Molecular and Cellular Biochemistry 183: 113–124, 1998. © 1998 Kluwer Academic Publishers. Printed in the Netherlands.
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Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate V. Natarajan,1 S. Vepa,1 R. Shamlal,1 M. Al-Hassani,1 T. Ramasarma,2 H.N. Ravishankar2 and W.M. Scribner1 1
Department of Medicine, Indiana University School of Medicine, Indianapolis, USA; 2Department of Biochemistry, Indian Institute of Science, Bangalore, India Received 14 July 1997; accepted 15 October 1997
Abstract Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [32P] phosphatidylbutanol (PBt) in a doseand time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20–200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [32P] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC α and ∈, the major isotypes of PKC in BPAECs, by TPA ( 100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [32P] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPVdependent protein tyrosine phosphorylation and PLD activation. (Mol Cell Biochem 183: 113–124, 1998) Key words: phospholipase D, diperoxovanadate, tyrosine kinase, protein tyrosine phosphorylation, intracellular Ca2+ Abbreviations: BAPTA - 1,2 bis (2-aminophenoxy) ethane N,N,N1,N1-tetracetic acid; BPAECs – bovine pulmonary artery endothelial cells; DAG – diacylglycerol; ECGF – endothelial cell growth factor; EGTA – ethylenebis (oxethylenenitrilo) tetracetic acid; NAC – N-acetylcysteine; PA – phosphatidic acid; PAO – phenylarsineoxide; PAF – platelet activating factor, PBt – phosphatidylbutanol; DPV – diperoxovanadate; PDTC – pyrrolidine dithiocarbamate; PKC – protein kinase C; PLC – phospholipase C; PLD – phospholipase D; SMC – smooth muscle cell; TPA – 12-0-tetradecanoyl phorbol 13-acetate; Tyrk – tyrosine kinase; PTP – protein tyrosine phosphatase
Introduction Phospholipase D (PLD) activation is recognized as an important signal transduction pathway in mammalian cells [1–3]. Receptor and non-receptor mediated stimulation of
PLD results in the generation of phosphatidic acid (PA) which is subsequently converted to lyso PA or diacylglycerol (DAG) catalyzed by phospholipase A1/A2 or phosphatidic acid phosphatase (PA Pase), respectively [4, 5].
Address for offprints: V. Natarajan, Department of Medicine, Pulmonary Division, Indiana University School of Medicine, 1001 W. 10th Street OPW 425, Indianapolis, IN 46202-2879, USA
114 We have recently reported that reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), fatty acid hydroperoxide and 4-hydroxynonenal activated endothelial cell (EC) PLD [6, 7]. The ROS-mediated PLD activation was insensitive to protein kinase C (PKC) inhibitors and downregulation of PKC by 12-0-tetradecanoylphorbol 1 3-acetate (TPA) as compared to agonist- or TPA-induced PLD activation [6)]. However, putative tyrosine kinase (tyrk) inhibitors genistein, tyrphostin and herbimycin attenuated H 2O2- or Ox-LDL-induced PLD activation [8, 9]. Addition of vanadate, a phosphatase inhibitor, synergistically increased ROSinduced PLD activation and protein tyrosine phosphorylation [8–10]. A combination of vanadate and H2O2, under neutral pH conditions, results in the generation of diperoxovanadate (DPV) [11], a potent protein tyrosine phosphatase inhibitor [12] and activator of tyrosine kineses [13]. Furthermore, H2O2 plus vanadate has been shown to be insulin mimetic [14], increase glucose transport [15], elevate intracellular Ca2+ [16)], stimulate PLD [17], phospholipase C [18], PI-3 kinase [19], MAP kinase [20], and enhance protein tyrosine phosphorylation [16, 17]. In the present study we have investigated the effect of diperoxovanadate, a specific model of experimentally produced reactive oxygen species, on protein tyrosine phosphorylation and PLD activation in vascular ECs. Employing bovine pulmonary artery endothelial cells (BPAECs) in monolayers, we observed that DPV stimulated PLD and protein tyrosine phosphorylation whereas vanadate was unable to stimulate PLD and only at higher concentrations was able to enhance protein tyrosine phosphorylation. The DPV-induced PLD activation was attenuated by tyrosine kinase inhibitors, chelators of intracellular Ca 2+ and antioxidants suggesting a role for protein tyrosine phosphorylation, Cai2+ and the redox state of the cell in DPVmediated PLD activation.
Materials and methods Materials 12-0-tetradecanoylphorbol-13-acetate, Minimum essential medium, hydrogen peroxide, sodium orthovanadate, nonessential amino acids, trypsin/EDTA, myelin basic protein, Penicillin/streptomycin, DMEM (phosphate free medium), catalase and fetal bovine serum were from Sigma (St. Louis, MO, USA). Phosphatidylbutanol (PBt) was from Avanti Polar Lipids (Alabaster, AL, USA). Silica gel precoated TLC plates were purchased from Analtech (Newark, DE, USA). BPAECs (CCL-209) were from American Type Culture Collection (Rockville, MD, USA). [32P] Orthophosphate (carrier-free) was purchased from New England Nuclear (Wilmington, DE, USA). Genistein, herbimycin, tyrphostin
and bisindolylmaleimide were from Calbiochem (San Diego, CA, USA). Endothelial cell growth factor (ECGF) and affinity purified monoclonal antiphosphotyrosine antibody (4G10) were obtained from Upstate Biotechnology Incorporated (Lake Placid, NY, USA). Enhanced chemiluminescence (ECL) kit for the detection of tyrosine phosphorylated proteins was from Amersham, (Arlington Heights, IL, USA). Polyclonal antibodies to ERK-1 and ERK-2 were obtained from Santa Cruz Biotechnology Inc., (Santa Cruz, CA, USA). Diperoxovanadate (potassium salt) was prepared by bubbling SO2 gas through a solution of triperoxovanadate and characterized as described earlier [11]. BPAECs were cultured in MEM containing 10% fetal bovine serum, 1 µg/ mL ECGF and 10 mL/1L of penicillin/streptomycin at 37°C in a humidified atmosphere of air/CO 2 (95:5) [22]. Cells were maintained in the above medium and grown in 35 mm dishes or T-75 cm2 flasks and cells at passage 18–20 were used in all the experiments.
Labelling of BPAECs with [32P] orthophosphate For labelling studies, BPAECs in 35 mm dishes were incubated with [32P] orthophosphate (5 µCi/mL) in DMEM phosphate free medium containing 2% fetal bovine serum for 18–24 h at 37°C [23]. About 1% of the added label was incorporated into total phospholipids [6].
Assay of PLD activation in intact cells BPAECs labelled with [32P] orthophosphate were washed in 1 mL of serum-free MEM. Cells were incubated with MEM or MEM containing various agents, at concentrations and time periods as indicated, in the presence of 0.05% butanol [9]. Incubations were terminated by the addition of 1 mL of methanol:HCl (100:1 v/v) followed by extraction of lipids in chloroform:methanol [9]. [ 32P] Phosphatidylbutanol (PBt) formed, an index of PLD activation, was separated by TLC as described previously [8, 22] and quantified by liquid scintillation counting. Radioactivity associated with [32P] PBt from different experiments varied due to differences in batches of BPAECs from ATCC and [32P] labelling of phospholipids.
Measurement of diacylglycerol BPAECs grown in T-75 cm2 flasks were washed in 1 mL of serum-free MEM before incubation for specified times with various agents in serum-free MEM. The reaction was terminated by addition of 2 mL of ice-cold methanol and lipids were extracted [22]. Diacylglycerol levels in the
115 lipid extract were measured using γ- 32P ATP and DAG kinase from E. coli [24]. Appropriate sample blanks and dioleoylglycerol (25–500 pmoles), as standards, were included in all the assays. DAG levels in the lipid extracts were expressed as nmols of [ 32P] PA formed/min/mg protein.
Measurement of inositol phosphates BPAECs grown in 35 mm dishes were incubated for 18 h in 1 mL of MEM containing 5 µCi of [2-3H] myo-inositol (Sp. Ac.9.25 MBq). The cells were washed in MEM and treated for 30 min with MEM or MEM containing H2O2 (1 mM) or vanadate (100 µM) or DPV (5 µM). The cells were extracted under acidic conditions and [3H] inositol phosphates were separated by anion exchange chromatography on AG 18 columns as described previously [25]. Radiolabelled inositol phosphate standards were used to identify the [3H] inositol phosphate radioactive peaks.
Cytosolic Ca2+ measurements BPAECs grown on glass cover slips were loaded with Fura2 AM as described earlier [9]. Fura-2 loaded BPAECs were challenged with different agents and studied in a thermostat regulated sample compartment of an SLM spectrofluorometer controlled by a PC-program to alternately excite the Fura-2 loaded cells at 340 and 380 nm and fluorescence monitored at 510 nm at a rate of 2/sec. The ratios of fluorescence intensities at 340 and 380 nm were calculated and [Ca2+i] were estimated [9]. In all experiments, measurements were corrected for the autofluorescence from cells not loaded with Fura-2.
and one portion of the immunocomplex was dissociated by boiling in 1 × SDS sample buffer for 5 min. Another aliquot of the immunocomplex was washed twice with kinase buffer (50 mM PIPES, pH 7.0; 10 mM MgCl2; 3 mM MnCl2 and 0.1 mM dithiothreitol). The kinase assays were initiated by the addition of 1 µg of myelin basic protein and 50 µM [γ-32P] ATP (10 Ci/mmol) in a final volume of 100 µ1. The reaction was terminated after various time periods at 30°C by the addition of 10 mM ATP and Laemmli sample buffer. The phosphorylation of myelin basic protein was examined by SDS-PAGE followed by autoradiography. For Western blotting, to 200 µl of the lysate, 40 µl of 6 × Laemmli SDSPAGE buffer was added [8, 26] and samples were boiled for 5 min and stored at –20°C. Cell lysates, adjusted to equal protein, were subjected to SDS-PAGE on 8 or 14% gels and were electrotransferred onto PVDF membranes for Western blotting. Membranes were blocked with blocking buffer (GIBCO-BRL) for 1 h followed by incubation with 4G10 monoclonal antiphosphotyrosine antibody (1:1000 dilution) for 2 h. The blots were washed with TBST (50 mM Tris base, 200 mM NaCl and 0.1% Tween 20) and were then incubated with goat antimouse IgG (H and L) horseradish peroxidase (1:3000 dilution) for 1 h. Subsequently, the blots were washed in TBST and the phosphotyrosine containing proteins were immunodetected using ECL (Amersham).
Assessment of cell injury Release of preincorporated (3H) deoxyglucose from BPAECs in the absence or presence of ROS was used as an index of cytotoxicity [27].
Statistical analysis Immunoprecipitation, SDS-PAGE and Western blotting Cells treated with MEM or MEM containing DPV or vanadate were rinsed thrice in ice-cold phosphate buffered saline (PBS) containing 1 mM vanadate. Cells were scraped in 250 µl of lysis buffer (20 mM Tris-HCl, pH 7.4; 1% NP40, 137 mM NaCl, 0.5% Triton X-100) supplemented with 2 µg/mL leupeptin, 2 µg/mL pepstatin, 1 µg/mL aprotinin, 1 mM PMSF and 1 mM vanadate. Cell lysates were sonicated and centrifuged at 14,000 × g for 15 min at 4°C. An aliquot of the supernatant was used for protein estimation by Pierce BCA assay. The cell lysates (equal protein of about 0.5–1 mg) were subjected to immunoprecipitation with anti ERK- 1 plus ERK-2 (1–2 µg/ml) at 4°C for 4–12 h. Protein A/G (20 µl) was then added and incubated for an additional 4–6 h at 4°C. The antibody complex was pelleted
All values are mean ± ranges (n = 2) or mean ± S.E.M. (n ≥ 3) where n represents the number of independent experiments in triplicate. Statistical significance (p < 0.05) was determined by student’s t-test and ANOVA was performed when comparing different treatments to control treatment.
Results Diperoxovanadate treatment enhances PLD activity In solution, sodium ortho or metavanadate combines with H2O2 to form peroxovanadate (V4+-00–) [13, 14], which is a potent inhibitor of tyrosine phosphatases [12]. Recently, diperoxovanadate (DPV) was identified as a major peroxovanadium compound generated from a solution of H2O2
116 plus sodium vanadate [11]. To further characterize V4+-OOH and DPV-induced signalling, (32P) orthophosphate labelled BPAECs were treated with these two agents. Formation of (32P) phosphatidylbutanol (PBt), an index of PLD activation [1], was similar with (V4+-OOH) and DPV (Fig. 1), however, with these agents the PLD activation was about 2 fold higher as compared to H2O2 alone [8]. Furthermore, the H2O2 but not V4+-OOH or DPV-induced (32P) PBt formation was attenuated by catalase (20 µg/mL) (Fig. 1). Based on these observations, DPV was used to further characterize its mechanism of PLD activation. The DPV-induced ( 32P) PBt formation was dose- and timedependent with maximal accumulation occurring with 10 µM DPV and at 30 min of incubation (Figs 2 and 3). Under identical conditions, the basal (32P) PBt formation was minimal and ROS treatment showed no cytotoxicity as determined by (3H) deoxyglucose release (data not shown).
PKC inhibitors do not affect diperoxovanadate-induced PLD activation Previous studies from our laboratory and others have demonstrated that (a) agonist-induced PLD stimulation was dependent on changes in intracellular free Ca2+ and PKC activation [22, 23], and (b) H2O2-induced PLD activation was PKC and Ca2+ independent but was dependent on tyrosine kineses [6]. Treatment of BPAECs with bisindoylmaleimide (BIM), an inhibitor of PKC [28], had no effect on DPVinduced (32P) PBt formation however, the TPA (100 nM)induced PLD activation was inhibited by BIM (Table 1). Since PKC is the intracellular receptor for phorbol esters, these data suggest that DPV-induced PLD activation does not involve PKC. Unlike H2O2 and TPA, which activated the PKCα isotype [24] as determined by translocation to the membrane from the cytosol, DPV had no effect on PKCα translocation (data not shown). Furthermore, downregulation of PKCα and ∈, the two major PKC isotypes in BPAECs [29], by prolonged treatment with TPA (100 nM) for 18 h attenuated the TPA- but not the DPV-induced ( 32P) PBt
Fig. 1. Effect of H2O2, pervanadate and DPV on [32P] PBt formation. BPAECs (5 × 105 cells/35 mm dish) were labelled with [32P] orthophosphate (5 µCi/dish) in DMEM-phosphate free medium for 18 h. Cells were washed in MEM without serum and were challenged with MEM or MEM containing H2O2 (1 mM) or V4+OOH, prepared by mixing equal volumes of 1 mM H2O2 and vanadate, (25 µM) or DPV (25 µM) with or without catalase (20 µg/ml) and 0.05% butanol for 30 min. Lipids were extracted under acidic condition and [32P] PBt formed was separated by TLC and quantified by scintillation counting. Values are mean ± S.D. (n = 3).
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Fig. 2. Dose-dependent formation of [32P] PBt by DPV. BPAECs (5 × 105 cells/35 mm dish) were labelled with [32P] orthophosphate as described in Fig. 1. Cells were washed in MEM without serum and were challenged with varying concentrations of DPV in the presence of 0.05% butanol for 30 min. Lipids were extracted under acidic condition and [32P] PBt formed was quantified as described under ‘Materials and methods’. Data are expressed as % control (n = 3).
Fig. 3. Time-course of DPV-induced [32P] PBt formation. BPAECs (5 × 10 5 cells/35 mm dish) were labelled with [32P] orthophosphate as described in Fig. 1. Cells were washed and challenged with DPV (10 µM) for varying time periods in the presence of 0.05% butanol. Lipids were extracted under acidic condition and [32P] PBt formed was quantified as described under Fig. 2. Values are mean ± S.D. (n = 3).
118 Table 1. Effect of bisindolylmaleimide on DPV-, H2O2- and TPA-induced PLD activation Pretreatment
Treatment
[32P] PBt formed (dpm)
Veh Veh Veh Veh BIM (2 µM) BIM (2 µM) BIM (2 µM) BIM (2 µM)
Veh H2O2 (1 mM) DPV (10 µM) TPA (100 nM) Veh H2O2 (1 mM) DPV (10 µM) TPA (100 nM)
242 ± 39 707 ± 45 1543 ± 233 2909 ± 161 362 ± 108 698 ± 58 1326 ± 105 628 ± 90
[32P] orthophosphate (5 µCi/dish in DMEM-phosphate free medium) labelled BPAECs (18 h) were pretreated with bisindoylmaleimide (2 µM) in MEM for 30 min. Cells were challenged with DPV (10 µM) or H2O2 (1 mM) or TPA (100 nM) in MEM containing 0.05% butanol for 30 min. Lipids were extracted under acidic condition and [32P] PBt was separated by TLC and quantified as described under “Materials and methods”. Values are mean ± S.D. of 3 independent experiments in triplicate.
formation (Fig. 4). In fact, down regulation of PKC by TPA potentiated the DPV-induced and [32P] PBt formation. These data further suggest that DPV-induced PLD activation is independent of PKC.
Intracellular Ca2+ is involved in diperoxovanadateinduced PLD activation One of the early responses of DPV was an increase in intracellular (Ca 2+i) (Fig. 5). The time course of DPVinduced (Ca2+i) showed an initial increase in (Ca2+i) which was followed by a second phase of slow decrease. Furthermore, DPV-treatment of ( 3H) inositol labelled BPAECs resulted in a significant increase in IP3 and diacylglycerol (Table 2) indicating an activation of PIP2 specific PLC [30]. As changes in (Ca2+i) are known to activate PLD [30], we investigated the role of Ca 2+ in DPV-induced ( 32P) PBt formation. Pretreatment of BPAECs with BAPTA/AM (10– 25 µM), a chelator of intracellular free Ca2+ , attenuated DPV-induced (32P) PBt formation (Table 3). Chelating the extracellular free Ca2+ by preincubating the cells with EGTA (5 mM) also resulted in a significant reduction in (32P) PBt formation (data not shown). These results suggest that changes in intracellular (Ca2+i) modulate DPV-induced PLD activation in BPAECs.
Involvement of protein tyrosine phosphorylation in diperoxovanadate-induced PLD activation We next investigated the role of tyrosine kineses/protein tyrosine phosphatases in DPV-induced PLD activation. If DPV increases [32P] PBt formation by inhibiting phosphotyrosine
Fig. 4. Effect of down regulation of PKC on DPV-induced [32P] PBt formation. BPAECs were labelled with [32P] orthophosphate (5 µCi/dish) as described in Fig. 1. Cells were washed in MEM without serum and were challenged with MEM or MEM containing TPA (100 nM) for 18 h. Cells were washed in MEM and were treated with TPA (100 nM) or DPV (5 µM) for 30 min in the presence of 0.05% butanol. [32P] PBt formed was quantified as described under ‘Materials and methods’. Values are mean ± S.D. (n = 3).
phosphatase activity [12], its effect should also be attenuated by tyrosine kinase inhibitors. As shown in Table 4, pretreatment with tyrosine kinase inhibitors such as genistein, herbimycin and tyrphostin attenuated DPV- but not TPAinduced [ 32P] PBt formation. The inhibitory effect of genistein was dose-dependent (Table 5) with 50% inhibition occurring at 100 µM. This effect of genistein was not mimicked by genistine, an analog which is inactive on tyrosine kineses (data not shown). These results indicate that DPV-induced activation of PLD involves tyrosine kineses. Inhibition of phosphotyrosine phosphatases or activation of tyrosine kineses or both by DPV may increase protein tyrosine phosphorylation of endothelial cells, in a manner similar to other mammalian cell types [31–33]. Treatment of BPAECs with varying concentrations of DPV caused a marked dose- dependent increase in tyrosine phosphorylation of several proteins as determined by immunoblotting with antiphosphotyrosine antibody (Fig. 6). At lower con-
119 Table 2. Effect of H 2O 2, vanadate, DPV and TPA on PBt, inositol trisphosphates and diacylglycerol generation Addition
[32P] PBt (dpm)
[3H]IP3 dpm/106 cells
Vehicle 285 ± 74 (100) 274 ± 45 (100) H2O2 (1 mM) 708 ± 96 (248) 1093 ± 262 (398) Vanadate (100 µM) 299 ± 42 (105) 475 ± 174 (174) Vanadate (10 µM) + H2O2 (10 µM) 2694 ± 170 (945) 1620 ± 218 (591) DPV (5 µM) 2782 ± 134 (976) 1746 ± 218 (613) TPA (100 nM) 3216 ± 217 (1128) 375 ± 77 (137)
DAG pmoles/106 cells 12 ± 1 (100) 28 ± 4 (233) 15 ± 5 (125) 42 ± 7 (350) 48 ± 7 (400) 31 ± 8 (258)
BPAECs (5 × 106 cells/T-75 cm2 flask) were labelled with [2-3H] inositol (5 µCi/ml, Sp. Ac 9.25 MBq) for 48 h. Cells were washed in MEM and were challenged with MEM or MEM containing H2O2 (1 mM) or vanadate (100 µM) or DPV (5 µM) for 30 min. Lipids were extracted under acidic conditions and water soluble inositol trisphosphate was analyzed after separation on AG1-X8 formate columns. (3H) IP3 was eluted with 0.1 M formic acid/1.0 M ammonium formate as described under ‘Materials and methods’. For DAG mass measurement, BPAECs after treatment with MEM or MEM containing H2O2 (1 mM) or vanadate (100 µM) or DPV (5 µM) were extracted under nonacidic conditions with chloroform: methanol (1:2 v/v) and DAG levels were quantified using DAG kinase and (γ-32P) ATP. Values are mean ± ranges (n = 2) and numbers in the parenthesis represent % of total.
centrations of DPV (0.5–1 µM) only three prominent tyrosine phosphorylated bands at Mr 110,000–130,000, Mr 65,000– 82,000 and Mr 40,000–50,000 were immunodetected. However, at higher concentrations of DPV (5–10 µM), additional components were detected in the Mr 175,000–
Table 3. Effect of BAPTA on DPV-induced [32P] PBt formation Addition
[32P] PBt (dpm) (–)DPV (+) DPV
∆dpm
% control
– BAPTA: 1 µM 10 µM 25 µM
370 ± 40 299 ± 35 409 ± 187 334 ± 60
2455 2365 1356 699
100 96 55 28
2825 ± 357 2664 ± 348 1765 ± 123 1033 ± 87
BPAECs were labelled with [32P] orthophosphate (5 µCi/35 mm dish) in DMEM-phosphate free medium for 18 h. Cells were washed in MEM and pretreated with MEM or MEM containing BAPTA (1–25 µM) for 60 min. Cells were washed in MEM and challenged with MEM or MEM containing DPV (5 µM) for 30 min in the presence of 0.05% butanol. Lipids were extracted under acidic condition and [32P] PBt was separated by TLC and was quantified as described under ‘Materials and methods.’ Values are mean ± S.D. (n = 3).
203,000 and Mr 25,000–35,000. No significant increase in tyrosine phosphorylation of proteins was detected below 0.5 µM DPV. As shown in Fig. 7, DPV (µM) increased tyrosine phosphorylation of Mr 110,000–130,000 band as early as 10 min, reached a maximum after 30 min and was sustained for up to 60 min of DPV treatment. In addition to increased protein tyrosine phosphorylation, the tyrosine kinase activity in total cell lysates from DPV-treated BPAECs was higher as compared to control cells (data not shown). These results suggest that DPV is an activator of protein tyrosine phosphorylation of several proteins in BPAECs. To confirm the observation that at least part of the DPV-mediated protein tyrosine phosphorylation was through tyrosine kineses, we
Fig. 5. Increase of [Ca2+i] by DPV. BPAECs were grown on coverslips and were loaded with Fura 2/AM, challenged with DPV and changes in intracellular Ca 2+ were measured by SML-AMINCO fluorospectrometer. Changes in intracellular Ca2+ are expressed as ratio of 340/380 nm.
120 Table 4. Effect of tyrosine kinase inhibitors on DPV- and TPA-induced PLD activation Addition
[32P] PBt (dpm) (–) DPV (+) DPV
[32P] PBt (dpm) (–)TPA (+) TPA
– Genistein (50 µM) Herbimycin (10 µM) Tyrphostin (10 µM)
416 ± 58 309 ± 38 472 ± 42 306 ± 29
416 ± 58 309 ± 38 472 ± 42 306 ± 29
2705 ± 75 1911 ± 99 2325 ± 120 2105 ± 192
3935 ± 235 3760 ± 70 4008 ± 149 3708 ± 126
BPAECs were labelled with [32P] orthophosphate as described in Table 1. Cells were washed in MEM and pretreated with genistein (100 µM) or herbimycin (10 µM) or tyrphostin (10 µM) for 60 min and were challenged with DPV (5 µM) or TPA (25 nM) for 30 min in the presence of 0.05% butanol. Lipids were extracted and [32P] PBt was quantified as described under ‘Materials and methods’. Values are mean ± ranges of triplicate determination (n = 2).
Table 5. Effect of genistein on DPV-induced PLD activation Addition – Genistein:
25 µM 50 µM 100 µM
[32P] PBt (dpm) (–)DPV (+) DPV
∆dpm
% control
729 ± 12 873 ± 30 690 ± 170 940 ± 133
1917 1497 1284 436
100 78 67 23
2646 ± 402 2370 ± 83 1974 ± 260 1376 ± 242
BPAECs were labelled with [32P] orthophosphate (5 µCi/35 mm dish) in DMEM-phosphate free medium for 18 h. Cells were washed in MEM and pretreated with MEM or MEM containing varying concentrations of genistein for 60 min. Cells were washed in MEM and were challenged with MEM or MEM containing DPV (5 µM) for 30 min in the presence of .05% butanol. Lipids were extracted under acidic condition and [32P] PBt was separated by TLC as described under ‘Materials and methods’. Values are mean ± S.D. (n = 3).
Fig. 6. Dose-dependent stimulation of protein tyrosine phosphorylation by DPV in BPAECs. BPAECs (35 mm dishes) were challenged with varying concentrations of DPV for 15 min, cells were washed in ice cold phosphate buffered saline containing 1 mM vanadate, and scraped into RIPA buffer (300 µl). Cell lysates (10 µg protein) were subjected to SDS-PAGE (8% gel) followed by transfer to PVDF membrane and immunoblotted with antiphosphotyrosine antibody (1:2000 dilution). Tyrosine phosphorylated proteins were detected using anti IgG-horseradish peroxidase and enhanced chemiluminescence. The immunoblot is representative of 3 separate experiments.
examined the effect of tyrk inhibitors on DPV-induced protein tyrosine phosphorylation. As shown in Fig. 8 pretreatment of BPAECs with tyrosine kinase inhibitors attenuated DPVinduced increase in tyrosine phosphorylation of proteins. These results suggest that DPV not only acts as an inhibitor of protein tyrosine phosphatases [12] but also stimulates tyrosine kineses thereby enhancing protein tyrosine phosphorylation and PLD activation. In contrast to DPV, treatment of [32P] labelled BPAECs with vanadate (1–10 µM) did not result in increased protein tyrosine phosphorylation (data not shown) or [32P] PBt formation [8].
Thiol agents attenuate diperoxovanadate-induced PLD activation and protein tyrosine phosphorylation Antioxidants and agents that alter the redox/thiol status of cells also modulate ROS-mediated tyrosine phosphorylation of proteins [41]. Therefore, we next investigated the effect of N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC) on DPV-induced PLD activation and protein tyrosine
Fig. 7. Time-dependent protein tyrosine phosphorylation by DPV in BPAECs. BPAECs (35 mm dishes) were challenged with DPV/(1 µM) for varying time periods and cell lysates (10 µg protein) were subjected to SDSPAGE and immunoblotted with 4G10 antiphosphotyrosine antibody as described in Fig. 6. Tyrosine phosphorylated proteins were detected using anti IgG-horseradish peroxidase and enhanced chemiluminescence. The immunoblot is representative of 3 separate experiments.
121 Table 6. Effect of N-acetylcysteine and pyrrolidine dithiocarbamate on DPVinduced PLD activation Addition
[32P] PBt (dpm) (–) DPV (+) DPV
∆dpm
% control
– NAC (5 mM) PDTC (1 mM)
169 ± 20 155 ± 46 143 ± 38
1091 174 371
100 16 34
1260 ± 61 329 ± 17 514 ± 32
BPAECs in 35 mm dishes were labelled with [32P] orthophosphate (5 µCi/ dish) in DMEM-phosphate free medium for 24 h. Cells were washed and treated with NAC (5 mM) or PDTC (1 mM) for 60 min. Cells were washed and challenged with DPV (5 µM) for 30 min in the presence of 0.05% butanol. Lipids were extracted under acidic condition and [32P] PBt was separated by TLC and quantified as described under ‘Materials and methods’. Values are mean ± S.D. (n = 3).
Fig. 8. Tyrosine kinase inhibitors attenuate DPV-induced protein tyrosine phosphorylation. BPAECs (35 mm dishes) were pretreated with genistein (100 µM) or herbimycin (10 µM) or tyrphostin (10 µM) for 60 min. Cells were washed in MEM without serum and were challenged with MEM or MEM containing DPV (5 µM) for 15 min. Cell lysates were prepared as described in Fig. 6 and 10 µg of cell lysates were subjected to SDS-PAGE, transferred onto membranes, incubated with 4G10 antiphosphotyrosine antibody and IgG HRP and were detected by enhanced chemiluminescence.
phosphorylation. As shown in Table 6, both NAC (5 mM) and PDTC (1 mM) attenuated the DPV-induced [32P] PBt formation. The effects of NAC and PDTC were dose- and timedependent (data not shown). In addition, both NAC and PDTC inhibited the DPV-induced increase in protein tyrosine
phosphorylation as determined by immunoblotting with antiphosphotyrosine (Fig. 9). These results suggest that DPVinduced PLD activation and protein tyrosine phosphorylation are sensitive to the redox/thiol status of the endothelial cells.
Discussion The present study is a continuation of our investigation into the mechanism(s) of regulation of ROS-induced PLD activation in vascular endothelial cells. In our earlier work, we have demonstrated that H2O2, 4-Hydroxynonenal and oxidized-LDL were potent stimulators of endothelial and smooth muscle cell PLD [6, 7, 9]. It was also observed that pretreatment of endothelial cells with vanadate enhanced the H2O2–, 4-HNE- and oxidized LDL-induced PLD activation
Fig. 9. N-acetylcysteine and pyrrolidine dithiocarbamate attenuate DPV-induced protein tyrosine phosphorylation. BPAECs (35 mm dish) were pretreated with varying concentrations of N-acetylcysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) for 60 min. Cells were washed in MEM without serum and were challenged with DPV (5 µM) for 30 min. Cell lysates (10 µg protein) in RIPA buffer were subjected to SDS-PAGE, membrane transfer and immunoblotted with 4G10 antiphosphotyrosine antibody. Tyrosine phosphorylated proteins were detected using enhanced chemiluminescence.
122
Fig. 10. Time course of DPV-induced tyrosine phosphorylation of mitogen activated protein kinases. Confluent BPAECs were treated with DPV (5 µM) for different time periods as indicated and cell lysates were subjected to immunoprecipitation with anti ERK-1 and anti ERK-2 polyclonal antibodies (1 µg/ml of each) followed by immunoblotting of equal amounts of immunoprecipitates with antiphosphotyrosine or anti ERK-1/ERK-2 as described under ‘Materials and methods’. A portion of the immunocomplex was also assayed for MAP kinase activity using myelin basic protein and [γ-32P] ATP. Tyrosine phosphorylated proteins were detected using ECL while 32P labelled myelin basic protein was detected on X-ray film after 5% SDS-PAGE.
[8–10]. Vanadate is not only a potent inhibitor of phosphatases, including protein tyrosine phosphatases, but can also interact with H2O2 to generate pervanadate (V4+-00–) [34]. Recent studies by Shankar and Ramasarma have shown that the major product of H2O2 and vanadate under neutral pH conditions is DPV [11]. Our data demonstrate that DPV, as compared to H2O2, is a potent activator of PLD, and DPVinduced PLD activation was insensitive to PKC inhibitors and down-regulation of PKC by TPA thereby indicating a PKC-independent mechanism. Although DPV and H2O2induced PLD activation share a common mechanism of PKC-independent pathway, there are differences in their mode of stimulation. Activation of PLD by DPV, but not by H2O2 was abolished by chelation of either extracellular Ca2+ with EGTA or intracellular Ca 2+ with BAPTA, thereby suggesting a role for Ca2+. Furthermore, DPV-treatment of BPAECs increased Ca2+i by PIP2 hydrolysis catalyzed by PLC and the subsequent release of IP3 and DAG. While H 202 treatment of BPAECs also caused changes in Ca2+i, DAG and PKC activation [24], DPV treatment failed to activate PKCα as determined by immunoblotting with PKCα antibodies. It is not clear why DPV failed to activate EC PKCα, although changes in intracellular calcium and DAG are known to activate PKCα, β and γ. It is possible that the molecular species of DAG generated by DPV is different as compared to agonist such as thrombin or bradykinin in ECs. Generation of DAG or phosphatidic acid with 1 alkyl/alkenyl moiety as compared to 1 acyl moiety may actually inhibit DAG induced PKC activation [50]. Another significant difference was that catalase treatment affected H2O2 but not DPV-induced PLD
activation suggesting that the DPV action was not due to its breakdown to H2O2. At this time very little is known about the metabolism of peroxovanadium compounds including DPV in intact cells but recent studies suggest that higher concentrations of catalase metabolizes DPV in vitro, however, at a much slower rate as compared to H2O2 (< 50 times compared to H2O2) [35]. Our results also demonstrate that DPV-induced PLD activation is reduced by tyrosine kinase inhibitors suggesting a role for protein tyrosine phosphorylation. DPV has been shown to be a powerful inhibitor of phosphatases [36] and, therefore, the DPV-mediated PLD activation and protein tyrosine phosphorylation may be due to inhibition of PTPases or activation of tyrosine kineses or both. The PTPase inhibitors had no effect on the basal PLD activity, however, at higher concentrations the inhibitors enhanced protein tyrosine phosphorylation suggesting the involvement of tyrosine kineses in DPV-induced PLD activation. The mechanism(s) involved in PKC- or tyrosine kinase mediated PLD activation is unclear. Studies by Conricode et al. suggest that PKC-mediated PLD activation may not involve protein phosphorylation of the enzyme but may depend on protein-protein interaction [37]. In neutrophils, the TPA-mediated PLD activation seems to depend on phos– phorylation of a 50 kD plasma membrane protein [38]. In vascular smooth muscle cells, the PDGF-mediated PLD activation seems to be downstream to tyrosine phosphorylation of PLC γ - 1 with subsequent activation and involvement of PKC [39]. While there is increasing evidence for the involvement of protein tyrosine phosphorylation in growth factor, oxidants, PAF and IgE-mediated PLD activation, the nature of the tyrosine phosphorylated proteins or the role tyrosine phosphorylation of PLD in stimulating the activity has not been identified. Studies by Vepa et al. in BPAECs suggest that caveolin (22 kDa) and focal adhesion kinase (125 kDa) [42] are targets for H2 O 2 -induced tyrosine phosphorylation. In the present study, ERK1/ERK2 was also identified as another potential target for DPV-induced increased tyrosine phosphorylation (Fig. 10) confirming the earlier observations with ROS and vanadate in mammalian cells [20, 41–42]. As MAP kinases have potential serine/ threonine and tyrosine phosphorylation sites, its activation may be involved in PLD stimulation. However, our studies using the MEK inhibitor PD 098059 suggest that DPVinduced PLD activation may not involve MAPK (Natarajan V, unpublished data). N-Acetylcysteine and PDTC function as antioxidants, prevent ROS-induced apoptosis, activate transcriptional factors NF-kB and AP-1 [46] and vascular cell adhesion molecules-1 [44–47]. The effect of the two structurally different thiol containing agents, NAC and PDTC on protein tyrosine phosphorylation and [32P] PBt formation suggest that the integrity of the sulfhydryls is critical in ROSmediated signalling.
123 The physiological relevance of DPV as a modulator of signal transduction pathways in mammalian cells is unclear. Although the occurrence of peroxovanadium compound(s) inside mammalian cells has not been established, there is evidence for the presence of vanadyl ion (V4+) (0.1–1 µM) inside mammalian cells [48]. This small concentration of vanadium in the cells may be sufficient to react with endogenously generated H 2O 2 or exogenously provided H2O2 to form peroxovanadium compounds, which in turn, could modulate protein kinases or phosphatases and signalling pathways. Recent studies in cell free preparations from rat adipocytes indicate that the conversion of vanadyl to vanadate by H2O2 activates cytosolic protein tyrosine kinases [49]. In summary, the data reported here show that the DPVinduced PLD activation is modulated by protein tyrosine phosphorylation, changes in intracellular Ca2+ and the redox status of the endothelial cells. An oxidation prevented by reduction with thiol compounds may be involved in DPVinduced activation of PLD. The physiological implication of the DPV-induced modulation of protein kinases/phosphatases and the generation of second messengers such as PA, lyso PA and DAG may be the alteration of endothelial cell permeability and subsequent barrier dysfunction. As ROS have been implicated in the pathophysiology of a number of vascular disorders including atherosclerosis, a better understanding of the mechanism(s) of oxidant-induced signal transduction pathways should provide new insights into endothelial cell function under normal and pathological conditions.
Acknowledgements We wish to thank Beverly Clark for her secretarial assistance. This study was supported in part by grants from the National Institutes of Health (HL-4767 1 and K04-HL03095) and an American Lung Association Career Investigator Award (VN). TR is a senior scientist of the Indian National Science Academy, New Delhi, India and received funding from Bhoruka Charitable Trust, Jaipur, India for a short visit to Indiana University School of Medicine to participate in this work.
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