Alcohol-induced interactive phosphorylation of Src and toll-like receptor regulates the secretion of inflammatory mediators by human astrocytes

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

Research output: Contribution to journalArticlepeer-review

41 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)533-545
Number of pages13
JournalJournal of Neuroimmune Pharmacology
Volume5
Issue number4
DOIs
StatePublished - Dec 2010
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Neuroscience (miscellaneous)
  • Immunology and Allergy
  • Immunology
  • Pharmacology

Keywords

  • cyclooxygenase
  • cytochrome P450-2E1
  • human astrocytes
  • phospholipase A2
  • reactive oxygen species

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