479

ARTICLE[

Protein Kinase C-Dependent Stimulation of Phospholipase D in Phospholipase C-Treated Fibroblasts Zoltan Kiss* and Nandor Garamszegi The Hormel Institute, University of Minnesota, Austin, Minnesota 55912

Treatment of [14C]choline- or [14C]ethanolamine-labeled NIH 3T3 fibroblasts with Bacillus cereus phosphatidylcholine-specific phospholipase C (PLC) enhanced phospholipase D (PLD)-mediated hydrolysis of the respective 14Clabeled phospholipids. PLD activity was stimulated by 1.5 U/mL of PLC and by 100 nM of the protein kinase C (PKC) activator phorbol 12-myristate l~acetate (PMA) to similar extents. Treatment of [14C]palmitic acid-labeled fibrc~ blasts with PLC in the presence of ethanol also enhanced PLD-mediated formation of phosphatidylethanol; the effects of PLC and PMA were nonadditive. PLC had no effect on PLD activity in fibroblasts in which PKC was down-regulated by prolonged (24 h) treatment with 300 nM PMA. These data indicate that treatment of fibrob|asts with exogenous PLC results in PKC
(20), this may not automatically lead to the activation of PLD because regulation of this latter enzyme by PKC al> pears to involve a nonphosphorylating mechanism (21). Ftm thermora regulation of PtdCho synthesis by exogenous PLC, which is mediated by 1,2-diacylglycerol, was shown to occur by a PKC-independent mechanism (22). Hera we show that exposure of NIH 3T3 fibroblasts to PLC leads to enhanced, PKC,dependent hydrolysis of phospholipids by PLD. MATERIALS AND METHODS

Materials. PLC from B. cereus, PMA and Dowex-50W(H + form) were purchased from Sigma Chemical Ca (St. Louis, MO); [rnethyl-~4C]choline chloride (50 mCi/mmol), [2-14C]ethanolamine (50 mCi/mmol) and [1-14C]palmitic acid (60 mCi/mmol) were from Amersham (Arlington Heights, IL); and tissue culture reagents were bought from Gibco (Grand Island, NY). Phosphatidylethanol (PtdEtOH) was prepared by PLD-catalyzed reaction from PtdCho and ethanol as described earlier (23). Treatment of [14C]choline-prelabeled fibroblasts. NIH 3T3 fibroblasts were grown to =80-90% confluency in 12-well culture dishes in the presence of [rnethyl-14C]choline (0.5 ~Ci]mL) for 48 h. Fibroblasts were washed twice and then incubated in fresh medium for another 3-h period (needed to lower intracellular levels of free [14C]choline). Fibroblasts were then treated with PLC and]or PMA in the presence of 20 mM unlabeled choline (final vol, 0.3 mL) for 5-20 min as indicated. After treatments with 0.75 or 1.5 U/mL of PLC or 100 nM PMA for 20 min, 95-98% of cells were viable, determined by the Trypan Blue dye exclusion assay. Incubations were terminated by adding 1 mL of ice-cold methanol to the wells. The methanol extracts were transferred to tubes containing 2 mL chloroform. The wells were washed twice with 0.5 mL of methanol. Treatment of [14C]ethanolamine- or [14C]palmitic acidlabeled fibroblasts. Fibroblasts were grown in 150 mmdiameter plastic dishes for 48 h in the presence of [2-~4C]ethanolamine (0.25 ~Ci/mL), or for 24 h in the presence of [1-14C]palmitic acid (0.25 gCi/mL). Fibroblasts were washed and then incubated in fresh medium for 3 h (to decrease the cellular level of unincorporated radiolabeled precursors; see Refs. 23-25). Fibroblasts were harvested by gentle scraping from 2 to 4 dishes. Washed fibroblasts (0.9-1.1 • l0 s cells/mL) were incubated (final vol 0.25 mL) in an incubator at 37~ in the presence of agents as indicated. In the case of [14C]ethanolaminelabeled fibroblasts, the incubation medium contained 2 mM unlabeled ethanolamine to prevent metabolism of newly formed [~4C]ethanolamine (24,25). Incubations were terminated by the addition of 4 mL of chloroform/methanol (1:1, vol/vol). Separation of 14C-labeled hydrolytic products. PtdEtOH was separated from other phospholipids on LIPIDS, Vol. 28, no. 6 (1993)

480

z. KISS AND N. GARAMSZEGI potassium oxalate (l%)-impregnated silica gel H plates (Analtech, Newark, DE) by using the solvent system of chloroform/methanol/acetone/acetic acid/water (50:10:15: 10:2, by vol). The choline and ethanolamine metabolites were fractionated on Dowex-50-W(H+)-packed columns (Bio-Rad Econo columns, Richmond, CA; 1-mL bed volume) as described by Cook and Wakelham (26) with the modifications described previously (27). The metabolites of [~4C]-ethanolamine and [~4C]choline were further identified by thin-layer chromatography (28). Contamination of the [~4C]ethanolamine fraction by [~4C]choline was less than 1%. RESULTS

Concentration-dependent effects of PLC on the formation of choline and choline phosphate. We have shown (28) that in N I H 3T3 fibroblasts, labeled with [14C]choline until radioisotopic equilibrium was achieved (48 h), PMA-induced formation of [~4C]choline from the prelabeled cellular PtdCho occurs by a PLD-mediated mechanism. In addition, we have demonstrated (23) that treatment of fibroblasts with PLC results in a significant increase in 1,2-diacylglycerol. As shown in Figure la, 0.25-1.5 U/mL concentrations of B. cereus PLC enhanced the formation of [14C]choline from [~4C]PtdCho in a concentration-dependent manner. At an optimal stimulatory concentration of PMA (100 nM), the stimulatory effects of PLC were nonadditive with that of PMA, suggesting that these agents were acting through the same mechanism (Fig. la). N I H 3T3 fibroblasts contain the a-, &, ~- and ~-PKC isozymes. Treatment of fibroblasts with 300 nM PMA for 24 h almost completely down-regulates PKC-a (29}, and also decreases the cellular levels of PKC-d and PKC-e by 75-90% (data not shown). In contrast, prolonged (24 h) PMA-treatment had no effect on the cellular level of PKC-~

(N. Garamszegi and Z. Kiss, unpublished data). Prolonged (24 h) treatment of fibroblasts with PMA (300 riM) significantly enhanced the formation of [14C]choline from labeled PtdCha indicating partial activation of PLD (Fig. la). After chronic (24 h) treatment with PMA, neither newly added PMA (not shown) nor PLC (Fig. la) had any further effect on PtdCho hydrolysis. Treatment of fibroblasts with PMA for 20 min or 24 h caused only little, if any, changes in PLC-catalyzed formation of choline phosphate (Fig. lb). Thus, the inability of PLC to enhance [14C]choline formation in the presence of PMA was not due to PMA-induced inactivation of PLC. In the presence of ethanol, activated PLD catalyzes the formation of the metabolically more stable PtdEtOH. As shown in Figure 2, 0.75-1.5 U/mL concentrations of exogenous PLC greatly enhanced the formation of PtdEtOH in the presence of 200 mM ethanol. In agreement with the data in Figure 1, PLC failed to enhance P t d E t O H synthesis in the presence of PMA (Fig. 2). In NIH 3T3 fibroblasts, activated PLD was shown to hydrolyze PtdEtn in addition to PtdCho (24,25,28-30). Thus, it was of interest to examine possible stimulation of PtdEtn hydrolysis by exogenous PLC. For this study, suspended [14C]ethanolamine-labeled fibroblasts were used, because they were found to contain much lower background levels of unincorporated [14C]ethanolamine compared to attached fibroblasts (24,27). Both in [~4C]choline-labeled attached fibroblasts (Fig. 3a) and [~4C]ethanolamine-labeled suspended fibroblasts (Fig. 3b), 0.75-1.5 U/mL concentrations of PLC rapidly, and to a similar extent, stimulated PLD-mediated hydrolysis of the respective labeled phospholipids. However, the stimula-

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FIG. 1. Concentration-dependent effects of phospholipase C (PLC) on phosphatidylcholine (PtdCho) hydrolysis in N I H 3T3 fibroblasts. Fibroblasts were labeled with [14C]choline for 48 h, followed by incubation of labeled fibroblasts for 20 rain in the absence ( o ) or presence of 100 nM phorboi 12-myristate 13-acetate (PMA) (&). In one set of experiments (m), cells were treated with 300 nM P M A for the last 24 h of the labeling period. [14C]Choline (a) and [14C]choline ~4hosphate (b) were separated by ion~xehange chromatojgraphy. The C content of PtdCho was 883000 and 819000 dpm/10 ~ cells in the untreated and PMA-pretreated fibroblasts, respectively. Each point represents the mean ___SE of three incubations. Similar results were obtained in two other experiments. L I P I D S , Vol. 28, no. 6 (1993)

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FIG. 2. Stimulatory effects of PLC and P M A on the formation of phosphatidylethanol (PtdEtOH) in [14C]p_-Imltate-labeled N I H 3T3 fibroblasts. Suspended [14C]pAtmltate-labeled N I H 3T3 fibroblasts were incubated in the presence of 200 mM ethanol for 10 min. PLC was absent (open bar) or present at a concentration of 0.75 U/mL (hatched bar) or 1.5 U/mL (closed bar). When present, the concentration of P M A was 100 nM. Data are the mean _ SE of four incubations. Similar results were obtained in two other experiments. Abbreviations as in Figure 1.

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acid in the mediation of mitogenic effect of PLC is unlikely. However, in view of the present finding that prolonged treatment of fibroblasts with PMA results in permanent partial activation of PLD, the possibility that increased formation of phosphatidic acid by PLD is a prerequisite for the mitogenic action of PLC cannot be excluded. Further experiments are required to distinguish between these possibilities.

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ACKNOWLEDGMENTS This work was supported by The Hormel Foundatiop_ We are grateful to K.S. Crilly for technical assistance and to C. Perleberg for secetarial assistance

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REFERENCES u_

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FIG. 3. Comparison of the time-dependent effects of PLC on the hydrolysis of PtdCho and PtdEtn in N I H 3T3 fibroblasts. Fibroblasts were labeled with [14C]choline (a} or [14C]ethanolamine {b} for 48 h. Attached [14C]choline-labeled and suspended [14C]ethanolaminelabeled fibrohlasts were incubated for 5-20 min in the absence { O) or presence of 0.75 UfmL of PLC (&}, or 1.5 U/mL of PLC {11). The [14C]content of PtdCho and PtdEtn was 0.966 X 106 and 1.29 X 106 dpm/10 ~ cells, respectively. Each point represents the mean +__ SE of three incubations. Similar results were obtained in three other experiments. Abbreviations as in Figure 1.

t o r y effect of P L C on P t d E t n hydrolysis a p p e a r e d to be r a t h e r t r a n s i e n t (lasting o n l y for 10 re_in) c o m p a r e d to P L C - i n d u c e d P t d C h o hydrolysis. O n e possible, a n d t h e m o s t likely, e x p l a n a t i o n for this difference is t h a t scraping decreased the sensitivity of fibroblasts to PLC. I n supp o r t of this possibility, t h e s t i m u l a t o r y effects of P L C on P t d C h o hydrolysis in [14C]choline-labeled s u s p e n d e d fibroblasts also lasted for only 10-15 min (data n o t shown).

In N I H 3T3 fibroblasts, the methylation process is a relatively minor pathway. Thus, after labeling of fibroblasts with [14C]ethanolamine for 48 h, only 3% of total phospholipid-associated 14C-activity was present in PtdCho (data not shown). This, coupled with efficient separation of t4C-labeled metabolites, ensured that contamination of the [14C]ethanolamine fraction by [14C]ch~ line was minimal (less than 1%). DISCUSSION We have s h o w n t h a t t r e a t m e n t of N I H 3T3 fibroblasts with e x o g e n o u s P L C leads to P K C - d e p e n d e n t a c t i v a t i o n of PLD. This indicates t h a t 1,2-diacylglycerol, g e n e r a t e d t h r o u g h t h e action of e x o g e n o u s PLC, can s t i m u l a t e n o t only t h e p h o s p h o r y l a t i n g a c t i v i t y of P K C (20), b u t it can also enhance the ability of P K C to activate P L D b y a nonp h o s p h o r y l a t i n g m e c h a n i s m (21). B e c a u s e activated P L D generates the p o t e n t m i t o g e n p h o s p h a t i d i c acid (13-19), this p h o s p h o l i p i d p r o d u c t could be involved, a t least in principle, in t h e m e d i a t i o n of m i t o g e n i c effects of PLC. While chronic t r e a t m e n t of cells w i t h P M A c o m p l e t e l y abolished t h e s t i m u l a t o r y effect of P L C on P L D a c t i v i t y (this work), such t r e a t m e n t failed to prevent the mitogenic effect of P L C (1). Therefore, a direct role of p h o s p h a t i d i c

1. Larrodera, P., Comet, M.E., Diaz-Mec~ M.T, Lopez-Barahona, M., Diaz-Laviada, I., Guddal, RH., Johansen, T., and Moscat, J. (1990) Cell 61, 1113-1120. 2. De Herreros, A.G., Dominquez, I., Diaz-Meco, M.T., Graziani, G., Comet, M.E., Guddai, RH., Johansen, T., and Moscat, J. (1991) J. BioL Chem. 266, 6825-6829. 3. Diaz-Meco, M.T, Dominquez, I., Sanz, L., Munici(~ M.M., Berra, E., Comet, M.E., De Herreros, A.G., Johansen, T., and Moscat, J. (1992) MoL CelL BioL 12, 302-308. 4. Cai, H., Erhardt, R, Szeberenyi, J., Diaz-Meco, M.T., Johansen, T., Moscat, J., and Cooper, G.M. (1992) MoL CelL Biol. 12, 5329-5335. 5. Choudhury, G.G., Sylvia, V.L., and Sakaguchi, A.Y. (1991)J. BioL Chem. 266, 23147-23151. 6. Fisher, G.J., Henderson, RA., Voorhees, J.J., and Baldassare, J.J. (1991) J. CelL PhysioL 146, 309-317. 7. Osada, S., Nakashima, S~, Saji, S., Nakamura, T., and Nozawa, Y. (1992) F E B S Lett. 297, 271-274. 8. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, V., and Nishizuka, Y. (1982) J. BioL Chem. 257, 7847-7851. 9. Nishizuka, Y. (1984) Nature 308, 693-698. 10. Exton, J.H. {1990}J. Biol. Chem. 265, 1-4. 11. Billah, M.M., and Anthes, J.C. (1990}Biochem. J. 269, 281-291. 12. Kiss, Z. (1990) Prog. Lipid Res. 29, 141-166. 13. Moolenaar, W.H., Kruijer, W., Tilly, B.C., Verlaan, I., Bierman, A.J., and De Laat, S.W. (1986) Nature (London) 323, 171-173. 14. Yu, C.L., Tsai, M.H., and Stacey, D.W. (1988) Cell 52, 63-71. 15. Imagawa, W., Bandyopadhyay, G.K., Wallace, D., and Nandi, S. (1989) Proa NatL Acad ScL USA 86, 4122-4126. 16. van Corven, E.J., Groenink, A., Jalink, K., Eichholtz, T., and Moolenaar, W.H. (1989) Cell 59, 45-54. 17. Knanss, T.C, Jaffer, EE., and Abbound, H.E. (1990)J. BioL Chem. 265, 14457-14463. 18. van Corven, E.J., van Rijswijk, A., Jalink, K., van der Bend, R.L., van Blitterswijk, W.J., and Moolenaar, W.H. {1992) Biochem. J. 281, 163-169. 19. Fukami, I~, and Takenawa, T. (1992) J. BioL Chem. 267, 10988-10993. 20. Kiss, Z., and Steinberg, R.A. (1985) CancerRes. 45, 2732-2740. 21. Conricode, K.M., Brewer, K.A., and Exton, J.H. (1992) J. BioL Chem. 267, 7199-7202. 22. Jones, G.A., and Kent, C. (1991) Arch. Biochem. Biophys. 288, 331-336. 23. Kiss, Z., Chattopadhyay, J., and Pettit, G.R. (1991) Biochem. J. 273, 189-194. 24. Kiss, Z. (1991) Lipids 26, 321-323. 25. Kiss, Z. (1992} Biochern. J. 285, 229-233. 26. Cook, SJ., and Wakelam, M.J.O. (1989) Biochem. J. 263, 581-587. 27. Kiss, Z., and Dell, E. (1992) Biochem. J. 288, 853-858. 28. Kiss, ~, and Anderson, W.B. (1989)J. BioL Chem. 264, 1484-t487. 29. Kiss, Z. (1992) Eur. J. Biochem. 209, 467-473. 30. Kiss, Z., and Anderson, W.B. (1990)J. BioL Chem. 265, 7345-7350.

[Received January 7, 1993; Revision accepted April 16, 1993] LIPIDS, Vol. 28, no. 6 (1993)