Activation of protein kinase C is coupled to prostaglandin F2α synthesis in the ovary: studies in cultured swine granulosa cells

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Molecular and Cellular Endocrinology, 49 (1987) 249-254 Elsevier Scientific Publishers Ireland, Ltd.

MCE 01600

Activation of protein kinase C is coupled to prostaglandin F,, synthesis in the ovary: studies in cultured swine granulosa cells Johannes D. Veldhuis ’ and Lawrence M. Demers 2, with the technical assistance of Paula Azimi, Diana Juchter and James Garmey ’ Division of Endocrinology and Metabolism, Department of Iniernal Medicine, Universiry of Virginia School of Medicine, Charlottesville,

VA 22908, and ’ Pennsylvania State Universi& School of Medicine, Departmenf of Pathology, Milton S. Hershey Medical Center, Hershey, PA I7033, U.S.A. (Received 28 July 1986; accepted 14 October 1986)

Key words: Protein kinase C; (Activation); Prostaglandin Fza syn thesis; (Ovary); Granulosa cell

Summary We have investigated the role of phospholipid-sensitive calcium-dependent protein kinase (protein cultures of swine granulosa cells. In this system, kinase C) in prostaglandin Fza syn thesis by monolayer specific phorbol ester derivatives known to activate protein kinase C significantly augmented the with the ionophore A23187 synergistically production of prostaglandin Fza. Phorbol ester in conjunction increased prostaglandin Fzu production. These stimulatory actions were dose- and time-dependent, and could be abolished by the cyclooxygenase inhibitor, indomethacin, or the protein synthesis inhibitor, cycloheximide. Moreover, the rank order of potency of phorbol esters in enhancing prostaglandin Fza production was concordant with that demonstrated for activation of protein kinase C in the swine ovary. In addition, a nonphorbol stimulator of protein kinase C, l-octanoyl-2-acetylglycerol, also significantly enhanced prostaglandin F2,, biosynthesis. The synthesis of immunoassayable prostaglandin Fza was confirmed by high-pressure liquid chromatographic purification of this radiolabeled metabolite of [3H]arachidonic acid. Thus, the present studies indicate that the protein kinase C effector pathway in the swine granulosa cell is functionally coupled to prostaglandin F,, production,

Introduction A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) participates in the expression of differentiated function in a variety of cell lines (Takai et al., 1979; Kuo et al., 1980). Although this specific phosphorylating sys-

Address for correspondence: J.D. Veldhuis, Box 202, Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, VA 22908, U.S.A. 0303-7207/87/$03.50

tern has also been identified recently in the mammalian ovary (Davis and Clark, 1983; Veldhuis and Demers, 1986), the precise relevance of such an effector pathway to ovarian cellular function has not been elucidated fully. Studies in cultured granulosa cells have indicated that presumptive stimulators of protein kinase C can inhibit hormonally stimulated progesterone biosynthesis, and stimulate prostaglandin E, production (Welsh et al., 1984; Kawai and Clark, 1985; Veldhuis and Demers, 1986). Since prostaglandin Fzrr functions as an effective inhibitor of steroidogenesis in a

6 1987 Elsevier Scientific Publishers Ireland, Ltd.

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variety of luteal tissues (Horton and Poyser, 1976), we have investigated the possibility that activation of the protein kinase C pathway stimulates prostaglandin F,, production. To this end, we used an in vitro system of cultured swine granulosa cells in which there is demonstrable protein kinase C activity and prominent steroidogenic responsiveness to protein kinase C activation (Veldhuis and Demers, 1986; Wheeler and Veldhuis, 1986).

Methods Culture conditions Granulosa cells were harvested by fine-needle aspiration of small Graafian follicles in the ovaries of immature swine as described earlier by Channing and Ledwitz-Rigby (1975). Cells were cultured at an approximate density of lo6 viable cells (as determined by the exclusion of Trypan blue) per cm* in Falcon multiwells containing Eagle’s minimum essential medium buffered with. bicarbonate and supplemented with 1% fetal calf serum (v/v), as reported previously (Veldhuis et al., 1982). Cultures were maintained for indicated intervals before the medium was replenished and/or harvested for the subsequent determination of progesterone or prostaglandin F201content. Except where indicated otherwise, assays were performed on the extracts of cells combined with medium after sonication to determine total prostaglandin F2a content, which was assayed by specific radioimmunoassay (Demers et al., 1974). HPLC analysis was performed on a radial compression column (Waters M-6000A) using a linear gradient of increasing concentrations of acetonitrile (23-70% v/v in aqueous buffer) on a 5 pm C-18 PBondapak system. The sample comprised cyclohexane/ethylacetate (1: 1) extracts of medium from 10’ granulosa cells incubated with 7.5 pCi/ml of [3H]arachidonic acid for 4 h with or without an effector agent present. Data were submitted to analysis of variance to ascertain significant overall treatment effects. The Newman-Keuls’ procedure was used to define individually significant differences (Winer, 1971). Data are expressed as means f SEM. Nonlinear least-squares curve-fitting of the dose-response curves was used to estimate the

mean (and 95% confidence limits) for the values of the half-maximally effective stimulatory concentrations (ED,,) (Johnson, 1983). Each experiment was performed at least twice using a separate batch of 200-250 ovaries to test reproducibility of the results. Materials Tissue culture materials were from Grand Island Biological Corporation, ovine FSH (NIHFSH-S15) from the National Hormone Distribution Office, and individual specific phorbol esters and A23187 from Sigma Chemical Corporation (St. Louis, MO). [3H]Arachidonic acid (specific activity, 240 Ci/mmol) was from New England

2100

7 HR

1600 3 HR

1500

1200

BOO

600

300

mtrol

Control

FSH

TPA

FSH

TPA

Control FSH

TPA

FSH TPA

;

he Fig. 1. Time-course of the stimulatory effect of FSH an phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on prostaglandin FIu production by swine granulosa cells. Swine granulosa cells were cultured for the times indicated in each panel (3 h, 7 h, 24 h, and 48 h) in the presence or absence of FSH (200 ng/ml) and/or TPA (10 ng/ml). Data are presented as prostaghmdin F,, accumulated per 2x lo6 cells per time interval. Data are means f SEM (n = 4 separate determinations). Differing superscripts denote significantly different means by analysis of variance.

Nuclear (Boston, MA). 1-Octanoyl-2-acetylglycer01 was provided by Dr. James Garrison, Dept. of Pharmacology, University of Virginia School of Medicine.

RMlltS As depicted in Fig. 1, initial time-course experiments demonstrated that the treatment with the potent tumor-promoter agent, 12-O-tetradecanoylphorbol-13-acetate (TPA, 10 ng/ml) elicited a time-dependent stimulation of prostaglandin Fza production by monolayer cultures of swine granulosa cells. Significant stimulatory effects of TPA (10 ng/ml).were observed within 3-7 h and continued to be expressed over 48 h of observation. Moreover, TPA was able to enhance the stimulatory effect of a maximally effective concentration of FSH (200 ng/ml). Based upon these results, further studies were performed at 48 h. The stimulatory effect of TPA was dose-dependent, and exhibited an ED,, of 0.54 (0.36-0.62) ng/ml (Fig. 2A). In the presence of FSH, the ED,, of TPA’s stimulation was 0.41 (0.33-0.58) ng/ml, which did not differ significantly from that in the absence of FSH. For comparison, the relative potency of other activators of protein kinase C, viz., phorbol-12,13dibutyrate (PDB) and its presumptively less effective counterpart, phorbol-12,13-diacetate (PDA), were tested for their ability to stimulate prostaglandin Fzu production. As shown in Fig. 2B, PDB also evoked dose-dependent increases in prostaglandin Fzol production by cultured swine granulosa cells. The apparent EDso for PDB was 1.9 (1.8-2.1) ng/ml. The less active tumor-promotor substance PDA stimulated prostaglandin Fzu production to a lesser degree and exhibited an ED,, of 42 (36-50) ng/ml. Moreover, 4cY-phorbol-12,13-decanoate and pure phorbol base, which have minimal ability to activate protein kinase C (Wheeler and Veldhuis, 1986) were inactive in stimulating prostaglandin Fzu production even at concentrations of 100 ng/ml (n = 3 experiments). The combined effects of TPA and the calcium ionophore, A23187, were tested by incubating serum-free monolayer cultures of swine granulosa cells with TPA (30 ng/ml) without or with in-

TABLE 1 INFLUENCE OF THE SYNTHETIC DIACYLGLYCEROL, l-OCTANOYL-2-ACETYLGLYCEROL (OAG), ON PROSTAGLANDIN F2. PRODUCTION BY CULTURED SWINE GRANULOSA CELLS Swine granulosa cells (2 X 106) were cultured for 4 h in serumfree Eagle’s minimum essential medium. Cells and medium were harvested, sonicated, and the total content of PGF2, determined by RIA. Data are means f SEM (n = 4 determinations). Treatment

PGF,, (pg/4 h)

None OAG (30 pg/d) FSH (200 ng/ml) FSH f OAG

30* 7a 16Ok 18 b 60f17a 310+27’

‘-’ Differing superscripts denote significantly different means by analysis of variance.

creasing concentrations of A23187 (0.03-3.0 pg/ ml) for 4 h. As depicted in Fig. 3, the combined actions of these two agents were synergistic in stimulating prostaglandin Fza production (panel A) whereas in the same cultures progesterone biosynthesis was significantly inhibited (panel B). The basis for the biphasic interaction between A23187 and TPA is not known. To investigate the mechanisms subserving the stimulatory effects of phorbol esters on prostaglandin F,, production by granulosa cells, cultures were treated with TPA in the presence or absence of the cyclooxygenase inhibitor, indomethacin (100 PM). As shown in Fig. 4, indomethacin abolished detectable prostaglandin F,, TABLE 2 INFLUENCE

OF THE

PHORBOL

ESTER,

TPA, ON

[ 3H]ARACHIDONIC ACID CONVERSION TO HPLC-SEPARATED [‘HIPROSTAGLANDIN SWINE GRANULOSA CELLS

F2,, BY CULTURED

Data are mean cpm/lO’ cells (k range for two separate determinations) eluting with the prostaglandin Fza standard after purification by HPLC.

Exp. 1 Exp. 2

Control

TPAB

686 f 12 748 f 14

1768k 95 * 2358+180 *

a Treatment with 30 ng/ml TPA for 4 h. * P < 0.02 vs. control.

B

0

0.1

0.3

1.0

3.0

10

Basal

FSH

Dose of TPA (w/ml)

0

0.1 1.0 10 100 FSH + PDB (ng/ml)

Fig. 2. Panel A : Dose-dependent stimulatory effect of TPA alone and/or combined with FSH on prostaglandin F,, production by swine granulosa cells. Granulosa cells were cultured for 48 h (Methods) in the absence or presence of increasing doses of TPA with or without FSH (200 ng/ml). Subsequent accumulation of prostaglandin Fsa was measured in cells combined with media. Panel B: Dose-dependent stimulatory effect of phorbol-12,13-~butyrate (PDB) on prost~l~din F,, accumulation by swine granulosa cells. Data in pg/106 cells/48 h are otherwise as presented in the legend of Fig. 1.

production in control and TPA-treated cultures, indicating a complete dependence of measured prosta~~~n F2* accumulation upon de novo

synthesis. In addition, when cultures were treated with the protein synthesis inhibitor, cycloheximide (1 ~g/&l), the induction of prostaglandin F24%

1700 B

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1500

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1100

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900

+TPA

700 0 0 DO%3 A-23187

&ugfml)

0.03

0.10

Dose of A-23187

0.30

1.0

3.0

(ug/mf)

Fig. 3. Acute synergistic effects of the phorbol ester, TPA, and the calcium ionophore, A23187, on prostaglandin F,, production. Panel A depicts total prostaglandin F,, production (pg/4 h.106 cells) in response to TPA (30 ng/ml) and/or A23187 (0.03-3.0 ,ug/mi) administration acutely in monolayer cultures of swine granulosa cells. Panel B presents the corresponding total progesterone accumulation in the same cultures. Data are means f SEM (n = 4 separate determinations).

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yl-2-acetylglycerol (OAG) was added to short-term incubations of swine granulosa cells. As shown in Table 1, OAG resulted in prompt and significant stimulation of prostaglandin Fza production in the absence or presence of FSH (P -e0.01). To test more directly the ability of swine granulosa cells to synthesize prostaglandin Fzcr at increased rates in response to TPA treatment, cells were cultured for 48 h, washed, and then exposed to [3H]arachidonic acid with or without TPA (30 ng/ml) for 4 h. The subsequent medium was extracted and subjected to HPLC to separate specific radiolabeled prostaglandins. As shown in Table 2, prior incubation with TPA resulted in a marked enhancement in the synthesis of HPLCpurified [3H]prostaglandin Fzcrby cultured granulosa cells (P -c0.02 vs. control). Discussion

Fig. 4. Influence of inhibitors of protein synthesis or cyclooxygenase on TPA-stimulated prostaglandin Fs,, production. Gramtlosa cells were cultured for 48 h in the absence or presence of indomethacin (100 PM, INDO) or cycloheximide (1 pg/ml, CYCLO), and/or TPA (10 ng/ml). The total content of prostaglandin Fsol in sonicates of cells combined with medium was determined. Data are otherwise as presented in the legend of Fig. 2.

synthesis by TPA was also inhibited. These observations, interpreted with the recognition that no inhibitors are entirely specific, suggest a need for ongoing protein synthesis in the stimulatory actions of TPA. To determine whether the effect of phorbol compounds was simply due to the diterpine structure, or rather reflected their mimicry of diacylglycerol, a synthetic diacylglycerol, l-octano-

The present results provide the first demonstration to our knowledge that the protein kinase C effector pathway is functionally coupled to the synthesis of prostaglandin Fza in ovarian cells. In the current studies with swine granulosa cells, prostaglandin Fzo: synthesis could be stimulated by both phorbol and nonphorbol activators of protein kinase C. Moreover, such stimulation of prostaglandin Fza! production occurred with a rank order of potency similar to that recently described for the activation of protein kinase C in these cells (Kawai and Clark, 1985; Veldhuis and Demers, 1986). Treatment of intact swine granulosa cells with the phorbol compound TPA has been shown to promote phosphorylation of discrete cytosolic proteins (Kawai and Clark, 1985). Thus, this potent stimulator of protein kinase C is capable of activating endogenous phosphorylation mechanisms in pig granulosa cells. Although the precise substrate(s) for protein kinase C in the ovary have not yet been identified, the present results indicate that the protein kinase C effector system is functionally coupled to prostaglandin F,, synthesis. The mechanisms subserving this putative coupling between the protein kinase C pathway and prostaglandin synthesis presumably involve calcium ions, since the effects of TPA and the divalent cation ionophore A23187 were synergistic, as well

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as ongoing protein synthesis, since the stimulatory actions of TPA were abolished by cycloheximide. In addition, TPA’s stimulation of prostaglandin F 2a accumulation reflects increased de novo synthesis of this prostaglandin, since an inhibitor of cyclooxygenase, indomethacin, eliminated this effect. Although no inhibitors are completely specific, this inference was supported by the observation that incubation of granulosa cells with TPA resulted in increased accumulation of HPLC-purified radiolabeled prostaglandin Fza by granulosa cells exposed to [ 3H]arachidonic acid. Since prostaglandin Fzo: represents a luteolysin in many species (Thomas et al., 1978), our results provide a possible link between the calcium-dependent protein kinase C pathway and luteolysis in the ovary. Moreover, recent studies by Leung et al. (1986) indicate that this prostaglandin can also initiate phosphoionositide hydrolysis, which in turn may participate in activation of the protein kinase C pathway. Accordingly, the present studies suggest that local intraovarian autofeedback mechanisms may operate, in which activation of protein kinase C promotes prostaglandin Fzcr synthesis and release, which in turn further activate the phospholipid-dependent, calcium-sensitive protein kinase C pathway. In conclusion, our results with a range of phorbol ester compounds that express differential potency in activating protein kinase C implicate this specific effector pathway in granulosa-cell efprostaglandin Fza synthesis. The stimulatory fects of phorbol ester were amplified by the calcium ionophore, A23187. Moreover, when the nonditerpine compound, l-octanoyl-2-acetylglycerol, was used as a presumptive activator of protein kinase C, prostaglandin F,, synthesis was also augmented significantly. Collectively, these observations provide strong evidence for specific functional coupling between the calcium-dependent protein kinase C pathway and prostaglandin Fza synthesis in the ovary.

Acknowledgements We thank Chris McNett for the skillful preparation of the manuscript, the Gwaltney-Smithfield Packing Corporation for providing swine ovaries, Paula P. Azimi for the artwork, and Dr. Julianne J. Sando for her expert advice and assistance in the conduct of this work. This work was supported in part by NIH Grant No. R 01 HD16806 and RCDA No. 1 K04 HD 00634 (J.D.V.).

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