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Endocrinology Vol. 140, No. 3 1054-1059
Copyright © 1999 by The Endocrine Society

ARTICLES

Cloning of Thyrotropin-Releasing Hormone Precursor and Receptor in Rat Thymus, Adrenal Gland, and Testis

Jean-Jacques Montagne, Ali Ladram, Pierre Nicolas and Marc Bulant

Laboratoire de Bioactivation des Peptides, Institut J. Monod, 75251 Paris Cedex 05, France

Address all correspondence and requests for reprints to: Dr. Marc Bulant, Laboratoire de Bioactivation des Peptides, Institut J. Monod, 2 place Jussieu, 75251 Paris Cedex 05, France. E-mail: bulant{at}infobiogen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRH IS a major product of hypothalamic pro-TRH that stimulates the secretion of TSH (1, 2). TRH acts on the anterior pituitary via a high affinity receptor to activate phospholipase C and generate inositol triphosphate and diacylglycerol. TRH receptor (TRH-R) complementary DNA (cDNA) has been cloned and expressed in mouse thyrotropic TtT cells and in rat, human, sheep, and bovine pituitary gland or pituitary cell lines (for a review, see Ref. 3). The predicted protein contained seven putative membrane- spanning domains, as do all G protein-coupled receptors, but there are different subtypes of TRH-R in cells outside the pituitary, and exogenous TRH has a variety of biological activities (4, 5). Moreover, it is difficult to determine the physiological role for the TRH until the sites of TRH precursor biosynthesis have been identified. For example, TRH functions as a neurotransmitter in the central nervous system in addition to the hypophysiotropic effects on the adenohypophysis (6, 7, 8). TRH is involved in stimulation of gastric acid secretion mediated by autonomic nerve fibers (9). However, the presence of TRH in many organs suggests that TRH acts via pathways other than the hypothalamic-pituitary-thyroid axis or neuron regulation. TRH might act via peripheral secretion. Some of the TRH in the peripheral circulation appears to come from extraneuronal source (10), although the peripheral sources of TRH gene expression, except for the pancreas and testis, remain obscure. Pro-TRH products have been detected in the rat pancreas, where their concentrations are extremely high during the neonatal period (11, 12, 13, 14). Prepro-TRH messenger RNA (mRNA) is present in neonatal ß-cells, indicating that the endocrine pancreas is a major site of TRH precursor in young rats (15, 16). The testis of adult rats contains high concentrations of TRH immunoreactivity (17), produced from a prepro-TRH mRNA longer than that in the hypothalamus (18). The Leydig cells are the site of TRH biosynthesis in testis (18, 19). Finally, peptides from pro-TRH have been detected in numerous organs (20). For instance, we have found pro-TRH products in the adrenal glands of adult rats (21). However, there is as yet no concrete evidence for expression of the TRH gene in these organs. This study demonstrates the endogenous origin of the TRH precursor in several peripheral organs together with the presence of TRH-R.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Polyadenylated [poly(A)+] RNA isolation
Adult male Sprague-Dawley rats (Dépré, France), weighing 300 µg, were killed by decapitation, and fresh tissues were immediately removed. Poly(A)+ RNA was isolated from the hypothalamus, testis, spleen, thymus, adrenal gland, intestine, lung, heart, epididymis, and cells of peritoneal fluid using the FastTrack mRNA isolation kit (Invitrogen). Poly(A)+ RNA extracted from rat neuroblastoma cells BN1010 (22) and mouse hybridoma K9 (23) cells were used as positive and negative controls.

PCR primers
Primers were constructed from published sequences of TRH and TRH-R cDNA (24, 25): TRH1 upstream, 5'-TCTGCAGAGTCTCCACTTCGCAGACTCCAG-3'; TRH2 downstream, 5'-GGTGACATCAGACTCCATCCAGGGGAAGGA-3'; TRH3 upstream, 5'-GATGAGGAGGACAGTGACTGGATGCCACGG-3'; TRH4 downstream, 5'-AGCATCTAAGAGAGGACAGCTAGTGAAGGG-3'; TRH-R1 upstream, 5'-AAACTGCCGCTCTGAAGCCTGAACCTCTGC-3'; and TRH-R2 downstream, 5'-TTGCTTCCTTATTGTGCCACCCTGTACCAT-3'.

All oligonucleotides were synthesized by Genset (Paris, France). Three sets of oligonucleotide primers (first set, TRH1 and TRH2; second set, TRH3 and TRH4; third set, TRH1 and TRH4) generate products of 538, 485, and 886 bp, respectively, from TRH cDNA (see Fig. 1AGo), whereas TRH-R1 and TRH-R2 generate a product of 1643 bp from TRH-R cDNA (Fig. 1BGo). A set of primers corresponding to the rat glyceraldehyde-3-phosphate dehydrogenase (G3PDH; Clontech, Palo Alto, CA) that generates a 983-bp product was used as an internal control.



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  		Figure 1. Diagram of the rat gene, cDNA, and PCR primers for TRH precursor (A) and TRH-R (B). Exons are shown as boxes. A, Stippled, hatched, and solid regions represent sequences encoding the signal peptide, the pro-TRH-connecting peptides, and the TRH progenitor peptides, respectively. The relative positions of specific primers TRH1, TRH2, TRH3, and TRH4 and the amplified segments along the TRH cDNA are shown. B, The relative position of the amplified segment along the TRH-R cDNA is shown. The hatched and solid regions represent sequences encoding hydrophilic and membrane-spanning domains of the TRH-R, respectively. A possible deleted sequence and its boundaries are indicated just before the end of the coding region.

 
RT-PCR analysis
Poly(A)+ RNA (1 µg) were reverse transcribed into cDNA using random hexamers (20 pmol) in 20 µl containing 1 x reaction buffer (75 mM KCl; 50 mM Tris-HCl, pH 8.3; and 3 mM MgCl2), each deoxy-NTP at 0.5 mM, ribonuclease inhibitor (1 U/µl), and Moloney murine leukemia virus reverse transcriptase (Clontech; 10 U/µl). The mixture was incubated for 60 min at 42 C, boiled for 5 min, and diluted with water to 100 µl. One tenth (10 µl) of each cDNA mixture was amplified by PCR in 50 µl containing 1 x PCR buffer, 0.2 mM of each deoxy-NTP, upstream and downstream TRH primers (0.4 µM each), and 2 U AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT). Amplification was performed using a 30-cycle program consisting of 94 C for 45 sec, 61 C for 1 min, and 72 C for 2 min, followed by a final extension for 7 min at 72 C. PCR products were analyzed by electrophoresis on 1.2% preparative agarose gels. Another amplification was performed using 15 µl DNA templates with the TRH-R primer set and a hybridization temperature of 65 C. The efficiency of cDNA synthesis was estimated using the G3PDH primer set. One twentieth of the cDNA was used at a hybridization temperature of 60 C. The PCR-amplified products were purified on gel agarose, cloned into the vector pGEM-Teasy (Promega Corp.) and sequenced using fluorescent dye-labeled dideoxynucleotide with the T7 and SP6 primers and Taq polymerase. Products were analyzed on a Biosystems 370A automated DNA sequencer (PE Applied Biosystems, Foster City, CA). The sequences obtained from the PCR products were identical to the published sequences of TRH and TRH-R cDNA.

Quantitative competitive PCR
TRH mRNA was quantified by competitive RT-PCR, which required the construction of a control PCR template with primer sites identical to those of the target template but of a different size (26). The restriction map of the rat TRH cDNA indicated the presence of BstEII restriction sites at positions 506 and 787, which are not found in the vector pGEM-Teasy. Cloned pGEM-TRH, which contained the entire translated region of the TRH cDNA (see Fig. 3AGo), was digested with BstEII, purified from the excised fragment by gel extraction, and religated to generate a deletion clone of the PCR target. Cloned pGEM-TRH-del was then digested with EcoRI to excise the competitive standard. The deletion fragment was purified and quantified spectrophotometrically to determine a range of concentrations suitable for RT-PCR. The appropriate amount of TRH competitor to use in the PCR was determined by amplifying 10-fold serial dilutions with a constant amount of cDNA. Competitive PCRs were then performed with 5 µl of cDNA templates and 10 µl of serially increasing (2-fold) known amounts of the TRH competitor using the conditions described above.



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  		Figure 3. Quantitative competitive PCR of TRH cDNA. A, Construction of an internal standard for competitive PCR. Clone pGEM-TRH contains the PCR product generated with oligonucleotide primers TRH1 and TRH4. The derived clone pGEM-TRH-del was obtained by digestion with BstEII. The TRH-del standard corresponds to the excised insert of clone pGEM-TRH-del. B, Quantitative analysis of the TRHc DNA in the hypothalamus (lanes 1–3), testis (lanes 4–6), thymus (lanes 7–9), and adrenal gland (lanes 10 and 11). Lane 12 contains a 100-bp DNA ladder as size markers. Aliquots of cDNA (5 µl) were amplified with 2-fold dilutions of the TRH-del standard. Twenty percent portions of the PCR products were then resolved on a 1.2% ethidium bromide-agarose gel. Lanes 1–3, 0.1, 0.05, and 0.025 amol TRH-del standard; lanes 4–6, 0.05, 0.025, and 0.0125; lanes 7–9, 0.0062, 0.0031, and 0.0016; lanes 10 and 11, 0.0016 and 0.0008.

 
Extraction of pro-TRH-related peptides
The thymus and spleen were removed from four adult rats, immediately immersed in 10% acetic acid at 95 C for 10 min, homogenized, and extracted at 4 C using a Polytron (Brinkmann Instruments, Westbury, NY). The resulting suspensions were centrifuged at 3000 x g at 4 C for 30 min and lyophilized. Crude extracts were filtered through Sep-Pak C18 cartridges (Alltech, Templemars, France) and evaporated to dryness. The samples were dissolved in 200 µl water containing 0.1% trifluoroacetic acid (TFA) and placed on a 5-µm Lichrospher C18 reverse phase column (3.9 x 250 mm; Interchrom, Asnières, France) equilibrated with the same solvent at a flow rate of 0.75 ml/min. Three minutes after injection the bound material was eluted with a linear gradient (1%/3 min) of acetonitrile (0.07% TFA) in water (0.1% TFA). Fractions (750 µl) were collected and lyophilized. Aliquots of each fraction were reconstituted in enzyme immunoassay buffer and assayed for TRH or prepro-TRH-(160–169) (Ps4) immunoreactivity (21).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The distribution of mRNA for prepro-TRH was examined by RT-PCR using specific oligonucleotide primers. The cDNA from each organ was amplified with two set of primers (TRH1-TRH2 and TRH3-TRH4), which could generate two PCR products (538 and 485 bp) covering the entire coding region of the TRH cDNA (Fig. 1AGo). The first set spans two introns and can identify artifactual amplification of genomic DNA. Finally, a third set (TRH1-TRH4) generated an 886-bp product that encompasses the translated sequence of the TRH cDNA. Hypothalamus and BN1010 cells, which synthesize TRH, served as positive controls, whereas K9 cells, which do not synthesize TRH, served as a negative control. The expected products were detected with the three set of primers in the hypothalamus and BN1010 cells, but no signal was found in the K9 cells (Fig. 2Go, A–C). A strong amplification signal was obtained in the testis, adrenal gland, and thymus, with a more moderate amplification in the spleen and a barely detectable signal in the peritoneal fluid. PCR-amplified products from the spleen and peritoneal fluid were diluted 1:50 and reamplified using the first set of primers (TRH1-TRH2). The expected product was then found in both cases (Fig. 2AGo). No additional larger products were seen with the TRH1-TRH2 and TRH1-TRH4 primers (Fig. 2Go, A and C), indicating that genomic DNA was not present in the samples studied. The cDNA templates were amplified with a set of primers corresponding to G3PDH cDNA as internal controls of the poly(A)+ RNA. Each sample contained almost the same total quantity of mRNA (Fig. 2DGo). To certify that the amplified fragments were derived from the TRH mRNA, the purified PCR products generated with the TRH1-TRH2 and TRH3-TRH4 sets were ligated to a linearized plasmid with 3'-deoxythymidine overhangs. The isolated clones had the same sequence as the rat hypothalamic TRH cDNA.



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  		Figure 2. Detection of TRH and TRH-R gene activities in various tissues by RT-PCR. A–C, Amplification of 10-µl DNA mixtures with specific primers of TRH cDNA: sets TRH1-TRH2 (A), TRH3-TRH4 (B), and TRH1-TRH4 (C; see Fig. 1AGo). D, Amplification of 5-µl DNA mixtures with specific primers of G3PDH cDNA. E, Amplification of 15-µl DNA mixtures with specific primers of TRH-R cDNA, set TRH-R1-TRH-R2. The relative positions of the amplified segments along the TRH cDNA and the TRH-R cDNA are schematically shown in Fig. 1Go. RT-PCR reaction mixtures (10 µl) were resolved on a 1.2% ethidium bromide-agarose gel. cDNA templates were from: 1, hypothalamus; 2, peritoneal fluid; 2', reamplified peritoneal fluid; 3, spleen; 3', reamplified spleen; 4, testis; 5, adrenal gland; 6, thymus; 7, BN1010 cells; and 8, K9 cells. Flanking lanes show the positions of size markers. *, Rat neuroblastoma; **, mouse Leydig cells hybridoma. F, RT-PCR analyses of gene expression in lung, epididymis, intestine, and heart. Lanes 9, 12, 15, and 18, RT-PCR-amplified gene product for G3PDH-specific primers; lanes 10, 13, 16, and 19, RT-PCR-amplified gene product for TRH cDNA-specific primers (set TRH1-TRH4); lanes 11, 14, 17, and 20, RT-PCR-amplified gene product for TRH-R cDNA-specific primers (set TRH-R1-TRH-R2). RT-PCR reaction mixtures (20 µl) were resolved on a 1.2% ethidium bromide-agarose gel.

 
Peripheral organs were also assayed for TRH-R gene expression by RT-PCR. PCR products of the predicted size were obtained from the hypothalamus, testis, adrenal gland, and thymus (Fig. 2EGo). The most intense signal was generated by the thymus. The partial sequence of this amplified fragment, obtained by DNA cloning, was identical to that of the TRH-R gene. The other tissues or organs studied (intestine, heart, lung, and epididymis) gave no signal for either TRH or the TRH-R genes by RT-PCR (Fig. 2FGo). Amplification with the G3PDH primers demonstrated that the DNA mixtures could be amplified efficiently by PCR.

The amounts of TRH mRNA were estimated by competitive PCR (Fig. 3Go). A competitive PCR fragment whose length differed from that of the natural target PCR product was generated by deleting the clone pGEM-TRH of a short fragment (Fig. 3AGo). Known quantities of the TRH-del insert were added to PCR amplification reactions containing the experimental cDNA samples (Fig. 3BGo). The hypothalamus contained 1.086 attomoles (amol)/µg TRH mRNA, the testis contained 0.357 amol/µg mRNA, the thymus contained 0.066 amol/µg mRNA, and the adrenal gland contained 0.018 amol/µg mRNA.

Reverse phase HPLC analysis of adult rat thymus and spleen extracts gave the same two major peaks of immunoreactive material. The first peaks had the same retention times as synthetic TRH (26 min), and the second, which cross-reacted with the Ps4 antiserum, eluted earlier than synthetic Ps4 (Fig. 4Go). This second peak was not further characterized because of the small amount available. The amounts of TRH in the thymus (0.42 fmol/mg protein) and spleen (0.47 fmol/mg protein) were similar.



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  		Figure 4. Fractionation of TRH- and Ps4-immunoreactive peptides extracted from the rat thymus (A and B) and spleen (C and D) on a Lichrospher OD2 HPLC column. Eluted TRH (A and C) and Ps4 (B and D) immunoreactivities were monitored by enzyme immunoassay on an aliquot of each of the 0.75-ml fractions. The column was postcalibrated with synthetic TRH and Ps4 (arrows).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although the distribution of TRH in almost all peripheral tissues is well described, little is known about the source of the TRH outside of the central nervous system. TRH immunoreactivity has been detected in almost all of the peripheral tissues tested, with large amounts in the gastrointestinal tract and the reproductive system (4). However, apart from hybridization studies with cDNA probes, TRH cDNA has not yet been cloned from peripheral tissues. We have used PCR coupled with cDNA cloning and sequencing to demonstrate TRH mRNA in extraneuronal tissues and to show that the TRH in the periphery is derived from a precursor molecule similar to hypothalamic prepro-TRH. We have also correlated the mRNA distribution with the distribution of TRH-R mRNA by PCR.

The highest concentration of TRH mRNA was detected in the testis. The testis appears to contain about one third the TRH mRNA as in the hypothalamus. Thus, the testis is one of the main sites of TRH biosynthesis in mammals. However, several points remain obscure. Previous studies using Northern blot analysis detected a prepro-TRH mRNA 0.4 kb larger than that in the hypothalamus. It has been suggested that the larger testicular TRH mRNA results from an extended poly(A)+ tail (18). Three TRH-related peptides have also been detected, including authentic TRH and a [Phe2]TRH-like peptide (19, 27). We found no differences in the 5'- and 3'-untranslated sequences, but our study of the coding region indicates that TRH is generated from a precursor identical to that of the hypothalamus. The biosynthesis of the natural TRH analogs modified in position 2 remains unclear. It has been suggested that the Leydig cells are the only source of authentic TRH in the rat testis, which may act in a paracrine or autocrine fashion. We have confirmed the presence of a mRNA coding for the TRH-R in the testis (28, 29). Two cDNA isoforms of the TRH-R, generated by alternative splicing, have been isolated from GH3 rat anterior pituitary (30). We detected only the longer variant of the receptor.

We have recently described the distribution of TRH immunoreactivity in the adrenal gland and showed that the protein was in the connective mast cells of the adipose tissue surrounding the gland (21). We also found two rat pro-TRH-derived peptides, TRH and Ps4, in extract of rat adrenal glands. This present work confirms that the TRH in rat adrenal gland is synthesized in situ from a precursor similar to that found in the rat hypothalamus. The amount of TRH in mast cells is probably too low to have any endocrine effects on distant tissues, but it could be adequate for local paracrine or autocrine effects. The TRH-R gene is expressed in adrenal glands, as the PCR experiments amplified the coding region of the receptor cDNA.

We have also show that there is TRH mRNA in thymus and spleen. There are significantly higher levels of TRH mRNA in thymus than in spleen. TRH and Ps4 enzyme immunoassays plus HPLC identification provide strong evidence that there are peptides structurally related to TRH and Ps4 in thymus and spleen. However, the Ps4-like material eluted earlier than the authentic Ps4, and the amounts of TRH in the two organs are similar. The conversion of pro-TRH to TRH may be rate limiting, and there may be intermediate steps in the TRH biosynthesis. There seems to be a tissue-specific regulation of the endoproteases involved in the posttranslational activation of pro-TRH in lymphoid organs. The thymus seems to be the main source of TRH-R mRNA outside the pituitary, but we detected no receptor mRNA in the spleen. The physiological function of thymic TRH remains to be established, but Fukusumi et al. (29) suggested that it acts in a paracrine fashion during an early stage in the development of the T cells. Both TRH and T3 can control thymocyte differentiation (31); TRH enhances bromodeoxyuridine uptake by thymus cell suspensions. TRH also causes T cells to make and release TSH, which ultimately increases the production of antibody by B cells (32, 33, 34, 35). The concept of a reciprocal communication between the immune and the neuroendocrine systems is supported by evidence that classical neuroendocrine hormones and their receptors are present in tissues and cells of the immune and inflammatory systems (36, 37). The results presented here show that the TRH gene is active in lymphoid organs, although the amounts of TRH in the thymus and spleen are very small.

The other tissues studied (intestine, heart, lung, and epididymis) contain substantial amounts of TRH (38), but we detected no signals by ethidium bromide staining. Perhaps the TRH gene expression is too low to allow detection of a signal, or perhaps the TRH is released from nerve fibers in these tissues.

In conclusion, our results suggest that TRH acts in an autocrine or paracrine fashion rather than as a true hormone in the testis, adrenal gland, and thymus. The specific humoral regulators of peripheral TRH are not known, but the overall action of hypothalamic TRH, acting via the neuroendocrine system, could be locally modified by TRH synthesized in peripheral tissues.

Received July 30, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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W. Savino and M. Dardenne
Neuroendocrine Control of Thymus Physiology
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