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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 8 2709-2713
Copyright © 2000 by The Endocrine Society

Original Studies

Analysis of Thyrotropin-Releasing Hormone-Signaling Components in Pituitary Adenomas of Patients with Acromegaly1

Jan Ehrchen, Anne Peters2, Dieter K. Lüdecke, Theo Visser and Karl Bauer

Max Planck Institut fur Experimentelle Endokrinologie (J.E., A.P., K.B.), D-30625 Hannover, Germany; Neurochirugische Klinik, Universitäts Krankenhaus Eppendorf (D.K.L.), 20246 Hamburg, Germany; and Department of Internal Medicine III and Clinical Endocrinology, Medical Faculty, Erasmus University Rotterdam (T.V.), NL-3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Dr. Karl Bauer, Max Planck Institut fur Experimentelle Endokrinologie, Feodor Lynen Strasse 7, D-30625 Hannover, Germany.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results and Discussion
 References
 
In many acromegalic patients the paradoxical release of GH in response to TRH has been well documented, but the mechanisms underlying this phenomenon are not understood. It has been suggested that aberrant GH secretion may result from TRH endogenously synthesized by the adenoma. In 32 adenomas from acromegalic patients, TRH-like immunoreactivity (TRH-LI) was measured using 2 well characterized antisera. TRH-LI was not detectable in 10 samples, and in 19 samples, TRH-LI was measured only by the less specific antibody. With the TRH-specific antibody, TRH-LI was identified only in 3 samples, 2 of which contained exceedingly high concentrations (40 and 96 pg/mg tissue). In the latter 2 samples, prepro-TRH messenger ribonucleic acid was identified by Northern blot analysis, but not in the control tissue sample of a patient without pituitary disease and also not in the other adenomas analyzed by this technique. Transcripts of the TRH receptor were almost undetectable in all adenomas analyzed. For the TRH-degrading ectoenzyme, a potential regulator of TRH signals at adenohypophyseal target sites, transcripts were significantly expressed only in the TRH-producing adenomas. We conclude that the TRH-signaling elements examined are, in general, not directly involved in the mechanisms causing paradoxical GH secretion in acromegalic patients.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results and Discussion
 References
 
TRH (pyroGlu-His-Pro-NH2) was originally isolated as a hypothalamic neuropeptide hormone that stimulates the release of TSH. Subsequent studies then demonstrated TRH to be very potent in releasing PRL from the anterior pituitary. Surprisingly, after TRH administration GH release has been observed in patients with a variety of disorders (for review, see Refs. 1, 2), most notably in many patients with acromegaly (3, 4), but not in healthy subjects. Some researchers suggested that this paradoxical GH response to TRH might result from inappropriate expression of TRH receptors (TRH-R) (5), but this hypothesis has not been verified by others (6, 7). Alternatively, it has been suggested that TRH produced and released locally by pituitary adenomas could act as an autocrine and/or paracrine regulator and affect hormone release or tumor growth (8, 9), either directly or indirectly.

Early reports on the persistence of immunoreactive TRH in long term primary anterior pituitary cultures (10) supported the hypothesis that TRH could be synthesized endogenously by the anterior pituitary. Furthermore, in subsequent studies authentic TRH and TRH precursor peptides as well as prepro-TRH messenger ribonucleic acid (mRNA) could be detected in rat anterior pituitary cells that were cultured as monolayers in the presence of 10% FCS for long periods of time, up to 3 weeks (11, 12). As TRH synthesis was not observed when rat pituitary cells were kept as reaggregate cultures (12), these studies indicated that TRH synthesis may reflect derepression of the TRH gene as a result of disrupted cell to cell communication. As alterations of cell to cell interactions are also crucial steps in tumor growth, we were interested in analyzing TRH synthesis and TRHsignaling components (expression of the TRH-R and the TRH-degrading ectoenzyme) in GH-producing pituitary adenomas.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results and Discussion
 References
 
Patients

Thirty-two GH-secreting adenomas were collected in liquid nitrogen during transsphenoidal adenomectomy and stored at -80 C. All operations were performed by an experienced neurosurgeon (D.K.L.) to ensure that only adenomatous material was used. Diagnosis was based on clinical criteria, analytical data [clearly elevated GH and insulin-like growth factor I (IGF-I) plasma levels and nonsuppression of GH levels in the oral glucose tolerance test] and magnetic resonance imaging. The diagnosis was confirmed by immunohistochemical staining of tumor fragments for GH after surgery. Most of the patients had been treated with either a short-acting somatostatin analog (octreotide) or a dopamine agonist (bromocriptine) before surgical intervention. Treatment was stopped within the last week, at least 24 h before surgery.

Sixteen patients were men, aged 19–57 yr, and 16 patients were women, aged 21–70 yr. Plasma levels of GH, IGF-I, PRL, TSH, and cortisol were measured before surgery. The average plasma levels were 13.5 µg/L for GH and 885 µg/L for IGF-I.

The control anterior pituitary was obtained from The Netherlands Brain Bank (no. 95–023, autopsy no. 95/076, obtained from a 66-yr-old female suffering from Alzheimer’s disease) and was included in the Northern blot analysis.

RIA

Tumor fragments were weighed and homogenized in 70% methanol/30% 2 N acetic acid. The suspension was sonicated and centrifuged at 10,000 x g for 10 min. The supernatants were lyophilized and reconstituted for RIA measurements in 0.5 mol/L sodium phosphate buffer (pH 7.4). The RIAs for TRH-immunoreactive material were performed as described previously (12) using [125I]TRH as tracer and either the TRH-specific antiserum 8880 at a final dilution of 1:10,000 or the less specific antiserum 4319 at a final dilution of 1:2,500. The characteristics of the antisera have been described previously (13, 14). The sensitivity of the method was about 1–2 pg/tube (no. 4319) and 3–5 pg/tube (no. 8880), respectively.

Northern blot analysis

Adenomatous tissue was homogenized in 4 mL buffer consisting of 0.1 mol/L Tris, 0.5 mol/L LiCl, 10 mmol/L ethylenediamine tetraacetate, 5 mmol/L dithiothreitol, and 1% SDS, pH 8. Polyadenylated [poly(A)+] enriched RNA was isolated using magnetic oligo(deoxythymidine)25 polystyrene beads (Deutsche Dynal, Hamburg, Germany) following the manufacturer’s instructions.

Approximately 1.5–10 µg poly(A)+ enriched RNA was size-fractionated by electrophoresis in a denaturing formaldehyde/agarose gel (2.2 mol/L formaldehyde and 1.25% agarose). The RNA was capillary transferred to a nylon membrane (Nytran NY 12 N, Schleicher & Schuell, Inc., Dassel, Germany) and cross-linked by UV irradiation.

Approximately 50 ng of the complementary DNA (cDNA) fragments used as probes were randomly labeled with [32P]deoxy-CTP to high specific activity (>109 cpm/µg) using the random primed labeling kit (Stratagene, La Jolla, CA) following the manufacturer’s instructions.

Hybridizations were performed at 42 C in 50% formamide containing 0.5% SDS, 100 µg/mL salmon sperm DNA, 0.5 mol/L NaCl, 12 mmol/L ethylenediamine tetraacetate, and 0.09 mol/L sodium phosphate, pH 7.4. After washing under high stringency conditions [59 C, 0.3% SDS/0.2 x SSPE (75 mM NaCl, 5 mM NaH2PO4, and 0.5 mM EDTA [pH 7.4])], the membranes were exposed to x-ray films (X-OMAT, Eastman Kodak Co., Rochester, NY).

The following cDNA fragments were used: a 219-bp fragment corresponding to nucleotides 331–550 of the cDNA encoding human hypothalamic prepro-TRH (15), the complete cDNA encoding the human TRH-degrading ectoenzyme (16), an 830-bp fragment (nucleotides 557-1387) of the cDNA encoding the human TRH-R (5), and the complete cDNA encoding rat cyclophilin (17).


   Results and Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results and Discussion
References
 
Determination of TRH-like material by RIA

The well characterized antibodies 8880 and 4319, previously described (14, 13), were used to measure TRH-like immunoreactivity (TRH-LI) in the collected surgical specimens that were classified as GH-secreting pituitary adenomas. Although Le Dafniet et al. (18) originally reported on the existence of TRH-LI in all GH-secreting pituitary adenomas tested (n = 18), TRH-LI was not detectable by either antiserum in 10 of the 32 adenomas examined, and in 19 tissue samples TRH-LI was not measured by the TRH-specific antibody 8880 but only with antibody 4319, a relatively nonspecific antiserum that recognizes TRH as well as TRH-related peptides of the general structure pyroGlu-X-Pro-NH2 (Table 1Go). This result is not surprising, because more recent studies have meanwhile demonstrated that in rat and human anterior pituitaries most of the adenohypophyseal TRH-LI is accounted for by pyroGlu-Glu-Pro-NH2 (12, 19, 20, 21), a peptide with unknown pituitary function. Considerable variations in the content of this material were noticed, ranging from very low levels to exceedingly high concentrations (up to 39 pg/mg tissue; Table 1Go). Only in 3 adenomas was TRH-LI measured with the TRH-specific antiserum 8880 and in comparable amounts with antiserum 4319, indicating that in these samples most of the TRH-LI may represent authentic TRH. In 2 of these adenomas the peptide content was very high (40 and 96 pg/mg tissue), reaching values comparable to those reported for the concentration of TRH in human hypothalamus (72–131 pg/mg tissue) (22, 23). No clinical or biochemical effects of the high TRH content were evident. For both female patients, the TSH levels (0.3 and 0.7 mU/L) and the PRL levels (5.6 and 9.8 µg/L) compared well with control values (0.3–0.7 mU/L for TSH; 3.0–26 µg/L for PRL), indicating that TRH is not effectively released, but presumably is stored intracellularly to give rise to the high TRH content. Unfortunately, TRH stimulation tests were not performed in these 2 patients. Due to the limited diagnostic information from these tests, the improvement of other methods, and the problem of apoplexy (for review, see Refs. 24, 25), TRH stimulation tests are no longer performed routinely. Only 6 (no. 4, 14, 15, 17, 21, and 26 in Table 1Go) of the 32 patients underwent this test, and a significant rise in GH levels after TRH was clearly detectable in 4 patients (no. 4, 17, 21, and 26 in Table 1Go; mean increase from 19.8 to 90.5 µg/L).


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  		Table 1. Content of TRH-LI in GH-secreting adenomas as measured by the TRH-specific antibody 8880 and the less specific antibody 4319, which recognizes TRH as well as TRH-like peptides

 
Our results are in agreement with the findings of Levy and Lightman (26). By in situ hybridization histochemistry, these researchers identified clusters of TRH transcript-positive cells in only 3 of 10 corticotroph adenomas and overall in 8 of 56 tumors examined. Taken together, these data clearly demonstrate that TRH is not a general constituent of GH-secreting adenomas. The discrepancy between these findings and a previous report on the presence of TRH in all GH-secreting adenomas (18) is most likely explained by the specificity of the antibodies used.

Northern blot analysis

Based on the known specificity of the antisera, only the TRH-LI measured by antiserum 8880 in 3 of the 32 samples can be considered to represent authentic TRH. This idea is fully supported by the results of the Northern blot analysis, which could be performed with 10 tumor specimens for which sufficient amounts of tissue were available. Prepro-TRH mRNA was not detectable in the nontumorous control pituitary, although the gel was loaded with large amounts of poly(A)+-enriched mRNA. Hybridization signals were not visible in 8 of 10 adenomas. However, as expected, distinct bands (Fig. 1Go, lanes 3 and 5) were readily visualized in the very tissue samples in which TRH was recognized by antibody 8880 (Fig. 1Go). The size of these transcripts (~1.6 kb) corresponds to the values reported for human and rat hypothalamic TRH mRNA (15, 27, 28). These results strongly indicate that TRH is not effectively transcribed in either nontumorous pituitary or most GH-secreting adenomas, although we cannot rule out that minute amounts of TRH mRNA may be present that have been detected by the extremely sensitive RT-PCR method (9).



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  		Figure 1. Northern blot analysis of TRH and TRH-signaling components (TRH-R and TRH-degrading ectoenzyme) in GH-secreting adenomas (no. 1–10) and in the anterior pituitary of a patient without pituitary disease (C). The numbers refer to the patient numbers as listed in Table 1Go. Poly(A)+ RNA was extracted from the tissue samples and blotted onto nylon membranes as described in Subjects and Methods. The same blot was successively probed with 32P-labeled cDNA fragments of prepro-TRH (ppTRH), TRH-R, TRH-degrading ectoenzyme (TRH-DE), and subsequently with the cyclophilin probe to provide information about the relative amounts and the quality of the mRNA preparations analyzed. The positions of the 28S and 18S ribosomal RNAs are indicated by closed and open arrowheads, respectively. The asterisk marks the TRH-R signals that are revealed after short exposure to the film, whereas in the adenomas, TRH-R signals could only be detected after very long exposure periods.

 
To our knowledge and surprise, Northern blot data on the human TRH-R (TRH-R1, the only human TRH-R identified to date) have not yet been reported despite extensive studies on the genomic, structural, and functional organization of the human TRH-R gene (5, 29, 30, 31, 32, 33) as well as cDNA cloning and functional expression of the TRH-R or splice variants thereof (34, 35) in brain and pituitary tissue. In the rat, a major transcript, 3.6 kb in size, has been reported by several investigators (36, 37, 38), which we also easily detected in rat anterior pituitary as well as in GH3 cells, a cell line derived from rat pituitaries (Fig. 2Go). In addition, we observed a less intensively labeled band of about 7.5 kb in size. Previously, a TRH-R transcript approximately of the same size has also been detected in rat pituitary (39, 36).



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  		Figure 2. Northern blot analysis of the TRH-R. Poly(A)+ RNA was extracted from a human anterior pituitary of a patient without pituitary disease (Hum. AP), from rat anterior pituitary (Rat AP), and from GH3 cells (GH3). After blotting to nylon membranes as described in Subjects and Methods, the blot was probed with 32P-labeled cDNA fragment of the TRH-R and subsequently with the cyclophilin probe to assess the amount and quality of the mRNA preparation. The arrows indicate the positions of the ribosomal RNAs as described in Fig. 1Go.

 
In the nontumorous human pituitary, a high molecular weight transcript of about 7.5 kb was detected as the major band, in addition to some bands of lower molecular weight that were labeled less intensively (Figs. 1Go and 2Go). Although in this tissue preparation TRH-R transcripts were readily detectable, this was not the case when we analyzed the adenomatous material. Only when the film was exposed for very long time periods (several weeks) was the transcript of about 7.5 kb barely detectable as a very faint band in some adenomas (Fig. 1Go), but not in all. Even in the TRH-producing adenomas (lanes 3 and 5), only very faint signals were observed, whereby in adenoma 5 a band of exceptionally high molecular weight was noticed. Elevated TRH-R expression was also not noticed in patient 4 (Table 1Go), in whom GH levels increased from 4.9 to 48 µg/L after the injection of TRH.

The extremely low expression of TRH-R transcripts does not support the concept that TRH signaling via the TRH-R is generally involved in the pathology of GH-secreting adenomas. Only in selected cases may inappropriate expression of the receptor be the cause of the paradoxical GH response (5). Extensive studies on the structure of TRH-R (7) and careful analysis of TRH-R levels by competitive RT-PCR (6) with large collections of pituitary adenomas also consistently demonstrated that in adenomas the hormonal responsiveness to TRH is not assessable by the TRH-R and suggested that other components of the pathway controlling TRHsignaling events may be implicated in pituitary tumorigenesis and the paradoxical GH secretion.

Therefore, we were interested in analyzing the TRHdegrading ectoenzyme in the GH-secreting adenomas, because this peptidase may represent the third element of TRH signaling at adenohypophyseal target sites (38, 40). In the rat, previous studies demonstrated that this TRH-specific peptidase is stringently regulated by estradiol (41) and thyroid hormones (42, 43) in mirror image to the TRH-R, thus indicating that this enzyme may represent a regulatory element that may serve an integrative function in modulating the response of adenohypophyseal target cells to TRH. As in the rat (44, 41), multiple transcripts could be detected at very low signal intensity in the nonadenomatous pituitary and in some adenomas. Interestingly, comparatively strong signals were visualized in the TRH-producing tumors (no. 3 and 5), although both female patients had normal thyroid gland functions and, as mentioned above, TSH and PRL levels within the normal range. Thus, it seems most likely that if released by these tumors, TRH would be rapidly inactivated by the TRH-degrading ectoenzyme on the surface of the tumor cells. Together with the very low TRH-R mRNA levels, it seems unreasonable to assume that TRH produced by these tumor cells may act as an autocrine and/or paracrine factor that could be implicated in pituitary dysfunction.

In summary, most studies reported to date indicate that the paradoxical release of pituitary hormones (GH and the {alpha}- and ß-subunits of LH and FSH) (45, 46) by TRH, in general, is neither related to the synthesis of TRH by adenoma cells or to the principal components at the signal-receiving site, the TRH-R and the TRH-degrading ectoenzyme. In this context it should be noted that paradoxical GH secretion is not only induced by TRH, but also by other hypothalamic releasing factors (e.g. CRH and LH-releasing hormone) (47, 48) as well as by centrally acting stimuli. Moreover, a vast body of literature describes the TRH-induced GH release in many pathophysiological conditions ranging from renal failure, diabetes, liver diseases, drug addiction, to various mental disorders (for review, see Ref. 2). In addition, this phenomenon is observed under normal biological conditions [e.g. in postmenopausal (49) or pregnant women (50)]; moreover, depending on the physical conditions (rest and inactivity, sleep cycles, etc.) (2), paradoxical GH release has also been observed in healthy individuals.

In most of these conditions, the pituitary gland per se seems to be normal, and as shown by our results for the adenomas studied, the TRH-signaling compounds within the pituitary are probably not affected directly. Taken together, these data indicate that adenoma formation and altered physiological/pathophysiological conditions may result in the formation of a vast array of chemical mediators that are produced locally (paracrine factors) by the intrapituitary signaling network (51). Thus, rather than TRH or the components of the TRH signaling system, paracrine factors produced locally may be the important elements mediating the effect of TRH on GH secretion.


   Acknowledgments
 
We thank Dr. E. Fliers (Academic Medical Center, Amsterdam, The Netherlands), Prof. Dr. D. Swaab (Netherlands Institute for Brain Research, Amsterdam, The Netherlands), and Dr. R. Ravid (Coordinator at the Netherlands Brain Bank, Amsterdam, The Netherlands) for providing the control anterior pituitary. We also thank Prof. Dr. W. Saeger (Department of Pathology, Marienkrankenhaus Hamburg, Hamburg, Germany) for the histological examinations, including the immunohistology of the adenomas, and V. Ashe for linguistic help and typing the manuscript.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (Grant Ba 593/4–3). Back

2 Present address: Pharmacia & Upjohn, Inc. GmbH, Am Wolfsmantel 46, D-91058 Erlangen, Germany. Back

Received December 14, 1999.

Revised April 18, 2000.

Accepted April 21, 2000.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results and Discussion
 References
 

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