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Originally published In Press as doi:10.1074/jbc.M602219200 on March 30, 2006

J. Biol. Chem., Vol. 281, Issue 23, 16058-16067, June 9, 2006
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Nitric Oxide Signaling via Nuclearized Endothelial Nitric-oxide Synthase Modulates Expression of the Immediate Early Genes iNOS and mPGES-1*Formula

Fernand Gobeil, Jr.{ddagger}1, Tang Zhu§, Sonia Brault§, Antoinette Geha§, Alejandro Vazquez-Tello§, Audrey Fortier{ddagger}, David Barbaz{ddagger}, Daniella Checchin§, Xin Hou§, Moni Nader, Ghassan Bkaily, Jean-Philippe Gratton||, Nikolaus Heveker§, Alfredo Ribeiro-da-Silva**, Krishna Peri{ddagger}{ddagger}, Harry Bard§, Alzbeta Chorvatova§, Pedro D'Orléans-Juste{ddagger}, Edward J. Goetzl§§, and Sylvain Chemtob§2

From the {ddagger}Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada, §Departments of Pediatrics, Ophthalmology, and Pharmacology, Research Center of Hôpital Sainte-Justine, Montréal, Québec H3T 1C5, Canada, Department of Anatomy and Cell Biology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada, ||Institut de Recherches Cliniques de Montréal, Québec H2W 1R7, Canada, **Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec H3G 1Y6, Canada, {ddagger}{ddagger}Theratechnologies, Montréal, Québec H4S 2A4, Canada, and §§Department of Medicine and Microbiology-Immunology, University of California, San Francisco, California 94143-0711

Received for publication, March 9, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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-{kappa}B 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Nitric oxide (NO) is a short-lived uncharged free radical involved in numerous complex biological processes such as blood pressure regulation, vascular inflammation, cell-mediated cytotoxicity, and survival (1, 2). One of the most biologically relevant actions of NO is its binding to the heme moiety in the heterodimeric enzyme soluble guanylate cyclase (sGC),3 which leads to the production of the intracellular second messenger molecule cGMP and activation of cGMP-dependent protein kinases G (PKG). However, NO can also interact with and modify the bioactivity of a number of protein macromolecules through a series of reversible and nonreversible chemical reactions (e.g. S-, N-, and hemenitrosylation, tyrosine and tryptophan nitration) providing multifaceted regulatory mechanisms for cellular functions (3, 4). NO is synthesized from L-arginine by a tripartite family of nitric-oxide synthase (NOS) isozymes composed of NOS-1 (nNOS), NOS-2 (iNOS), and NOS-3 (eNOS) originally found in brain, macrophages, and endothelium, respectively, and later discovered, for each NOS, in many other cell types (5).

NOS isozymes, especially eNOS, are highly regulated by a number of mechanisms. Emerging findings show that eNOS bioactivity and mobilization to specific organelles are dictated by multiple post-translational modifications (e.g. phosphorylation, myristoylation, palmitoylation, S-nitrosylation) and numerous protein-protein interactions (e.g. Akt, caveolin, Hsp-90, calcium/calmodulin, NOSIP, NOSTRIN). These latter allosteric interactions, which impart either inhibitory or stimulatory effects on the eNOS enzyme, have been postulated to ensure specificity of eNOS signaling in different parts of the same cell (6–8).

Because of the significant reactivity of NO and its limited diffusion, its cellular actions are likely triggered in the immediate vicinity of its site of synthesis (3, 6, 8). There is now convincing evidence that eNOS is targeted from cell membrane plasmalemmal caveolae (and to a lesser extent lipid rafts) to specific intracellular regions through stimulus-evoked translocation (7–10). In this context, the presence of native eNOS has recently been detected at the nucleus of endothelium (11) and other cell types (10, 12, 13) paralleling, in some instances, NO-sensitive guanylate cyclase localization (14, 15). However, the physiological consequences of eNOS routing to such distinct intracellular locales is poorly understood and rather speculative. In line with the presence of intracellular eNOS, we and others have recently disclosed the presence of functional G protein-coupled receptors at the nuclear/perinuclear envelope, including LPA/LPA1Rs (16) along with a number of their signaling mediators, such as some that interact with eNOS (17, 18). However, whether natural eNOS is active at the nucleus and whether its products can stimulate nuclear functions have never been addressed.

Because LPA is a G protein-coupled receptor agonist that exerts actions on the plasma membrane and separately on intracellular compartments via its cognate receptor, and because it is a recognized eNOS activator, it has been used to study NO-related responses in cells and in the nuclear organelles isolated from these cells (16, 19). Our initial experiments on freshly isolated rat hepatocytes stimulated with physiologically relevant concentrations of LPA demonstrated a conspicuous NO-dependent increase in inducible mPGES-1 (microsomal prostaglandin E synthase-1) and iNOS gene expression. In the present study, we tested and confirmed the hypothesis that eNOS is distributed and active at the nucleus, and at this site it can promote (via G protein-coupled receptor-evoked mechanisms) NO-mediated signal transduction cascades within the nucleus to modulate gene expression.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemical Reagents and Antibodies—Materials and chemicals were obtained from the following sources. Steroyl-lysophosphatidic acid (sLPA) was from Avanti%20Polar%20Lipids">Avanti Polar Lipids Inc; fura-2-AM, KT5823, and PD98059 from Calbiochem; SK&F96365 from Biomol; fluo-4-AM from Molecular Probes; sodium nitroprusside (SNP), EGTA, BAPTA-AM, 1H-(1, 2, 4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), ammonium pyrrolidine-dithiocarbamate (PDTC), NG-nitro-L-arginine methyl ester (L-NAME), 3-isobutyl-1-methylxanthine, creatinine phosphate, creatine phosphokinase, L-arginine, (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride ((6R)-BH4), calmodulin from bovine brain, flavin adenine dinucleotide (FAD), and nitrate reductase (from Aspergillus niger) from Sigma; GTP from Amersham Biosciences; NADPH from Roche Applied Science; and bis(sulfosuccinimidyl)suberate (BS3) from Pierce. THG1603 (LPA1R antagonist; PCT WO 00/17348) was a gift from Theratechnologies Inc. (Montréal, Québec, Canada). All other chemicals were analytical reagents and were purchased from Fisher Scientific.

Antibodies and their sources were as follows. Anti-eNOS/NOS type III monoclonal and polyclonal antibodies, anti-iNOS monoclonal antibody, anti-nNOS monoclonal antibody, and anti-beta1 integrin monoclonal antibody were from Transduction Laboratories; anti-guanylate cyclase soluble polyclonal antibodies from Cayman and Fabgennix Inc.; anti-beta-actin monoclonal antibody from Novus Biological; anti-phosphorylated MAPK (p-Erk1/2) polyclonal antibody from Promega; anti-MAPK (Erk 1/2) polyclonal antibody and anti-phosphorylated eNOS (Ser-1177) monoclonal antibody from Upstate%20Biotechnology">Upstate Biotechnology; anti-lamin B1 monoclonal antibody from Serotec; and goat anti-rabbit Alexa-Fluor 594-conjugated IgG from Molecular Probes.

Animals—Animal housing and experimental protocols were carried out in accordance with regulations set by the Canadian Council of Animal Care Committee and approved by local Animal Care Committee. Experiments were performed on hepatocytes isolated from the livers of adult Sprague-Dawley male rats (Charles River; St. Constant, Québec, Canada) as well as of eNOS–/– (20) and corresponding wild-type mice (provided by P. D'Orléans-Juste, Université de Sherbrooke).

Cell Fractionation and Nuclear Isolation—Isolation of rat liver nuclei was carried out by discontinuous sucrose gradient ultracentrifugation as described previously (16). The purity of subcellular fractions was validated using biochemical, immunological, and ultrastructural techniques described in several previous reports from our laboratories (16, 17). Prepared nuclear fractions demonstrated low specific activity of plasma membrane enzyme 5'-nucleotidase (<2%), lack of immunoreactivity with organelle-specific antibodies to alkaline phosphatase and cytochrome C, and high immunoreactivity with lamin A/C, which are marker antigens for plasma membrane, mitochondria (and cytosol), and nuclei, respectively. Highly purified mouse liver nuclei were prepared as indicated above (16) with slight modifications. For the latter, crude nuclear fractions were resuspended in buffer containing 1.2 M sucrose and layered on top of a buffer solution containing 1.8 M sucrose prior to centrifugation at 60 000 x g for 60 min at 4 °C. Porcine cerebral microvascular endothelial cell (pCMVEC) fractionation and nuclear isolation were achieved by the hypotonic/Nonidet P-40 lysis method (17). Purity of organellar fractions from pCMVEC was assessed by immunoblotting with anti-beta1 integrin (1:250) and anti-lamin B1 (1:50) monoclonal antibodies for detection of plasma membrane- and nuclear-specific marker antigens, respectively, as portrayed in supplemental Fig. S1. The purity and integrity of hepatic as well as endothelial nuclear fractions were confirmed by gold standard electron microscopic technique (16, 17) as well as by high resolution scanning electron microscopy (see Fig. 1C). For the latter, isolated nuclei laid on polylysine-treated coverslips were fixed, dehydrated by standard techniques, and visualized with a Hitachi S4700 (coldFEG) scanning electron microscope.

Cell Culture and Transfection—Primary endothelial cells obtained from porcine cerebral microvessels (17) and COS-1 and stable LPA1R/HTC4 cells were cultured and passaged as reported previously (17, 21). COS-1 cells were transfected with the cDNA for GFP-eNOS (kindly provided by Dr. J. P. Gratton, Institut de Recherches Cliniques de Montréal) using Lipofectamine as per the manufacturer's instructions.

Gene Induction and Semiquantification of iNOS and mPGES-1 Transcripts—Hepatocytes were harvested following digestion of rat liver with collagenase (type II, 0.05% Sigma) as described previously (16). Hepatocytes were counted, and 250,000 cells were used per reaction tube. Freshly isolated hepatocytes were left to stabilize for 1 h at 37 °C in stimulation buffer consisting of Hepes 20 mM, pH 7.4, dextrose 20 mM, KH2PO4 1.18 mM, MgSO4 1.18 mM, MgSO4, 1.18 mM, CaCl2, 2.5 mM, KCl 4.7 mM, and NaCl 118 mM. Hepatocyte suspensions were pretreated for 20 min with vehicle, nonselective NO synthase inhibitor L-NAME (1 mM), or LPA1R peptide antagonist THG1603 (100 µM) and then stimulated for 3 h with sLPA (10 µM). Cells were then centrifuged (600 x g, 5 min at 4 °C) and the resulting pellet snap-frozen for subsequent RNA extraction. Quiescent pCMVEC (90% confluency) cells were incubated in Dulbecco's modified Eagle's medium buffer and treated for 1 h at 37°C with either vehicle (bidistilled and deionized water) or s-LPA (10 µM). Cells were then washed twice with cold phosphate-buffered saline (PBS), harvested in the same buffer by gentle scraping, and collected following centrifugation. Total RNA from purified hepatocytes and cultured pCMVEC were isolated by the standard guanidine isothiocyanate method and by using the RNeasy Protect Mini kit (Qiagen), respectively. Rat and porcine iNOS and beta-actin mRNAs were quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) method as described elsewhere (16, 22). The sequences of rat mPGES-1 primers are: sense, 5'-CTG GCT AAG CTA GGT GTG TGG; antisense, 5'-GCT AAG GCA GGA CAG AGA GGT. Those for pig mPGES-1 primers are: sense, 5'-ACC AGT ACA AGA CAT GTC CCT TC; antisense, 5'-CAC TTC ATC TCC TCC GTC CTG. For mPGES-1 cDNA amplification, the reaction was heated at 94 °C for 40 s, annealed at 58 °C for 40 s, and extended at 68 °C for 1 min 20 s for 30 repetitive cycles.

iNOS gene expression was also studied in a cell-free nuclear system essentially as described (16). In brief, highly purified rat and mouse liver nuclei (~106 nuclei/assay) were preincubated at 37 °C for 15 min with L-NAME (1 mM) or THG1603 (100 µM) prior to stimulation with sLPA (10 µM) for 60 min. In another set of experiments, isolated rat liver nuclei were incubated at 37 °C for 60 min with either vehicle (buffer) or SNP (1 µM) in the presence or absence of ODQ (0.3 µM), KT5823 (1 µM), EGTA (100 µM), and PD98059 (1 µM) or NF-{kappa}B inhibitor PDTC (100 µM) (15 min preincubation). Total nuclear RNA was isolated as described (16). Expression of rat iNOS and beta-actin and porcine iNOS and beta-actin, as well as murine iNOS and 18S mRNAs, was analyzed by RT-PCR. The oligonucleotide primer pairs and PCR amplification protocols used for assessment of iNOS and beta-actin as well as 18S (Ambion, catalog no. 1718) message steady-state levels were as described (16, 22, 23). PCR products were quantified using an Agilent 2100 bioanalyzer in conjunction with DNA 7500 Labchip kits and by electrophoresis on precast 1% agarose gels stained with SYBR® Gold nucleic acid stain (Agilent Technologies). Gels were imaged and analyzed with a fluorescence gel imaging system (16).

For cross-linking experiments, freshly isolated rat hepatocytes (7 x 105 cells/assay) were incubated with the membrane-impermeable, water-soluble cross-linker reagent BS3 (1 mM) for 1 h at 37°C. Thereafter, 50 mM Tris-HCl was added to the incubation medium to stop the reaction as per the manufacturer's instruction (Pierce). Hepatic cells were then challenged with sLPA (10 µM). Reaction was terminated by freezing samples in liquid N2. Western blots of MAPK activation after sLPA (10 µM) exposure (0 to 30 min) and RT-PCR for iNOS gene induction (1 h) were then performed. For the former assay, prepared cytosolic proteins (50 µg) were resolved by SDS-PAGE on a 9% gel, transferred onto polyvinylidene difluoride (PVDF) membranes, and then probed with Erk1/2 and phospho-Erk(1/2) antibodies (diluted 1:1000). Autoradiograms were scanned and analyzed by densitometry (ImagePro 4+ software).

RT-PCR Detection of LPA/LPA1R in COS-1 Cells—The presence of basal endogenous LPA/LPA1R (among others) in several host expression systems often used to study LPA signaling has recently been documented (24). We investigated whether COS-1 cells, used for functional experiments (see below), expressed endogenous LPA1R mRNA; LPA1R stably transfected HTC4 rat hepatoma cells were used as positive control (21). For this purpose, cells were cultured on 10-cm diameter Petri dishes to 80% confluency, and then total RNA was extracted using the Qiagen RNeasy Mini kit. Total RNA (2.5 µg) was reverse-transcribed using random hexamers, and the cDNA was amplified using specific primers designed from highly conserved regions of the LPA1R gene across species. The sequences of LPA1R primers are as follows: forward, 5'-ATC TWG CCA CAG AAT GGA ACA CAG T (where W = A or T), reverse, 5'-CAG ATR CAG TTC CAG CCC ACA CTG GG (where R = G or A). The reaction mixture was heated at 94 °C for 40 s, annealed at 58 °C for 45 s, and extended at 68 °C for 1 min 20 s for 35 repetitive cycles. Controls were carried out by omitting the reverse transcriptase in total RNA samples before the amplification reaction. Resulting PCR products were submitted to gel electrophoresis and detected as described above. The expected amplified cDNA fragment, corresponding to a 450 base-pair product, was observed in both COS-1 and LPA1R/HTC4 cells (not shown).

Immunocytochemical and Confocal Microscopic Analysis of eNOS Trafficking—pCMVEC and COS-1 cells grown on coverslips (VWR Int.) to quasi-confluency were serum-starved overnight in Dulbecco's modified Eagle's medium prior to beginning the experiments. Cells were incubated for various periods of time (0, 15, 30, and 60 min) with sLPA (10 µM). Control experiments, performed by Western blotting on total cell lysates, showed that the eNOS expression level in pCMVEC remained unchanged over the time frame of the experiments (see Fig. 3). After the incubation, cells were washed twice with cold PBS and then fixed with PBS plus 4% paraformaldehyde for 20 min at room temperature. For eNOS immunofluorescence, pCMVECs were additionally blocked and permeabilized with PBS containing 5% goat serum, 5% fetal calf serum, and Triton X-100 (0.1%) for 10 min at room temperature. pCMVECs were then incubated overnight at 4 °C with a rabbit polyclonal anti-eNOS antibody (1:100) diluted in PBS, 5% goat serum, and 5% fetal calf serum followed by a 1-h incubation at room temperature with a goat anti-rabbit Alexa Fluor 594-conjugated IgG (1:400). Nuclear staining was achieved with 4',6-diamidino-2-phenylindole (300 nM) (Molecular Probes) added 15 min prior to LPA stimulation. pCMVEC and COS-1 cells were examined by a Multi Probe 2001 confocal argon laser scanning system (Amersham Biosciences). Scant fluorescence labeling was observed in the absence of primary antibody (not shown). All acquired images were contrasted and brightened in equal modes.

Electron and Cryomicroscopic Immunohistochemistry of eNOS and sGC—Both experimental procedures were based on previously reported methods with slight modifications (16, 22). Briefly, rat livers were perfused with 30 ml of isotonic saline through the portal vein followed by a series of perfusion/fixation solutions (22). Rat livers were removed and immersed in 30% sucrose/0.1 M phosphate buffer, pH 7.4, overnight at 4 °C. The tissues were then frozen in liquid nitrogen, thawed, and cut into sections (50 µm). Sections were permeabilized further by immersing samples in 0.1 M phosphate buffer, pH 7.4, containing 0.1% Triton X-100 for 10 min at 4 °C. Tissue sections were then incubated with rabbit polyclonal anti-eNOS (1:10) followed by incubation with a goat anti-rabbit gold (1 nm)-conjugated IgG (1:100).

Post-embedding immunolabeling of sGC was performed on cryomicroscopic sections as described (15, 16). Sections were incubated with rabbit polyclonal anti-bovine pulmonary beta1-subunit sGC antibody (1:10) followed by incubation with a goat anti-rabbit gold (10 nm)-conjugated IgG (1:20) (Sigma). Sections were contrasted, embedded, and viewed as described (16). Negative controls were carried out by omitting primary antibodies.

Western Blot Analysis of eNOS and sGC in Rat Hepatic Subfractions—SDS-PAGE and Western blot were performed as described previously with minor modifications (16). Briefly, equal amounts (50 µg) of plasma membrane, cytosolic, whole nuclei, and nuclear envelope protein fractionated from harvested hepatocytes were separated by 7.5% (for eNOS) or 9% (for sGC) reducing SDS-PAGE and electroblotted onto PVDF membranes. Membranes were then blocked and subsequently incubated overnight at 4 °C with a monoclonal anti-eNOS (1:500), anti-iNOS (1:250), or anti-nNOS (1:2000) or with an anti-soluble guanylate cyclase antiserum (1:500). Membranes were incubated thereafter with horseradish peroxidase-conjugated secondary antibodies and developed as reported (16).

For Western blot analysis of eNOS phosphorylation (and resultant activation), isolated nuclei (200 µg of protein) from rat liver were treated or not with sLPA (10 µmol/liter) for 0, 5, and 15 min in incubation buffer consisting of Hepes (25 mM), pH 7.2, KCl (125 mM), K2HPO4 (2 mM), MgCl2 (4 mM), and CaCl2 (400 nM). Reaction was terminated by freezing in liquid N2. Protein samples were dissolved using the NUPAGE system (Novex/Invitrogen) supplemented with dithiothreitol (50 mM), resolved by SDS-PAGE on a 7% gel, transferred onto PVDF membranes, and then probed with anti-phospho-eNOS antibody (diluted 1:500). Aliquots of individual samples were run in parallel and membranes probed instead with anti-eNOS monoclonal antibody (dilution 1:1000) for protein normalization. Autoradiograms were scanned and analyzed by densitometry (ImagePro 4+ software).

Measurement of Nitrite Production in Isolated Rat Liver Nuclei—Isolated rat liver nuclei (400 µg of protein) were placed in an incubation buffer of the following composition: Tris-HCl (10 mM), pH 7.5, KCl (10 mM), MgCl2 (3 mM), CaCl2 (100 nM). L-Arginine (100 µM), NADPH (1 mM), BH4 (15 µM), calmodulin (1 µM), FAD (1 µM), and nitrate reductase (80 units/liter) were added and bubbled with O2 (reaction medium volume of 500 µl) in sealed containers. Nuclei were incubated for 15 min at 37 °C with vehicle (buffer alone) or with serial concentrations of sLPA (0.1, 1, and 10 µM). Similar experiments were conducted with sLPA (10 µM) in the presence or absence of L-NAME or peptide THG1603 preincubated for 10 min. Nitrite production (reduced back to NO with acetic acid and potassium iodide) was then measured by chemiluminescence (Sievers NO analyzer). In addition, nuclear eNOS activity was determined by the conversion of L-[3H]arginine to L-[3H]citrulline using a NOS assay kit (Calbiochem) as per the manufacturer's instructions. For this purpose, isolated nuclei (500 µg) were incubated with sLPA in the presence or absence of the above indicated inhibitors at 37 °C for 15 min. Protein concentration was determined by Bradford protein assay using bovine serum albumin as standard. Nitrite production and L-[3H]citrulline counts were normalized according to protein concentration.

Measurement of cGMP Production in Isolated Rat liver Nuclei by Radioimmunoassay—Isolated rat liver nuclei (100 µg of protein) were placed in the buffer as indicated above for nitrite production assays (reaction medium volume of 50 µl). Nuclei were preincubated for 5 min at 37 °C with 40 µl of the spike buffer containing 3-isobutyl-1-methylxanthine (1.25 mM), GTP (2.5 mM), creatinine phosphate (37.5 mM), and creatine phosphokinase (462.5 units/ml) and stimulated with SNP (50 µM) with or without the NO-sensitive guanylate cyclase inhibitor ODQ (0.3 µM). cGMP levels in the supernatant were measured by a commercial radioimmunoassay kit (Amersham Biosciences) and normalized for protein concentration as above.

Measurement of Calcium Signals in Isolated Nuclei—Nuclear Ca2+ signals were measured by the fura-2-AM technique essentially as described (16). Briefly, isolated rat liver nuclei loaded with fura-2-AM were stimulated with graded concentrations of SNP (1, 10, or 50 µM). In another set of experiments, nuclei were treated with SNP (1 µM) in the presence or absence of the calcium chelators EGTA (100 µM) and BAPTA-AM (100 µM), putative receptor-operated Ca2+ channel blocker SK&F96365 (10 µM), ODQ (0.3 µM), or cGMP-dependent protein kinase inhibitor KT5823 (1 µM) for 15 min at 37 °C. The intranuclear calcium concentration was measured with a spectrofluorometer (model LS50, PerkinElmer Life Sciences) and the fluorescent signal appropriately calibrated (16). In another set of experiments, confocal microscopy was used to monitor spatiotemporal movements of nuclear Ca2+ induced by SNP within a single nucleus using the calcium indicator dye fluo-4-AM as described (16, 25). Intensity of fluorescence of the calcium-fluo-4 complex was converted into absolute calcium concentration as reported (26). Stimulated nuclei were recorded at a rate of 330 ms/scan (3 scans/s) for a total of 200–250 frames. Each frame consisted of 32 lines/scan (512 pixels) and a pixel size of 0.17 µm. Individual image was obtained from a single optical midsection in the z axis (0.5 µm) with a step size of 0.5 µm. Image acquisition settings were identical for all experiments.

Western Blot of MAPKs—Rat liver derived-nuclei (50 µg protein) were treated or not with SNP (1 µM) for 0–40 min. In concomitant experiments, nuclear suspensions were pretreated with or without SNP (1 µM) in the presence or absence of ODQ (0.3 µM), KT5823 (1 µM), SK&F96365 (10 µM), EGTA (100 µM), or the MEK1 inhibitor PD98059 (1 µM) for 15 min at 37 °C; inhibitors were applied 10 min prior to SNP challenge. Proteins were resolved by SDS-PAGE on a 9% gel, transferred onto PVDF membranes, and then probed with Erk1/2 and phospho-Erk(1/2) antibodies (diluted 1:1000), as described (17). Autoradiograms were scanned and analyzed by densitometry (ImagePro 4+ software).

Gene Transcription Assays in Cell-free Nuclear System—Nuclear RNA (nRNA) expression was assessed as described previously (16) with slight modifications. Briefly, isolated rat liver nuclei were incubated at 37 °C for 60 min with either vehicle (buffer) or SNP (1 µM) in the presence or absence of ODQ (0.3 µM), KT5823 (1 µM), EGTA (100 µM), and PD98059 (1 µM) or F-{kappa}B inhibitor PDTC (100 µM). Total RNA from purified nuclei was isolated using TRIzol reagent according to the manufacturer's instructions. 1 µg of nRNA was transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) and cDNA (100 ng) amplified following a 35-cycle protocol (94 °C for 30 s, 57.5 °C for 45 s, and 68 °C for 1 min) using TaqDNA polymerase (Fermentas) and specific primers for rat iNOS, mPGES-1, and beta-actin (16). PCR products were separated and quantified as described above.

Electrophoretic Mobility Shift Assay and Binding of NF-{kappa}B to DNA Consensus Sequence—Electrophoretic mobility shift assay conditions were essentially as described (22). Rat liver isolated nuclei were stimulated with SNP (1 µM) for 1 h. Nuclei were then lysed in high salt buffer and centrifuged for 30 min at 21,000 x g. Nuclear extracts were dialyzed against buffer containing 20 mM Hepes, 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.5 dithiothreitol, pH 7.8. Binding reactions were conducted in a mixture containing 5 mM Hepes, 80 mM KCl, 8% glycerol, 2 mM dithiothreitol, pH 7.8, 20 µg of nuclear extract, 2.5 µg of double-stranded poly(dI-dC), and 30 pmol of double-stranded 32P-labeled oligonucleotide probe of the NF-{kappa}B recognition consensus sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3'). Binding competition was assayed by incubating a 20–50-fold molar excess of nonradioactive double-stranded oligonucleotides either with wild type or mutated (5'-AGTTGAGCTCACTTTCCCAGGC-3'). After incubation (20 min at 30 °C), the complexes were resolved by PAGE on 4% gel in 0.5x Tris borate-EDTA buffer. Quantification was done by PhosphorImager (GE Healthcare).

Statistical Analysis—Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test or the Tukey-Kramer method as appropriate. Statistical significance was set at p < 0.05. Data are presented as means ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Endogenous NO Contributes to LPA-mediated Immediate Early Gene Induction in Vivo and in Intact Nuclear Organelles—Stimulation of freshly isolated rat hepatocytes or pCMVEC with sLPA (10 µM) resulted in LPA1R-mediated and NO-dependent up-regulation of the immediate early genes iNOS and mPGES-1, as these effects were negated by LPA1R peptide antagonist THG1603 and by NOS inhibitor L-NAME as assessed by RT-PCR (Fig. 1, A and B). Because LPA is both an extra- and intracellular mediator, we repeated these experiments on highly purified rat hepatic nuclei to assess whether these organelles are capable on their own of inducing iNOS gene expression upon activation of nuclear eNOS and ensuing NO generation. We also used hepatic nuclei from mice lacking eNOS to confirm this hypothesis; purity of nuclei can be appreciated by high resolution scanning electron microscopy (Fig. 1C) as documented previously with transmission electron microscopy and cell fraction immunological and biochemical markers (16). Again, exposure of the nuclei of rats and wild-type mice to LPA resulted in a significant increase in iNOS expression inhibitable by L-NAME; correspondingly, this increase in iNOS expression was abrogated in eNOS–/– mice (Fig. 1D). Hence, local nuclear eNOS activity played a major role in regulating iNOS gene expression.


Figure 1
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  		FIGURE 1.
Endogenous NO contributes to LPA-mediated iNOS expression in vivo and in intact nuclear organelles. Rat liver isolated hepatocytes (A) and cultured pCMVEC (B) were treated with sLPA (10 µM) with and without the NOS inhibitor L-NAME (1 mM). Rat and porcine iNOS and beta-actin mRNAs were quantified by RT-PCR. Autoradiographic and histographic representations are presented. C, field emission scanning electron microscopy of highly purified rat liver nuclei (RLN; upper panel) and mouse liver nuclei (MLN; lower panel) at lower and higher (from left to right) magnifications, presenting the purity and integrity of these organelles. Note the remnants of cytoskeleton-like structures neighboring highly dense nuclear pores. D, rat and mouse (wild type (WT) and eNOS–/–) nuclei of isolated hepatocytes were treated with sLPA (10 µM) with and without the NOS inhibitor L-NAME (1 mM). Autoradiographic and histographic representations are presented. Values in the histogram are means ± S.E. of 4–7 experiments. *, p < 0.05, and **, p < 0.01, compared with control untreated cells; {dagger}, p < 0.05, and {dagger}{dagger}, p < 0.01 versus sLPA. E, inhibition of cell surface LPA-mediated responses using membrane-impermeable cross-linker reagent BS3 in hepatic cells. Western blots of MAPK activation (left panel) after sLPA (10 µM) exposure (0–30 min) and RT-PCR for iNOS gene induction (1 h) (right panel).

 


Figure 2
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  		FIGURE 2.
Spatiotemporal distribution of native (pCMVEC) and GFP-conjugated eNOS (COS-1, HTC4 cells) in sLPA (10 µM)-stimulated cells assessed by fluorescence confocal microscopy. Left panels, anti-eNOS antibody staining (red) in pCMVECs. Center panels, GFP-eNOS fluorescence in COS-1 cells (green). Nucleus counterstained with 4',6-diamidino-2-phenylindole (blue); note the merge of red fluorescence of eNOS with blue nuclear stain. Right panel, live cell imaging of GFP-eNOS fluorescence in a single HTC4 cell. Note the time-dependent fluorescence of eNOS specifically within the nucleus (white arrowheads). The same z-plane is presented in all four panels. Individual cell imaging was obtained from a single optical midsection in the z axis (1 µm). Results are representative of three experiments performed in duplicate. Scale bar, 50 µm.

 
Because of its lipid character, we tested the hypothesis that extracellular LPA could partially cross biological membranes and elicit direct nuclear actions involved in iNOS expression. For this purpose, we used the nonpermeable cross-linker reagent BS3 to block cell surface signaling events, including those evoked by sLPA in intact hepatocytes (Fig. 1E). The effectiveness of this treatment was shown by the inability of exogenous sLPA to induce cytosolic Erk1 kinase phosphorylation (Fig. 1E). Under the same experimental conditions, exogenous sLPA was still able, albeit to a lesser extent, to promote iNOS induction (Fig. 1E), inferring that following its diffusion to the intracellular milieu, sLPA triggers nuclear signal transduction pathways complementary to those elicited at the cellular membrane. The molecular mechanism for cell permeability of LPA remains unknown.

eNOS Immigrates to the Nucleus in Response to Cell Stimulation—The presence of basal endogenous LPA1R expression in COS-1 cells was demonstrated by semiquantitative RT-PCR in agreement with Chun et al. (24) (not shown). Earlier studies have also shown that vascular endothelial cells, including pCMVEC, express predominantly LPA1R (16). We therefore used these cells to study LPA receptor-mediated intracellular (re)localization of eNOS. In addition, to examine the possibility that NO genotropic responses arise from nuclear translocation of surface-bound eNOS and/or from resident nuclear activable eNOS, in vitro cell-based models expressing either native or chimeric green fluorescent protein (GFP)-tagged eNOS were used. Under resting unstimulated conditions (time zero), prominent eNOS fluorescent signals were evidenced on peripheral membranes in both cell types, as expected (Fig. 2, left and center panels); eNOS fluorescent signals were also detected in Golgi-like structures in COS-1 cells (Fig. 2, center panels). However, upon cell stimulation with sLPA, there was a rapid and robust intracellular redistribution of surface-bound eNOS onto (COS-1 and pCMVECs) and within the nucleus (pCMVEC), peaking at 30 min and partially (pCMVEC) or fully reverting at 60 min (COS-1 cells) (Fig. 2, left and center panels). It is noteworthy to mention that the pattern of eNOS distribution differed in the two cell types, appearing more punctuate and discrete in pCMVEC (Fig. 2, left panels), which suggests vesicular structures, and appearing mostly continuous in COS-1 cells (Fig. 2, center panels). This may reflect distinct endocytotic machinery in eNOS translocation in these cells upon stimulation with LPA. Dynamic intranuclear dissemination of eNOS was also appreciated using live cell fluorescence imaging of sLPA-treated single HTC4 cell containing GFP-tagged eNOS (Fig. 2, right panels).


Figure 3
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  		FIGURE 3.
LPA- and bradykinin-induced nuclear translocation of eNOS in pCMVEC. Serum-starved confluent pCMVEC were treated with sLPA (10 µM) or bradykinin (BK)(1 µM) for 0, 30 or 60 min, homogenized, fractionated, and nuclear fractions were prepared as described under "Materials and Methods." Nuclear extracts were subjected to SDS-PAGE and Western blotting. Top panel, representative autoradiograms. Bottom panel, densitometric analysis of eNOS relative to nuclear and plasma membrane-specific markers lamin B1 and beta1 integrin, respectively, using ImagePro 4+ software. The level of eNOS expression did not change in either of these two cell compartments in unstimulated or vehicle-treated cells over the time frame of the experiments (not shown). Values in histogram are means ± S.E. of 4–5 experiments. *, p < 0.05 compared with base line (time zero) respective to the plasma membrane fraction; {dagger}, p < 0.05 versus base line (time zero) respective to the nuclear fraction; ##, p < 0.01 versus base line (time zero) of the plasma membrane fraction. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test. WCL, whole cell lysates (prepared using conventional radioimmune precipitation buffer); PM, plasma membrane; WN, whole nuclei.

 
To consolidate and confirm the results obtained by confocal imaging depicting plasmalemmal eNOS translocation kinetics into the nucleus of pCMVEC (Fig. 2), we performed Western blot analyses of nuclear fractions issued from cells previously challenged with the eNOS activators sLPA and bradykinin (Fig. 3, BK). We validated the purity of organellar fractions from pCMVEC by immunoblotting with anti-beta1 integrin and anti-lamin B1 monoclonal antibodies for detection of plasma membrane- and nuclear-specific marker antigens, respectively (supplemental Fig. S1), as shown previously for purity of hepatocyte nuclei by electron microscopy and cell fraction-specific markers (16). Essentially, we noted a concurrent time-dependent increase and decrease of eNOS expression, respectively, in nuclear and plasma membrane subcellular fractions (Fig. 3).


Figure 4
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  		FIGURE 4.
In situ ultrastructural localization of eNOS and sGC proteins on rat liver tissue sections. Shown is eNOS immunoreactivity on plasma membrane (A) and endoplasmic reticulum (B) and within the hepatocyte nucleus (C) (arrowheads) using transmission electron microscopy. Preimbedded immunohistochemistry of eNOS was achieved by incubating specimens with rabbit polyclonal anti-eNOS antibody followed by incubation with a goat anti-rabbit gold-conjugated IgG. No labeling was observed in the absence of primary antibody (D). E, cryo-immunogold electron microscopy of sGC in hepatocyte. Post-embedding immunolabeling of sGC was performed on cryostat sections incubated with rabbit polyclonal anti-bovine pulmonary beta1-subunit sGC antibody followed by incubation with a goat anti-rabbit gold-conjugated antibody. Note the constitutive localization of sGC in cytoplasmic and nuclear spaces (arrowheads). No sGC immunoreactivity could be detected in the absence of primary antibody (not shown). N, nucleus; C, cytosol. Scale bar, 0.5 µm.

 
eNOS/sGC Exhibit Nuclear Localization in Vivo—To substantiate our in vitro observations depicting eNOS nuclear localization, immunogold labeling of endogenous eNOS was performed in vivo on rat liver sections. Immunoreactive eNOS was identified at the plasma membrane (Fig. 4A), rough endoplasmic reticulum (Fig. 4B) of hepatocytes, and notably at nuclear regions (Fig. 4B) mostly confined to transcriptionally active euchromatin structures. This nuclear-associated eNOS accounts for ~15% of overall cellular immunoreactivity and doubles in cells stimulated with LPA or bradykinin (Fig. 3). Marked eNOS plasma membrane and nuclear immunoreactivity was also detected in vascular endothelial cells adjacent to hepatocytes (not shown). Negative controls revealed negligible specific labeling when the primary antibody was omitted (Fig. 4D).

In the process of assessing whether eNOS can coordinate its activity with downstream effector sGC at the cell nucleus, we first examined the distribution of sGC in rat liver hepatocytes. A remarkably dense labeling of sGC was found in the cytoplasm as well as in the nuclear regions (Fig. 4E), consistent with previous reports (14, 15). The identification of eNOS and sGC enzymes at the nucleus in exogenously unstimulated rat liver does not of course exclude ongoing endogenous regulation of enzyme distribution.

To strengthen these findings, we performed Western blots of rat liver subcellular fractions with anti-eNOS monoclonal antibody and with anti-sGC polyclonal antibody. Both immunoreactive enzymes were revealed in plasma membrane and cytosolic fractions, respectively, as well as in whole nuclear preparations (supplemental Fig. S2); iNOS and nNOS immunoreactivity was not detected in rat liver nuclear fractions (not shown).


Figure 5
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  		FIGURE 5.
Functional activities of eNOS and sGC in purified rat liver nuclei. A, net nitrite production of nuclear suspensions stimulated with sLPA in the presence or absence of NO synthase inhibitor L-NAME or LPA1-R antagonist THG1603 was measured by chemiluminescence as described under "Materials and Methods." Inset in A depicts Western blots of phosphorylated and total eNOS in isolated nuclei challenged with sLPA (10 µM) at different time points. Mean -fold increments of phosphorylated eNOS relative to total eNOS following sLPA challenge from 0 time point (base line) versus at 5 and 15 min were 2.0 ± 0.2 and 1.8 ± 0.2, respectively (n = 3). B, conversion of L-[3H]arginine to L-[3H]citrulline. C, net cGMP generation of isolated nuclei stimulated with SNP in the absence and presence of the sGC inhibitor ODQ determined by radioimmunoassay. Data are means ± S.E. of 4–7 separate experiments. #, p < 0.05 compared with vehicle; *, p < 0.05, and **, p < 0.01 compared with sLPA (10 µM); {dagger}, p < 0.05 compared with vehicle; {dagger}{dagger}, p < 0.01 compared with SNP (50 µM). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test.

 
Both eNOS and sGC Operate at the Nucleus—NO can undergo autoxidation (i.e. reaction with O2) in aqueous solutions to produce N2O3, which can hydrolyze and in turn form nitrite. To determine whether nuclear eNOS is functionally active, we measured nitrite production and conversion of arginine to citrulline in isolated nuclei under basal and stimulated conditions (Fig. 5, A and B). sLPA increased nitrite production in a concentration-dependent manner. sLPA also caused increased eNOS phosphorylation (Fig. 5A, inset). These responses could be completely repressed by inhibiting eNOS activity with L-NAME or by antagonizing LPA1R with THG1603; these inhibitors also repressed sLPA-induced citrulline formation (Fig. 5B). Along with the presence of constitutive nuclear sGC (Fig. 4E), cGMP levels in isolated nuclei increased upon stimulation with NO donor SNP; this effect was prevented with the sGC inhibitor ODQ (Fig. 5C). Altogether, these results demonstrate that both eNOS and sGC are functional enzymes at the nuclear compartment.

NO Evokes Nuclear Signaling Cascade Leading to iNOS Expression in Cell-free Nuclei—The calcium ion is a quintessential mediator for many physiological processes including gene transcription (16). Moreover, it is now becoming obvious that nuclear and cytosolic calcium are regulated independently (25, 27), and a number of Ca2+ channels and pumps have been identified on the nuclear envelope (18). We therefore examined whether changes in nuclear Ca2+ could be elicited by SNP stimulation by means of live imaging confocal microscopy and spectrofluorometry using the fluorescent Ca2+-sensitive probes fluo-4-AM and fura-2-AM, respectively (16). Rapid line scan imaging from a z axis optical section of a single nucleus was used to monitor temporal oscillation of intranuclear calcium during SNP stimulation (Fig. 6, A and B). A rapid and reversible nuclear calcium mobilization in response to SNP was visualized (Fig. 6A). Live imaging of varied depth sections from a single nucleus clearly illustrate differential transients in Ca2+ movement within the nucleus in response to SNP (100 µM; Fig. 6B); responses to lower concentrations of SNP (1–10 µM) were of smaller magnitude and relatively delayed compared with higher concentrations (not shown). Incubation of isolated nuclei with SNP induced a concentration-dependent increase in nuclear Ca2+ levels by spectrofluorometry (Fig. 6C). Ca2+ chelating agents EGTA and BAPTA-AM and the nonspecific Ca2+ channel blocker SK&F96365 prevented the SNP-induced Ca2+ transients. In addition, sGC and PKG inhibitors ODQ and KT5823 prevented SNP-induced Ca2+ nuclear transients, suggesting a role for the sGC/PKG signaling axis in modulating nuclear calcium status (Fig. 6C).


Figure 6
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  		FIGURE 6.
Nitric oxide-induced calcium mobilization in isolated rat liver nuclei. A, intranuclear calcium mobilization induced by SNP (100 µM) in a single nucleus (one midsection) of rat hepatocyte loaded with fluo-4-AM measured by laser scanning confocal microscopy. The live calcium fluorescent intensity was automatically converted to numerical data using the GE Healthcare software. B, similar sets of experiments performed on four different confocal planes. Bottom row, three-dimensional reconstruction of the four optical z-sections (top view projection). Nuclear identity and delineation were established at the end of experiments with the nucleic acid fluorescent dye Syto-11. The pseudocolor intensity refers to calcium levels. Images are representative of 5–7 experiments. Scale bar, 2 µm. C, transient calcium signals were obtained from fura-2-AM-loaded nuclei of rat liver and detected by spectrofluorometry. Data are means ± S.E. of five separate experiments. {dagger}, p < 0.05, and {dagger}{dagger}{dagger}, p < 0.001 versus vehicle; **, p < 0.01, and ***, p < 0.001 versus SNP (50 µM). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test.

 
Activation of MAPK signaling can be induced by calcium (17), and inhibition of the MEK1/p42/p44 pathway (using PD98059) does not alter calcium transients (Fig. 6C). We therefore proceeded to determine the time-dependent effects of SNP on p42 and p44 MAPK phosphorylation (and consequential activation) in isolated nuclei (Fig. 7). Under our experimental conditions, the total immunoreactive MAPK found in the nuclear fraction relative to the homogenate fraction was estimated at 20% as determined by Western blotting (not shown). SNP elicited a phasic increase in p42 MAPK phosphorylation, which was sensitive to blockers of calcium mobilization as well as inhibitors of sGC, PKG, and MEK1/p42/p44 pathway (Fig. 7B). These results conformed to a recent report demonstrating the presence of constitutive nuclear p42 MAPK in mammalian cells (28). Moreover, stimulation of nuclei with SNP also evoked a dose-dependent increase in iNOS (and mPGES-1) RNA congruent with findings on intact rat hepatocytes (not shown). These effects were calcium-, sGC-, PKG-, and MEK1/p42/44-sensitive (Fig. 8, A and B), consistent with results from calcium mobilization and MAPK assays (see Figs. 6C and 7B). Finally, an electrophoretic mobility shift assay using oligonucleotide probes corresponding to the consensus sequence of the NF-{kappa}B DNA binding site, which is present on rat iNOS and the mPGES-1 promoter, showed that nuclear stimulation with SNP enhanced NF-{kappa}B binding to DNA (Fig. 8C). Corresponding gene induction was inhibited by the NF-{kappa}B blocker PDTC (Fig. 8B). Collectively, observations using exogenous NO (donor) are consistent with endogenous nuclear NO from eNOS, which was also found to elicit iNOS induction upon agonist (LPA) stimulation (Fig. 1D).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Studies reported to date primarily relate the role of endogenous NO in gene regulation upon extracellular stimuli, through activation of cytosolic cGMP-dependent protein kinase-mediated signal transduction followed by shuttling of transcription factors to the nucleus (29–31). The present study has uncovered an alternative noncanonical pathway for intracrine NO signaling associated with immediate early gene expression, specifically iNOS and mPGES-1, which depends upon a nuclearized eNOS/sGC network. A number of observations presented herein, obtained using various methodologies, support this novel paradigm. 1) sLPA-driven amplification of inducible genes iNOS and mPGES-1 in hepatocytes and pCMVEC (Fig. 1, A and B) and in derived isolated nuclei (Fig. 1D) was considerably dependent on the contribution of endogenous NO. Such an effect on mPGES-1 gene induction has also been demonstrated recently in human colonocytes (32). 2) Participation of nuclear-generated NO in gene transcription was clearly shown by the absence of iNOS expression in eNOS-deficient mice (Fig. 1D). 3) A nuclear pool of eNOS, incremented upon agonist stimulation, was revealed in vitro in cultured pCMVEC carrying native eNOS and in host cells over-expressing recombinant GFP-tagged eNOS (Figs. 2 and 3). 4) More importantly, nuclear compartmentalization of both eNOS and sGC enzymes was demonstrated in vivo in rat liver specimens (Fig. 4) and in subcellular nuclear fractions of the same origin (supplemental Fig. S2). Accordingly, stimulation of isolated nuclei with the potent eNOS activator sLPA and the NO donor SNP correlated with increased nitrite, citrulline, and cGMP formation (Fig. 5, A, B, and C, respectively). Moreover, the dual in situ nuclear residence of eNOS and sGC enzymes has provided an appropriate functional coordination for cGMP-dependent kinase (PKG) actions related to gene transcription (Figs. 6C, 7B, and 8B).


Figure 7
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  		FIGURE 7.
SNP-induced MAPK kinase activation in purified nuclei of rat liver. A, time course of p42/44 MAPK kinase phosphorylation (pp42/pp44) triggered by SNP (1 µM). Because the p42 signal was more robust, data analysis was focused on this immunoblot band. Top panel, autoradiograms representative of three independent experiments. Bottom panel, increase of p42 MAPK phosphorylation relative to total p42 MAPK. Total MAPK (p42 and p44) level did not change over the time frame of the experiments. *, p < 0.05, and **, p < 0.01 versus time zero. Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. B, calcium, sGC, and PKG dependence of SNP-induced MAPK p42 phosphorylation. Data are means ± S.E. of four experiments. #, p < 0.05 versus SNP (0 min); **, p < 0.01, and ***, p < 0.001 versus SNP (10 min). Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison test.

 


Figure 8
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  		FIGURE 8.
SNP-induced iNOS gene transcription in isolated hepatic nuclei. A, concentration dependence of SNP-triggered iNOS RNA expression. Top panel, autoradiograms representative of four experiments. Bottom panel, increase of iNOS RNA expression relative to constitutive beta-actin. **, p < 0.01 versus vehicle. B, calcium, sGC, PKG, MAPK, and NF-{kappa}B dependence of SNP-induced iNOS gene expression. {dagger}{dagger}{dagger}, p < 0.001 versus vehicle; **, p < 0.01 versus SNP (1 µM). Data were analyzed by one-way ANOVA followed by Dunnett's multiple comparison test (A) or Tukey's multiple comparison test (B). C, NF-{kappa}B binding to DNA in response to nuclear stimulation with SNP (1 µM). Nuclear extracts were incubated with 32P-labeled oligonucleotide NF-{kappa}B DNA binding sequence. SNP elicited a marked increase in binding (in absence of competitors). Excess cold consensus sequence (specific competitors) entirely abolished the binding, whereas the mutant sequence abolished only nonspecific binding.

 
The constitutive presence of phosphorylated eNOS in peri- and intranuclear regions, demonstrated by confocal imaging, has been found to be associated with intense NO formation at these locations in human umbilical vein endothelial cells stimulated with vascular endothelial growth factor (9). Similarly, a dominant nuclear eNOS protein localization in different human mast cell lines was shown and, upon stimulation, evoked nuclear NO formation as assessed by fluorometry (33). Although many of these reports agree with our observations, the present study goes further by providing the first clear evidence for the involvement of nuclearized eNOS and derived NO in the control of signaling cascades leading to gene expression. While this manuscript was under revision, Jagandan et al. (34) demonstrated a relatively reduced activity of the nuclear eNOS fusion protein, and ensuing low NO production, using cultured COS-7 cells transfected with cDNAs encoding various organelle-targeting constructs. Thus, the presence and roles of eNOS within the nucleus are apparently not universally applicable; or they may be cell-specific and/or dependent on cellular conditions or context.

An increasing body of evidence suggests that numerous signaling molecules traditionally considered to be cell membrane, including tyrosine kinase and serpentine receptors (e.g. for growth hormone, peptide and lipid ligands) and their attendant cytosolic enzymes (e.g. G proteins, adenylyl cyclase, phospholipases, beta-arrestins, MEK, and MAPK) along with activable transcription factors (e.g. NF-{kappa}B), may also be found in the nuclei of resting as well as stimulated cells (18, 28, 35–37). In line with this notion, the presence of constitutive LPA/LPA1R on nuclear membranes of rat liver hepatocytes has previously been documented by binding and immunoreactivity (16). Direct stimulation of rat liver isolated nuclei with LPA leads to an increase of eNOS phosphorylation and, nitrite and citrulline formation (Fig. 5, A and B) as well as iNOS expression (16). These effects could be attenuated by L-NAME. This genomic regulation of iNOS by LPA was mimicked by the NO donor SNP (Fig. 8A). NO orchestrated the induction of the iNOS gene through an ordered cascade of biochemical events. In accordance with the increasing armamentarium of serpentine receptor downstream effectors (see above) along with co-factors and proteins regulating eNOS activity (Ca2+ ions, BH4-biosynthetic enzymes, Akt, calmodulin) (16, 38, 39) that are found in the nucleus, our results indicate that in the nuclear compartment eNOS-derived NO stimulates a sGC/cGMP/PKG pathway to induce nuclear calcium transients, consistent with activation of calcium channels by cGMP/PKG in hepatocytes (40, 41). This action in turn initiates MAPK (p42/44)-sensitive NF-{kappa}B activation and finally induces iNOS gene expression (Figs. 7 and 8).

NO can also undergo oxidation or a reduction in biological systems and be converted into a number of reactive nitrogen species, which exert a plethora of activities including gene expression (42). The occurrence of these processes is achieved mainly at a high concentration of NO (>1 µM) and is mostly dependent on the chemical environment (3, 43). At low NO concentrations (≤1 µM), the most physiologically relevant target of NO is the heme-containing protein sGC (3, 43). Because most, if not all, SNP-triggered responses involved in gene induction in isolated nuclei were observed at low SNP concentrations (0.01–1 µM) (see Fig. 8), the likelihood of an indirect effect of NO via reactive nitrogen species would appear questionable. Nonetheless, we cannot totally rule out the possibility that NO adducts are actually produced in the nuclear compartment and contribute, at least in part, to the overall action of NO associated with gene expression; this would warrant further investigation.

The positive feedback mechanism of eNOS derived-NO on NO synthesis via the modulation of iNOS expression may have a number of (patho)physiological consequences, as NO per se regulates gene expression both positively and negatively at transcriptional as well as post-transcriptional levels (31). From this perspective, an instrumental and proinflammatory role of eNOS-derived NO as a modulator of iNOS expression and activity pertaining to septic shock was recently proposed based on in vitro findings and subsequently validated in vivo in a murine model of experimental sepsis (44). In our study, the stimulatory effect of NO on isolated hepatic nuclei yielded an increased expression of iNOS. One could thus envisage inflammatory and cytocidal secondary effects in response to excess NO generated by iNOS in liver tissue (45).

In summary, using primary mammalian cells, we have described the novel localization of nuclear eNOS and its functional correlates in gene expression. The regulated localization of eNOS may be essential in controlling enzyme activity and maintaining the spatiotemporal organization of intracrine NO signaling in specific organelles. From the inferences of the distinct effects of calcium fluxes in cytoplasmic and nuclear compartments on gene expression (46), one would be tempted to surmise different functional outcomes of NO signaling from these compartments.


   FOOTNOTES
 
* This study was supported in part by grants from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Québec, and the March of Dimes. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 Recipient of a Junior 1 scholarship from the Fonds de la Recherche en Santé du Québec and a researcher with the Canada Foundation for Innovation. To whom correspondence may be addressed. E-mail: Fernand.Gobeil{at}USherbrooke.ca. 2 Recipient of a Canada Research Chair. To whom correspondence may be addressed. E-mail: sylvain.chemtob{at}umontreal.ca.

3 The abbreviations used are: sGC, soluble guanylate cyclase; LPA, lysophosphatidic acid; LPA1R, LPA1 receptor; sLPA, steroyl-lysophosphatidic acid; NOS, nitric-oxide synthase; iNOS, inducible NOS; eNOS, endothelial NOS; nNOS, neuronal NOS; mPGES-1, microsomal prostaglandin E synthase-1; SNP, sodium nitroprusside; PKG, protein kinases G; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; PDTC, ammonium pyrrolidine-dithiocarbamate; L-NAME, NG-nitro-L-arginine methyl ester; (6R) BH4, (6R)-5,6,7,8-tetrahydrobiopterin dihydrochloride; pCMVEC, porcine cerebral microvascular cell; PBS, phosphate-buffered saline; GFP, green fluorescent protein; PVDF, polyvinylidene difluoride; MAPK, mitogen-activated protein kinase; Erk, extracellular signal-regulated kinase; MEK, MAPK/Erk kinase; RT, reverse transcriptase; ANOVA, analysis of variance; BS3, bis(sulfosuccinimidyl)suberate; BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. Back


   ACKNOWLEDGMENTS
 
We thank H. Fernandez, V. Bovenzi, and J. Ouellette for technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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