Modulation of pro-inflammatory gene expression by nuclear lysophosphatidic acid receptor type-1.
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.
Gobeil
Fernand
F
Departments of Pediatrics, Ophthalmology and Pharmacology, Research Center of Hôpital Sainte-Justine, Montréal, Québec H3T 1C5, Canada. Fernand.Gobeil@USherbrooke.ca
Bernier
Sylvie G
SG
Vazquez-Tello
Alejandro
A
Brault
Sonia
S
Beauchamp
Martin H
MH
Quiniou
Christiane
C
Marrache
Anne Marilise
AM
Checchin
Daniella
D
Sennlaub
Florian
F
Hou
Xin
X
Nader
Mony
M
Bkaily
Ghassan
G
Ribeiro-da-Silva
Alfredo
A
Goetzl
Edward J
EJ
Chemtob
Sylvain
S
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D002474:Cell-Free System / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation; D015151:Immunoblotting; D008099:Liver / Q000378:metabolism; D008833:Microcirculation; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D037342:Pertussis Toxin / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010741:Phospholipases A / Q000378:metabolism; D054467:Phospholipases A2; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism* / Q000502:physiology*; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D000077191:Wortmannin
D000730:Androstadienes; C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D002614:Chelating Agents; D002966:Clathrin; D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D004533:Egtazic Acid; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium; D000077191:Wortmannin