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Cytosolic Phospholipase A₂-Mediated Regulation of Phospholipase D2 in Leukocyte Cell Lines¹

Jae Ho Kim^*,, Byoung Dae Lee, Yong Kim, Sang Do Lee, Pann-Ghill Suh and Sung Ho Ryu^2,

^* National Creative Research Initiative Center for Calcium and Learning, Department of Life Science and School of Environmental Engineering, Pohang University of Science and Technology, Pohang, South Korea

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phospholipase D (PLD) has been implicated in a variety of cellular processes, including inflammation, secretion, and respiratory burst. Two distinct PLD isoforms, designated PLD₁ and PLD₂, have been cloned; however, the regulatory mechanism for each PLD isoform is not clear. In our present study we investigated how PLD₂ activity is regulated in mouse lymphocytic leukemia L1210 cells, which mainly contain PLD₂ , and in PLD₂ -transfected COS-7 cells. Intriguingly, A23187, a calcium ionophore that induces calcium influx, potently stimulates PLD activity in these two cell lines, suggesting that Ca²⁺ might be implicated in the regulation of the PLD₂ activity. In addition to the A23187-induced PLD₂ activation, A23187 also increases PLA₂-mediated arachidonic acid release, and the A23187-stimulated PLD₂ and PLA₂ activities could be blocked by pretreatment of the cells with cytosolic calcium-dependent PLA₂ (cPLA₂) inhibitors, such as arachidonyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate in these two cell lines. Moreover, the A23187-induced PLD₂ and PLA₂ activities could be inhibited by cotransfection with antisense cPLA₂ oligonucleotide. These results suggest a role for cPLA₂ in the regulation of PLD₂ activity in vivo. The inhibitory effect of arachidonyl trifluoromethyl ketone on the A23187-induced PLD₂ activity could be recovered by addition of exogenous lysophosphatidylcholine. This study is the first to demonstrate that PLD₂ activity is up-regulated by Ca²⁺ influx and that cPLA₂ may play a key role in the Ca²⁺-dependent regulation of PLD₂ through generation of lysophosphatidylcholine.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phospholipase D (PLD)³ catalyzes the hydrolysis of phosphatidylcholine to produce choline and phosphatidic acid (PA). PA may act as a signaling molecule to activate specific targets that include protein kinases, protein tyrosine phosphatase, phosphatidylinositol-4-phosphate kinase, phospholipase C, and Ras GTPase-activating proteins (1). In addition, PA can be further metabolized to form diacylglycerol (DAG) by PA phosphohydrolase. DAG is a well-characterized activator of certain protein kinase C (PKC) isoforms (1). Thus, the PA and DAG formed via PLD activation have been implicated in a wide range of physiological processes, including inflammation, secretion, mitogenesis, and the respiratory burst in neutrophils (2, 3, 4).

PLD activity is stimulated by a great variety of hormones, growth factors, cytokines, and chemoattractants (5). Although it has been suggested that PKC, calcium, GTP-binding proteins, and protein tyrosine kinases are involved in the agonist-stimulated PLD activation (6), the molecular mechanism of PLD activation is still unknown. Recently, two distinct PLD isoforms, PLD₁ and PLD₂, have been cloned (7). The first cloned human PLD, hPLD₁, has low basal activity and is activated by ARF1, RalA, RhoA, and PKC in the presence of PIP₂ in vitro (7, 8, 9). PKC activates the PLD₁ activity by directly interaction with PLD₁ in the presence of PMA in PLD₁ -transfected COS-7 cells (10). In addition, it has been reported that PLD₁ activity is up-regulated by tyrosine phosphorylation in certain cell types, such as HL-60 granulocytes and Swiss 3T3 fibroblasts (11, 12).

A second isoform of PLD, PLD₂, also essentially requires PIP₂ for its enzymatic activity. In contrast to PLD₁, it is constitutively active in vitro, and its activity is not affected by PLD₁-activating factors, such as ARF1, RhoA, and PKC in vitro (13). Colley et al. reported that PLD₂ was highly active when it was transiently overexpressed in COS-7 cells (13). A recent report suggested that PLD₂ activity may be regulated by epidermal growth factor receptor through an unknown mechanism in PLD₂ -transfected HEK293 cells (14). However, it remains unclear how PLD₂ activity is regulated in vivo.

In addition to the PLD-activating factors, including PKC, GTP binding proteins, and protein tyrosine kinases, Ca²⁺ ions, too, have been linked to PLD activation in several cell types, such as mast cells, neutrophils, platelets, macrophages, and erythroleukemia cells (6). There is good evidence implicating Ca²⁺-dependent PLD activation in diverse physiological responses, such as superoxide generation and secretion in hemopoietic cell lines (4, 15). However, the molecular identity of the Ca²⁺-dependent PLD isoform and the molecular mechanism of Ca²⁺-induced PLD activation remain unknown.

In this report we show evidence that PLD₂ is highly expressed in both P388D1 macrophages and lymphocytic leukemic L1210 cells. In addition, for the first time we demonstrate that PLD₂ can be specifically activated by A23187-induced calcium influx and that Ca²⁺-dependent cytosolic PLA₂ (cPLA₂) is involved in Ca²⁺-dependent PLD₂ activation in L1210 and P388D1 cells. We, therefore, suggest the possible participation of PLD₂ in cellular responses that involve cPLA₂ activation in these cell lines.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents

[5,6,8,9,11,12,14,15-³H]Arachidonic acid ([³H]AA; 100 Ci/mmol) and [³H]myristic acid were purchased from DuPont-New England Nuclear (Boston, MA) and Amersham International (Aylesbury, U.K.), respectively. Arachidonyltrifluoromethyl ketone (AACOCF₃), methyl arachidonyl fluorophosphonate (MAFP), and phosphorothioate cPLA₂ antisense and control oligonucleotides were purchased from Biomol (Plymouth Meeting, PA). DMEM and FCS were obtained from Life Technologies (Gaithersburg, MD). PMA, mepacrine, and A23187 were purchased from Calbiochem-Novabiochem (La Jolla, CA). AA was obtained from Cayman Chemical (Ann Arbor, MI). Palmitoyl-lysophosphatidylcholine (lysoPC) and palmioyl-lysophosphatidylethanolamine (lysoPE) were purchased from Sigma (St. Louis, MO). The hPLD₂ cDNA and anti-cPLA₂ Ab were provided by Dr. J. David Lambeth (Emory University, Atlanta, GA) and D. K. Kim (Chung-ang University, Seoul, Korea), respectively. Polyclonal antisera that recognized both PLD₁ and PLD₂ were produced by immunization of a rabbit with a synthetic peptide corresponding to aa residues 1063–1072 of the sequence of human PLD₁ as described previously (16).

Cell culture

Four different hemopoietic cell lines, human monocytic leukemic U937, mouse lymphocytic leukemic L1210, mouse P388D1 macrophage, and human erythroleukemic K562, were originally obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 medium containing 10% FBS and 2 mM L-glutamine at 37°C in a humidified, CO₂-controlled (5%) incubator. COS-7 cells were maintained in DMEM containing high glucose, 10% bovine calf serum (HyClone, Logan, UT), and 2 mM L-glutamine at 37°C in a humidified, CO₂-controlled (5%) incubator.

Transfection and transient expression

For transient expression of PLD isoforms, COS-7 cells were plated at a density of 4 x 10⁵ cells/well in six-well plates and incubated overnight in a 5% CO₂ incubator. One microgram of DNA and 6 µl of LipofectAmine (Life Technologies, Gaithersburg, MD) were then used according to the manufacturer’s protocol. In antisense studies, COS-7 cells and L1210 cells were exposed to 1 µM phosphorothioate antisense or control cPLA₂ oligonucleotides in the presence of LipofectAmine. Twenty-four hours later, the cells were starved in DMEM or RPMI containing high glucose and 2 mM L-glutamine for 21 h, after which PLD and PLA₂ activities were measured.

Measurement of phosphatidylbutanol (PBtOH)

PLD activity was assayed by measuring the formation of PBtOH, the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol as previously described with a slight modification (17). The four hemopoietic cell cultures were harvested, resuspended at a density of 1 x 10⁶ cells/ml, starved in serum-free RPMI for 21 h, and then labeled with [³H]myristic acid (1 µCi/ml) for 3 h. Transfected COS-7 cells were also starved in serum-free DMEM for 21 h and then labeled with [³H]myristic acid. Unincorporated [³H]myristic acid was removed by washing with PBS. The cells were then treated with fresh serum-free medium with (or without for the control) agonists in the presence of 1% 1-butanol for the indicated times at 37°C. After the incubation the medium was aspirated, and 0.5 ml of ice-cold methanol was added to each well. The cell debris was then scraped into an Eppendorf tube, and chloroform and H₂O were added, resulting in a final chloroform/methanol/H₂O ratio of 1/1/1 (v/v/v). After vortexing, the tubes were centrifuged at 15,000 x g for 1 min, and the organic phase was harvested, dried, and spotted onto a Silica Gel 60 TLC plate, which was then developed with chloroform/methanol/acetic acid (90/10/10, v/v/v). The amounts of labeled PBtOH and total lipids were determined with a Fuji BAS-2000 image analyzer (Tokyo, Japan).

Measurement of AA release

The cells of four hemopoietic cells lines (1 x 10⁶ cells/ml) were incubated with [³H]AA (1 µCi/1 ml) in RPMI for 24 h as previously reported with a slight modification (18). COS-7 cells grown in six-well plates were loaded with [³H]AA (1 µC/1 ml) in DMEM. After three washes with fresh medium, 1 ml of serum-free medium with or without agonist was added, and the amount of free [³H]AA released into the supernatant was measured by liquid scintillation counting. The percentage of AA released was calculated according to the formula (S/(S + P)) x 100, where S and P are the radioactivities measured in equal portions of the supernatant and the cell pellet, respectively.

Immunoprecipitation and Western blotting

PLDs were immunoprecipitated essentially as previously reported (19). In brief, the cells were solubilized with ice-cold lysis buffer (20 mM HEPES-NaOH (pH 7.5), 2% cholic acid, 1 mM EGTA, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM PMSF) and precleared by centrifugation at 15,000 x g for 10 min at 4°C. Immunoprecipitations were performed with the addition of 30 µl of protein A-agarose (Pierce, Rockford, IL) coupled to 2 µg of anti-PLD Ab for 3 h, followed by a brief centrifugation. The precipitates were washed three times with washing buffer (20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.5% cholic acid, 1 mM EGTA, 1 mM EDTA, and 1 mM PMSF). For gel electrophoresis, the immunoprecipitates were boiled in Laemmli sample buffer, and the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany). Blocking was performed with TBS buffer (10 mM Tris/HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween-20) containing 5% skimmed milk powder. The membranes were then incubated with the primary Ab at the concentration recommended by the manufacturer for 2 h at room temperature. The immunoblots were subsequently washed and incubated with HRP-linked secondary Ab (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 1 h at room temperature, rinsed four times in TBS buffer, and developed with HRP-dependent chemiluminescence reagents (Amersham International).

Statistical analysis

Results are expressed as the mean ± SD from the number of determinations indicated. Student’s t test was used for comparison of individual treatments with their respective control values; p < 0.05 was accepted as a significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Distribution of PLD isozymes and effects of PMA and A23187 on the PLD activity in leukocyte cell lines

To elucidate how the PLD₂ activity is regulated in leukocytes, we examined agonist-induced PLD activities in several cell lines that exclusively expressed either PLD₁ or PLD₂. Employing immunoprecipitation with anti-PLD Ab and subsequent immunoblot analysis, we found that PLD₁ and PLD₂ were differentially expressed in four hemopoietic cell types: promonocytic U937, erythroleukemic K562, P388D1 macrophage, and lymphocytic leukemic L1210 cells. As shown in Fig. 1A, U937 and K562 cells mainly contained PLD₁, whereas PLD₂ was barely detectable in these cells. In contrast, mainly PLD₂ was detected in both P388D1 and L1210 cells. The expression levels of PLD₁ were far less than those of PLD₂; thus, P388D1 and L1210 cells made excellent models in which to study the regulatory mechanism pertaining to PLD₂ activity. To delineate the signaling pathways involved in PLD₂ activation, we examined the effects of PMA and A23187, substances that have been implicated in PLD activation in a variety of cell lines, on PLD activities in the four cell lines. PLD activity in U937 and K562 cells, which mainly expressed PLD₁, was potently stimulated by treatment with PMA (Fig. 1B), and PMA-induced PLD activity was higher than A23187-induced PLD activity in these two cell lines, suggesting a major role for PMA-dependent PKC in PLD₁ activation, which is consistent with previous reports (10, 13). On the other hand, both P388D1 and L1210 cells, which strongly express PLD₂, have a higher basal PLD activity than U937 and K562 cells. These results are in agreement with a previous report that stated that PLD₂ maintains high basal activity (13). Intriguingly, the PLD activities of both P388D1 and L1210 cells were more potently activated by treatment with A23187 than by treatment with PMA (Fig. 1B). The A23187-induced PLD activities in these cell lines were completely blocked by the presence of 2 mM EGTA in the medium, suggesting that A23187 is implicated in PLD activation via Ca²⁺ influx (data not shown). These results suggest that PLD₁ and PLD₂ must be differentially regulated by the two PLD-activating signals, PMA and A23187, and that the A23187-induced Ca²⁺ influx may be implicated in the up-regulation of PLD₂ in both L1210 and P388D1 cells.

View larger version (36K): [in this window] [in a new window]   FIGURE 1. Distribution of PLD isozymes and effects of PMA and A23187 on PLD activities in hemopoietic cell lines. A, Distribution of PLD isozymes in the indicated hemopoietic cell lines was measured by immunoprecipitation from lysates (20 mg of protein) of the cells and subsequent immunoblot analysis of the precipitates with anti-PLD Ab as described in Materials and Methods. The data are representative of four separate experiments. B, [3H]myristic acid-labeled cells were treated with 0.1% DMSO (control), 0.1 µM PMA, or 2 µM A23187 for 15 min at 37°C in the presence of 1% 1-butanol. The PLD activities were determined by measuring the formation of [3H]PBtOH using TLC. Data represent the mean ± SD from four separate experiments, each conducted in duplicate. An asterisk indicates a statistically significant difference (p < 0.05 vs control).

Despite the differential distribution of the PLD isoforms and the distinctive PLD activations in response to PMA or A23187 in the four hemopoietic cell lines, it remained unclear whether PLD₂ was specifically activated by the A23187-induced Ca²⁺ influx in both L1210 and P388D1 cell lines. To gain more insight into the effect of a Ca²⁺ increase on PLD₂ regulation, we transiently expressed either hPLD₁ or hPLD₂ in COS-7 cells, where we then measured PLD activity in response to PMA or A23187. As shown in Fig. 2A, overexpression of PLD₂ resulted in high basal activity (up to 5-fold higher than in the vector-transfected COS-7 cells), which could be further stimulated by treatment with 2 µM A23187. The A23187-specific activation of PLD₂ (3-fold above the basal level) was more evident when the endogenous component of the response was subtracted. PMA treatment did not affect the PLD₂ activity in PLD₂-transfected COS-7 cells (Fig. 2B). In contrast to PLD₂, PLD₁ had low basal activity, although the expression level of PLD₁ was comparable with that of PLD₂, and the PLD₁ activity could be further stimulated by treatment with PMA as well as with A23187 (Fig. 2A). Thus, the stimulatory effect of A23187 on the PLD activity in PLD₂-transfected COS-7 cells supports the conclusion that PLD₂ activity is selectively up-regulated following A23187-induced Ca²⁺ influx in both P388D1 and L1210 cells (Fig. 1B).

View larger version (23K): [in this window] [in a new window]   FIGURE 2. Effects of PMA and A23187 on the activities of hPLD1 and hPLD2. COS-7 cells were transiently transfected with expression vector, hPLD1, or hPLD2 using LipofectAmine as described in Materials and Methods. A, The transfected COS-7 cells were loaded with [3H]myristic acid and treated with 0.1% DMSO (control), 0.1 µM PMA, or 2 µM A23187 for 15 min at 37°C in the presence of 1% 1-butanol. The formation of [3H]PBtOH was measured as described in Fig. 1. Inset, Expression levels of the PLD isoforms were measured by Western blot analysis with anti-PLD Ab as described in Materials and Methods. The data are representative of four separate experiments. B, The average basal (endogenous) PLD activity (vector transfectant) was subtracted from the averaged experimental values for the hPLD1 and hPLD2 transfectants to obtain the activity specifically generated by the overexpressed hPLD1 and hPLD2 proteins. The numbers indicate the x-fold increase attributable to the stimulation by PMA or A23187. Data represent the mean ± SD from four separate experiments. *, p < 0.05.

In contrast to the Ca²⁺-dependent PLD₂ activation in the PLD₂-containing cell lines, including P388D1, L1210, and the PLD₂-transfected COS-7 cells, purified PLD₂ activity could not be stimulated by Ca²⁺ in vitro (data not shown). This suggests that a Ca²⁺-dependent activating factor may be working in regulating PLD₂ activity. Recently, it had been reported that PLA₂ was involved in the up-regulation of PLD activity in rat heart sarcolemmal membranes (20) and in L1210 cells (21). Ca²⁺ has been known to induce translocation of cPLA₂ and subsequent activation of cPLA₂ (22, 23, 24, 25) This suggests the possibility that cPLA₂ might be involved in the regulation of PLD₂ activity. We therefore examined the effects of PMA and A23187 on PLA₂-mediated AA release in the four hemopoietic cell lines. As shown in Fig. 3A, A23187, but not PMA, potently increased AA release in both P388D1 and L1210 cells. In K562 and U937 cells neither A23187 nor PMA had a stimulatory effect on AA release. In agreement with these observations of AA release is the fact that while cPLA₂ was detected by immunoblot analysis in both L1210 and P388D1 cells, it was not detected in K562 and U937 cells (Fig. 3B). The results raise the possibility that Ca²⁺-dependent cPLA₂ activation may be involved in A23187-induced PLD activation in both P388D1 and L1210 cells.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Effects of PMA and A23187 on the AA release and distribution of cPLA2 in hemopoietic cell lines. A, The four indicated cell lines were preloaded with [3H]AA and then treated with 0.1 µM PMA or 2 µM A23187 for 15 min at 37°C. The released [3H]AA was determined as described in Materials and Methods. Data represent the mean ± SD of four separate experiments, each conducted in duplicate. *, p < 0.05 vs control. B, Distribution of cPLA2 in the indicated hemopoietic cell lines was measured by immunoblot analysis using anti-cPLA2 Ab. The results shown are representative of four separate experiments.

To assess the contribution of cPLA₂ to A23187-induced PLD activation, we studied what effect AACOCF₃, a specific inhibitor of cytosolic Ca²⁺-dependent PLA₂, had on PMA- or A23187-induced PLD activity in L1210 cells. As shown in Fig. 4A, A23187-induced PLD activity could be inhibited by treatment of the cells with 20 µM AACOCF₃, whereas PMA-dependent PLD activity was not affected. Consistent with these results is the finding that PLA₂-mediated AA release was specifically stimulated by treatment with A23187, but not by treatment with PMA, and that A23187-induced AA release was markedly suppressed by 20 µM AACOCF₃ treatment (Fig. 4B). These results suggest that the A23187-stimulated PLD₂ activity is up-regulated by prior activation of the Ca²⁺-dependent PLA₂, whereas PMA-stimulated PLD activity, which may be caused via activation of PLD₁, is not mediated by cPLA₂ in L1210 cells.

View larger version (20K): [in this window] [in a new window]   FIGURE 4. Effects of AACOCF3 on PLD activity and AA release of L1210 cells. A, [3H]myristic acid-preloaded L1210 cells were treated with 20 µM AACOCF3 for 15 min and then stimulated with 0.1 µM PMA or 2 µM A23187 for 15 min at 37°C in the presence of 1% 1-butanol. PLD activities were determined by measuring the formation of [3H]PBtOH as described in Materials and Methods. B, [3H]AA-preloaded L1210 cells were pretreated with 20 µM AACOCF3 for 15 min and then stimulated with 0.1 µM PMA or 2 µM A23187 for 15 min at 37°C. Released [3H]AA was measured. Data represent the mean ± SD of four separate experiments. *, p < 0.05 compared with the absence of AACOCF3.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Effects of various PLA2 inhibitors on A23187-induced PLD and PLA2 activities in L1210 cells. A, [3H]myristic acid-preloaded L1210 cells were pretreated with 20 µM AACOCF3, 20 µM MAFP, or 20 µM mepacrine for 15 min, then treated with 2 µM A23187 for 15 min in the presence of 1% 1-butanol, and the formation of [3H]PBtOH was measured. B, [3H]AA-preloaded L1210 cells were pretreated with 20 µM AACOCF3, 20 µM MAFP, or 20 µM mepacrine for 15 min, then treated with 2 µM A23187 for 15 min, and the released [3H]AA was determined. Data represent the mean ± SD of four separate experiments, each conducted in duplicate. *, p < 0.05 vs control.

View larger version (19K): [in this window] [in a new window]   FIGURE 6. Effect of AACOCF3 on PLD activity in K562 cells. [3H]myristic acid-preloaded K562 cells were pretreated with 20 µM AACOCF3 for 15 min and then treated with 0.1 µM PMA or 2 µM A23187 for 15 min at 37°C in the presence of 1% 1-butanol. PLD activities were determined by measuring the formation of [3H]PBtOH as described in Materials and Methods. Data represent the mean ± SD of four separate experiments, each conducted in duplicate.

To further establish whether the PLD₂ activity is regulated by PLA₂ activation in a Ca²⁺-dependent manner, we measured the PLD and PLA₂ activities in PLD₂-transfected COS-7 cells. As shown in Fig. 7A, the A23187-induced PLD₂ activity (3-fold above the basal level, after subtraction of the endogenous component of the response as described in Fig. 2B) was specifically inhibited by pretreatment with 20 µM AACOCF₃. In contrast to A23187-induced PLD₂ activity, PMA- or A23187-stimulated PLD₁ activity was not affected by pretreatment with AACOCF₃, indicating that PLD₁ activity was not linked to PLA₂ activation. Treatment with A23187 increased PLA₂-mediated AA release up to about 3% of the total lipids in the COS-7 cells, and A23187-mediated AA release was not affected by overexpression of the PLD isoforms (Fig. 7B). Consistent with the inhibitory effect of AACOCF₃ on A23187-stimulated PLD₂ activity, AACOCF₃ also inhibited A23187-induced AA release (Fig. 7B). In addition to AACOCF₃, treatment with MAFP or mepacrine also inhibited the A23187-stimulated PLD₂ and PLA₂ activation (Fig. 8, A and B). These findings support the conclusion that PLD₂ activity is regulated by Ca²⁺-dependent PLA₂ activation in L1210 cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 7. Effects of AACOCF3 on PLD activity and AA release in hPLD1- and hPLD2-transfected COS-7 cells. COS-7 cells were transiently transfected with control expression plasmid, hPLD1, or hPLD2 using Lipofectamine as described in Materials and Methods. A, The transfected COS-7 cells were loaded with [3H]myristic acid, pretreated with 20 µM AACOCF3 for 15 min, and then treated with 0.1 µM PMA or 2 µM A23187 for 15 min at 37°C in the presence of 1% 1-butanol. The average basal (endogenous) PLD activity (control) was subtracted from the averaged experimental values for cells transfected with hPLD1 or hPLD2 to yield the activity specifically generated by the overexpressed hPLD1 and hPLD2 proteins as described in Fig. 2. The numbers indicate the x-fold increase in the presence of PMA or A23187. B, [3H]AA-preloaded COS-7 cells were pretreated with 20 µM AACOCF3 for 15 min, then treated with 0.1 µM PMA or 2 µM A23187 for 15 min, and released [3H]AA was determined. Data represent the mean ± SD from four separate experiments, each conducted in duplicate. *, p < 0.05.

View larger version (18K): [in this window] [in a new window]   FIGURE 8. Effects of PLA2 inhibitors on PLD2 activity and AA release in hPLD2-transfected COS-7 cells. A, Control vector- or hPLD2-transfected COS-7 cells preloaded with [3H]myristic acid were pretreated with 20 µM AACOCF3, 20 µM MAFP, or 20 µM mepacrine for 15 min and then treated with 2 µM A23187 for 15 min in the presence of 1% 1-butanol. The formation of [3H]PBtOH was measured. The average endogenous PLD activity (control) was subtracted from the averaged experimental values for the cells transfected with hPLD2 to obtain the activity specifically generated by the overexpressed hPLD2 protein. B, The hPLD2-transfected COS-7 cells were preloaded with [3H]AA; pretreated with 20 µM AACOCF3, 20 µM MAFP, or 20 µM mepacrine for 15 min; and then treated with 2 µM A23187 for 15 min. [3H]AA release was determined. Data represent the mean ± SD from four different experiments, each conducted in duplicate. *, p < 0.05 vs control.

To corroborate the finding that cPLA₂ is required for A23187-induced PLD₂ activation, we took another approach to the regulatory activity of cPLA₂. In these studies we used cPLA₂-specific antisense oligonucleotides to inhibit cPLA₂ expression, and a cPLA₂ sense oligonucleotide as a control in parallel cultures as described in Materials and Methods. Western blot analysis using a cPLA₂-specific Ab (Fig. 9, A and D) showed that cPLA₂ protein expression was inhibited in the presence of cPLA₂-specific antisense oligonucleotides in both L1210 cells and PLD₂-transfected COS-7 cells, whereas the sense oligonucleotide had no effect on cPLA₂ protein expression. We also examined both A23187-induced PLD and PLA₂ activities in L1210 cells and PLD₂-transfected COS-7 cells containing either antisense or sense oligonucleotides. As shown in Fig. 9, B and E, A23187-induced PBtOH formation was substantially inhibited by the presence of antisense oligonucleotides. In contrast, the sense oligonucleotide did not affect PBtOH formation. Consistent with the inhibitory effect of the cPLA₂ antisense oligonucleotide on A23187-induced PLD activity, A23187-induced AA release was also attenuated by the presence of the cotransfected cPLA₂ antisense oligonucleotide (Fig. 9, C and F). This supports the conclusion that cPLA₂ is implicated in the A23187-induced PLD₂ activation.

View larger version (43K): [in this window] [in a new window]   FIGURE 9. Effect of cPLA2 antisense oligonucleotide on A23187-induced PLD and PLA2 activities. A, L1210 cells were transfected with 1 µM cPLA2-specific antisense (AS) or sense oligonucleotide (SS), and cPLA2 expression of the L1210 cells was measured by Western blot analysis with anti-cPLA2 Ab. The data are representative of four independent experiments. B, The transfected L1210 cells were labeled with [3H]myristic acid and treated with 2 µM A23187 for 15 min at 37°C in the presence of 1% 1-butanol. The formation of [3H]PBtOH was determined. C, The transfected L1210 cells were preloaded with [3H]AA and incubated with 2 µM A23187 for 15 min, and the amount of released [3H]AA was determined. D, Control expression plasmid- or hPLD2-transfected COS-7 cells had been cotransfected with 1 µM cPLA2-specific antisense (AS) or sense oligonucleotide (SS) as indicated. cPLA2 expression in COS-7 cells was measured by Western blot analysis using anti-cPLA2 Ab. The data are representative of four independent experiments. E, A23187-induced PBtOH formation was measured for 15 min in the transfected COS-7 cells. The average basal (endogenous) PLD activity (control) was subtracted from the averaged experimental values for cells transfected with hPLD2 to yield the activity specifically generated by the overexpressed hPLD2 protein as described in Fig. 2. F, [3H]AA-preloaded COS-7 cells were incubated with 2 µM A23187 for 15 min, and the amount of released [3H]AA was determined. Data represent the mean ± SD from four separate experiments, each conducted in duplicate. *, p < 0.05.

Since AACOCF₃ could inhibit PLD₂ activity, we explored whether metabolites such as AA and lysoPC, which result from PLA₂ activity, would overcome the inhibitory effect of AACOCF₃ on the stimulation of PLD activity in both L1210 and PLD₂-transfected COS-7 cells. As shown in Fig. 10A, exogenous addition of 20 µM AA in the absence of A23187 did not stimulate PBtOH formation by L1210 cells. Moreover, the inhibitory effect of AACOCF₃ on PLD activity could not be restored by the addition of exogenous AA. In addition to AA, other fatty acids, including oleic acid and palmitic acid, did not activate PLD activity in L1210 cells (data not shown). On the other hand, exogenous addition of 20 µM lysoPC did stimulate PLD activity in the absence of A23187. Lysophosholipids are natural amphiphiles, affecting membrane permeability as a detergent. Thus, this detergent property may be responsible for PLD₂ activation. However, this is unlikely, because, in contrast to lysoPC, lysoPE had no effect on PLD₂ activity in either cell type (Fig. 10, A and B). Intriguingly, the lysoPC stimulation of PLD activity was not affected by pretreatment with AACOCF₃, and exogenously added lysoPC could overcome the inhibitory effect of AACOCF₃ on PLD activity. This implicates lysoPC, which is generated by cPLA₂, in the A23187-dependent regulation of PLD activity in L1210 cells. To confirm the lysoPC interference in the inhibitory effect of AACOCF₃ on PLD₂ activity, we next examined what effect lysoPC would have on the PLD activity of PLD₂-transfected COS-7 cells. As shown in Fig. 10B, exogenous addition of 20 µM lysoPC, but not of 20 µM AA, could restore the AACOCF₃-mediated inhibition of A23187-stimulated PLD₂ activity. Based on these observations, we suggest that lysoPC could be a participant in the cPLA₂-dependent regulation of PLD₂ activity.

View larger version (25K): [in this window] [in a new window]   FIGURE 10. Effect of exogenous AA or lysoPC on AACOCF3-mediated PLD2 inhibition. A, [3H]myristic acid-labeled L1210 cells were pretreated with 20 µM AACOCF3 (+AACOCF3) or 0.1% DMSO (-AACOCF3) for 15 min and incubated with the indicated concentration of AA, lysoPC, or lysoPE in the presence (+) or absence (-) of 2 µM A23187. [3H]PBtOH formation was measured in the presence of 1% 1-butanol. B, COS-7 cells were transfected with control expression plasmid or hPLD2, labeled with [3H]myristic acid, and pretreated with 20 µM AACOCF3. COS-7 cells were then treated with 2 µM A23187, 20 µM AA, 20 µM lysoPE, or 20 µM lysoPC as indicated. The average basal (endogenous) PLD activity (control) was subtracted from the averaged experimental values for cells transfected with hPLD2 as described in Fig. 2. Data represent the mean ± SD from four separate experiments, each conducted in duplicate. *, p < 0.01 vs AACOCF3; **, p < 0.05 (compared with the absence of AACOCF3).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

PLA₂ has been implicated in cellular responses such as inflammation and superoxide generation through the generation of AA and lyso-PAF, which are the precursors of a wide spectrum of proinflammatory mediators, such as PGs, thromboxanes, leukotrienes, and platelet-activating factor, in hemopoietic cells (23, 24, 25). A large number of agonists that stimulate PLA₂ activity also stimulate PLD activity (27), suggesting that the activation of PLA₂ and PLD may be linked. PA generated by PLD has been reported to be essential for agonist-induced PLA₂ activation (28, 29, 30), and PLA₂-mediated AA release has been shown to be inhibited by treatment with ethanol, which blocks PLD-mediated PA formation (28, 29). Balboa et al. (30) recently reported that PA phosphohydrolase generates diacylglycerol from PA, which is produced during PMA-dependent PLD activation. Diacylglycerol plays a key role in PMA-dependent PLA₂ activation in human amniotic WISH cells. Thus, these various reports suggest that PLA₂ activity is up-regulated by PA and diacylglycerol, which are produced by PLD activity. However, for COS-7 cells the participation of PLD in agonist-induced PLA₂ activation can be ruled out based on the following findings. First, overexpression of the two PLD isozymes does not result in an increase in PLA₂-mediated AA release in COS-7 cells (Fig. 7B). Second, A23187-induced PLA₂ activation is not affected by the presence of 1% ethanol, which would have blocked the production of PLD-mediated PA (data not shown). Third, treatment with PMA, which induces PKC-dependent PLD₁ activation, does not increase AA release in COS-7 cells (Fig. 7B), suggesting that PLA₂ activation does not require prior PLD activation. These results are consistent with a previous report that demonstrated that PMA induces PLD activity, but not PLA₂-mediated AA release, in P388D1 cells (31). The regulation of PLD₂ activity by Ca²⁺-dependent PLA₂ activation therefore suggests a possible involvement of PLD₂ in PLA₂-dependent cellular responses such as inflammation and superoxide generation.

PLD₂ activity in PLD₂ -transfected COS-7 cells was specifically stimulated by A23187-induced Ca²⁺ influx (Fig. 2). This suggests that PLD₂ activity may become up-regulated in response to a rise in intracellular Ca²⁺. However, purified PLD₂ does not require Ca²⁺ for optimal activation in vitro (data not shown), and it does not have any Ca²⁺ binding domains such as the calcium-dependent phospholipid binding domain or C2 domains. The data, therefore, suggest that Ca²⁺ is unlikely to directly modulate the catalytic activity of PLD₂ and that the Ca²⁺-induced PLD₂ activation may be mediated by prior stimulation of a Ca²⁺-dependent signaling enzyme, possibly cPLA₂. cPLA₂ contains an N-terminal calcium-dependent phospholipid binding domain that shares homology with the C2 domains in the conventional isoforms of PKC, phospholipase C, synaptotagmin, and p120_GAP (22). Thus, cPLA₂ translocates from the cytosol to the membrane in response to increased cytosolic free Ca²⁺ mobilized from intracellular stores and/or increased influx of extracellular Ca²⁺. The Ca²⁺-dependent translocation then induces cPLA₂-mediated AA release. In our study we demonstrated inhibition of Ca²⁺-stimulated PLD₂ activity by cPLA₂-specific inhibitors, AACOCF₃ and MAFP, in both L1210 and COS-7 cells. Our results, therefore, indicate that PLD₂ activity is up-regulated following the intracellular calcium rise induced by A23187 and that cPLA₂ participates in the Ca²⁺-dependent PLD₂ activation.

Conventional isoforms of PKC, which can be activated by an intracellular Ca²⁺ rise as well as treatment with PMA, have been seen to regulate PLD activity in a variety of cell types (32). It has been reported previously that the enzymatic activity of purified PLD₁ is directly regulated by PKC, a conventional isoform of PKC, in a PMA-dependent manner (33, 34). In agreement with these observations, we found that A23187-induced Ca²⁺ influx, and that PMA stimulated the enzymatic activity of PLD₁ in PLD₁-transfected COS-7 cells (Fig. 2) and also in U937 and K562 cells, which mainly contain PLD₁ (Fig. 1B). However, both PMA and Ca²⁺ ions have been implicated in the up-regulation of cPLA₂ activity in certain other cell types, such as macrophages and neutrophils (32, 33), although in many cell types PMA is only effective when combined with a calcium-mobilizing agonist (34). Thus, it is difficult to determine whether the PMA- or Ca²⁺-induced PLD activation derives from PKC-dependent PLD₁ activation or cPLA₂-dependent PLD₂ activation. In this study we show that the cells of four cell lines (U937, K562, P388D1, and L1210) responded differentially to PMA or A23187 in terms of PLD activity as well as AA release. In contrast to the stimulatory effect of either PMA or A23187 on PLD₁ activity in both U937 and K562 cells, PMA and A23187 had no effect on AA release in these two cell lines. Moreover, the PMA- or A23187-induced PLD activities of these two cell lines were not affected by pretreatment with AACOCF₃. This suggests a PLA₂-independent regulation of PLD₁. PLA₂-independent PLD₁ regulation is also supported by the finding that PMA- or A23187-stimulated PLD₁ activity was not inhibited by pretreatment of PLD₁-transfected COS-7 cells with AACOCF₃ (Fig. 7A). On the other hand, treatment with A23187, but not PMA, specifically induced both PLD₂ activity and AA release in P388D1 and L1210 cells. This indicates that PKC is not implicated in PLA₂-mediated AA release and PLD₂ activation in P388D1 and L1210 cells. Consistent with our results is the reported existence of a PKC-independent PLA₂ activation pathway (31). Balsinde et al. demonstrated that PLA₂-mediated AA release in P388D1 macrophages is not affected by treatment of the cells with PMA. Therefore, the four cell lines, U937, K562, P388D1, and L1210, make useful models in which to study the two independent signal transduction pathways, one leading to PKC-mediated PLD₁ activation and the other to PLA₂-mediated PLD₂ activation, which appears not to require PKC activation. Our research also showed that it is possible to use PLA₂ inhibitors to study physiological responses related to agonist-induced PLD₂ activation.

We showed that Ca²⁺ influx leads to PLD₂ activation in L1210 and P388D1 cells. However, PMA also increases PLD activity in these two cell types, indicating the existence of a PMA-dependent PLD₁ in these cell types, albeit the expression levels of PLD₁ are far less than those of PLD₂ as shown in Fig. 1A. It is not likely that PMA-dependent PLD₁ activity is regulated via the PLA₂-dependent pathway, because PMA does not increase PLA₂-mediated AA release in these cells, and PMA-stimulated PLD activity was not affected by treatment with AACOCF₃, not only in L1210 cells (Fig. 4A) but also in P388D1 macrophages (data not shown). Thus, the PMA-dependent PLD₁ activity must be differentiated from the PLD₂ activity, which is regulated by Ca²⁺-dependent PLA₂ activation in both L1210 and P388D1 cells.

cPLA₂-mediated PC hydrolysis leads to the concomitant generation of AA and lysoPC, which act as second messengers, activating diverse signaling enzymes (22, 23, 24, 25). Various reports suggested that unsaturated fatty acids, including oleic acid and AA, directly stimulate the enzymatic activity of certain PLD isotypes in vitro (35, 36, 37, 38, 39), whereas lysoPC has been reported to inhibit the enzymatic activities of both PLD₁ and PLD₂ in vitro (40). Intriguingly, we found that unsaturated fatty acids specifically activate the enzymatic activity of PLD₂, but not of PLD₁, in vitro (16). These results suggest that AA, which is generated via cPLA₂ activation, may be implicated in the regulation of PLD₂ activity in vivo. However, PLD₂ activity was affected by treatment with exogenous lysoPC, but not AA, in both intact L1210 cells and PLD₂-transfected COS-7 cells (Fig. 10). Moreover, the inhibitory effect of AACOCF₃ on PLD activity can be restored by the exogenous addition of lysoPC, but not by the addition of AA. However, it remains unclear whether PLD₂ activity can be directly stimulated by lysoPC in intact cells, because lysoPC does not stimulate PLD₂ activity in vitro. Thus, it will be necessary to investigate exactly how PLD₂ activity may be regulated by lysoPC in intact cells.

In the present report we show evidence that PLD₂ activity is affected by A23187-induced Ca²⁺ influx in both P388D1 and L1210 cells, and that cPLA₂ activation is implicated in this Ca²⁺-dependent PLD₂ activation. The discovery of this novel signaling mechanism for the regulation of PLD₂ will perhaps lead us to a full understanding of such physiological phenomena as inflammation, in which PLD may be implicated.

	Acknowledgments

 
We thank Drs. M. Murakami (Showa University, Tokyo, Japan), D. K. Kim, and S. H. Baek for helpful discussions, and G. Hoschek for editorial assistance with the preparation of this manuscript.

	Footnotes

 
¹ This work was supported in part by the program of the Korea Science and Engineering Foundation, the Highly Advanced National Project from the Ministry of Science and Technology, and the Center for Cell Signaling Research in the Republic of Korea.

² Address correspondence and reprint requests to Dr. Sung Ho Ryu, Department of Life Science, Pohang University of Science and Technology, Pohang, 790-784, Republic of Korea. E-mail address:

³ Abbreviations used in this paper: PLD, phospholipase D; PA, phosphatidic acid; DAG, diacylglycerol; PIP₂, phosphatidylinositol 4,5-bisphosphate; ARF, ADP-ribosylation factor; PKC, protein kinase C; cPLA₂, cytosolic phospholipase A₂; lysoPC, lysophosphatidylcholine; lysoPE, lysophosphatidylethanolamine; AACOCF₃, arachidonyltrifluoromethyl ketone; MAFP, methyl arachidonyl fluorophosphonate; AA, arachidonic acid; PBtOH, phosphatidylbutanol; [³H]AA, [5,6,8,9,11,12,14,15-³H]AA.

Received for publication June 1, 1999. Accepted for publication September 7, 1999.

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