evidence for g-protein-dependent and g-protein-independent activation of phospholipase d in...

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 229, 630–634 (1996)ARTICLE NO. 1855

Evidence for G-Protein-Dependent and G-Protein-IndependentActivation of Phospholipase D in Lymphocytes

Yu-Zhang Cao, Padala V. Reddy, Lorraine M. Sordillo,George R. Hildenbrandt, and C. Channa Reddy1

Environmental Resources Research Institute and Department of Veterinary Science, The Pennsylvania StateUniversity, University Park, Pennsylvania 16802

Received October 24, 1996

Previously we reported that tumor-promoting phorbol esters stimulate phospholipase D (PLD) independentof protein kinase C (PKC) activation in bovine lymph node lymphocytes. (Cao et al., Biochem. Biophys.Res. Commun. 171, 955–962, 1990; 217, 908–915, 1995). In the present study, we examined the effectsof prostagladins (PGs), E2 , F2a , D2, and H2 on PLD activity as measured by conversion of [1-14C] arachidonicacid-labeled phospholipids into phosphatidylethanol (PEt) in bovine lymph node lymphocytes. Prostaglan-dins stimulated the formation of PEt at an optimal concentration of 10 mM with relative stimulatory effecton the order of PGE2 ú PGF2a ú PGH2 ú PGD2. The PGE2-stimulated formation of PEt was dose-dependent in the range of 0.1 to 10 mM and was not inhibited by PKC inhibitors staurosporine and K252a.When both PGE2 and 12-0-tetradecanoylphorbol-13-acetate (TPA) were included, their effect on the PLDactivation was additive. Furthermore, NaF, a G-protein activator, stimulated the PEt formation. Interestingly,the stimulatory effects of PGE2 and NaF were not additive; however, the formation of PEt by NaF andTPA was additive. These results suggest that similar to TPA, PGs increase PLD activity independent ofPKC and the stimulation by PGs and TPA in lymphocytes may involve both G-protein-dependent and G-protein-independent signaling pathways. q 1996 Academic Press, Inc.

Phospholipase D (EC 3.1.4.4.) (PLD) activation has been recognized as an important routeof signal transduction in a number of cell types. It has been implicated in the regulation ofDNA synthesis, cell proliferation and many other cellular functions (1). Several factors, likeGTP-binding protein (G-protein), protein kinase C (PKC), protein-tyrosine kinase and calciumions have been reported to regulate PLD activation. Phospholipase D activity was shown tobe enhanced by the non-hydrolyzable GTP analogue GTP-g-S in a number of systems, includ-ing HL-60 cells (1), human neutrophils (2), dog brain microsomes (3), and rat liver plasmamembranes (4). Recently, it was reported that prostaglandin (PG) F2a , an arachidonic acidmetabolite, stimulates the activation of PLD in osteoblast-like cells(5) and in chinese hamsterovary (CHO) cells (6).

In the previous studies (7,8), we demonstrated that the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) activates PLD via a PKC-independent mechanism in bovine lymphnode lymphocytes. However, the precise mechanism(s) of PLD activation by TPA in immunecells have not been established. The present study was undertaken to delineate the molecularmechanisms of PLD activation by TPA as well as the role of PGs and NaF, a G-proteinactivator, in PLD activation in bovine lymphocytes.

MATERIALS AND METHODSMaterials. [1-14C] arachidonic acid ( specific activity 51.7 mCi/mmol) was purchased from Dupont NEN, Boston,

MA. DMSO, TPA, PGE2, PGD2, PGF2a , staurosporine, NaF, and calcium ionophore A23187 were purchased from

1 Corresponding author. Fax: (814) 863-1696. E-mail: [email protected].

0006-291X/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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Vol. 229, No. 2, 1996 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. Relative effects of various agonists on PLD activity. Autoradiography of TLC separation of lipids extractedfrom cell cultures prelabeled with 1-14C arachidonic acid and incubated with various test compounds: (1) 0.1% DMSO;(2) 10 mM PGE2; (3) 100 nM TPA; (4) 0.2 mM 15-HPETE; (5) 40 mM NaF; (6) 1 mM Calcium ionophore A23187;(7) 3 mM Staurosporine / 100 nM TPA; (8) 1 mM K252a / 100 nM TPA. Incubation was for 3 h in the presenceof 1% ethanol or 0.1% DMSO and indicated test compounds.

Sigma Chemical Co., St. Louis, MO. PGH2 was prepared in our laboratory as previously described (9). K252a wasobtained from Kamiya Biochemical Co., Thousand Oaks, CA. RPMI-1640 with L-glutamine medium was purchasedfrom Mediatech Co., Washington, DC. Precoated silica gel 150A plates were from Whatman Laboratory, Clifton, NJ.

Preparation of bovine lymph node lymphocytes. Lymphocytes were prepared from bovine retropharyngeal lymphnodes and cultured as described previously (10). Cell cultures (108 cells/5 ml) were prelabeled with [1-14C] arachidonicacid (0.1 mCi/ml) at 377C for 1 h, washed once with RPMI-1640 medium and resuspended in fresh medium.

Measurement of phospholipase D activity. Quantitation of radioactive phosphatidylethanol (PEt) formed from theendogenous prelabeled phospholipids in the presence of ethanol was used to measure PLD activity. All incubationson prelabeled cultures were conducted in presence of test compounds and 1.0 % ethanol for 3 h or as indicated inthe legends of Figure and Tables. Test compounds were included in the 1.0 % ethanol additions except for TPAwhich was added in DMSO (0.1 % final concentration). In the experiments containing PKC inhibitor, the inhibitorwas added 30 min prior to the addition of test compound and 1.0 % ethanol. The cells were centrifuged at 300 1 gfor 10 min and washed once with phosphate-buffered saline (PBS) and the lipids were extracted. The lipid extractswere evaporated and applied to precoated silica gel 150A plates and the chromatograms developed with the organicphase of ethyl acetate-2,2,4-trimethylpentane-acetic acid-water (11:5:2:10, v/v/v/v). The lipids were visualized byiodine vapors and the radioactivity in PEt was determined by scraping the PEt band and subjecting it to liquidscintillation spectrometry as described before (7).

Preparation of PEt standard. A PEt standard was synthesized using cabbage PLD and egg lecithin as describedby Kobayashi and Kanfer (11).

Data analysis. Data were analyzed by the Student’s t test and the statistical significance was assigned at p õ 0.05.

RESULTS

Effect of various agonists on PEt formation catalyzed by PLD. It is well recognized thatthe assay of PEt formation has many advantages over the assay of phosphatidic acid (PA) inthe measurement of PLD activity. Double-labeling method demonstrated that PEt was formedfrom phospholipids exclusively by the action of PLD and it was metabolically stable, lastingup to 12 h after agonist treatment (12,13). Thus, in the present study, the PEt formation wasmeasured to evaluate the activity of PLD in intact cells. As shown in Figure 1, TPA greatlystimulated PEt formation in lymphocytes. PGE2 and NaF also stimulated the PEt formation. Aspreviously reported by us, the time course of PEt accumulation in TPA stimulated lymphocytesindicates that significant PEt accumulated within 15 min. The rate of increase remained rela-tively linear for 3 h. The increase in PEt with TPA was approximately 10 times greater thanthat with 0.1% DMSO alone with the highest stimulation of PEt synthesis seen at 1 1 1007

M TPA (7).The relative effects of different PGs on PLD-mediated PEt formation. As shown in Table

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TABLE 1Relative Effects of PGs on PLD-Mediated

PEt Formation

Test compounds PEt formation (CPM)

Control 320 { 35PGE2

a 1123b { 115PGF2a

a 729b { 103PGD2

a 469b { 37PGH2

a 561b { 45

a All incubations on prelabeled cultures wereconducted in the presence of test compoundsat 10 mM concentration and 1% ethanol for3 h.

b The values with a superscript are signifi-cantly different from the control value (p õ0.05). Data are expressed as mean { S.D. forthree individual experiments.

1, at 10 mM concentration, PGE2, PGF2a , PGD2, and PGH2 caused significant increase (põ0.05)in PLD activity in bovine lymph node lymphocytes with PGE2 showing the highest stimulatoryeffect followed by PGF2a ú PGH2 ú PGD2. Time course of PGE2-stimulated PEt formationin bovine lymphocytes revealed that significant PEt accumulated within 1 h and the linearincrease continued up to 3 h. A dose response study (not shown) revealed that PGE2 stimulatedthe formation of PEt in a concentration dependent manner in the range between 0.1-10 mMwith the maximum stimulation of PEt formation achieved at 10 mM. At this concentration thePEt formation by PGE2 was increased approximately to 3 fold over the control (Table 1).

The effect of PKC inhibitors on the activation of PLD by TPA and PGE2. We examinedthe effects of two potent PKC inhibitors K252a and staurosporine on TPA- and PGE2-stimulatedPEt formation in intact lymphocytes. These two compounds have been shown to be veryselective in inhibiting PKC activity in different cells (14). Both K252a and staurosporine failedto affect PEt formation stimulated by TPA and PGE2 (Figure 1 and Table 2). These resultsstrongly suggest that PKC is not involved in the activation of PLD by TPA and PGE2 inbovine lymph node lymphocytes.

The comparative effects of PGE2, NaF, and TPA on PEt formation. Sodium fluoride (NaF),a nonspecific activator of G-protein which has been widely used to activate G-protein (4,15),was employed to check its effect on activation of PLD. It was found that NaF at the concentra-tion of 10-40 mM stimulates the formation of PEt in bovine lymphocytes with the maximumstimulation observed at 40 mM. At the maximum stimulatory concentration , PGE2 (10 mM)and NaF (40 mM) together did not result in additive effect (Table 3). However, the formationof PEt by a combination of 100 nM TPA and 40 mM NaF was additive. Furthermore, wefound that the stimulation of PEt formation by TPA and by PGE2 at their maximum stimulatoryconcentration was additive (Table 3).

DISCUSSION

It has been reported that arachidonic acid, but not other fatty acids, stimulates lymphocyteproliferation (16). Ecosanoids, enzymatic oxidation products of arachidonic acid, areformed by virtually every tissue in the body and play crucial roles in blood clotting,inflammation, control of vascular tone, renal function and reproductive functions (17).Moreover, lymphocytes were shown to respond and produce ecosanoids in a variety of

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TABLE 2The Effect of Protein Kinase C Inhibitors on the Activation

of PLD Activity by TPA or PGE2

Test compounds Concentration PEt formation (CPM)

DMSO 0.1% 340 { 24TPA 1007 M 2540* { 310K252a 1006 M 328 { 52Staurosporine 3 1 1006 M 343 { 64PGE2 1005 M 958* { 110K252a 1006 M/TPA 1007 M 2348* { 228Staurosporine 3 1 1006 M/TPA 1007 M 2643* { 289K252a 1006 M/PGE2 1005 M 833* { 134Staurosporine 3 1 1006 M/PGE2 1005 M 1072* { 115

Means with a superscript * are significantly different from thecontrol (0.1% DMSO) value (p õ 0.05). In the last four treatments,K252a or Staurosporine was added 30 min before TPA or PGE2.The results are expressed as mean { S.D. (n Å 3).

disease conditions (18,19). However, the precise mechanism(s) by which eicosanoids affectthese processes remains to be fully understood. In the present study, we examined theeffect of PGs on PLD activity in bovine lymph node lymphocytes. Prostaglandins stimulatedthe formation of [1-14C]-labeled PEt at an optimal dose of 10 mM with relative stimulatoryeffect in the order of PGE2 ú PGF2a ú PGH2 ú PGD2 . The PGE2-stimulated formationof PEt was not inhibited by PKC inhibitors staurosporine and K252a. Although PGE2 wasreported to stimulate PLD in human erythroleukemia cells, other PGs including PGF2a hadno effect on PLD (20). Recently PGF2a was reported to activate PLD in Osteoblast-likecells (5). Our results in this study indicate that several PGs have the ability to activate

TABLE 3Effect of TPA and NaF on the PGE2-Induced Formation

of PEt in Bovine Lymphocytes

Test compounds PEt formation (CPM)

Control (no addition) 287 { 54a

PGE2 10 mM 984 { 116b

NaF 40 mM 1210 { 158b

TPA 100 nM 2524 { 225c

NaF 40 mM / PGE2 10 mM 1236 { 125b

TPA 100 nM / PGE2 10 mM 3347 { 336d

TPA 100 nM / NaF 40 mM 3466 { 373d

Note. The pre-labeled cells were incubated with 1% etha-nol, 10 mM PGE2, 40 mM NaF, 100 nM TPA, or combina-tions thereof as indicated. Each value represents the mean {S.D. of three individual experiments (n Å 3). Means withdifferent superscript letters are significantly different fromone another (p õ 0.05).

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PLD in lymphocytes. To our knowledge, this is the first report that shows PLD wasstimulated by different PGs in one type of cells.

We report here an activation of PLD by PGE2 which is independent of TPA with maximumstimulation of PEt formation by PGE2 being 1

3 of that observed for TPA. The effects of PGE2

and TPA on the formation of PEt were additive. Likewise the stimulatory effects of TPA andNaF were additive; however, the formation of PEt by NaF and PGE2 was not additive. Inconclusion, we found that, similar to TPA, PGs activate PLD independent of PKC and thestimulation of PLD by PGs and TPA in lymphocytes may involve both G-protein-dependentand G-protein-independent signaling pathways.

ACKNOWLEDGMENTSThis research was supported by the National Institutes of Health Grants HL31245 and AI06347.

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