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Expression of Protein Kinase C-β Promotes the Stimulatory Effect of Phorbol Ester on Phosphatidylethanolamine Synthesis
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 347, No. 1, November 1, pp. 37–44, 1997 Article No. BB970308
Expression of Protein Kinase C-b Promotes the Stimulatory Effect of Phorbol Ester on Phosphatidylethanolamine Synthesis Zoltan Kiss1 The Hormel Institute, University of Minnesota, 801 16th Avenue N.E., Austin, Minnesota 55912
Received March 27, 1997, and in revised form July 23, 1997
zymes, i.e., PKC-b and PKC-a, respectively. Stimulation of phosphatidylethanolamine (PtdEtn) synthesis by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) has reportedly been found only in hepatocytes expressing the a-, bII-, e-, and z-PKC isozymes. In contrast, stimulation of phosphatidylcholine synthesis by PKC activators, known to be mediated by PKC-a, is widespread in mammalian cells. In this work, various cell lines exhibiting characteristic differences in their PKC systems were used to determine the role of specific PKC isozymes in the mediation of PMA effect on PtdEtn synthesis. In NIH 3T3 fibroblasts, which express high levels of PKC-a but none of the b (bI or bII) isoforms, PMA did not stimulate PtEtn synthesis. In contrast, in Rat-6 fibroblasts overexpressing PKC-bI, 10 – 100 nM PMA considerably (1.7- to 2.6-fold) enhanced PtdEtn synthesis. In wild-type or multidrug resistant MCF-7 human breast carcinoma cells, which express PKC-a and PKC-bII (to varying extents) but not PKC-bI, PMA had only small or no effects on PtdEtn synthesis. In contrast, in MCF-7 cells overexpressing PKC-a, and as a consequence also expressing the bI- and bII-PKC isoforms, PMA effectively stimulated the synthesis of PtdEtn. Finally, in HL60 human leukemia cells, which contains PKC-bII as the major PKC isoform, PMA again stimulated PtdEtn synthesis. The results establish that while stimulation of PtdEtn synthesis by PMA occurs only in selected cell lines, this phenomenon is not restricted to hepatocytes. Furthermore, the data indicate that expression of either PKC-bI or PKC-bII, but not PKC-a, correlates with the effect of PMA on PtdEtn synthesis. Overall, these observations strongly suggest that regulation of PtdEtn and PtdCho synthesis by PMA involves separate PKC iso-
1 Address correspondence to author. Fax: (507) 437-9606. E-mail: [email protected].
Key Words: phosphatidylethanolamine synthesis; phorbol ester; protein kinase C-bII; protein kinase C-a.
In most mammalian cells the protein kinase C (PKC)2 system is a major regulator of phosphatidylcholine (PtdCho) synthesis (1–3). Activators of PKC, including phorbol 12-myristate 13-acetate (PMA), increase the rate of PtdCho synthesis via stimulating CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), the key regulatory enzyme of the PtdCho-synthesizing pathway (3, 4). Although the mechanism by which PMA regulates cytidylyltransferase activity has not been clarified yet, it seems clear that its effect is mediated by PKC-a (5). This isozyme also mediates the stimulatory effect of PMA on choline uptake (5), but these two effects of PMA are unrelated (6). PMA-stimulated PKC-a is also a major regulator of phospholipase D-mediated hydrolysis of PtdCho (7–10) and phosphatidylethanolamine (PtdEtn) (11). Thus, in PMA-treated cells the hydrolysis of PtdCho is usually followed by its resynthesis, so that PMA does not cause significant long-term changes in the cellular content of this phospholipid. In comparison to PtdCho synthesis, regulation of PtdEtn synthesis by the PKC system is much less understood. So far, a stimulatory effect of PMA on PtdEtn 2 Abbreviations used: PKC, protein kinase C; PMA, phorbol 12myristate 13-acetate; PtdEtn, phosphatidylethanolamine; PtdCho, phosphatidylcholine; DMEM, Dulbecco’s modified Eagle’s medium; MCF-7/PKC-a and MCF-7/vector cells are stably transfected with PKC-a and the corresponding empty vector, respectively; MCF-7/WT and MCF-7/MDR1 cells are wild-type and multidrug-resistant MCF7 cells, respectively; and R6/PKC-bI cells are R6 fibroblasts overexpressing PKC-bI.
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0003-9861/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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synthesis has been described only in isolated hepatocytes (12). Since PtdEtn is usually the second major membrane phospholipid, it would be important to know how its synthesis is regulated by PKC, a major regulatory enzyme system. In this work, the effects of PMA on PtdEtn synthesis were examined in various cell lines exhibiting different PKC isozyme compositions. The results indicate that the stimulatory effect of PMA on PtdEtn synthesis is not restricted to hepatocytes and that it requires cellular expression of either PKC-bI or PKC-bII, but not PKC-a. MATERIALS AND METHODS Materials. PMA and Dowex-50W[H/] were purchased from Sigma; [methyl-14C]choline chloride (50 mCi/mmol), [2-14C]ethanolamine (55 mCi/mmol), L-[U-14C]serine (150 mCi/mmol), and carrier-free phosphorus-32 orthophosphate (32Pi) were bought from Amersham; and tissue culture reagents were purchased from GIBCO BRL. The polyclonal antibody raised against PKC-a was kindly provided by Dr. Yusuf A. Hannun (Duke University, Durham, NC). Cell culture. NIH 3T3 clone-7 cells, obtained from Dr. Douglas R. Lowy (NCI, National Institutes of Health, Bethesda, MD), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin/streptomycin (50 units/ ml and 50 mg/ml, respectively) and glutamine (2 mM). MCF-7/PKCa cells, kindly donated by Dr. D. Kirk Ways (Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN), were generated by stably cotransfecting wild-type MCF-7 cells with PKC-a subcloned into the pSV2M(2)6 vector and a neomycin-resistant plasmid (19). Cells stably transfected with the empty pSV2M(2)6 vector (MCF-7/ vector cells) (19) were also used as a control cell line in this study. Because of the altered PKC isozyme composition, MCF-7/PKC-a cells, unlike the vector control cells, were unable to attach to the substratum; accordingly, MCF-7/PKC-a cells were grown in suspension. MCF-7/MDR1 cells, overexpressing MDR1 P-glycoprotein, was generously provided by Dr. Kenneth Cowan (National Cancer Institute, NIH, Bethesda, MD). Each MCF-7 line was maintaned in pyruvate-free DMEM supplemented with 2 mM glutamine, 10 mM Hepes, 10% fetal calf serum, 200 mg/ml G418 (in case of MCF-7/ PKC-a and MCF-7/vector cells), 50 units/ml penicillin, and 50 mg/ml streptomycin. R6/PKC-bI cells, Rat-6 fibroblasts highly overexpressing PKC-b1 (20), and the corresponding vector cells were generously provided by Dr. I. Bernard Weinstein (Institute of Cancer Research, Columbia University, New York, NY). The R6 cell lines were maintained in DMEM supplemented with 10% fetal calf serum, 1 mM glutamine, 50 units/ml penicillin, 50 mg/ml streptomycin, and 50 mg/ml G418. The human leukemia HL60 cells were maintained in RPMI 1640 medium as previously described (21). Measurement of incorporation of [14C]ethanolamine into cells and PtdEtn. For these experiments MCF-7/PKC-a and HL60 cells, both grown in suspension, were transferred (Ç4–8 1 106 cells/tube) to 15-ml plastic tubes (incubation volume, 0.5 ml), while NIH 3T3 and R6 fibroblasts as well as MCF-7/WT and MCF-7/MDR1 cells were grown in 12-well plates up to 85–95% confluency. Incubation of cells with 25 mM [14C]ethanolamine (0.8–1 mCi/ml) or 32Pi (1 mCi/ml) were performed in the absence or presence of PMA for the time indicated in the figure legends. At the end of incubation of attached cells, the incubation medium was aspirated and then cells were rapidly (within 20 sec) washed with 5 ml medium followed by the addition of icecold methanol (2 ml) to the wells. Then, cells were scraped into methanol, and the methanol extracts were rapidly transferred to 2 ml of choroform. In the case of MCF-7/PKC-a and HL60 cells, the incubations were stopped by adding 14 ml medium to the tubes, followed by pelleting the cells at 500g for 5 min. After aspirating the superna-
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tant, the tight cell pellets were gently, but rapidly, rinsed with 5 ml medium followed by the addition of 4 ml of chloroform/methanol (1:1, v/v) to the tubes. The background values for cell-bound free [14C]ethanolamine (i.e., when cells were immediately washed or pelleted after the addition of radioactive precursor) were always less than 10% of that observed after incubation of cells for 30 min in the absence of PMA; under these conditions there was no detectable radiolabel in the phospholipids. After phase separation, PtdEtn present in the chloroform phase was separated from other phospholipids on silica gel H plates by onedimensional TLC using a chloroform/methanol/28% ammonia (65/25/ 5, by volume) mixture as solvent. Fractionation of radiolabeled water-soluble ethanolamine metabolites, present in the upper water/ methanol phase, was performed on Dowex-50W[H/]-packed columns (Bio-Rad Econo-columns, 0.75-ml bed volume) with minor modifications of the procedure described by Cook and Wakelam (22). The initial flowthrough (5 ml) along with a following wash by 3.5 ml of water contained glycerophosphoethanolamine. Phosphoethanolamine and ethanolamine were eluted by 15 ml of water and 1 M HCl, respectively. [14C]CDP-ethanolamine was separated by a TLC system described previously (13). Western blot analysis of PKC-a. Cells from varius subclones of MCF-7 line were collected in 1.5 ml homogenization buffer containing 20 mM Tris–HCl pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 100 mg/ml leupeptin, and 25 mg/ml aprotinin. After homogenization, homogenates (40 mg protein each) were subjected to SDS–PAGE (10% acrylamide minigel) and proteins were transblotted from gels to nitrocellulose membranes. The membranes were reacted with polyclonal antibody to PKC-a (used at 1:1000 dilution), and the immunoreactive proteins were stained and analyzed as described earlier (11). Data analysis. Results are expressed either as means { SEM for the stated number of experiments, or as means { SE for the stated number of determinations in one experiment. Statistical analyses were performed with Student’s t test. Statistical significance was determined at the 0.05 level.
RESULTS
In hepatocytes, which contain the a-, bII-, e-, and zPKC isoforms (23, 24), PMA was previously shown to stimulate PtdEtn synthesis (12). NIH fibroblasts also contain the a-, e-, and z-PKC isoforms, but in these cells the bII-isoform is replaced by the d-isoform of PKC (25, 26). Thus, a study of PMA effects on PtdEtn synthesis and on the uptake of precursor [14C]ethanolamine by NIH 3T3 cells was performed first to obtain preliminary information on the regulatory role of various PKC isozymes. Treatment of attached NIH 3T3 fibroblasts with PMA (100 nM) for 30 min resulted in a nearly twofold increase in the amount of cell-bound unesterified [14C]ethanolamine (Fig. 1). The stimulatory effect of PMA lasted for about 1 h; after 2 h treatments with PMA, the cellular level of [14C]ethanolamine returned to the basal level (data not shown). In other experiments, NIH 3T3 cells were first detached from the substratum by trypsin, and the suspended cells were then treated with PMA in the presence of radiolabeled ethanolamine for 30 min. Despite the relatively long (5 min) centrifugation performed in a large (14 ml) volume of DMEM (to reduce background radioactivity), the stimulatory effects of PMA on the cellular uptake of [14C]-
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FIG. 1. Stimulatory effects of PMA on the cellular uptake of [14C]ethanolamine in attached and suspended NIH 3T3 fibroblasts. Attached (I) or suspended (II) NIH 3T3 fibroblasts were labeled for 30 min with [14C]ethanolamine in the absence (h) or presence (j) of 100 nM PMA. Data are means of four independent incubations in a representative experiment with the same passage of cells. The difference between the two extreme values was always less than 14%. Similar results were obtained in two other experiments each performed in triplicate.
ethanolamine (Fig. 1) was maintained during this procedure. The stimulatory effect of PMA on ethanolamine uptake was similarly maintained in suspended cells when cells were collected by scraping. These results indicated that the technique used in case of suspended NIH 3T3 cells should also be suitable for the study of PMA effects on [14C]ethanolamine uptake and [14C]PtdEtn formation in cells maintained and used in suspension cultures. Despite the stimulatory effect of PMA on [14C]ethanolamine uptake, PMA failed to enhance the incorporation of [14C]ethanolamine (25 mM) into PtdEtn in attached fibroblasts (Fig. 2) or in suspended fibroblasts (not shown) over an 80-min incubation period. Since in hepatocytes PMA appeared to maximally stimulate PtdEtn synthesis in the 50– to 100-mM range of ethanolamine (12), the above experiments were repeated by using these higher concentrations of ethanolamine; PMA also failed to enhance the synthesis of PtdEtn at 50–100 mM [14C]ethanolamine (data not shown). Since the experiments with NIH 3T3 cells indicated that the presence of a-, d-, e-, and z-PKC isoforms does not promote the effect of PMA on PtdEtn synthesis, in
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the following experiments the possible role of PKC-b was studied in more detail. First, the effects of PMA were examined in R6/PKC-bI cells which highly express PKC-bI, as well as in vector control R6 cells which do not express this PKC isoform (20). Treatments of cells with 100 nM PMA for 30 min enhanced the cellular contents of [14C]ethanolamine in vector control cells and in R6/PKC-bI cells about 1.3- and 1.5fold, respectively (data not shown). During shorter (30– 60 min) periods of incubation, in R6/PKC-bI cells 10 or 100 nM PMA stimulated PtdEtn synthesis 1.7- to 1.9-fold or 2.1- to 2.6-fold, respectively (Fig. 3). During the same incubation period, in vector control cells 100 nM PMA (but not 10 nM PMA) had detectable, but considerably smaller (Ç1.4-fold), stimulatory effects on PtdEtn synthesis (Fig. 3). In R6/PKC-bI cells the relative stimulatory effects of PMA were considerably decreased when the incubation time was extended to 2 h (1.2- to 1.3-fold stimulation). It still should be noted that at various time points the ‘‘basal’’ level of PtdEtn synthesis in R6/PKC-bI cells was about two to three times higher than in the vector control cells (Fig. 3). This may indicate that in these cells PKC-bI is partially activated even in the absence of PMA resulting in partial stimulation of PtdEtn synthesis. This possibility remains to be experimentally verified. Next the effects of PMA on PtdEtn synthesis were determined in (i) wild-type MCF-7 human breast carcinoma cells (MCF-7/WT), which contain no PKC-bI (27),
FIG. 2. PMA has no effect on PtdEtn synthesis in NIH 3T3 fibroblasts. Attached NIH 3T3 fibroblasts were incubated without (l) or with 100 nM PMA (m) in the presence of [14C]ethanolamine for up to 80 min. Each point represents the mean { SEM of three separate experiments each performed in triplicate.
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FIG. 3. Expression of PKC-bI in R6 fibroblasts promotes the stimulatory effect of PMA on PtdEtn synthesis. Attached vector control R6 cells (open symbols) and R6/PKC-bI cells (closed symbols) were incubated for up to 120 min with [14C]ethanolamine in the absence (s, l) or presence of 10 nM PMA (n, m), or 100 nM PMA (h, j). Each point represents the mean { SE of four determinations from separate wells in a single experiment. Similar results were obtained in two other experiments each performed in the absence or presence of 100 nM PMA in triplicate.
express little PKC-a (Fig. 4; Ref. 27), and contain significant amount of PKC-bII (27); (ii) multidrug-resistant MCF-7 cells (MCF7-7/MDR1) which express large amounts of PKC-a (Fig. 4; Ref. 27), contain no PKC-bI (27), and express only small amount of PKC-bII (27); and (iii) MCF-7/PKC-a cells which not only contain PKC-a at high level (Fig. 4; Ref. 19) but they also express significant amount of PKC-b (19), mostly PKCbI (11). Finally, MCF-7/vector contor cells, containing only the empty vector and developed as controls for MCF-7/PKC-a cells, have PKC isozyme composition practically identical with that of wild-type cells. Of interest to note is that MCF-7/MDR1 and MCF-7/PKC-a cells express PKC-a at similar levels (Fig. 4). It was expected that these four cell lines, showing characteristic differences in the expression of PKC-b and PKC-a isoforms, will provide additional important information on the PKC isozyme requirement of PMA effect on PtdEtn synthesis. In MCF-7/PKC-a cells, but not in vector control or MCF-7/MDR1 cells, PMA at 5–100 nM concentrations significantly (Ç1.5–1.7-fold) increased the incorporation of [14C]ethanolamine into PtdEtn over a 30-min
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incubation period (Fig. 5A). At this incubation time, PMA had no effect on PtdEtn synthesis in MCF-7/WT cells (data not shown). Interestingly, PMA also increased the cellular content of free [14C]ethanolamine only in MCF-7/PKC-a cells, but not in the vector control or MCF-7/MDR1 cells (Fig. 5B). Increased synthesis of [14C]PtdEtn in PMA-treated MCF-7/PKC-a cells was accompanied by decreased cellular level of [14C]phosphoethanolamine (Fig. 6), reflecting increased utilization of this precursor for PtdEtn synthesis. While in MCF-7/PKC-a cells both radiolabeled ethanolamine and phosphoethanolamine were easily detectable both in the absence and presence of PMA, [14C]CDP-ethanolamine formation could only be observed in the presence of PMA and only at a very low level (Ç50–80 dpm/106 cells). In contrast to the shorter-term (30 min) experiments, when the incubation time was extended to 2 h PMA had clearly detectable (Ç1.3-fold) stimulatory effects on PtdEtn synthesis in MCF-7/WT (Fig. 7C) and MCF7/vector control cells (Fig. 7B), but not in MCF-7/MDR1 cells (Fig. 7D). Also, in contrast to R6/PKC-bI cells (Fig. 3), in MCF-7/PKC-a cells (Fig. 7A) the effect of PMA on PtdEtn synthesis was somewhat increased, rather than decreased, when the incubation time was extended from 45 min (1.9-fold stimulation) to 120 min (2.2-fold stimulation). Since MCF-7/WT cells contain significantly more PKC-bII than MCF-7/MDR1 cells (27), the small stimulatory effects of PMA in the former cells could be mediated by this isozyme. This possibility led to the study of human leukemic HL60 cells in which PKC-bII is the major PKC isoform and PKC-bI is absent (28–30). In the experiments with HL60 cells, both [14C]ethanolamine and 32Pi were employed as labeling agents. The incubation time was sufficiently long (2 h) to achieve appropriate 32P labeling of the cellular ATP pool, and thereby the cellular PtdEtn pool. As shown in Fig. 8, in the presence of both radiolabeled precursors PMA significantly (Ç2- to 2.3-fold) enhanced the labeling of PtdEtn. When examined over a 30-min incubation period, PMA enhanced the cellular content of free [14C]ethanolamine only Ç1.2-fold, and it had no
FIG. 4. Western blot analysis of PKC-a in MCF-7 lines. PKC-a in homogenates from vector control MCF-7 (lane 1), MCF-7/PKC-a (lane 2), MCF-7/WT (lane 3), and MCF-7/MDR1 cells (lane 4) was determined by Western blot analysis as described under Materials and Methods. Lane 5 represents the PKC-a standard from rat brain.
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FIG. 5. PMA stimulates PtdEtn synthesis and [14C]ethanolamine uptake in MCF-7/PKC-a cells but not in vector control MCF-7 or MCF7/MDR cells. Vector control MCF-7 cells (l), MCF-7/PKC-a cells (m), and MCF-7/MDR1 cells (j) were incubated with 0–100 nM PMA for 30 min in the presence of [14C]ethanolamine. The cellular contents of [14C]PtdEtn (A) and free [14C]ethanolamine (B) were determined as described under Materials and Methods. Each point represents the mean { SEM of three experiments each performed in triplicate or quadruplicate (n Å 10). *Indicates significantly different from the control (P õ 0.05-0.01).
detectable effect on the uptake of 32Pi . Also, at this earlier time point 100 nM PMA had only a relatively small (Ç1.4-fold) stimulatory effect on the incorporation of [14C]ethanolamine into PtdEtn (data not shown). In mammalian cells, decarboxylation of phosphatidylserine is known to be a significant pathway to synthesize PtdEtn. However, in HL60 cells, incorporation of [14C]serine into PtdEtn was not significantly affected by PMA after an incubation for 2 h. This suggests that in these cells PMA specifically stimulated the de novo synthetic pathway of PtdEtn synthesis. Since in HL60 cells PMA also has significant stimulatory effect on phospholipase D-mediated hydrolysis of PtdEtn (13), it was of interest to determine how PMA affects the cellular content of PtdEtn in these cells. After a treatment for 2 h with 100 nM PMA, there was no significant change in the inorganic phosphate content of PtdEtn, indicating that PMA did not cause a detectable change in the cellular content of PtdEtn. DISCUSSION
In mammalian cells, the PKC system appears to be a nearly universal regulator of PtdCho synthesis (1– 3). This may reflect widespread occurrence of PKC-a,
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which has been reported to mediate the effect of PMA on PtdCho synthesis (5). In contrast, stimulation of PtdEtn synthesis by PMA has so far been reported to occur only in rat hepatocytes (12), although this phenomenon may not have been so well studied as PMAstimulated PtdCho synthesis. Considering the importance of PtdEtn as the second major membrane phospholipid and the major role of the PKC system as a regulator of cell growth and differentiation, it was considered to be important to know whether the PKC system regulates PtdEtn synthesis in other cell types as well and which PKC isoform(s) may mediate the stimulatory effect of PMA. Accordingly, the major goal of this work was to compare the effects of PMA on PtdEtn synthesis in a number of cell lines which show characteristic differences in their PKC isozyme composition. While hepatocytes are known to express the a-, bII-, e-, and z-PKC isoforms (23, 24), NIH 3T3 fibroblasts (25) and R6 fibroblasts (31) similarly express the a-, d-, e-, and z-PKC isoforms but do not contain the bI and bII isoforms. Thus, the present observations that PMA had significant stimulatory effect on PtdEtn synthesis in R6/PKC-bI cells but not in vector control R6 cells or in NIH 3T3 cells strongly suggest that in the overex-
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found to significantly stimulate PtdEtn synthesis (12; this work). However, the role of PKC-bII in the mediation of PMA effect remains to be verified by a more direct method. The mechanism of PMA effect on PtdEtn synthesis remains to be determined. In case of PtdCho synthesis, it was shown that PMA stimulation of choline uptake is independent of its effect on PtdCho synthesis (6), and that CTP-phosphocholine cytidylyltransferase is the most likely target of activated PKC (reviewed in Refs. 2–4). In NIH 3T3 cells PMA enhanced the uptake of ethanolamine, but not PtdEtn synthesis. This indicates that PMA-induced increase in the rate of ethanolamine uptake is not directly related to the stimulatory effect of PMA on PtdEtn synthesis. Since the cytidylyltransferase enzymes which are involved in the synthesis of CDP-choline and CDP-ethanolamine are different (32), and the present data show that PtdCho and PtdEtn synthesis are regulated by different PKC isozymes, it seems likely that PMA stimulates PtdEtn synthesis through the stimulation of CTP:phosphoethanolamine cytidylyltransferase. This would require that this enzyme represents the rate-limiting step of PtdEtn synthesis. Others have already suggested that FIG. 6. PMA decreases the level of cellular [14C]phosphoethanolamine in MCF-7/PKC-a cells, but not in vector control MCF-7 or MCF-7/MDR1 cells. The effects of PMA on the cellular level of [14C]phosphoethanolamine in vector control MCF-7 cells (l), MCF-7/ PKC-a cells (m), and MCF-7/MDR1 cells (j) were determined in the same three separate experiments that were described in the legend to Fig. 5.
pressor cells the effect of PMA was mediated by PKCbI. The results also suggest that the PKC isozymes present in normal fibroblasts, including PKC-a, are not effective mediators of PMA effect on PtdEtn synthesis. However, it should be added here that in vector control R6 cells 100 nM PMA had a detectable stimulatory effect on PtdEtn synthesis despite the absence of PKCb isoforms. This may mean that in certain cell lines PKC-a can mediate a small stimulatory effect of PMA on PtdEtn synthesis. The observations that PMA had only small or no effects on PtdEtn synthesis in MCF-7/WT and MCF-7/ MDR1 cells are generally in agrement with the conclusion that PKC-a (which is highly expressed in MCF-7/ MDR cells) is not an important mediator, while PKCbI (which is absent from both cell lines) is an effective mediator of this PMA effect. However, the small but consistent effects of PMA in MCF-7/WT cells suggested that an additional PKC isozyme may also be able to mediate the effect of PMA on PtdEtn synthesis. The obvious candidate is PKC-bII because this isozyme is present in MCF-7/WT cells (27), and it is also a major PKC isozyme in both HL60 cells (28–30) and hepatocytes (23, 24), the two cell types in which PMA was
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FIG. 7. Time dependence of PMA effects on PtdEtn synthesis in MCF-7 cell lines. MCF-7/PKC-a (A), vector control MCF-7 (B), MCF7/WT (C), and MCF-7/MDR1 cells (D) were incubated for up to 120 min with [14C]ethanolamine in the absence (l) or presence (m) of 100 nM PMA. Each point represents the mean { SE of four determinations (from separate wells) in a single experiment. Similar results were obtained in two other experiments performed in triplicate.
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hepatocytes. Furthermore, evidence has been presented that PKC-bI, and possibly PKC-bII, can mediate the stimulatory effects of PMA on PtdEtn synthesis. In contrast to the major role of PKC-a in the regulation of PtdCho synthesis, PKC-a is clearly not a major mediator of PMA effect on PtdEtn synthesis, although in certain cell lines this isozyme may have a partial regulatory role. The differential regulation of PtdEtn and PtdCho synthesis by the PKC system can clearly provide a mechanism to independently modify the cellular levels of these major membrane phospholipids. Furthermore, the observations that PMA stimulation of PtdEtn hydrolysis (11), but not PtdEtn synthesis, is mediated by PKC-a also suggest that differences in PKC isozyme composition will determine whether in a certain cell type activation of PKC results in an increase, a decrease, or no change (like in HL60 cells) in the cellular level of PtdEtn. The physiological significance of these findings remains to be determined. ACKNOWLEDGMENT
FIG. 8. Stimulation by PMA of PtdEtn synthesis in HL60 cells. HL60 cells were incubated for 2 h with [14C]ethanolamine (I) or 32Pi (II) in the absence (h) or presence (j) of 100 nM PMA. Data are means { SEM of three separate experiments each performed in triplicate. *Indicates significantly different from the control (P õ 0.01).
this may well be the case (32). The observations in this work that PMA also stimulated incorporation of 32Pi into PtdEtn, i.e., de novo synthesis of PtdEtn, yet there was only a very small accumulation of CDP-ethanolamine, strongly suggest that formation of CDP-ethanolamine by the corresponding cytidylyltransferase may indeed represent the rate-limiting step of PtdEtn synthesis. However, this possibility remains to be verified. Interestingly, PMA also enhanced the cellular level of ethanolamine (presumably reflecting increased uptake) in NIH 3T3, vector control R6, and MCF-7/PKCa cells, but not in the MCF-7/MDR1 cells which also highly express PKC-a. The negative results obtained with MCF-7/MDR1 cells do not support the role of PKCa in the mediation of stimulatory effects of PMA on the cellular level of ethanolamine. Thus, not only PtdCho and PtdEtn synthesis, but also the uptake of choline and ethanolamine may be mediated by different PKC isozymes. Clarification of the mechanism by which PMA increases the cellular level of ethanolamine also requires further studies. In summary, it has been shown that regulation of PtdEtn synthesis by the PKC system is not confined to
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I am grateful to Mrs. Karan S. Crilly and Mr. Wayne Anderson for their expert technical help and to Drs. Yusuf A. Hannun, I.Bernard Weinstein, D. Kirk Ways, and Kenneth Cowan for providing polyclonal antibody against PKC-a, the R6 cell lines, the MCF-7/PKC-a cell line, and the MCF-7/MDR1 cell line, respectively. This work was supported by NIH Grant AA09292 and by the Hormel Foundation.
REFERENCES 1. Pelech, S. L., and Vance, D. E. (1984) Biochim. Biophys. Acta 779, 217–251. 2. Kent, C. (1990) Prog. Lipid Res. 29, 87–105. 3. Kiss, Z. (1990) Prog. Lipid Res. 29, 141–166. 4. Vance, D. E. (1989) in Phosphatidylcholine Metabolism (Vance, D. E., Ed.), pp. 225–239, CRC Press, Boca Raton, FL. 5. Slack, B. E., Breu, J., Livneh, E., Eldar, H., and Wurtman, R. J. (1995) Biochem. J. 305, 621–626. 6. Kiss, Z. (1996) Arch. Biochem. Biophys. 335, 191–196. 7. Conricode, K. M., Smith, J. L., Burns, D. J., and Exton, J. H. (1994) FEBS Lett. 342, 149–153. 8. Balboa, M. A., Firestein, B. L., Godson, C., Bell, K. S., and Insel, P. A. (1994) J. Biol. Chem. 269, 10511–10516. 9. Singer, W. D., Brown, H. A., Jiang, X., and Sternweis, P. C. (1966) J. Biol. Chem. 271, 4504–4510. 10. Ohguchi, K., Banno, Y., Nakashima, S., and Nozawa, Y. (1996) J. Biol. Chem. 271, 4366–4372. 11. Mukherjee, J. J., Chung, T., Ways, D. K., and Kiss, Z. (1996) J. Biol. Chem. 271, 28912–28917. 12. Tijburg, L. B. M., Houweling, M., Geelen, M. J. H., and van Golde, L. M. G. (1987) Biochim. Biophys. Acta 922, 184–190. 13. Kiss, Z., and Anderson, W. B. (1989) J. Biol. Chem. 264, 1483– 1487. 14. Kiss, Z., and Deli, E. (1992) Biochem. J. 288, 853–858. 15. Hii, C. S. T., Edwards, Y. S., and Murray, A. W. (1991) J. Biol. Chem. 26, 20238–20243. 16. Fisk, H. A., and Kano-Sueoka, T. (1992) J. Cell. Physiol. 153, 589–595.
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17. Mizunuma, M., Tanaka, S., Kudo, R., and Kanoh, H. (1993) Biochim. Biophys. Acta 1168, 213–219. 18. Natarajan, V., Taher, M. M., Roehm, B., Parinandi, N. L., Schmid, H. H. O., Kiss, Z., and Garcia, J. G. N. (1993) J. Biol. Chem. 268, 930–937. 19. Ways, D. K., Kukoly, C. A., de Vente, J., Hooker, J. L., Bryant, W. O., Posekany, K. J., Fletcher, D. J., Cook, P. P., and Parker, P. J. (1995) J. Clin. Invest. 95, 1906–1915. 20. Housey, G. M., Johnson, M. D., Hsiao, W-L. W., O’Brian, C. A., Murphy, J., Kirschmeier, P., and Weinstein, I. B. (1988) Cell 52, 343–354. 21. Kiss, Z., Deli, E., and Kuo, J. F. (1987) Bichem. J. 248, 649–656. 22. Cook, S. J., and Wakelam, M. J. O. (1989) Biochem. J. 263, 581– 587. 23. Tang, E. Y., Parker, P. J., Beattie, J., and Houslay, M. D. (1993) FEBS Lett. 326, 117–123.
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24. Ducher, L., Croquet, F., Gil, S., Davy, J., Feger, J., and Brehier, A. (1995) Biochem. Biophys. Res. Commun. 217, 546–553. 25. Szallasi, Z., Smith, C. B., Pettit, G. R., and Blumberg, P. M. (1994) J. Biol. Chem. 269, 2118–2124. 26. Kiss, Z. (1992) Eur. J. Biochem. 209, 467–473. 27. Blobe, G. C., Sachs, C. W., Khan, W. A., Fabbro, D., Stabel, S., Wetsel, W. C., Obeid, L. M., Fine, R. L., and Hannun, Y. A. (1993) J. Biol. Chem. 268, 658–664. 28. Tonetti, D. A., Horio, M., Collart, F. R., and Huberman, E. (1992) Cell Growth Differ. 3, 739–745. 29. Hocevar, B. A., and Fields, A. P. (1991) J. Biol. Chem. 266, 28– 33. 30. Macfarlane, D. E., and Manzel, L. (1994) J. Biol. Chem. 269, 4327–4337. 31. Borner, C., Guadagno, S. N., Fabbro, D., and Weinstein, I. B. (1992) J. Biol. Chem. 267, 12892–12899. 32. Vermeulen, P. S., Geelen, M. J. H., and van Golde, L. M. G. (1994) Biochem. Biophys. Acta 1211, 343–349.
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Vol. 347, No. 1, November 1, pp. 37–44, 1997 Article No. BB970308
Expression of Protein Kinase C-b Promotes the Stimulatory Effect of Phorbol Ester on Phosphatidylethanolamine Synthesis Zoltan Kiss1 The Hormel Institute, University of Minnesota, 801 16th Avenue N.E., Austin, Minnesota 55912
Received March 27, 1997, and in revised form July 23, 1997
zymes, i.e., PKC-b and PKC-a, respectively. Stimulation of phosphatidylethanolamine (PtdEtn) synthesis by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) has reportedly been found only in hepatocytes expressing the a-, bII-, e-, and z-PKC isozymes. In contrast, stimulation of phosphatidylcholine synthesis by PKC activators, known to be mediated by PKC-a, is widespread in mammalian cells. In this work, various cell lines exhibiting characteristic differences in their PKC systems were used to determine the role of specific PKC isozymes in the mediation of PMA effect on PtdEtn synthesis. In NIH 3T3 fibroblasts, which express high levels of PKC-a but none of the b (bI or bII) isoforms, PMA did not stimulate PtEtn synthesis. In contrast, in Rat-6 fibroblasts overexpressing PKC-bI, 10 – 100 nM PMA considerably (1.7- to 2.6-fold) enhanced PtdEtn synthesis. In wild-type or multidrug resistant MCF-7 human breast carcinoma cells, which express PKC-a and PKC-bII (to varying extents) but not PKC-bI, PMA had only small or no effects on PtdEtn synthesis. In contrast, in MCF-7 cells overexpressing PKC-a, and as a consequence also expressing the bI- and bII-PKC isoforms, PMA effectively stimulated the synthesis of PtdEtn. Finally, in HL60 human leukemia cells, which contains PKC-bII as the major PKC isoform, PMA again stimulated PtdEtn synthesis. The results establish that while stimulation of PtdEtn synthesis by PMA occurs only in selected cell lines, this phenomenon is not restricted to hepatocytes. Furthermore, the data indicate that expression of either PKC-bI or PKC-bII, but not PKC-a, correlates with the effect of PMA on PtdEtn synthesis. Overall, these observations strongly suggest that regulation of PtdEtn and PtdCho synthesis by PMA involves separate PKC iso-
1 Address correspondence to author. Fax: (507) 437-9606. E-mail: [email protected].
Key Words: phosphatidylethanolamine synthesis; phorbol ester; protein kinase C-bII; protein kinase C-a.
In most mammalian cells the protein kinase C (PKC)2 system is a major regulator of phosphatidylcholine (PtdCho) synthesis (1–3). Activators of PKC, including phorbol 12-myristate 13-acetate (PMA), increase the rate of PtdCho synthesis via stimulating CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15), the key regulatory enzyme of the PtdCho-synthesizing pathway (3, 4). Although the mechanism by which PMA regulates cytidylyltransferase activity has not been clarified yet, it seems clear that its effect is mediated by PKC-a (5). This isozyme also mediates the stimulatory effect of PMA on choline uptake (5), but these two effects of PMA are unrelated (6). PMA-stimulated PKC-a is also a major regulator of phospholipase D-mediated hydrolysis of PtdCho (7–10) and phosphatidylethanolamine (PtdEtn) (11). Thus, in PMA-treated cells the hydrolysis of PtdCho is usually followed by its resynthesis, so that PMA does not cause significant long-term changes in the cellular content of this phospholipid. In comparison to PtdCho synthesis, regulation of PtdEtn synthesis by the PKC system is much less understood. So far, a stimulatory effect of PMA on PtdEtn 2 Abbreviations used: PKC, protein kinase C; PMA, phorbol 12myristate 13-acetate; PtdEtn, phosphatidylethanolamine; PtdCho, phosphatidylcholine; DMEM, Dulbecco’s modified Eagle’s medium; MCF-7/PKC-a and MCF-7/vector cells are stably transfected with PKC-a and the corresponding empty vector, respectively; MCF-7/WT and MCF-7/MDR1 cells are wild-type and multidrug-resistant MCF7 cells, respectively; and R6/PKC-bI cells are R6 fibroblasts overexpressing PKC-bI.
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synthesis has been described only in isolated hepatocytes (12). Since PtdEtn is usually the second major membrane phospholipid, it would be important to know how its synthesis is regulated by PKC, a major regulatory enzyme system. In this work, the effects of PMA on PtdEtn synthesis were examined in various cell lines exhibiting different PKC isozyme compositions. The results indicate that the stimulatory effect of PMA on PtdEtn synthesis is not restricted to hepatocytes and that it requires cellular expression of either PKC-bI or PKC-bII, but not PKC-a. MATERIALS AND METHODS Materials. PMA and Dowex-50W[H/] were purchased from Sigma; [methyl-14C]choline chloride (50 mCi/mmol), [2-14C]ethanolamine (55 mCi/mmol), L-[U-14C]serine (150 mCi/mmol), and carrier-free phosphorus-32 orthophosphate (32Pi) were bought from Amersham; and tissue culture reagents were purchased from GIBCO BRL. The polyclonal antibody raised against PKC-a was kindly provided by Dr. Yusuf A. Hannun (Duke University, Durham, NC). Cell culture. NIH 3T3 clone-7 cells, obtained from Dr. Douglas R. Lowy (NCI, National Institutes of Health, Bethesda, MD), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, penicillin/streptomycin (50 units/ ml and 50 mg/ml, respectively) and glutamine (2 mM). MCF-7/PKCa cells, kindly donated by Dr. D. Kirk Ways (Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN), were generated by stably cotransfecting wild-type MCF-7 cells with PKC-a subcloned into the pSV2M(2)6 vector and a neomycin-resistant plasmid (19). Cells stably transfected with the empty pSV2M(2)6 vector (MCF-7/ vector cells) (19) were also used as a control cell line in this study. Because of the altered PKC isozyme composition, MCF-7/PKC-a cells, unlike the vector control cells, were unable to attach to the substratum; accordingly, MCF-7/PKC-a cells were grown in suspension. MCF-7/MDR1 cells, overexpressing MDR1 P-glycoprotein, was generously provided by Dr. Kenneth Cowan (National Cancer Institute, NIH, Bethesda, MD). Each MCF-7 line was maintaned in pyruvate-free DMEM supplemented with 2 mM glutamine, 10 mM Hepes, 10% fetal calf serum, 200 mg/ml G418 (in case of MCF-7/ PKC-a and MCF-7/vector cells), 50 units/ml penicillin, and 50 mg/ml streptomycin. R6/PKC-bI cells, Rat-6 fibroblasts highly overexpressing PKC-b1 (20), and the corresponding vector cells were generously provided by Dr. I. Bernard Weinstein (Institute of Cancer Research, Columbia University, New York, NY). The R6 cell lines were maintained in DMEM supplemented with 10% fetal calf serum, 1 mM glutamine, 50 units/ml penicillin, 50 mg/ml streptomycin, and 50 mg/ml G418. The human leukemia HL60 cells were maintained in RPMI 1640 medium as previously described (21). Measurement of incorporation of [14C]ethanolamine into cells and PtdEtn. For these experiments MCF-7/PKC-a and HL60 cells, both grown in suspension, were transferred (Ç4–8 1 106 cells/tube) to 15-ml plastic tubes (incubation volume, 0.5 ml), while NIH 3T3 and R6 fibroblasts as well as MCF-7/WT and MCF-7/MDR1 cells were grown in 12-well plates up to 85–95% confluency. Incubation of cells with 25 mM [14C]ethanolamine (0.8–1 mCi/ml) or 32Pi (1 mCi/ml) were performed in the absence or presence of PMA for the time indicated in the figure legends. At the end of incubation of attached cells, the incubation medium was aspirated and then cells were rapidly (within 20 sec) washed with 5 ml medium followed by the addition of icecold methanol (2 ml) to the wells. Then, cells were scraped into methanol, and the methanol extracts were rapidly transferred to 2 ml of choroform. In the case of MCF-7/PKC-a and HL60 cells, the incubations were stopped by adding 14 ml medium to the tubes, followed by pelleting the cells at 500g for 5 min. After aspirating the superna-
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tant, the tight cell pellets were gently, but rapidly, rinsed with 5 ml medium followed by the addition of 4 ml of chloroform/methanol (1:1, v/v) to the tubes. The background values for cell-bound free [14C]ethanolamine (i.e., when cells were immediately washed or pelleted after the addition of radioactive precursor) were always less than 10% of that observed after incubation of cells for 30 min in the absence of PMA; under these conditions there was no detectable radiolabel in the phospholipids. After phase separation, PtdEtn present in the chloroform phase was separated from other phospholipids on silica gel H plates by onedimensional TLC using a chloroform/methanol/28% ammonia (65/25/ 5, by volume) mixture as solvent. Fractionation of radiolabeled water-soluble ethanolamine metabolites, present in the upper water/ methanol phase, was performed on Dowex-50W[H/]-packed columns (Bio-Rad Econo-columns, 0.75-ml bed volume) with minor modifications of the procedure described by Cook and Wakelam (22). The initial flowthrough (5 ml) along with a following wash by 3.5 ml of water contained glycerophosphoethanolamine. Phosphoethanolamine and ethanolamine were eluted by 15 ml of water and 1 M HCl, respectively. [14C]CDP-ethanolamine was separated by a TLC system described previously (13). Western blot analysis of PKC-a. Cells from varius subclones of MCF-7 line were collected in 1.5 ml homogenization buffer containing 20 mM Tris–HCl pH 7.5, 1 mM phenylmethylsulfonyl fluoride, 100 mg/ml leupeptin, and 25 mg/ml aprotinin. After homogenization, homogenates (40 mg protein each) were subjected to SDS–PAGE (10% acrylamide minigel) and proteins were transblotted from gels to nitrocellulose membranes. The membranes were reacted with polyclonal antibody to PKC-a (used at 1:1000 dilution), and the immunoreactive proteins were stained and analyzed as described earlier (11). Data analysis. Results are expressed either as means { SEM for the stated number of experiments, or as means { SE for the stated number of determinations in one experiment. Statistical analyses were performed with Student’s t test. Statistical significance was determined at the 0.05 level.
RESULTS
In hepatocytes, which contain the a-, bII-, e-, and zPKC isoforms (23, 24), PMA was previously shown to stimulate PtdEtn synthesis (12). NIH fibroblasts also contain the a-, e-, and z-PKC isoforms, but in these cells the bII-isoform is replaced by the d-isoform of PKC (25, 26). Thus, a study of PMA effects on PtdEtn synthesis and on the uptake of precursor [14C]ethanolamine by NIH 3T3 cells was performed first to obtain preliminary information on the regulatory role of various PKC isozymes. Treatment of attached NIH 3T3 fibroblasts with PMA (100 nM) for 30 min resulted in a nearly twofold increase in the amount of cell-bound unesterified [14C]ethanolamine (Fig. 1). The stimulatory effect of PMA lasted for about 1 h; after 2 h treatments with PMA, the cellular level of [14C]ethanolamine returned to the basal level (data not shown). In other experiments, NIH 3T3 cells were first detached from the substratum by trypsin, and the suspended cells were then treated with PMA in the presence of radiolabeled ethanolamine for 30 min. Despite the relatively long (5 min) centrifugation performed in a large (14 ml) volume of DMEM (to reduce background radioactivity), the stimulatory effects of PMA on the cellular uptake of [14C]-
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FIG. 1. Stimulatory effects of PMA on the cellular uptake of [14C]ethanolamine in attached and suspended NIH 3T3 fibroblasts. Attached (I) or suspended (II) NIH 3T3 fibroblasts were labeled for 30 min with [14C]ethanolamine in the absence (h) or presence (j) of 100 nM PMA. Data are means of four independent incubations in a representative experiment with the same passage of cells. The difference between the two extreme values was always less than 14%. Similar results were obtained in two other experiments each performed in triplicate.
ethanolamine (Fig. 1) was maintained during this procedure. The stimulatory effect of PMA on ethanolamine uptake was similarly maintained in suspended cells when cells were collected by scraping. These results indicated that the technique used in case of suspended NIH 3T3 cells should also be suitable for the study of PMA effects on [14C]ethanolamine uptake and [14C]PtdEtn formation in cells maintained and used in suspension cultures. Despite the stimulatory effect of PMA on [14C]ethanolamine uptake, PMA failed to enhance the incorporation of [14C]ethanolamine (25 mM) into PtdEtn in attached fibroblasts (Fig. 2) or in suspended fibroblasts (not shown) over an 80-min incubation period. Since in hepatocytes PMA appeared to maximally stimulate PtdEtn synthesis in the 50– to 100-mM range of ethanolamine (12), the above experiments were repeated by using these higher concentrations of ethanolamine; PMA also failed to enhance the synthesis of PtdEtn at 50–100 mM [14C]ethanolamine (data not shown). Since the experiments with NIH 3T3 cells indicated that the presence of a-, d-, e-, and z-PKC isoforms does not promote the effect of PMA on PtdEtn synthesis, in
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the following experiments the possible role of PKC-b was studied in more detail. First, the effects of PMA were examined in R6/PKC-bI cells which highly express PKC-bI, as well as in vector control R6 cells which do not express this PKC isoform (20). Treatments of cells with 100 nM PMA for 30 min enhanced the cellular contents of [14C]ethanolamine in vector control cells and in R6/PKC-bI cells about 1.3- and 1.5fold, respectively (data not shown). During shorter (30– 60 min) periods of incubation, in R6/PKC-bI cells 10 or 100 nM PMA stimulated PtdEtn synthesis 1.7- to 1.9-fold or 2.1- to 2.6-fold, respectively (Fig. 3). During the same incubation period, in vector control cells 100 nM PMA (but not 10 nM PMA) had detectable, but considerably smaller (Ç1.4-fold), stimulatory effects on PtdEtn synthesis (Fig. 3). In R6/PKC-bI cells the relative stimulatory effects of PMA were considerably decreased when the incubation time was extended to 2 h (1.2- to 1.3-fold stimulation). It still should be noted that at various time points the ‘‘basal’’ level of PtdEtn synthesis in R6/PKC-bI cells was about two to three times higher than in the vector control cells (Fig. 3). This may indicate that in these cells PKC-bI is partially activated even in the absence of PMA resulting in partial stimulation of PtdEtn synthesis. This possibility remains to be experimentally verified. Next the effects of PMA on PtdEtn synthesis were determined in (i) wild-type MCF-7 human breast carcinoma cells (MCF-7/WT), which contain no PKC-bI (27),
FIG. 2. PMA has no effect on PtdEtn synthesis in NIH 3T3 fibroblasts. Attached NIH 3T3 fibroblasts were incubated without (l) or with 100 nM PMA (m) in the presence of [14C]ethanolamine for up to 80 min. Each point represents the mean { SEM of three separate experiments each performed in triplicate.
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FIG. 3. Expression of PKC-bI in R6 fibroblasts promotes the stimulatory effect of PMA on PtdEtn synthesis. Attached vector control R6 cells (open symbols) and R6/PKC-bI cells (closed symbols) were incubated for up to 120 min with [14C]ethanolamine in the absence (s, l) or presence of 10 nM PMA (n, m), or 100 nM PMA (h, j). Each point represents the mean { SE of four determinations from separate wells in a single experiment. Similar results were obtained in two other experiments each performed in the absence or presence of 100 nM PMA in triplicate.
express little PKC-a (Fig. 4; Ref. 27), and contain significant amount of PKC-bII (27); (ii) multidrug-resistant MCF-7 cells (MCF7-7/MDR1) which express large amounts of PKC-a (Fig. 4; Ref. 27), contain no PKC-bI (27), and express only small amount of PKC-bII (27); and (iii) MCF-7/PKC-a cells which not only contain PKC-a at high level (Fig. 4; Ref. 19) but they also express significant amount of PKC-b (19), mostly PKCbI (11). Finally, MCF-7/vector contor cells, containing only the empty vector and developed as controls for MCF-7/PKC-a cells, have PKC isozyme composition practically identical with that of wild-type cells. Of interest to note is that MCF-7/MDR1 and MCF-7/PKC-a cells express PKC-a at similar levels (Fig. 4). It was expected that these four cell lines, showing characteristic differences in the expression of PKC-b and PKC-a isoforms, will provide additional important information on the PKC isozyme requirement of PMA effect on PtdEtn synthesis. In MCF-7/PKC-a cells, but not in vector control or MCF-7/MDR1 cells, PMA at 5–100 nM concentrations significantly (Ç1.5–1.7-fold) increased the incorporation of [14C]ethanolamine into PtdEtn over a 30-min
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incubation period (Fig. 5A). At this incubation time, PMA had no effect on PtdEtn synthesis in MCF-7/WT cells (data not shown). Interestingly, PMA also increased the cellular content of free [14C]ethanolamine only in MCF-7/PKC-a cells, but not in the vector control or MCF-7/MDR1 cells (Fig. 5B). Increased synthesis of [14C]PtdEtn in PMA-treated MCF-7/PKC-a cells was accompanied by decreased cellular level of [14C]phosphoethanolamine (Fig. 6), reflecting increased utilization of this precursor for PtdEtn synthesis. While in MCF-7/PKC-a cells both radiolabeled ethanolamine and phosphoethanolamine were easily detectable both in the absence and presence of PMA, [14C]CDP-ethanolamine formation could only be observed in the presence of PMA and only at a very low level (Ç50–80 dpm/106 cells). In contrast to the shorter-term (30 min) experiments, when the incubation time was extended to 2 h PMA had clearly detectable (Ç1.3-fold) stimulatory effects on PtdEtn synthesis in MCF-7/WT (Fig. 7C) and MCF7/vector control cells (Fig. 7B), but not in MCF-7/MDR1 cells (Fig. 7D). Also, in contrast to R6/PKC-bI cells (Fig. 3), in MCF-7/PKC-a cells (Fig. 7A) the effect of PMA on PtdEtn synthesis was somewhat increased, rather than decreased, when the incubation time was extended from 45 min (1.9-fold stimulation) to 120 min (2.2-fold stimulation). Since MCF-7/WT cells contain significantly more PKC-bII than MCF-7/MDR1 cells (27), the small stimulatory effects of PMA in the former cells could be mediated by this isozyme. This possibility led to the study of human leukemic HL60 cells in which PKC-bII is the major PKC isoform and PKC-bI is absent (28–30). In the experiments with HL60 cells, both [14C]ethanolamine and 32Pi were employed as labeling agents. The incubation time was sufficiently long (2 h) to achieve appropriate 32P labeling of the cellular ATP pool, and thereby the cellular PtdEtn pool. As shown in Fig. 8, in the presence of both radiolabeled precursors PMA significantly (Ç2- to 2.3-fold) enhanced the labeling of PtdEtn. When examined over a 30-min incubation period, PMA enhanced the cellular content of free [14C]ethanolamine only Ç1.2-fold, and it had no
FIG. 4. Western blot analysis of PKC-a in MCF-7 lines. PKC-a in homogenates from vector control MCF-7 (lane 1), MCF-7/PKC-a (lane 2), MCF-7/WT (lane 3), and MCF-7/MDR1 cells (lane 4) was determined by Western blot analysis as described under Materials and Methods. Lane 5 represents the PKC-a standard from rat brain.
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FIG. 5. PMA stimulates PtdEtn synthesis and [14C]ethanolamine uptake in MCF-7/PKC-a cells but not in vector control MCF-7 or MCF7/MDR cells. Vector control MCF-7 cells (l), MCF-7/PKC-a cells (m), and MCF-7/MDR1 cells (j) were incubated with 0–100 nM PMA for 30 min in the presence of [14C]ethanolamine. The cellular contents of [14C]PtdEtn (A) and free [14C]ethanolamine (B) were determined as described under Materials and Methods. Each point represents the mean { SEM of three experiments each performed in triplicate or quadruplicate (n Å 10). *Indicates significantly different from the control (P õ 0.05-0.01).
detectable effect on the uptake of 32Pi . Also, at this earlier time point 100 nM PMA had only a relatively small (Ç1.4-fold) stimulatory effect on the incorporation of [14C]ethanolamine into PtdEtn (data not shown). In mammalian cells, decarboxylation of phosphatidylserine is known to be a significant pathway to synthesize PtdEtn. However, in HL60 cells, incorporation of [14C]serine into PtdEtn was not significantly affected by PMA after an incubation for 2 h. This suggests that in these cells PMA specifically stimulated the de novo synthetic pathway of PtdEtn synthesis. Since in HL60 cells PMA also has significant stimulatory effect on phospholipase D-mediated hydrolysis of PtdEtn (13), it was of interest to determine how PMA affects the cellular content of PtdEtn in these cells. After a treatment for 2 h with 100 nM PMA, there was no significant change in the inorganic phosphate content of PtdEtn, indicating that PMA did not cause a detectable change in the cellular content of PtdEtn. DISCUSSION
In mammalian cells, the PKC system appears to be a nearly universal regulator of PtdCho synthesis (1– 3). This may reflect widespread occurrence of PKC-a,
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which has been reported to mediate the effect of PMA on PtdCho synthesis (5). In contrast, stimulation of PtdEtn synthesis by PMA has so far been reported to occur only in rat hepatocytes (12), although this phenomenon may not have been so well studied as PMAstimulated PtdCho synthesis. Considering the importance of PtdEtn as the second major membrane phospholipid and the major role of the PKC system as a regulator of cell growth and differentiation, it was considered to be important to know whether the PKC system regulates PtdEtn synthesis in other cell types as well and which PKC isoform(s) may mediate the stimulatory effect of PMA. Accordingly, the major goal of this work was to compare the effects of PMA on PtdEtn synthesis in a number of cell lines which show characteristic differences in their PKC isozyme composition. While hepatocytes are known to express the a-, bII-, e-, and z-PKC isoforms (23, 24), NIH 3T3 fibroblasts (25) and R6 fibroblasts (31) similarly express the a-, d-, e-, and z-PKC isoforms but do not contain the bI and bII isoforms. Thus, the present observations that PMA had significant stimulatory effect on PtdEtn synthesis in R6/PKC-bI cells but not in vector control R6 cells or in NIH 3T3 cells strongly suggest that in the overex-
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found to significantly stimulate PtdEtn synthesis (12; this work). However, the role of PKC-bII in the mediation of PMA effect remains to be verified by a more direct method. The mechanism of PMA effect on PtdEtn synthesis remains to be determined. In case of PtdCho synthesis, it was shown that PMA stimulation of choline uptake is independent of its effect on PtdCho synthesis (6), and that CTP-phosphocholine cytidylyltransferase is the most likely target of activated PKC (reviewed in Refs. 2–4). In NIH 3T3 cells PMA enhanced the uptake of ethanolamine, but not PtdEtn synthesis. This indicates that PMA-induced increase in the rate of ethanolamine uptake is not directly related to the stimulatory effect of PMA on PtdEtn synthesis. Since the cytidylyltransferase enzymes which are involved in the synthesis of CDP-choline and CDP-ethanolamine are different (32), and the present data show that PtdCho and PtdEtn synthesis are regulated by different PKC isozymes, it seems likely that PMA stimulates PtdEtn synthesis through the stimulation of CTP:phosphoethanolamine cytidylyltransferase. This would require that this enzyme represents the rate-limiting step of PtdEtn synthesis. Others have already suggested that FIG. 6. PMA decreases the level of cellular [14C]phosphoethanolamine in MCF-7/PKC-a cells, but not in vector control MCF-7 or MCF-7/MDR1 cells. The effects of PMA on the cellular level of [14C]phosphoethanolamine in vector control MCF-7 cells (l), MCF-7/ PKC-a cells (m), and MCF-7/MDR1 cells (j) were determined in the same three separate experiments that were described in the legend to Fig. 5.
pressor cells the effect of PMA was mediated by PKCbI. The results also suggest that the PKC isozymes present in normal fibroblasts, including PKC-a, are not effective mediators of PMA effect on PtdEtn synthesis. However, it should be added here that in vector control R6 cells 100 nM PMA had a detectable stimulatory effect on PtdEtn synthesis despite the absence of PKCb isoforms. This may mean that in certain cell lines PKC-a can mediate a small stimulatory effect of PMA on PtdEtn synthesis. The observations that PMA had only small or no effects on PtdEtn synthesis in MCF-7/WT and MCF-7/ MDR1 cells are generally in agrement with the conclusion that PKC-a (which is highly expressed in MCF-7/ MDR cells) is not an important mediator, while PKCbI (which is absent from both cell lines) is an effective mediator of this PMA effect. However, the small but consistent effects of PMA in MCF-7/WT cells suggested that an additional PKC isozyme may also be able to mediate the effect of PMA on PtdEtn synthesis. The obvious candidate is PKC-bII because this isozyme is present in MCF-7/WT cells (27), and it is also a major PKC isozyme in both HL60 cells (28–30) and hepatocytes (23, 24), the two cell types in which PMA was
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FIG. 7. Time dependence of PMA effects on PtdEtn synthesis in MCF-7 cell lines. MCF-7/PKC-a (A), vector control MCF-7 (B), MCF7/WT (C), and MCF-7/MDR1 cells (D) were incubated for up to 120 min with [14C]ethanolamine in the absence (l) or presence (m) of 100 nM PMA. Each point represents the mean { SE of four determinations (from separate wells) in a single experiment. Similar results were obtained in two other experiments performed in triplicate.
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hepatocytes. Furthermore, evidence has been presented that PKC-bI, and possibly PKC-bII, can mediate the stimulatory effects of PMA on PtdEtn synthesis. In contrast to the major role of PKC-a in the regulation of PtdCho synthesis, PKC-a is clearly not a major mediator of PMA effect on PtdEtn synthesis, although in certain cell lines this isozyme may have a partial regulatory role. The differential regulation of PtdEtn and PtdCho synthesis by the PKC system can clearly provide a mechanism to independently modify the cellular levels of these major membrane phospholipids. Furthermore, the observations that PMA stimulation of PtdEtn hydrolysis (11), but not PtdEtn synthesis, is mediated by PKC-a also suggest that differences in PKC isozyme composition will determine whether in a certain cell type activation of PKC results in an increase, a decrease, or no change (like in HL60 cells) in the cellular level of PtdEtn. The physiological significance of these findings remains to be determined. ACKNOWLEDGMENT
FIG. 8. Stimulation by PMA of PtdEtn synthesis in HL60 cells. HL60 cells were incubated for 2 h with [14C]ethanolamine (I) or 32Pi (II) in the absence (h) or presence (j) of 100 nM PMA. Data are means { SEM of three separate experiments each performed in triplicate. *Indicates significantly different from the control (P õ 0.01).
this may well be the case (32). The observations in this work that PMA also stimulated incorporation of 32Pi into PtdEtn, i.e., de novo synthesis of PtdEtn, yet there was only a very small accumulation of CDP-ethanolamine, strongly suggest that formation of CDP-ethanolamine by the corresponding cytidylyltransferase may indeed represent the rate-limiting step of PtdEtn synthesis. However, this possibility remains to be verified. Interestingly, PMA also enhanced the cellular level of ethanolamine (presumably reflecting increased uptake) in NIH 3T3, vector control R6, and MCF-7/PKCa cells, but not in the MCF-7/MDR1 cells which also highly express PKC-a. The negative results obtained with MCF-7/MDR1 cells do not support the role of PKCa in the mediation of stimulatory effects of PMA on the cellular level of ethanolamine. Thus, not only PtdCho and PtdEtn synthesis, but also the uptake of choline and ethanolamine may be mediated by different PKC isozymes. Clarification of the mechanism by which PMA increases the cellular level of ethanolamine also requires further studies. In summary, it has been shown that regulation of PtdEtn synthesis by the PKC system is not confined to
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I am grateful to Mrs. Karan S. Crilly and Mr. Wayne Anderson for their expert technical help and to Drs. Yusuf A. Hannun, I.Bernard Weinstein, D. Kirk Ways, and Kenneth Cowan for providing polyclonal antibody against PKC-a, the R6 cell lines, the MCF-7/PKC-a cell line, and the MCF-7/MDR1 cell line, respectively. This work was supported by NIH Grant AA09292 and by the Hormel Foundation.
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