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Endocrinology Vol. 138, No. 9 3587-3593
Copyright © 1997 by The Endocrine Society

ARTICLES

Regulation of the Adenohypophyseal Thyrotropin-Releasing Hormone-Degrading Ectoenzyme by Estradiol1

Lutz Schomburg and Karl Bauer

Max-Planck-Institut für experimentelle Endokrinologie, 30603 Hannover, Germany

Address all correspondence and requests for reprints to: Dr. Karl Bauer, Max-Planck-Institut für experimentelle Endokrinologie, POB 610309, 30603 Hannover, Germany. E-mail: 106001,2503{at}compuserve.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TRH is inactivated by the TRH-degrading ectoenzyme, a TRH-specific metallopeptidase. At the pituitary level, this enzyme is stringently regulated by thyroid hormones. We describe here gender-related differences and the effect of estradiol (E2) on the expression of this enzyme in the anterior pituitary.

Compared with male rats, only about one third of the enzymatic activities and the messenger RNA levels were found in the anterior pituitary of female rats, whereas the TRH receptor transcript levels were found inversely related. When male rats received a single injection of 0.5 µg E2/100 g BW, the enzymatic activity decreased to 65% of control values within 14 h, preceded by a decrease of the transcript levels to 25% of control within 6 h. Basal values were reached again 24–48 h after the injection. E2 had no effect on the expression of the enzyme in the brain. In vivo and with GH3 cells in vitro, E2 effectively counteracted the increase in enzymatic activity induced by T3, whereas neither testosterone nor progesterone, aldosterone, or dexamethasone showed any significant effects.

Because the expression of the adenohypophyseal TRH-degrading ectoenzyme is tightly regulated by both T3 and E2 with adequate dynamics, we conclude that this peptidase serves integrative functions for the control of TRH-stimulated hormone secretion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRH (pyroGlu-His-Pro-NH2) plays a central role in the regulation of TSH, PRL, and at times, GH secretion (for review, see Refs. 1–3). The association of TRH with TRH receptors on hypophyseal target cells leads to the activation of phospholipase C and the inositol lipid-signaling pathway. TRH receptor expression is tightly regulated by a variety of extracellular signals, including peripheral hormones such as thyroid hormones, glucocorticoids, and estrogens (for review, see Refs. 4–6). Thyroid hormones are known to decrease the density of pituitary TRH receptors in vivo and in pituitary cells in culture (7, 8, 9). In parallel, the TRH response is diminished. Estrogens, by contrast, increase TRH receptor levels in vitro and in vivo, which may account for the heightened sensitivity of pituitary cells to TRH in the presence of elevated estrogens (7, 10). Thus, these hormones not only affect the synthesis and secretion of pituitary hormones but also influence the sensitivity of adenohypophyseal target cells to the hypothalamic neuropeptide TRH.

The response of TRH-target cells conceivably might be modulated also by the rate of degradation of the tripeptideamide at specific target sites. This mechanism could effectively control the intensity of stimulation and/or the duration of action of the peptidergic-releasing factor. The biochemical data strongly suggest that the hypothalamic neuropeptide TRH is inactivated by a peptidase (for review, see Refs. 11 and 12) that is preferentially localized on the surface of lactotropic cells (13). This ectoenzyme exhibits a high degree of substrate specificity, as does the TRH-degrading serum enzyme (14).

Further studies also demonstrated that the activity of the adenohypophyseal TRH-degrading ectoenzyme is stringently regulated by thyroid hormones (15, 16, 17) and seems to be influenced by estrogens, because, with ovariectomized rats, an increase in the activity of the adenohypophyseal enzyme was noticed, which decreased again when these animals were substituted with estradiol (E2) benzoate (18). Furthermore, in rat pituitaries, fluctuations of the enzymatic activity were observed during the estrous cycle. However, these fluctuations were not in phase with the E2 levels, and the activity of the adenohypophyseal TRH-degrading ectoenzyme was not modified during lactation (19). We demonstrated previously that the effects of thyroid hormones are exerted mainly at the pretranslational level (20). Because estrogens are well known to act not only at the transcriptional level, but also to regulate a variety of metabolic functions, we were interested in studying the effects of E2 on the expression of this enzyme. In addition, because the numbers of lactotrophs and somatotrophs in the pituitary are influenced by sex steroids, we also determined the activity and the messenger RNA (mRNA) levels of this enzyme in male and female rats. For comparison, the mRNA levels of the TRH receptor also were determined in some experiments.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Dexamethasone, progesterone, testosterone, and T3 were purchased from Sigma-Aldrich Chemie (Deisenhofen, Germany); 17ß-E2 and all other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany). [3H]glutamine (43.9 Ci/mmol) was purchased from New England Nuclear (Dreieich, Germany), Ready Flow III scintillation cocktail from Beckman Instruments (Munich, Germany) and {alpha}-32P 2-deoxycytidine 5'-triphosphate from Hartmann Analytic (Braunschweig, Germany). The random-primed DNA labeling kit was purchased from Stratagene GmbH (Heidelberg, Germany). HB 101 medium was obtained from Irvine Scientific, Santa Ana, CA, through Laboserv (Giessen, Germany).

Determination of the activity of the TRH-degrading ectoenzyme
[pyroGlu-3H]TRH was synthesized, and the enzyme assay was performed, as described previously (13). Briefly, aliquots (10–70 µl) of washed membrane preparations were incubated at 30 C in a final reaction mixture of 100 µl containing 2 µM [pyroGlu-3H]TRH (10 Ci/mmol) and the inhibitors of the cytosolic TRH-degrading enzymes [2 µM pGluCHN2 (21) and 4 µM Cbz-Gly-Pro-CHN2 (22)]. The initial rate of TRH-degradation as a measure of enzyme activity was determined by a four-point kinetic test. Protein was determined by a modification of the Lowry method (23), using BSA as standard.

In vivo studies
Adult male and female Sprague-Dawley rats (4–6 months old) were used. The animals, maintained according to the guidelines of the Animal Welfare Committee of the Medizinische Hochschule Hannover, had access to water and standard laboratory chow ad libitum. An ambient temperature of 22 C and alternating 12-h light, 12-h dark cycles were controlled automatically.

The animals received sc injections of dexamethasone (30 µg/100 g BW), progesterone (0.6 mg/100 g BW), testosterone (70 µg/100 g BW), T3 (5 µg/100 g BW), E2 (0.5 µg/100 g BW), or vehicle (sesame oil) and were killed after the time periods indicated. Immediately after decapitation, the tissues of interest were removed and frozen in liquid nitrogen.

In vitro studies
GH3 cells were propagated in Ham’s F-10 medium supplemented with 15% horse serum and 2.5% FCS (24). The cells grown as monolayers on petri dishes (80 cm2) were then cultured in serum-free HB101 medium for 2 days and subsequently in the same medium, containing T3 and/or E2, as indicated. After given periods of time, the medium was aspirated, and the cells were washed twice with PBS, scraped off the plates with PBS, and collected by centrifugation at 200 x g for 5 min. The cell pellet was immediately frozen in liquid nitrogen and stored at -80 C until assayed.

Northern blot analysis
The frozen tissues [a pool of six anterior pituitaries (APs) or 100 mg pooled hypothalamic fragments per preparation] or pellets containing approximately 5 x 107 GH3 cells were homogenized in 5 ml of an SDS-containing Tris-based buffer (0.1 M Tris, 0.5 M LiCl, 10 mM EDTA, 1% SDS, and 5 mM dithiothreitol, pH 8.0) with the aid of a teflon-glass homogenizer. Poly-A+-enriched RNA was isolated directly from the homogenates by using magnetic oligo-dT25 polystyrene-beads (Deutsche Dynal, Hamburg, Germany), according to the manufacturer’s instructions.

Samples of 10 µg poly-A+-enriched RNA were separated by electrophoresis in denaturating agarose gels (2.2 M formaldehyde, 1.5% agarose), capillary transferred to nylon membranes (Nytran NY 12 N, Schleicher & Schuell, Dassel, Germany), and cross-linked by UV-irradiation.

Hybridizations were performed, as described previously (20), with the following 32P-labeled complementary DNA (cDNA)-probes: a 1.2-kb SphI-fragment of rat cDNA encoding the TRH-degrading ectoenzyme (25), a 1.1-kb EcoRV/SstI-fragment of the mouse cDNA encoding TRH receptor (26), and as standard, a 1.1-kb fragment of human cDNA encoding glyceraldehyde-3-phosphate dehydrogenase (Clontech, Heidelberg, Germany).

The nylon membranes were washed to a final stringency of 0.2 x SSPE/0.3% SDS (0.2 x SSPE = 30 mM NaCl, 2 mM Na-phosphate, 0.2 mM EDTA, pH 7.4) for 30 min at 60 C [TRH receptor and glyceraldehyde-3-phosphate dehydrogenase (GPDH)] or 65 C (TRH-degrading ectoenzyme). Autoradiographic signals were analyzed by a phosphoimager (Fujix BAS 1000), in combination with densitometric software (Mac BAS, Fuji Photo Film Co., both from Tokyo, Japan, supplied through Raytest, Sprockhövel, Germany). For photographic reproduction, autoradiograms were developed after exposure to x-ray films (XOMat, Kodak, Rochester, NY). All data were corrected for variability in loading by calculation as a ratio to the values obtained with glyceraldehyde-3-phosphate dehydrogenase.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Sex-related differences
A tissue-specific gender difference was observed in the expression of the TRH-degrading ectoenzyme from rat APs. Considerably higher (3.3-fold) enzymatic activities were found in the adenohypophyseal tissue preparations of male, compared with female, rats. Membranes prepared from the posterior pituitaries, hypothalamus, or total brain contained considerably higher enzymatic activities, but sex-related differences were not observed (Table 1Go).


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  		Table 1. Specific activity of the TRH-degrading ectoenzyme from rat tissues

 
The steady-state mRNA levels displayed similar sex-related differences (Fig. 1Go). Preparations from male APs contained 2.9 ± 0.4 times more transcripts of the TRH-degrading ectoenzyme, compared with those from female rats (mean ± SD, n = 5). Inversely related, the steady-state levels of the adenohypophyseal TRH receptor mRNA were found to be 2.6 ± 0.4 times higher in female than in male rats (mean ± SD, n = 5). Again, these gender differences were not observed with hypothalamic mRNA preparations (not shown).



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  		Figure 1. Northern-blot analysis of steady-state mRNA levels in male and female rat APs. The APs from six male or six female rats (6 months old) were isolated, pooled, and analyzed by Northern-blot, as described in Materials and Methods. This experiment was repeated four times with almost identical results. The GPDH signals were used as an internal standard.

 
Regulation by E2: time-course in vivo
A single injection of E2 (0.5 µg/100 g BW) into male rats modulated the expression of the TRH-degrading ectoenzyme and the mRNA levels of the TRH receptor. After an initial lag phase of 4 h, the enzymatic activity decreased to 65% of control values 14 h after the injection (Fig. 2Go A). The decrease was transient and the activity returned to basal levels within 96 h. This effect was specific for E2. Single injections of the steroid hormones dexamethasone (30 µg/100 g BW), progesterone (0.6 mg/100 BW), or testosterone (70 µg/100 g BW) did not significantly affect the enzyme’s activity assayed 12 h, 24 h, or 48 h after the injection.



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  		Figure 2. Time course of the effect of a single injection of E2 on the expression of the adenohypophyseal TRH-degrading ectoenzyme. Male rats (350–450 g BW) received a single sc injection of 0.5 µg E2 per 100 g BW or vehicle (0.5 ml sesame oil). After the indicated periods of time, the animals were killed. A, Effect on the enzymatic activity. Washed membranes from the APs ({circ}) or posterior ({blacktriangleup}) pituitaries were prepared and the enzymatic activities were determined, as described in Materials and Methods. The numbers indicate the number of experiments performed and the bars indicate the SD. B, Effect on mRNA levels. The APs were prepared and the mRNA levels were analyzed by Northern-blot, as described in Materials and Methods. Each lane represents the signals obtained after pooling the APs from six animals. The same pattern was observed when this experiment was repeated. C, Control.

 
The effects of E2 on the transcript levels of the adenohypophyseal TRH-degrading ectoenzyme were faster and more pronounced than on the enzymatic activities. Already, 4 h after a single injection of E2 (0.5 µg/100 g BW), a significant decrease became evident (Fig. 2BGo). The nadir was obtained after 6 h, displaying 25% (mean ± 12% SD; n = 4) of control values. The steady-state mRNA levels returned almost to basal levels (90 ± 8% of control values; mean ± SD, n = 4) 24 h after the injection.

As expected, E2 significantly increased the mRNA levels of the TRH receptor. After the injection of E2, the transcript concentrations increased, within 6 h, to 1.8 ± 0.3 times control levels (mean ± SD, n = 4) and returned to 1.3 ± 0.2 times basal values (mean ± SD; n = 4) 24 h after E2 application (Fig. 2BGo). When the hypothalami of these animals were analyzed by Northern blot 6 h and 24 h after the injection of E2, the mRNA levels of both the TRH-degrading ectoenzyme and of the TRH receptor were found to be unaltered (data not shown).

E2 counteracts the effects of thyroid hormones on the expression of the TRH-degrading ectoenzyme
In vivo studies.
Thyroid hormones are known to increase considerably the activity and mRNA levels of the TRH-degrading ectoenzyme (13, 15, 16, 17, 20). When female rats received multiple injections of T3 (5 µg/100 g BW) every 8 h, the enzymatic activity increased drastically and reached plateau levels about 36-fold above control values after 3–4 days (Fig. 3Go). When male rats were subjected to the same experimental protocol, the specific enzymatic activities were almost identical to those of T3-treated female rats. However, these values only accounted for a 12-fold increase in enzyme activity caused by the 3-fold higher basal levels (data not shown). When female rats received multiple injections of T3 in combination with E2 (0.5 µg/100 g BW) for 96 h, the enzyme activity increased only 15-fold above control values. E2, given in combination with T3 to rats that were first treated with T3 alone for 48 h, also very effectively counteracted the effects induced by T3 (Fig. 3Go).



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  		Figure 3. Effects of T3 and E2 on the activity of the TRH-degrading ectoenzyme from the APs of female rats. Female rats (six animals per group; 240–280 g BW) received multiple injections of T3 (5 µg/100 g BW) every 8 h, either alone ({circ}) or in combination with E2-3-benzoate (0.5 µg/100 g BW) ({blacktriangleup}). Alternatively, the animals were first treated for 48 h with T3 only and subsequently with the combination of the two hormones (•). Washed membranes were prepared, and the enzymatic activities were determined, as described in Materials and Methods. The values are the mean of two experiments.

 
In vitro studies.
The TRH-responsive somatomammotropic GH3 cells were used to study the hormonal effects on the expression of the enzyme in vitro. The cells were propagated in growth medium containing FCS and were then kept in a defined, serum-free medium. Among the serum-free media tested, the HB101 medium proved to be the most suitable. In this medium, GH3 cells not only survived but also grew, albeit less rapidly. As expected, cells kept in HB101 medium exhibited very low basal enzymatic activities, which increased about 15-fold within 48–72 h after addition of 1 nM T3 to the culture medium (Fig. 4Go). E2 (1 nM), added to the cell culture medium simultaneously with T3 or after preincubation for 24 h with T3 alone, effectively counteracted the T3-induced increase in the enzymatic activity. The T3-induced increase in enzymatic activity was completely blocked when cycloheximide (1 µg/ml), a protein synthesis inhibitor, was added to the culture medium simultaneously with T3.



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  		Figure 4. Effects of T3 and E2 on the activity of the TRH-degrading ectoenzyme of GH3 cells. As described in Materials and Methods, GH3 cells were propagated in serum-containing medium and then cultured in serum-free HB101 medium supplemented with 1 nM T3 ({circ}), 1 nM T3 and 1 nM E2 ({blacktriangleup}), or 1 nM T3 and 1 µg/ml cycloheximide ({blacksquare}). Alternatively, the cells were first cultured for 24 h in medium containing 1 nM T3 and were then switched to medium containing both hormones at 1 nM concentrations (•). Enzyme activity was determined as described in Materials and Methods. n = 4, mean values ±SD).

 
To study the effect of E2 on the TRH-degrading enzyme in more detail, GH3 cells were first cultured for 96 h in HB101 containing 1 nM T3. E2 added to the T3-containing medium decreased the enzymatic activity in a dose- (Fig. 5Go) and time-dependent (Fig. 6AGo) manner. At 1 nM concentration, E2 decreased the enzymatic activity after an initial lag phase of about 6 h, and 50% of the control values were reached after 48 h (Fig. 6AGo). The transcript levels of this enzyme decreased considerably faster and displayed about 50% of control values already 4 h after the addition of E2 (Fig. 6BGo).



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  		Figure 5. Concentration-dependent effect of E2 on the activity of the TRH-degrading ectoenzyme of GH3 cells. As described in Materials and Methods, the cells were first cultured for 3 days in serum-free medium containing 1 nM T3. The medium was then replaced by fresh medium containing 1 nM T3 and the indicated concentrations of E2. After incubation for 48 h, the cells were harvested, and the enzymatic activity was determined, as described in Materials and Methods. n = 4; mean values ± SD).

 


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  		Figure 6. Time course of the effect of E2 on the expression of the TRH-degrading ectoenzyme of GH3 cells. After culturing GH3 cells in the presence of 1 nM T3 for 3 days, as described in Materials and Methods, the medium was replaced by fresh medium containing 1 nM T3 and 1 nM E2. The cells were harvested at given time intervals. The enzymatic activities (n = 4, mean values ± SD) (Fig. 6AGo) and the mRNA levels (Fig. 6BGo) were determined as described in Materials and Methods. C, Control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The synthesis and secretion of adenohypophyseal hormones are known to be strongly influenced by peripheral hormones, such as thyroid and steroid hormones, either directly via the classical feedback regulatory systems or by indirect mechanisms. In addition, these hormones are known to strongly affect the transduction of hypothalamic signals at adenohypophyseal target sites. Within the hypothalamic-pituitary-gonadal axis, estrogens are well known not only to affect the synthesis and secretion of LH and FSH, as well as the synthesis of LHRH by hypothalamic neurons (27), but also the responsiveness of the gonadotrophs toward LH-RH (for review, see 28 , partly by modulating the density and the mRNA level of the GnRH receptor (29, 30). In addition, estrogens also play an important role in the heterologous regulation of other target cells. For example, estrogens are well known to exert pronounced effects on lactotrophs (31, 32). The gene-transcription and synthesis of PRL are directly affected by E2 (32, 33). Moreover, stimulatory effects of E2 on basal and TRH-induced PRL secretion (7, 34) are clearly established and seem to be partly caused by alteration in the responsiveness of lactotrophic cells. Because the responsiveness to TRH might conceivably be affected by the TRH-degrading ectoenzyme, we studied the effects of steroid hormones on the expression of this enzyme. The present study clearly demonstrates that the expression of the adenohypophyseal TRH-degrading ectoenzyme is down-regulated by E2. The decrease in the enzymatic activity is preceded by a decrease in the mRNA levels, suggesting that the enzyme is regulated at the pretranslational level.

In agreement with the findings that the number of TRH-binding sites in female rat pituitaries are significantly higher than in male rats (35), we observed a significant gender difference in the mRNA levels of the TRH receptor. Inversely related, the expression of the TRH-degrading ectoenzyme differed considerably among the sexes.

The enzymatic activity and the mRNA levels correlated well, and this sex-related difference was observed only at the pituitary level. Because this difference also might be related to the difference in the number of lactotrophic cells, we studied the acute effect of E2 after injection into male rats. A rapid decrease in the enzymatic activity, preceded by a profound decrease in the mRNA levels, could be demonstrated, suggesting that the expression of the TRH-degrading ectoenzyme is directly regulated by E2.

The effects of E2 on the expression of the adenohypophyseal TRH receptors in rat pituitaries in vivo (7) and in GH and GC cells in vitro (10, 34) have been analyzed before, and recently, it has been demonstrated that in GH3 cells, E2 up-regulates TRH receptor mRNA levels by increasing both the rate of transcription and mRNA stability (34). In good agreement with these data, we even observed a rapid increase of adenohypophyseal TRH receptor mRNA levels in vivo within hours after treating male rats with E2.

Compared with the TRH receptor, the impact of E2 on the transcript levels of the adenohypophyseal TRH-degrading enzyme is more striking. The effects at the pretranslational level are characterized by a faster kinetic and a higher amplitude. Diminished mRNA levels preceded reduced enzymatic activity, and basal levels were regained within physiologically meaningful time periods. This tissue-specific effect was observed only with E2 but not with other steroid hormones. Whereas pituitary TRH receptors also are known to be regulated by hydrocortisone (36), an injection of dexamethasone or adrenalectomy had no effect on the activity of the TRH-degrading ectoenzyme.

Because the adenohypophyseal TRH-degrading ectoenzyme is strongly up-regulated by thyroid hormones (13, 15, 16, 17, 20), we were interested in studying whether E2 would counteract the effects of thyroid hormones. This might be expected, because the receptors of both hormones belong to the same superfamily of ligand-dependent nuclear transcription factors (37, 38) and especially because our previous results pointed to a similar mechanism for T3 and E2, i.e. regulation at the pretranslational level. Indeed, only E2 (but not the other steroid hormones tested) effectively counteracted the T3-induced up-regulation of the enzyme in vivo and in vitro using the TRH-responsive GH3 cells.

GH3 cells have been widely used to study TRH-induced PRL secretion. Because our previous studies had demonstrated that the TRH-degrading ectoenzyme is preferentially localized on lactotrophic cells (13), we assumed that GH3 cells would be an interesting model system. To our surprise, however, we detected neither significant enzymatic activities nor mRNA levels when we tested the GH3 cells provided by the American Tissue Culture Collection. Other subclones of this cell line that were provided by several laboratories exhibited very different properties. There were some subclones that exhibited very high specific activities and mRNA levels, but the TRH-degrading ectoenzyme was not up-regulated by thyroid hormones, whereas other subclones (such as the one used in this study) exhibited properties that are similar to those of pituitary cells in primary culture. As with primary pituitary cells in culture (13), the activity of the TRH-degrading ectoenzyme is very low when these GH3 cells are kept in serum-free culture medium. In serum-containing medium, the enzymatic activity is considerably higher, at least partly because of the thyroid hormones present in these serum preparations. By screening a variety of synthetic culture media, we observed that the HB101 medium, originally designed by Sato et al. (39) as a hybridoma culture medium, can substitute the serum-containing culture medium normally required to grow GH3 cells. Under these conditions, we observed a rapid up-regulation of the enzyme by thyroid hormones and a decrease by E2 but not by other steroid hormones.

In conclusion, the stringent, tissue-specific, and steroid hormone-specific regulation of the adenohypophyseal TRH-degrading enzyme by E2 lends further support to the hypothesis that this enzyme might act as a regulatory control element. The very rapid regulation of the TRH-degrading ectoenzyme by E2 and thyroid hormones, in mirror image to the regulation of the TRH receptor, indicates that both elements cooperate on the signal-receiving site of TRH target cells, reinforcing the control of adenohypophyseal hormone secretion.


   Acknowledgments
 
We thank Prof. Dr. P. W. Jungblut and Prof. Dr. H. Jäckle for continuous support. We also thank P. Affeldt, B. Kühlein, A. Rosebrock, H.-O. Bader, and S. Thiele for excellent technical assistance; V. Ashe for typing and especially for linguistic help; R. Ehlers and A. Peters for the graph work; and H. Heuer, S. Turwitt, and J. Ehrchen for stimulating discussions. Our thanks also to Dr. D. Gourdji, Collège de France, and Drs. W. Meyerhof and D. Richter, University of Hamburg, for generously providing us with various subclones of GH3 cells; and Dr. M. C. Gershengorn, Cornell University, NY, for kindly providing the TRH receptor-containing plasmid.


   Footnotes
 
1 This work was supported by a grant from the Deutsche Forschungs-gemeinschaft. Back

Received February 23, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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