Characterization of a G Protein-activated Phosphoinositide 3-Kinase in Vascular Smooth Muscle Cell Nuclei*

Daniel BacquevilleDagger , Paul DélérisDagger , Christiane Mendre§, Marie-Thérèse Pieraggi, Hugues ChapDagger , Gilles Guillon§, Bertrand PerretDagger , and Monique Breton-DouillonDagger ||

From the Dagger  Institut Claude de Préval, INSERM Unité 326, Hôpital Purpan, 31059 Toulouse Cedex, the § INSERM Unité 469, CCIPE, 34094 Montpellier Cedex 5, and the  Institut Louis Bugnard, INSERM Unité 466, Hôpital Rangueil, 31043 Toulouse Cedex, France

Received for publication, December 21, 2000, and in revised form, March 20, 2001

    ABSTRACT
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma -thio)triphosphate (GTPgamma S) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), whereas guanosine 5'-(beta -thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgamma S-induced PtdIns(3,4,5)P3 synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P3 accumulation, suggesting that nuclear Gi/G0 proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha 0 proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma . These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the contol of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.

    INTRODUCTION
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INTRODUCTION
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Vascular smooth muscle cells (VSMCs)1 play a central role in the fibroproliferative response during the development of atherosclerosis and of restenosis after angioplasty (1, 2). Recently, we have demonstrated that phosphoinositide 3-kinase (PI3K) was essential for the progression of VSMCs throughout the G1 phase of the cell cycle (3), implying that a better understanding of the PI3K signaling pathway might be of pathophysiological relevance. PI3K phosphorylates the D3 position of the inositol ring in phosphoinositides (PI) to generate the putative second messengers PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (4). In addition to proliferation, the PI3K products have also been involved in cell transformation, apoptosis, vesicle trafficking, and cytoskeleton organization (5, 6).

Three distinct classes of PI3Ks have now been identified on the basis of their in vitro substrate specificity, structure, and mode of regulation (7, 8). The most studied are class I PI3Ks, which in vitro phosphorylate PtdIns, PtdIns(4)P, and PtdIns(4,5)P2, and display a preference for PtdIns(4,5)P2 in vivo. Class I PI3Ks form a heterodimeric complex and are subdivided according to the adaptor protein associated with the catalytic subunit. Class IA PI3Ks consist of a 110-kDa catalytic subunit (p110 alpha , beta , delta ) (9) and a 85-kDa adaptor protein (p85 alpha , beta ) (10) containing Src homology 2 (SH2) domains that link them to tyrosine kinase signaling. In contrast, class IB PI3K or PI3Kgamma defines a G protein-coupled receptor-regulated PI3K (11). It is made of a p110gamma catalytic subunit and a p101 regulatory subunit unrelated to p85 (12). The p110gamma can be activated in vitro by both the alpha  and beta gamma subunits of heterotrimeric G proteins (11-13). This stimulation is considerably enhanced by the p101 adaptor (12).

Moreover, there is now considerable evidence that a nuclear PI cycle, apart from that occurring in the plasma membrane, is involved in the regulation of nuclear functions (14). Indeed, it has been demonstrated that nuclei contain almost all the enzymes involved in the classical PI cycle, including kinases required for the synthesis of PtdIns(4,5)P2, phospholipases C, and diacylglycerol kinase (15, 16). Furthermore, specific changes in the nuclear levels of PI have been implicated in both cell growth and differentiation (17-19). To date, information concerning the role and the regulation of nuclear PI3K are still very limited. Immunocytochemical and biochemical analyses demonstrate the presence of the p85alpha regulatory subunit in the nuclei of rat and human cells (20-22) and the growth factor-dependent nuclear translocation of the p110beta catalytic subunit in osteoblast-like cells (23), suggesting that class IA PI3Ks exist in the nucleus. Recently, a study based on the immunolocalization of epitope-tagged p110gamma in HepG2 cells reported that PI3Kgamma translocates to the nucleus after serum stimulation (24). Since a nuclear G protein-regulated PI3K activity has not yet been demonstrated, we investigated whether GTP-binding proteins directly modulate 3-phosphorylated phosphoinositide (3-PI) metabolism in membrane-free nuclei isolated from pig aorta VSMCs. Our data provide the first evidence for the existence of a pertussis toxin (PTX)-sensitive PI3K inside VSMC nucleus. This enzyme, which might be related to PI3Kgamma , phosphorylates an intranuclear pool of PtdIns(4,5)P2, suggesting that nuclear PtdIns(3,4,5)P3 may regulate protein kinases involved in vascular proliferative disorders.

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Chemicals and Antibodies-- All culture reagents were obtained from Life Technologies Inc. U73122, wortmannin, and LY294002 were obtained from Biomol (Plymouth Meeting, PA). GTPgamma S, GDPbeta S, and RNase-free DNase I were from Roche Molecular Biochemicals. [gamma -32P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Monoclonal anti-p110gamma , fluorescein isothiocyanate-conjugated anti-rabbit antibodies, and the enhanced chemiluminescence (ECL) system were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Polyclonal anti-p110gamma was kindly provided by S. Roche. Specific polyclonal anti-Galpha 0 antibodies directed against the last 10 amino acids of the common carboxyl-terminal sequence of the Galpha 01 and G alpha 02 (anti-Galpha 0(C-ter) or against the amino acid 291-302 of the alpha  subunit of G01 (anti-Galpha 01) were obtained and characterized as previously described (25). Horseradish peroxidase-conjugated anti-rabbit/mouse antibodies and polyclonal anti-p85alpha antibody were, respectively, supplied from New England Biolabs Inc. (Saint Quentin, France) and Upstate Biotechnology Inc. (Lake Placid, NY). Monoclonal anti-nucleoporin p62 antibody was from BD Transduction Laboratories (San Diego, CA). Lactate dehydrogenase and 5'-nucleotidase kits, polyclonal anti-tubulin antibodies, and all other reagents were obtained from Sigma. COS-7 cells overexpressing human p110gamma or p110alpha were a generous gift of A. Yart and P. Raynal.

Cell Culture and Isolation of VSMC Nuclei-- VSMCs were prepared from 6-week-old pig thoracic aorta using the explant technique (26) and cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, as previously described (27). For all the experiments, VSMCs were used from the third to the sixth passage.

Growing VSMCs were washed twice with ice-cold calcium- and magnesium-free PBS and once with a hypotonic buffer containing 5 mM Tris-HCl, 1.5 mM KCl, 2.5 mM MgCl2, pH 7.4. All subsequent procedures were carried out at 4 °C. Medium was then switched to hypotonic buffer supplemented with 200 µM Na3VO4, 1 mM NaF, 1 mM EDTA, 1 mM EGTA, 100 µM phenylmethylsulfonylfluoride, 10 µg/ml each aprotinin, benzamidin, and leupeptin for 1 min, and VSMCs were lyzed by the addition of 1% Nonidet-P40 and 1% deoxycholic acid. Cells were allowed to swell for 1 min and were sheared by three passages through a 25-gauge needle. The cell lysate was layered over a 0.3 M/2 M sucrose discontinuous gradient and was centrifuged at 500 × g for 15 min in polypropylene tubes pretreated with a siliconizing agent (Sigmacote) in a further attempt to reduce nuclei adsorption. Then, nuclei were recovered at the interface of 0.3 M/2 M sucrose and washed once with the assay buffer containing 1 mg/ml fatty-acid free bovine serum albumin, 40 mM Hepes, pH 7.5, 2 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 50 mM NaCl, 4 mM MgCl2, 200 µM Na3VO4, 1 mM NaF, and proteases inhibitors, as above. As to the yield of the nuclear isolation, an average of 0.5 × 106 nuclei were obtained from 1 × 106 cells. 1 × 106 cells and 1 × 106 nuclei contained 300 µg and 30 µg of proteins, respectively.

Lipid Kinase Assay-- Nuclei were disrupted by sonication (10 kHz for 3 × 1 s) using an ultrasonic cell disrupter (Branson Sonifier 250) and treated for 1 h at 4 °C with 10 units/1.5 × 106 nuclei RNase-free DNase I. All assays were conducted in a final volume of 100 µl of assay buffer containing 5 × 106 nuclei, 1 µM thapsigargin (ATPase inhibitor), and 5 µM U73122 (phospholipase C inhibitor). For experiments in the presence of PI3K inhibitors and a Gi/G0 inhibitor, nuclei were preincubated with 20 nM wortmannin or 10 µM LY294002 and 5 ng/ml pertussis toxin, respectively, for 15 min on ice. When indicated, 100 µM GTPgamma S or 100 µM GDPbeta S was added for another 15 min. The assays were then started by the addition of 1 mM ATP (10 µl) containing 65 µCi of [gamma -32P]ATP, and the 32P incorporation was allowed for 15 min at 30 °C under shaking. For exogenous lipid phosphorylation, 30 µl of lipid vesicles containing 100 µM PtdIns and 200 µM phosphatidylserine were added 5 min before starting the assay. Reactions were stopped by the addition of 1360 µl of chloroform/methanol (1:1, v/v), 300 µl of 2 N HCl, and 280 µl of 200 mM EDTA. Lipids were immediately extracted after the modified procedure of Bligh and Dyer (28). Lipids were then analyzed either on oxalate-coated thin-layer chromatography plates (Silica Gel 60, Merck) developed in isopropanolol:acetic acid:H2O (65:1:34) or by HPLC on a Partisphere SAX column (Whatman International Ltd, U. K.) after deacylation, as previously described (29). The synthesis of radioactive standard PtdIns(3)P, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 was performed from specific anti-p85alpha immunoprecipitates, essentially as described in Payrastre et al. (30).

Gel Electrophoresis and Immunoblotting-- Proteins from whole cells or purified nuclei were resuspended in electrophoresis sample buffer, incubated for 1 h at room temperature with 10 units/105 cells RNase-free DNase I, boiled for 10 min, separated by SDS-PAGE using 7.5 and 10% polyacrylamide gel, transferred onto nitrocellulose membrane (Schleicher & Schuell), and immunoblotted as previously described (30). Immunodetection was achieved using the relevant antibody, anti-p85alpha (1/1000) for 1 h at room temperature and anti-tubulin (1/1000), anti-nucleoporin p62 (1/1000), monoclonal and polyclonal anti-PI3Kgamma (1/100 and 1/1500, respectively), anti-Galpha 0(C-ter) and anti-Galpha 01 (1/500) overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies (1/2500) were incubated of 1 h at room temperature, and immunoreactive proteins were visualized with ECL reagents according to the manufacturer's instructions.

Immunofluorescence-- VSMCs were plated on glass coverslips, stimulated for 3 days with 10% fetal calf serum, and fixed in 70% ethanol for 30 min at 4 °C. Cells were then permeabilized, and the DNA was denaturated for 30 min in a solution of 2 N HCl, 0.5% Triton X-100, 0.5% Tween 20 in PBS. Cells were washed extensively with PBS and were blocked for 1 h in PBS containing 0.5% bovine serum albumin (w/v) and 0.5% Tween 20. Coverslips were incubated with the anti-p85alpha antibody (1/20, overnight at 4 °C) then with fluorescein isothiocyanate-conjugated anti-rabbit antibody (1/100, 1 h), mounted in Mowiol, and examined under epifluorescent illumination using a Zeiss microscope coupled to a Micromax camera (Princeton Instruments Inc.).

Electron Microscopy-- The purified nuclei were immediately fixed in 3% glutaraldehyde in PBS, postfixed in osmium tetroxide, dehydrated in graded ethanol series, and embedded in Epon 812. Ultrathin sections were then cut (Reichert ultratome), placed on 300 mesh copper grids, counterstained with uranyl acetate and lead citrate, and examined in a Hitachi 300 transmission electron microscope.

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Purity of VSMC Nuclear Preparations-- Membrane-depleted nuclei from pig aorta VSMCs were isolated by hypotonic shock combined with detergents. The purity of nuclear preparations was evaluated by biochemical and immunochemical analyses. Lactate dehydrogenase and 5'-nucleotidase activities, recognized as markers for cytoplasm and plasma membrane were, respectively, found to be 0.29 ± 0.12% (n = 3) and 0.18 ± 0.08% (n = 3) of the activity in the total homogenate. Furthermore, Western blot analysis using anti-tubulin antibody showed the absence of immunoreactivity to the cytoskeletal proteins in the purified nuclei (Fig. 1A). In addition, the nuclear fraction was highly enriched with nucleoporin p62, a protein of nuclear pore complex (Fig. 1B). Finally, electron microscopy analysis confirmed that the isolation procedure yielded nuclei of high purity (Fig. 1C). No appreciable morphological change of the nuclei was noted during the isolation procedure, the nucleolar structure was maintained, and the lysis procedure completely removed the nuclear envelope to leave the naked laminar layer and nuclear pore remnants (Fig. 1C, right panel).


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Fig. 1.   Purity of VSMC nuclear preparations. Membrane-depleted nuclei were obtained by hypotonic shock combined with detergents, separated from lysate, and washed once time as detailed under "Experimental Procedures." A and B, detection of markers by Western blotting analyses. 15 µg of proteins from 5 × 104 cells (lane 1) and 5 × 105 purified nuclei (lane 2) were fractionated on SDS-PAGE, transferred and probed with anti-tubulin antibody (A). 3 µg of proteins from 1 × 104 cells (lane 1) and 1 × 105 purified nuclei (lane 2) were probed with anti-nucleoporin p62 antibody (B). The immunoblots shown are representative of three independent experiments. C, transmission electron micrographs of purified nuclei. Nuclei were fixed and embedded, and ultrathin sections were examined at different magnifications (left panel, ×10,000; middle panel, ×12,000; right panel, ×20,000). Note the presence of the lamina layer (L) and nuclear pore remnants (P) and the absence of the nuclear envelope. The black bar represents the width of 1 µm.

VSMC Nuclei Contain a GTP-dependent PI3K Activity-- Previous studies suggest the existence of a nuclear PI3K pathway (20-24), but the intranuclear location and regulation of a PI3K activity has not clearly been demonstrated. Therefore, we first investigated whether membrane-free nuclei were able to produce 3-PI from endogenous precursors. We assessed nuclear PI3K activity in vitro by phosphate incorporation from [gamma -32P]ATP into inositol lipids. All assays were conducted in the presence of 2 mM EGTA and 5 µM U73122, a phospholipase C (PLC) inhibitor, so that nuclear PLC activity would not interfere with PI3K activity.

As shown in Fig. 2A (upper panel), HPLC analyses revealed a peak of [32P]phosphatidic acid, suggesting a residual PLC activity, but we never detected radiolabeled 3-PI. To look for the possible presence of a G protein-regulated PI3K (11-13), we next tested whether 100 µM GTPgamma S, a nonhydrolyzable GTP analogue, could trigger the production of 3-PI in isolated nuclei (Fig. 2A, middle panel). Under this condition, we detected the synthesis of only one PI3K product, the PtdIns(3,4,5)P3, and the incorporation into [32P]PtdIns(3,4,5)P3 was 49073 ± 2000 cpm/107 nuclei (n = 3) (Fig. 3A). This peak coincided with the HPLC profile of pure [32P]PtdIns(3,4,5)P3 used as a control (Fig. 2B). In addition, the [32P]phosphatidic acid peak was always present, and we never observed the production of any other inositol lipids, especially PtdIns(3)P and PtdIns(3,4)P2. No further stimulation of PtdIns(3,4,5)P3 was observed with 200 µM GTPgamma S (data not shown). Moreover, 100 µM GDPbeta S was unable to activate PtdIns(3,4,5)P3 synthesis, as presented in Fig. 2A (lower panel). These results were confirmed by TLC showing the specific activation of only PtdIns(3,4,5)P3 synthesis by GTPgamma S in isolated nuclei (Fig. 3B, lanes 1 and 3). A weak wortmannin-insensitive production of PtdIns(3,4,5)P3 was observed in the absence of GTPgamma S (Fig. 3B, lanes 1 and 2), but in general, the PI3K activity was too small to be detectable. These data strongly suggest that a nuclear GTP-dependent PI3K phosphorylates a pre-existing intranuclear pool of PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3. We next investigated the effects of two specific PI3K inhibitors on GTPgamma S-induced nuclear PI3K activation. Both HPLC (Fig. 3A) and TLC studies (Fig. 3B) showed that accumulation of PtdIns(3,4,5)P3 was reduced by 60-80% after preincubation of nuclei with 20 nM wortmannin or 10 µM LY294002, demonstrating that the nuclear GTP-dependent PI3K is sensitive to PI3K inhibitors in a classical range of concentrations.


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Fig. 2.   GTPgamma S induces a PtdIns(3,4,5)P3 synthesis in VSMC nuclei. A, membrane-free nuclei were obtained as indicated in Fig. 1. Nuclear lipid kinase activity was assayed with 5 × 106 nuclei (N) without adding exogenous lipids. Before starting the reaction, nuclei were preincubated for 15 min in the absence (upper panel) or in the presence of 100 µM GTPgamma S (middle panel) or with 100 µM GDPbeta S (lower panel). The assays were started by the addition of [gamma -32P]ATP, and incorporation was achieved for 15 min at 30 °C with shaking. Then radioactive lipids were extracted and analyzed by HPLC after deacylation. The data shown are representative of three different experiments. B, HPLC profile of purified [32P]PtdIns(3,4,5)P3 control after deacylation. PA, phosphatidic acid; Pi, inorganic phosphate; PIP3, PtdIns(3,4,5)P3.


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Fig. 3.   Effects of PI3K inhibitors on GTPgamma S-induced nuclear PtdIns(3,4,5)P3 synthesis. Membrane-depleted nuclei were isolated, and nuclear lipid kinase activity was assayed as described in Fig. 2. Nuclei were pretreated for 15 min with 20 nM wortmannin or 10 µM LY294002 before incubation with 100 µM GTPgamma S. A, lipids were extracted and analyzed by HPLC. Quantification of [32P]PtdIns(3,4,5)P3 synthesis is expressed as cpm/107 nuclei. Data for GTPgamma S alone are the mean ± S.E. from three independent experiments. Data for PI3K inhibitors represent the mean of two independent experiments. B, lipids were extracted and analyzed by TLC. The data shown are representative of two different experiments. The position of standard radioactive 3-PI is shown as the control. PI3P, PtdIns(3)P; PI3,4P2, PtdIns(3,4)P2; PIP3, PtdIns(3,4,5)P3.

Substrate Specificity of the Nuclear GTP-dependent PI3K-- The above data suggest either that the GTP-dependent PI3K is selective for nuclear PtdIns(4,5)P2 or that PtdIns and PtdIns(4)P present in the nuclear membrane were removed during nuclei isolation. To address this question, vesicles containing PtdIns/phosphatidylserine (100 µM/200 µM, final concentrations) were added to nuclear PI3K assays, and the formation of 32P-labeled lipids derived from PtdIns was analyzed by HPLC (Fig. 4). Incubation of nuclei with exogenous PtdIns in the absence of GTPgamma S resulted in the synthesis of [32P]PtdIns(4)P, as already described (15), and also of [32P]PtdIns(3)P (36,963 ± 4105 cpm/107 nuclei, n = 3) but not of any other 3-PI (Fig. 4A, upper panel, and Fig. 4B). This result suggests that membrane-free VSMC nuclei also contain a GTP-independent PI3K phosphorylating in vitro exogenous PtdIns to PtdIns(3)P but unable to phosphorylate the intranuclear pool of PtdIns(4,5)P2. In the presence of GTPgamma S, [32P]PtdIns(3)P synthesis was increased by 40% (57114 ± 572 cpm/107 nuclei, n = 3, p < 0.01), and under that condition, we also measured PtdIns(3,4,5)P3 synthesis (Fig. 4A, lower panel). These results indicate that the nuclear GTP-dependent PI3K can phosphorylate both endogenous nuclear PtdIns(4,5)P2 and exogenous PtdIns.


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Fig. 4.   Exogenous PtdIns phosphorylation by nuclear GTP-dependent PI3K. Membrane-depleted nuclei were isolated, and nuclear lipid kinase activity was assayed in the presence of exogenous lipids. Nuclei (N) were preincubated in the absence (A, upper panel, and B) or in the presence (A, lower panel, and B) of 100 µM GTPgamma S. Vesicles of PtdIns/phosphatidylserine at the final concentrations 100 µM/200 µM were added 5 min before starting the reaction. A, representative HPLC profiles of three different experiments. B, quantification of [32P]PtdIns(3)P synthesis is expressed as cpm/107 nuclei. Data are the mean ± S.E. from three independent experiments. The asterisk indicates significant difference using the Student's t test (p < 0.01). PA, phosphatidic acid; Pi, inorganic phosphate; PI3P, PtdIns(3)P; PI4P, PtdIns(4)P; PIP3, PtdIns(3,4,5)P3.

A 117-kDa PI3Kgamma but Not p85alpha Is Expressed Inside VSMC Nuclei-- We next performed immunoblot experiments to identify the two PI3K isoforms expressed in isolated nuclei (Figs. 5 and 6). The existence of a nuclear PI3K that phosphorylates both PtdIns and PtdIns(4,5)P2 in a GTP-dependent manner suggests that it could be a class IB PI3K. Indeed, we detected the presence of the catalytic subunit of the G protein-regulated PI3K by using two different anti-PI3Kgamma antibodies (Fig. 5). The analysis with a monoclonal antibody against the amino-terminal region of human p110gamma revealed a 117-kDa protein in both the total cell homogenate (Fig. 5A, lane 1) and the nuclear fraction (Fig. 5A, lane 2), assayed here in similar amounts in terms of nuclei number. A positive control with human p110gamma overexpressed in COS-7 cells also showed a single band at 117 kDa (Fig. 5A, lane 3). Moreover, the p110gamma detection was specific, as the monoclonal antibody did not cross-react with human p110alpha overexpressed in COS-7 and as no signal was detected in VSMC nuclei when the primary antibody was omitted (Fig. 5A, lanes 4 and 5). The polyclonal anti-p110gamma antibody also confirmed the presence of a 117-kDa protein in VSMC nuclei (Fig. 5B). Unfortunately, as previously noted in HepG2 cells (24), all available antibodies were unable to detect endogenous PI3Kgamma by immunofluorescence in VSMCs.


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Fig. 5.   Expression of PI3Kgamma in VSMCs. A, proteins from 1 × 105 whole VSMCs (lane 1) and 1 × 105 purified nuclei (lane 2) were fractionated on 7.5% SDS-PAGE, transferred, and probed with monoclonal anti-p110gamma antibody. As a positive control, proteins from 1 × 105 COS-7 cells overexpressing human p110gamma were also probed (lane 3). The specificity was tested by probing proteins from 1 × 105 COS-7 cells overexpressing human p110alpha (lane 4) and proteins from 1 × 105 VSMC nuclei without the primary antibody incubation (lane 5). B, proteins from VSMC purified nuclei (1 × 105 to 5 × 105 nuclei) were immunoblotted with polyclonal anti-p110gamma antibody. The data are from a single experiment representative of at least three others.


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Fig. 6.   Expression of p85alpha in VSMCs. A, Western blotting analysis of p85alpha . 15 µg of proteins from 5 × 104 cells and 5 × 105 nuclei were fractionated on 7.5% SDS-PAGE, transferred, and immunoblotted with polyclonal anti-p85alpha antibody. The data are from a single experiment representative of at least three others. B, p85alpha subcellular localization by immunofluorescence. Cells were plated on glass coverslips, fixed, permeabilized, and incubated with polyclonal anti-p85alpha antibody. The bar represents the width of 10 µm.

Having shown that a nuclear PI3K phosphorylates exogenous PtdIns in the absence of GTPgamma S and considering that p85alpha has been reported in rat liver nuclei (20), we checked whether class IA PI3Ks would also be present in VSMC nuclei (Fig. 6). We used an anti-p85alpha antibody to detect this adaptor protein and loaded the same amount of proteins (15 µg) from cell homogenate or from purified nuclei (Fig. 6A). In contrast to previous reports, p85alpha was undetectable in VSMC nuclei when compared with total cell homogenates, although 10 times as much nuclei were assayed. The absence of nuclear p85alpha expression was further confirmed by indirect immunofluorescence microscopy. As shown in Fig. 6B, the p85alpha labeling was evident as a ring at the perinuclear region and as fluorescent dots in the cytoplasm and the plasma membrane, whereas the nuclear interior remained unstained. These results strongly suggest that the heterodimeric PI3K p85alpha /p110 is absent from our nuclear preparations but could be present in the nuclear envelope, whereas a 117-kDa PI3K gamma -like kinase is, significantly, located inside VSMC nuclei.

The Nuclear GTP-dependent PI3K Could Be Regulated by G Proteins-- We next sought to determine whether nuclear heterotrimeric Gi/G0 proteins could be responsible for the nuclear PI3K activation by using PTX. Pretreatment of isolated nuclei with 5 ng/ml PTX inhibited about 50% of the GTPgamma S-induced PI3K activity (Fig. 7A). Moreover, to identify PTX-sensitive G proteins inside VSMC nuclei, we performed immunoblots using specific antibodies directed against both Galpha 01/alpha 02 subunits (anti-Galpha 0(C-ter)) or against the alpha  subunit of G01 (anti-Galpha 01). As shown in Fig. 7B, a protein of 42-43 kDa is recognized by both antibodies in our nuclear preparations. These results suggest that PTX-sensitive G proteins are present in VSMC nuclei and could be involved in nuclear PI3K activation.


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Fig. 7.   Effects of PTX on GTPgamma S-induced nuclear PtdIns(3,4,5)P3 synthesis and expression of Galpha 0 proteins in VSMCs. Membrane-depleted nuclei were isolated, and nuclear lipid kinase activity was assayed as described in Fig. 2. A, nuclei were pretreated for 15 min with 5 ng/ml pertussis toxin before incubation with 100 µM GTPgamma S. Lipids were extracted and analyzed by HPLC. Quantification of [32P]PtdIns(3,4,5)P3 synthesis is expressed as cpm/107 nuclei. Data for GTPgamma S alone are the mean ± S.E. from three independent experiments. Data for PTX represent the mean of two independent experiments. B, Western blotting analysis of heterotrimeric Galpha 0 protein. Protein from 1 × 105 whole cells or 5 × 105 purified nuclei were fractionated on 10% SDS-PAGE, transferred, and probed with polyclonal anti-Galpha 0(C-ter) or anti-Galpha 01 antibodies. Experiments illustrated are representative of at least three distinct experiments. PIP3, PtdIns(3,4,5)P3.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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In the present study, we provide the first evidence that a GTP-dependent PI3K generates the second messenger PtdIns(3,4,5)P3 from a pre-existing nuclear pool of PtdIns(4,5)P2, directly within VSMC nucleus. Furthermore, we showed that this PI3K activity, which could be related to a PI3Kgamma , is coupled to nuclear Gi/G0 heterotrimeric G proteins.

PtdIns(3,4,5)P3 Synthesis in VSMC Nuclei-- We prepared highly purified VSMC nuclei stripped of their nuclear envelope. Our data demonstrated that the GTPgamma S-responsive PI3K and the PtdIns(4,5)P2 resist treatment with non-ionic detergents, suggesting that this enzyme and its substrate are tightly associated with non-membrane nuclear structures. In agreement with these results, PtdIns(4,5)P2 was localized in nucleolus-associated heterochromatin in rat pancreas (31), and picomole-sensitive mass assays have revealed that 35% of nuclear PtdIns(4,5)P2 (about 30 pmol/mg of protein) remained in rat liver nuclei treated with Triton X-100 (32). In addition, recent data from Boronenkov et al. (33) provide new insights about the localization of PtdIns(4,5)P2 within the nucleus. They demonstrated that PtdIns(4,5)P2 is spatially organized to "nuclear speckles" in mammalian cells. Interestingly, nuclear speckles are separated from known membrane structures and contain pre-mRNA processing factors, and their morphology is tightly linked to the status of mRNA transcription. These observations suggest that speckles might function as centers for PI-signaling pathways in nuclei. However, PI3K activity was not addressed in these studies. In this respect, our finding of a nuclear G protein-regulated PI3K is of special interest, and we can speculate that phosphorylation of PtdIns(4,5)P2 into PtdIns(3,4,5)P3 might modulate speckle functions.

The PtdIns(3,4,5)P3-dependent signaling pathways in the nucleus are still unknown. However, PtdIns(3,4,5)P3 synthesis was associated with activation of the serine/threonine kinases, protein kinase Czeta , and Akt/protein kinase B (34, 35), and the nuclear translocation of these two PtdIns(3,4,5)P3 effectors was demonstrated upon mitogenic stimulation (36, 37). More recently, Neri et al. (38) show that nuclear PtdIns(3,4,5)P3 production correlates both with p85alpha /p110 PI3K and protein kinase Czeta translocations to the nucleus of nerve growth factor-treated PC12 cells. Thus, the G protein-activated PI3K could produce PtdIns(3,4,5)P3 inside VSMC nuclei to recruit and/or activate downstream effectors. In this respect, we also observed the presence of protein kinase Czeta and active Akt/protein kinase B in VSMC nuclei (data not shown). The nuclear targets of protein kinase Czeta and Akt/protein kinase B only begin to be described. Indeed, a major component of the nucleolus, nucleolin, has been shown to be phosphorylated by protein kinase Czeta (36), and Akt/protein kinase B has been reported to promote phosphorylation of the nuclear transcription factors CREB (cAMP-response element-binding protein) (39) and FKHR1 (forkhead in rhabdomyosarcoma 1) (40). Finally, evidence has been provided that PIP3BP, a PtdIns(3,4,5)P3-binding protein, is exported out of the nucleus by the expression of constitutively activated PI3K (41).

Nuclear GTP-dependent and -independent PI3Ks-- In response to GTPgamma S, we never detected nuclear PtdIns(3)P or PtdIns(3,4)P2 synthesis from endogenous substrates, suggesting that PtdIns and PtdIns(4)P, which have been reported located in the nuclear membrane (14, 32), were totally removed during nuclei isolation. On the other hand, the GTP-dependent PI3K might be PtdIns(4,5)P2-selective. To address this question, we added exogenous PtdIns in assays. We found that GTPgamma S stimulated a PtdIns(3)P synthesis, demonstrating that the nuclear GTP-dependent PI3K uses PtdIns as a substrate in vitro. This result suggests that GTP-binding proteins activate a class IB PI3K in VSMC nucleus. Accordingly, immunoblot analysis revealed the presence of a 117-kDa catalytic subunit of G protein-regulated PI3Kgamma inside VSMC nuclei, suggesting that the nuclear GTP-dependent PI3K could be PI3Kgamma or a PI3Kgamma -like kinase. In this respect, Stephens et al. (12) purified two G protein-activated PI3Ks from pig neutrophils. Both were heterodimers composed of the 101-kDa regulatory protein and either a 120-kDa or a 117 kDa catalytic subunit. Only, the p120 cDNA was cloned and was shown to be highly related to PI3Kgamma . Furthermore, two recent reports demonstrated the presence of a PI3Kgamma -mediating ion channel stimulation in smooth muscle cells (42, 43). However, the mechanism governing the nuclear targeting of the GTP-dependent PI3K is unclear. Nevertheless, Metjian et al. (24) showed that serum induces translocation of tagged p110gamma into HepG2 nuclei and suggested that p101 could regulate this relocation. In contrast with this observation, preliminary data from our laboratory seem to indicate that serum does not modify nuclear PI3Kgamma -like kinase expression.2

We further showed that VSMC nuclei contain a PI3K activity able to generate PtdIns(3)P from exogenous PtdIns in the absence of GTPgamma S. Immunoblots and immunofluorescence analyses failed to detect the adaptor protein p85alpha inside the VSMC nuclei, suggesting that the GTP-independent PI3K is different from p85alpha /p110 PI3Ks (class IA). This conflicts with the data from Lu et al. (20) and Marchisio et al. (44), who found p85alpha in the nuclear matrix of rat liver and of differentiated HL60 cells, respectively. Such a discrepancy might be due to differences in cell type or in nuclei isolation. However, p85alpha /p110 could be expressed in the nuclear envelope of VSMCs. We also cannot exclude the presence of a class IA PI3K containing the p85beta adaptor protein or class II/III PI3K inside VSMC nuclei. These hypotheses are currently under investigation. However, it is noteworthy that this GTP-independent PI3K was unable to phosphorylate the pool of PtdIns(4,5)P2 within the nucleus.

Our experiments with bacterial toxin and the nuclear identification of Galpha 01 allowed us to propose that PTX-dependent G proteins are present in the nucleus and participate in the activation of the GTP-dependent PI3K. In support of this hypothesis, the association between Galpha 0 and the mitotic spindle was observed in certain cancer cell lines (45). It has also been shown that growth factor-induced cell division is paralleled by the translocation of alpha i to the nucleus (46). Moreover, Gbeta gamma subunits might also play an important role in GTP-dependent PI3K activation, since Gbeta gamma directly stimulates PI3Kgamma activity in vitro (47), and a beta gamma subunit-like activity has been reported in rat liver nuclei (48). Interestingly, PI3Kgamma was implicated in both Galpha i- and beta gamma -mediated survival pathways elicited by G protein-coupled receptors in COS-7 cells (49).

Clearly, further investigations will be required to answer important points, in particular to clarify the role of the nuclear heterotrimeric G protein-activated PI3K in VSMC pathophysiology. It will be of special interest to study the regulation of this PI3K throughout the cell cycle. In this regard, the recent demonstration that serum-induced VSMC proliferation is mediated primarily via Gbeta gamma in vitro and that targeted inhibition of Gbeta gamma reduces intimal hyperplasia and limits restenosis in vivo (50) appears essential.

    ACKNOWLEDGEMENTS

We appreciate the expert technical assistance of J. C. Thiers for the electron microscopy studies. We thank S. Roche for providing the polyclonal antibody to p110gamma , A. Yart and P. Raynal for COS-7 cells transfected with plasmid encoding human p110gamma or p110alpha . We are grateful to B. Payrastre and C. Racaud-Sultan for helpful discussions and critical reading of the manuscript.

    FOOTNOTES

* This work was supported by grants from the Association pour la Recherche contre le Cancer, the Ligue Nationale contre le Cancer, and the Fondation pour la Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom all correspondence should be addressed. Tel.: 33-5-61-77-94-15; Fax: 33-5-61-77-94-01; E-mail: monique.douillon@purpan. inserm.fr.

Published, JBC Papers in Press, April 12, 2001, DOI 10.1074/jbc.M011572200

2 D. Bacqueville, P. Déléris, and M. Breton-Douillon, unpublished observation.

    ABBREVIATIONS

The abbreviations used are: VSMC, vascular smooth muscle cell; PI, phosphoinositides; PI3K, PI 3-kinase; 3-PI, 3-phosphorylated phosphoinositides; PtdIns, phosphatidylinositol; PtdIns(4)P, Ptd 4-monophosphate; PtdIns(3)P, Ptd 3-monophosphate; PtdIns(4, 5)P2, Ptd 4,5-bisphosphate; PtdIns(3, 4)P2, Ptd 3,4-bisphosphate; PtdIns(3, 4,5)P3, Ptd 3,4,5-trisphosphate; PLC, phospholipase C; PAGE, polyacrylamide gel electrophoresis; GTPgamma S, guanosine 5'-(gamma -thio)triphosphate; GDPbeta S, guanosine 5'-(beta -thio)diphosphate; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PTX, pertussis toxin; PBS, phosphate-buffered saline; HPLC, high performance liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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