6). (D) mLMVECs were stimulated with IL-1β (5 ng/mL) for the indicated time. Effect of IL-1β treatment on CREB binding to the mouse VE-cadherin promoter regions (CRE2 and CRE5) in mLMVECs was investigated by ChIP assays (n = 3). (E) mLMVECs were transiently transfected with the 2.5-kb VE-cadherin promoter luciferase construct, CREB expression vector, and the internal control p-RL-TK vector by lipofectamine 3000 reagent for 2 days. Cells were then treated with IL-1β (5 ng/mL) over time. CREB-regulated VE-cadherin transcription activity was measured using the dual luciferase reporter assay system (n = 6). (F–H) mLMVECs were transfected with CREB plasmids or control for 2 days, and stimulated with IL-1β (5 ng/mL) for 1 day. Immunofluorescence staining of VE-cadherin (F); arrows denote disruption of endothelial barrier integrity. Permeability to HRP (n = 6) (G) and protein expression by Western blot (H) were determined. **P < 0.01; ***P < 0.001; NS, no significant difference. Statistics obtained from ANOVA. Scale bars: 10 μm.