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Am J Physiol Heart Circ Physiol 290: H46-H54, 2006. First published August 19, 2005; doi:10.1152/ajpheart.00769.2005
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ANG II stimulates phospholipase D through PKC{zeta} activation in VSMC: implications in adhesion, spreading, and hypertrophy

Jean-Hugues Parmentier, Zoran Pavicevic, and Kafait U. Malik

Department of Pharmacology, University of Tennessee Health Science Center, Memphis, Tennessee

Submitted 20 July 2005 ; accepted in final form 11 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ANG II stimulates phospholipase D (PLD) activity and growth of vascular smooth muscle cells (VSMC). The atypical protein kinase C-{zeta} (PKC{zeta}) plays a central role in the regulation of cell survival and proliferation. This study was conducted to determine the relationship between ANG II-induced activation of PKC{zeta} and PLD and their implication in VSMC adhesion, spreading, and hypertrophy. ANG II stimulated PKC{zeta} activity with maximal activation at 30 s followed by a decline in its activity to 45% above basal at 5 min. Inhibition of PKC{zeta} activity with a myristoylated pseudosubstrate peptide or overexpression of a kinase-inactive form of PKC{zeta} decreased ANG II-induced PLD activity. Moreover, depletion of PKC{zeta} with selective antisense oligonucleotides also decreased ANG II-induced PLD activity. Interaction between PLD2 and PKC{zeta} in VSMC was detected by coimmunoprecipitation. ANG II-induced PLD activity was inhibited by the primary alcohol n-butanol but not the tertiary alcohol t-butanol. The functional significance of PKC{zeta} and PLD2 in VSMC adhesion, spreading, and hypertrophy was investigated. Inhibition of PKC{zeta} and PLD2 activity or expression attenuated VSMC adhesion to collagen I and ANG II-induced cell spreading and hypertrophy. These results demonstrate that ANG II-induced PLD activation is regulated by PKC{zeta} and suggest a crucial role of PKC{zeta}-dependent PLD2 in VSMC functions such as adhesion, spreading, and hypertrophy, which are associated with the pathogenesis of atherosclerosis and malignant hypertension.

vascular smooth muscle cells; protein kinase C{zeta}; angiotensin II

PHOSPHOLIPASE D (PLD) catalyzes the hydrolysis of phosphatidylcholine into phosphatidic acid and choline. Activation of PLD by neurotransmitters, hormones, or growth factors has been implicated in a wide range of biological actions, including cellular trafficking, inflammatory and immune response, mitogenesis, cellular differentiation, and apoptosis (23). To date, two PLD isoforms, PLD1 and PLD2, have been cloned and characterized (7, 11). PLD1 is activated by direct binding of classical PKCs and small G proteins of Arf and Rho family (13, 26). PLD2 requires phosphatidylinositol 4,5-bisphosphate and is not or is less responsive to Arf, Rho, and protein kinase C (PKC) than PLD1 (4, 19).

ANG II contributes to the regulation of vascular tone and blood pressure (5). Hypertrophy of vascular smooth muscle cells (VSMC) induced by ANG II is an important feature of hypertension, and the structural changes in the vessel wall contribute to the increase in vascular resistance (25). VSMC response to ANG II, i.e., hyperplasia vs. hypertrophy, is determined by autocrine expression of transforming growth factor-{beta} (TGF-{beta}) (12). ANG II in most cases does not increase DNA synthesis in VSMC. However, it increases protein synthesis and causes hypertrophy of confluent quiescent VSMC (3). A key event in neointima formation and atherogenesis is the migration of VSMC and fibroblasts into the intima. This is controlled by cytokines and extracellular matrix components within the microenvironment of the diseased vessel wall. The extracellular matrix is an important factor for the regulation of cell behavior and the functions of VSMC such as adhesion and spreading (40). A variety of cytokines and growth factors accompanying vascular diseases such as hypertension and atherosclerosis are able to influence cell adhesion and spreading (15, 32). ANG II enhances adhesion and spreading of human VSMC (17).

ANG II stimulates PLD activity in VSMC through the angiotensin type 1 (AT1) receptor (2, 10). ANG II-induced PLD stimulation in VSMC has been involved in the activation of NADPH oxidase (37), ERK (34), Akt (21), and cell growth (8). PLD may contribute to the functional effects of ANG II on VSMC through the formation of phosphatidic acid and/or its metabolites (30). Several proteins have been involved in mediating PLD activation elicited by ANG II in VSMC such as heterotrimeric and small G proteins (1, 38) and phospholipase A2 (27). PLD may contribute to the functional effects of ANG II on VSMC through the formation of phosphatidic acid and/or its metabolites (27–30). ANG II selectively activates PLD2 isoform in VSMC (27). However, the exact signaling pathway regulating ANG II-induced PLD2 activation and its functional significance in VSMC has not yet been established.

The atypical PKC isoforms are both calcium and diacylglycerol independent. PKC{zeta} is a critical component of mitogenic signaling in many cell types, mediating ANG II-induced activation of ERK in VSMC (22). Moreover, PKC{zeta} mediates polymorphonuclear neutrophil adhesion and chemotaxis (20) and endothelial adhesion through phosphorylation of intercellular adhesion molecule-1 (16). However, the involvement of PKC{zeta} or PLD in VSMC adhesion or spreading is not known. The present study was conducted to determine the contribution of PKC{zeta} to PLD activation in response to ANG II in VSMC and their potential role in VSMC functions associated with atherosclerosis and malignant hypertension such as adhesion, spreading, and hypertrophy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. ANG II was from Bachem (King of Prussia, PA); myristoylated PKC{zeta} and PKC{alpha}/{beta}(20–28) peptide inhibitors were from Biomol (Plymouth meeting, PA); Ro-31-8220, Go-6976, and bisindolylmaleimide V were from Calbiochem (San Diego, CA); [3H]oleic acid (50 Ci/mmol) was from ARC (St. Louis, MO); [3H]leucine (40 Ci/mol), [3H]thymidine (20 Ci/mmol), and [{gamma}-32P]ATP (3,000 Ci/mmol) were from Amersham (Arlington Heights, IL).

Culture of VSMC. Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA), maintained according to our Institutional Guidelines and by the Animal Care Committee at the University of Tennessee Health Science Center, were anesthetized with pentobarbital sodium (Sigma); the aorta was removed, and VSMC were isolated and cultured as previously described (27).VSMC between passages 3 and 8 were used for experiments.

Transient transfections. VSMC were transfected with 200 nM antisense or scrambled oligonucleotides designed from the first six codons of rat PKC{zeta} cDNA sequence (GenBank NM_022507) (24). A BLAST search with PKC{zeta} confirmed its selectivity. Nuclease-resistant phosphorothioate oligonucleotides (IDT, Coralville, IA) were complexed with Oligofectamine reagent (Invitrogen-Life Technologies, Gaithersburg, MD) according to the manufacturer's instructions. The oligonucleotide mix was added to the cell or tissue as described in the experimental protocol specified for each type of experiment. Protein levels of PKC{zeta} were measured by Western blot analysis to show the selective decrease in the protein level. For experiments with pCMV5-FLAG-tagged PKC{zeta} plasmids (gift from Dr. A. Toker, Boston Biomedical Research Institute, Boston, MA; and R. Farese, Univ. of South Florida, Tampa, FL), cells in six-well plates were transfected with wild-type PKC{zeta} and kinase-deficient T410A PKC{zeta} for 48 h using Lipofectamine Plus (Invitrogen-Life Technologies). For experiments with PLD2 plasmids, pCGN-hemagglutinin (HA)-wild type-PLD2 and catalytically inactive K758R-PLD2 were used (gift from M. Frohman, State University of New York, Stony Brook, NY). Transfection of VSMC with PLD2 plasmids has been successfully achieved in our laboratory (27, 28). Transfection efficiencies were determined by Western blot analysis by using nPKC{zeta} antibody (Santa Cruz Biotechnology) or PLD2 antibody (gift from S Bourgoin, Universite Laval, Laval, Quebec, Canada), an anti-FLAG antibody (Sigma) or anti-HA antibody (Santa Cruz Biotechnology) for ectopic expression of PKC{zeta} or PLD2, respectively.

Western blot analysis. The efficiency of transient transfection with both oligonucleotides and plasmids was determined by Western blot analysis. Briefly, VSMC treated with oligonucleotides were washed twice with PBS and scraped in radioimmunoprecipitation assay (RIPA) buffer. Cell lysates were subjected to sonication, and Laemmli sample buffer was added. Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. Blocking was performed with TBS buffer (20 mM Tris·Cl, pH 7.6, 200 mM NaCl) containing 5% nonfat dry milk powder. The membrane was then incubated with nPKC{zeta} antibody (1:250) overnight. The membranes were subsequently washed, incubated with horseradish peroxidase-linked secondary antibodies, and rinsed and developed with enhanced chemiluminescence reagents (Amersham). To determine transfection efficiencies of VSMC with FLAG-tagged PKC{zeta} plasmids, the top aqueous layer and insoluble fraction obtained from samples for PLD assay were precipitated with four volumes of ice-cold acetone, incubated 1 h at –80°C, and pelleted and dried under nitrogen. The pellet was resuspended in boiling Laemmli buffer and treated as described above. Ectopic expression of FLAG-PKC{zeta} was detected with an anti-FLAG antibody. Protein level of PLD2 was determined by Western blot analysis as previously described (27, 28). PKC{alpha} was detected with a PKC{alpha} antibody (Santa Cruz Biotechnology).

PKC{zeta} assay. The activity of PKC{zeta} was determined according to the method described (29). VSMC in 100-mm dishes were washed twice with PBS and scraped in RIPA buffer. Cells lysates were incubated with rabbit polyclonal PKC{zeta} antibody (Santa Cruz Biotechnologies) for 3 h, and the immunocomplex was captured with a 50% slurry of protein A-agarose beads. PKC{zeta} immunoprecipitates were washed twice with high-salt (50 mM Tris·Cl, pH 7.5; 10 mM MgCl2; 0.5 M LiCl) and low-salt (50 mM Tris·Cl, pH 7.5; 10 mM MgCl2) buffers and incubated with a kinase buffer (50 mM Tris·Cl, pH 7.5; 10 mM MgCl2; 0.2 mM EGTA, 50 µM ATP) containing 50 µM {epsilon}-peptide and 3 µCi [{gamma}-32P]ATP at 30°C. The reaction was stopped by the addition of 200 mM EDTA, and the proteins were precipitated by the addition of 25% TCA. The solutions were centrifuged for 1 min at 14,000 rpm, and supernatants were spotted onto p81 phosphocellulose filters. Filters were washed with 1% (vol/vol) orthophosphoric acid and analyzed by Cerenkov counting. PKC{zeta} activity was calculated from the amount of 32P incorporated into the {epsilon}-peptide.

The phosphorylation of PKC{zeta} at Thr410, required for full PKC{zeta} activity, was measured on VSMC lysates after treatment with ANG II by Western blot analysis using a phospho-PKC{zeta}/{lambda} antibody (Cell Signaling Technologies) at 1:1,000 dilution. Densitometric analysis of the bands was performed using Image J (NIH).

PLD activity. PLD activity in VSMC was assayed as described previously (27, 28).

Immunoprecipitation assay. Cells lysates were incubated overnight with a rabbit polyclonal PLD2 antiserum (a kind gift from Dr. Sylvain Bourgoin), and the immunocomplex was captured with a 50% slurry of protein A-agarose beads. PLD2 immunoprecipitates were washed once with RIPA buffer and twice with PBS. Samples were resuspended in lysis buffer, and Laemmli sample buffer was added. Proteins were separated and transferred on membranes and incubated with PLD2 antiserum (1:1,000), PKC{zeta} (1:250), or PKC{alpha} (1:250) antibodies (Santa Cruz Biotechnologies). HA-PLD mix was a kind gift from Dr. Michael Frohman.

DNA and protein synthesis. DNA synthesis and protein synthesis were determined by quantitating the incorporation of [3H]thymidine or [3H]leucine, respectively. Cells (0.5 ml), adjusted to 80,000 cells/ml, were plated in 24-well plates for 24 h and incubated in serum-free medium for 48 h to induce mitotic quiescence. Treatments (alcohols or PLD2 transfection) are indicated in Fig. 9. [3H]thymidine (0.5 µCi/well) or [3H]leucine (0.25 µCi/well) was added 6 h before processing the cells. Cells were washed three times with PBS, 10% TCA, and ethanol/ether (2:1). 3H-labeled proteins were extracted with 0.1% SDS-0.1 N NaOH, and radioactivity was measured by scintillation spectroscopy. [3H]thymidine or [3H]leucine incorporation was measured as disintegrations per minute per well and expressed as percent change from basal.



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  		Fig. 9. Effect of PKC{zeta} and PLD inhibition on ANG II-induced protein synthesis. A: effect of PKC{zeta} depletion on leucine incorporation in rat VSMC. Cells in 24-well plates were transfected with 200 nM PKC{zeta} AS or SCR for 48 h, and ANG II was added during the last 24 h. Data are expressed as percent change in [3H]leucine incorporation from basal (11,051 ± 1,452 dpm/well); n = 24. *P < 0.05, ANG II + oligonucleotides vs. ANG II alone. B: effect of butanol alcohols on leucine incorporation. Cells in 24-well plates were serum deprived for 48 h and treated with n-but (0.4% vol/vol) or t-but (0.4% vol/vol) for 30 min before the addition of 100 nM ANG II (24 h). Basal = 8,943 ± 2,484 dpm/well, n = 20. *P < 0.05, ANG II + but vs. ANG II alone. C: effect of catalytically inactive PLD2 on leucine incorporation. Cells were transiently transfected with wt PLD2 or catalytically inactive PLD2 for 72 h, and ANG II was added during the last 48 h. [3H]leucine incorporation was determined as described in MATERIALS AND METHODS. Data are expressed as percent change in [3H]leucine incorporation from wt PLD2-transfected cells (14,306 ± 877 dpm/well); n = 12. *P < 0.05, wt PLD2 vs. K798R PLD2.

 
Cell adhesion assay and cell spreading. Collagen I-coated 24-wells plates (BD Biosciences) were washed three times with PBS and incubated for 2 h at room temperature. VSMC (0.5 ml), adjusted to 80,000 cells/ml, were added to 24-well plates. PLD inhibitor (0.4% vol/vol n-butanol) or its vehicle with or without ANG II was directly added to the cell suspension before addition to the 24-well plates. After 1 h at 37°C, cell adhesion was stopped by removal of the medium. Nonadherent cells were washed off, and attached cells were fixed with 0.5 ml acetone-methanol (1:1) and stained with 0.2% crystal violet (Fisher Scientific). For quantitation, residual cell-bound stain was solubilized in 2% SDS, and optical density was measured at 560 nm (41). Cell attachment was always measured after 1 h and in sextuplicate, and experiments were repeated three times. The ability of cells to spread was determined by measuring the ratio of cells that spread after a defined time (17, 41). Bright-field images of marked microscope fields were collected at x40 magnification after 5 h treatment with various agents and fixation and staining with hematoxylin. The analysis of cell spreading was based on cell morphology. Cells that have a recognizable nucleus with a developed cytoplasm or a noncircular shape with extended protrusions are considered spread cells, whereas nonspread cells retained a round shape with uneven outline. Cells were counted in three different fields, and experiments were repeated three times in duplicates.

Statistical analysis. Results of PKC{zeta} and PLD assays, thymidine and leucine incorporation, adhesion, and spreading are presented as the mean relative increase above the basal level (±SE) as the details are given in the figure legend for each assay. The n value in figure legends refers to the number of experiments. ANOVA and paired or unpaired t-test were performed for statistical analysis as appropriate. Values of P < 0.05 were considered statistically significant. The phospho-PKC{zeta}/total PKC{zeta} ratio was calculated from densitometric analysis of the bands and arbitrarily chosen as 100% for time 0 and expressed as fold change from the value obtained at time 0 for the subsequent time course during treatment with ANG II. Mean ± SD of the ratio was calculated from three different Western blot analyses.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
ANG II stimulates PKC{zeta} activity in rat VSMC. We measured the activity of immunoprecipitated PKC{zeta} using a selective peptide as substrate (Fig. 1A). ANG II stimulated PKC{zeta} activity in a time-dependent manner with maximal activation (105% above basal) at 30 s, followed by a gradual decline to ~45% above basal at 5 min (Fig. 1A). FBS (10%), which was included as positive control, stimulated PKC{zeta} activity at 5 min to the same extent as ANG II at 30 s. These data demonstrate a rapid activation of PKC{zeta} in response to ANG II in rat VSMC. The phosphorylation of PKC{zeta} at Thr410, a phosphorylation site required for PKC{zeta} activation, was also measured using a phospho-PKC{zeta}/{lambda} antibody (Fig. 1B). PKC{zeta} was already phosphorylated at Thr 410 in unstimulated cells, and treatment with ANG II (30 s to 10 min) did not increase this basal phosphorylation.



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  		Fig. 1. Effect of ANG II treatment on protein kinase C-{zeta} (PKC{zeta}) activity in rat vascular smooth muscle cells (VSMC). A: cells were serum deprived for 48 h and incubated with 100 nM ANG II for different periods of time. FBS (10%, 5 min) was used as control. Cells were lysed and immunoprecipitated with PKC{zeta} antibody for a kinase assay performed using [{gamma}-32P]ATP and a selective peptide substrate as described in MATERIALS AND METHODS. Values are means ± SE of 4 independent experiments and are expressed as the percent increase over basal PKC{zeta} activity. *P < 0.05 vs. basal. B: PKC{zeta} phosphorylation measured by Western blot analysis. Cells were treated as in A and lysed in radioimmunoprecipitation assay (RIPA) buffer, and samples were prepared for Western blot analysis and incubated with a phospho (p)-PKC{zeta}/{lambda} (Thr 410/403) or a PKC{zeta} antibody as described in MATERIALS AND METHODS. The phospho-PKC{zeta}/PKC{zeta} ratio (n = 3) was calculated from densitometric analysis as described in MATERIALS AND METHODS.

 
PKC{zeta} mediates ANG II-induced PLD activation. We have previously reported that ANG II stimulates PLD activity, which was due to selective activation of PLD2 isoform in VSMC (27). To assess the contribution of PKC{zeta} to PLD2 activation in rat VSMC, we examined the effects of myristoylated PKC{zeta} pseudosubstrate peptide inhibitor (PS{zeta}), a selective inhibitor of PKC{zeta} and Ro-31-8220, a broad range PKC inhibitor. PS{zeta} (5 µM, 1 h) and Ro-31–8220 (1 µM, 30 min) decreased ANG II-induced PLD activity by 62% and 58%, respectively (Fig. 2). Bisindolylmaleimide V, an inactive analog of Ro-31-8220, did not alter PLD activity. Classical PKC isoforms were not involved in PLD activation because the inhibitors of PKC{alpha}/{beta}/{gamma} isoforms, myristoylated PKC{alpha} pseudosubstrate peptide inhibitor (PS{alpha}, 5 µM, 1 h) and Go-6976 (10 µM), did not alter ANG II-induced PLD activity. To further determine the role of PKC{zeta} in PLD activation, rat VSMC were transfected with a PKC{zeta} antisense oligonucleotide to deplete the corresponding protein levels (Fig. 3A). ANG II-induced PLD activity was inhibited in cells transfected with the antisense (64% decrease vs. ANG II alone), whereas the scrambled oligonucleotide did not alter PKC{zeta} protein level (Fig. 3A) or ANG II-induced PLD activity (Fig. 3B). To further establish the contribution of the catalytic activity of PKC{zeta} to PLD activation, we examined the effect of ANG II on PLD activity in VSMC transiently transfected with wild-type and kinase-deficient T410A PKC{zeta}. Figure 4A shows the ectopic expression of FLAG-tagged PKC{zeta} constructs in rat VSMC. Overexpression of kinase-deficient PKC{zeta}, but not wild-type PKC{zeta}, decreased ANG II-induced PLD activation by 63% (Fig. 4B). These results suggest that ANG II activates PLD through PKC{zeta} in rat VSMC.



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  		Fig. 2. Effect of selective PKC inhibitors on PLD activity. [3H]oleic acid-labeled VSMC were preincubated with myristoylated pseudosubstrate peptide inhibitor for PKC{zeta} (PS{zeta}, 5 µM, 1 h) or for PKC{alpha} (PS{alpha}, 5 µM, 1 h), Ro-31-8220 (Ro, 1 µM, 30 min), bisindolylmaleimide V (Bis V, 1 µM, 30 min), or Go-6976 (10 µM, 30 min). Cells were then exposed to 100 nM ANG II for 15 min. PLD activity was measured as described in MATERIALS AND METHODS. Data are expressed as percent change in PLD activity from basal activity. Values are means ± SE of 3 independent experiments performed in duplicate. *P < 0.05 vs. vehicle. PEt, phosphatidylethanol.

 


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  		Fig. 3. Effect of PKC{zeta} antisense oligonucleotides on PLD activity. A: representative Western blot showing the effect of treatment with PKC{zeta} antisense (AS) and scrambled (SCR) oligonucleotide treatment on PKC{zeta} protein level. VSMC were incubated with 200 nM PKC{zeta} AS or SCR oligonucleotides or with vehicle (Oligofectamine) for 48 h, and samples were prepared for Western blot analysis as described in MATERIALS AND METHODS. Experiments were repeated 3 times. B: VSMC were incubated with oligonucleotides as described in A and labeled with [3H]oleic acid for 18 h. Cells were exposed to 100 nM ANG II for 15 min, and PLD activity was measured as described in MATERIALS AND METHODS. Data are expressed as percent change from basal PLD activity in unstimulated Oligofectamine-treated cells. Values are means ± SE of 3 independent experiments performed in duplicate. *P < 0.05, ANG II + oligonucleotides vs. ANG II alone.

 


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  		Fig. 4. Effect of kinase-inactive PKC{zeta} on PLD activity. A: representative Western blot showing the expression of FLAG-tagged PKC{zeta} plasmid after transfection. Cells were transfected with wild-type (wt) PKC{zeta}, kinase-deficient T410A PKC{zeta} (T410A), or vehicle (Lipofectamine) for 48 h, and samples were treated for Western blot analysis using a FLAG antibody as described in MATERIALS AND METHODS. Experiments were repeated 3 times. B: VSMC were transfected with plasmids as shown in A and labeled with [3H]oleic acid for 18 h. Cells were exposed to 100 nM ANG II for 15 min. PLD activity was measured as described in MATERIALS AND METHODS. Data are expressed as percent change from basal PLD activity in unstimulated Lipofectamine-treated cells. Values are means ± SE of 3 independent experiments performed in duplicate. *P < 0.05, ANG II + plasmids vs. ANG II alone.

 
Constitutive interaction of PKC{zeta} and PLD2 in VSMC. The possible interaction between PLD2 and PKC{zeta} after stimulation with ANG II was studied by coimmunoprecipitation without protein overexpression because we are using a physiologically relevant model of cultured VSMC. PKC{zeta} and PLD2 coimmunoprecipitated independently of ANG II stimulation (5 min) (Fig. 5). PKC{alpha} did not interact with PLD2 in VSMC. A mix of purified HA-tagged PLD1 and PLD2 proteins was used as positive control for PLD2. PKC{zeta} and PKC{alpha} were also detected in this sample mix (Fig. 5).



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  		Fig. 5. Coimmunoprecipitation of PKC{zeta} and PLD2 in VSMC. VSMC cultured in 100-mm dishes were serum deprived for 48 h before exposure to ANG II for 5 min. The medium was removed, and cells were washed twice with PBS and lysed for 10 min in RIPA buffer. Immunoprecipitation (IP) was carried out as described in MATERIALS AND METHODS. A mix of purified hemagglutinin-tagged PLD1 and PLD2 proteins (PLD lane) was used as control for the identification of PLD2. PKC{alpha} and PKC{zeta} were also found in the PLD mix.

 
Characterization of the selectivity of n-butanol as inhibitor of PLD activity in rat VSMC. The transphosphatidylation reaction occurs when primary alcohols interfere with PLD activity, leading to the synthesis of inactive phosphatidylethanol instead of the reactive phosphatidic acid. PLD inhibition with n-butanol, a primary alcohol that can act as an acceptor for the phosphatidyl moiety, generates inactive phosphatidylbutanol instead of phosphatidic acid (28, 39). Ethanol, like all primary alcohols, is similar in its inhibitory effect on PLD activity to n-butanol. We did not use ethanol to inhibit PLD activity because ethanol was used as the substrate for PLD assay. The tertiary alcohol t-butanol does not react with the phosphatidyl moiety of phosphatidylcholine and is incapable of blocking the synthesis of phosphatidate. In this study, n-butanol (0.4% vol/vol), but not t-butanol (0.4% vol/vol), reduced ANG II-induced PLD activation by 61% (Fig. 6). A higher concentration of n-butanol (1% vol/vol) further reduced ANG II-induced PLD activity but also decreased basal activity by 19% in the absence of ANG II. Moreover, t-butanol also reduced PLD activity at the same concentration most likely because of a nonspecific effect. These data show that n-butanol at a concentration of 0.4% (vol/vol) is selective in inhibiting PLD activity.



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  		Fig. 6. Effect of n-butanol (n-but) on PLD activity. [3H]oleic acid-labeled VSMC were preincubated with 0.4% or 1% (vol/vol) n-but or t-butanol (t-but) for 30 min. Cells were then exposed to 100 nM ANG II for 15 min, and PLD activity was measured as described in MATERIALS AND METHODS. Data are expressed as percent change in PLD activity from basal activity. Values are mean ± SE of 4 independent experiments performed in duplicate. *P < 0.05, ANG II + n-but vs. ANG II alone.

 
Contribution of PKC{zeta} and PLD activity to cell adhesion and spreading. Adhesion onto the matrix is an early event in the regulation of cell migration toward a gradient of chemoattractant (41). Adhesion of rat VSMC on collagen I-coated surface was measured 1 h after plating and quantified from dye absorbance at 560 nm. Cell adhesion remained unchanged by the addition of ANG II (Fig. 7). Inhibition of PKC{zeta} with 5 µM PS reduced cell adhesion by 63% in presence or absence of ANG II, whereas PS{alpha} did not alter basal cell adhesion (Fig. 7A). n-Butanol at noncytotoxic concentration (0.4% vol/vol) reduced cell adhesion by one-third either with or without addition of ANG II. t-Butanol did not alter VSMC adhesion on collagen (Fig. 7B). These data demonstrate that PKC{zeta} and PLD are involved in spontaneous cell adhesion of rat VSMC on collagen. To investigate the contribution of PKC{zeta} and PLD to the motility of VSMC, we monitored the change in cell morphology during spreading experiments (Fig. 8). The phase-contrast images of the selected fields were analyzed to evaluate the number of spread cells relative to the total number of attached cells. The basal rate of cell spreading on collagen was 73–78% after 5 h (Tables 1 and 2). Treatment with ANG II increased the ratio of cell spreading to 93%. Treatment with PS{zeta} with or without ANG II reduced the ratio of spread cells to 5% (Table 1), whereas PS{alpha} did not alter cell spreading (not shown). n-Butanol reduced the ratio of spread cells to 16 and 19% in the absence or presence of ANG II, respectively. t-Butanol did not alter basal spreading or reverse the effect of ANG II (Table 2). Catalytic activity of PKC{zeta} and PLD seems crucial for the spontaneous as well as ANG II-induced spreading of rat VSMC on collagen.



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  		Fig. 7. Effect of PKC{zeta} and PLD inhibition on rat VSMC adhesion. Cells (40,000 in 0.5 ml) were resuspended with 5 µM PS{zeta} or 5 µM PS{alpha} (A) or with 0.4% n-but or t-but (B) with or without 100 nM ANG II and incubated for 1 h in collagen-coated 24-wells; adhesion was determined by measuring the absorbance at 560 nm as described in MATERIALS AND METHODS. Data are expressed as percent change from adhesion in untreated cells (A: basal = 0.457 ± 0.048 OD560; B: basal = 0.406 ± 0.074 OD560; OD560 is optical density at 560 nm). *Significantly different from basal, P < 0.05; n = 12.

 


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  		Fig. 8. Effect of PKC{zeta} and PLD inhibition on cell spreading. Representative pictures of rat VSMC (40x magnification) spread on collagen after 5 h of incubation. Cells were incubated with vehicle or ANG II (100 nM) in the presence or absence of the designated alcohol (0.4% vol/vol) or PS{zeta} (5 µM). Wells were washed with PBS to remove nonadherent cells and were fixed and stained as described in MATERIALS AND METHODS. Quantification of spreading is shown in Tables 1 and 2.

 

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  		Table 1. Effect of PKC{zeta} inhibition on rat VSMC spreading

 

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  		Table 2. Effect of PLD inhibition on rat VSMC spreading

 
PKC{zeta} and PLD mediates ANG II-induced hypertrophy in rat VSMC. After adhesion and spreading, ANG II promotes cellular hypertrophy as a result of increased protein synthesis (31, 33). Protein synthesis measured by [3H]leucine incorporation was used as a marker for cell hypertrophy. Depletion of PKC{zeta} with antisense but not scrambled oligonucleotides abolished ANG II-induced protein synthesis (Fig. 9A), indicating an essential role for PKC{zeta} in mediating VSMC hypertrophy. Inhibition of PLD activity with n-butanol, but not t-butanol, attenuated ANG II-induced [3H]leucine incorporation (Fig. 9B). We have previously shown that the isoform PLD2, but not PLD1, mediates ANG II-induced PLD activation in VSMC (27). Moreover, the catalytically inactive K758R PLD2 mutant acts as dominant negative when overexpressed in VSMC (27). In rat VSMC, overexpression of K798R PLD2, but not wild-type PLD2, abolished ANG II-induced increase in [3H]leucine incorporation (Fig. 9C). ANG II did not increase DNA synthesis (vehicle = 708 ± 46 dpm vs. ANG II = 684 ± 56 dpm, P < 0.05). However, n-butanol, but not t-butanol, significantly reduced basal [3H]thymidine incorporation (n-butanol = 496 ± 60 dpm vs. t-butanol = 728 ± 68 dpm, P < 0.05). These results suggest that PLD2 isoform mediates cellular hypertrophy induced by ANG II in rat VSMC and that PLD2 contributes to basal DNA synthesis in these cells.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates that ANG II-induced PLD activation is regulated by PKC{zeta} in VSMC and that PKC{zeta} and PLD contribute to the adhesion, spreading, and hypertrophy of VSMC. ANG II stimulates PLD activity through AT1 receptors in VSMC (9). A multitude of signal transduction components including G proteins, kinases, and growth factor receptors have been shown to regulate PLD activation elicited by ANG II in VSMC (36). In contrast to PLD1, PLD2 activity is not directly regulated in vivo by classical PKC isoforms or small G proteins (4, 13, 19, 26). We have reported the selective activation of PLD2 isoform in response to ANG II in VSMC (27). In this study, we evaluated the role of PKC{zeta} in ANG II-induced PLD activation in rat VSMC using three different strategies. Pharmacological inhibition of atypical PKCs with PS{zeta}, a pseudosubstrate peptide inhibitor, or Ro-31-8220, a broad range PKC inhibitor, reduced ANG II-induced PLD activation. Classical PKC isoforms were not involved in PLD activation since PS{alpha} and Go-6976, selective for PKC{alpha}/{beta}/{gamma} isoforms, did not alter PLD activity. Depletion of PKC{zeta} with antisense oligonucleotides also reduced ANG II-induced PLD activation. Finally, ectopic expression of a kinase-inactive PKC{zeta} also reduced PLD activation. Therefore, PKC{zeta} mediates ANG II-induced PLD activation in rat VSMC.

Activation of PKC{zeta} in rat VSMC is rapid and occurs within seconds of stimulation by ANG II and is followed by a decline in activity. This rapid pattern of activation has also been reported in endothelial cells (16). However, we were unable to measure an increase in PKC{zeta} phosphorylation using phosphoantibodies due to an elevated basal phosphorylation in rat VSMC. PKC{zeta} is rendered fully active by two consecutive phosphorylations, Thr410 by PDK1 followed by autophosphorylation of Thr 560 in the activation loop of the catalytic site (14). Therefore, phosphorylation of Thr410 in intact VSMC did not correlate with increased PKC{zeta} activity measured after immunoprecipitation in our study. Our results reflect the possibility that ANG II may stimulate a pool of prephosphorylated and activated PKC{zeta} that is not able to correctly exert its kinase activity toward several substrates because of compartmentalization or steric constraint. For example, the catalytic site of PKC{zeta} could be blocked through an interaction with another protein, thus preventing substrate phosphorylation by an activated PKC{zeta}. In this model, stimulation of PKC{zeta} with ANG II could remove this inhibitory protein and therefore allow correct phosphorylation of PKC{zeta} substrates, including PLD2. The interaction between the PX domain of PLD2 and the kinase domain of PKC{zeta} in COS-7 cells has recently been reported (18). We also found native PKC{zeta} and PLD2 in the same complex in VSMC and in a purified mix of HA-tagged PLD1 and PLD2 proteins. However, the direct or indirect interaction of PKC{zeta} and PLD2 in VSMC is constitutive. Based on these observations, it appears that ANG II promotes a rapid and transient increase in PKC{zeta} activity of a Thr410-phosphorylated PKC{zeta} in VSMC. From these observations it follows that the activity of atypical PKC{zeta} is required for ANG II-induced PLD activation in VSMC in a manner that may be dependent on the interaction between PLD2 and PKC{zeta}.

Our study also shows that different stages of cell motility, namely adhesion and spreading, are dependent on PKC{zeta} and PLD activity. Cell attachment onto matrix proteins may be independent of signals initiated by cell membrane receptors (41), and a short-term stimulation with ANG II in our study did not increase cell adhesion onto collagen. However, the intrinsic activity of PKC{zeta} and PLD was important for VSMC adhesion. The functional effect of a basal activity of both PKC{zeta} and PLD would explain our results on cell adhesion showing that inhibition of PLD2 or PKC{zeta} attenuated spontaneous basal cell adhesion. However, the level of activity of PKC{zeta} or PLD in VSMC that are in suspension is not known. These cells in suspension may have different properties regarding PLD and PKC{zeta} regulation than adherent cells. Indeed, it seems likely that PLD and PKC{zeta} are activated in cells in suspension and their activities are required for cell adhesion because inhibition of the activity of these enzymes also markedly reduces cell adhesion. ANG II stimulates the spreading of VSMC on collagen and fibronectin (17) by a mechanism dependent on phosphatidylinositol 3-kinase and src (41). We found that ANG II increased the spontaneous spreading on collagen from 78% to 93% and that PKC{zeta} or PLD inhibition reduced both spontaneous and ANG II-induced VSMC spreading. Supporting this view is the report that phosphatidic acid, the product of PLD activity, stimulates cytoskeletal reorganization (35).

Hypertrophy of VSMC is an important feature of hypertension, and the structural changes in the vessel wall contribute to the increase in vascular resistance (25). ANG II exerts a direct trophic effect on VSMC, in addition to being a potent vasoconstrictor, thus contributing to the pathogenesis of hypertension and atherosclerosis (5). ANG II did not stimulate DNA synthesis under our experimental conditions. The stimulatory effect of ANG II on cardiac myocytes and VSMC hypertrophy, but not hyperplasia, are well documented (31, 33). Phosphatidic acid, the product of PLD activity, has been implicated as a potential signal transducer for cardiac hypertrophy (6). Moreover, the subsequent generation of diacylglycerol from phosphatidic acid may also contribute to sustained PLD activation in cardiomyocytes and VSMC (36). Inhibition of PLD activity with n-butanol or dominant negative PLD2 totally abrogated ANG II-induced protein synthesis, therefore indicating a critical role for PLD in smooth muscle cell hypertrophy. To our knowledge, this is the first report documenting the involvement of PLD as well as PKC{zeta} in ANG II-mediated VSMC hypertrophy. Increased adhesion of VSMC to the matrix and wide spreading could partly account for the hypertrophic response to ANG II and the lack of hyperplasia.

In conclusion, our study demonstrates for the first time that PKC{zeta} mediates ANG II-induced PLD activation in VSMC. In addition, we show that PLD contributes to some functional aspects involved in the pathophysiology of the cardiovascular system such as VSMC adhesion, spreading, and hypertrophy.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by a Beginning Grant-In-Aid of the American Heart Association (Southeast Affiliates) (J.-H. Parmentier) and National Heart, Lung, and Blood Institute Grant 19134–29 (K. U. Malik).


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Alex Toker and Dr. Robert Farese for providing us with the PKC{zeta} constructs, to Dr. Sylvain Bourgoin for the PLD2 antibodies, and to Dr. Michael Frohman for the PLD2 constructs. We thank Anne Estes for excellent technical skills and Dr. Lauren Cagen for editorial comments.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J.-H. Parmentier, Dept. of Pharmacology, Crowe Bldg., Rm. 211, The Univ. of Tennessee, 874 Union Ave., Memphis, TN 38163 (e-mail: jparmentier{at}utmem.edu)

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.


   REFERENCES
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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