- Email: [email protected]
Regulation of Prostaglandin E2 synthesis in human amnion by protein kinase C
PROSTAGLANDINS
REGULATION
SYNTBBSIS OF PROSTAGLANDIN E PROTEIN a INASE C J. Sander and L. Nyatt
Department
IN BUNAN ANNION
BY
of Obetetrics and Gynecology, Pediatrics, Physiology h Biophyeics, Perinatal Research Institute, University of Cincinnati, College of Wedicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0526
ABSTRACT: A role for protein kinase C (PKC)in mediation of prostaglandin E synthesis in human amnion cells has been suggested. We have investigated the specs‘f.rcity of the stimulation of PGE, synthesis by phorbol esters and employed putative PKC inhibitors to demonstrate the specificity of PKC stimulation. The three phorbol esters, tetradecanoyl phorbol-13-acetate, phorbol-12,13-dibutyrate and phorbol-12,13-didecanoate gave concentration-dependent (10“’ - 10m7M)increases in PGE synthesis when added to amnion cells in monolayer. however. no effect was seen with th e structurally similar phorbols phorbol- 12- 13zdiacetate, 4o phorbol- 12- 13-didecanoate or phorbol base. The stimulatory effect of TPA (lO‘*M) on amnion PGE, synthesis could be prevented bv coincubation with the putative protein kinase C inhibit&s I-(5isoquinoline sulphonyi) piperazine, 1-Ooctadecyl-2-0-methyl-rac-glycero-3-phosphocholine, sphingosine and chlopromazine at concentrations of 10M6-10e4M. Addition of the transcription inhibitor actinomycin D at 10~6-10-5M prevented TPA (lo-aM)-induced PGE synthesis. However paradoxically, a further increase in PGE, synthesis was seen when t 0 -9- 10e7M actinomycin D was added together with TPA. The phospholipase A, inhibitor quinacrine was able to prevent the TPA-induced increase in PGE, synthesis even in the presence of exogenous arachidonic acid suggesting that phospholipase Az may be a target for PKC action. INTRODUCTION: The amnion is recognized as a major site of increased PGE synthesis during labor (1) and increased release of arachidonic acid, the precursor of 8GE from membrane phospholipids is seen in amnion at the time of parturition. These T’ atter observations support the tenet that release of arachidonic acid from cell membrane phospholipid limits prostaglandin production. There is increasing awareness that paracrine mechanisms may operate in fetal membranes to regulate parturition (2), however, neither the specific agonists nor the intracellular signalling pathways that mediate prostaglandin production in amnion have been definitively identified. The actions of many hormones and neurotransmitters depends on the hydrolysis of a membrane phosphoinositide, phosphatidyl inositol 4,5 bisphosphate to give 1,2 diacylglycerol and inositol 1,4,5 triphosphate (3). These products act as second messengers down two parallel but independent signal pathwavs both of which result in activation of orotein kinase C. which mayphosphorgate particular substrates resulting in a physiological response within a cell. The PKC arm of phosphatidylinositol signalling pathway has been implicated in the control of PGE, synthesis in several cell types (4,5,6) and a phospholipid and calciumregulated PKC activity has been described in human amnion (7). The ohorbol ester. tetradecanoyl phorbol-13-acetate, which is a mimic of diacylglycerol, has been shown to stimulate PGE synthesis by human amnion (8,9) implying that PKC is involved in the regulation of +GE2 synthesis. We have extended these studies using a range of phorbol esters and putative inhibitors of PKC to demonstrate that PKC is specifically involved in amnion PGE2 synthesis.
APRIL 1990VOL. 39 NO. 4
355
PROSTAGLANDINS
MATERIALS AND METHODS: Human amnion tissue was obtained at elective cesarean section prior to labor. Amnion was stripped from underlying chorion and transported to the laboratory in phosphate buffered saline at 4°C. Amnion cells were then enzymatically dispersed and cultured in monolayer as previously described (10) in 12 well culture plates (Falcon) until confluency. At confluency, culture medium (Medium 199 t 10% fetal bovine serum) was replaced with fresh culture medium containing either tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-didecanoate (PDD), phorbol-12,13-diacetate (PDA), phorbol12,13-dibutyrate (PDB) or phorbol base (all at 10*‘“-10‘7M) for 24 hr at 4°C. The appropriate controls (without phorbol) were used and all concentrations were studied in duplicate wells within the same experiment. Each experiment was repeated four times employing cells from separate amnions. At the end of 24 hr culture medium was removed and stored at -20°C until radioimmunoassay for PGE, as previously described (10). The production of PGE, at each concentration of phorbol ester was expressed as a percentage of control production (no phorbol). In a separate series of experiments, the confluent cells were incubated with lOeM TPA in the presence or absence of various inhibitors of PKC namely I-(5isoquinoline sulphonyl) piperazine (IQSP), l-0-octadecyl-2-0-methyl-rac-glycerd-3-~hosphocholine (Et-18-OMe), sphingosine (Sph) or chlorpromazine (CPZ), all at 10~6-10~4M, for 24 hr at 37°C. PGE, production in the presence of inhibitors was expressed as a percentage of production in the presence of TPA (10W8M) alone. The mean + SEM of four to five separate experiments, each employing cells from a different amnion, are shown. Repeated measures analysis of variance was used to determine the significance of changes in PGEz production. To determine if PKC may be actinn by mechanisms other than covalent modification of substrates, the effect of transcription inhibition was examined by incubatin confluent amnion cells with TPA (10M8M) with or without actinomycin D In a final series of (10-9-10-&) for 24 hr at 37°C and measuring PGE synthesis. experiments, amnion cells were incubated with T b A (10-8M) plus or minus the phospholipase A2 inhibitor quinacrine (10T8- 10m4M) for 24 hr at 37”C, to examine the putative role of phospholipase A, as a taraet for PKC action. These experiments were performed in cells cultured to conlluency in the presence of medium containing 10% fetal bovine serum prior to addition of TPA and quinacrine, in medium either with or without serum for 24 hr. The serum normally acts as a source of exogenous arachidonic acid to cells. RESULTS: Of the phorbol esters examined, TPA, PDB and PDD were able to stimulate PGE synthesis by amnion cells in a concentration-dependent manner (Fig 1). PDA, phorbo t (Fig. 1) and 4a PDD (data not shown) which have similar structure were, however, unable to stimulate PGE, synthesis. The data shown is of a representative experiment using the five phorbols on cells from the same amnion. A time course of TPA-induced PGE, synthesis showed the effect to begin within 1 hr and to be maximal between 12 and 24 hr at 37°C (data not shown). All the inhibitors_gested (IQSP, Et-18-OMe, Sph and CPZ) were effective at preventing the TPA (10 M)-induced increase in PGE, synthesis in a significant concentration-dependent manner (Figures 2a-2d).
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
PHORBOL
ESTER
CONC (M)
Figure I. Stimulation of PGEa synthesis in human amnion by phorbol esters. PGE, synthesis in a representative experrment over a 24 hr period is expressed as a percentage of control synthesis in the absence of phorbol following addition of phorbol esters. 125’
.T; 2 e II ; a.
1 I
50.
TPA (10 _BU) + ICSP
\
I
25.
01
.-. 10-g
1 3x10-g
10-5 IQSP Cone” (M)
3x10-5
10-4
Figure 2a. Inhibition of TPA-induced PGE synthesis in human amnion cells by l-(5isoquinoline sulphonyl) piperazine (IQSP). I&P (10s6- 10-&M) added together with TPA (lOaM) significantly attenuated PGE, synthesis compared to TPA alone (p
APRIL 1990 VOL. 39 NO. 4
357
PROSTAGLANDINS
TPA (lo-%I)
125-
+ 18-EtOMc
” ;100.
‘\A
n.
1 04
- -. 10-6
3x1o-6
10’5
18-EtOMe
3x1 o-5
10-4
Cone” (M)
Figure 2b. Inhibition of TPA-induced PGEa synthesis in human amnion cells by I-_$octadecyl-2-0-methyl-rac-glycero-3-phosphocholine (Et-18-OMe). Et-18-OMe (10 10‘4M) added together with TPA (10W8M) significantly attenuated PGE, synthesis compared to TPA alone (~~0.025 repeated measures analysis of variance mean + SEM, n=4). 125
TPA (10%)
1
+ Sphingosine
, 10-6
3x1o-6 SphingoGne
1 o-5
3x10-5
10-4
Cone” (M)
Figure 2c. Inhibition of T$A-infuced PGE,. synthesis i_nghuman amnion cells by sphingosine. Sphingosine (10 - 10 MB added wrth TPA (10 M) significantly inhibited PGE, synthesis compared to TPA (lo- M) alone (p
358
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
150-
TPA (10 “‘U)
+ Chlorpromazine
125.
100.
75.
50.
25.
01
10-6
3x10-6
10’5
Chlorpromazine
3x10-5 Cone”
10”
(M)
2d. Inhibition of TPA-induced PGE synthesis in human amnion cells by chlorpromazine. Chlorpromazine (10S6-10-‘~ added with TPA (10m8M) significantly inhibited PGE, synthesis in a 24 hr incubation compared to TPA alone (p
TPA (lo-‘M)
+ Actinomycin
D
1
10-g
10-a Actinomycin
10-7 D Cone”
to-6
10-5
(M)
Figure 3. The effect of actinomycin D (10e9- 10‘5M) on PGE, synthesis in human amnion cells induced by TPA (IO-‘M). A significant concentratron-dependent effect of actinomycin D was seen (txO.005, repeated measures analysis of variance).
APRIL 1990 VOL. 39 NO. 4
359
PROSTAGLANDINS
The transcription inhibitor actinomycin D at low concentrations (10S9-10‘7M) paradoxically increased PGE, synthesis beyond that seen upon addition of TPA alone but actinomycin D at higher concentrations (10-6-10‘5M) significantly inhibited PGE, synthesis when added together with TPA (Fig. 3). TPA was able to increase PGE, synthesis in cells in both the presence or absence of serum (data not shown). However, the phospholipase Aa inhibitor quinacrine (1O-810m4M,Fig. 4) prevented this TPA-induced increase in PGE, synthesis in a concentrationdependent manner in either the presence or absence of serum. In the absence of serum, although inhibition of PGE, synthesis was seen, the effect did not reach statistical significance (~~0.1) unlike the inhibition seen in the presence of serum (~~0.005).
TPA (10S7M)
O-O+serum
O--O-serum
0-i
10-a
10-7 Quinacrine
10-g
10-5
-;zT-4
Cone” (M)
Figure 4. The effect of quinacrine on PGEa synthesis in human amnion cells induced by TPA (10e7M) in (a) the presence or (b) the absence of serum. Quinacrine (10~8-10~4M) inhibited TPA-induced PGE, synthesis over a 24 hr period of incubation compared to TPA alone (p
360
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
DISCUSSION: We have shown that the phorbol esters TPA, PDA and PDD were able to stimulate PGE, synthesis whereas the structurally related phorbols. PDA, 4aPDD (not shown) and phorboi base, which do not have biological activity on proteinkinase C,. were unable to stimulate production. By comparison of our data with that of Shoyab and Boaze (11) on the specificity of phorboi esters for the purified phorbol receptor which has protein‘kinase activity we conclude that we are observing a specific protein kinase C mediated effect of phorbol esters. These conclusions were strengthened by employing several different putative inhibitors of protein kinase C, which act via different mechanisms, and demonstrating significant concentration-dependent inhibition of the TPA-induced stimulation of PGE synthesis. A variety of inhibitors was used as none of them are completely specific f or orotein kinase C. The inhibitors used were IOSP which interacts with the catalytic center of the enzyme but also inhibits other protein kinases (12). Et18-OMe, which is unable to inhibit CAMP and cGMP protein kinases (13), sphingosine (14) and chlorpromazine (15) which interferes with the phospholipid-protein interaction of PKC. The concentrations of IQSP and Et-18-OMe that gave inhibition of PGE, synthesis were similar to those found to inhibit PKC in MDCK cells (5). As previously reported (13,14), Sphingosine and Chlorpromazine were less potent on a molar basis at inhibiting PKC than the other two inhibitors. Although a co-concentration-dependent inhibition by sphingosine was found, the effect only became marked at 10‘4M sphingosine. While PKC, presumably acting via phosphorylation of a regulatory element, may increase PGE synthesis in amnion, the precise target of PKC remains unknown. Proteins of 41 and 48kka, phosphorylated by a calcium-phospholipid-regulated PKC activity in human amnion, have been described (7). Casey et al (16) have shown that EGF (perhaps acting via PKC) may stimulate amnion PGE, synthesis by causing de novo synthesis of the PGH, synthase enzyme. Similarly, Zakar and Olson (8) have demonstrated TPAinduced stimulation of PGE, synthesis in amnion following irreversible acetylation of PGHz synthase suggesting that PKC activation results in de novo synthesis-of PGH synthase. Our findings that actinomycin D at 10e6M was able to prevent the TPA-induce 2 increase in PGE synthesis is in agreement with the findings of Zakar & Olson (8) suggesting that Pk C may be having an additional effect on gene regulation as well as acting via covalent modification (phosphorylation) of tar et substrates. However, the finding that lower concentrations of actinomycin D (10 5 -10 -7 M) apparently further stimulated the TPA-induced increase in PGEz synthesis was surprising. It is possible that at these low concentrations, actinomycin D may be inhibiting the synthesis of a protein that itself is inhibitory towards PGEz synthesis and that at higher concentrations actinomycin D then inhibits the de novo synthesis of enzymes directly involved in PGE, synthesis. In the experiment of Casey et al (16) although EGF was able to stimulate PGH, synthase in vitro a source of free arachidonic acid was required to see increased PGE, synthesis. Therefore in vivo, endogenous phospholipase A activity must increase to supply this arachidonic acid to the PGH, synthase. Both EdF (17) and TPA (18) have been reported to increase PLA activity m other systems. This suggests that PLA may be acutely regulated by direct pfiosphorylation/dephosphorylation. Gronich et al (16, also established that phorbol ester treatment changed the proportion of PLA, activity associated with the cell-membrane. Such association which occurs in a calcium-dependent manner may regulate phospholipase A, activity. We have found that the phospholipase A, inhibitor quinacrine gave a concentration-dependent decrease in the TPA-induced increase in PGE synthesis suggesting that PKC may be regulating PLA activity in amnion. Indeed,Barker et al (5) have shown identical dose response curvezfor the activation of PKC and the release of arachidonic acid by phorbol esters. Our inhibition studies with quinacrine were performed in culture medium either containing 10% fetal bovine serum or containing no serum. The presence of serum would expose cells to an exogenous source
APRIL 1990 VOL. 39 NO. 4
361
PROSTAGLANDINS
of arachidonic acid. The finding that quinacrine was able to prevent the TPA-induced increase in PGEz synthesis in these experiments in a significant concentration-dependent manner suggests that the exogenous arachidonic acid does not enter the cell and act directly as substrate for PGH synthase but rather has to be incorporated into the membrane phospholipid pool t;or subsequent liberation by phospholipase. Subsequent experiments employing tissue culture medium without fetal bovine serum and hence no exogenous arachidonic acid again showed that quinacrine was able to prevent the TPAinduced increase in PGE, synthesis. However, in this case the inhibition did not quite reach statistical significance. These studies have not addressed indirect regulation of PLAZ activity by the lipocortins. The lipocortins may inhibit PLAz activity in a substrate-dependent manner by binding to phospholipid on the cytofacial surface of the membrane (19,20). In turn the activity of lipocortins may be regulated hormonally at the transcription level or by covalent modification including phosphorylation by PKC. In summary, we have shown more clearly that stimulation of PKC activity in human amnion increases PGEz synthesis. The effect of PKC may be via covalent modification (phosphorylation) of a regulatory element in the arachidonic acid cascade e.g. phospholipase, to increase its activity or perhaps may include a transcription-dependent mechanism which then increases PLAZ activity. ACKNOWLEDGEMENT: This work was aided by Basic Research Dimes Birth Defects Foundation.
Grant No. l-l 120 from the March of
REFERENCES: 1.
Okazaki T, Sagawa N, Okita JR, Bleasdale JE, MacDonald PC, Johnston JM: Diacylglycerol metabolism and arachidonic acid release in human fetal membranes and decidua Vera. J Biol Chem 256:7316-7321, 1981.
2.
Challis JRG: Causes of parturition. Royal Society of Medicine Services, International Congress and Symposium Series No. 925-14, 1985.
3.
Berridge MJ: Inositol triphosphate and diacylglycerol: messengers. Ann Rev Biochem 56:158-193, 1987.
4.
Halenda SP, Zavoico GB, Feinstein MB: Phorbol esters and oleoyl acetoyl glycerol enhance release of arachidonic acid in platelets stimulated by Ca2+ ionophore A23187. J Biol Chem 260:12484-12491, 1985.
5.
Parker J, Daniel LW, Waite M: Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis. J Biol Chem 202:5385-5393, 1987.
6.
Veldhuis JD, Demers LM: Activation of protein kinase C is coupled to prostaglandin E, synthesis in swine granulosa cells. Prostaglandins 33:819-829, 1987.
I.
Okazaki T, Ban C, Johnson JM: The identification and characterization of protein kinase C activity in fetal membranes. Arch Biochem Biophys 229:27-32, 1984.
8.
Zakar T and Olson DM: Stimulation of human amnion prostaglandin E production by activators of protein kinase C. J Clin Endocr Metab 67:915-923, 5988.
362
two interacting
Ltd.
second
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
9.
Moore JJ, Dubyak GR, Moore RM, Vander Kooy D: Oxytocin activates the inositol-phospholipid-protein kinase-C system and stimulates prostaglandin production in human amnion cells. Endocrinology 123:1771-1777, 1988.
10.
Bennett PR, Rose MP, Myatt L, Elder MG: Preterm labor: stimulation of arachidonic acid metabolism in human amnion cells by bacterial products. Amer J Obstet Gynecol 156:649-665, 1987.
11.
Shoyab M, Boaze R. Isolation and characterization of a specific receptor for biologically active phorbol and ingenol esters. Arch Biochem Biophys 234:197205, 1984.
12.
Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic-nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036-5041, 1984.
13.
Helfman DM, Barnes KC, Kinkade JM, Vogler WR, Shoji M, and Kuo JF. Phospholipid sensitive Ca2+ -dependent protein phosphorylation system in various types of leukemic cells from human patients and in human leukemia cell lines HL60 and JK562, and its inhibition by alkyl-lysophospholipid. Cancer Res 43:2955-2961, 1983.
14.
Hannun YA, Bell RM. Lysosphingolipids inhibit protein kinase C: implications the sphingolipidoses. Science 235:670-674, 1987.
15.
Uratsuji Y, Nakanishi H, Takeyama Y, Kishimoto A, and Nishizuka Y. Activation of cellular protein kinase C and mode of inhibitory action of phospholipidinteracting compounds. Biochem Biophys Res Comm 130~654-661, 1985.
16.
Casey ML, Korte K, MacDonald PC: Epidermal growth factor stimulation of prostaglandin E, biosynthesis in amnion cells. J Biol Chem 262:7846-7854, 1988.
17.
Margolis BL, Holub BJ, Troyer DA, and Skorecki KL: Epidermal growth factor stimulates phospholipase A in vasopressin-treated rat glomerular mesangial cells. Biochem J 256:469-474, l&8.
18.
Gronich JH, Bonventre JV, and Nemenoff RA: Identification and characterization of a hormonally regulated form of phospholipase A, in rat renal mesangial cells. J Biol Chem 263:16645-16651, 1988.
19.
Davidson FF, Dennis EA, Powell M, Glenney JR Jr: lnhibition of phospholipase A2 by lipocortins and calpactins. J Biol Chem 262:1698- 1705, 1987.
20.
Aarsman AJ, Mynbeek G, Van den Bosch H et.al: Lipocortin extracellular and intracellular phospholipases A, is substrate dependent. Febs Lett 219176-180, 1988. Editor:
HR Behrman
APRIL 1990 VOL. 39 NO. 4
Received:
12-22-89
for
inhibition of concentration
Accepted:
363
2-2-90
REGULATION
SYNTBBSIS OF PROSTAGLANDIN E PROTEIN a INASE C J. Sander and L. Nyatt
Department
IN BUNAN ANNION
BY
of Obetetrics and Gynecology, Pediatrics, Physiology h Biophyeics, Perinatal Research Institute, University of Cincinnati, College of Wedicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0526
ABSTRACT: A role for protein kinase C (PKC)in mediation of prostaglandin E synthesis in human amnion cells has been suggested. We have investigated the specs‘f.rcity of the stimulation of PGE, synthesis by phorbol esters and employed putative PKC inhibitors to demonstrate the specificity of PKC stimulation. The three phorbol esters, tetradecanoyl phorbol-13-acetate, phorbol-12,13-dibutyrate and phorbol-12,13-didecanoate gave concentration-dependent (10“’ - 10m7M)increases in PGE synthesis when added to amnion cells in monolayer. however. no effect was seen with th e structurally similar phorbols phorbol- 12- 13zdiacetate, 4o phorbol- 12- 13-didecanoate or phorbol base. The stimulatory effect of TPA (lO‘*M) on amnion PGE, synthesis could be prevented bv coincubation with the putative protein kinase C inhibit&s I-(5isoquinoline sulphonyi) piperazine, 1-Ooctadecyl-2-0-methyl-rac-glycero-3-phosphocholine, sphingosine and chlopromazine at concentrations of 10M6-10e4M. Addition of the transcription inhibitor actinomycin D at 10~6-10-5M prevented TPA (lo-aM)-induced PGE synthesis. However paradoxically, a further increase in PGE, synthesis was seen when t 0 -9- 10e7M actinomycin D was added together with TPA. The phospholipase A, inhibitor quinacrine was able to prevent the TPA-induced increase in PGE, synthesis even in the presence of exogenous arachidonic acid suggesting that phospholipase Az may be a target for PKC action. INTRODUCTION: The amnion is recognized as a major site of increased PGE synthesis during labor (1) and increased release of arachidonic acid, the precursor of 8GE from membrane phospholipids is seen in amnion at the time of parturition. These T’ atter observations support the tenet that release of arachidonic acid from cell membrane phospholipid limits prostaglandin production. There is increasing awareness that paracrine mechanisms may operate in fetal membranes to regulate parturition (2), however, neither the specific agonists nor the intracellular signalling pathways that mediate prostaglandin production in amnion have been definitively identified. The actions of many hormones and neurotransmitters depends on the hydrolysis of a membrane phosphoinositide, phosphatidyl inositol 4,5 bisphosphate to give 1,2 diacylglycerol and inositol 1,4,5 triphosphate (3). These products act as second messengers down two parallel but independent signal pathwavs both of which result in activation of orotein kinase C. which mayphosphorgate particular substrates resulting in a physiological response within a cell. The PKC arm of phosphatidylinositol signalling pathway has been implicated in the control of PGE, synthesis in several cell types (4,5,6) and a phospholipid and calciumregulated PKC activity has been described in human amnion (7). The ohorbol ester. tetradecanoyl phorbol-13-acetate, which is a mimic of diacylglycerol, has been shown to stimulate PGE synthesis by human amnion (8,9) implying that PKC is involved in the regulation of +GE2 synthesis. We have extended these studies using a range of phorbol esters and putative inhibitors of PKC to demonstrate that PKC is specifically involved in amnion PGE2 synthesis.
APRIL 1990VOL. 39 NO. 4
355
PROSTAGLANDINS
MATERIALS AND METHODS: Human amnion tissue was obtained at elective cesarean section prior to labor. Amnion was stripped from underlying chorion and transported to the laboratory in phosphate buffered saline at 4°C. Amnion cells were then enzymatically dispersed and cultured in monolayer as previously described (10) in 12 well culture plates (Falcon) until confluency. At confluency, culture medium (Medium 199 t 10% fetal bovine serum) was replaced with fresh culture medium containing either tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-didecanoate (PDD), phorbol-12,13-diacetate (PDA), phorbol12,13-dibutyrate (PDB) or phorbol base (all at 10*‘“-10‘7M) for 24 hr at 4°C. The appropriate controls (without phorbol) were used and all concentrations were studied in duplicate wells within the same experiment. Each experiment was repeated four times employing cells from separate amnions. At the end of 24 hr culture medium was removed and stored at -20°C until radioimmunoassay for PGE, as previously described (10). The production of PGE, at each concentration of phorbol ester was expressed as a percentage of control production (no phorbol). In a separate series of experiments, the confluent cells were incubated with lOeM TPA in the presence or absence of various inhibitors of PKC namely I-(5isoquinoline sulphonyl) piperazine (IQSP), l-0-octadecyl-2-0-methyl-rac-glycerd-3-~hosphocholine (Et-18-OMe), sphingosine (Sph) or chlorpromazine (CPZ), all at 10~6-10~4M, for 24 hr at 37°C. PGE, production in the presence of inhibitors was expressed as a percentage of production in the presence of TPA (10W8M) alone. The mean + SEM of four to five separate experiments, each employing cells from a different amnion, are shown. Repeated measures analysis of variance was used to determine the significance of changes in PGEz production. To determine if PKC may be actinn by mechanisms other than covalent modification of substrates, the effect of transcription inhibition was examined by incubatin confluent amnion cells with TPA (10M8M) with or without actinomycin D In a final series of (10-9-10-&) for 24 hr at 37°C and measuring PGE synthesis. experiments, amnion cells were incubated with T b A (10-8M) plus or minus the phospholipase A2 inhibitor quinacrine (10T8- 10m4M) for 24 hr at 37”C, to examine the putative role of phospholipase A, as a taraet for PKC action. These experiments were performed in cells cultured to conlluency in the presence of medium containing 10% fetal bovine serum prior to addition of TPA and quinacrine, in medium either with or without serum for 24 hr. The serum normally acts as a source of exogenous arachidonic acid to cells. RESULTS: Of the phorbol esters examined, TPA, PDB and PDD were able to stimulate PGE synthesis by amnion cells in a concentration-dependent manner (Fig 1). PDA, phorbo t (Fig. 1) and 4a PDD (data not shown) which have similar structure were, however, unable to stimulate PGE, synthesis. The data shown is of a representative experiment using the five phorbols on cells from the same amnion. A time course of TPA-induced PGE, synthesis showed the effect to begin within 1 hr and to be maximal between 12 and 24 hr at 37°C (data not shown). All the inhibitors_gested (IQSP, Et-18-OMe, Sph and CPZ) were effective at preventing the TPA (10 M)-induced increase in PGE, synthesis in a significant concentration-dependent manner (Figures 2a-2d).
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
PHORBOL
ESTER
CONC (M)
Figure I. Stimulation of PGEa synthesis in human amnion by phorbol esters. PGE, synthesis in a representative experrment over a 24 hr period is expressed as a percentage of control synthesis in the absence of phorbol following addition of phorbol esters. 125’
.T; 2 e II ; a.
1 I
50.
TPA (10 _BU) + ICSP
\
I
25.
01
.-. 10-g
1 3x10-g
10-5 IQSP Cone” (M)
3x10-5
10-4
Figure 2a. Inhibition of TPA-induced PGE synthesis in human amnion cells by l-(5isoquinoline sulphonyl) piperazine (IQSP). I&P (10s6- 10-&M) added together with TPA (lOaM) significantly attenuated PGE, synthesis compared to TPA alone (p
APRIL 1990 VOL. 39 NO. 4
357
PROSTAGLANDINS
TPA (lo-%I)
125-
+ 18-EtOMc
” ;100.
‘\A
n.
1 04
- -. 10-6
3x1o-6
10’5
18-EtOMe
3x1 o-5
10-4
Cone” (M)
Figure 2b. Inhibition of TPA-induced PGEa synthesis in human amnion cells by I-_$octadecyl-2-0-methyl-rac-glycero-3-phosphocholine (Et-18-OMe). Et-18-OMe (10 10‘4M) added together with TPA (10W8M) significantly attenuated PGE, synthesis compared to TPA alone (~~0.025 repeated measures analysis of variance mean + SEM, n=4). 125
TPA (10%)
1
+ Sphingosine
, 10-6
3x1o-6 SphingoGne
1 o-5
3x10-5
10-4
Cone” (M)
Figure 2c. Inhibition of T$A-infuced PGE,. synthesis i_nghuman amnion cells by sphingosine. Sphingosine (10 - 10 MB added wrth TPA (10 M) significantly inhibited PGE, synthesis compared to TPA (lo- M) alone (p
358
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
150-
TPA (10 “‘U)
+ Chlorpromazine
125.
100.
75.
50.
25.
01
10-6
3x10-6
10’5
Chlorpromazine
3x10-5 Cone”
10”
(M)
2d. Inhibition of TPA-induced PGE synthesis in human amnion cells by chlorpromazine. Chlorpromazine (10S6-10-‘~ added with TPA (10m8M) significantly inhibited PGE, synthesis in a 24 hr incubation compared to TPA alone (p
TPA (lo-‘M)
+ Actinomycin
D
1
10-g
10-a Actinomycin
10-7 D Cone”
to-6
10-5
(M)
Figure 3. The effect of actinomycin D (10e9- 10‘5M) on PGE, synthesis in human amnion cells induced by TPA (IO-‘M). A significant concentratron-dependent effect of actinomycin D was seen (txO.005, repeated measures analysis of variance).
APRIL 1990 VOL. 39 NO. 4
359
PROSTAGLANDINS
The transcription inhibitor actinomycin D at low concentrations (10S9-10‘7M) paradoxically increased PGE, synthesis beyond that seen upon addition of TPA alone but actinomycin D at higher concentrations (10-6-10‘5M) significantly inhibited PGE, synthesis when added together with TPA (Fig. 3). TPA was able to increase PGE, synthesis in cells in both the presence or absence of serum (data not shown). However, the phospholipase Aa inhibitor quinacrine (1O-810m4M,Fig. 4) prevented this TPA-induced increase in PGE, synthesis in a concentrationdependent manner in either the presence or absence of serum. In the absence of serum, although inhibition of PGE, synthesis was seen, the effect did not reach statistical significance (~~0.1) unlike the inhibition seen in the presence of serum (~~0.005).
TPA (10S7M)
O-O+serum
O--O-serum
0-i
10-a
10-7 Quinacrine
10-g
10-5
-;zT-4
Cone” (M)
Figure 4. The effect of quinacrine on PGEa synthesis in human amnion cells induced by TPA (10e7M) in (a) the presence or (b) the absence of serum. Quinacrine (10~8-10~4M) inhibited TPA-induced PGE, synthesis over a 24 hr period of incubation compared to TPA alone (p
360
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
DISCUSSION: We have shown that the phorbol esters TPA, PDA and PDD were able to stimulate PGE, synthesis whereas the structurally related phorbols. PDA, 4aPDD (not shown) and phorboi base, which do not have biological activity on proteinkinase C,. were unable to stimulate production. By comparison of our data with that of Shoyab and Boaze (11) on the specificity of phorboi esters for the purified phorbol receptor which has protein‘kinase activity we conclude that we are observing a specific protein kinase C mediated effect of phorbol esters. These conclusions were strengthened by employing several different putative inhibitors of protein kinase C, which act via different mechanisms, and demonstrating significant concentration-dependent inhibition of the TPA-induced stimulation of PGE synthesis. A variety of inhibitors was used as none of them are completely specific f or orotein kinase C. The inhibitors used were IOSP which interacts with the catalytic center of the enzyme but also inhibits other protein kinases (12). Et18-OMe, which is unable to inhibit CAMP and cGMP protein kinases (13), sphingosine (14) and chlorpromazine (15) which interferes with the phospholipid-protein interaction of PKC. The concentrations of IQSP and Et-18-OMe that gave inhibition of PGE, synthesis were similar to those found to inhibit PKC in MDCK cells (5). As previously reported (13,14), Sphingosine and Chlorpromazine were less potent on a molar basis at inhibiting PKC than the other two inhibitors. Although a co-concentration-dependent inhibition by sphingosine was found, the effect only became marked at 10‘4M sphingosine. While PKC, presumably acting via phosphorylation of a regulatory element, may increase PGE synthesis in amnion, the precise target of PKC remains unknown. Proteins of 41 and 48kka, phosphorylated by a calcium-phospholipid-regulated PKC activity in human amnion, have been described (7). Casey et al (16) have shown that EGF (perhaps acting via PKC) may stimulate amnion PGE, synthesis by causing de novo synthesis of the PGH, synthase enzyme. Similarly, Zakar and Olson (8) have demonstrated TPAinduced stimulation of PGE, synthesis in amnion following irreversible acetylation of PGHz synthase suggesting that PKC activation results in de novo synthesis-of PGH synthase. Our findings that actinomycin D at 10e6M was able to prevent the TPA-induce 2 increase in PGE synthesis is in agreement with the findings of Zakar & Olson (8) suggesting that Pk C may be having an additional effect on gene regulation as well as acting via covalent modification (phosphorylation) of tar et substrates. However, the finding that lower concentrations of actinomycin D (10 5 -10 -7 M) apparently further stimulated the TPA-induced increase in PGEz synthesis was surprising. It is possible that at these low concentrations, actinomycin D may be inhibiting the synthesis of a protein that itself is inhibitory towards PGEz synthesis and that at higher concentrations actinomycin D then inhibits the de novo synthesis of enzymes directly involved in PGE, synthesis. In the experiment of Casey et al (16) although EGF was able to stimulate PGH, synthase in vitro a source of free arachidonic acid was required to see increased PGE, synthesis. Therefore in vivo, endogenous phospholipase A activity must increase to supply this arachidonic acid to the PGH, synthase. Both EdF (17) and TPA (18) have been reported to increase PLA activity m other systems. This suggests that PLA may be acutely regulated by direct pfiosphorylation/dephosphorylation. Gronich et al (16, also established that phorbol ester treatment changed the proportion of PLA, activity associated with the cell-membrane. Such association which occurs in a calcium-dependent manner may regulate phospholipase A, activity. We have found that the phospholipase A, inhibitor quinacrine gave a concentration-dependent decrease in the TPA-induced increase in PGE synthesis suggesting that PKC may be regulating PLA activity in amnion. Indeed,Barker et al (5) have shown identical dose response curvezfor the activation of PKC and the release of arachidonic acid by phorbol esters. Our inhibition studies with quinacrine were performed in culture medium either containing 10% fetal bovine serum or containing no serum. The presence of serum would expose cells to an exogenous source
APRIL 1990 VOL. 39 NO. 4
361
PROSTAGLANDINS
of arachidonic acid. The finding that quinacrine was able to prevent the TPA-induced increase in PGEz synthesis in these experiments in a significant concentration-dependent manner suggests that the exogenous arachidonic acid does not enter the cell and act directly as substrate for PGH synthase but rather has to be incorporated into the membrane phospholipid pool t;or subsequent liberation by phospholipase. Subsequent experiments employing tissue culture medium without fetal bovine serum and hence no exogenous arachidonic acid again showed that quinacrine was able to prevent the TPAinduced increase in PGE, synthesis. However, in this case the inhibition did not quite reach statistical significance. These studies have not addressed indirect regulation of PLAZ activity by the lipocortins. The lipocortins may inhibit PLAz activity in a substrate-dependent manner by binding to phospholipid on the cytofacial surface of the membrane (19,20). In turn the activity of lipocortins may be regulated hormonally at the transcription level or by covalent modification including phosphorylation by PKC. In summary, we have shown more clearly that stimulation of PKC activity in human amnion increases PGEz synthesis. The effect of PKC may be via covalent modification (phosphorylation) of a regulatory element in the arachidonic acid cascade e.g. phospholipase, to increase its activity or perhaps may include a transcription-dependent mechanism which then increases PLAZ activity. ACKNOWLEDGEMENT: This work was aided by Basic Research Dimes Birth Defects Foundation.
Grant No. l-l 120 from the March of
REFERENCES: 1.
Okazaki T, Sagawa N, Okita JR, Bleasdale JE, MacDonald PC, Johnston JM: Diacylglycerol metabolism and arachidonic acid release in human fetal membranes and decidua Vera. J Biol Chem 256:7316-7321, 1981.
2.
Challis JRG: Causes of parturition. Royal Society of Medicine Services, International Congress and Symposium Series No. 925-14, 1985.
3.
Berridge MJ: Inositol triphosphate and diacylglycerol: messengers. Ann Rev Biochem 56:158-193, 1987.
4.
Halenda SP, Zavoico GB, Feinstein MB: Phorbol esters and oleoyl acetoyl glycerol enhance release of arachidonic acid in platelets stimulated by Ca2+ ionophore A23187. J Biol Chem 260:12484-12491, 1985.
5.
Parker J, Daniel LW, Waite M: Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis. J Biol Chem 202:5385-5393, 1987.
6.
Veldhuis JD, Demers LM: Activation of protein kinase C is coupled to prostaglandin E, synthesis in swine granulosa cells. Prostaglandins 33:819-829, 1987.
I.
Okazaki T, Ban C, Johnson JM: The identification and characterization of protein kinase C activity in fetal membranes. Arch Biochem Biophys 229:27-32, 1984.
8.
Zakar T and Olson DM: Stimulation of human amnion prostaglandin E production by activators of protein kinase C. J Clin Endocr Metab 67:915-923, 5988.
362
two interacting
Ltd.
second
APRIL 1990 VOL. 39 NO. 4
PROSTAGLANDINS
9.
Moore JJ, Dubyak GR, Moore RM, Vander Kooy D: Oxytocin activates the inositol-phospholipid-protein kinase-C system and stimulates prostaglandin production in human amnion cells. Endocrinology 123:1771-1777, 1988.
10.
Bennett PR, Rose MP, Myatt L, Elder MG: Preterm labor: stimulation of arachidonic acid metabolism in human amnion cells by bacterial products. Amer J Obstet Gynecol 156:649-665, 1987.
11.
Shoyab M, Boaze R. Isolation and characterization of a specific receptor for biologically active phorbol and ingenol esters. Arch Biochem Biophys 234:197205, 1984.
12.
Hidaka H, Inagaki M, Kawamoto S, Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic-nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036-5041, 1984.
13.
Helfman DM, Barnes KC, Kinkade JM, Vogler WR, Shoji M, and Kuo JF. Phospholipid sensitive Ca2+ -dependent protein phosphorylation system in various types of leukemic cells from human patients and in human leukemia cell lines HL60 and JK562, and its inhibition by alkyl-lysophospholipid. Cancer Res 43:2955-2961, 1983.
14.
Hannun YA, Bell RM. Lysosphingolipids inhibit protein kinase C: implications the sphingolipidoses. Science 235:670-674, 1987.
15.
Uratsuji Y, Nakanishi H, Takeyama Y, Kishimoto A, and Nishizuka Y. Activation of cellular protein kinase C and mode of inhibitory action of phospholipidinteracting compounds. Biochem Biophys Res Comm 130~654-661, 1985.
16.
Casey ML, Korte K, MacDonald PC: Epidermal growth factor stimulation of prostaglandin E, biosynthesis in amnion cells. J Biol Chem 262:7846-7854, 1988.
17.
Margolis BL, Holub BJ, Troyer DA, and Skorecki KL: Epidermal growth factor stimulates phospholipase A in vasopressin-treated rat glomerular mesangial cells. Biochem J 256:469-474, l&8.
18.
Gronich JH, Bonventre JV, and Nemenoff RA: Identification and characterization of a hormonally regulated form of phospholipase A, in rat renal mesangial cells. J Biol Chem 263:16645-16651, 1988.
19.
Davidson FF, Dennis EA, Powell M, Glenney JR Jr: lnhibition of phospholipase A2 by lipocortins and calpactins. J Biol Chem 262:1698- 1705, 1987.
20.
Aarsman AJ, Mynbeek G, Van den Bosch H et.al: Lipocortin extracellular and intracellular phospholipases A, is substrate dependent. Febs Lett 219176-180, 1988. Editor:
HR Behrman
APRIL 1990 VOL. 39 NO. 4
Received:
12-22-89
for
inhibition of concentration
Accepted:
363
2-2-90