Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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Biology of Reproduction 61, 147-153 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Diverse Effects of Tyrosine Kinase Inhibitors on Follicle-Stimulating Hormone-Stimulated Estradiol and Progesterone Production from Rat Granulosa Cells in Serum-Containing Medium and Serum-Free Medium Containing Epidermal Growth Factor

Donna Haynes-Johnsona, Muh-Tsann Laia, Carolyn Campena, and Stephen Palmer1,a

a Reproductive Medicine, The R.W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey 08869


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidermal growth factor (EGF) has been shown to influence FSH-stimulated estradiol (E2) and progesterone (P4) production from granulosa cells. RG 50810, a tyrosine kinase inhibitor (TKI), has previously been shown to inhibit the EGF-receptor tyrosine kinase. RG 50810 has also been shown to inhibit FSH-stimulated increases in mRNA for steroidogenic enzymes, implying a functional role of tyrosine kinases in FSH action in granulosa cells. However, inhibition of FSH-stimulated steroidogenesis by TKIs has not been evaluated in connection with the effects of EGF in granulosa cells. In the present studies, FSH-stimulated E2 production was inhibited similarly by inhibitors of protein kinase A (H-89) and protein kinase C (calphostin C) and by TKIs, and none of the inhibitors were capable of reversing the EGF-induced inhibition of FSH-stimulated E2 production. FSH-stimulated P4 production was enhanced dramatically in serum-containing medium with concentrations of TKI that were near previously reported IC50s. The enhancing effect of TKIs was less evident in serum-free medium. Addition of EGF to serum-free medium enhanced FSH-stimulated P4 production, and the TKIs reversed EGF-enhanced P4 production, but in a manner similar to that of protein kinase A inhibitor H-89. Compared to results in serum-free medium, the potency of RG 50810 and genistein to inhibit the effects of EGF on P4 production was 3- to 8-fold greater relative to H-89. These studies have demonstrated that TKIs RG 50810 and genistein selectively inhibit the effects of EGF on FSH-stimulated P4 production in granulosa cell cultures. In contrast, these studies have demonstrated nonselective inhibition of FSH-stimulated E2 and P4 production by TKIs in serum-free medium, in which it is not clear which enzyme system is affected by the compounds tested.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
FSH, LH, and epidermal growth factor (EGF) are important regulators of ovarian follicular growth and differentiation. FSH and LH bind to adenylyl-cyclase-coupled receptors on granulosa cells within the follicle, resulting in cAMP, progesterone (P4), and estradiol (E2) production [1]. Although FSH and LH appear to act via similar pathways, granulosa cells need to be able to discriminate between an FSH and an LH signal on a biochemical basis as well as on a temporal basis for normal follicular growth and differentiation. Various growth factors have been proposed as agents that can influence granulosa cell functions. EGF has been shown to bind to receptors on granulosa cells and modify the effects of FSH on granulosa cell steroid production [2–5]. The precise mechanisms whereby EGF influences effects of FSH on granulosa cells from immature follicles are unclear.

Previous studies have shown that EGF inhibits FSH-stimulated E2 production under defined culture conditions [6–9]. However, the reported effects of EGF on FSH-stimulated P4 production differ. EGF has been shown to inhibit FSH-stimulated P4 production in rat [10], porcine [11, 12], and chicken granulosa cell cultures [13]. In contrast, several reports have shown that EGF enhances FSH-stimulated P4 production from rat, sheep, and mouse granulosa cell cultures [6, 14–16]. Similarly, EGF has been shown to enhance FSH-stimulated pregnenolone and 20-{alpha}-hydroxyprogesterone production and cholesterol side-chain cleavage enzyme amount and activity in rat granulosa cells [6, 14,17]. Still other studies have reported that EGF has no effect on FSH-stimulated P4 production in rat granulosa cells [7]. These studies suggest that the effects of EGF on FSH-stimulated P4 production are not well understood.

Recent studies have suggested that FSH activity in granulosa cells may be influenced by a tyrosine kinase-like activity [18] that is affected by tyrphostins, a class of compounds that have been shown to inhibit receptor tyrosine kinases. Two tyrphostins, RG 50810 (a.k.a. AG-18) and RG 50864 (a.k.a. AG-213), have been described in other systems as selective inhibitors of the EGF-receptor tyrosine kinase in both membrane preparations and whole cells [19]. In several recent studies in granulosa cells, RG 50810 and genistein (a general, not EGF-specific, tyrosine kinase inhibitor [TKI]), have been shown to reduce levels of steroidogenic enzymes, or their mRNA, suggesting that tyrosine kinases influence FSH action in a selective manner [20–22]. However, studies to date have not clearly demonstrated that TKIs inhibit the pharmacologic target in granulosa cells, that is, the EGF-dependent tyrosine kinase. Although RG 50810 has been shown to inhibit FSH-induced cytochrome P450 side-chain cleavage enzyme (P450scc) levels and FSH-induced mRNA for P450scc and P450 aromatase enzymes in granulosa cells [21, 22], these effects were not evaluated in the presence of EGF or in the presence of inhibitors of other intracellular pathways. Therefore, it is unclear whether RG 50810 was acting via the EGF-receptor tyrosine kinase, another tyrosine kinase, or pathways other than tyrosine kinases in granulosa cells. If the effects of RG 50810 reported in granulosa cells were, in fact, associated with the EGF receptor, then other TKIs with similar pharmacology [19, 23], but not other classes of protein kinase inhibitors, should also inhibit the effects of EGF on FSH-stimulated events such as E2 and P4 production. Information about the effects of TKIs with varying potency and selectivity on FSH-stimulated E2 and P4 production may help determine the influence of the EGF-receptor tyrosine kinase or other kinases on FSH action in the granulosa cell.

The present studies were conducted to determine whether selected TKIs inhibit EGF-dependent effects on FSH-stimulated E2 and P4 production in a manner consistent with their selectivity and potency for inhibition of EGF-receptor tyrosine kinase. Four TKIs, with a range of IC50s and selectivities for tyrosine kinases relative to protein kinases were used to determine whether they have selective effects on FSH-stimulated E2 and P4 production in serum-containing medium, known to contain growth factors that have been shown to stimulate receptor-tyrosine kinases, and in serum-free medium. The same four TKIs were then tested in the presence of EGF, in order to determine whether the TKIs exert their effects through the EGF-receptor tyrosine kinase. Protein kinase A (PKA) and protein kinase C (PKC) inhibitors were also used to assess specificity of the above TKIs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents

Insulin, diethylstilbestrol, androstenedione, forskolin, and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO). Fungizone, penicillin/streptomycin, charcoal-treated heat-inactivated fetal bovine serum (CT-HI-FBS), and Dulbecco's Modified Eagle's medium (DMEM):Ham's F-12 medium containing 15 mM Hepes and L-glutamine (F12; DMEM:F12) were purchased from GIBCO BRL (Grand Island, NY). Calphostin C (PKC inhibitor), H-89 (PKA inhibitor), erbstatin analogue (mixed activity as tyrosine kinase and PKA inhibitor), genistein, RG 50810, and RG 50864 (TKIs) were purchased from BIOMOL Inc. (Plymouth Meeting, PA). The selectivity and IC50s of the TKIs, as well as those of H-89 and calphostin C, have been determined previously in other systems [23,24], and for reference purposes are listed in Table 1.


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  		TABLE 1. Selectivity of protein kinase inhibitors from previous studies with various cells.*

Hormones

Ovine FSH (NIADDK-oFSH-17; FSH potency = 20 NIH-FSH-S1 U/mg; LH contamination = 0.04 times NIH-LH-S1) was a generous gift from Ogden Bioservices Corporation, Rockville, MD. EGF and platelet-derived growth factor were purchased from Upstate Biological Inc. (Lake Placid, NY).

Granulosa Cell Culture

Immature intact female rats (Wistar-derived strain; 21–25 days old) received implants of a single pellet (Innovative Research of America, Sarasota, FL) containing 2.5 mg diethylstilbestrol (DES) for 3 days. The care and use of the animals was approved by the Institutional Animal Care and Use Committee. On the third day, the animals were killed, the ovaries were removed, and the granulosa cells were isolated as described previously [25], except that DMEM:F12 replaced M199. Granulosa cells were expressed from the ovaries, the residual ovarian tissue was removed, and the cells were centrifuged at 500 x g and resuspended and plated in DMEM:F12 containing 2% serum (CT-HI-FBS), insulin (1 µg/ml), fungizone (0.75 µg/ml amphotericin), 100 U/ml penicillin, 100 µg/ml streptomycin, and 10-8 M DES. Granulosa cells were plated at a density of 300 000 cells per milliliter, and 0.2 ml was added to each well of 96-well culture dishes (Corning, NY). Cultures were incubated at 37°C in a humidified incubator (95% air:5% CO2) overnight (18 h).

The effects of FSH and the various inhibitors on granulosa cell steroidogenesis were compared under three different culture conditions: media with 2% CT-HI-FBS, serum-free media, or serum-free media containing EGF. Eighteen hours after plating, the spent medium was removed, the cells were washed, and fresh DMEM:F12 (without insulin) containing androstenedione (10-7 M) was added with experimental treatments. Granulosa cells were then cultured for an additional 48 h. Inhibitors (erbstatin analogue, genistein, RG 50810, RG 50864, H-89, and calphostin C) were dissolved in 100% DMSO and diluted to 400 µM with DMEM:F12, and 50 µl of serial dilutions of the inhibitors were added to the cultures (final concentration of DMSO was 0.25%). Control cells received DMEM:F12 medium containing 0.25% DMSO. Isobutylmethylxanthine (IBMX), commonly used in granulosa cell cultures, was omitted to reduce confounding drug effects in the study of protein kinase activity.

RIA of E2 and P4

Concentrations of E2 and P4 in media from the same culture wells were measured using 125I-P4 and 125I-E2 Coat-A-Count RIA kits (Diagnostic Products Corp., Los Angeles, CA). According to the specification sheets provided, the anti-P4 antibody cross-reacts 2% with 20{alpha}-dihydroprogesterone, 2.4% with 11-deoxycortisol, 1.7% with 11-deoxycorticosterone, and 1.3% with 5ß-pregnan-3,20-dione. The cross-reactivity of pregnenolone, 17{alpha}-hydroxyprogesterone, and testosterone was less than 0.4%. The assay detection limit was 0.03 ng/ml. The anti-E2 antibody cross-reacts 10% with estrone, 4.4% with equilenin, 1.8% with estrone glucuronide, 0.3% with estriol, and less than 0.1% with other estrogens and androgens. The assay detection limit was 8 pg/ml.

Data Analysis

Data were analyzed using the SuperAnova package containing general linear models (Abacus Concepts, Inc., Berkeley, CA). E2 and P4 production from cells incubated in the presence of inhibitor were compared to E2 and P4 production from cells incubated with FSH alone. Differences in E2 and P4 production at different concentrations of inhibitor were analyzed in a split-plot randomized complete block, in which the inhibitor was the whole plot, the concentration of inhibitor was the split plot, and repeats of the experiment were the blocks. Differences between means were considered significant if p < 0.05. Six separate experiments were performed comparing effects of inhibitors in medium containing 2% serum, medium containing no serum (serum-free), or serum-free medium containing EGF.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effects of TKIs on FSH-stimulated E2 production are shown in Figures 1 and 2 (6 replicate experiments). FSH (25 ng/ml) stimulated a 15-fold increase in E2 production from granulosa cells cultured in medium containing either 2% serum (Fig. 1A) or no serum (Fig. 1B). In serum-containing medium, RG 50810 (10 µM) slightly enhanced FSH-stimulated E2 production (Fig. 1A; 10 µM), but FSH-stimulated E2 production was inhibited at 30-µM concentrations of genistein and erbstatin and at 100-µM concentrations of all four of the TKIs. Calphostin C (selective PKC inhibitor) and H-89 (selective PKA inhibitor) decreased FSH-stimulated E2 production at 1- and 3-µM concentrations, respectively, and the inhibition was maximal at 30-µM concentrations of either inhibitor. The concentration response curves for inhibition of E2 production showed that H-89 and calphostin C were approximately 10-fold and 30-fold more potent, respectively, than either RG 50810 or genistein.



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  		FIG. 1. Effect of TKIs RG 50810, RG 50864, genistein, and erbstatin; PKA inhibitor H-89; and PKC inhibitor calphostin C (Calph C) on FSH-stimulated E2 production from granulosa cells cultured in 2% serum (A) or serum-free medium (B). C (control): 2% CT-HI-FBS or serum-free medium. F (FSH): 25 ng/ml oFSH. = p < 0.05 vs. control. *, +, §, £, ¶, #: p < 0.05 vs. FSH.



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  		FIG. 2. Effect of TKIs RG 50810, genistein, and RG 50864; PKA inhibitor H-89; and PKC inhibitor calphostin C (Calph C) on EGF-induced suppression of FSH-stimulated E2 production from granulosa cells. C (control): serum-free medium. F (FSH and serum-free medium): 25 ng/ml oFSH. F+E (FSH, serum-free medium + EGF) 5 ng/ml. {theta} = p < 0.05 vs. control; {xi} = p < 0.05 vs. 25 ng/ml FSH. *, +, §, £, ¶, #: p < 0.05 vs. FSH + EGF.

In serum-free medium (Fig. 1B), RG 50810 and erbstatin inhibited FSH-stimulated E2 production at 10-µM and higher concentrations, and genistein inhibited FSH-stimulated E2 production at 30-µM and 100-µM concentrations. Erbstatin, the most potent TKI in this experiment, is known to have a poor selectivity ratio between PKA and tyrosine kinase inhibition [26], suggesting that the inhibitory effects of the TKIs on FSH-stimulated E2 concentrations may include actions on other protein kinases. Both calphostin C (PKC inhibitor) and H-89 (PKA inhibitor) inhibited E2 production in serum-free media at approximately 3-fold lower concentrations compared to the TKIs (Fig. 1B). These data indicate that protein kinase inhibitors from a variety of pharmacologic classes at high concentrations (> 3 µM) inhibit FSH-stimulated E2 production similarly in serum-free medium. Although cell numbers were not affected by the various kinase inhibitors (data not shown), many cells (30–40%) incubated with a 100-µM concentration of inhibitors were rounded and smaller, and they had irregular shaped plasma membranes compared to the flattened appearance of healthy granulosa cells. After removal of the inhibitors, the viability returned to >= 80%, and the steroidogenic potential of the cells returned except for cells exposed to 100 µM inhibitor. The effects of all inhibitors on E2 production at 100-µM concentrations in serum-free medium were potentially toxic.

Addition of EGF (5 ng/ml) to granulosa cells inhibited FSH-stimulated E2 production (Fig. 2). None of the TKIs nor the A or C kinase inhibitors reversed the EGF-induced inhibition of FSH-stimulated E2 production. Higher concentrations (> 10 µM) of all inhibitors tested reduced FSH-stimulated E2 production below the levels of E2 measured in the presence of FSH and EGF, and in a manner similar to that seen in serum-free medium (Fig. 1B).

The effects of inhibitors and EGF on FSH-stimulated P4 production were more complicated (Figs. 3–4) than their effects on E2 production. FSH (25 ng/ml) caused a 15-fold stimulation of P4 production by rat granulosa cells cultured in 2% CT-HI-FBS (Fig. 3A) and in serum-free medium (Fig. 3B). In medium containing 2% serum (Fig. 3A), genistein (0.3–3 µM), RG-50810 (3–30 µM), and RG 50864 (30 µM) enhanced FSH-stimulated P4 production at concentrations that were close to their reported IC50 (Table 1). At higher concentrations (100 µM), RG 50810 and genistein inhibited FSH-stimulated P4 production. The PKA inhibitor H-89 enhanced P4 production at concentrations near its reported IC50 (0.01–0.1 µM; see Table 1), while P4 production was inhibited at 10-µM and higher concentrations of H-89. Relative to RG 50810, the concentrations of H-89 that increased (30- to 100-fold lower) or decreased (10-fold lower) FSH-stimulated P4 production were lower. In contrast to the biphasic effects of H-89 and RG 50810, calphostin C (PKC inhibitor) inhibited FSH-stimulated P4 production at concentrations greater than 1 µM.



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  		FIG. 3. Effects of TKIs RG 50810, RG 50864, genistein, and erbstatin; PKA inhibitor H-89; and PKC inhibitor calphostin C (Calph C) on FSH-stimulated P4 production from granulosa cells in 2% CT-HI-FBS medium (A) or serum-free medium (B). Granulosa cells were collected from DES-treated rats, and cultured and treated as describe in Materials and Methods. C (control): 2% CT-HI-FBS or serum-free medium. F (FSH): 25 ng/ml oFSH). {theta}: p < 0.05 vs. control; *, +, §, £, ¶, #: p < 0.05 vs. FSH.



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  		FIG. 4. Effects of TKIs on FSH-stimulated P4 production from granulosa cells cultured in serum-free medium in the presence of EGF (5 ng/ml). C (control): serum-free medium. F (FSH, serum-free medium) 25 ng/ml oFSH. F+E (FSH, serum-free medium + EGF): 5 ng/ml. {theta}: p < 0.05 vs. control; {xi}: p < 0.05 vs. 25 ng/ml FSH; *, +, §, ¶: p < 0.05 vs. FSH + EGF.

In cultures containing serum-free medium, genistein (0.1–3 µM), RG 50810 (0.01–10 µM), and RG 50864 (10–30 µM) minimally enhanced FSH-stimulated P4 production (Fig. 3B). At higher concentrations of several inhibitors (30–100 µM), the TKIs reduced FSH-stimulated P4 production; and erbstatin analogue, the least selective TKI, was the most potent inhibitor in this regard. The protein kinase inhibitors H-89 and calphostin C inhibited FSH-stimulated P4 production at all concentrations tested (0.01–100 µM; Fig. 3B). Therefore, the distinguishing and selective effect of TKIs (although minimal), compared to PKA or PKC inhibitors, was to enhance FSH-stimulated P4 production at low concentrations. The inhibition of FSH-stimulated P4 production in serum-free medium by all concentrations of PKA and PKC inhibitors, by > 3-µM concentrations of erbstatin analogue, and by 100-µM concentrations of the remaining TKIs suggests that inhibitory effects of TKIs on P4 production at 100-µM concentrations are not limited to the pharmacology of tyrosine kinases and may represent a nonspecific effect.

The impact of the TKIs on their pharmacologic target, the EGF-receptor tyrosine kinase, and their subsequent effects on FSH-stimulated P4 production was examined in granulosa cells cultured in serum-free medium containing EGF (5 ng/ml). EGF increased FSH-stimulated P4 production nearly 3-fold compared to cells receiving FSH alone (Fig. 4), a response to EGF that is consistent with some previous studies [6, 14–16]. Genistein and RG 50810, but not RG 50864, inhibited the EGF-dependent increase in FSH-stimulated P4 production at concentrations near their previously reported IC50s. The effect of the PKA inhibitor H-89 was biphasic, as had been seen in serum-containing medium (Fig. 3A). However, the concentration response curve for H-89 in medium containing EGF compared to serum-free medium was shifted markedly to the right, relative to RG 50810 or genistein. This shift in the concentration response curve suggests that EGF either increases FSH-stimulated PKA enzyme level or activity, or minimizes the impact of PKA on P4 production.

The calculated IC50s for inhibition of EGF-enhanced, FSH-stimulated P4 production and the potency ratios comparing serum-free and EGF-containing medium demonstrated clearly that the potency of RG 50810 and genistein were improved relative to H-89 (Table 2). In the presence of EGF, the potency of the TKIs increased 3- to 8-fold, but the potency of H-89 decreased 5- to 10-fold. Therefore, under conditions in which EGF enhanced FSH-stimulated P4 production in serum-free medium, the TKIs demonstrated a pharmacology consistent with their ability to inhibit EGF-dependent tyrosine kinase enzyme activity. However, at high concentrations (> 30 µM) and in the absence of EGF, the overlapping profiles of tyrosine kinase and PKA inhibitors tested in this experiment raised questions about the utility of the TKIs tested to clarify the influence of tyrosine kinase and PKA pathways on P4 production in long-term (>= 24 h) granulosa cell cultures.


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  		TABLE 2. Calculated IC50s for the inhibition of FSH-stimulated P4 production from granulosa cells in serum-free (SF) medium and EGF-enhanced medium.*


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Four tyrphostins with previously determined selectivity for tyrosine kinases and protein kinases were used in a comparison study to determine whether an EGF-receptor tyrosine kinase was responsible for the effects of TKIs on FSH-stimulated E2 and P4 production. The endpoints of E2 and P4 production were chosen to provide an overview of the pharmacology of TKIs on FSH-stimulated pathways in granulosa cells. Results from these studies emphasized the importance of evaluating the effects of TKIs relative to PKA inhibitors on the pharmacologic target (in this case the EGF-receptor tyrosine kinase) in order to determine their selective influence on the predominantly cAMP-dependent PKA pathway of FSH receptor activation. The significant findings presented in this work were 1) that none of the TKIs or other inhibitors tested reversed EGF-induced inhibition of FSH-stimulated E2 production; 2) that the TKIs enhanced FSH-stimulated P4 production from granulosa cells cultured in medium containing serum at concentrations that minimize nonspecific effects; and 3) that two TKIs were able to inhibit EGF-enhanced FSH-stimulated P4 production, albeit in a manner similar to that of the PKA inhibitor H-89.

The inhibitors tested provided a spectrum of selectivity for their respective pharmacologic target. RG 50864, genistein, and RG 50810 are reported to be selective inhibitors of tyrosine kinases in cell-free and in whole-cell systems [19, 23, 24]. The selectivity of RG 50810 and RG 50864 for the EGF-receptor tyrosine kinase has been reported to be better than genistein. Genistein has been shown to inhibit other growth factor tyrosine kinases, protooncogene tyrosine kinases, and also PKC in other cell systems [23,26]. In addition, genistein is an isoflavonoid, a phytoestrogen, that has been shown to have estrogen-like effects in granulosa cells [27, 28]. Erbstatin analogue, the least selective tyrphostin, has been shown to have nearly equal inhibition of tyrosine kinase and PKA enzymes [29]. PKA inhibitor H-89 and PKC inhibitor calphostin C have been used in many recent studies because they are more selective for their respective enzymes than previously used inhibitors staurosporine, H-7, and H-8 [30]. In other cell types, calphostin C has been shown to inhibit the PKC-related effects of serum on DNA synthesis, and mRNA and protein expression [31, 32], while calphostin C has been shown to inhibit specific isoforms of PKC [33], and in a manner dissociated from phorbol 12-myristate 13-acetate (TPA) stimulation of PKC [34].

The effects of diverse kinase inhibitors on FSH-stimulated steroid production in granulosa cells has not been evaluated previously in a concentration-dependent manner. Results from this study indicate that inhibition demonstrated by all the compounds at 100 µM are nonspecific. The responses of granulosa cells in the present studies to H-89 and calphostin C at higher concentrations (1–100 µM) and to the nonselective erbstatin (between 5 µM and 100 µM) were similar to effects seen with higher concentrations of the reportedly more selective tyrphostins. RG 50810 has previously been shown to inhibit FSH-, prostaglandin E2-, and forskolin-induced increases in steroidogenic enzyme mRNA or protein [21, 22]. Given that all three ligands work through the cAMP-dependent PKA pathway, these previous results suggest that effects of TKIs at high concentrations are not limited to tyrosine kinase inhibition, or unique to the FSH receptor, but are involved, directly or indirectly, in other intracellular protein kinase-mediated pathways. Since the tyrphostins are classified as inhibitors of tyrosine kinases, it seemed necessary to demonstrate that TKIs at lower concentrations perform in their predicted manner, i.e., to inhibit EGF-dependent tyrosine kinase activity in a profile distinguishable from other pharmacologic classes of inhibitors.

A well-characterized response of EGF (or TGF{alpha}) in granulosa cells is to inhibit FSH-stimulated E2 production from human [9] and rat [6–8, 35] granulosa cells. By definition, TKIs should have reversed EGF-induced inhibition of FSH-stimulated E2 production in the present studies. In contrast to the predicted response, all of the inhibitors tested failed to reverse EGF inhibition of E2 production, suggesting that effects of these compounds occurred independently of added EGF and may be nonselective effects. The effect of withdrawal of the inhibitors from the granulosa cell cultures on P4 and E2 production was determined in previous experiments, and recovery from the inhibitory effects of TKI required more than 24 h [21, 22]. In our own preliminary studies, FSH-stimulated E2 production from granulosa cells exposed to <= 30 µM inhibitors for 24 h followed by 24 h withdrawal of compound returned to normal (data not shown). However, cells exposed to 100 µM concentrations were unable to recover E2 production completely, suggesting that the effects of the inhibitors at this concentration may be toxic.

Although ineffective in reversing EGF-dependent inhibition of E2 production in this study, RG50810 and RG 50864 have previously been shown to inhibit phosphorylation of the EGF receptor in B2A4 and A431 squamous carcinoma cells. In addition, RG 50810 and RG 50864 antagonized EGF-stimulated proliferation of B2A4 cells. However, neither TKI was able to reverse the inhibitory effect of EGF on cell growth in A431 cells [36]. A common feature from the studies using squamous carcinoma cell lines and the present work in rat granulosa cells is that the TKIs tested were unable to reverse an inhibitory effect of EGF in either system. Previous studies that have examined TKI inhibition of an EGF effect in granulosa cells have shown that the TKIs inhibited a growth-promoting effect of EGF [20]. It is possible that intracellular pathways responsible for inhibitory effects of EGF in cells are regulated differently than are pathways for stimulatory effects of EGF.

A consistent but unexpected effect of the TKIs demonstrated in this work is the enhancement of FSH-stimulated P4 production in serum-containing medium (Fig. 3A) with lower concentrations of TKIs (< 10 µM). The enhancing effect of TKIs on FSH-stimulated P4 production in serum-containing, compared to serum-free, medium suggests that factors in serum modulate the effect of TKIs on P4 production. The enhanced production of P4 by TKIs (most evident with RG 50810) contrasts with previous studies that have shown that RG 50810 or genistein inhibited FSH-stimulated increases in P450scc enzyme at high concentrations (> 20 µM) [21, 22]. In the prior studies, the effects of higher concentrations of TKIs were evaluated in serum-free medium alone, while the present studies have explored the potential interaction of serum and growth factors with a broader concentration range of TKIs on FSH-stimulated P4 production. It is unclear whether the enhancing effects of TKIs affect cAMP-dependent pathways directly or indirectly.

The effects of EGF on P4 production from granulosa cells have varied among studies, but the present results as well as several previous studies [6, 8, 14–17] have shown that EGF enhances FSH-stimulated P4 production. The reasons for the variation in the response of granulosa cells to EGF is unclear; however, a number of growth factor-related effects have been proposed [8, 14, 15, 37, 38]. These reports suggest that the presence of other growth factors present in the conditioned medium of granulosa cell cultures may influence the effects of EGF and/or FSH on P4 production.

Several results from the present work suggested that the TKIs demonstrated selective pharmacology in the presence of EGF, but that the endpoint of inhibition may be similar among TKIs and the PKA inhibitor H-89. First, RG 50810 and genistein inhibited EGF-enhanced P4 production (Table 2) at concentrations consistent with previously reported IC50s (Table 1 [19, 23, 24, 26, 27]). Second, the rightward shift in the concentration-response curve of H-89 over RG 50810 and genistein in the presence of EGF versus serum-free medium (Table 2; potency ratio) indicates that these two TKIs have a greater impact on EGF-enhanced P4 production, at a concentration consistent with their IC50s, than the PKA inhibitor H-89. Third, the similar effects of RG 50810 and genistein at high concentrations (>= 30 µM) and of H-89 at lower concentrations (> 1 µM) suggest that the effects of the TKIs overlap with the effects of H-89 on the PKA pathway.

The extent of intracellular communication between FSH (cAMP-dependent) and EGF (tyrosine kinase-dependent) pathways within granulosa cells still remains unknown. The intracellular cascade from the EGF-receptor tyrosine kinase to nuclear transcription factor phosphorylation includes multiple enzymes [39–42] that may be potential links between the cAMP-dependent and receptor tyrosine kinase-dependent pathways. Both EGF and FSH (and cAMP), known to affect different intracellular pathways, have been shown to rapidly phosphorylate mitogen-activated protein kinase (MAPK: ERK1 and ERK2) in granulosa cells [14,43–45]. The pattern of ERK1 and ERK2 phosphorylation in nuclear and cytosolic fractions has been shown to differ after FSH or EGF treatments, suggesting subtle differences in MAPK activation mediated by cAMP and tyrosine kinase pathways. In rat granulosa cells, FSH has been shown to stimulate phosphorylation of MAPK within 30 min (ERK1 or ERK2), and 50 µM RG 50810 failed to inhibit this phosphorylation [45]. These latter results would confirm that early events following FSH receptor activation do not include an EGF-receptor-like tyrosine kinase. In cell culture studies of longer duration, RG 50810 has been shown to inhibit FSH-stimulated increases in steroidogenic enzyme mRNA and protein levels [21, 22] and to inhibit EGF-enhanced, FSH-stimulated P4 production (present study). Differences in the effects of TKIs among studies that compare rapid phosphorylation events versus studies that compare changes in steroidogenic enzyme levels or steroid production may reflect time-dependent responses of the inhibitors.

In summary, the present work has shown dual effects of TKIs that are concentration-dependent and diverse, and that share some similar actions with PKA inhibitors. The results of this study demonstrate the importance of evaluating the effects of TKIs in the presence of EGF, or other growth factors, in order to determine the selective pharmacologic effects of these inhibitors on tyrosine kinases in granulosa cells. In addition, these studies have highlighted potential differences in the effects of EGF and TKIs on E2 and P4 production. Although two TKIs reversed EGF-enhanced, FSH-stimulated P4 production, at a concentration consistent with their reported IC50, similar effects have also been shown with a PKA inhibitor. The cross-talk between tyrosine kinase and PKA pathways in granulosa cells [46] make it difficult to distinguish the individual effects of inhibitors of these two pathways on steroid production.


   FOOTNOTES
 
1 Correspondence: Stephen Palmer, The RW Johnson Pharmaceutical Research Institute, 1000 Rt 202, PO Box 300, Raritan, NJ 08869. FAX: 908 526 6469; spalmer{at}prius.jnj.com Back

Accepted: February 3, 1999.

Received: February 27, 1997.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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