Regulation of eNOS Expression in Brain Endothelial Cells by Perinuclear EP3 Receptors
We reported upregulation of endothelial nitric oxide synthase (eNOS) by PGE2 in tissues and presence of perinuclear PGE2 receptors (EP). We presently studied mechanisms by which PGE2 induces eNOS expression in cerebral microvessel endothelial cells (ECs). 16,16-Dimethyl PGE2 and selective EP3 receptor agonist M&B28767 increased eNOS expression in ECs and the NO-dependent vasorelaxant responses induced by substance P on cerebral microvessels. These effects could be prevented by prostaglandin transporter blocker bromcresol green and actinomycin D. EP3 immunoreactivity was confirmed on plasma and perinuclear membrane of ECs. M&B28767 increased eNOS RNA expression in EC nuclei, and this effect was augmented by overexpression of EP3 receptors. M&B28767 also induced increased phosphorylation of Erk-1/2 and Akt, as well as changes in membrane potential revealed by the potentiometric fluorescent dye RH421, which were prevented by iberiotoxin; perinuclear KCa channels were detected, and their functionality corroborated by NS1619-induced Ca2+ signals and nuclear membrane potential changes. Moreover, pertussis toxin, Ca2+ chelator, and channel blockers EGTA, BAPTA, and SK&F96365, as well as KCa channel blocker iberiotoxin, protein-kinase inhibitors wortmannin and PD 98059, and NF-κB inhibitor pyrrolidine dithiocarbamate prevented M&B28767-induced increase in Ca2+ transients and/or eNOS expression in EC nuclei. We describe for the first time that PGE2 through its access into cell by prostaglandin transporters induces eNOS expression by activating perinuclear EP3 receptors coupled to pertussis toxin-sensitive G proteins, a process that depends on nuclear envelope KCa channels, protein kinases, and NF-κB; the roles for nuclear EP3 receptors seem different from those on plasma membrane.
Endothelial nitric oxide synthase (eNOS) plays a crucial role in the maintenance of systemic as well as cerebral hemodynamics.1 The regulation of the constitutive eNOS and neuronal NOS gene expression is relatively stringent. We recently reported that some of the biological effects of prostaglandins, specifically prostaglandin E2 (PGE2), involve the induction of eNOS in cerebral microvascular endothelium and are mediated distinctly via EP3 receptors;2 this effect of PGE2 operates in the mature and the developing subject where it may be instrumental in controlling oxygenation of neuronal tissues.3
The biological actions of prostaglandins have been attributed to their interaction with cell surface G protein-coupled receptors. However, circumstantial evidence supports the idea that prostaglandins, either formed within the cell or captured from extracellular space, may also act intracellularly. For example, the enzymes involved in the biosynthesis of prostaglandins, namely COX-1, COX-2, and PLA2, have been found to be localized at the nuclear envelope of different cell types.4,5⇓ A specific prostaglandin transporter that facilitates the influx of prostaglandins has also been identified.6,7⇓ More importantly, in addition to plasma membrane EP3 receptors, presence of functional perinuclear EP3 receptors for PGE2 has been demonstrated in a variety of cells such as cerebral microvascular ECs, Swiss 3T3 cells, and host cells HEK293.8,9⇓ Genomic effects of PGE2 through its perinuclear receptor, notably on highly inducible genes such as mitogenic transcription factor c-fos and iNOS, have been recently reported.8,9⇓ However, the regulation of constitutive genes, such as eNOS by PGE2 acting through perinuclear receptors, has never been shown. Such a conjecture can be further extended with regards to the relative contribution of plasma membrane G protein-coupled receptors versus their perinuclear counterparts in the regulation of cellular physiological events.
Because COX-2 localizes principally to the nuclear envelope4 and this enzyme plays a dominant role in prostaglandin production in brain microvasculature in early postnatal development,10 one could speculate that in the presence of functional intracellular prostaglandin receptors, PGE2-induced upregulation of eNOS gene might involve EP3 receptors localized intracellularly, specifically at the cell nucleus.2 The present study was undertaken to test the hypothesis that PGE2 receptor EP3 on nuclear envelope exhibits different functions from those on plasma membrane, such that PGE2-induced eNOS expression is largely mediated by activation of nuclear EP3 receptors in brain microvascular ECs. Our findings support this hypothesis and reveal that PGE2-induced eNOS expression is mediated by a pertussis toxin-sensitive EP3, which involves activation of Ca2+-dependent K+ channels (KCa), phosphatidylinositol 3-kinase (PI-3 kinase), MAP kinase kinase (MEK), and NF-κB pathways.
Material and Methods
M&B28767 was a gift from Rhone-Poulenc Rorer, Dagenham Essex, UK; EP3α antibody was a gift of Dr H. Shichi (Kresge Eye Institute, Detroit, Mich); and human EP3α cDNA was obtained from Merck (Pointe-Claire, Québec, Canada). The following agents were purchased: 16,16-dimethyl-PGE2, U46619, sulprostone, and 17-phenyl trinor-PGE2 (Cayman); ibuprofen, glibenclamide, soybean trypsin inhibitor, bromcresol green, bromosulfophthalein, substance P, arachidonic acid, phenylmethylsulfonyl fluoride, actinomycin D, 3-aminopropyltriethoxysilane, 17β-estradiol, Nonidet P-40, and pyrrolidine dithiocarbamate (PDTC) (Sigma); iberiotoxin, cromakalim, EGTA, pertussis toxin (PTX), fura-2-AM, BAPTA-AM, wortmannin, and PD 98059 (Calbiochem); SK&F96365, methylcarbamyl-platelet-activating factor (C-PAF) (Biomol); NS1619 (Research Biochemicals International); PGE2 RIA kits (Advanced Magnetics); anti-Big KCa polyclonal antibody (Alomone Labs); anti-FLAG monoclonal antibody (Santa Cruz Biotechnology); anti-phospho-MAP kinase (Erk1/2) polyclonal antibody (Promega); anti-MAP kinase (Erk1/2) polyclonal antibody (Upstate Biotechnology); anti-phospho-Akt (Ser473) and anti-Akt polyclonal antibodies (New England BioLabs); horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce); styryl dye RH421 (Molecular Probes); pCMV-Tag2 vector containing FLAG (Strategene); RNA guard RNase inhibitor (Amersham Pharmacia Biotech Inc); human pulmonary artery ECs and brain microvessel endothelial growth media (BioWhittaker); Dulbecco’s modified Eagle’s medium (Life Technologies); [3H]PGE2 (Amersham); and all other chemicals were purchased from Fisher Scientific.
Experiments were performed on cells from brain microvasculature from Yorkshire piglets (L’Ange Gardien, Québec, Canada) anesthetized with halothane (2%) and euthanized with intracardiac pentobarbital (120 mg/kg) in accordance with regulations of the Canadian Council of Animal Care Committee and approval of the Sainte-Justine Hospital Animal Care Committee.
Cell Culture and Transfection
Cerebral microvessels were isolated and primary EC cultures established as previously described.2,11⇓ ECs from passages 5 to 13 were used in the present study. The full-length human EP3α receptor cDNA was subcloned into the pCMV-Tag2 vector downstream to the FLAG epitope; framing and orientation were confirmed by sequencing of the construct. Positioning of the small FLAG epitope at the N-terminus does not alter ligand-induced function12 (see Results). ECs (60% confluence) grown on glass coverslips were transfected with the plasmid using the FuGENE 6 Transfection Reagent (Roche) or PolyFect (Qiagen) according to manufacturer’s instructions.
Cell Fractionation and Nuclear Isolation
Cell fractionation was achieved by the hypotonic/Nonidet P-40 lysis method. Briefly, ECs were washed 3 times with ice-cold PBS, gently scraped, and pelleted at 500g for 5 minutes. The cell pellet (for ≈50×106 cells as starting material) was resuspended in 2 mL lysis buffer (10 mmol/L Trizma/Base, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, 100 μg/mL soybean trypsin inhibitor, and 1 mmol/L phenylmethylsulfonyl fluoride [PMSF]), homogenized (100 gentle strokes) with a Dounce tissue grinder (tight pestle; Bellco Glass), and then centrifuged at 600g for 10 minutes at 4°C. The pellet was resuspended in 2 mL lysis buffer containing 0.1% (v/v) NP-40, left on ice for 5 to 10 minutes and sedimented thereafter at 600g for 10 minutes at 4°C. Nuclear pellet was washed 3 times with 10-mL lysis buffer. The morphological integrity and purity (>98%) was assessed by light microscopy after trypan blue staining and by electron microscopy (Figure 1). Essentially, isolated nuclei were fixed for 4 hours at 4°C in Tris-HCl buffer (50 mmol/L) pH 7.0 containing 3% glutaraldehyde, MgCl2 (5 mmol/L), and sucrose (250 mmol/L). Samples were then washed, fixed with osmium tetroxide (1%), dehydrated with ethyl alcohol, and embedded in Epon. Ultrathin sections were examined using a transmission electron microscope (Philips 410 LS). The nuclear fraction contained <7% of the total cellular activity of plasma membrane marker 5′ nucleotidase (Sigma assay kit).
Gene Transcription Assays and Detection of eNOS RNA
Porcine primary cerebral ECs (80% confluence) were incubated for 18 hours in Dulbecco’s modified Eagle’s medium in the absence or presence of the following agents: ibuprofen (10 μmol/L), 16,16 dimethyl PGE2 (1 μmol/L, stable PGE2 analog), prostaglandin transporter inhibitor bromcresol green (50 μmol/L),6,13⇓ and/or selective EP3 agonist M&B28767 (1 μmol/L).14 Ribonuclease protection assays were performed to detect eNOS and destrin (control) mRNA as previously described.2,15⇓
For experiments using isolated nucleus, eNOS and 18S nuclear RNA was quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) method as utilized.15 For this purpose, isolated nuclei (200 μg protein, 50 μL) were placed in buffer of the following composition: Tris-HCl (10 mmol/L; pH 7.4), KCl (135 mmol/L), MgCl2 (3 mmol/L), CaCl2 (100 nmol/L), ATP, UTP, GTP, and CTP (500 μmol/L), and RNase guard (100 U). Nuclei were incubated at 37°C for 60 minutes with saline or 17-phenyl trinor-PGE2 (1 μmol/L) or sulprostone (1 μmol/L) or M&B28767 (0.1 μmol/L) in the presence of membrane permeable and impermeable Ca2+ chelators EGTA (100 μmol/L) and BAPTA-AM (100 μmol/L), preactivated PTX (20 μg/mL, 60 minutes preincubation),9 nonspecific receptor-operated Ca2+ channel blocker SK&F96365 (10 μmol/L), selective Big Ca2+-sensitive K+ channel blocker [BKCa] iberiotoxin (0.1 μmol/L), ATP-sensitive K+ channel [KATP] blocker glibenclamide (10 μmol/L), PI-3-kinase inhibitor wortmannin (50 nmol/L), MEK inhibitor PD 98059 (10 μmol/L), or with NF-κB binding inhibitor PDTC (100 μmol/L).16 Total RNA was purified by standard guanidine isothiocyanate method and subjected to RT-PCR for eNOS and 18S RNA detection using methods and primers reported.2,15⇓
Effect of EP3 Receptor Agonist M&B28767 on NO-Dependent Vasorelaxation of Cerebral Microvessels
To assess whether modulation of M&B28767-induced eNOS expression by prostaglandin transporter inhibitor bromcresol green was reflected into function, vasorelaxation to eNOS-dependent substance P was studied17 on cerebral microvessels in situ using video-imaging technique.2,15⇓
Western Blot of MAP Kinases and Akt Activation
Isolated nuclei (100 μg protein) from ECs were treated or not with M&B28767 (0.1 μmol/L) for 0 and 15 minutes in the above-mentioned buffer. Freezing samples in liquid N2 terminated the reaction. Proteins were resolved by SDS-PAGE on 9% gel, transferred onto PVDF membranes, and then probed in immunoblots with Erk, phospho-Erk (1/2), Akt, and phospho-Akt antibodies (diluted 1:1000) according to manufacturer’s instructions. Autoradiograms were scanned and analyzed by densitometry (ImagePro 4+ software).
Detection of EP3 Receptors and KCa Channels
EP3 receptors and BKCa channels were immunodetected on ECs and derived isolated nuclei as described8,9⇓; immunogold technique for EP3 was used as reported.8 Briefly, after fixation, cells and nuclei were incubated for 1 hour with appropriate antibody diluted in PBS containing 5% goat serum and 5% fetal calf serum. Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit or Texas Red-conjugated donkey anti-rabbit IgG was used as the secondary antibody (1:200 dilution) (Santa Cruz Biotechnology). In separate experiments, omission of primary antibodies was used as negative controls. Nuclei were stained with propidium iodide (PI; 3 ng/mL) or Sytox green (100 nmol/L). Samples were then mounted on slides with fluoroguard solution (Bio-Rad) and analyzed with fluorescent microscope (Zeiss) or a Multi Probe 2001 confocal argon laser scanning system (Molecular Dynamics). In other experiments, [3H]PGE2 binding and displacement by specific EP ligands was performed as described.8
Measurement of Calcium Signals and Potentiometric Changes in Nuclei
Nuclear calcium [Ca2+] signals were measured by fura-2-acetoxymethyl ester technique as described.8,9⇓ The fluorescent potentiometric styryl dye RH421 was used to ascertain change of nuclear membrane potential and fluorescence was measured as reported.18 Nuclei were placed in HEPES/Tris (20 mmol/L, pH 7.0) containing CaCl2 (10 nmol/L), sucrose (300 mmol/L), and either 100 mmol/L K2SO4 or 1 mmol/L K2SO4. Nuclei were stimulated with NS1619 or with M&B28767 with or without iberiotoxin. Excitation and emission wavelengths were 475 nm and 645 nm, respectively.
cAMP and Inositol 1,4,5-Triphosphates (IP3) Assays
Membrane preparations (200 μg protein) were incubated in the absence or presence of M&B28767 (0.1 μmol/L),11,19⇓ and cAMP and inositol 1,4,5-triphosphates (IP3) generation was measured on nuclear and plasma membranes as described.11
Data were analyzed by 1-way ANOVA factoring for treatments with the exception of vasomotor responses, which were analyzed by 2-way ANOVA factoring for concentration and treatment groups. Comparison among means was performed by Tukey-Kramer method. Statistical significance was set at P<0.05. Data are presented as mean±SEM.
Intracellular Role of PGE2 in Regulation of eNOS Expression
Inhibition of endogenous prostaglandin formation with ibuprofen (12 to 18 hours) caused a marked reduction of eNOS mRNA in microvascular ECs (Figure 2A). Concurrent treatment with stable PGE2 analog 16,16-dimethyl PGE2 or selective EP3 agonist M&B28767 prevented the effect of ibuprofen, as documented.2 Prostaglandin transporter inhibitor bromcresol green prevented 16,16-dimethyl PGE2- and M&B28767-induced upregulation of eNOS expression; comparable effects were observed with a distinct prostaglandin transporter inhibitor, bromosulfophthalein (100 μmol/L).6 These prostaglandin transporter inhibitors per se had no effect on eNOS mRNA expression. Data suggest that PGE2 and analogs do not seem to induce eNOS mRNA expression by activating the readily accessible plasma membrane EP3 receptor but rather requires intracellular transport for this purpose.
To test whether the effect of PGE2 analogs on eNOS expression modulated by prostaglandin transporter inhibitors is reflected functionally, we exposed porcine brain slices to M&B28767 (4 to 6 hours) and measured the NO-dependent vasorelaxant responses of microvessels to substance P. Substance P-induced vasorelaxation, which as anticipated could be fully inhibited by the NOS blocker l-nitro-arginine (L-NA) (1 mmol/L), was augmented in tissues incubated with M&B28767 (Figure 2B). This enhanced vasorelaxation to substance P was prevented by concomitant treatment (4 to 6 hours) with bromcresol green or transcription inhibitor actinomycin D; treatment simply with bromcresol green (without M&B28767) did not alter vasorelaxation to substance P. In contrast, treatment with bromcresol green or actinomycin D did not interfere with vasoconstriction to M&B28767 (Figure 2C); M&B28767 does not evoke relaxation of brain parenchymal vessels.19 Hence, findings imply distinct functions for intracellular and plasma membrane EP3 receptors, such that the former induces eNOS expression and the latter mediates vasoconstriction.
Nuclear Immunolocalization of EP3 Receptors and Their Effect on eNOS Expression
We investigated if direct stimulation of nuclear EP3 receptors can elicit eNOS expression. EP3 immunoreactivity on plasma membrane and perinuclear region of ECs was confirmed (Figures 3A and 3B) and similarly shown on isolated nuclei (Figure 3C); furthermore, high-resolution electron microscopy revealed dominance of EP3 more specifically on the outer nuclear membrane (Figure 3A). Stimulation with M&B28767 elicited eNOS RNA expression in isolated nuclei (Figure 3E). Similar results were obtained using sulprostone, an EP1/EP3 receptor agonist, whereas the EP1 selective agonist 17-phenyl trinor-PGE2 was ineffective.
To further establish that the effect of M&B28767 was specifically dependent on stimulation of EP3 receptors, this effect was tested on nuclei of ECs in which we overexpressed EP3 by transfection with a cDNA of EP3α fused to FLAG (Figure 3D); nontransfected cells do not exhibit immunoreactivity to FLAG (not shown). Nuclear eNOS expression induced by M&B28767 (0.1 μmol/L) was significantly enhanced in transfected cells (Figure 3E); unrelated lipid, platelet-activating factor was ineffective. In addition, on ECs from pulmonary artery where EP3 receptor binding sites were not detectable, but which do contain the other 3 EP receptors, M&B28767 did not induce upregulation of eNOS RNA as opposed to the well known eNOS inducer 17β-estradiol (10 nmol/L),20 which caused a 65±5% increase in eNOS RNA.
Effect of EP3 Stimulation on Nuclear Ca2+ Transients and eNOS Transcription: Role for G Proteins, KCa Channels, and Kinases
PTX inhibited nuclear eNOS expression evoked by EP3 agonist M&B28767, suggesting coupling to Gi/o (Figure 4A). Despite presence of functional phospholipase C and adenylate cyclase at the nuclear membrane,21,22⇓ EP3 stimulation did not reduce cAMP generation induced by forskolin or elicit IP3 formation, whereas on plasma membrane, M&B28767 decreased net forskolin-induced cAMP formation (from 39±3 to 30±2 pmol/mg protein/min, P<0.05). Because G protein signaling can occur through Ca2+ channels23 and nuclear Ca2+ participates in controlling gene transcription,24 we investigated if changes in nuclear Ca2+ can be elicited by EP3 stimulation. Incubation of isolated nuclei from ECs with M&B28767 induced a concentration-dependent increase in nuclear Ca2+ levels (Figure 4B). Ca2+ chelators EGTA and BAPTA and nonspecific Ca2+ channel blocker SK&F96365 prevented M&B28767-induced Ca2+ transients and induction of eNOS RNA, as seen with PTX (Figures 4A and 4B).
Upregulation of eNOS gene expression by PI-3 kinase/Akt and MEK-1-dependent pathways has recently been reported in human ECs.25 These protein kinases have been localized in the nucleus where they can regulate binding of transcription factors onto promoter regions of targeted genes.24,25,26⇓⇓ Our findings show phosphorylation (and resultant activation) of Akt and Erk1/2 by direct treatment of nuclei with M&B28767 and suppression of eNOS gene with PI-3 kinase-activated protein kinase Akt27 and MEK inhibitors wortmannin and PD98059, respectively (Figure 4A); NF-κB binding inhibitor PDTC also prevented M&B28767-induced eNOS expression.
We also tested if EP3-induced Ca2+ transients can be elicited by opening ion channels23 identified at the nuclear envelope.28 We focused on K+ channels because (1) they are present in endothelium including of neurovascular tissue,15 (2) their activation can lead to Ca2+ transients in ECs, and (3) they have been detected at the nuclear membrane.28 Isolated nuclei of neurovascular ECs exhibited perinuclear immunostaining to BKCa detected by confocal microscopy imaging (Figure 5A). On nuclei, KCa channel opener NS1619 stimulated Ca2+ transients and potentiometric changes (in high K+ buffer) detected with the styryl dye RH421 (Figures 5B and 5C); these signals were inhibited by iberiotoxin. EP3 stimulation induced nuclear Ca2+ transients, fluorescence-detected potentiometric changes, and nuclear eNOS expression, which were all prevented by iberiotoxin but not by KATP blocker glibenclamide (Figures 4 and 5⇓C).
The present study was intended to determine the cellular site of action of PGE2 in regulating eNOS expression in brain microvascular ECs. In this process, we have unveiled a previously undescribed concept for the G protein-coupled receptor EP3, which exhibits on the nuclear envelope functions that are different from those on plasma membrane. We have provided direct evidence that stimulation of the perinuclear EP3 receptor can induce the expression of a constitutive gene, namely eNOS. This EP3 induction of eNOS expression is coupled to a BKCa channel, formerly never shown in nuclear envelope of ECs.
Evidence for perinuclear G protein-coupled receptors is accumulating. Receptors for PTH, endothelin, angiotensin II, opioids, and prostanoids have been found at the perinuclear membrane.8,9,29–31⇓⇓⇓⇓ Physiological relevance of perinuclear prostanoid receptors is strengthened by localization of ligand-generating enzymes, namely cPLA2, COX-1, and COX-2, in the vicinity of receptor sites at the nuclear envelope.4,5⇓ Furthermore, functionality of perinuclear G protein-coupled receptors has only been described for PGE2 receptors including EP3, which was reported to evoke immediately early gene transcription.8,9⇓ The present findings extend the scope of nuclear EP3 actions to include the regulation of constitutive genes, namely of eNOS.
A major feature of this study is the suggestion for different roles for EP3 receptors on nuclear envelope and plasma membrane. This inference is supported by a number of observations. (1) The presence of EP3 receptors on plasma and nuclear membranes of brain ECs has previously been documented by binding and immunoreactivity9; these observations have been substantiated (Figure 3A). (2) Effects of ibuprofen on eNOS expression in ECs imply a role for endogenous prostaglandins; accordingly, an inhibitable net synthesis of PGE2 (1.8±0.4 pg/mg protein/min) by nuclear membranes was observed during incubation with arachidonic acid (1 μmol/L). Inhibition of endogenous prostaglandins with ibuprofen allowed to investigate the effects of exogenous PGE2 analogs on eNOS expression, which were reversed by PGE2 and selective EP3 agonist M&B28767.14 But prostaglandin transporter inhibitor bromcresol green (and bromosulfophthalein) prevented the PGE2- and M&B28767-induced increase of eNOS expression and associated NO-dependent relaxation to substance P (Figure 2). The prostaglandin transporter is present on ECs.32 The transporter inhibitors utilized block uptake of prostaglandins into cells but per se do not interfere with prostanoid receptor-mediated events7,13⇓ or with eNOS expression (Figures 2A through 2C). (3) Vasoconstrictor effects evoked by direct stimulation of EP3 receptors were unaltered by prostaglandin transporter inhibitors (Figure 2C), suggesting effects on plasma membrane receptors.11 (4) EP3 receptors on nuclear envelope and plasma membrane are coupled to different signals: adenylate cyclase in the latter but not in the former. (5) More convincingly, a role for nuclear EP3 receptors in inducing eNOS expression was obtained by direct stimulation of isolated nuclei from ECs with the EP3 agonist M as anticipated from the receptor occupancy theory, these effects were augmented by overexpressing the EP3 receptor (Figure 3D). Collectively, these findings suggest a major role for perinuclear EP3 receptors in the regulation of eNOS expression, which seems distinct from actions mediated by plasma membrane EP3.
The nucleus is a dynamic calcium and ion barrier28; in the unstimulated state, our nuclear preparations exhibited stable Ca2+ concentrations and potentiometry (Figure 5). Nuclear calcium plays an instrumental role in DNA repair, chromatin condensation and regulation of gene transcription.24 A number of Ca2+ channels and pumps have been identified on the nuclear envelope. IP3, IP4 and ryanodine receptors, and Ca2+-ATPase pump, are some of these nuclear Ca2+ trafficking mechanisms.33,34⇓ Our findings conform to these reports. Specifically, chelation of extranuclear and intranuclear Ca2+ or blockade of Ca2+ channels prevented EP3 stimulation-induced transcription of eNOS in isolated nuclei.
It has been suggested that ion currents regulate nuclear Ca2+ channels.28 A variety of ion channels have been found localized at the nuclear envelope mostly on the outer membrane.28 We have identified functional KCa channels on the nuclear envelope, which are coupled to EP3 receptors, regulate nuclear Ca2+ channels, and in turn affect eNOS expression (Figures 4 and 5⇑). This deduction is supported by effects of KCa channel openers on nuclear Ca2+ transients and potentiometry, and more importantly by effects of selective BKCa blockers on these parameters and eNOS expression induced by EP3 stimulation (Figures 4 and 5⇑); of relevance, changes in nuclear membrane potential have been associated with DNA replication.24 The actions, we observed for EP3 mediated by BKCa, are dependent on G protein activation but independent of cAMP and IP3 formation. EP3 receptors can couple to Gq and/or PTX-sensitive Gi/Go proteins.35 This suggests that in brain vascular ECs, induction of eNOS gene by PGE2 via EP3 receptors seems linked to subsequent activation of Go proteins; through their β/γ subunits these G proteins may directly control ion channels36 independently of cAMP or IP3.37 In addition, this nuclear EP3 receptor-evoked increase in eNOS expression was found to be mediated via PI-3 kinase/Akt and Erk-MAP kinase-dependent pathways and via NF-κB activation (Figure 4), providing an alternate explanation for similar finding on whole cells.25 In line with these observations, transcription factors of the NF-κB as well as AP-1 systems can be regulated by MAP and PI-3 kinases.25,38⇓ These along with other factors (such as AP-2, CRE, ER, GATA-1, and SP-1) may bind onto cis elements of known consensus sequences on the human eNOS promoter.39 Altogether the findings set forth new perspectives in elucidating the signaling of nuclear G protein-coupled receptors such as EP3 in controlling gene expression.
In summary, the present study identifies for the first time distinct functional roles for the nuclear envelope G protein-coupled EP3 receptor of PGE2 from those on plasma membrane. In so doing, the data provide a mechanism for the regulation of the constitutive eNOS gene on brain microvascular cells acting via nuclear EP3 receptors by PGE2, that involves formerly undescribed perinuclear KCa channels as well as PI-3 kinase, Erk-MAP kinase, and NF-κB. This concept alluding to intracrine effects of PGE2 applies to conditions which exhibit high endogenous prostaglandin synthesis via COX-2 pathways localized at the nuclear membrane,4,5⇓ such as in inflammation and in the developing subject.10,15⇓
This study was supported by grants from the Canadian Institute of Health Research, the Heart and Stroke Foundation of Québec, and the March of Dimes. I. Dumont is a recipient of a studentship from the Ministry of Indian and Northern Affairs, Canada. F. Gobeil Jr, and A.M. Marrache are recipients of fellowship and studentship awards, respectively, from the Canadian Institute of Health Research. S. Chemtob is recipient of a Canada Research Chair. We are thankful to Hendrika Fernandez for technical assistance and to Les Fermes Ménard Inc (L’Ange Gardien, Québec, Canada) for their generous supply of piglets.
Original received May 30, 2001; resubmission received December 13, 2001; revised resubmission received January 24, 2002; accepted February 6, 2002.
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- ↵Bhattacharya M, Peri K, Ribeiro-da-Silva A, Almazan G, Shichi H, Hou X, Varma DR, Chemtob S. Localization of functional prostaglandin E2 receptors EP3 and EP4 in the nuclear envelope. J Biol Chem. 1999; 274: 15719–15724.
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- ↵Neuschafer-Rube F, Oppermann M, Moller U, Boer U, Puschel GP. Agonist-induced phosphorylation by G protein–coupled receptor kinases of the EP4 receptor carboxyl-terminal domain in an EP3/EP4 prostaglandin E2 receptor hybrid. Mol Pharmacol. 1999; 56: 419–428.
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