Staurosporine Stimulates Progesterone Production by Bovine ...

BIOLOGY OF REPRODUCTION 51, 146-151 (1994)

Staurosporine Stimulates Progesterone Production by Bovine Placental Cells M. SHEMESH,'

2

E. HAREL-MARKOWITZ, M. GUREVICH,' and L.S. SHORE'

Department of Hormone Research,' Kimron Veterinary Institute, Bet Dagan, Israel and Koret School of Veterinary Medicine,2 Hebrew University of Jerusalem, Jerusalem, Israel ABSTRACT Progesterone (P4 ) production by the bovine placenta differs from that of other steroidogenic tissue in two important respects: 1) it is calcium-dependent but cyclic nucleotide-independent and 2) it is suppressed by an endogenous inhibitor for most of the life span of the placenta. This natural refractory state of the placenta can be overcome in in vitro incubations of fetal cotyledon cells by agents that increase intracellular calcium (3-isobutylmethylxanthine [MIX], calcium ionophore (A23187)), addition of substrate (pregnenolone, hydroxycholesterol), and stimulators of protein kinase C (PKC) such as phorbol ester (TPA). We therefore tested, in cultures of cotyledonary cells, two compounds that have been reported to inhibit protein kinases: 1) staurosporine (STA), an inhibitor of PKC, cAMP-dependent kinase, tyrosine kinase (TK), and the epidermal growth factor (EGF) receptor TK, and 2) genistein, an inhibitor of TK It was found that STA stimulated steroidogenesis in a dose-dependent manner in both the absence and presence of added calcium. STA (10 -9 M) stimulated at least a twofold increase in P4 production by cultured fetal cotyledon cells throughout the first half of gestation (50-130 days). EGF was also found to cause a twofold stimulation of P4 production, and the effect was additive to that of STA. Both basal and EGF- or STA-stimulated production were inhibited by genistein. In contrast, two inhibitors of PKC and PKA (H-7, H-8) had no effect on P4 production. We conclude that STA-induced steroidogenesis in the bovine placenta is not related to its reported ability to inhibit PKC, TK, or EGF receptor TK

INTRODUCTION

tested two compounds that have been reported to inhibit protein kinases: 1) staurosporine (STA), a potent microbial inhibitor of PKC, cAMP-dependent kinase (PKA), tyrosine kinase (TK), and the epidermal growth factor (EGF) receptor TK [10, 11] and 2) genistein, a plant isoflavone that specifically inhibits TK [12]. In addition, we examined other potent inhibitors of PKC and PKA, H-7 and H-8. Although both these compounds are inhibitors of PKC and PKA [13], H-7 is a more potent inhibitor of PKC while H-8 more markedly inhibits the cAMP-dependent protein kinase. The activity of these compounds was tested in placental cells obtained from 60-120 days of gestation, since the endogenous inhibitor of P4 in the maternal caruncle is maximal in early gestation (declining to low levels by 200 days of gestation) [5] in order to determine which factors could overcome this inhibition.

The bovine placenta is a unique steroidogenic tissue in that it is calcium-dependent but cyclic nucleotide-independent [1]. However, the placentome generally produces very low amounts of progesterone (P4 ), and luteectomy in pregnant cows at up to 200 days of gestation results in abortion [2-4]. We have reported [5] that this refractory state is due to an endogenous inhibitor of steroidogenesis that is present in extracts of maternal and fetal portions of the placentome as well as in the media from cultures of dispersed fetal and maternal cells. This inhibitor was shown to inhibit both basal and stimulated synthesis by dispersed cell preparations of bovine CL as well as placental and granulosa cells. Since the inhibitor was not present in extracts of term placental cells, i.e., at a time when the placenta becomes steroidogenically active (when luteectomy does not result in abortion), it was suggested that this inhibitor could play a role in the physiological regulation of P4 synthesis in the bovine placenta. The site of action of the inhibitor is not known. The refractoriness of the placentome can be overcome by agents that increase intracellular calcium, i.e., 3isobutyl-methylxanthine (MIX) and the calcium ionophore A23187, both of which can cause a twofold increase in P4 production [6-8]. Furthermore, since phorbol ester (TPA), a stimulator of protein kinase C (PKC), stimulates steroidogenesis, it was hypothesized that the effect of the endogenous inhibitor is mediated through an inhibitory protein kinase pathway that reduces steroidogenesis. Therefore, we

MATERIALS AND METHODS Tissues Placental tissues were collected from Holstein-Frisian cows at an abattoir after slaughter (n = 28). In accordance with the protocol approved by the Animal Care and Use Committee of the Kimron Veterinary Institute, prior to slaughter the animals were kept at a large farm (kibbutz) with excellent husbandry standards. Gestational age (60-120 days) was estimated by the crown-rump length of the fetus (1035 cm). Maternal caruncles and fetal cotyledons were separated by blunt dissection. Finger-like projections of cotyledon tissue were not found in the caruncle up to 100 days of gestation. As pregnancy progressed, the finger-like projections were readily apparent, but the dissection com-

Accepted March 10, 1994. Received May 11, 1993. 'Correspondence: Mordechai Shemesh, Department of Hormone Research, Kimron Veterinary Institute, Bet Dagan, P.O.B. 12, Israel. FAX: 972-3-9681-753.

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STAUROSPORINE STIMULATED PLACENTAL STEROIDOGENESIS

pletely separated them from the caruncle as shown by histological sections [14]. Chemicals MIX, STA, and EGF were purchased from Sigma (St. Louis, MO). H-7 and H-8 were obtained from Calbiochem (LaJolla, CA) and the genistein from ICN Biomedicals (Costa Mesa, CA). 25-OH-cholesterol (25-OH-C) was purchased from Steraloids (Wilton, NH). The medium was Dulbecco's modified Eagle's medium (DMEM) without calcium, prepared at our Institute. The inorganic salts were Fe(NO 3)9 H20 (0.10 mg/L); KCI (400 mg/L); MgSO 4 (anhydrous, 97.67 mg/L); NaCl (6.4 g/L), and NaH 2PO4'H 2O (125 mg/L). Incubation of DispersedPlacentalCells Tissues (placentome or CL) were dissected in a laminar flow hood with the aid of sterile instruments. The tissues were then minced and incubated at 37°C with 0.13% collagenase (type 1; Sigma) in Eagle's medium for 10 min. The supernatant was discarded, and the tissue was resuspended in 0.13% collagenase and incubated at 37°C for 45 min. The dispersed cells were subsequently collected from the incubation mixture by centrifugation at 500 x g for 10 min. Cells were then washed three times with DMEM and incubated for 1 h at 5°C to release endogenous P4. The cell pellets were resuspended in DMEM. The cells (3.0 x 105 cells/ml) were plated in 1.5 ml of medium and incubated for periods of 3 to 15 h at 37 0C in an atmosphere of 5% CO 2:95% air. Unless other noted, 1 mM Ca2+ was added to the medium. The phrase "absence of calcium in the medium" is used even though the medium was not absolutely calcium-free, as no chelating agents were used. P4 levels were determined in the cells at 0 time (range, 70-150 pg/ 3 x 105 cells) and again in the incubation medium after the selected times of incubation. After removal of the aliquots (50 or 100 pul) for analysis, the aliquot was replaced with an equal volume of medium. Each treatment group consisted of five replicates. Assay of Progesterone P4 secreted into the culture medium was quantified directly in aliquots of the culture medium. Specific polyclonal antibodies to P4 were prepared in our laboratory as described by Shemesh [15]. The inter- and intraassay coefficients of variation were 11% and 9%, respectively. The sensitivity of the assay was 10 pg/tube. In all cases, net P4 in the medium was determined by subtracting the concentration in the incubation medium at 0 time (120 + 28 pg/3 x 105 cells, mean + SEM; range, 70-150 pg) from the value at the end of the indicated incubation period. The endogenous P4 levels were determined in the cells at 0 time by diethyl ether extraction as previous described [1]. Synthesis was calculated as the level found in the medium minus this intracellular concentration.

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Treatments STA was added to the incubation medium in the range of 10-12 to 10- 8 M. Incubations were generally carried out in 10-9 M STA, as this dose produced maximal effects (not significantly different from those obtained with 10-8 M) and was not toxic as determined by cell counts and trypan blue exclusion. MIX was added at 0.5 mM and the 25-OH-C at 10 Ig/ml, as these concentrations had been shown previously to increase placental cell P4 production by twofold [1]. H-7 and H-8 were added at 50, 100, and 150 ,uM, as at 100 ,uM they decreased steroidogenesis by dispersed luteal cells by 50%. Genistein was used at 1 Rg/ml (3.7 ,uM), as this dose was shown to completely abolish P4 production by the fetal cotyledon cells. Genistein caused a 50% inhibition below control values at 0.1 jLg/ml. EGF was added at 10 ng/ml, since preliminary studies showed that this was the maximal stimulatory dose for increasing P4 over the range of 1 to 50 ng/ml. Cycloheximide was used at 20 or 50 Ig/ ml. Data are expressed as net P4 per 3 x 105 cells and are the mean ± SEM of tissues from four cows, each treatment having been done in five replicates unless otherwise indicated. Significant differences from control values were determined using analysis of variance, followed by either Dunnett's test, Student's t-test or, when appropriate, a paired t-test was used. RESULTS

To define the role of protein kinases in the regulation of P4 synthesis by bovine placental cells, the time course of P4 production by dispersed fetal cotyledon cells in the presence of STA was determined (Fig. 1). A concentration of 10 -9 M STA, which causes maximal stimulation, produced a fourfold enhancement of P4 secretion by the cells with the P4 production being linear during the first 15 h of incubation. Basal P4 secretion progressed at a low rate during this period. Stimulation of P4 synthesis by fetal cotyledon cells in the presence of increasing concentrations of STA is shown in Figure 2. Maximal stimulation was achieved by 10 - 9 M STA, a nontoxic dose as measured by cell counts and trypan blue exclusion. It was of particular interest that 10- 9 M STA increased P4 by both caruncular and cotyledon cells of early gestation (50-80 days; from basal values for both tissues of 0.5 ± 0.1 ng/3 x 105 cells/6 h to 1.0 ± 0.1 ng and 1.3 ± 0.6 ng [n = 5], respectively; Fig. 3). P4 production by bovine placental cells was not affected by 50, 100, or 150 viM concentrations of either H-7 or H8, in contrast to the luteal cells, in which steroidogenesis was significantly reduced (50%) in the presence of H-8 (Fig. 4, A and B). We next examined the effects of the TK inhibitor genistein on placental steroidogenesis stimulated by STA. The time course of P4 production by dispersed placental cells in the presence of STA and genistein was determined (Fig. 5). A concentration of 1 jfg/ml genistein, which caused

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In order to further characterize the site of STA action, we incubated placental cells with cycloheximide in the presence or absence of calcium in the medium. The stimulation by STA of P4 synthesis in the presence of calcium was abolished by cycloheximide. However, the stimulation

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of P4 synthesis in the absence of calcium was only partially inhibited (30 + 3%; mean of three experiments) by the same concentration of cycloheximide. DISCUSSION The data presented here provide the first evidence that STA can stimulate steroidogenesis. This stimulation was significant both in the absence and in the presence of calcium in the incubation medium, while hydroxysterols stimulated P4 production only in the presence of calcium. This result was not expected, as STA has been reported to inhibit PKC, cAMP-dependent kinase, TK, and the EGF receptor TK [10, 11].

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Effect of Inhibition of TK Genistein, a specific inhibitor of TK, completely suppressed steroidogenesis by the fetal cotyledon cells at the concentration used (1 ,g/ml). It also suppressed the stimulated steroidogenesis caused by STA, EGF, or cholesterol. Fifty percent inhibition of P4 production by the fetal cotyledon cells occurred at a dose of genistein (0.1 pug/ml) that caused a 50% increase in P4 production by cultured granulosa cells [16]. It therefore appears that TK activity is essential for steroidogenesis by placental cells and that STA does not inhibit the activity of this enzyme. Effect of EGF

Effect of Inhibition of PKA or PKC

Neither of the two inhibitors of PKC and PKA (H-7, H8) had any facet on steroidogenesis by the placental cells in concentrations that reduced P4 production by 50% in similarly incubated luteal cells. This was in agreement with our previous observation that TPA, a stimulator of PKC, has only a transient effect on stimulation of steroidogenesis in this tissue. It was also expected that inhibition of PKA would have no effect, as the bovine placenta is a cyclic nucleotideindependent steroidogenic tissue.

The action of EGF in increasing steroidogenesis probably encompasses at least two events: 1) the binding of EGF to the membrane and 2) the activation of EGF receptor TK. STA has been reported [11] to have two apparently conflicting effects on EGF action: 1) increasing EGF binding to its high-affinity receptor and 2) decreasing the activity of EGFstimulated TK. Since in our experiments, STA potentiated the increased steroidogenesis elicited by EGF, it would appear that the second action, decreasing TK activity, is not expressed in this tissue.

STAUROSPORINE STIMULATED PLACENTAL STEROIDOGENESIS

The data therefore suggest that STA can affect tissue processes without affecting any of the kinases reported in the literature as its site of inhibition. Similar results have been reported by Levine [17] for rat liver cells with respect to stimulation of prostacyclin synthesis. Specifically, Levine found that STA enhanced prostacyclin production stimulated by TPA, PAF, and calcium ionophore but not prostacyclin formation stimulated by arachidonic acid. Similarly, Rasouly et al. [18] showed that the effects of STA on neurite outgrowth in PC12 cells are independent of PKC inhibition. Effect of Calcium Concentration In order to further characterize the site of STA action, we incubated placental cells with cycloheximide in the presence or absence of calcium in the medium. The stimulation by STA of P4 synthesis in the presence of calcium was abolished by cycloheximide. However, the stimulation of P4 synthesis in the absence of calcium was only partially inhibited (30%) by the same concentration of cycloheximide. This indicated that STA acts through different mechanisms depending on the calcium concentration. Similarly, EGF production of P4 was slightly (20%) but significantly higher in cells incubated in the absence of calcium than in those incubated in the presence of 1 mM Ca2+. In contrast, hydroxysterols increase P4 production only in the presence of Ca+. The bovine placenta is only a potential steroidogenic tissue for most of pregnancy, since luteectomy in pregnant cows at up to 200 days of gestation results in abortion [24]. Indeed, the preponderance of evidence indicates that throughout most of pregnancy, this large mass of tissue (5 kg) does not produce significant amounts of P4 [19]. This is in spite of the fact that all the necessary elements for steroidogenesis are present [20, 21]. We have previously reported evidence that this lack of steroidogenic activity is due to the presence of an endogenous inhibitor that gradually decreases toward 200 days of gestation [5]. This suggests two possible mechanisms of the stimulatory effect of STA on P4 production: it could 1) act indirectly to inhibit kinase involved in the regulation of the inhibitor or 2) act directly on steroidogenic enzymes. However, since a stimulatory effect has not yet been described for another steroidogenic tissue, it is more probable that the action in the placental tissue is through affecting the inhibitor rather than steroidogenic enzymes. However, direct evidence for this hypothesis would require a study of the effect of STA on enzyme phosphorylation in the bovine placenta. Finally, since both the study of Levine [17] on the stimulatory effect of STA on prostacyclin stimulation in rat liver cells and the present paper indicate that stimulatory effects of STA are

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independent of PKC inhibition, the possibility that STA mediates a new protein kinase that has not yet been defined cannot be excluded. REFERENCES 1. Shemesh M, Hansel W, Strauss III JF. Calcium-dependent, cyclic nucleotide-independent steroidogenesis in the bovine placenta. Proc Natl Acad Sci USA 1984; 81:6403-6407. 2. Estergreen VL, Frost OL, Gomes WR, Erb RE, Bullard JF. Effect of ovariectomy on pregnancy maintenance and parturition in dairy cows. J Dairy Sci 1967; 50:12931295. 3. McDonald LE, McNutt SH, Nichols RE. On the essentiality of bovine corpus luteum of pregnancy. Am J Vet Res 1953; 14:529-541. 4. Tanabe TY. The role of progesterone during pregnancy in dairy cows. Penn Agric Exp Sta Bull 1970; 774:1-61. 5. Shemesh M, Hansel W, Strauss III JF. Bovine placentomes contain factors which

decrease progesterone secretion. Biol Reprod 1983; 29:856-862. 6. Shalem Z, Izhar M, Shore LS, Shemesh M, Hansel W, Strauss III JF. Control of bovine placental progestin synthesis: calcium dependent steroidogenesis is modulated at the site of the cholesterol side chain cleavage enzyme. J Steroid Biochem 1988; 31:835-838. 7. Shemesh M, Strauss III JF, Hansel W, Shore LS, Izhar M. Control of bovine placental progesterone synthesis: role of cholesterol availability and calcium-activated systems. J Steroid Biochem 1988; 29:21-25. 8. Shemesh M, Hansel W, Strauss III JF, Shore LS. Regulation of side chain cleavage +2 and 3,-OH steroid dehydrogenase by Ca second messenger systems in bovine placenta. J Reprod Fertil Suppl 1989; 27:163-172. 9. Shemesh M. Production and regulation of progesterone in bovine corpus luteum and placenta in mid and late gestation: a personal review. Reprod Fertil Dev 1990; 2:129-135. 10. Tamakaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F. Stauros2 porine, a potent inhibitor of phospholipid/Ca+ -dependent protein kinase. Biochem Biophys Res Commun 1986; 135:387-402. 11. Friedman BA, Fujuki J, Rosner MR. Regulation of the epidermal growth factor receptor by growth-modulating agents: effects of staurosporine, a protein kinase inhibitor. Cancer Res 1990; 50:533-538. 12. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S-I, Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem 1987; 262:5592-5595. 13. Hidaka H, Inagaki M, Kawamotos S, Sasaki Y. Isoquinoline-sulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 1984; 23:5036-5041. 14. Ben-David E, Shemesh M. Ultrastructural localization of cytochrome P450, in the bovine placentome using protein A-gold technique. Biol Reprod 1990; 42:131138. 15. Shemesh M. Inhibitory action of follicular fluid on progesterone and prostaglandin synthesis in bovine follicles. J Endocrinol 1979; 82:27-31. 16. Kaplanski O, Shemesh M, Berman A Effects of phyto-estrogens on progesterone synthesis of isolated bovine granulosa cells. J Endocrinol 1991; 89:343-348. 17. Levine L. Effects of the protein kinase inhibitors, staurosporine and K-252a, on PG12 production by rat liver cells (the C-9 cell line). Prostaglandins 1990; 40:260267. 18. Rasouly D, Rahamim E, Lester D, Matsuda Y, Lazarovici P. Staurosporine induced neurite outgrowth in PC12-cells is independent of protein kinase-C inhibition. Mol Pharmacol 1992; 42:35-43. 19. Conley AJ, Ford SP. Effect of prostaglandin F2a-induced luteolysis on in vivo and in vitro progesterone production by individual placentomes of cows. J Anim Sci 1987; 65:500-507. 20. Conley AJ, Head JR, Stirling DT, Mason JI. Expression of steroidogenic enzymes in the bovine placenta and fetal adrenal glands throughout gestation. Endocrinology 1992; 130:2641-2650. 21. Izhar M, Pasmanik M, Shemesh M. Bovine placental progesterone synthesis: comparison of first and second trimesters of gestation. Biol Reprod 1992; 46:846852.