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Alcohol-Induced Interactive Phosphorylation of Src and Toll-like Receptor Regulates the Secretion of Inflammatory Mediators by Human Astrocytes

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Abstract

Secretion of pro-inflammatory molecules by astrocytes after alcohol treatment was shown to be associated with neuroinflammation. We hypothesized that activation of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX-2) by ethanol in astrocytes enhanced the secretion of inflammatory agents via the interactive tyrosine phosphorylation of toll-like receptor 4 (TLR4) and Src kinase. To test this hypothesis, we treated primary human astrocytes with 20 mM ethanol for 48 h at 37°C. Ethanol exposure elevated cytochrome P450-2E1 activity, reactive oxygen species levels, and secretion of prostaglandin E2 (PGE2) in these cells. Secretion of PGE2 was associated with induction of cPLA2 activity and protein content as well as COX-2 protein level in a Src phosphorylation-dependent manner that occurred by enhanced transcription. Immunoprecipitation and Western blot analyses indicated that the interactive tyrosine phosphorylation of TLR4–Src complex at the cell membrane triggered the activation of cPLA2 and COX-2 in the cytoplasm through a Src signaling intermediate. Inhibition of ethanol metabolism, blockage of Src activity, or inactivation of TLR4 prevented the activation of cPLA2 and COX-2 as well as diminished PGE2 production, suggesting that interactive phosphorylation of TLR4–Src regulated the pro-inflammatory response in astrocytes. Experiments with small interfering RNA knockdown of TLR4 in human astrocytes confirmed that silencing expression also abolished the interactive phosphorylation of both TLR4 and Src in the presence of ethanol.

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Abbreviations

AA:

arachidonic acid

AACOCF3:

arachidonyltriflouromethyl ketone

ACD:

actinomycin D

Ach:

acetaldehyde

ADH:

alcohol dehydrogenase

APC:

apocynin

Chx:

cycloheximide

COX-2:

cyclooxygenase

cPLA2:

cytosolic phospholipase A2

CYP2E1:

cytochrome P450-2E1

EtOH:

ethanol

4-MP:

4-methylpyrazole

NOX:

NADPH oxidase

PGE2:

prostaglandin E2

PP2:

4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine

ROS:

reactive oxygen species

Src:

Src kinase

TLR4:

toll-like receptor 4

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Acknowledgments

This work was supported in part by NIH grants AA017398-01 (to YP and JH) and AA016403-01A2 (to JH).

Conflict of interest statement

The authors have no financial or other conflicts of interest.

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Authors and Affiliations

  1. Neurovascular Oxidative Injury Laboratory, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5215, USA

    Nicholas A. Floreani, Travis J. Rump, P. M. Abdul Muneer, Saleena Alikunju, Brenda M. Morsey, Michael R. Brodie & James Haorah

  2. Department of Pathology and Lab Medicine, Temple University School of Medicine, 3401 N Broad St., Philadelphia, PA, 19140, USA

    Yuri Persidsky

  3. Laboratory of Neurovascular Oxidative Injury, Department of Pharmacology and Experimental Neuroscience, 985215 Nebraska Medical Center, Omaha, NE, 68198-5215, USA

    James Haorah

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  1. Nicholas A. Floreani
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Correspondence to James Haorah.

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Floreani, N.A., Rump, T.J., Muneer, P.M.A. et al. Alcohol-Induced Interactive Phosphorylation of Src and Toll-like Receptor Regulates the Secretion of Inflammatory Mediators by Human Astrocytes. J Neuroimmune Pharmacol 5, 533–545 (2010). https://doi.org/10.1007/s11481-010-9213-z

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