Role of NF-κB-dependent Caveolin-1 Expression in the Mechanism of Increased Endothelial Permeability Induced by Lipopolysaccharide

Chinnaswamy Tiruppathi,Jun Shimizu,Kayo Miyawaki-Shimizu,Stephen M Vogel,Angela M Bair,Richard D Minshall,Dan Predescu,Asrar B Malik

doi:10.1074/jbc.m703153200

Chinnaswamy Tiruppathi, Jun Shimizu + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m703153200

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Abstract

We investigated the role of NF-kappaB activation by the bacterial product lipopolysaccharide (LPS) in inducing caveolin-1 (Cav-1) expression and its consequence in contributing to the leakiness of the endothelial barrier. We observed that LPS challenge of human lung microvascular endothelial cells induced concentration- and time-dependent increases in expression of Cav-1 mRNA and protein. The NEMO (NF-kappaB essential modifier binding domain)-binding domain peptide (IkB kinase (IKK)-NEMO-binding domain (NBD) peptide), which prevents NF-kappaB activation by inhibiting the interaction of IKKgamma with the IKK complex, blocked LPS-induced Cav-1 mRNA and protein expression. Knockdown of NF-kappaB subunit p65/RelA expression with small interfering RNA also prevented LPS-induced Cav-1 expression. Caveolae open to the apical and basal plasmalemma of endothelial cells increased 2-4-fold within 4 h of LPS exposure. IKK-NBD peptide markedly reduced the LPS-induced increase in the number of caveolae as well as transendothelial albumin permeability. These observations were recapitulated in mouse studies in which IKK-NBD peptide prevented Cav-1 expression and interfered with the increase in lung microvessel permeability induced by LPS. Thus, LPS mediates NF-kappaB-dependent Cav-1 expression that results in increased caveolae number and thereby contributes to the mechanism of increased transendothelial albumin permeability.

Highlights

Cav-1, the structural protein of caveolae in endothelial cells and other cell types, regulates the formation of caveolae, the vesicle carriers involved in the transcytosis of albumin across the endothelial barrier (7)
LPS Induces NF-␬B-dependent Increase in Caveolae Number— we addressed whether the increased Cav-1 protein expression in response to LPS challenge was coupled to the formation of caveolae by electron microscopy
It has been shown that LPS exposure of endothelial cells induces the expression of a broad array of proteins involved in the host defense and inflammatory responses (12)

Summary

EXPERIMENTAL PROCEDURES

Materials—Human lung microvascular endothelial cells (HLMVECs) and microvascular endothelial growth medium (EGM-2 MV) were obtained from Cambrex Bio Science (Walkersville, MD). To study the effect of antibodies on DNA-protein binding, nuclear extracts were first incubated with NF-␬B protein-specific antibodies (2 ␮g/assay) for 15 min at 25 °C, and labeled double-stranded probe was added, and the incubation was continued for an additional 20 min After this incubation, non-denaturing sample buffer added, and the DNA-protein complexes were separated as described above. Before the experiment a confluent endothelial monolayer was kept in 2% FBS containing medium for 2 h, and cells were challenged with LPS, IKK-NBD, or mutant IKK-NBD. Albumin permeability-surface area product was calculated as described from vascular counts, tissue counts, and tracer exposure time (22) In another set of experiments lungs were harvested for immunoblotting to determine Cav-1 expression

RESULTS

62 Ϯ 9 58 Ϯ 12 44 Ϯ 7 69 Ϯ 11 35 Ϯ 8 32 Ϯ 4

DISCUSSION