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J Biol Chem, Vol. 274, Issue 39, 27385-27391, September 24, 1999


Differential Phospholipase D Activation by Bradykinin and Sphingosine 1-Phosphate in NIH 3T3 Fibroblasts Overexpressing Gelsolin*

Yoshiko BannoDagger §, Hisakazu Fujita, Yoshitaka Onoparallel , Shigeru NakashimaDagger , Yuzuru ItoDagger , Noboru Kuzumaki, and Yoshinori NozawaDagger

From the Dagger  Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500-8705,  Laboratory of Molecular Genetics, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060-8638, and parallel  Department of Biology, Faculty of Science, Kobe University, Kobe 657, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP2). Here we showed in in vitro experiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP2 concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca2+ ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-alpha and PKC-beta 1 but not PKC-epsilon was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from Gi for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Hydrolysis of phosphatidylcholine (PC)1 by phospholipase D (PLD) to generate choline and phosphatidic acid (PA) has been implicated in a variety of cellular functions, including secretion, vesicle trafficking, mitosis, and meiosis (1, 2). PA has also been reported to cause activation of the growth factor signal transduction, which includes protein-tyrosine phosphatase (3), phospholipase Cgamma (PLCgamma ) (4), Ras-GTPase-activating protein (5), Raf-1 translocation (6), and sphingosine kinase (7). Furthermore, several lines of evidence suggest that PA induces actin polymerization based on its ability to bind to actin-binding proteins (8-10). Two mammalian PLD genes, PLD1 (11) and PLD2 (12, 13), have recently been cloned. PLD1 is regulated by low molecular weight GTP-binding proteins such as ADP-ribosylation factor (ARF) and the Rho family and also by protein kinase C (1, 2). The mechanisms that regulate PLD2 activity are still undefined. PLD2 is constitutively active and requires merely phosphatidylinositol 4,5-bisphosphate (PIP2) as cofactor (12, 13).

Gelsolin is a Ca2+- and polyphosphoinositide-regulated actin-binding protein (14-16). In vitro studies have shown that gelsolin modulates the activities of several important signaling enzymes including PLC (17, 18) and PLD (19) through interaction with PIP2. We have previously demonstrated that gelsolin inhibited PLCgamma activity via its strong binding to PIP2 in vitro (17). Moreover, it has been reported that agonist-stimulated PLCbeta activity was also inhibited in gelsolin-overexpressed cells (18). On the other hand, gelsolin has been known to enhance PLD activity through physical association (19). Hydrolysis of exogenous substrate PC by PLD1 and PLD2, but not by oleate-dependent enzyme, requires the presence of PIP2 (11-13, 20). Thus, PLC, PLD, and gelsolin are thought to compete for available PIP2 in agonist-stimulated cells. In the present study, to determine whether gelsolin regulates PLD activity via binding to PIP2 in vivo, we examined bradykinin- and S1P-induced PLD activation in NIH 3T3 cells overexpressing gelsolin.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Phosphatidylethanolamine (PE), PC, PIP2, sodium oleate, geneticin (G418), A23187, and phorbol myristate acetate (PMA) were purchased from Sigma. Sphingosine 1-phosphate (S1P) was from Matreya, Inc. (Pleasant Gap, PA). [9,10-3H]Palmitic acid (54.0 Ci/mmol), myo-[3H]inositol (90 Ci/mmol), and [choline-methyl-3H]dipalmitoyl-PC (26.5 Ci/mmol) were from NEN Life Science Products. LipofectAMINE was from Life Technologies, Inc. Rabbit polyclonal antibodies were prepared to the carboxyl-terminal 15 residues of human PLD1a (Ab-224) and carboxyl-terminal 16 residues of rat PLD2 (Ab-226). Polyclonal antibodies to PKC isozymes were from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibody to phosphorylated mitogen-activated protein (MAP) kinase was from New England BioLabs (Boston, MA). Anti-rabbit antibody conjugated with horseradish peroxidase and chemiluminescence kit (ECL system) were from Amersham Pharmacia Biotech.

Cell Culture and Transaction of Gelsolin cDNA-- NIH 3T3 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamate supplemented with 10% (v/v) fetal bovine serum, 100 units of penicillin/ml, and 100 µg of streptomycin/ml at 37 °C in a humidified, CO2-controlled (5%) incubator.

A HindIII-StuI fragment of human cytoplasmic gelsolin cDNA (21) was cloned into HindIII-HpaI site of pLNCX retroviral vector (a generous gift from Dr. A. D. Miller, Fred Hutchinson Cancer Research Center) (22). The resulting construct, pLNChGsn or pLNCX alone was transfected into Bosc 23, a highly efficient ecotropic virus-packaging cell line (23), using LipofectAMINE. Two days after lipofection, the virus-containing supernatant was collected and centrifuged to remove cells and debris. NIH 3T3 cells were infected with the virus-containing supernatant. The vector and gelsolin-transfected cells were maintained in the presence of 0.5 mg/ml G418. The expression of gelsolin or PLD proteins was examined by Western blotting with specific antibodies using the ECL detection system.

Baculovirus Expression and Assay of PLD Activity-- PLD1 and PLD2 were overexpressed in Sf9 cells that had been infected with recombinant baculoviruses harboring human PLD1 or PLD2 cDNAs (kindly supplied by Dr. M. A. Frohman, State University of New York) as described previously (11). Cells were suspended in ice-cold lysis buffer (20 mM HEPES, pH 7.4, 1 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucyl-agmatine, E-64) and lysed by sonication. The lysates were centrifuged at 1,000 × g for 5 min, and the resulting supernatant was centrifuged at 100,000 × g for 60 min to obtain the membrane fraction.

PLD1 activity was assayed essentially as described previously (20) by measuring the generation of 3H-labeled choline from [choline-methyl-3H]dipalmitoyl-PC. Briefly, 20 µl of lipid vesicles containing PE, PIP2, and PC in a molar ratio of 16:1.4:1 with [choline-methyl-3H]dipalmitoyl-PC (total 4 × 105 cpm/assay) were added to 100 µl of a mixture containing PLD source, 10 µM ARF, 5 µM GTPgamma S, 50 mM HEPES-NaOH, pH 7.5, 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2, and 2 mM CaCl2. PLD2 activity was assayed using the procedure used for PLD1 except for omitting PLD1, ARF, and GTPgamma S. Oleate-dependent PLD activity was assayed by measuring the generation of 3H-labeled choline from [choline-methyl-3H]dipalmitoyl-PC in the presence of 0.5 mM oleate (20).

Measurement of Total Inositol Phosphates-- NIH 3T3 cells in 6-well plates were prelabeled with 1 µCi/ml myo-[3H]inositol for 24 h in inositol-free DMEM medium containing 0.3% bovine serum albumin (BSA). Cells were then washed twice with the HEPES/Tyrode buffer (10 mM HEPES/NaOH, pH 7.4, 134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.36 mM NaH2PO4, 1.0 mM MgCl2, 1.8 mM CaCl2, 1 mg/ml BSA, and 1 mg/ml glucose) containing 20 mM LiCl and further incubated for 15 min at 37 °C before stimulation with agonists. Cells were then stimulated with agonists for the indicated time intervals, and the reactions were terminated by the addition of ice-cold 10% perchloric acid. Inositol phosphates were separated using AG 1 × 8 anion exchange resin (formate form, 200-400 mesh, Bio-Rad) as described by Berridge et al. (24).

Measurement of Phosphatidylbutanol Formation-- Subconfluent cells were labeled overnight with 1 µCi/ml [3H]palmitic acid in DMEM containing 0.3% BSA. Cells were washed and preincubated in HEPES-Tyrode buffer containing 0.3% 1-butanol (v/v) for 10 min. After stimulation of cells with agonists, the reactions were terminated by removing the assay buffer followed by immediate addition of 1 ml of an ice-cold phosphate-buffered saline/methanol (2:5, v/v) mixture to the culture dishes. Lipids were then extracted by the method of Bligh and Dyer (25) and separated on a Silica Gel 60 plate by one-dimensional thin-layer chromatography in a solvent system using an upper phase of ethyl acetate/2,24-trimethylpentane/acetic acid/water (13:2:3:10, v/v/v/v) as described previously (26). The area corresponding to [3H]phosphatidylbutanol (PBut), identified by co-migration with PBut standard, was scraped off the plate, and radioactivity was determined in a liquid scintillation counter (Beckman LS-6500).

Determination of MAP Kinase Phosphorylation-- Cells were grown in 100-mm dishes to near confluence and subjected to serum-free DMEM containing 0.3% BSA for 24 h. Washed cells were then stimulated with agonists, and harvested in ice-cold lysis buffer (1% Triton X-100, 0.1% SDS, 0.5% sodium cholate, 1 mM EDTA, 1 mM EGTA, 50 mM NaCl, 25 mM HEPES, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml E-64, 20 mM beta -glycerophosphate, 1 mM sodium fluoride, and 1 mM sodium orthovanadate, pH 7.4), and then homogenized by sonication. Protein concentrations were assayed with Bradford protein assay reagent using BSA as the standard. Total cell lysates (20 µg) were subjected to electrophoresis on 10% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA. Phosphorylation of MAP kinase 1/2 was determined by immunoblotting with a polyclonal antibody (anti-phospho-MAP kinase) that recognizes only activated MAP kinase 1/2. Total MAP kinase 1/2 was detected by blotting with an antibody against MAP kinases. After washing in 50 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl and 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-linked secondary antibody. After repeated washings, the bound antibody was detected by using the ECL Western blotting detection system.

PKC Translocation-- For measurement of PKC translocation, NIH 3T3 cells were subjected to serum-free DMEM containing 0.3% BSA for 24 h before stimulation. The washed cells were stimulated and harvested in ice-cold buffer A (25 mM HEPES, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin) and then lysed by sonication. Cell lysates were centrifuged at 1,500 × g for 5 min, and the supernatants were further centrifuged at 100,000 × g for 30 min. The pellets were resuspended in buffer A. For Western blot analysis, these membranes were separated on 8% gels by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. After blocking in 5% (w/v) BSA, the membranes were incubated with specific antibodies for PKC isozymes. The membranes were incubated with horseradish peroxidase-linked secondary antibody. After repeated washings, the bound antibody was detected by using the ECL Western blotting detection system.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effects of Gelsolin on Activities of Phospholipase D Isoforms in Vitro-- Since gelsolin is a potential actin-binding protein that has high affinity for PIP2 (15, 16), we examined whether gelsolin inhibits PLD activity by binding the cofactor PIP2 using Sf9 cells overexpressing PLD1 and PLD2. Gelsolin (5 µM) inhibited ARF-dependent PLD1 and PLD2 activities of membrane fractions from transfected Sf9 cells using PE-PIP2-PC micelles as substrate (Fig. 1). On the other hand, gelsolin had no effect on oleate-dependent PLD activity abundant in PC12 cells. Further examination showed that PLD2 activity was inhibited by gelsolin in a concentration-dependent manner with maximum inhibition at 5 µM (Fig. 2A). On the other hand, PLD2 activity was stimulated by adding PIP2 to PE/PC vesicles in a concentration-dependent manner with a maximum at 40 µM in the absence of gelsolin (Fig. 2B). The inhibitory effect of gelsolin (1 µM) was not abrogated by increasing PIP2 concentration, suggesting that gelsolin did not inhibit PLD2 activity by binding PIP2.


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  		Fig. 1.   Effects of gelsolin on activities of PLD isoforms. cDNA of hPLD1 and PLD2 were overexpressed in Sf9 cells, and the membranes were isolated. The activities of PLD1 and PLD2 in membrane fractions were measured without (CONT) or with (GSL) gelsolin (5 µM) using PE/PIP2/[choline-methyl-3H]dipalmitoylPC as substrate. PLD1 activity was measured with or without 50 nM ARF. Oleate-dependent PLD activity was measured in membrane fractions from PC12 cells with or without gelsolin (5 µM) using [choline-methyl-3H] dipalmitoyl-PC in the absence (NONE) or presence of 0.5 mM oleate (OL). After incubation for 30 min at 37 °C, the released [3H]choline was measured as described under "Experimental Procedures." Results are expressed as the mean ± S.E. of duplicate determinations from three independent experiments.


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  		Fig. 2.   Concentration-dependent PLD2 inhibition by gelsolin and effects of PIP2 on the inhibition. A, PLD2 activity was measured with various concentrations of gelsolin using PE/PIP2/[choline-methyl-3H]dipalmitoylPC as substrate as described under "Experimental Procedures." B, the effects of PIP2 were examined without or with gelsolin (Gsl) (0.5 µM) at different concentrations of PIP2. Results are expressed as mean ± S.E. of duplicate determinations from three similar experiments.

Overexpression of Gelsolin in NIH 3T3 Cells-- -To examine the effect of gelsolin on PLD activation in vivo, we studied various clones stably overexpressing gelsolin in NIH 3T3 fibroblasts. Cells were transfected with a construct containing gelsolin cDNA, and two clones (G2 and G6) were obtained. The expression level of gelsolin was higher in G2 clone than G6 clone, as inferred by Western blot analysis (Fig. 3A). A clone of vector-transfected NIH 3T3 cells (Vect) was used as control. No significant differences were observed in the growth rate and morphology between Vect, G2, and G6 cells (data not shown). To examine the expression levels of PLD isoforms in the transfected NIH3T3 cells, the lysates were subjected to Western blot analysis by using two PLD antibodies (Ab-224 and Ab-226). Both antibodies could react with recombinant human PLD1a, PLD1b, and mouse PLD2 used as standards. Ab-224 can detect PLD1a, PLD1b, and PLD2, and Ab-226 detected PLD1b and PLD2 but not PLD1a in HaCaT cell lysates (Fig. 3B, upper panels, 1 and 2). Western blot analysis using these two antibodies revealed the presence of a significant amount of PLD2 and much less PLD1a (23% of PLD2) but not PLD1b in vector-transfected control NIH 3T3 cells (Vect) (Fig. 3B, lower panel). However, there were no significant differences in the amount of these PLD isoforms among Vect, G2, and G6 clones. Expression levels of other signaling enzymes, such as PKC isozymes (alpha , beta 1, delta 1, and epsilon ) and PLC isozymes (PLCbeta 1, beta 3, and gamma 1) were also not different between Vect- and gelsolin-overexpressing clones (data not shown).


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  		Fig. 3.   Western blotting of gelsolin and PLD in gelsolin overexpressing clones. NIH 3T3 cells were transfected with human cytosolic gelsolin expression vector or empty vector as described under "Experimental Procedures." Vector (VECT) and gelsolin-transfected cells (G2 and G6) were lysed by sonication. A, the cell lysates (50 µg protein) were subjected to 8% SDS-polyacrylamide gel electrophoresis and immunostained with anti-gelsolin antibody. Recombinant human gelsolin was used as standard (upper panel). Relative amounts of gelsolin in gelsolin-overexpressing (G2 and G6) cells shown in the upper panel were quantified by scanning densitometry and expressed as relative % to vector cells (VECT) (lower panel). B, as for PLD expression, the cell lysates (300 µg protein) were subjected to Western blot analysis using Ab-224 (1) and Ab-226 (2) (upper panels), and relative amounts of PLD isoforms were expressed as relative % to PLD2 (lower panel). Recombinant PLD1a, PLD1b, PLD2, and HaCaT cell lysates (HA) were used as standards. A blot representative of three independent experiments is shown (upper panels), and the expression levels (relative %) are shown as mean of three independent experiments (lower panels).

Effects of Gelsolin Overexpression on Bradykinin-induced PLD Activation-- To examine the effects of gelsolin overexpression on PLD activation, formation of PBut was examined in Vect, G2, and G6 cells stimulated by bradykinin. The time course of PBut formation by bradykinin (2 µM)-stimulated Vect, G2, and G6 cells is shown in Fig. 4. Gelsolin overexpression reduced PBut formation induced by bradykinin stimulation. G2 clone with the highest level of gelsolin expression showed the lowest PBut formation compared with control (Vect) cells. PLD activation was reduced to a lesser extent in G6 clone, which had a lower gelsolin expression. These results suggest that inhibition of bradykinin-stimulated PBut formation appear to correlate with the expression level of gelsolin.


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  		Fig. 4.   Effects of gelsolin overexpression on bradykinin-induced PLD activation. Vector (Vect) or gelsolin (G2 and G6) overexpressing NIH 3T3 cells were labeled for 24 h with [3H]palmitic acid in DMEM containing 0.3% BSA. Labeled cells were stimulated with bradykinin (2 µM) in the presence of 0.3% butanol, and the levels of [3H]PBut were measured as described under "Experimental Procedures." Results represent the mean ± S.E. of three independent experiments.

Involvement of PLC Activation in Bradykinin-induced PLD Activation in Gelsolin-overexpressing Cells-- PLD activation is known to be dependent on phosphoinositide hydrolysis by PLC, since PKC activation by diacylglycerol derived from PIP2 breakdown is involved in the activation of PLD by agonist stimulation (27, 28). We have previously demonstrated that gelsolin inhibited PLC activity in vitro (17), and Sun et al. (18) also demonstrate that gelsolin overexpression suppresses bradykinin-stimulated PLCbeta activity. To examine the effect of gelsolin overexpression on PLC activation in NIH 3T3 cells, we measured the formation of inositol phosphates (InsPs) in response to bradykinin. As shown in Fig. 5, bradykinin stimulation increased InsP formation in a concentration-dependent manner in control cells (Vect), whereas gelsolin overexpression reduced PLC response to bradykinin by approximately 50% in G2 cells (Fig. 5A). Similar repression of PBut formation was observed in G2 cells stimulated with bradykinin at all concentrations tested (Fig. 5B).


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  		Fig. 5.   Effects of gelsolin overexpression on bradykinin-induced PLC and PLD activation. Vector (Vect)- or gelsolin (G2)-overexpressing NIH 3T3 cells were labeled with [3H]inositol in inositol-free DMEM or [3H]palmitic acid in DMEM containing 0.3% BSA for 24 h. [3H]Inositol phosphate generation (A) and [3H]PBut formation (B) were measured at various concentrations of bradykinin as described under "Experimental Procedures." Results represent the mean ± S.E. of three independent experiments.

To examine whether inhibition of PLD activation by gelsolin overexpression is a downstream event in the PLC-PKC pathway, we examined the effect of Ca2+ ionophore, A23187, and PMA on PLD activation. PBut formation was increased by stimulation with 1 µM A23187 (Fig. 6A) or 100 nM PMA (Fig. 6B) in a time-dependent manner. PBut formation induced by either stimulator was not affected by gelsolin overexpression. These observations suggested that bradykinin-mediated PLD activation was dependent upon PLC activation in NIH 3T3 fibroblasts.


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  		Fig. 6.   Effects of gelsolin overexpression on PLD activation induced by ionophore A23187 or PMA. Vector (Vect) or gelsolin (G2) overexpressing NIH 3T3 cells were labeled with [3H]palmitic acid in DMEM containing 0.3% BSA for 24 h. The labeled cells were stimulated with A23187 (1 µM) or PMA (100 nM) in the presence of 0.3% butanol. At indicated time intervals, [3H]PBut formation was measured as described under "Experimental Procedures." Results represent the mean ± S.E. of three independent experiments.

Effects of Gelsolin Overexpression on PLD Activation Induced by Sphingosine 1-Phosphate-- S1P is known to stimulate PLD activity in various cells (29-31). To examine the effect of gelsolin on S1P signaling in NIH 3T3 cells, we investigated PLC and PLD activation by S1P in G2 cells. InsPs formation was increased by S1P stimulation in a concentration-dependent manner in control cells (Vect) but was reduced in G2 cells at the concentrations examined (Fig. 7A). PBut formation by S1P stimulation increased in a concentration-dependent manner in control cells, reaching a peak level with 5 µM S1P at 2 min. In sharp contrast to InsPs formation, S1P-stimulated PBut formation was not inhibited in G2 cells (Fig. 7B). These results suggested that gelsolin overexpression repressed PLC activation but not PLD activation in S1P-stimulated cells.


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  		Fig. 7.   Effects of gelsolin overexpression on sphingosine 1-phosphate-induced PLC and PLD activation. Vector (Vect)- or gelsolin (G2)-overexpressing NIH 3T3 cells were labeled with [3H]inositol in inositol-free DMEM or [3H]palmitic acid in DMEM containing 0.3% BSA for 24 h. [3H]Inositol phosphates generation (A) and [3H]PBut formation (B) were measured at various concentrations of S1P as described under "Experimental Procedures." Results represent the mean ± S.E. of three independent experiments.

Effects of Pertussis Toxin on Agonist-induced PLC and PLD Activation----- The bradykinin receptor has been known to couple to Gq (32), whereas the S1P receptor associates with a heterotrimeric G protein (EDG-1) (33, 34). A number of EDG receptor subfamilies are coupled to G proteins such as pertussis toxin (PTX)-sensitive Gi/Go or -insensitive Gq/G11, or G12/13 proteins (35). As shown in Fig. 8, PTX pretreatment (200 ng/ml, for 24 h) of Vect and G2 cells markedly reduced InsP formation induced by S1P but not by bradykinin, suggesting that PLC activation by S1P may be mediated via PTX-sensitive G protein. On the other hand, PBut formation induced by S1P was not affected by pretreatment with PTX, even at a high concentration of PTX (2 µg/ml). These results suggest that the G protein involved in S1P-evoked PLD activation is distinct from that in PLC activation.


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  		Fig. 8.   Effects of pertussis toxin pretreatment on PLC and PLD activation induced by sphingosine 1-phosphate or bradykinin. Vector (Vect) or gelsolin (G2) overexpressing NIH 3T3 cells were labeled with [3H]inositol in inositol-free DMEM or [3H]palmitic acid in DMEM containing 0.3% BSA for 24 h. The labeled cells were treated without or with PTX (200 ng/ml) for 24 h and stimulated with S1P (5 µM) or bradykinin (BK) (2 µM). [3H]Inositol phosphate generation and [3H]PBut formation were measured as described under "Experimental Procedures." Results represent the mean ± S.E. of three independent experiments.

Effects of Gelsolin Overexpression on Translocation of Protein Kinase C Isozymes Induced by Agonists-- PKC is one of the most potent stimulators of agonist-induced PLD activation (1). To study the involvement of PKC in the agonist-induced PLD activation, membrane fractions from bradykinin or S1P-stimulated Vect and G2 cells were examined by using antibodies to PKC isozymes. Treatment of Vect cells with bradykinin or S1P induced translocation of PKC-alpha , PKC-beta 1, and PKC-epsilon (Fig. 9). The addition of EGTA inhibited the translocation of PKC-alpha and PKC-beta 1 but not PKC-epsilon . Furthermore, S1P- but not bradykinin-induced translocation of PKC isozymes was suppressed by PTX treatment. PKC-alpha and PKC-beta 1 levels did not increase in membranes from G2 cells incubated with bradykinin or S1P. On the other hand, translocation of PKC-epsilon induced by both agonists was not affected in gelsolin overexpressing cells.


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  		Fig. 9.   Membrane translocation of PKC isozymes induced by bradykinin and sphingosine 1-phosphate and effects of EGTA and PTX treatment. A, vector (Vect)- or gelsolin (G2)-overexpressing NIH 3T3 cells with or without pretreatment of PTX (200 ng/ml) (lanes 4 and 7) for 24 h were stimulated with bradykinin (2 µM) (lanes 2-4) for 1 min or S1P (5 µM) (lanes 5-7) for 2 min and lysed by sonication. In some cases, EGTA (3 mM) (lanes 3 and 6) was added in the buffer. Lane 1 is a control membrane. Membrane fractions were prepared as described under "Experimental Procedures." Membrane fractions (5-20 µg of proteins) were subjected to 8% SDS-polyacrylamide gel electrophoresis and immunostained with the indicated antibodies. A, blot representative of three independent experiments. B, relative amounts of PKC isozymes in vector (Vect)- or gelsolin-overexpressing (G2) cells shown in A were quantified by scanning densitometry, and the mass of all membranes in each PKC isozyme was designated as 100%. Results are shown as mean of three independent experiment. , control membranes; , bradykinin-membranes; black-square, S1P membranes.

Agonist-induced Mitogen-activated Protein Kinase Activation-- Treatment of various cells with bradykinin or S1P resulted in activation of the MAP kinase pathway. Recently, Rizzo et al. (6) reported that PA formation via activation of PLD by insulin induced stimulation of the MAP kinase pathway via Raf-1 kinase activation. We examined the effect of gelsolin overexpression on the agonist-induced MAP kinase activation using an antibody that recognized the phosphorylated form of MAP kinase. Stimulation of Vect cells with S1P caused a marked phosphorylation of MAP kinase (Fig. 10). PKC down-regulation by long term treatment with PMA (300 nM for 24 h) and PKC inhibitor Ro31-8220, but not tyrosine kinase inhibitor ST638, reduced S1P-induced MAP kinase phosphorylation by 80-90%. Pretreatment of Vect cells with PTX abolished S1P-induced MAP kinase phosphorylation, but EGTA had no effect. These results suggest that S1P-induced MAP kinase activation was PKC-dependent and PTX-sensitive. As shown in Fig. 10C, MAP kinase phosphorylation induced by PMA, bradykinin, and S1P was similar in Vect and G2 cells, suggesting that gelsolin overexpression had no effect on agonist-stimulated MAP kinase activation.


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  		Fig. 10.   Effects of gelsolin overexpression and various inhibitors on phosphorylation of MAP kinase A, vector-transfected NIH 3T3 cells were pretreated with PMA (300 nM) or PTX (200 ng/ml) for 24 h and with ST638, EGTA (3 mM) or Ro31-8220 (RO, 50 µM) for 15 min and stimulated with S1P (5 µM) for 2 min. The cell lysates (20 µg protein) were analyzed by Western blotting using anti-MAP kinase (MAPK 1/2) or -phospho-specific MAP kinase (P-MAPK 1/2) antibodies. A, blot representative of three independent experiments. CONT, control. B, amounts of phosphorylated MAP kinase 2 shown in B were quantified by scanning densitometry and expressed as fold increase relative to unstimulated control. Results represent the mean ± S.E. of three separate experiments. C, vector (lanes 1, 3, 5, and 7) or gelsolin-overexpressed NIH 3T3 cells (lanes 2, 4, 6, and 8) were stimulated with PMA (100 nM) for 2 min, bradykinin (BK) (2 µM) for 1 min, and S1P (5 µM) for 2 min. Cell lysates were analyzed by Western blotting using anti-phospho-specific MAP kinase (P-MAPK 1/2). A blot representative of three independent experiments is shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We showed in the present study that gelsolin inhibited recombinant PLD1 and PLD2 activities but not oleate-dependent PLD, which requires PIP2 as a cofactor in the exogenous substrate in vitro assay (1, 2). There are a number of inhibitory proteins for PLD activity such as synaptojanin and fodrin. These are not direct inhibitors of PLD activity but are related to reduced PIP2 in agonist-stimulated cells (36, 37). Ceramide also modulates agonist-mediated PLD activation by inhibiting PKC-alpha , ARF, or RhoA translocation (28, 38, 39). Our previous study, however, indicated that C2-ceramide directly inhibited both GTPgamma S-dependent and -independent PLD activities in membrane fractions from HaCaT cells when assayed using the exogenous substrate PE/PIP2/PC (40). This action of ceramide was probably due to competition with PIP2, since the inhibitory action of ceramide on PLD activity could be restored by increasing PIP2 (38). Gelsolin is also known as a potential PIP2-binding protein and inhibits PLC activity by competing with PIP2 (17, 18). However, this is unlikely for PLD inhibition, since in our experiment a gelsolin-induced decrease in PLD2 activity could not be abrogated by increasing PIP2 concentration. Sun et al. (18) recently reported that PLD is physically associated with gelsolin based on experiments using partially purified PLD from rabbit brain. However, we could not find any association with gelsolin when PLD1 or PLD2 from overexpressed NIH 3T3 cell lysates was immunoprecipitated using specific antibodies.

Our study demonstrated that overexpression of gelsolin in NIH 3T3 cells modified agonist-stimulated PLC and PLD activation. Thus, gelsolin overexpression repressed bradykinin-mediated PLC and PLD activation but did not affect PLD activation by PMA, ionophore A23187, and S1P. Several lines of evidence indicate that PKC plays a major role in agonist-induced PLD activation in a variety of cell types (1, 2). Furthermore, recent studies have demonstrated that PKC-alpha and PKC-beta are the principal regulator of PLD activity (2, 41, 42) and that the regulatory domain of PKC-alpha itself is an effective activator of PLD (43). PKC activation is thought to be due to activation of PLC isozymes, which hydrolyze PIP2 to generate inositol 1,4,5-trisphosphate and diacylglycerol. The resultant increase in diacylglycerol and Ca2+ leads to activation and translocation of conventional PKC isozymes. Thus, the interaction of PKC with PLD in membranes may be sufficient to induce PLD activation. PLD activation was abolished in PKC-down-regulated cells, suggesting that PLC acts upstream of PLD. The involvement of PLC/PKC in PLD regulation by growth factors has been shown by studies using mouse embryonic fibroblasts with disrupted PLCgamma 1 gene (44). Our studies demonstrated here that gelsolin overexpression inhibited bradykinin-stimulated PLCbeta activity, thereby leading to repression of activation of PKC-alpha and PKC-beta 1 but not PKC-epsilon . Therefore, the reduced PLD response to bradykinin in gelsolin-overexpressed cells may be secondary to repressed PKC-alpha activation. This notion was further supported by the finding that PMA- or A23187-induced PLD activation was unaffected by gelsolin overexpression. Considered together, these results suggest that bradykinin-mediated PLD activation may be attributed to PKC-dependent PLD1. PLD1 activation is mediated by several factors such as PKC (-alpha , -beta ) and small G proteins (ARF, Rho family, and Ral) (1, 2, 42, 45). We also noted that bradykinin stimulation increased RhoA level in membranes of control cells, whereas its level was decreased in gelsolin-overexpressed cells (data not shown). Therefore, reduced RhoA levels might be also responsible for the reduced PLD response to bradykinin in gelsolin overexpressed cells.

On the other hand, PLD2 is independent of PLC activation, since it is not activated by PKC and small G proteins (1, 2). S1P stimulation induced PLC and PLD activation and translocation of PKC (-alpha , -beta 1) in NIH 3T3 cells. Gelsolin overexpression reduced PLC response to S1P and translocation of PKC-alpha and PKC-beta 1 but did not affect PLD activation. These results indicated that S1P-induced PLD activation was PKC-independent and involved PLD2, which was abundantly present in NIH3T3 cells. Furthermore, our preliminary experiments gave supportive data suggesting that PLD2 is involved in S1P-induced PLD activation. In NIH 3T3 cells transiently transfected with hemagglutinin-tagged PLD2 cDNA, there was 1.5-fold increase in S1P-induced PLD activation, whereas cells transfected with catalytically inactive PLD2 mutants showed reduced PLD activation in response to S1P (data not shown). These observations suggest that gelsolin represses bradykinin-induced PLD1 activation via PLC/PKC suppression but does not affect S1P-induced PLD2 activation.

Our findings that S1P-mediated signaling involving activation of PLC, PKC-alpha , and MAP kinase was sensitive to PTX treatment but PLD activation was insensitive to such treatment indicate that PLD activation is independent of these signaling components. This notion is consistent with the S1P signaling pathway in Swiss 3T3 fibroblasts (30). There are three subfamilies of S1P receptor (EDG1, EDG3, and EDG5) (35). EDG1 is coupled to Gi/o protein, which mediates the signaling pathway involving PLC, adenylate cyclase, and MAP kinase (47). In contrast, EDG3 and EDG5 mediate the pathways via the Gi, Gq, and G12/13 (35, 46). Furthermore, it has been reported that in Chinese hamster ovary cells stably expressing EDG1 receptor, S1P induces MAP kinase activation in a PTX- and genistein-sensitive but PKC-independent manner (30). In comparison, our study demonstrated that S1P- stimulated MAP kinase activation was PTX-sensitive and PKC-dependent but tyrosine kinase-insensitive. These results indicate that in NIH3T3 fibroblasts, S1P-induced MAP kinase activation was mediated via Gi but not EDG1. PLC activation induced by bradykinin and S1P stimulation were suppressed by gelsolin overexpression, whereas MAP kinase activation by both agonists was not affected. These results suggest that S1P induced PLD activation via certain G proteins distinct from Gi for PLC and the MAP kinase pathway in NIH 3T3 fibroblasts. Recently, PLD2 was demonstrated to be involved in insulin-dependent MAP kinase pathway (8). Our results, however, suggested that neither PLC nor PLD is associated with MAP kinase activation in S1P-stimulated NIH 3T3 fibroblasts.

    ACKNOWLEDGEMENT

We thank Dr. M. A. Frohman (State University of New York) for providing cDNA of human PLD1 and PLD2.

    FOOTNOTES

* This work was supported in part by Grants-in-aid for Scientific Research on Priority Areas 09273104 and 10212204 and Grants-in-aid for Scientific Research (B) 09480162 and (C) 09670150 from the Ministry of Education, Science, Sports, and Culture of Japan. A special Coordination Fund for Promoting Science and Technology was from the Science and Technology Agency of Japan.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.

§ To whom correspondence should be addressed: Dept. of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan. Tel.: 81-58-267-2230; Fax: 81-58-265-9002.

    ABBREVIATIONS

The abbreviations used are: PC, phosphatidylcholine; ARF, ADP-ribosylation factor; DMEM, Dulbecco's modified Eagle's medium; G2 and G6, gelsolin-overexposing clones 2 and 6, respectively; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; InsP, inositol phosphate; MAP kinase, mitogen-activated protein kinase; PA, phosphatidic acid; PBut, phosphatidylbutanol; PE, phosphatidylethanolamine; PIP2, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; PLC and PLD, phopholipase C and D, respectively; PMA, phorbol 12-myristate 13-acetate; PTX, pertussis toxin; S1P, sphingosine 1-phosphate; Vect, vector-transfected; Ab, antibody; MAP, mitogen-activated protein; BSA, bovine serum albumin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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