Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1

J Biol Chem. 2003 Oct 3;278(40):38875-83. doi: 10.1074/jbc.M212481200. Epub 2003 Jul 7.

Abstract

Lysophosphatidic acid (LPA) is a bioactive molecule involved in inflammation, immunity, wound healing, and neoplasia. Its pleiotropic actions arise presumably by interaction with their cell surface G protein-coupled receptors. Herein, the presence of the specific nuclear lysophosphatidic acid receptor-1 (LPA1R) was revealed in unstimulated porcine cerebral microvascular endothelial cells (pCMVECs), LPA1R stably transfected HTC4 rat hepatoma cells, and rat liver tissue using complementary approaches, including radioligand binding experiments, electron- and cryomicroscopy, cell fractionation, and immunoblotting with three distinct antibodies. Coimmunoprecipitation studies in enriched plasmalemmal fractions of unstimulated pCMVEC showed that LPA1Rs are dually sequestrated in caveolin-1 and clathrin subcompartments, whereas in nuclear fractions LPA1R appeared primarily in caveolae. Immunofluorescent assays using a cell-free isolated nuclear system confirmed LPA1R and caveolin-1 co-localization. In pCMVEC, LPA-stimulated increases in cyclooxygenase-2 and inducible nitric-oxide synthase RNA and protein expression were insensitive to caveolea-disrupting agents but sensitive to LPA-generating phospholipase A2 enzyme and tyrosine kinase inhibitors. Moreover, LPA-induced increases in Ca2+ transients and/or iNOS expression in highly purified rat liver nuclei were prevented by pertussis toxin, phosphoinositide 3-kinase/Akt inhibitor wortmannin and Ca2+ chelator and channel blockers EGTA and SK&F96365, respectively. This study describes for the first time the nucleus as a potential organelle for LPA intracrine signaling in the regulation of pro-inflammatory gene expression.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Androstadienes / pharmacology
  • Animals
  • Blotting, Western
  • Calcium / metabolism
  • Caveolin 1
  • Caveolins / metabolism
  • Cell Nucleus / metabolism*
  • Cell-Free System / metabolism
  • Cells, Cultured
  • Chelating Agents / pharmacology
  • Clathrin / metabolism
  • Egtazic Acid / pharmacology
  • Endothelium, Vascular / cytology
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation*
  • Immunoblotting
  • Liver / metabolism
  • Microcirculation
  • Microscopy, Electron
  • Microscopy, Fluorescence
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type II
  • Pertussis Toxin / pharmacology
  • Phosphoinositide-3 Kinase Inhibitors
  • Phospholipases A / metabolism
  • Phospholipases A2
  • Precipitin Tests
  • Protein Binding
  • Protein-Tyrosine Kinases / metabolism
  • Rats
  • Receptors, Cell Surface / metabolism*
  • Receptors, Cell Surface / physiology*
  • Receptors, G-Protein-Coupled*
  • Receptors, Lysophosphatidic Acid
  • Subcellular Fractions / metabolism
  • Swine
  • Time Factors
  • Transfection
  • Tumor Cells, Cultured
  • Wortmannin

Substances

  • Androstadienes
  • Cav1 protein, rat
  • Caveolin 1
  • Caveolins
  • Chelating Agents
  • Clathrin
  • Enzyme Inhibitors
  • Phosphoinositide-3 Kinase Inhibitors
  • Receptors, Cell Surface
  • Receptors, G-Protein-Coupled
  • Receptors, Lysophosphatidic Acid
  • Egtazic Acid
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, rat
  • Pertussis Toxin
  • Protein-Tyrosine Kinases
  • Phospholipases A
  • Phospholipases A2
  • Calcium
  • Wortmannin