Nitric oxide signaling via nuclearized endothelial nitric-oxide synthase modulates expression of the immediate early genes iNOS and mPGES-1.
Stimulation of freshly isolated rat hepatocytes with lysophosphatidic acid (LPA) resulted in LPA1 receptor-mediated and nitricoxide-dependent up-regulation of the immediate early genes iNOS (inducible nitric-oxide synthase (NOS)) and mPGES-1 (microsomal prostaglandin E synthase-1). Because LPA is a ligand for both cell surface and intracellular receptor sites and a potent endothelial NOS (eNOS) activator, we hypothesized that NO derived from activated nuclearized eNOS might participate in gene regulation. Herein we show, by confocal microscopy performed on porcine cerebral endothelial cells expressing native LPA1-receptor and eNOS and on HTC4 rat hepatoma cells co-transfected with recombinant human LPA1-receptor and fused eNOS-GFP cDNA, a dynamic eNOS translocation from peripheral to nuclear regions upon stimulation with LPA. Nuclear localization of eNOS and its downstream effector, soluble guanylate cyclase, were demonstrated in situ in rat liver specimens by immunogold labeling using specific antibodies. Stimulation of this nuclear fraction with LPA and the NO donor sodium nitroprusside resulted, respectively, in increased production of nitrite (and eNOS phosphorylation) and cGMP; these separate responses were also correspondingly blocked by NOS inhibitor L-NAME and soluble guanylate cyclase inhibitor ODQ. In addition, sodium nitroprusside evoked a sequential increase in nuclear Ca2+ transients, activation of p42 MAPK, NF-kappaB binding to DNA consensus sequence, and dependent iNOS RNA. This study describes a hitherto unrecognized molecular mechanism by which nuclear eNOS through ensuing NO modulates nuclear calcium homeostasis involved in gene transcription-associated events. Moreover, our findings strongly support the concept of the nucleus as an autonomous signaling compartment.
Gobeil
Fernand
F
Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada. Fernand.Gobeil@USherbrooke.ca
Zhu
Tang
T
Brault
Sonia
S
Geha
Antoinette
A
Vazquez-Tello
Alejandro
A
Fortier
Audrey
A
Barbaz
David
D
Checchin
Daniella
D
Hou
Xin
X
Nader
Moni
M
Bkaily
Ghassan
G
Gratton
Jean-Philippe
JP
Heveker
Nikolaus
N
Ribeiro-da-Silva
Alfredo
A
Peri
Krishna
K
Bard
Harry
H
Chorvatova
Alzbeta
A
D'Orléans-Juste
Pedro
P
Goetzl
Edward J
EJ
Chemtob
Sylvain
S
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D017931:DNA Primers; D015870:Gene Expression / Q000187:drug effects; D017781:Genes, Immediate-Early; D006162:Guanylate Cyclase; D006801:Humans; D007249:Inflammation / Q000235:genetics*; D008099:Liver / Q000378:metabolism; D008246:Lysophospholipids / Q000494:pharmacology; D018613:Microscopy, Confocal; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D000071876:Prostaglandin-E Synthases; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics*; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D049368:Receptors, Lysophosphatidic Acid / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D000071756:Soluble Guanylyl Cyclase; D013552:Swine
D017931:DNA Primers; D008246:Lysophospholipids; D018160:Receptors, Cytoplasmic and Nuclear; D049368:Receptors, Lysophosphatidic Acid; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011451:Prostaglandin-Endoperoxide Synthases; D006162:Guanylate Cyclase; D000071756:Soluble Guanylyl Cyclase; D000071876:Prostaglandin-E Synthases; C497818:Ptges protein, rat; C032881:lysophosphatidic acid