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Am. J. Respir. Cell Mol. Biol., Volume 17, Number 2, August 1997 251-259

Phosphatase Inhibitors Potentiate 4-Hydroxynonenal-induced Phospholipase D Activation in Vascular Endothelial Cells

Viswanathan Natarajan, William M. Scribner, and Suryanarayana Vepa

Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously reported that endothelial cell phospholipase D (PLD), activated by 4-hydroxynonenal (4-HNE), was independent of protein kinase C activation. To determine whether PLD stimulation by 4-HNE is related to protein tyrosine phosphorylation, the effects of tyrosine kinase (Tyrk) and protein tyrosine phosphatase (PTPase) inhibitors on PLD activation were investigated. Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with Tyrk inhibitors, such as genistein, erbstatin, and herbimycin attenuated 4-HNE-induced PLD activation. Furthermore, vanadate, phenylarsine oxide, and diamide, inhibitors of PTPases, markedly increased the 4-HNE-induced PLD activation. The effects of Tyrk and PTPase inhibitors were specific towards the 4-HNE, as these agents had no effect on the agonist- or TPA- induced PLD activation. In addition to PLD activation, treatment of BPAEC with 4-HNE increased tyrosine phosphorylation of proteins including bands of molecular weights 40,000-60,000, 70,000-90,000, and 110,000-130,000. The 4-HNE-mediated increase in protein tyrosine phosphorylation was partly inhibited by genistein (100 µM). Vanadate (10 µM) pretreatment also potentiated 4-HNE-induced protein tyrosine phosphorylation. These data suggest that 4-HNE-mediated stimulation of PLD may occur as a result of activation of tyrosine kinases.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

4-Hydroxynonenal (4-HNE), a major metabolite of lipid peroxidation, exhibits cytotoxic and stimulatory properties when added to mammalian cells in culture (1). The cytotoxic effects were observed at higher concentrations while low levels of 4-HNE stimulated enzymes involved in the generation of second messengers such as adenylate cyclase (7) and phospholipases (8). Stimulation of adenylate cyclase increases cAMP levels while the activation of phospholipases C and D results in the accumulation of diacylglycerol (DAG)/inositol (1, 4, 5) trisphosphate and phosphatidic acid (PA), respectively.

The mechanism(s) of 4-HNE-induced activation of adenylate cyclase and phospholipases are unknown. We and others have previously reported that agonist- induced PLD activation in human umbilical vein and bovine pulmonary artery endothelial cells (BPAEC) was protein kinase C (PKC) and Ca2+ dependent (12). However, the 4-HNE- and H2O2-mediated PLD activation in BPAEC was insensitive to PKC inhibitors, downregulation of PKC and Ca2+ chelators (11, 16). Recent studies in platelets, neutrophils, and mast cells suggest a possible role for protein tyrosine phosphorylation in PLD activation mediated by growth factors IgE and reactive oxygen species (17).

The preceding findings prompted us to examine the effect of tyrosine kinase (Tyrk) and protein tyrosine phosphatase (PTPase) inhibitors on 4-HNE-induced PLD activation. The results presented here demonstrate that Tyrk inhibitors such as genistein, herbimycin, and erbstatin attenuate 4-HNE-mediated PLD activation while PTPase inhibitors, such as vanadate, phenylarsine oxide, and diamide potentiate the 4-HNE-induced PLD activity. In addition to PLD activation, 4-HNE and vanadate plus 4-HNE also increased protein tyrosine phosphorylation of endothelial cellular proteins.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

12-O-tetradecanoylphorbol-13-acetate, sodium orthovanadate, genistein, phenylarsine oxide, diamide, leupeptin, aprotinin, trypsin, fetal bovine serum, minimal essential medium (MEM) and dioleoyl phosphatidic acid were purchased from Sigma (St. Louis, MO). 4-Hydroxynonenal was obtained from Biomol (Plymouth Meeting, PA). [32P]Orthophosphate (carrier free) was from Du Pont NEN (Boston, MA). Monoclonal antiphosphotyrosine antibody, 4G10, was obtained from UBI (Lake Placid, NY). Phosphatidylethanol was purchased from Avanti Polar Lipids (Alabaster, AL). Bovine pulmonary artery endothelial cells (CCL-209) were from ATCC (Rockville, MD). Enhanced chemiluminescence kit was procured from Amersham (Arlington Heights, IL). Pefabloc, a protease inhibitor, was obtained from Centerchem Inc. (Stamford, CT). Precoated silicagel plates were purchased from Analtech (Newark, DE).

Cell Culture

Bovine pulmonary artery endothelial cells (BPAEC) were grown in MEM supplemented with 10% fetal bovine serum, 20 µg/ml streptomycin, 0.05 µg/ml fungizone and 0.2 ml sodium heparin/100 ml of medium. Cultures were maintained in a humidified 95% air and 5% CO2 atmosphere at 37°C (16). All experiments were carried out at cell passage number 18 through 20.

Labeling of Cells and Determination of Phospholipase D Activation

Confluent monolayers of BPAEC in 35-mm dishes (5 × 105 cells/dish) were labeled with [32P]orthophosphate (5 µCi/dish) in 1 ml of DMEM phosphate-free media supplemented with 2% fetal bovine serum and antibiotics for 18- 24 h. (12, 13, 16). Cells were washed twice with MEM and were challenged with MEM or MEM containing 4-HNE or other agents in the presence of 0.5% ethanol as indicated. In experiments involving TyrK or PTPase inhibitors, cells were preincubated for 15-60 min with the inhibitors before challenging with 4-HNE. Cells were incubated at 37°C for the indicated time periods and lipids were extracted under acidic conditions as described earlier (12, 13, 16). Phosphatidylethanol (PEt) was separated on layers of silica gel G or silica gel H containing 1% potassium oxalate and quantified by liquid scintillation counting.

SDS-PAGE and Western Blotting

Upon completion of treatment with MEM or MEM containing 4-HNE, cells were rinsed twice in ice cold phosphate buffered saline (PBS) containing pefabloc (1 mM) and once in PBS plus vanadate (1 mM). Cell lysates were prepared by adding 250 µl of lysis buffer (1% NP-40; 20 mM Tris-HCl, pH 8.0; 137 mM NaCl; 10% glycerol) supplemented with 15 µg/ml leupeptin, 0.15 U/ml aprotinin, 1 mM pefabloc and 1 mM vanadate. Cells were scraped on ice with a cell scraper, sonicated, transferred to microfuge tubes and centrifuged at 12,000 × g for 15 min. at 4°C. To 200 µl of the supernatant was added 40 µl of 6× SDS-PAGE laemmli buffer (21) and samples were boiled for 5 min. Cell lysates adjusted to equal protein were subjected to SDS-PAGE on 8% gels and proteins were electrotransferred onto immobilon-P membranes for Western blotting. After the transfer, membranes were blocked with blocking buffer for 1 h and incubated with 4G10 antiphosphotyrosine antibody (1:1,000 dilution) for 4-18 h. The blots were washed extensively with TBST (50 mM Tris-base; 200 mM NaCl and 0.1% Tween-20), incubated with goat antimouse IgG (H + L) Horseradish peroxidase (1:3,000 dilution) for 1 h. Subsequently, the blots were washed in TBST and the binding was detected using enhanced chemiluminescence Western blotting detection reagents according to manufacturer's recommendation. Band intensities were quantified by densitometric scanning of the X-ray films and were normalized to total cell lysate protein as determined by Pierce BCA assay.

Measurement of Tyrk Activity In Vitro

BPAEC in T-75 flasks (5 × 106 cells) were preincubated in MEM or MEM containing vanadate (10 µM) for 15 min. The medium was aspirated, washed once in MEM and challenged with 4-HNE (50 µM). At the end of 30 min, cells were washed in PBS and PBS plus 1 mM vanadate. Cells were scraped into 1 ml of cell lysis buffer (50 mM Tris-HCl pH 7.5, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.5 mM pefabloc, 100 U/ml Aprotinin) and sonicated (3 × 5 s). The lysate was centrifuged at 12,000 rpm for 15 min in a microfuge centrifuge and supernatant was stored at -20°C. Tyrosine kinase activity was assayed in a reaction mixture (50 µl final volume) containing: 50 mM Tris-HCl buffer, pH 7.4; 2 mM MnCl2; 20 mM MgCl2, 0.5 mM Na3VO4 and 25 µM [gamma 32P] ATP (specific activity [Sp. Ac.] 400 cpm/pmol) and 50 µg of poly (Glu4-Tyr) for 15 min at 30°C. The reaction was terminated by addition of 1 ml of 5% TCA and [32P]-incorporation with poly (Glu4Tyr) was determined by collecting the phosphorylated poly (Glu4Tyr) onto phosphocellulose paper (Whatman P-81). The phosphocellulose was washed thrice in cold 75 mM phosphoric acid dried and radioactivity counted in a liquid scintillation counter. Tyrosine kinase activity was expressed as pmol of [32P] incorporated into substrate/15 min/mg protein.

Other Methods

The concentration of 4-HNE was determined by measuring the optical density (O.D.) in water at 223 nm and using a molar absorptivity of 13750. All experiments were done in triplicate and data expressed as mean ± SD. Statistical comparisons were made by Student's t-test and by analysis of variance. Statistical significance was accepted at P < 0.05.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Tyrosine Kinase Inhibitors on 4-HNE-induced PLD Activation

We have previously demonstrated that 4-HNE-induced PLD activation was insensitive to PKC inhibitors and changes in Ca2+ (11). To further characterize the mechanism of 4-HNE-mediated PLD activation in endothelial cells, we examined the effect of Tyrk inhibitors. Pretreatment of [32P]orthophosphate-labeled BPAEC with putative Tyrk inhibitors such as genistein (100 µM) or herbimycin (10 µM) or erbstatin (10 µM) attenuated 4-HNE-induced [32P] PEt formation (Table 1), an index of PLD activation (14). The inhibitory effect of genistein and erbstatin was dose- dependent with IC50 of 35 µM and 11 µM, respectively (Figure 1). Further, the effect of genistein was specific for 4-HNE, as TPA (100 nM)-mediated [32P] PEt formation was not altered by genistein (Figure 2). Further, as shown in Table 2, genistein did not affect the bradykinin- or ionomycin-mediated PLD activation as compared with 4-HNE. Similarly, the TPA-induced [32P] PEt-formation was unaffected by herbimycin or erbstatin (data not shown). These data suggest that 4-HNE-induced PLD activation in BPAEC involves Tyrks.

                              
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TABLE 1
Inhibition of 4-HNE-induced PLD activation by tyrosine kinase inhibitors


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  		Figure 1.   Inhibition of 4-HNE induced PLD activation by genistein and erbstatin. BPAEC labeled with [32P]orthophosphate (5 µCi/ dish, 24 h) in DMEM-PO4 free media containing 2% fetal bovine serum were preincubated with varying concentrations of genistein (panel A) or erbstatin (panel B) for 60 min. Cells were washed and challenged with MEM or MEM containing 4-HNE (50 µM) and 0.5% ethanol for 30 min. Lipids were extracted under acidic conditions and [32P] PEt was quantified by TLC as described under MATERIALS AND METHODS. The effect of genistein and erbstatin on 4-HNE-induced PLD activation is expressed as Activity (% control). In the absence of Tyrk inhibitors, the radioactivity in [32P] PEt from control and 4-HNE treated cells was 180 ± 36 and 528 ± 47, respectively. Values are mean ± SD of triplicate determination.


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  		Figure 2.   Influence of genistein on 4-HNE and TPA-induced PLD activation. BPAEC were labeled with [32P] orthophosphate (5 µCi/dish, 24 h) in DMEM-PO4 free media containing 2% fetal bovine serum. Cells were washed, pretreated with genistein (35 µM) for 60 min and then challenged with 4-HNE (50 µM) or TPA (100 nM) in the presence of 0.5% ethanol for 30 min. Lipids were extracted under acidic conditions and [32P] PEt formed was quantified after separation by TLC as described under MATERIALS AND METHODS. Values are mean ± SD from two independent experiments in triplicate.

                              
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TABLE 2
Effect of genistein on 4-HNE-, bradykinin-, and ionomycin-induced PLD activation

Effect of Tyrosine Phosphatase Inhibitors on 4-HNE-induced PLD Activation

BPAEC were treated with 100 µM vanadate or 10 µM phenylarsine oxide (PAO) or 1 mM diamide as inhibitors of phosphatase activity (22). All three phosphatase inhibitors significantly potentiated the 4-HNE-mediated formation of [32P] PEt by 250%, 200%, and 315%, respectively (Figure 3). The dose-dependent effect of vanadate, PAO, and diamide on basal and 4-HNE-induced [32P] PEt formation are shown in Figure 4 (panels A, B, and C), respectively. Pretreatment of BPAEC with vanadate, as low as 1 µM for 15 min followed by the addition of 4-HNE, resulted in a 1.5- to 2.0-fold increase in [32P] PEt as compared with control cells not pretreated with vanadate (Figure 4, panel A). Higher concentrations of vanadate (10 µM-100 µM) also exhibited a 2- to 3-fold increase in [32P] PEt accumulation (Figure 4, panel A). However, vanadate treatment alone showed only a small increase in the basal accumulation of [32P] PEt (Figure 4, panel A). The concentration of vanadate used was limited by its cytotoxicity which became apparent at concentrations greater than 250 µM. Vanadate at higher concentrations induced morphological changes as observed under phase contrast microscopy, with elongation and gap formation in cells and eventual detachment and rounding up of cells from the culture dish (data not shown). Treatment of cells with PAO, another inhibitor of phosphatase activity (23), also potentiated the 4-HNE-induced [32P] PEt formation (Figure 4, panel B). At 1 µM concentration, in contrast to vanadate, PAO showed only a very slight increase in [32P] PEt accumulation (Figure 4, panel B). However, at 10 µM concentration, PAO showed a 3-fold increase in PLD activation (Figure 4, panel B). Diamide at 0.5 mM concentration potentiated the 4-HNE-induced PLD activation 2-fold as compared with 4-HNE alone (Figure 4, panel C). To further clarify the specificity of phosphatase inhibitors on 4-HNE-induced PLD activation, BPAEC were pretreated with vanadate (10 µM) or PAO (10 µM) for 15 min before challenging with 4-HNE (25 µM) or TPA (100 nM) for 30 min. Under these incubation conditions, vanadate and PAO showed no effect on TPA-induced [32P] PEt accumulation (Figure 5). In addition to 4-HNE, H2O2 and linoleic acid hydroperoxide also stimulated PLD activity in BPAEC (Table 3). However, the potentiating effect of vanadate was observed with H2O2 and 4-HNE, not with linoleic acid hydroperoxide. The above data suggest that the effect of phosphatase inhibitors was specific to 4-HNE-mediated PLD activation.


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  		Figure 3.   Effect of tyrosine phosphatase inhibitors on 4-HNE- induced PLD activation. BPAEC were labeled with [32P]orthophosphate as described in Figure 1. Cells were washed in MEM and were pretreated with vanadate (100 µM) or PAO (10 µM) or diamide (1 mM) for 60 min. Cells were rinsed with MEM and were challenged with 4-HNE (50 µM) in the presence of 0.5% ethanol for 30 min. PLD activation was quantified by measuring the formation of [32P] PEt as described under MATERIALS AND METHODS. Values are mean ± SD of triplicate determination. Radioactivity associated with [32P] PEt (dpm/dish): vehicle, 144 ± 42 and 4-HNE, 391 ± 43.


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  		Figure 4.   Dose-dependent effect of vanadate, phenylarsine oxide, and diamide on 4-HNE-induced PLD activation. [32P]orthophosphate-labeled BPAEC (5 µCi/dish, 18 h) were pretreated with varying concentrations of vanadate (panel A) or diamide (panel B) or PAO (panel C) for 60 min. Cells were washed in MEM and were challenged with 4-HNE (50 µM) in the presence of 0.5% ethanol for 30 min. [32P] PEt was quantified as described under MATERIALS AND METHODS. Values are mean ± SD of triplicate determination.


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  		Figure 5.   Effect of vanadate and phenylarsineoxide on 4-HNE and TPA-induced PLD activation. [32P]Orthophosphate labeled BPAEC (5 µCi/dish, 18 h) were pretreated with vanadate (10 µM) or PAO (10 µM) for 15 min. Cells were washed in MEM and were challenged with 4-HNE (25 µM) or TPA (100 nM) in the presence of 0.5% ethanol for 30 min. [32P] PEt was quantified as described under MATERIALS AND METHODS. Values are mean ± SD of two independent experiments in triplicate.

                              
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TABLE 3
Effect of vanadate on H2O2, 4-HNE, and linolenic acid hydroperoxide-induced PLD activation

To determine whether Tyrk inhibitors would modulate the effect of vanadate plus 4-HNE-induced PLD activation, BPAEC were pretreated with genistein (35 µM) for 45 min followed by the addition of vanadate (10 µM) for 15 min. The cells were then challenged with MEM alone or MEM containing 4-HNE (50 µM) for 30 min. As shown in Table 4, genistein attenuated the 4-HNE and vanadate plus 4-HNE-induced PLD activation. These data further imply a role for Tyrks in 4-HNE-induced PLD stimulation.

                              
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TABLE 4
Effect of genistein on vanadate plus 4-HNE-induced PLD activation

Effect of 4-HNE on Protein Tyrosine Phosphorylation

To study the effect of 4-HNE on cellular protein tyrosine phosphorylation, BPAEC were treated with 4-HNE and cell proteins (10 µg of protein) in whole cell lysates were separated by SDS-PAGE followed by Western blot analysis with antiphosphotyrosine antibody. Exposure of BPAEC to varying concentrations 4-HNE resulted in a small but significant increase in protein tyrosine phosphorylation (Figure 6). However, in cells preincubated with vanadate, addition of 4-HNE (25 µM-100 µM) increased protein tyrosine phosphorylation of several cellular proteins including major bands between 40-60 kDa, 70-90 kDa, 110- 130 kDa. Vanadate (1-10 µM), in the absence of 4-HNE, exhibited only a small increase in tyrosine phosphorylation of proteins as compared to basal phosphorylation. Immunodetection of 4-HNE plus vanadate-induced protein tyrosine phosphorylation was prevented by co-incubating the blots with phosphotyrosine (2 mM) and antiphosphotyrosine antibody (Figure 7). However, co-incubation with phosphoserine (2 mM) or phosphothreonine (2 mM) had no effect on the 4-HNE plus vanadate-mediated increase in protein tyrosine phosphorylation (data not shown). These data suggest that the antiphosphotyrosine antibody used for the immunodetections was specific for tyrosine-phosphorylated proteins. To further demonstrate that the 4-HNE-induced protein phosphorylation was tyrosine kinase mediated, the effect of genistein (a putative tyrosine kinase inhibitor) was tested. Pretreatment of BPAEC with genistein (100 µM) attenuated the 4-HNE and 4-HNE plus vanadate-induced protein tyrosine phosphorylation (Figure 8). The effect of genistein was dose-dependent with an IC50 of approximately 150 µM (data not shown). To further demonstrate that 4-HNE modulated Tyrk, BPAEC were treated with MEM alone or MEM containing either 4-HNE (50 µM) or vanadate (10 µM) or vanadate (10 µM) plus 4-HNE (50 µM) for 30 min. Analysis of the Tyrk activity in cell lysates revealed that [32P] phosphorylation of poly (Glu4Tyr) was increased 235% by 4-HNE, while vanadate plus 4-HNE exhibited a 677% elevation in phosphorylation (Table 5). These results are highly suggestive of modulation of Tyrk activity by 4-HNE, vanadate, and 4-HNE plus vanadate.


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  		Figure 6.   Concentration-dependency of 4-HNE and vanadate plus 4-HNE-induced protein tyrosine phosphorylation. BPAEC were pretreated with MEM or MEM containing vanadate (10 µM) for 60 min and were challenged with varying concentrations of 4-HNE for 30 min. Cell lysates were subjected to SDS-PAGE and immunodetected with 4G10 antiphosphotyrosine antibody as described under MATERIALS AND METHODS. The immunoblot is representative of two similar experiments.


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  		Figure 7.   Effect of phosphotyrosine on immunodetection of 4-HNE plus vanadate-induced protein tyrosine phosphorylation. BPAEC were pretreated with MEM or MEM containing vanadate (10 µM) for 60 min. The cells were treated with MEM alone or MEM containing 4-HNE (50 µM) for an additional 30 min. Cell lysates were separated by SDS-PAGE and proteins containing phosphotyrosine were immunodetected with 4G10 antiphosphotyrosine antibody as described under MATERIALS AND METHODS. A typical immunoblot is shown, and lane marked phosphotyrosine was immunodetected in the presence of 2 mM phosphotyrosine.


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  		Figure 8.   Effect of genistein on 4-HNE and 4-HNE plus vanadate-induced protein tyrosine phosphorylation. BPAEC were incubated with MEM or MEM containing vanadate (10 µM) for 30 min followed by genistein (100 µM), as indicated, for an additional 60 min. Incubations were continued in the presence of 4-HNE (50 µM) for an additional 30 min. Cell lysates were subjected to SDS-PAGE and tyrosine phosphorylated proteins were immunodetected as described under MATERIALS AND METHODS.

                              
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TABLE 5
Effect of 4-HNE and vanadate on tyrosine kinase activity in cell lysates

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously demonstrated that 4-HNE-mediated activation of PLD in endothelial cells was insensitive to PKC inhibitors and downregulation of PKC and was independent of exogenous Ca2+ and intracellular changes in free Ca2+ (11). In this report, our findings indicate that 4-HNE-mediated activation of PLD is attenuated by putative Tyrk inhibitors such as genistein, erbstatin, and herbimycin and potentiated by PTPase inhibitors, suggesting Tyrk involvement in the regulation of PLD activation. In BPAEC, Tyrk inhibitors did not attenuate the TPA-induced PLD activation as compared with 4-HNE treatment. However, it has recently been shown that genistein and tyrphostin suppressed the TPA-induced activation of PLD in osteoblast-like cells suggesting protein tyrosine kinase regulation of PLD downstream from PKC (25).

As protein tyrosine phoshorylation is a balance between Tyrks and PTPases (26), we used PTPase inhibitors to offset this balance favoring Tyrks and thereby enhance protein tyrosine phosphorylation. The biological effect of vanadates are due to its properties as a phosphate analog while phenylarsine oxide complexes with vicinal thiol groups inhibiting the phosphatase activity (23). An important finding in the present study is that the potentiating effect of vanadate or PAO or diamide was specific for 4-HNE as these inhibitors showed no effect on TPA or agonist-mediated PLD activation. However, vanadate potentiated the stimulatory action of TPA on PLA2 activation in thioglycollate elicited mouse peritoneal- and bone marrow-derived macrophages (27). The mechanism(s) of vanadate-induced enhancement of 4-HNE-mediated PLD activation in BPAEC may be due to inhibition of PTPase or activation of Tyrk activity or both. Recent studies in neutrophils have shown that vanadate by itself or in the presence of H2O2 stimulated tyrosine phosphorylation of a number of cellular proteins (28). Further, qualitative similarity between protein tyrosine phosphorylation in response to H2O2 plus vanadate and to H2O2 plus insulin were observed in FAO cells (29). These data suggest that either these protein substrates are phosphorylated by the same Tyrk or by different Tyrks having similar substrate specificities. Similar to H2O2-induced increase in tyrosine phosphorylation of endothelial cell proteins (30, 31), 4-HNE or 4-HNE in the presence of vanadate also enhanced tyrosine phosphorylation of several proteins including 40-60 kDa, 70-90 kDa, and 110-130 kDa.

Our results suggest different mechanisms for agonist- and oxidant-induced stimulation of PLD (32). While agonist-induced PLD activation was dependent on cellular PKC stimulation (33), H2O2 and 4-HNE-mediated PLD activation was PKC and Ca2+-independent (11, 16). There is increasing evidence suggesting a role for protein tyrosine phosphorylation in the regulation of PLD activation. Platelet-derived growth factor (PDGF) has been shown to increase PLD activation in NIH 3T3 fibroblasts (34, 35), smooth muscle cells (36) and in TRMP cells, a canine kidney epithelial cell line (37). Albeit, PDGF signaling pathway involves autophosphorylation of its receptor, the mechanism of PDGF-mediated PLD activation is unclear. In NIH 3T3 fibroblasts, PDGF-mediated activation seems to be dependent on PLCgamma -1 tyrosine phosphorylation (34). In these cells, overexpression of PLCgamma -1 dramatically increased PLD activity and inhibition of PLCgamma -1 tyrosine phosphorylation by staurosporine and genistein decreased PLD activity (34). Direct evidence for the downstream effect of PLCgamma -1 in PDGF-mediated PLD activation was demonstrated in Ki-ras transfected NIH 3T3 cells (35). However, PLD activation in PDGF-stimulated vascular smooth muscle cells appears to be receptor-kinase independent (36). In human platelets, activation of PLD by thrombin was inhibited by protein tyrosine kinase inhibitors suggesting a role for protein tyrosine kinases (38).

The precise mechanism by which PKC or tyrosine kinases regulate PLD activation is unclear. Although PKC activation is critical to TPA-and agonist-induced PLD activation (33), studies of Conricode and associates (39) have provided evidence for no direct role for ATP-dependent protein phosphorylation in the TPA-mediated PLD activation. However, recent studies in human neutrophils suggest that a conventional isoform of PKC phosphorylates a target protein in the plasma membrane in ATP[gamma ]s/TPA-mediated PLD activation (40). The nature of the PKC-phosphorylated protein in neutrophil plasma membranes is yet to be identified. The small molecular weight GTP-binding proteins, ADP-ribosylation factor and Rho, have been identified in PLD activation (41). It is possible that these small molecular weight GTP-binding proteins may be modulated by 4-HNE through Tyrks as part of the PLD signaling pathway. In this regard, it is interesting to note that genistein inhibited the ability of ATP to stimulate PLD in GTPgamma S-treated leukocytes implicating G-proteins in Tyrk activation (44). Recently, activation of tyrosine kinases and PLD in intact small arteries by noradrenaline was demonstrated to be linked to G-protein and tyrosine phosphorylation (45).

In conclusion, the present study indicates that 4-HNE-mediated activation of endothelial cell PLD may involve protein tyrosine phosphorylation. Further studies on the cloning and expression of PLD are in progress to understand the mechanism(s) of 4-HNE-induced PLD activation and signal transduction in endothelial cells.

    Footnotes

Address correspondence to: Dr. Viswanathan Natarajan, Department of Medicine, Pulmonary Division, Indiana University School of Medicine, 1001 W. 10th Street, WD OPW 425, Indianapolis, IN 46202

(Received in original form April 18, 1996 and in revised form December 2, 1996).

Acknowledgments: The writers thank Ms. Beverly Clark for her excellent administrative support and Rita Shamlal for her technical assistance. This study was supported by grants HLBI 47671, PHS KO4 03095 from the National Institute of Health and the American Lung Association Career Investigator Award (V.N.).

Abbreviations BPAEC, bovine pulmonary artery endothelial cells; DAG, diacylglycerol; PA, phosphatidic acid; PAO, phenylarsineoxide; PBS, phosphate buffered saline; PKC, protein kinase C; PLC, phospholipase C; PLD, phospholipase D; PEt, phosphatidylethanol; PTPase, protein tyrosine phosphatase; SDS-PAGE, sodium dodecylsulphate-polyacrylamide gel electrophoresis; TBST, Tris buffered saline/Triton X-100; TPA, 12-O-tetradecanoylphorbol 13-acetate; 4-HNE, 4-hydroxynonenal.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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4. Curzio, M., H. Esterbauer, C. DiMauro, and M. U. Dianzani. 1992. Influence of the lipid peroxidation product 4-hydroxynonenal in human neutrophil migration. Int. J. Tissue React. 6: 13-18 .

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