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Biology of Reproduction 67, 1003-1012 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Regulation of Granulosa Cell-Derived Insulin-Like Growth Factor Binding Proteins (IGFBPs): Role for Protein Kinase-C in the Pre- and Posttranslational Modulation of IGFBP-4 and IGFBP-51

Diran Chamouna, DooSeok Choib, Adriano B. Tavaresc, Laurence C. Udoffd, Eliahu Levitase, Carol E. Resnickd, Ron G. Rosenfeldf, and Eli Y. Adashi2,,c

a 95 Bulldog Blvd., Suite 204, Melbourne, Florida 32901 b Department of Obstetrics and Gynecology, Sungkyunkwan University, School of Medicine, Samsung Medical Center, Seoul, Korea c Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Huntsman Cancer Institute, Salt Lake City, Utah 84112 d Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, Maryland 21201 e Fertility and IVF Unit, Department of Obstetrics and Gynecology, Soroka University Medical Center, Beer Sheva, Israel f Department of Pediatrics, University of Oregon Health Sciences Center, Portland, Oregon 97201


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A growing body of information suggests antigonadotropic and atretogenic roles for granulosa cell-derived insulin-like growth factor binding proteins (IGFBPs) 4 and 5 during ovarian folliculogenesis. Activation of protein kinase-A (PKA) in rat granulosa cells has been shown to modulate the relative expression of IGFBP-4 and -5 transcripts and proteins. In this article, we assess the role of protein kinase-C (PKC) in this regard. Provision of granulosa cells with phorbol 12-myristate 13-acetate (PMA) (but not 4{alpha}PMA, an inert analogue), a tumor-promoting phorbol ester and an established activator of PKC, was without significant effect on the expression of IGFBP-4 transcripts but resulted in biphasic dose-dependent alterations in IGFBP-5 transcripts and in the accumulation of the IGFBP-4 and -5 proteins. Comparable effects were noted for GnRH, an established PKC agonist. Provision of staurosporine, a potent inhibitor of the catalytic subunit of PKC, produced significant dose-dependent decrements in the relative expression of IGFBP-5 transcripts. Treatment with FSH (presumptively PKA-mediated) markedly attenuated the ability of PMA or GnRH to upregulate the accumulation of the IGFBP-5 (but not IGFBP-4) protein. Taken together, our present findings indicate that the modulation of rat ovarian IGFBP-4 and -5 is PKC as well as PKA dependent and that these two signaling pathways interact in a diametrically opposed and antagonistic fashion.

female reproductive tract, granulosa cells, insulin-like growth factor receptor, kinases, ovary


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite the well-accepted role of gonadotropins in ovarian folliculogenesis, the variable fate of follicles afforded comparable gonadotropic support suggests the existence of additional intraovarian modulatory systems [1]. Among potential intraovarian regulators, insulin-like growth factor-I (IGF-I) has been the subject of particularly intense investigation [2, 3] and seems to serve as the central signal of a complete ovarian intrafollicular IGF-I system comprised of a receptor (type-I), binding proteins (IGFBP-4 and -5), and IGFBP (-4 and -5)-directed endopeptidases (IGFBPases).

Based on current knowledge, the role of intrafollicular IGF-I is the amplification of FSH action at the level of the granulosa cell [4, 5], a facilitatory function in keeping with the often exponential nature of follicular development. It is the FSH-amplifying property of IGF-I, its attendant antiatretic effect, and its selective expression in healthy but not atretic follicles that underlie the hypothesis that the ovarian intrafollicular IGF-I system is a determinant of the otherwise enigmatic phenomena of follicular selection and dominance in the ovary [6–8]. In fact, it is the net bioavailable intrafollicular IGF-I, defined by the IGF-I/IGFBP ratio and determined by the relative intrafollicular representation of the predominant IGFBPs (4 and 5) and their specific proteases, that may distinguish follicles destined to ovulate from those that will succumb to atresia.

As might be expected, a growing body of information supports antigonadotropic [9–15] and atretogenic [16–21] roles for granulosa cell-derived IGFBP-4 and -5. The IGFBPs are antigonadotropins by virtue of their IGF-I-sequestering property, a property that may be causally related to the failure and demise of atretic follicles. Given in situ hybridization studies, IGFBP-4 and -5 were noted to be differentially expressed in atretic and healthy antral follicles. Specifically, healthy antral follicles proved IGFBP deplete whereas atretic follicles proved IGFBP replete. The net IGFBP accumulation may well be determined by the opposing influences of multiple regulatory signals, among which both FSH and IGF-I may play a dominant but opposite role. Treatment with FSH (A-kinase dependent) inhibits the accumulation of granulosa cell-derived IGFBPs, while treatment with IGF-I stimulates their accumulation.

On the other hand, the corresponding specific proteases have been recognized as markers of healthy follicles [22, 23]. The pregnancy-associated plasma protein-A (PAPP-A) has recently been firmly established as an IGFBP-4 protease [24–27]. Other proteins have been implicated in IGFBP-5 proteolysis, including C1s, a protein member of the classical pathway of the complement cascade [28], the human heat shock protein HtrA [29], and the pregnancy-associated plasma protein-A2 (PAPP-A2) [30].

Despite the projected central role of the intraovarian IGFBPs in folliculogenesis, the precise cellular mechanisms underlying the regulation of the locally derived IGFBP-4 and -5 remain largely unstudied. In this regard, note must be made of the now-established role of protein kinase-A (PKA) in the regulation of granulosa cell-derived IGFBP-4 and -5 transcripts and proteins. Indeed, treatment of rat granulosa cells with FSH has been shown to exert a biphasic effect on the relative expression of IGFBP-4 and -5 transcripts and proteins [13, 31–34]. In contrast, little is known about the role of protein kinase-C (PKC) in the regulation of granulosa cell-derived IGFBPs. Although the precise role of PKC in ovarian physiology remains uncertain, there can be little doubt about the existence of such an intraovarian signal transduction pathway [35–41]. It was the purpose of this article to assess the role of PKC in the modulation of granulosa cell-derived IGFBP-4 and -5. Our findings reveal that the modulation of ovarian IGFBP-5 (but not IGFBP-4) transcripts and proteins is PKC dependent. Specifically, activation of PKC signaling has been shown to upregulate IGFBP-5 transcripts and proteins, an effect counteracted by the activation of the PKA pathway.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Note must be made that all animal experimentation described in this article was conducted in accordance with accepted standards of humane animal care, the Animal Welfare Act, and the Institutional Animal Care and Use Committee (IACUC) protocols.

Animals

Immature (23–25 days old) intact Sprague-Dawley female rats were purchased from Zivic-Miller Laboratories (Zelienople, PA) following 4 days of diethylstilbestrol (DES) priming by the vendor. The latter was achieved by the s.c. of 10-mm silastic DES-containing capsules. The animals were killed on Day 25 of life by CO2 asphyxiation. All protocols were approved by IACUC.

Hormones and Reagents

McCoy 5a medium (modified, serum free), penicillin-streptomycin solution, L-glutamine, and trypan blue stain (0.4%; wt/vol) were from Life Technologies, Inc. (Grand Island, NY). Ovine FSH (NIH-oFSH-S18; FSH potency 65 NIH FSH-S1 U/mg; LH activity 0.1 NIH LH-S1 U/mg; prolactin activity less than 0.1% by weight) was generously provided by the National Pituitary Agency, Pituitary Hormone Distribution Program, NIDDK. Phorbol 12-myristate 13-acetate (PMA), 4{alpha}-phorbol-12,13-didecanoate (4{alpha}PMA), and DES were obtained from Sigma Chemical Co. (St. Louis, MO). GnRH was from Peninsula Laboratories (Belmont, CA). Staurosporine (Streptomyces sp.) was from Calbiochem-Novabiochem Corporation (La Jolla, CA). Recombinant human activin-A was generously provided by Jennie P. Mather (Genentech, Inc., South San Francisco, CA).

In Vitro Studies

Granulosa cells were obtained by follicular puncture, as previously described [42], and plated onto tissue culture dishes (35 x 10 mm; Falcon Plastics, Oxnard, CA) at a density of 5 x 105 viable cells per dish. The cells were cultured for up to 72 h at a temperature of 37°C under a water-saturated atmosphere of 95% air and 5% CO2. Use was made of 1 ml of serum-free McCoy 5a medium supplemented with 2 mmol/L of L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. All reagents were dissolved in sterile culture media. All treatments were added in 50-µl aliquots. At the conclusion of the incubation period, media were removed and the corresponding cellular pellets subjected to RNA extraction and to Northern blot analysis as described below.

Nucleic Acid Probes

The rat IGFBP-4 [43] and -5 [44] cDNAs were generously provided in pBluescript SK+ by Dr. Shunichi Shimasaki (Department of Obstetrics and Gynecology and Reproductive Sciences, University of California, San Diego). Membranes were probed with a 444-base pair SmaI-HindIII restriction fragment of the rat IGFBP-4 cDNA and a 300-base pair SacII-HindIII restriction fragment of the rat IGFBP-5 cDNA. To generate quantitative data, gels were also exposed to a phosphor screen (Molecular Dynamics, Sunnyvale, CA). The resultant digitized data were analyzed with Image Quant Software (Molecular Dynamics).

RNA Extraction

Total cellular RNA was extracted with RNAZOL-B as recommended by the manufacturer (Tel Test, Inc., Friendswood, TX). Briefly, cells grown in 35 x 10-mm culture dishes were lysed at 4°C with 1 ml of RNAZOL-B solution before transfer to a microfuge tube. Phase separation was achieved by mixing and incubating with chloroform at 4°C for 5 min and centrifugation at 1200 x g for 15 min at 4°C. After transferring the upper phase to a second microfuge tube, RNA was precipitated by the addition of 0.6 ml isopropanol and freezing for 30 min at -70°C. The RNA pellet was recovered by centrifugation for 15 min at 12 000 x g at 4°C, washing once with 1 ml 75% ethanol, air drying, and resuspending in 10 µl diethyl pyrocarbonate-treated distilled deionized water. The integrity of the resulting RNA was assessed by visual inspection of the ethidium bromide-stained 28S and 18S rRNA bands after electrophoresis through a 1.0% agarose/2.2 mol/L formaldehyde gel.

Progesterone Assay

The coat-a-count progesterone procedure is a solid-phase radioimmunoassay (RIA) in which 125I-labeled progesterone competes with the progesterone in the conditioned medium for the antibody. Radioactivity was counted in a DPC gamma counter (Diagnostic Products Corp., Los Angeles, CA) and progesterone levels in the samples calculated from the counts by conventional techniques of RIA.

Northern Blot Analysis

Glyoxal-denatured RNA was electrophoresed and transferred to nylon membranes (Magna Graph, MSI, Westboro, MA) using 10x saline-sodium citrate (SSC). Prehybridization was carried out at 42°C for 12–24 h in a final buffer composition of 1.5x sodium chloride, sodium phosphate, EDTA (SSPE), 10x Denhardt (0.2% BSA, 0.2% polyvinylpyrrolidone, 0.2% Ficoll), 50% formamide, 1% sodium dodecyl sulfate, and 67 µg/ml denatured salmon sperm DNA. Probes were labeled with 50 µCi of [32P]deoxy-CTP, prepared using the random hexanucleotide-primed second strand synthesis method. Membranes were hybridized overnight at 42°C with 32P-labeled IGFBP-4 and -5 probes in 15 ml of hybridization buffer (same as prehybridization buffer save the addition of 10% dextran sulfate). Membranes were washed sequentially at room temperature in 5x SSPE/0.5% SDS followed by 1x SSPE/0.75% SDS and finally at 65°C in 0.1x SSPE/1% SDS. Membranes were stripped by heating to 95°C in 0.2x SSC/0.5% SDS before hybridization with another probe. To quantify the extent of hybridization, membranes were further exposed to a phosphor screen (Molecular Dynamics) and the resultant digitized data analyzed with Image Quant software (Molecular Dynamics). After the membranes were stripped, the corresponding blots were reprobed with the hamster CHOB [45] or human ß-actin cDNA to correct for possible variation in RNA loading and/or transfer. However, attempts at normalizing the data with the above "housekeeping" genes failed due to significant changes in the relative expression of CHOB and ß-actin transcripts in PMA-treated cells (data not shown). Similar effects of PMA were noted for other housekeeping genes (i.e., cyclophilin and GADPH), in keeping with previous reports [46]. Due to the inability to identify PMA-independent transcripts, strict quantitative normalization of Northern blots was not possible. Instead, accuracy of RNA loading relied on the estimation of the concentration of 18S and 28S mRNAs using UV-illuminated ethidium bromide-stained gels. Moreover, each experiment was carried out at least three times in an effort to minimize possible errors introduced by a given individual experiment.

Western Ligand Blot Analysis

Synthetic IGF-II was iodinated by a modification of the chloramine-T technique to specific activities of up to 250 µCi/µg. The iodinated peptide was then purified by gel filtration over a Sephadex G-50 column (1.0 x 120 cm) at 4°C and eluted with 100 mM Hepes buffer, pH 7.4, containing 0.5% BSA, 120 mM NaCl, 1.2 mM MgSO4, 5 mM KCl, 50 mM Na acetate, and 10 mM dextrose.

Conditioned media were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE; 10%) under nonreducing conditions. The size-fractionated proteins were then electroblotted onto nitrocellulose for 1 h. Thereafter, the filter-immobilized proteins were blocked, incubated overnight at 4°C with 1 x 106 cpm of [125I]IGF-II, washed, and visualized by autoradiography according to the method of Hossenlopp et al. [47]. Molecular weights were estimated using prestained protein standards.

Statistical Analysis

Except as noted, each experiment was replicated a minimum of three times. Data points are presented as mean ± SEM. Statistical significance (Fisher protected least significant difference) was determined by ANOVA and Student t-test. Statistical values were calculated using Statview 512+ for Macintosh (BrainPower, Inc., Calabasas, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Treatment with Phorbol 12-Myristate 13-Acetate on IGFBP-4 and IGFBP-5 Transcripts: Dose Requirements

To examine the role of the PKC pathway in the regulation of IGFBP-4 and -5 gene expression by cultured granulosa cells, use was made of PMA, a tumor-promoting phorbol ester and an established potent activator of PKC [34]. Specifically, granulosa cells were cultured for 72 h in the absence or presence of increasing concentrations of PMA (10-9–10-6 M). Cellular pellets corresponding to control and PMA-treated cells were then subjected to Northern blot analysis as described in the Materials and Methods. As shown in Figure 1, provision of PMA resulted in modest dose-dependent but (statistically) nonsignificant increments in the relative expression of IGFBP-4 transcripts, maximal stimulation (47%) being noted at the 10-7 M dose of the agonist. In contrast, treatment with PMA produced biphasic dose-dependent modulation of IGFBP-5 transcripts (Fig. 1). Specifically, treatment with low concentrations (10-9–10-8 M) of PMA resulted in dose-dependent increments in IGFBP-5 transcripts, a 3-fold increase (P < 0.05) being noted at the 10-8 M dose of the agonist. In contrast, provision of higher doses (10-7 and 10-6 M) of PMA resulted in progressive albeit (statistically) nonsignificant decrements in IGFBP-5 transcripts (relative to peak levels). Treatment with activin-A (50 ng/ml) resulted in modest but (statistically) nonsignificant decrements in the relative expression of IGFBP-4 and -5 transcripts, respectively, in keeping with a previous report [35]. These findings indicate that the modulation of ovarian IGFBP-5 (but not IGFBP-4) transcripts is PKC dependent. Accordingly, all subsequent experiments were limited to the evaluation of IGFBP-5 transcripts.



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  		FIG. 1. Effect of treatment with phorbol 12-myristate 13-acetate (PMA) on IGFBP-4 and -5 transcripts: dose requirements. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured for 72 h under serum-free conditions in the absence or presence of increasing concentrations of PMA (10-9–10-6 M) or activin-A (50 ng/ml). At the conclusion of the incubation period, cellular pellets from four dishes were pooled for a total of 2 x 106 cells per treatment and subjected to RNA extraction and Northern blot analysis using 32P-labeled probes corresponding to the rat IGFBP-4 and -5 genes as described. The intensity of the signals was quantified as described. The upper panels display, in bar graph form, the mean ± SEM of three experiments normalized relative to control (no treatment), to which an arbitrary value of one was assigned. The lower panel reflects a representative experiment

Effect of Treatment with PMA on IGFBP-5 Transcripts: Time Requirements

To assess the time requirements of the upregulatory component of the above-mentioned PMA effect on IGFBP-5 gene expression, granulosa cells were cultured for the duration indicated (up to 72 h) in the absence or presence of PMA (10-8 M). As shown in Figure 2, cultures of untreated granulosa cells displayed a sharp increase in the expression of IGFBP-5 transcripts at 24 h (as compared with the 0 time point), modest but (statistically) nonsignificant additional increments being noted at 48 and 72 h. Treatment with PMA resulted in a modest but (statistically) nonsignificant increase in IGFBP-5 transcripts (relative to untreated controls) by 48 h of culture, a significant (P < 0.02) 2.6-fold increase over the relevant control group being noted at the 72-h time point.



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  		FIG. 2. Effect of treatment with PMA on IGFBP-5 transcripts: time requirements. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured under serum-free conditions for the duration indicated (up to 72 h) in the absence or presence of PMA (10-8 M). At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described for Figure 1. The intensity of the signals was quantified as described. The upper panel displays, in bar graph form, the mean ± SEM of three experiments normalized relative to the 0 time point, to which an arbitrary value of one was assigned. The lower panel reflects a representative experiment.

Effect of Treatment with PMA on the Accumulation of the IGFBP-4 and -5 Proteins in Media Conditioned by Cultured Granulosa Cells

To determine whether the PMA effect is also manifest at the protein level, granulosa cells were cultured for the duration indicated (up to 72 h) in the absence or presence of PMA (10-8 M). Conditioned media corresponding to control and to PMA-treated cells were then subjected to Western ligand blot analysis as described in Materials and Methods. Media conditioned by untreated granulosa cells displayed progressive time-dependent increments in the signals corresponding to IGFBP-4 and -5 over the 72-h incubation period (Fig. 3). Treatment with PMA resulted in further enhancement of the accumulation of the IGFBP-4 and -5 proteins at all time points studied. These findings demonstrate the ability of PMA to stimulate the accumulation of granulosa cell-derived IGFBP-4 and -5 proteins in a time-dependent fashion in media so conditioned. Given that treatment with PMA was without significant effect on IGFBP-4 transcripts, the present observations suggest a posttranscriptional effect of PMA on this IGF-binding protein species.



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  		FIG. 3. Effect of treatment with PMA on the accumulation of the IGFBP-4 and -5 proteins in media conditioned by cultured granulosa cells. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured under serum-free conditions for the duration indicated (up to 72 h) in the absence or presence of PMA (10-8 M). At the conclusion of the incubation period, conditioned media corresponding to control and PMA-treated cells were subjected to Western ligand blot analysis as described. Molecular weights were estimated using prestained protein standards. The figure reflects a representative experiment. Duplicates are shown for each time point

PKC-Mediated Regulation of IGFBP-5 Transcripts: Specificity Studies

To further validate the PMA effect, granulosa cells were cultured for 72 h in the absence or presence of PMA (an established activator of PKC) or 4{alpha}PMA, an inert phorbol ester [48]. As expected (Fig. 4), treatment with 10-7 and 10-8 M concentrations of PMA resulted in 2.0- and 3.2-fold increases (P = 0.04) in the relative expression of IGFBP-5 transcripts as compared with untreated controls. In contrast, similar concentrations of 4{alpha}PMA were without effect on the steady-state levels of IGFBP-5 transcripts. These findings support the specificity of the PMA effect and thus its ability to modulate the expression of IGFBP-5 transcripts.



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  		FIG. 4. PKC-mediated regulation of IGFBP-5 transcripts: specificity studies. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured under serum-free conditions for 72 h in the absence or presence of 10-8 and 10-7 M concentrations of PMA or 4{alpha}PMA. At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described. The intensity of the signals was quantified as described. The upper panel displays, in bar graph form, the mean ± SEM of three experiments normalized relative to control (no treatment), to which an arbitrary value of one was assigned. The lower panel reflects a representative experiment

Effect of Treatment with Staurosporine on IGFBP-5 Transcripts: Inhibition of the PKC Pathway

To further examine the role of the PKC pathway in the regulation of IGFBP-5 gene expression, use was made of staurosporine, a microbial alkaloid and a potent inhibitor of the catalytic subunit of PKC [49]. Specifically, granulosa cells were cultured for 72 h in the absence or presence of increasing concentrations of staurosporine (10-12–10-8 M). As shown in Figure 5, treatment with low doses of staurosporine (10-12 and 10-11 M) resulted in modest but nonsignificant stimulation (18% and 11%, respectively) of the steady-state levels of IGFBP-5 transcripts. In contrast, provision of higher concentrations of the inhibitor produced progressive, dose-dependent, and significant decrements in the relative expression of IGFBP-5 transcripts (42 [P < 0.05] and 98% [P < 0.05] inhibition at 10-9 and 10-8 M concentrations, respectively). These findings further establish the intermediary role of PKC in the regulation of IGFBP-5 transcripts.



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  		FIG. 5. Effect of treatment with staurosporine on IGFBP-5 transcripts: inhibition of the PKC pathway. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured under serum-free conditions for 72 h in the absence or presence of increasing concentrations (10-12–10-8 M) of staurosporine. At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described. The intensity of the signals was quantified as described. The upper panel displays, in bar graph form, the mean ± SEM of three experiments normalized relative to control (no treatment), to which an arbitrary value of one was assigned. The lower panel reflects a representative experiment

Effect of Treatment with GnRH on IGFBP-5 Transcripts

To further evaluate the impact of PKC activation on the regulation of IGFBP-5 transcripts, use was made of GnRH, an established agonist of the PKC pathway in rat granulosa cells [50]. Specifically, granulosa cells were cultured for 72 h in the absence or presence of increasing concentrations of GnRH (10-8–10-6 M). As shown in Figure 6, the provision of GnRH resulted in dose-dependent increments in the relative expression of IGFBP-5 transcripts, a significant (P = 0.004) 2.1-fold increase over untreated controls being noted at the 10-6 M dose level. These findings provide further support for an intermediary role of PKC in the regulation of IGFBP-5 transcripts in cultured rat granulosa cells.



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  		FIG. 6. Effect of treatment with GnRH on IGFBP-5 transcripts. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured under serum-free conditions for 72 h in the absence or presence of increasing concentrations (10-8–10-6 M) of GnRH. At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described. The intensity of the signals was quantified as described. The upper panel displays, in bar graph form, the mean ± SEM of four experiments normalized relative to the control (no treatment), to which an arbitrary value of one was assigned. The lower panel reflects a representative experiment

Effect of Treatment with GnRH on the Accumulation of the IGFBP-4 and -5 Proteins in Media Conditioned by Cultured Granulosa Cells

To determine whether the GnRH effect is also manifest at the protein level, granulosa cells were cultured for the duration indicated (up to 72 h) in the absence or presence of GnRH (10-6 M). Conditioned media corresponding to control and to GnRH-treated cells were then subjected to Western ligand blot analysis. Media conditioned by untreated granulosa cells displayed progressive time-dependent increments in their IGFBP-4 and -5 content over the 72-h incubation period (Fig. 7). Treatment with GnRH resulted in additional enhancement of the accumulation of the IGFBP-4 and -5 proteins at all the points studied. These findings indicate that GnRH, like PMA, is capable of stimulating the accumulation of the IGFBP-4 and -5 proteins in media so conditioned.



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  		FIG. 7. Effect of treatment with GnRH on the accumulation of the IGFBP-4 and -5 proteins in media conditioned by cultured granulosa cells. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured under serum-free conditions for the duration indicated (up to 72 h) in the absence or presence of GnRH (10-6 M). At the conclusion of the incubation period, conditioned media corresponding to control and GnRH-treated cells were subjected to Western ligand blot analysis as described. Molecular weights were estimated using prestained protein standards. The figure reflects a representative experiment. Duplicates are shown for each time point

Effect of Treatment with PMA on IGFBP Expression: Role of FSH

To examine the potential interaction between the (presumptively PKA-mediated) FSH effect and the PKC-mediated PMA action, granulosa cells were cultured for 72 h in the absence or presence of maximally effective concentrations of FSH (100 mIU/ml), PMA (10-8 or 10-7 M), or both. As previously demonstrated (Figs. 1 and 3), treatment with 10-8 or 10-7 M concentrations of PMA resulted in significant (P < 0.05) 4.4- and 1.7-fold increases in IGFBP-5 transcripts as compared with untreated controls (Fig. 8A). In contrast, treatment with FSH was without effect on the steady-state levels of IGFBP-5 transcripts as compared with untreated controls. The application of FSH proved likewise without significant effect on the ability of PMA to upregulate IGFBP-5 transcripts.



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  		FIG. 8. Effect of treatment with PMA: role of FSH. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured for 72 h under serum-free conditions in the absence or presence of FSH (100 mIU/ml), PMA (10-8 or 10-7 M), or both. At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described. The intensity of the signals was quantified as described. Panel A represents, in bar graph form, the mean ± SEM of three experiments normalized relative to the control (no treatment), to which an arbitrary value of one was assigned. Conditioned media corresponding to control and to hormone-treated cells were subjected to Western ligand blot analysis as described. Molecular weights were estimated using prestained protein standards. Panel B reflects a figure of a representative experiment

To examine potential interactions of the PKA and PKC systems at the IGFBP protein level, conditioned media corresponding to control and to hormone-treated cells were subjected to Western ligand blot analysis. As shown in Figure 8B, treatment with FSH produced a marked inhibitory effect on the accumulation of both the IGFBP-4 and -5 proteins. Treatment with 10-8 and 10-7 M of PMA in turn resulted in the stimulation of the accumulation of the IGFBP-4 and -5 proteins as shown above. The concurrent provision of FSH markedly attenuated the ability of PMA to upregulate the accumulation of the IGFBP-5 (but not IGFBP-4) protein. These findings document the ability of FSH to modulate PMA action, thereby suggesting a relatively dominant role for PKA in the modulation of the accumulation of the IGFBP-5 (but not IGFBP-4) protein. Unlike the modulation of IGFBP-5 protein, FSH exerts a predominant up-regulatory effect on progesterone biosynthesis (P = 0.04) (Fig. 9), being markedly attenuated by PMA (87.5% and 92% inhibition at 10-8 and 10-7 M doses of PMA, respectively), the effect not reversed by the addition of FSH.



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  		FIG. 9. Effect of treatment PMA on the accumulation of progesterone: role of FSH. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured for 72 h under serum-free conditions in the absence or presence of FSH (100 mIU/ml), PMA (10-8 or 10-7 M), or both. At the conclusion of the incubation period, media conditioned by granulosa cells were collected and assayed for their immunoreactive progesterone content as described. The figure represents, in bar graph form, the mean ± SEM of progesterone content obtained from five experiments. The accumulation of progesterone is measured in ng/ml

Effect of Treatment with GnRH on IGFBP-5 Transcripts: Role of FSH

To further explore the effect of FSH on PKC-mediated GnRH hormonal action at the IGFBP-5 protein level, granulosa cells were cultured for 72 h in the absence or presence of FSH (100 IU/ml), GnRH (10-6 M), or both. As shown in Figure 10, treatment with FSH by itself resulted in a modest but nonsignificant reduction of the steady-state levels of IGFBP-5 transcripts. In contrast, treatment with GnRH resulted in a significant (>3-fold) increase in IGFBP-5 transcripts as compared with untreated controls. The addition of FSH was without significant effect on the GnRH-stimulated increase on IGFBP-5 transcripts. Conditioned media corresponding to control and to hormone-treated cells were then subjected to Western ligand blot analysis. Treatment with FSH (Fig. 11) resulted in a marked decrease in the IGFBP-4 and -5 content, as previously described [16–18]. Treatment with GnRH resulted in the previously observed (Fig. 7) stimulation of the IGFBP-4 and -5 content (Fig. 11). The concurrent provision of FSH resulted in modest attenuation of the ability of GnRH to upregulate the IGFBP-5 (but not IGFBP-4) content. These findings further document the limited ability of FSH to modulate GnRH hormonal action and suggest a relatively dominant role for PKC in the modulation of granulosa cell-derived IGFBP-5 transcripts.



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  		FIG. 10. Effect of treatment with GnRH on IGFBP-5 transcripts: role of FSH. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured for 72 h under serum-free conditions in the absence or presence of FSH (100 mIU/ml), GnRH (10-6 M), or both. At the conclusion of the incubation period, cellular pellets were subjected to RNA extraction and Northern blot analysis as described. The intensity of the signals was quantified as described. The upper panel represents, in bar graph form, the mean ± SEM of three experiments normalized relative to the control (no treatment), to which an arbitrary value of one was assigned. The lower panel reflects a representative experiment



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  		FIG. 11. Effect of treatment with GnRH on the accumulation of the IGFPB-4 and -5 proteins in media conditioned by cultured granulosa cells: role of FSH. Granulosa cell cultures (5 x 105 viable cells/dish), obtained and maintained as described, were cultured for 72 h under serum-free conditions in the absence or presence of FSH (100 mIU/ml), GnRH (10-6 M), or both. At the conclusion of the incubation period, conditioned media corresponding to control and to hormone-treated cells were subjected to Western ligand blot analysis as described. Molecular weights were estimated using prestained protein standards. The figure reflects a representative experiment on the accumulation of granulosa-cell derived IGFBPs in cultures without treatment (C, Control) and under the effect of FSH, GnRH, or both


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In studying the role of PKC in the regulation of the steady-state levels of IGFBP-4 and IGFBP-5 transcripts, note was made of the ability of PMA to exert a biphasic dose-dependent modulation of IGFBP-5 (but not IGFBP-4) transcripts (Fig. 1). On balance, a stimulatory effect was noted for the 10-9 and 10-8 M dose levels, decrements induced by higher doses (10-7 and 10-6 M) proving (statistically) nonsignificant. The PMA effect proved time dependent, a 2.6-fold increase over control being noted at the 72-h time point (Fig. 2). The specificity of the PMA effect was confirmed by demonstrating the inability of 4{alpha}PMA, an inert form [48], to alter the steady-state levels of IGFBP-5 transcripts (Fig. 4). Moreover, the addition of staurosporine, a potent inhibitor of the catalytic subunit of PKC [49], produced dose-dependent and significant decrements in the relative expression of IGFBP-5 transcripts. Taken together, these observations establish the intermediary role of PKC in the regulation of IGFBP-5 transcripts.

The phenomenon in question appears to be dose and time dependent as well as specific. As regards the specificity of PMA as a PKC activator, however, note must be made of the fact that non-PKC proteins can also bind this pharmacologic substance [51]. Thus, one must exercise caution when interpreting the current results. In the absence of additional information, the current findings cannot distinguish between an apparent stimulation of IGFBP-5 transcription as distinct from stabilization of the corresponding transcript.

Our present findings also reveal the ability of PKC signaling to exert nonselective regulatory effects at the IGFBP protein level (as distinct from transcript). Specifically, treatment with PMA resulted in enhancement of the accumulation of the IGFBP-4 and -5 proteins at all time points studied in media conditioned by cultured granulosa cells (Fig. 3). Consequently, the selectivity observed for PMA at the level of IGFBP transcripts could not be duplicated at the level of the corresponding proteins. In this respect, our findings disclose that the activation of PKC signaling results in consistent upregulation of granulosa cell-derived IGFBP-5 transcripts and proteins. The apparent upregulation of the IGFBP-5 protein could reflect enhanced transcriptional efficiency, stabilization of the corresponding transcript, or posttranslational intervention. In this context, it is tempting to speculate that activation of the PKC pathway inhibits the activity of the IGFBP-5-directed endopeptidase, the role of which in regulating IGFBP content has been amply documented [13, 52–54]. Additional studies would be required to distinguish between the above possibilities.

Our present findings also stand out in terms of the selective role played by PKC in the regulation of IGFBP-4 transcripts and proteins. On the one hand, activation of PKC signaling proved without significant effect on the steady-state levels of IGFBP-4 transcripts (Fig. 1). On the other hand, treatment with PMA produced time-dependent increments in the signal corresponding to the IGFBP-4 protein (Fig. 3). One must conclude therefore, by inference, that the upregulatory effect of PMA on the IGFBP-4 protein content reflects a posttranscriptional and, by all likelihood, a posttranslational phenomenon. Once again, it is tempting to speculate that activation of the PKC pathway results in the inhibition of the previously reported IGFBP-4-directed endopeptidase, the role of which in determining the intraovarian IGFBP-4 content has been well documented [13]. Additional studies would be required to determine the precise posttranslational effects of PMA on the granulosa cell-derived IGFBP-4 protein.

To further buttress the above observations, use was made of yet another agonist of the PKC pathway, i.e., GnRH. Indeed, a growing body of information establishes GnRH as an agonist of the PKC pathway in the rat granulosa cell [50]. Importantly, our present observations as they relate to GnRH are identical to those elicited with PMA. Specifically, the provision of GnRH resulted in dose-dependent increments in the relative expression of IGFBP-5 transcripts (Fig. 6). Moreover, GnRH, like PMA, proved capable of stimulating the accumulation of the IGFBP-4 and -5 proteins in conditioned media (Fig. 7). As such, these observations reaffirm the ones obtained with PMA and further establish the PKC pathway as an intermediary in the regulation of granulosa cell-derived IGFBPs.

The present observations shed additional light on the complementary yet antagonistic interactions between the intraovarian PKA and PKC systems. Whereas previous studies have clearly established that activation of the PKA pathway will result in the reduction of the intraovarian content of the IGFBP-4 and IGFBP-5 proteins [31–33], interactions between the PKC and PKA pathways remain largely untested. Our current observations reveal limited regulatory interactions between the PKA and PKC pathways, the endpoint being the steady-state levels of IGFBP-5 transcripts (Fig. 8A). In contrast, strong regulatory interactions were noted at the IGFBP-4 and IGFBP-5 protein levels (Fig. 8B). The concurrent provision of FSH, an established PKA activator [55], markedly attenuated the ability of PMA to upregulate the accumulation of the IGFBP-5 (but not IGFBP-4) protein. These findings document the ability of FSH to modulate PMA action, thereby suggesting a relatively dominant role for PKA in the modulation of the accumulation of the IGFBP-5 (but IGFBP-4) protein. Similar observations were made for GnRH (Figs. 10 and 11). As such, these findings further document the limited ability of FSH to modulate GnRH hormonal action and suggest a relatively dominant role for PKC in the modulation of granulosa cell-derived IGFBP-5 transcripts. In this respect, the current observations are confirmatory of those of Onoda et al. [56], wherein GnRH has been shown to overcome the ability of FSH to inhibit IGFBP-5 synthesis and the promotion of its degradation.


   ACKNOWLEDGMENTS
 
The authors wish to thank Ms. Cornelia T. Szmajda, Ms. Linda Elder, and Ms. Michelle S. Lewandowski for their valuable assistance in the preparation and completion of this manuscript.


   FOOTNOTES
 
First decision: 28 November 2001.

1 This research was supported in part by NIH Research grant HD 30288 (E.Y.A.), CAPES/Brazil BEX 1007/99-8 (A.B.T.), and CNPq/Brazil 870.313/97-5 (A.B.T.). Back

2 Correspondence: Eli Y. Adashi, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Utah Health Sciences Center, Huntsman Cancer Institute, 2000 Circle of Hope, Room 5221, Salt Lake City, UT 84112. FAX: 801 585 9256; eadashi{at}hsc.utah.edu Back

Accepted: April 23, 2002.

Received: November 7, 2001.


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