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Endocrinology Vol. 138, No. 2 574-579
Copyright © 1997 by The Endocrine Society

Articles

Cryptic Gonadotropin-Releasing Hormone Receptors of Rat Pituitary Cells in Culture Are Unmasked by Epidermal Growth Factor

P. Leblanc, A. L’Héritier and C. Kordon

Unité de Dynamique des Systèmes Neuroendocriniens, INSERM U-159, Centre Paul Broca, Paris, France

Address all correspondence and requests for reprints to: Dr. Claude Kordon, INSERM U-159, Unité de Dynamique des Systèmes Neuroendocriniens, Centre Paul Broca, 2ter rue d’Alésia, Paris F-75014, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Protein kinase activators as well as several neuropeptides are able to increase the GnRH-binding capacity of cultured adenohypophyseal cells. To determine whether such up-regulation of GnRH-binding sites can be achieved by a substance(s) endogenous to the pituitary, binding experiments were performed after exposure of cells to increasing amounts of medium conditioned by incubation with primary cultures of adenohypophyseal cells for 4 days. Addition of the conditioned medium elicited a 50% increase in GnRH binding. Characterization of the agent(s) responsible for the effect was attempted by submitting the conditioned medium to molecular sieve filtration, adding or immunoprecipitating endogenous substances, and comparing the susceptibilities of the responses to various inhibitors of transduction processes. Fractionation of the medium indicated that active molecules were of a proteic nature, with Mr ranging from 5,000–10,000. Among major endogenous moieties corresponding to these criteria [epidermal growth factor (EGF), transforming growth factor-{alpha}, and insulin-like growth factors I and II), only the first two exhibited properties similar to those of the conditioned medium. EGF stimulated binding with an EC50 of 3.6 ± 0.8 pM. Immunoprecipitation of EGF, but not transforming growth factor-{alpha}, inactivated the conditioned medium. The effects of both conditioned medium and EGF were inhibited by herbimycin, a tyrosine kinase inhibitor; U73122, a phospholipase C inhibitor; and prior desensitization of protein kinase C. In contrast, both were insensitive to pertussis toxin pretreatment. In parallel, EGF did not increase LH secretion by itself, but potentiated its response to GnRH in a concentration range of 1 pM to 1 nM, resulting in a shift of the curve toward lower values of GnRH. It is concluded that EGF is able to control the accessibility of binding sites to GnRH and to potentiate the responsiveness of gonadotropes to the decapeptide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN PITUITARY cells sampled from male adult rats and maintained in primary culture, high affinity binding of GnRH is increased at low temperatures (1); the number of binding sites is greater when binding is performed at 4 C than at 21 C. The additional population of binding sites, called cryptic receptors, can be unmasked at 21 C by activators of protein kinase C (2, 3), but other protein kinases (3) are also involved. In addition, cryptic receptors can also be up-regulated by peptides endogenous to the pituitary; neuropeptide Y (NPY), for instance, a neuropeptide of mixed hypothalamic and pituitary origin (4, 5), exhibits the same property as activators of protein kinase C (PKC) on this population of receptors (6).

In the present work, we investigated whether paracrine factors endogenous to the pituitary could play a role in up-regulating GnRH-binding sites. We thus tested the capacity of medium conditioned by long term incubation with pituitary cells to affect GnRH binding in a freshly washed population of adenohypophyseal cells and attempted to identify endogenous factors involved in the effect by combining molecular sieve filtration and immunological methods. Epidermal growth factor (EGF) could be identified as the endogenous substance accounting for the effect of the conditioned medium (CM).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male rats (Charles River Breeding Laboratories, St. Aubin les Elbeuf, France) were kept under controlled temperature and light conditions (21 ± 1 C; 14 h of light, 10 h of darkness; lights on, 0500–1900 h). Food and water were supplied ad libitum.

Cell preparation and culture
Anterior pituitary glands were removed and dispersed as previously described (1). Briefly, anterior lobes were cut into small cubes and successively incubated after initial enzymatic digestion with trypsin (Sigma Chemical Co., St. Louis, MO; 5 mg/ml for 20 min) and deoxyribonuclease (Sigma; 2 mg/ml for 2 min), with a trypsin inhibitor (Sigma; 1 mg/ml for 6 min), and finally in a Ca2+/Mg2+-free medium supplemented with 0.6 mg/ml EDTA (pH 7.4 for 16 min). Fragments were mechanically dispersed with a siliconed Pasteur pipette for 30 min, and cells were plated in 35-mm diameter plastic culture wells (Nunc, Roskilde, Denmark). The volume was completed at a 3-ml final concentration with DMEM (Boehringer Mannheim, Mannheim, Germany) containing phenol red (15 mg/ml), 1% glutamine (0.29 mg/ml), antibiotics (penicillin G sulfate; 83 U/ml), streptomycin sulfate (37 U/ml; Sigma), and 8% FCS treated with 1% Norit-A charcoal (Sigma) and 0.1% Dextran T-70 (Pharmacia, Uppsala, Sweden) for 12 h at 4 C to remove steroids. Cell cultures were maintained in a humidified atmosphere containing 7% CO2 at 37 C for 4 days.

Ligand iodination
The GnRH agonist des-Gly10-(D-Ala6)-LHRH ethylamide (GnRHa; Peninsula Laboratories, Belmont, CA) was used as ligand. The compound was labeled with [125I]MS-30 (Amersham) by chloramine-T (7). Specific activity ranged from 1100–1500 Ci/mmol, and binding to pituitary membranes reached 40%.

Binding assay
Binding was performed on intact attached cells (1). Culture wells were washed three times with 1 ml Krebs-Ringer medium (KRM) containing NaCl (120 mM), KCl (3 mM), CaCl2 (2.6 mM), MgCl2 (0.67 mM), KH2PO4 (1.2 mM), glucose (1.2 mM), and HEPES (25 mM), pH 7.4, at 21 C to remove unattached cells. Cells were subsequently incubated for 25 min at 21 C in the presence of final radioligand concentrations ranging from 0.3–9 nM. The final volume was 650 µl unless otherwise indicated in the figure legends. Each experimental point was run in triplicate. Nonspecific binding was assessed in the presence of 0.3 µM unlabeled GnRHa. After incubation, the incubation medium was discarded, and the wells were washed three times with 1 ml cold KRM supplemented with 0.3% BSA and twice with cold KRM alone. One milliliter of 0.5 N NaOH with 0.1% SDS was then added. After 1-h digestion at room temperature, the solution was transferred to 3-ml plastic tubes for determination of radioactivity by {gamma}-spectroscopy (Rack gamma 1270, LKB, Uppsala, Sweden). After determination of binding, the digested cell solution was assayed for protein content.

PKC desensitization
Cells were preincubated with phorbol 12-myristate 13-acetate (1 µM; Sigma) in 1% (wt/vol) BSA-DMEM for 24 h before the GnRHa binding test, as described by Strulovici et al. (8).

Drugs and inhibitors
U73122, a phospholipase C (PLC) inhibitor (2 mM final dilution; Biomolecular Research, Plymouth Meeting, PA) was added without preincubation. Herbimycin A, a tyrosine kinase inhibitor (0.1 mM final dilution; Sigma), was preincubated with the cells for 24 h before experimentation (9). Pertussis toxin (Sigma; 15 ng/ml) was also added to the culture medium 24 h before the experiment.

Fractionation procedure
Separation was performed by ultrafiltration with Centri/Por centrifuge concentrators (Spectrum, Medical Industries) with Mr cut-offs of 30,000, 10,000, and 5,000. After centrifugation of CM at 50,000 x g for 20 min to sediment cellular fragments, the supernatant was successively filtered through the concentrators. Fractions retained the filter containing moieties with Mr higher than either 30,000 or 10,000 were recovered, adjusted to the initial volume with DMEM, and filtered again. The process was repeated three times for eliminating molecules of lower Mr. The whole procedure yielded four CM fractions: CM >30,000, 30,000 < CM > 10,000, CM <10,000, and CM <5000.

Proteolysis of CM <10,000
CM <10,000 fractions were incubated for 60 min at 30 C in the presence or absence of thermolysine type X (200 mg/ml; Sigma) and pronase (200 mg/ml; Boehringer) and subsequently filtered across a 10,000 Mr cut-off centriport to eliminate proteolytic enzymes. Unconditioned medium (UCM) incubated with proteolytic enzymes and filtered was used as the control.

Immunoprecipitation
Fractions derived from CM and UCM were treated in the same way. The CM <30,000 Mr fraction was divided into four aliquots and incubated either with or without specific antibodies: rabbit antimouse EGF IgG (Becton Dickinson, Mountain View, CA; 2.4 µl/ml corresponding to a 417-fold dilution), mouse antihuman transforming growth factor-{alpha} (TGF{alpha}) Mab IgG1 (Santa Cruz Biotechnology, Santa Cruz, CA; 18 µl/ml corresponding to a 56-fold dilution), or both for 90 min at 21 C. The UCM <30,000 Mr fraction was incubated with both antibodies as a control. After incubation, fractions were filtered though <10,000 Mr Centri/Por concentrators to eliminate antibodies. The capacity of both antibodies to cross-react with rat EGF and TGF{alpha} was tested. At a 1:5,000 dilution, rat [125I]EGF bound to antimouse EGF IgG was displaced by rat EGF with an IC50 of 0.6 nM. No cross-reaction was observed with rat TGF{alpha} up to 100 nM. Rat TGF{alpha}, which only differs from human TGF{alpha} by four amino acid residues, was recognized by mouse antihuman TGF{alpha} on a Western blot. It did not cross-react with rat EGF.

LH release
Half a million cells per well were incubated at 37 C for 2 h in KRM (1 ml) with or without GnRHa and/or EGF. Each point was assessed on seven wells. Incubates were sampled, centrifuged at 1500 x g for 10 min, assayed for LH by double antibody RIA according to the method of Niswender et al. (10), and expressed as milligrams of rat LH RP-1 standard per ml.

Statistics
Dissociation constants at equilibrium (Kd) and the maximal number of binding sites (Bmax) were calculated from specific binding curves by nonlinear regression using a one-site cooperative model (11). Specific binding was represented on a Scatchard plot (12). For determination of EC50 values, all points were analyzed by the computerized iterative least square method.

LH responses to GnRH and EGF were calculated as a percentage of the maximal response amplitude (Vmax) obtained for saturating concentrations of GnRH within each experiment and submitted to parametric ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of CM on GnRH binding
Addition of increasing amounts of medium conditioned by a 4-day preincubation with dispersed pituitary cells induced a marked augmentation of GnRH binding (Fig. 1Go). The effect was dose dependent. Binding constants calculated from saturation curves obtained at 21 C indicated that the Bmax increased by 54 ± 5% in the presence of CM. EC50 values were obtained for a volume of 3.2 ± 0.9 ml CM corresponding to a 200-fold final dilution in the binding medium. Binding Kd was also increased (5.1 ± 0.6 vs. 2.9 ± 0.3 nM; Fig. 1Go).



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  		Figure 1. Scatchard plot of the effects of CM on [125I]GnRHa binding to pituitary cells from male rats (5 x 105 cells/well; incubation time, 25 min at 21 C). {square}, Control incubation with 40 µl UCM at a final volume of 690 µl. Bmax = 14.6 ± 0.8 fmol/mg proteins, Kd = 2.9 ± 0.3 nM. •, Incubation with 40 ml CM. Bmax = 23.9 ± 1.9 fmol/mg proteins; Kd = 5.1 ± 0.6 nM. Bmax, Kd, and bound/free ratio (B/F) values were calculated from saturation isotherm curves. Specific binding was about 30% of total binding. Inset, Effect of increasing concentrations of CM on Bmax values. Each point represents a Bmax estimation based on at least three independent saturation isotherm curves. Each CM volume was adjusted to 60 µl with UCM in a final incubation volume of 710 µl.

 
Molecular sieve filtration of the CM
Spinning the CM on calibrated filters yielded four fractions of Mr ranges between more than 30,000 and less than 5,000. Using the same end point as in the previous experiment (increased Bmax of GnRHa binding), the activity present in the unfractionated medium was recovered only from 5,000–10,000 Mr fractions (Fig. 2aGo). Binding up-regulation induced by these fractions was eliminated upon incubation with proteases (Fig. 2bGo).



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  		Figure 2. Distribution of the GnRH binding up-regulating activity in fractionated CM and effects of pronase and thermolysine. a, The activity was recovered from CM fractions obtained by ultrafiltration on centrifuge concentrators. Cells were incubated for 25 min at 21 C at a density of 5 x 105 cells/well. Twenty-microliter aliquots of each fraction or of UCM were added to a final volume of 670 µl. b, Effects of proteolytic enzymes. CM <10,000 Mr fractions were incubated for 60 min at 30 C with or without proteases and filtered. UCM was incubated with proteases and filtered. Twenty microliters of each fraction per well were used in a final volume of 670 µl. The number of independent saturation isotherm curves is shown in parentheses. Differences are analyzed with respect to controls (a) and to the fraction incubated without proteases (b). By Student’s t test: *, P <= 0.05; **, P <= 0.01.

 
Effect of growth factors on GnRH binding
In the next experiment, four substances [EGF, TGF{alpha}, insulin-like growth factor I (IGF-I), and IGF-II] were selected on the basis of the following criteria: of a proteic nature, corresponding to a Mr in the range of 5,000–10,000, and endogenously produced by adenohypophyseal cells (13, 14, 15, 16, 17, 18). Two substances corresponding to all criteria, EGF and TGF{alpha}, were able to increase the number of GnRHa-binding sites, whereas IGF-I and IGF-II were ineffective (Fig. 3Go, a and b). The action of EGF was concentration dependent, with an EC50 of 3.6 ± 0.8 pM; the maximal effect was comparable to that obtained with the CM itself (52.3 ± 2.3%; Fig. 3cGo).



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  		Figure 3. Effects of EGF, TGF{alpha}, IGF-I, or IGF-II on GnRHa binding. Cells were incubated at a density of 5 x 105 cells/well in the presence of the test substances (0.1 nM) for 25 min at 21 C without prior preincubation. a and b, Bmax calculated from three independent isotherm curves. c, Dose dependency of [125I]GnRHa binding at increasing EGF concentrations. Each point represents the mean of three independent Bmax determinations.

 
Immunoprecipitation of EGF and TGF{alpha}
To determine whether EGF or TGF{alpha} was able to account for the effect of the CM, we attempted to eliminate them from the medium by immunoprecipitation. Immunoprecipitation of EGF and TGF{alpha} in the CM with a mixture of antibodies directed against both growth factors abolished the activity of the filtrate (Fig. 4Go). Immunoprecipitation of EGF alone was almost as effective, whereas immunoprecipitation of TGF{alpha} did not prevent the increase in GnRH binding.



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  		Figure 4. Effect of immunoprecipitation of CM <10,000 Mr fractions with anti-EGF or anti-TGF{alpha} on [125I]GnRHa binding. Cells were incubated at a density of 5 x 105 cells/well for 25 min at 21 C with 20 µl UCM or CM and treated or not with antiserum for 90 min at 21 C. The [125I]GnRHa concentration was 6 nM. Increments represent difference between specific binding observed with CM and UCM. Values shown are the mean of three independent experiments, with seven measures per test. Immunoprecipitation with 10-fold lower concentrations of antibodies yielded identical results.

 
Effect of tyrosine kinase and PLC inhibitors or of PKC desensitization and pertussis toxin pretreatment
To determine whether similar mechanisms are involved in the effects of CM and EGF, GnRHa binding was measured in the presence of various transduction inhibitors (Table 1Go). Herbimycin, a tyrosine kinase inhibitor (19), as well as U73122, a PLC inhibitor (20), prevented unmasking of cryptic GnRH receptors by both CM and EGF. Desensitization of PKC by pretreatment of the cells with large concentrations of phorbol ester also abolished the effects of both CM and EGF, whereas preincubation with pertussis toxin was ineffective. In contrast, increased binding induced by NPY (6) was not affected by herbimycin or U73122.


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  		Table 1. Effect of tyrosine kinase or phospholipase C inhibitors, of PKC desensitization, or pertussis toxin on the capacity of CM, EGF, and NPY to up-regulate GnRHa binding

 
Effect of EGF on GnRHa-induced LH release
Figure 5aGo presents a dose-response curve of LH stimulation by GnRHa or EGF. The EC50 of the GnRHa response was 85 ± 20 pM. EGF (1 pM to 1 nM) had a slight, but insignificant, effect on LH release (Fig. 5aGo). In contrast, at a 100-pM concentration, EGF potentiated the GnRHa-stimulated LH response without modifying its maximal amplitude by inducing a 2-fold, significant reduction in the EC50, as shown in Fig. 5bGo.



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  		Figure 5. Potentiation by EGF of the LH response to GnRHa. Cells were incubated at a density of 5 x 105 cells/well for 2 h at 37 C in a final volume of 1 ml. a, LH secretion (•) stimulated by GnRHa, average of four independent experiments. Data are expressed as the maximum increase ({Delta}; stimulated - basal) in secretion in micrograms per ml. EC50 = 85 ± 21 pM; maximal stimulation = 21.3 ± 1.2 µg; basal secretion = 4.9 ± 0.3 µg/ml LH/well. {circ}, Effect of EGF. Maximal stimulation = 0.9 ± 0.4 mg LH/well for a concentration of 10 pM EGF. The response to EGF is not significant. b, Dose-response curve of LH secretion in the presence or absence of EGF. A pool of three to five independent experiments is presented. Average LH basal secretion = 6.2 ± 0.6 µg/ml; maximal secretion in the presence of 1 nM GnRHa = 15.8 ± 0.9 in the control group and 16.8 ± 1.1 µg/ml in the presence of EGF (0.1 nM). For statistical assessment, responses were calculated as a percentage of the maximal response of control cells (Bmax - basal values) for each experiment. Parametric variance analysis indicated a highly significant difference (P < 0.001; F = 46.5 for 7 and 111 degrees of freedom) between cells exposed or unexposed to EGF for GnRHa concentrations in the range of parallelism of the curves (10–300 pM).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data suggest that EGF and TGF{alpha} are the most likely candidates to account for the GnRH binding-stimulating activity recovered from CM of pituitary cells. Both factors are able to mimic the effect of CM on GnRH binding; their Mr (between 5,000–10,000) is compatible with that of active fractions obtained after sieve filtration of the medium. Their equipotency on GnRH binding up-regulation is not surprising because they are known to share the same receptor (21, 22).

Both growth factors are endogenous to the adenohypophysis. The presence of EGF has been documented in somatotropes, gonadotropes, thyrotropes, and corticotropes (13, 14), whereas TGF{alpha} seems mostly present in somatotropes and lactotropes (15). The growth factors may not be actually produced by all these cell types, as EGF mRNA has only been detected in somatotropes and gonadotropes, and TGF{alpha} mRNA has only been found in somatotropes, gonadotropes, and lactotropes (23). Under our experimental conditions, however, only EGF is present in amounts compatible with the action of CM on GnRH binding, as immunoprecipitation of both factors or of EGF alone, but not of TGF{alpha} alone, was able to block it.

We also tested two other factors endogenous to the pituitary, IGF-I and IGF-II (16, 17, 18), the Mr of which correspond to that of the active fraction of the CM. Neither was able to affect GnRH binding, despite the fact that the IGF-I (but not the IGF-II) receptor also exhibits intrinsic tyrosine kinase activity (24).

Identification of EGF with the factor responsible for up-regulating GnRH binding in the CM is further substantiated by the finding that both treatments involve a comparable mode of action. The activity of CM as well as that of EGF is blocked by herbimycin, an observation consistent with the presence of a tyrosine kinase domain in the EGF/TGF{alpha} receptor (25, 26, 27). In addition, the intrinsic tyrosine kinase activity of the growth factors is positively coupled to PLC{gamma} (28, 29, 30, 31); abolition of the response to EGF and the medium by U73122, an inhibitor of PLC (20), thus provides an additional argument that EGF accounts for the effect of the medium. Finally, activation of PLC{gamma} and subsequent release of diacylglycerol result in PKC stimulation (32, 33). This can explain why treatment with CM or EGF failed to unmask GnRH receptors when PKC was down-regulated by prior exposure of the cells to large concentrations of phorbol esters; under these conditions, the effects of both EGF and CM were blocked to a comparable extent as after treatment with a PLC inhibitor.

NPY, another factor endogenous to the pituitary (34), has been shown to be able to unmask cryptic GnRH-binding sites (6). When added to adenohypophyseal cells in culture, NPY also results in binding up-regulation. The effect of NPY, however, is mediated by a different mechanism; in contrast to our observation concerning CM and EGF, its action persists after PKC desensitization, but disappears after pretreatment with pertussis toxin, an agent that ADP-ribosylates and subsequently inactivates G{alpha}i and G{alpha}o proteins. We also report here that binding stimulation by NPY is insensitive to herbimycin and U73122, in contrast to the effect observed under the influence of EGF or CM. This also rules out participation of NPY in binding up-regulation after exposure to the CM.

Taken together, these results identify EGF as the active endogenous factor present in the CM and able to up-regulate GnRH-binding sites. This does not necessarily preclude a possible additional effect of TGF{alpha} under different physiological or experimental conditions, but suggests that effective concentrations of TGF{alpha} were not achieved under our incubation conditions.

As previously observed (1, 3, 6), other agents unmasking GnRH receptors also induce a parallel increase in Kd and Bmax. The effect on the Kd cannot be accounted for by distinct affinities of naive and cryptic receptors, as binding isotherms best fit a single binding site without cooperativity. Although we have no interpretation for the change in Kd, increased Bmax can be considered more relevant than increased Kd from a functional point of view, because it is accompanied by a greater sensitivity of the GnRH response; the contrary should have been expected if the response was determined by changes in Kd.

By itself, EGF was not able to induce a significant stimulation of LH release. This observation contrasts with that by Przylipiak et al. (35), who reported stimulation by EGF of basal LH release from dispersed pituitary cells. In the latter study, however, the concentration of EGF used (10 nM) was significantly higher than that in our study (100 pM).

In contrast, the gonadotropin response to GnRHa is potentiated by EGF. Addition of EGF does not affect the maximal amplitude of the LH response, so it does not further stimulate LH release induced by maximally active concentrations of GnRH, but at lower than submaximal doses of GnRHa, the growth factor shifts the response toward higher sensitivities to GnRHa, an effect that results in a 2-fold decrease in the EC50.

These data point to an independent, potentiating, but not additive role of EGF on GnRHa-induced LH release. Under different experimental conditions, Mikaye et al. (36) also reported potentiation by low concentrations of EGF (100 pM) of estradiol-induced LH release from pituitary fragments, suggesting that the actions of EGF on gonadotropes may depend on the hormonal status of the pituitary. In this respect, it is of interest to note that cryptic receptors are spontaneously unmasked in pituitaries sampled from castrated male rats (1, 3).

In conclusion, endogenous EGF can be assumed to regulate GnRH receptor numbers and, subsequently, the responsiveness of gonadotropes to submaximal decapeptide concentrations, suggesting that the cryptic subpopulation of GnRH receptors described in the pituitary can be functionally relevant. Paracrine and possibly also autocrine processes intrinsic to the pituitary may thus participate in the adaptation of gonadotropes to their predominantly episodic mode of stimulation (37).

Received July 15, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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