Title

Interleukin-1β-Induced Barrier Dysfunction Is Signaled Through Pkc-θ in Human Brain Microvascular Endothelium

Document Type

Article

Publication Date

2012

Keywords

neuroinflammatory conditions, edema, blood-brain barrier, microvascular permeability, tight junction proteins

Digital Object Identifier (DOI)

https://doi.org/10.1152/ajpcell.00371.2011

Abstract

Blood-brain barrier dysfunction is a serious consequence of inflammatory brain diseases, cerebral infections, and trauma. The proinflammatory cytokine interleukin (IL)-1β is central to neuroinflammation and contributes to brain microvascular leakage and edema formation. Although it is well known that IL-1β exposure directly induces hyperpermeability in brain microvascular endothelium, the molecular mechanisms mediating this response are not completely understood. In the present study, we found that exposure of the human brain microvascular endothelium to IL-1β triggered activation of novel PKC isoforms δ, μ, and θ, followed by decreased transendothelial electrical resistance (TER). The IL-1β-induced decrease in TER was prevented by small hairpin RNA silencing of PKC-θ or by treatment with the isoform-selective PKC inhibitor Gö6976 but not by PKC inhibitors that are selective for all PKC isoforms other than PKC-θ. Decreased TER coincided with increased phosphorylation of regulatory myosin light chain and with increased proapoptotic signaling indicated by decreased uptake of mitotracker red in response to IL-1β treatment. However, neither of these observed effects were prevented by Gö6976 treatment, indicating lack of causality with respect to decreased TER. Instead, our data indicated that the mechanism of decreased TER involves PKC-θ-dependent phosphorylation of the tight junction protein zona occludens (ZO)-1. Because IL-1β is a central inflammatory mediator, our interpretation is that inhibition of PKC-θ or inhibition of ZO-1 phosphorylation could be viable strategies for preventing blood-brain barrier dysfunction under a variety of neuroinflammatory conditions.

brain inflammation is a pathological consequence of trauma, stroke, cerebral infection, multiple sclerosis, and other inflammatory conditions (4, 10, 30, 34). A serious consequence of brain inflammation is microvascular leakage and brain edema, leading to brain swelling, neuronal injury, and death. Microvascular leakage occurs due to misregulation of the protective interface between the blood and the brain tissue known as the blood-brain barrier (BBB; for review see Refs. 1, 12, 17, 22). During brain inflammation, microvascular barriers become compromised in response to changes in expression or organization of endothelial tight junction proteins permitting plasma components to leak across the BBB into the brain tissue interstitial space. This BBB hyperpermeability occurs in response to proinflammatory agents that are present in the brain tissue during inflammation.

The proinflammatory cytokine interleukin (IL)-1β is produced in the brain during neuroinflammation and contributes to brain microvascular leakage and brain edema (8, 10, 13, 30, 34). The proedematous effects of IL-1β are mediated through the IL-1 receptor (IL1-R1), in that direct inhibition of IL-1β binding to IL1-R1 prevented both brain edema and brain tissue injury in rodent models of experimental cerebral ischemia (38). Brain edema resulting from hypoxic/ischemic injury was also prevented in IL1-R1 knockout mice compared with wild-type mice (27). In addition, vascular effects result from direct exposure of endothelial cells to IL-1β, as demonstrated in studies (14) with cultured human brain microvascular endothelial cells where IL-1β treatment induced hyperpermeability in the absence of all other brain parenchymal cell types. While the effects of IL-1β on brain endothelial hyperpermeability have been clearly demonstrated, most of the intermediate cellular signaling events leading to hyperpermeability are unknown.

In many tissue types, microvascular hyperpermeability is signaled through classical (α, βI, βII, and γ), novel (δ, ε, η, θ, and μ), or atypical (ζ and λ/ι) protein kinase C (PKC) isoforms (32, 3941). For example, microvascular leakage was mediated by PKC-βII (2, 3), -δ (25), and -ζ (6) at the blood-retinal barrier. Likewise, the isoform-selective PKC inhibitor Gö6976 [12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole] prevented microvascular leakage during ischemia-reperfusion injury in the rat heart, as well as hyperpermeability in human coronary microvascular endothelium in response to IL-1β exposure (36). In addition, BBB dysfunction was mediated by PKC-α in response to tumor necrosis factor (TNF)-α exposure in mouse brain endothelium (29) and involved activation of PKC-θ and possibly other PKC isoforms during hypoxia in the rat brain (18, 37). Based on these observations, we hypothesized that select PKC isoforms mediate hyperpermeability in human brain microvascular endothelium during IL-1β exposure.

In the present study, we examined the role of PKC in mediating barrier dysfunction in human brain microvascular endothelium in response to IL-1β exposure. Our data indicated that novel PKC isoforms (δ, θ, and μ) were activated in response to IL-1β exposure in human brain microvascular endothelial cells (hBMECs). Evidence from PKC isoform-specific inhibitors and gene silencing indicated that PKC-θ was necessary for IL-1β-induced barrier dysfunction [measured as decreased transendothelial electrical resistance (TER)] in hBMEC monolayers. We found that decreased TER was not accompanied by altered expression of junction proteins, increased cell contractility, or apoptotic mechanisms. Furthermore, immunoprecipitation experiments demonstrated that decreased TER in response to IL-1β involved PKC-θ-dependent phosphorylation of zona occludens (ZO)-1. Therefore, selective inhibition of PKC-θ under inflammatory conditions may prevent BBB leakage that is due to posttranslational modification of tight junction proteins and conformational modification of tight junctions in the brain microvascular endothelium.

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Yes

Citation / Publisher Attribution

American Journal of Physiology-Cell Physiology, v. 302, issue 10, p. C1513-C1522

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