Published online before print June 15, 2006, doi: 10.1161/01.RES.0000232323.86227.8b
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© 2006 American Heart Association, Inc.
Molecular Medicine |
Selective Activation of Nuclear Phospholipase D-1 by G Protein–Coupled Receptor Agonists in Vascular Smooth Muscle Cells
From the Département Lipoprotéines and Médiateurs Lipidiques (S.G, M.L., J.-P.S., B.P., M.R., M.B.-D.), CPTP, INSERM Unité 563, CHU Purpan, BP 3028, 31024 Toulouse Cedex 3, France; the laboratoire de Signalisation et Croissance Cellulaire (P.D.), Institut de Recherche en Immunovirologie et Cancérologie, Université de Montréal, Canada; and Département Biochimie (K.L.), Université Sciences II, Genève, Switzerland.
Correspondence to Monique Breton-Douillon, Département Lipoprotéines and Médiateurs lipidiques, CPTP, INSERM Unité 563, Bâtiment C, CHU Purpan, BP 3028, 31024 Toulouse Cedex 3, France. E-mail monique.douillon{at}toulouse.inserm.fr
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Abstract |
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Recent studies highlight the existence of an autonomous nuclear lipid metabolism related to cellular proliferation. However, the importance of nuclear phosphatidylcholine (PC) metabolism is poorly understood. Therefore, we were interested in nuclear PCs as a source of second messengers and, particularly, nuclear phospholipase D (PLD) identification in membrane-free nuclei isolated from pig aorta vascular smooth muscle cells (VSMCs). Using immunoblot experiment, in vitro PLD assay with fluorescent substrate and confocal microscopy analysis, we demonstrated that only PLD1 is expressed in VSMC nucleus, whereas PLD1 and PLD2 are present in VSMC. Inhibition of RhoA and protein kinase C
(PKC) by C3-exoenzyme and PKC pseudosubstrate inhibitor, respectively, conducted a decrease of phosphatidylethanol production. On the other hand, treatment of intact VSMCs, but not nuclei, with phosphoinositide 3-kinase (PI3K) inhibitors prevented partially nuclear PLD1 activity, indicating for the first time that PI3K may have a role in nuclear PLD regulation. In addition, lysophosphatidic acid (LPA) or angiotensin II treatment of VSMCs resulted in an increase of intranuclear PLD activity, whereas platelet-derived growth factor and epidermal growth factor have no significant effect. Moreover, pertussis toxin induced a decrease of LPA-stimulated nuclear PLD1 activity, suggesting that heterotrimeric Gi/G0 protein involvement in intranuclear PLD1 regulation. We also show that LPA-induced nuclear PLD1 activation implied PI3K/PKC pathway activation and PKC nuclear translocation as well as nuclear RhoA activation. Thus, the characterization of an endogenous PLD1 that could regulate PC metabolism inside VSMC nucleus provides a new role for this enzyme in control of vascular fibroproliferative disorders.
Key Words: PLD1 • nucleus • smooth muscle cells
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Introduction |
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Atherosclerotic plaques typically consist of a lipid-rich core covered by a fibrous cap that arises from the migration and the proliferation of vascular smooth muscle cells (VSMCs) and from matrix deposition. The balance between VSMCs and inflammatory cells has recently been emphasized as a strong determinant of plaque rupture. In stable plaques, a thick cap consisting of VSMCs and extracellular matrix covers the entire lipid core. These lesions are clinically silent, and they rarely rupture. By contrast, fibrous cap in unstable plaques is thin, especially at the shoulder lesion, and contains few VSMCs but numerous inflammatory cells. These plaques are prone to rupture, leading to thrombosis followed by either occlusion or by episodic plaque expansion. Moreover, the treatment of occlusive atherosclerotic lesions by percutaneous balloon angioplasty, transplant vasculopathy, and vein bypass graft failure leads to restenosis involving VSMC proliferation as the primary pathophysiological mechanism. Thus, the understanding of signaling pathways leading to VSMC migration and proliferation is important to prevent these pathologies.1,2
It is now becoming clear that the activation of phospholipase D (PLD) is a major component of signal-transduction cascades and that many of the external agents that promote proliferation and migration of VSMCs activate PLD.3,4 In mammalian cells, PLD catalyzes the hydrolysis of the principal membrane lipid phosphatidylcholine (PC), producing phosphatidic acid (PA), and choline. PA can be metabolized to diacylglycerol (DAG) by PA phosphohydrolase or to lysophosphatidic acid (LPA) by phospholipase A2. Although the physiological role of PLDs remains unclear, multiple functions have been proposed based on their involvement in signaling pathway and on the roles of PA and its derivatives DAG or LPA in a wide range of cellular functions, including vesicle trafficking, inflammatory and immune response, cellular proliferation, and apoptosis.5,6 To date, 2 PLD genes, PLD1 and PLD2, have been cloned and characterized,7 and 2 splice variants of each isoform have been identified.8 PLD1 has a low basal activity and is upregulated by small GTP-binding proteins (Rho, Ral, and ARF), protein kinase C (PKC) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in vitro. In contrast, PLD2 has a high basal activity, requires PtdIns(4,5)P2, and is upregulated by ARF and PKC.6,9
Over the past 10 years, evidence has accumulated highlighting that an internal nuclear lipid metabolism, especially the inositol lipid metabolism, is regulated independently from that of the plasma membrane.10 Thus, we identified a phosphoinositide 3-kinase 
(PI3K) and the two 3-phosphoinositide phosphatases SHIP-2 and PTEN in VSMC membrane-free nuclei, suggesting an intranuclear PtdIns(3,4,5)P3 cycle in VSMCs.11,12 However, the importance of other nuclear lipids, in particular PC, is poorly understood. PC has been found associated with the nuclear envelope, the chromatin, and nuclear matrix.13,14 Moreover, studies from several laboratories demonstrated the presence of a PLD activity associated with nuclei. G protein–dependent and oleate-dependent PLD activities have been identified in intact nuclei from MDCK cells,15 IIC9 fibroblasts,16 rat liver and hepatoma cells,17,18 and LA-N-1 neuroblastoma cells.19,20 The nuclear RhoA- and ARF-activated PLD was suggested to reside in the nuclear envelope.16,18 Furthermore, immunohistochemical analyses indicated that PLD1 localizes to nuclear membranes in pituitary GH3 cells21 and that an intranuclear PLD2 is expressed in human renal cancer cells.22 In addition, a 90-kDa PLD, distinct from PLD1 or PLD2, was identified in nuclei of human leukemia HL-60 cells.23 Thus, these studies reported PLD activities in the nucleus of some mammalian cells, but the isoforms responsible and their activation have yet to be established. Therefore, we investigated whether an active PLD is localized inside membrane-free nuclei isolated from pig aorta VSMC.
Our results show that VSMC membrane-depleted nuclei only contain an endogenous active PLD1 regulated by the small G protein RhoA and by PKC, whereas VSMC possess both PLD1 and PLD2. Moreover, the present study demonstrates for the first time that a nuclear PLD was regulated by PI3K and was specifically stimulated by heterotrimeric G protein–coupled receptors (GPCR) but not by receptor tyrosine kinases (RTK). Thus, these observations suggest that an intranuclear PLD1 activity might regulate specific signaling pathways involved in vascular proliferative disorders.
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Materials and Methods |
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Chemicals and Antibodies
The following chemicals were used: human recombinant epidermal growth factor (EGF), Ro-31-8220, Go 6976, and PKC
pseudosubstrate inhibitor (PS-PKC) (Calbiochem); platelet-derived growth factor (PDGF) (R&D Systems); LPA, angiotensin II, wortmannin, and pertussis toxin (PTX) (Sigma); PtdIns(4,5)P2 (Avanti Polar Lipids Inc); 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Bodipy)-PC (D-3771) (Molecular Probes); and purified Clostridium botulinum C3-exoenzyme and LY-294002 (Biomol Research Laboratories Inc). The following antibodies were used: monoclonal anti-PLD1/2 (SKB2 clone) (Upstate Biotechnology); specific polyclonal anti-PLD1 (gift from S. Bourgoin)24; specific polyclonal antibodies for RhoA (sc-179) and RhoB (sc-180), polyclonal anti-p42/p44 mitogen-activated protein kinase (MAPK), polyclonal anti-PKC, and monoclonal anti–lamin A/C (Santa Cruz Biotechnology Inc); monoclonal anti–phospho-p42/p44 MAPK and horseradish peroxidase–conjugated anti-rabbit (Promega); horseradish peroxidase–conjugated anti-mouse antibody (Cell Signaling); monoclonal anti–tubulin 
and tetramethylrhodamine isothiocyanate (TRITC)-conjugated anti-rabbit antibody (Sigma). Cell culture reagents were from Gibco. Enhanced chemiluminescence (ECL) system was from Amersham Biosciences.
Cell Culture and Isolation of VSMC Nuclei
VSMCs were prepared from 6-week-old pig (INRA, Toulouse, France) thoracic aorta using an explant technique and cultured as previously described.11 For experiments, VSMCs at 80% confluence from passages 2 to 5 were used. All animal care and procedures were in accordance with institutional guidelines.
Highly purified VSMC nuclei stripped of their nuclear envelope were obtained according to the method described previously.11 Our isolation procedure yields nuclei of high purity and stripped of their nuclear envelope as observed by electron microscopic analysis, by Western blot analysis of cytoskeleton protein (tubulin) and nuclear pore protein (nucleoporin, laminin A/C), and by determining marker enzymes for cytoplasm (lactate dehydrogenase) and plasma membrane (5'-nucleotidase).11,12 An average of 0.5x106 nuclei were obtained from 1x106 cells (1x106 cells and 1x106 nuclei contained 300 µg and 30 µg of proteins, respectively).
Gel Electrophoresis and Immunoblotting
Proteins from whole cells or purified nuclei were separated by SDS-PAGE, transferred onto nitrocellulose membrane (Schleicher & Schuell), and immunoblotted as described previously.12 Immunodetection was achieved using the relevant primary antibody, anti-PLD1/2 (1:500), anti-PLD1 (1:2000), anti-RhoA (1:500), anti-RhoB (1:500), anti-PKC (1:500), anti–tubulin (1:1000), and anti–lamin A/C (1:250) overnight at 4°C or anti–extracellular signal-regulated kinase (anti-ERK) (1:1000), and anti-phospho-ERK (1:10000) 1 hour at room temperature. Horseradish peroxidase–conjugated secondary antibodies (anti-mouse, 1:5000; anti-rabbit, 1:10000) were incubated for 1 hour at room temperature, and immunoreactive proteins were visualized with ECL reagents according to the instructions of the manufacturer.
Immunochemistry
Growing VSMCs were cultured at low confluence on glass coverslips, fixed, and permeabilized as described.12 After 3 washes in PBS, cells were blocked with 1% BSA for 30 minutes, followed by incubation with the primary antibody anti-PLD1 (1/1000) in 0.05% saponin and 0.1% BSA, overnight at 4°C. After washing, coverslips were incubated with TRITC-conjugated anti-rabbit (1:200) for 1 hour at room temperature, mounted in Mowiol, and examined by confocal scanning laser immunofluorescence microscopy using a LSM510 ZEISS microscope.
PLD Activity Assay
PLD activity was assessed by measuring accumulation of the unambiguous PLD activity marker phosphatidylethanol (PEt) that is generated in the presence of ethanol (EtOH) by transphosphatidylation reaction. Purified nuclei were incubated 1 hour at 37°C in PBS buffer with 1.17 µmol/L Bodipy-PC as a PLD substrate, 0.1% DMSO with or without 1% EtOH to measure PEt production. All assays were conducted in a final volume of 1 mL. Total lipids were immediately extracted with 1 mL of butanol-1 and separation of Bodipy-PC–derived products was realized according to procedure described by Kemken et al.25
For experiments in the presence of PLD regulators, nuclei were preincubated with oleic acid (from 25 to 200 µmol/L) or PtdIns(4,5)P2 (2.5 µmol/L) for 15 minutes at 37°C before adding EtOH. For experiments in which C3-exoenzyme was used, nuclei were treated with the indicated concentrations of C3-exoenzyme for 15 minutes at 37°C in a buffer consisting of 100 mmol/L Tris-HCl, 5 mmol/L MgCl2, 10 mmol/L thymidine, 1 mmol/L dithiothreitol (DTT), 10 µmol/L nicotinamide-adenine dinucleotide (NAD). Experiments with PKC inhibitors, Ro-31-8220, Go 6976, and PS-PKC, or with PI3K inhibitors, LY-294002 and wortmannin, were conducted as detailed in Figures 4 and 5
, respectively. For PLD activity following cell activation with agonists, VSMCs were previously incubated in fresh serum-depleted medium for 48 hours, then stimulated with LPA (10 µmol/L), angiotensin II (100 nmol/L), PDGF (10 µmol/L), or EGF (10 µmol/L) for 5 minutes to 120 minutes, at 37°C.
Statistical Analysis
Data are expressed as mean±SD. Differences were analyzed by Student unpaired t test. Western blots shown are representative of 3 or more independent experiments. PLD activity is expressed as percentage of control. Difference were considered significant at P<0.05.
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Results |
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VSMC Membrane-Free Nuclei Contain PLD Activity
Recent studies have reported that apart from the lipids present in the nuclear envelope, the nucleus also contains lipids located further inside, and evidence is being accumulated on the importance of internal nuclear lipid metabolism in cellular signaling. Therefore, we first investigated whether VSMC membrane-free nuclei contained PLD activity able to hydrolyze PC to produce second messengers.
PLD activity was assessed by measuring its transphosphatidylation product, PEt. Thus, VSMC nuclei were incubated with the fluorescent substrate Bodipy-PC in absence (Figure 1, top) or presence (Figure 1, bottom) of EtOH, and the products were then analyzed by high-performance liquid chromatography (HPLC). As shown in Figure 1, the presence of EtOH induced the formation of PEt and a concomitant decrease of PA production, indicating a nuclear PLD activity. Moreover, both HPLC profiles revealed 2 additional peaks corresponding to diglyceride (DG), and LysoPC suggesting that various enzymes are able to degrade PC inside VSMC nuclei. Indeed, DG production may be the consequence of PC–phospholipase C (PC-PLC) activity and/or the consequence of the sequential action of PLD and phosphatidate phosphohydrolase, whereas lysophosphatidylcholine (LPC) could result from PLA2 activity. These data indicate an active intranuclear PC catabolism and reveal the presence of a PLD activity inside VSMC nuclei.
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PLD1, but Not PLD2, Is Expressed in VSMC Nuclei
To determine which type of PLD was expressed in VSMC nuclei, we performed immunoblot experiments using an antibody that recognizes both PLD1 and PLD2 (Figure 2A). We loaded the same amount of proteins from cell homogenates and from membrane-free nuclei. In total cell homogenate, we detected 116- and 96-kDa proteins corresponding to the molecular mass reported for PLD1 and PLD2, respectively. By contrast, only a single band at 116 kDa was observed in nuclear fraction, suggesting that only PLD1 was expressed in nuclear compartment. To confirm the nuclear localization of PLD1, growing VSMCs were stained with specific anti-PLD1 antibody21,24 and analyzed by confocal scanning laser microscopy (Figure 2B). In agreement with immunoblot studies, PLD1 was found both in nuclei and cytosol. No red labeling was observed when the primary antibody was omitted (data not shown).
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Additional experiments were undertaken to confirm the presence of PLD1 inside VSMC nuclei. In fact, PLD1 and PLD2 are activated by PtdIns(4,5)P2, but PLD1 is inhibited, whereas PLD2 is activated by oleic acid in vitro.6,9 Thus, purified VSMC nuclei were incubated with Bodipy-PC, EtOH, and either PtdIns(4,5)P2 (2.5 µmol/L) (Figure 2C) or increasing concentrations of oleic acid (Figure 2D). Our results show that PtdIns(4,5)P2 treatment increased PEt production (+350%, P<0.001, n=3), whereas oleic acid treatment decreased intranuclear PLD activity in a dose-dependent manner: inhibition was 40% with 50 µmol/L oleic acid and nearly 100% with 200 µmol/L oleic acid. Taken together, these results indicate that both isoforms are present in VSMCs, but only PLD1 is expressed and active in VSMC nucleus.
RhoA and PKC Regulate Intranuclear PLD1 Activity
There is now strong evidence that PLD1 is specifically regulated by monomeric G protein of Rho family and PKC.6,9 However, PLD1 regulation in nucleus remains largely undefined. To address this question, we first investigated the expression of RhoA and RhoB within VSMCs and membrane-depleted nuclei. We used specific polyclonal anti-RhoA (Figure 3A) or anti-RhoB (Figure 3B) antibodies and loaded the same amount of protein from cell homogenate and nuclei. Western blot analysis showed that RhoA and RhoB were present in VSMC, whereas only RhoA was expressed in nuclear compartment. To assess the role of RhoA on PLD1 activity, VSMC nuclei were pretreated with C botulinum C3-exoenzyme, an ADP-ribosyl transferase that blocks the activity of small G proteins of the Rho family.26 Figure 3C shows that PEt production dramatically decreased (83% inhibition, P<0.01, n=3) in the presence of the bacterial toxin (25 µg/mL), indicating that RhoA regulates intranuclear PLD1 activity.
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To test a possible role of PKC in nuclear PLD1 activity, nuclei were pretreated with Ro-31-8220 (0.1 to 10 µmol/L), a broad-range inhibitor of PKC. Figure 4A shows that Ro-31-8220 inhibited PEt production in a dose-dependent manner. A significant inhibition (47.5% inhibition, P<0.001, n=4) was observed in presence of 1 µmol/L Ro-31-8220. To further evaluate the involvement of classical and/or atypical PKC, nuclei were pretreated for 15 minutes with Go 6976, a selective inhibitor for classical PKC and -ß (Figure 4B), or with PS-PKC, a selective inhibitor of PKC (Figure 4C). We observed that Go 6976 did not modify PLD activity significantly, whereas PS-PKC inhibited PEt production in a dose-dependent manner. These data indicate that RhoA and PKC regulate intranuclear PLD1 activity.
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Cellular PI3K Regulates Intranuclear PLD1 Activity
It has been reported that PtdIns(3,4,5)P3 can stimulate PLD activity.27 As we have previously identified an active PI3K in VSMC nuclei,11 we tested whether specific PI3K inhibitor modified nuclear PLD activity. Thus, we preincubated membrane-free nuclei with LY-294002 (0 to 10 µmol/L), at 4°C for 15 to 60 minutes, or at 37°C for 15 or 30 minutes. In these conditions, we never observed a significant inhibition of PEt production, whereas the treatment of VSMC nuclei with 10 µmol/L LY-294002 for 15 minutes at 4°C reduced nuclear PtdIns(3,4,5)P3 production by 80%11 (data not shown). To study the possible involvement of cytosolic PI3K on nuclear PLD1 activity, VSMCs were pretreated with wortmannin (100 nmol/L) for different times, at 37°C. Figure 5A shows a significant inhibition of nuclear PLD1 after a 30 minute exposure to wortmannin. Moreover, VSMCs treatment with LY-294002 (0 to 15 µmol/L) (Figure 5B) or wortmannin (0 to 100 nmol/L) (Figure 5C) for 4 hours, inhibited PEt production in a concentration-dependent manner, with a maximal effect (
60% inhibition) at 10 µmol/L and 100 nmol/L, respectively. These results show for the first time that PI3K-dependent signaling is implicated in nuclear PLD1 activity and suggest that cytosolic PI3K is important for this activity.
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GPCR Agonists Specifically Stimulate PLD1 Activity
Several studies have shown that cytosolic PLD is activated in VSMCs via different classes of cell-surface receptor including RTK and GPCR.6 However, the specific role of RTK and GPCR agonists on nuclear PLD activity remains unclear. Therefore, we investigated whether PDGF and LPA, RTK and GPCR agonists, respectively, could activate intranuclear PLD1 (Figure 6).
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Quiescent VSMCs were stimulated by LPA (10 µmol/L) or PDGF (10 µmol/L) for different times, then nuclei were isolated, and PLD activity was estimated after HPLC analysis. As shown in Figure 6A, LPA activated nuclear PLD in a time-dependent manner and mediated a maximal increase in PEt production at 1 hour of exposure (+110%, P<0.001, n=3). In contrast, PDGF did not stimulate intranuclear PLD1 activity (Figure 6B). These results suggest that GPCR agonists, but not RTK agonists, stimulate intranuclear PLD activity. To examine this hypothesis, the same experiment was performed in presence of various agonists. Thus, intact VSMCs were stimulated for 1 hour with 10 µmol/L LPA, 100 nmol/L angiotensin II, 10 µmol/L PDGF, or 10 µmol/L EGF. As shown in Figure 6C, the GPCR agonist angiotensin II also stimulated PLD1 activity, with a lower level of PEt production as compared with LPA (+67%, P<0.05, n=3). EGF, like PDGF, had no significant effect on nuclear PLD1 activity, although these RTK agonists induced signal transduction cascade and activated p42/44 MAPK, as shown by Western blot analysis using anti–phosphorylated p42/44 MAPK antibodies (Figure 6D). Taken together, these results demonstrate for the first time a selective activation of a nuclear PLD isoform by signaling cascade downstream GPCR.
LPA-Induced Activation of Nuclear PLD1
We next investigated the mechanism by which LPA could stimulate the nuclear PLD1 activity. LPA activates 3 distinct GPCRs (LPA1, LPA2, and LPA3). LPA1 and LPA2 interact with Gi/0, Gq, and G12/13 proteins, whereas LPA3 combines with Gi/0 and Gq proteins.28 Thus, quiescent VSMCs were incubated with PTX (100 ng/mL) for 16 hours. Then, nuclei were isolated and PLD1 activity was estimated. Figure 7A shows that PTX reduced PLD1 activity by 55% below the basal activity (P<0.001, n=3), suggesting that heterotrimeric Gi/0 protein is implicated in both LPA-induced nuclear PLD1 activity and basal activity. Moreover, to assess the involvement of cytosolic PI3K in nuclear PLD1 activation, quiescent VSMCs were pretreated with LY-294002 or wortmannin for 3 hours before stimulation with LPA. We observed that the PI3K inhibitors induced a decrease in LPA-stimulated nuclear PLD1 activity by 60% below the basal activity (Figure 7B). To further establish the contribution of nuclear RhoA and PKC to PLD1 activation, nuclei isolated from LPA-stimulated VSMCs were treated with C3-exoenzyme or PS-PKC (Figure 7B). C3-exoenzyme blocked LPA-induced nuclear PLD1 activity, whereas PS-PKC reduced PLD1 activity to the same extent as the PI3K inhibitors (60% below the basal activity). Also, the inhibition of LPA-induced PEt production by the addition of both PI3K inhibitor (LY-294002 or wortmannin) and PS-PKC was not significantly different from that obtained by each inhibitor (Figure 7B), indicating that PI3K could regulate nuclear PLD1 activity via PKC.
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We next investigated whether LPA induced the nuclear translocation of PLD1, PKC, and RhoA by using Western blot analysis (Figure 7C). We did not observe significant translocation of both PLD1 and Rho-A, although the expression of these enzymes was always higher in nuclei isolated from LPA-treated VSMCs than in nuclei from control cells. In contrast, LPA induced an increase of nuclear PKC expression by 109±37%. The nuclear translocation of PKC was inhibited when VSMCs were treated with the PI3K inhibitors LY-294002 or wortmannin, whereas PLD1 and RhoA translocation were not significantly modified (Figure 7C). Thus, the LPA-promoted nuclear translocation of PKC is dependent on PI3K.
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Discussion |
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The present work clearly demonstrates the presence of an endogenous active PLD1 inside VSMC nuclei isolated from pig aorta, whereas PLD1 and PLD2 have been identified in VSMC lysate. Although previous work has reported PLD enzyme activity in cell nuclei,5 the isoform responsible was not characterized. Moreover, in most studies, nuclear PLD has been identified only indirectly through overexpression of enzyme that could lead to a distribution of the PLD significantly different from endogenous enzyme.21 In this study, intranuclear localization of PLD1 was determined by cell fractionation and also by immunofluorescence using highly sensitive antibodies to PLD1.21,24 As an additional control, we showed that nuclear PLD activity is inhibited by oleic acid in a concentration-dependent manner. This result confirmed the presence of PLD1 inside VSMC nucleus, as PLD2 is stimulated by oleic acid.29
PLD1 is activated in vitro by small GTPases of Arf and Rho families and by direct interaction with PKC.9 Arf-dependant18 and Rho-dependant16 PLD activities have been identified in nuclear envelope of liver and hepatocytes18 and IIC9 fibroblasts,16 respectively. Our data demonstrate that RhoA is expressed in membrane-free VSMC nuclei, and it could be a dominant activator of nuclear PLD1 because C3-exoenzyme inhibited more than 80% of its activity. However, we cannot exclude a role for Arf in PLD activity, although we were unable to identify Arf protein inside pig VSMC nucleus by immunoblot experiments with antibodies used (data not shown).
We also observed that direct treatment of nuclei with the selective inhibitor for classical PKC and -ß, Go 6976, did not modify PLD1 activity, whereas the selective inhibitor of PKC, PS-PKC, induced a dramatic decrease in nuclear PLD1 activity. In a range of cells and tissues, PKC 30,31 and PKCß30 regulate PLD1 activity. However, a critical role for PKC in PLD activation induced by norepinephrine in rabbit VSMC32 or by IA-adrenergic receptor agonist in Rat-1 fibroblasts has been recently demonstrated,33 but the PLD isoform responsible was not determined. All PKC isoforms, except the conventional PKC and the novel PKCµ, are expressed in human aortic VSMCs.34 Thus, in VSMC nuclei, PKC is required for nuclear PLD1 activity. However, it is possible that cytosolic PLD1 is regulated by other PKC isoform(s) in these cells.
An interesting finding in the present study was the observation that angiotensin II and LPA induced nuclear PLD1 activation, whereas PDGF and EGF had no significant effect on this activity. Our results show that nuclear PLD1 activation by LPA involves heterotrimeric Gi/G0 proteins, PKC, and RhoA. Moreover, we present the first evidence for the involvement of PI3K in nuclear PLD1 activity. Very recently, Stahelin et al35 demonstrated that PLD1 PX domain binds PdtIns(3,4,5)P3 with high specificity and affinity and that this binding could be an important factor in spatiotemporal regulation and activation of PLD1. However, a direct interaction between nuclear PdtIns(3,4,5)P3 and PLD1 seems unlikely because our data showed that the pretreatment of nuclear fraction with PI3K inhibitors had no effect on nuclear PLD1 activity. On the other hand, VSMC treatment by the PI3K inhibitors LY-294002 or wortmannin inhibited LPA-induced nuclear PLD1 activity to the same extent as PS-PKC, suggesting that PI3K could regulate nuclear PLD1 activity via PKC. Indeed, our results showed that the inhibition of LPA-induced nuclear PLD1 activity obtained with both PS-PKC and PI3K inhibitor or with each of inhibitor was very similar and that LPA-induced PKC translocation into nucleus is inhibited by LY-294002 or wortmannin. Thus, in LPA-stimulated VSMCs, the nucleocytoplasmic shuttling of PKC is dependent on cytosolic PI3K. However, in nerve growth factor (NGF)-stimulated PC-12 cells36 and in ceramide-treated hepatocytes,37 nuclear increase in PI3K activity and PdtIns(3,4,5)P3 synthesis seems necessary for the subsequent nuclear translocation of PKC. These discrepancies might be attributable to differences in cell type and/or agonist-activated signaling pathway.
Furthermore, RhoA appears to regulate only LPA-induced nuclear PLD1 activity, whereas PKC regulates both basal and LPA-induced nuclear PLD1 activity, suggesting that RhoA and PKC could activate nuclear PLD1 via different pathways. Figure 8 summarizes the different pathways for nuclear PLD1 activation by LPA based on the data presented here.
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Interestingly, our finding that nuclear PLD1 is selectively activated by GPCR, but not by RTK, is in favor of a very specific role of nuclear phospholipids mediators generated by this enzyme, suggesting an emerging role of nuclear localization in PLD1 functions and, more importantly, in pathological processes as hypertension and atherosclerosis that involve differentiation and hyperplasia of VSMCs.
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Acknowledgments |
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Sources of Funding
This work was supported by grants from the Ligue Nationale contre le Cancer, the Fondation pour la Recherche Médicale, and the Nouvelle Société française d’Athérosclérose.
Disclosures
None.
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Footnotes |
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Original received July 22, 2005; resubmission received November 23, 2005; revised resubmission received May 18, 2006; accepted June 6, 2006.
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References |
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