Phospholipase D-derived products in the regulation of 12-O-tetradecanoylphorbol-13-acetate-stimulated prostaglandin synthesis in madin-darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of ethanol synthesize phosphatidylethanol (PEt) instead of phosphatidic acid (PA) and diglyceride (DG). We have used ethanol to block the production of phospholipase D (PLD)-derived PA and DG (from PA hydrolysis) to study their role in signal transduction. In MDCK cells, TPA-stimulated prostaglandin E2 (PGE2) synthesis was inhibited by ethanol at concentrations which inhibit PA and DG formation. In addition, TPA elicited a prolonged increase in PGE2 synthesis that is dependent upon continuous activation of PLD. The TPA-stimulated translocation of protein kinase Calpha (PKCalpha) from cytosol to membrane was unaffected by ethanol. This suggests that PLD-derived products act downstream of PKC in TPA-stimulated prostaglandin synthesis. The calcium ionophore, A23187, did not activate PLD, and PGE2 synthesis in response to A23187 was unaffected by ethanol. TPA increased prostaglandin endoperoxide H synthase (PGHS) activity and increased the amount of immunodetectable prostaglandin endoperoxide H synthase 2 (PGHS-2). A23187 did not induce PGHS-2 and A23187-stimulated PGE2 synthesis appears to be due to the constitutively expressed PGHS-1. Blocking the formation of PLD-derived products, PA and DG, inhibited the induction of PGHS-2 by TPA. These results indicate that prolonged PGE2 synthesis in response to TPA is due to the continuous induction of PGHS-2, which is dependent upon PLD activation. In contrast, induction of PGHS-2 by epidermal growth factor was not affected by ethanol. Epidermal growth factor did not induce PKCalpha translocation nor activate PLD. Taken together, these data suggest that PLD-derived PA or DG act as second messengers in the induction of PGHS-2 by PKC-dependent pathways. The demonstration that inhibition of TPA-induced PA formation inhibits Raf-1 translocation in MDCK cells (Ghosh, S., Strum, J. C., Sciorra, V. A., Daniel, L. W. , and Bell, R. M. (1996) J. Biol. Chem. 271, 8472-8480) suggests that PA is the active PLD metabolite in TPA-stimulated signaling.

that PA is the active PLD metabolite in TPA-stimulated signaling.
The tumor promoter, TPA, 1 stimulates PC turnover in MDCK cells, resulting in the generation of DG, the major endogenous activator of PKC (1). Agonist-induced hydrolysis of PI was once considered to be the sole mechanism by which DG is generated (2). However, hormones, growth factors, and serum stimulate PC turnover in a variety of cell types; including fibroblasts (3,4), neutrophils (5)(6)(7)(8)(9)(10), and HL-60 cells (11,12). It is now recognized that PC degradation is an important source of lipid-derived second messengers in cell signaling (for review, see Ref. 13). Because PI-derived DG is transient, a function of PC-derived DG may be to maintain PKC activation (14,15). However, some groups have reported that DG derived from PC turnover does not activate PKC (16,17).
The generation of DG from PC has been attributed to the direct action of PLC (18,19) or to the sequential actions of PLD and PAP (20 -24). In neutrophils (5,6), HL-60 granulocytes (25), platelets (26), endothelial cells (27), and hepatocytes (22,28), diverse agents that cause rapid hydrolysis of PC by PLD to generate PA and subsequently DG. Huang and Cabot (29) have demonstrated that TPA induces a time-dependent increase in the formation of PA prior to DG formation as a result of PLD activation in MDCK cells. As a result of its rapid and transient formation during cell activation, PA is thought to have a biological function in cell signaling. PA has been shown to stimulate DNA synthesis and cell division in fibroblasts (30) and has been shown to have a role in the activation of NADPH oxidase in human neutrophils (31)(32)(33). More recently, Khan et al. (34) have identified a PA-activated protein kinase in human platelets. However, a specific physiological function for this PAactivated kinase has not been determined.
In addition to these potential biological functions, PA can be rapidly converted to DG by PAP. Unlike PI-derived DG, DGs generated from PC degradation contain both acyl and alkyl linkages. Recently, we have demonstrated that TPA-stimulated PC hydrolysis by PLD is selective for alkyl-PC in MDCK cells, thus generating both alkyl-PA and -DG (35). The alkyl-DG generated did not activate PKC derived from MDCK cells, suggesting an alternative role for alkyl-PC turnover. Alkyl-DG has been shown to activate HL-60 acute myelocytic leukemia cell differentiation (36). In addition, it has been demonstrated that acyl-DG and alkyl-DG have differential effects on the metabolism of released 20:4 (37) and on the activation of NADPH oxidase (38). However, the significance of agonistinduced PLD activation has been unclear.
To determine a role for PLD activation, we used ethanol to block the production of PA and DG derived from the PLD/PAP pathway. In the presence of ethanol, PLD catalyzes a transphosphatidylation reaction generating PEt at the expense of PA and DG. Pai et al. (25) first demonstrated that PEt is formed exclusively by PLD in cultured cells, and utilized PEt formation as an indication of PLD activation by a variety of agonists. Huang et al. (29) have shown that ethanol completely blocks the formation of PA and DG by PLD in TPA-treated MDCK cells. In order to identify potential PLD dependent signaling events, ethanol was used to block PA and DG formation. We report herein that the inhibition of PA and DG synthesis by ethanol blocks TPA-induced PGE 2 production in MDCK cells. Prolonged PGE 2 synthesis by TPA is due to the induction of PGHS-2. This induction is dependent upon TPAinduced PLD activation. Taken together, these findings indicate that the products of PC degradation via the PLD/PAP pathway, are required for downstream signaling events in TPA-stimulated prostaglandin synthesis.
Cell Culture and Harvest-MDCK cells were cultured in DMEM supplemented with 10% (v/v) FBS, 100 units of penicillin/ml, 100 g of streptomycin/ml, 2 mM L-glutamine, and 0.22% NaHCO 3 (completed-DMEM). The cells were maintained in 75-cm 2 flasks and subcultured at 3-4-day intervals. For the experiments herein, MDCK cells were used between passages 60 and 75. Cells were grown in 10-mm plastic Petri dishes at a concentration of 5 ϫ Lipid Extraction and Analysis-Cells were harvested by scraping directly from the plates into 2 ml of methanol, 2% acetic acid. Extracellular fluids were harvested by transferring the medium from each plate to 2 ml of methanol, 2% acetic acid. The lipids were extracted by a modification of the method of Bligh and Dyer (39) as described previously (1). Extracted lipids were dried under nitrogen and resuspended in chloroform/methanol (9:1, v/v). PC, PE, PI/PS, and neutral lipids were separated on Silica Gel 60 plates developed in a solvent system consisting of chloroform/methanol/acetic acid/water (50:25:8:2, v/v). Prostaglandins and free 20:4 were resolved on Silica Gel 60 plates developed in the organic phase of ethyl acetate/isooctane/acetic acid/ water (80:50:20:100, v/v) as described by Flower and Blackwell (40). PEt was resolved on Silica Gel 60 plates developed in the system described above for prostaglandins. The R f values for PGE 2 and PEt were approximately 0.20 and 0.14, respectively. Lipids were located by autoradiography using EN 3 HANCE spray and Kodak SB-5 film, were scraped from the plates, and quantitated by scintillation counting.
Prostaglandin Endoperoxide H Synthase (PGHS) Activity Assay in MDCK Cell Sonicates-MDCK cells were grown for 2-3 days (or until 80% confluent) at 37°C. After TPA stimulation, cells were detached with trypsin, and washed with ice-cold buffer consisting of 5 mM Tes, 5.3 mM KCl, 2.5 mM MgCl 2 , 138 mM NaCl, and 5.5 mM glucose, pH 7.4. The cells were pelleted by centrifugation at 800 ϫ g for 8 min at 4°C. The pellet was resuspended in 2 ml of buffer and sonicated on ice for 3 ϫ in 5-s bursts with a Branson probe-type sonicator set at 10% of maximum energy. The sonicates were assayed immediately. Protein content was determined by the method of Bradford (41) using bovine serum albumin as a standard. One mg of total protein from sonicates was incubated for 3 min at room temperature in 1 ml of assay buffer consisting of 100 mM Tris-HCl, pH 8.5, 2 mM reduced glutathione, 5 mM L-tryptophan, 1 M hematin, 0.1% Triton X-100, and 10 M 20:4 containing 0.5 Ci of [ 3 H]20:4 per sample to be assayed as described by Beaudry et al. (42). The reaction was stopped by the addition of 2 ml of methanol, 2% acetic acid. Lipids were extracted and quantitated as described previously. Specific activities were calculated by determining the percent conversion of [ 3 H]20:4 to all stable prostaglandin products. Specific activites were expressed as pmol of 20:4 converted to prostaglandins/min/mg of total protein.
Prostaglandin Endoperoxide H Synthase 1 and 2 Analysis-MDCK cells were grown for 2-3 days (or until 80% confluent) at 37°C. After stimulation, cells were washed with cold phosphate-buffered saline and lysed in 100 l of lysis buffer consisting of 100 mM Tris, pH 7.5, 100 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 10 g/ml leupeptin on ice for 10 min. Cell lysates were centrifuged at 16,000 ϫ g for 10 min at 4°C. Protein content was determined by the method of Bradford (41) using bovine serum albumin as a standard. 100 g of protein from each cell lysate sample were separated by SDS-polyacrylamide gel electrophoresis (5% stacking, 7% running gel) according to Laemmli (43), and transferred onto nitrocellulose membranes (Schleicher and Schuell) at 100 mAmps, at room temperature overnight. Membranes were blocked with phosphate-buffered saline containing 5% non-fat dry milk and 1% Tween 20 for 1 h; and then hybridized with primary antibodies in phosphate-buffered saline containing 5% bovine serum albumin and 0.2% sodium azide for an additional hour. After washing, the membranes were incubated with goat anti-rabbit horseradish peroxidase (Transduction Labs) for 1 h. PGHS-1 and -2 were detected using enhanced chemiluminesence (Du-Pont NEN) according to the instructions of the manufacturer.
Translocation of PKC␣-MDCK cells were grown for 2-3 days (or until 80% confluent) at 37°C. Cells were incubated with serum-free DMEM overnight at 37°C prior to stimulation. Cells were then stimulated with 10 nM TPA or 100 nM EGF in the presence or absence of 1% ethanol. After stimulation, cells were washed with cold phosphatebuffered saline and scraped in buffer H (10 mM Hepes, pH 7.4, 2 mM EDTA, 1 mM Na 3 VO 4 , and 1 mM phenylmethylsulfonyl fluoride). Cells were pelleted and resuspended in 500 l of buffer H plus 50 mM NaF, 10 g/ml aprotinin, and 10 g/ml leupeptin. Cells were lysed by sonication on ice for 3 ϫ 15-s bursts with a Branson probe-type sonicator set at 10% of maximum energy. Unbroken cells were pelleted by centrifugation at 2,940 ϫ g for 2 min at 4°C. Cell sonicates were then centrifuged (Beckman) at 120,000 ϫ g for 80 min at 4°C. The cytosol was removed and saved. Membrane pellets were sonicated as before in buffer H plus 50 mM NaF, 10 g/ml aprotinin, 10 g/ml leupeptin, 100 mM NaCl, and 1% Triton X-100 and placed on ice for an additional 30 min. Sonicates were centrifuged (Beckman) at 120,000 ϫ g for 80 min at 4°C. Onehundred g of protein from cytosol and membrane were separated by SDS-polyacrylamide gel electrophoresis (5% stacking, 7% running) according to Laemmli (43) and transferred to nitrocellulose membranes (Schleicher and Schuell) overnight. Membranes were analyzed for PKC␣ by methods as described above using a monoclonal mouse PKC␣ antibody (Upstate Biotechnology Inc.) as the primary antibody and a goat anti-mouse horseradish peroxidase antibody (Transduction Labs) for detection.

RESULTS
When we used ethanol to block PA and DG formation by PLD, TPA (10 nM)-induced PEt formation increased with increasing concentrations of ethanol (0 -2%, v/v), while the formation of TPA-induced PGE 2 was inversely proportional to the concentration of ethanol (Fig. 1). Inhibition of PGE 2 synthesis was seen at pharmacological concentrations (0.1%) of ethanol. Similar results were obtained using butanol (0 -50 mM, data not shown). From these data, we chose 1% ethanol to determine the time dependent effect of ethanol on PGE 2 synthesis. TPAstimulated PGE 2 synthesis was inhibited by ethanol (Fig. 2) at concentrations which inhibit PA and DG formation (29,35). After approximately 6 h, ethanol appeared to be without effect upon the rate of PGE 2 synthesis (Fig. 2). By adding ethanol at the time of stimulation, it was previously assumed that PEt accumulation defined the time course of PLD activation (Fig.  2). However, we observed that ethanol was lost from the media by evaporation.
In order to define the time course of PLD activation by TPA, we added ethanol (1%) at different times after stimulation. MDCK cells were stimulated with TPA (10 nM), and the media was removed and replaced with fresh completed-DMEM containing ethanol (1%) for a 2-h pulse prior to harvesting at the indicated times (Fig. 3). These data indicate that PLD remains activated for a much longer time than previously assumed. The data presented in Fig. 3 illustrate that PLD activation is required for new PGE 2 synthesis, but not necessary for 20:4 release. Together, these data indicate that prolonged PLD activation by TPA is required for continued PGE 2 synthesis in response to TPA.
To determine if ethanol was affecting 20:4 availability by altering reincorporation into phospholipids, we added ethanol simultaneously with 20:4 (Fig. 4). Alterations in 20:4 distribution will affect 20:4 release and prostaglandin production. The incorporation and distribution of 20:4 was not affected by the presence of ethanol (1%), indicating that inhibition of PGE 2 production is not due to a change in 20:4 reincorporation. These data provide additional evidence that ethanol is not causing alterations in cellular lipid metabolism as a result of nonspe-cific membrane or metabolic perturbations. In order to determine if ethanol is acting on a specific signaling pathway, we compared ethanol's effect on 20:4 release and PGE 2 production induced by the calcium ionophore, A23187, or TPA (Fig. 5). PGE 2 synthesis was inhibited by ethanol in response to TPA (Fig. 5) but not in response to 10 M A23187 (Fig. 5). Ethanol caused a small reduction in TPA-mediated 20:4 release (Fig. 5); however, the release of free 20:4 induced by A23187 was not affected by the presence of ethanol (Fig. 5). In addition, A23187 is not a good activator of PC-PLD (data not shown) and does not stimulate prolonged PGE 2 synthesis (49). Ethanol had no effect on PGE 2 production nor 20:4 release in unstimulated cells (Fig. 5).
To further characterize ethanol's effect on PGE 2 synthesis, we determined if ethanol was inhibiting TPA-induced PGHS activity (Fig. 6). MDCK cells were stimulated with 10 nM TPA in the presence or absence of ethanol (1%) for 4 h. Sonicates were prepared and assayed for PGHS activity as described previously (42). These data show that there is a reduced stimulation of PGHS activity in intact MDCK cells stimulated with TPA in the presence of ethanol (Fig. 6A). In contrast, ethanol had no effect on induced PGHS activity in response to 100 nM EGF (Fig. 6B). When sonicates (control or TPA-treated) were assayed in ethanol (1%), PGHS activity was not affected, indicating that ethanol did not inhibit the conversion of 20:4 to prostaglandins in vitro (data not shown).
Beaudry et al. (42) demonstrated that inhibition of protein synthesis with cycloheximide prevented the induction of PGHS activity by TPA. This indicated that the increased PGHS activity in MDCK cells involves de novo synthesis of the enzyme. To obtain direct evidence that TPA stimulates de novo synthesis of PGHS in MDCK cells, immunodetection was employed using polyclonal antibodies against PGHS-1 and PGHS-2. MDCK cells were stimulated with TPA in the presence or absence of ethanol for 6 h. Cell lysates were prepared and analyzed for PGHS-1 and -2. TPA caused a marked stimulation of PGHS-2 (Fig. 7 bottom). When PLD product formation was blocked by ethanol, TPA-induced PGHS-2 was inhibited (Fig. 7,  bottom). MDCK cells express low levels of PGHS-1, and this expression was not inhibited by the presence of ethanol (Fig. 7,  top). PGHS-1 was not detected in TPA-treated cells (in the presence or absence of ethanol). Increased PGE 2 synthesis by TPA (Fig. 2) correlated with the marked induction of PGHS-2 (Fig. 7, bottom, and Fig. 10, top) without any significant effect on PGHS 1 (Fig. 7, top). By 4 h, PGHS-2 synthesis resumed in cells stimulated with TPA in the presence of ethanol (Fig. 10, top), which correlates with the rate of PGE 2 synthesis seen in Fig. 2. When ethanol is added at the time of stimulation, by 4 h the ethanol is apparently depleted; and PA and DG formation as well as PGHS-2 synthesis resumes. In contrast, A23187 did not cause induction of PGHS-2 (data not shown).
To determine if the induction of PGHS-2 seen in cells not stimulated with TPA was due to serum stimulation (Fig. 7,  bottom), MDCK cells were made quiescent (1% FBS/DMEM for 48 h) followed by serum starvation for 3 h prior to stimulation. PGHS-2 was induced by serum (10% FBS) and serum stimulation was inhibited by the presence of ethanol (Fig. 8). We also found that serum-stimulated PLD activity in MDCK cells made quiescent by serum reduction. 2 In addition, ethanol did not affect PGHS-2 in unstimulated cells (Fig. 8).
Previous work from this laboratory suggested that PKC activation by TPA is involved in the increased prostaglandin production (44). In addition, it has been reported that PKC␣ mediates PLD activation and PGE 2 synthesis induced by TPA in MDCK cells (45,46). We next determined if blocking PLD product formation with ethanol inhibited PKC␣ translocation. The translocation of PKC␣ from cytosol to membrane in response to TPA was unaffected by ethanol (Fig. 9). This suggests that PLD products may act downstream of PKC␣ activation by TPA. Significant translocation of PKC␣ by EGF was not detected at the times indicated (Fig. 9), which is in agreement with observations made by other investigators (47). However, EGF was shown to induce PGHS-2, and this induction was not affected by ethanol (Fig. 10, bottom). EGF did not activate PLD (data not shown). Together, these data suggest that PLD-derived products participate in the induction of PGHS-2 by PKCdependent pathways. DISCUSSION The degradation of PC by the PLD/PAP pathway is a potential source of lipid-derived second messengers; however, the role of PC-derived products has not been determined (13). It 2 C. Huang, V. Sciorra, and L. Daniel, unpublished observations. has been suggested that PC-derived DG acts to sustain PKC activation (48); however, alkyl-linked DGs are also generated by PC degradation, and neither stimulate PKC nor inhibit its activation by diacylglycerol (35). It is possible that PA generated directly from PLD serves as a second messenger in cellular signaling. In this report, we have used ethanol to examine if PLD-derived products, PA and DG, participate in TPA-stimulated PGE 2 synthesis. In MDCK cells, TPA-stimulated PGE 2 synthesis was inhibited by ethanol at concentrations which inhibit PA and DG formation. Previously, we reported that TPA stimulates prolonged PGE 2 production in MDCK cells (49). We report here that TPA also stimulates prolonged activation of PLD. PGE 2 synthesis in response to TPA is dependent on continuous activation of PLD. Thus, it appears that PA and/or DG derived from PC degradation by the PLD/PAP pathway act as second messengers in the regulation of TPA-stimulated PGE 2 synthesis.
Regulation of prostaglandin synthesis involves the regulation of both 20:4 mobilization and PGHS synthesis. It is believed that phospholipase A 2 (PLA 2 ) is the primary enzyme involved in regulating 20:4 liberation. Balsinde et al. (50) showed that ethanol inhibited prostaglandin synthesis in zymosan-stimulated macrophages, but did not directly inhibit PLA 2 . This report suggested that ethanol inhibited a process leading to PLA 2 activation. In addition, Ferná ndez et al. (51) demonstrated that PA induces 20:4 mobilization in mouse peritoneal macrophages. The mechanisms by which PA potentiates 20:4 release remain in question. Ethanol caused a small reduction in TPA-mediated 20:4 release; however, this effect cannot fully explain the complete inhibition of PGE 2 synthesis in response to TPA. In addition, 20:4 release in response to A23187 and 20:4 incorporation into phospholipids was not affected by ethanol. It is possible that ethanol inhibits a process leading to PLA 2 activation, for example, alterations in cPLA 2 phosphorylation may result in reduced activity.
In addition to our observations in MDCK cells, Diez et al. (52) have shown that ethanol has no effect on PGHS or lipoxygenase activities in intact macrophages or in a cell-free system. PGHS activity is inhibited in TPA-treated MDCK cells grown in the presence of ethanol. PGHS activity induced by TPA can also be blocked by protein synthesis inhibitors (42). This suggested that TPA stimulates the de novo synthesis of PGHS. Recently, two PGHS genes were identified (53). PGHS-1 was first purified from ovine seminal vesicles (54) and is present in virtually all mammalian tissues (55). In contrast, PGHS-2 was originally isolated as a v-src inducible gene product in chicken fibroblasts (56). PGHS isozymes from a single species are about 60% identical, with protein regions believed to be important for enzyme function are conserved (57). A major difference between the two gene products lies in the factors that regulate expression (58). PGHS-2 is up-regulated in inflammation and mitogenesis (59). PGHS-1 was once considered to be constitutively expressed in mammalian cells (53); however, there have been recent reports that the expression of PGHS-1 and not PGHS-2 is modulated in some cell types (60 -64).
We report that MDCK cells constitutively express PGHS-1, and that TPA stimulates the induction of PGHS-2 and not PGHS-1. PGHS-1 expression remains constant in unstimulated cells grown in the presence or absence of ethanol; while the induction of PGHS-2 in response to TPA is inhibited by ethanol. The increase in PGHS-2 correlated with the elevated PGE 2 synthesis. Furthermore, prolonged PGE 2 synthesis and the continuous induction of PGHS-2 are dependent on PLDderived product formation, since both of these events are inhibited by blocking PLD product formation with ethanol.
Balboa et al. (45) have demonstrated that PKC␣ mediates PLD activation by TPA in MDCK cells. It has been previously shown in this laboratory that PKC activation by TPA is required for TPA-stimulated 20:4 release and PGE 2 synthesis (44). In addition, Godson et al. (46) have demonstrated that PKC␣ is involved in TPA-mediated 20:4 release. We have demonstrated that blocking PLD-derived products with ethanol does not inhibit the translocation of PKC␣ in response to TPA. These data indicate that PLD-derived products act further FIG. 6. TPA-stimulated PGHS activity in MDCK cell sonicates. MDCK cells were grown 2-3 days at 37°C or to 80% confluency. Cells were stimulated with 10 nM TPA (A) or 100 nM EGF (B) with or without 1% ethanol for 4 and 2 h, respectively. Cell sonicates were prepared and assayed immediately for prostaglandin endoperoxide H synthase activity (40). One mg of protein from cell sonicates was incubated for 3 min at 25°C in 1 ml of assay mixture consisting of: 2 mM reduced glutathione, 5 mM L-tryptophan, 1 M hematin, 0.1% Triton X-100, 100 mM Tris-HCl, pH 8. MDCK cells were grown to 80% confluency at 37°C. Cells were treated with media alone, with 1% ethanol, with 10 nM TPA or with 10 nM TPA and 1% ethanol for 6 h (in duplicate). Cell lysates were prepared as described under "Experimental Procedures." Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and probed for PGHS-2 (bottom). The membrane was stripped in buffer containing 62.5 mM Tris, pH 6.8, 2% SDS, and 100 mM 2-mercaptoethanol for 30 min at 50°C, and then probed for PGHS-1 (top). The location of PGHS-1 and -2 was confirmed using recombinant PGHS-1 and -2 (David DeWitt, Michigan State University) as standards (data not shown).
FIG. 8. Induction of PGHS-2 by serum in quiescent MDCK cells. MDCK cells were grown to 80% confluency and made quiescent by growing in 1% FBS for 48 h. Cells were serum-starved for 3 h prior to stimulation with 10% FBS in the presence or absence of ethanol (1%) for 6 h. Lysates were prepared and analyzed for PGHS-2 as described in the legend to Fig. 7. downstream from PKC. We also have shown that EGF did not cause significant translocation of PKC␣ (other isozymes have not been examined) nor cause activation of PLD. However, the effects of EGF on phospholipid metabolism appear to be cellspecific (65)(66)(67). EGF induces PGHS-2 synthesis, and this induction was not affected by ethanol. It appears that EGF stimulates PGHS-2 synthesis by a PKC-independent pathway not requiring PLD activation.
It is possible that the signaling pathways in response to both TPA and EGF converge downstream of PKC␣ activation, leading to the induction of PGHS-2. There is now evidence that activation of the mitogen-activated protein kinase pathway is required for PGHS-2 induction by v-src (68) and both TPA and EGF activate mitogen-activated protein kinase (69,70). Therefore, the pathways may differ in their activation of upstream kinases (for example, Raf-1). Activation of Raf-1 can trigger a protein kinase cascade which results in mitogen-activated protein kinase activation (71). Once activated, mitogen-activated protein kinase can translocate to the nucleus and activate a variety of transcription factors (72).
We have recently shown that TPA stimulates Raf-1 translocation to the membrane in MDCK cells (73). Further studies found that ethanol, used under the same conditions as described herein, inhibits TPA-induced translocation of Raf-1 (73). In addition, PA, but not diacylglycerol or PEt, binds to Raf-1 and stimulates Raf-1 binding to model membranes (73). These data indicate that PA may be the active metabolite of PC-PLD-dependent signaling in TPA-stimulated MDCK cells. FIG. 9. Translocation of PKC␣ in response to TPA but not EGF. MDCK cells were grown to 80% confluency and then incubated with serum-free DMEM overnight at 37°C. Cells were stimulated with 10 nM TPA (T), 100 nM EGF (E), or not stimulated (C) in the presence or absence of ethanol (1%) for the indicated times. Cytosol and membrane proteins were prepared and analyzed for PKC␣ as described under "Experimental Procedures. "   FIG. 10. Ethanol inhibits the induction of PGHS-2 by TPA but not EGF. MDCK cells were grown to 80% confluency and then incubated with serum-free DMEM overnight at 37°C. Cells were stimulated with 10 nM TPA (top) or 100 nM EGF (bottom) in the presence or absence of ethanol (1%) for the indicated times (hours). Lysates were prepared and analyzed for PGHS-2 as described in the legend to Fig. 7.