Structure and Regulation of the Human Growth Hormone-Releasing Hormone Receptor Gene

Stephan Petersenn, Anja C. Rasch, Maria Heyens and Heinrich M. Schulte

IHF Institute for Hormone and Fertility Research (S.P., A.C.R., M.H., H.M.S.) University of Hamburg 22529 Hamburg, Germany
Department of Medicine (S.P.) University of Hamburg 20246 Hamburg, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The GHRH receptor (GHRH-R) acts as a critical molecule for proliferation and differentiation of somatotrophic pituitary cells. A role in the pathogenesis of GH hypersecretion and GH deficiency has been implicated. We investigated structure and regulation of the human GHRH-R gene. A genomic clone including approximately 12 kb of 5'-flanking region was isolated. The gene is of complex structure consisting of more than 10 exons. Two kilobase pairs of the promoter were sequenced, and putative transcription factor binding sites were identified. The transcription start site was defined by ribonuclease protection assay. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 108-1456 bp. GHRH-R promoter (1456 bp) directed high levels of luciferase expression in GH4 rat pituitary cells whereas no activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal 202-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells is enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHRH-R promoter by forskolin, phorbol-myristate-acetate, or T3. Glucocorticoids lead to a significant stimulation, and estrogen leads to a significant inhibition. Further mapping suggests a glucocorticoid-responsive element between -1456 and -1181 and an estrogen-responsive element between -202 and -108. These studies demonstrate the complex nature of the human GHRH-R gene and identify its 5'-flanking region. Furthermore, specific activity of the promoter and regulation by various hormones are demonstrated.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human GH is a major regulator of linear growth and participates in the regulation of protein and fat metabolism. The GH molecule is synthesized, stored, and secreted by somatotrophic cells of the pituitary under control of a variety of hormonal agents, including GH-releasing hormone (GHRH), somatostatin, insulin-like growth factor I, thyroid hormone, and glucocorticoids (1). GHRH is expressed predominantly in the arcuate nuclei of the hypothalamus, but also in cells and tissue outside the brain, where GHRH may have diverse biological activities unrelated to the control of GH secretion (2). It has been found in the placenta (3), where it might participate in the regulation of fetal GH secretion during the embryonic period and is expressed in ovary (4) and testis (5), possibly acting as an intragonadal regulatory factor (6, 7). Furthermore, it has been detected in lymphocytes, where it may be involved in immune modulation (8), and in pancreas and gastrointestinal tract (9, 10), where it possibly regulates the secretion of hormones from both the exocrine and endocrine pancreas (11).

The GHRH receptor (GHRH-R) belongs to the family of Gs-protein-coupled cell surface receptors that activates adenylate cyclase, resulting in increased cAMP levels and activation of protein kinase A (12). Other members of this family include the receptors for secretin, vasoactive intestinal peptide, glucagon, GLP-1, PACAP, and gastric inhibitory peptide. GHRH-R cDNAs from rat (13, 14), mouse (13), swine (15), and humans (14, 16) have been identified. The amino acid alignment deduced from the human cDNA revealed that the GHRH-R consists of 423 amino acids containing seven hydrophobic domains with the potential to serve as membrane-spanning helices. The human GHRH-R gene has been assigned to chromosome 7p14 by in situ hybridization by one group (17) and to chromosome 7p15 by another group (18). It is expressed predominantly in the anterior pituitary gland, as expected based on its functional role in the regulation of GH secretion. It is not clear whether there are separate receptors for GHRH expressed in nonpituitary tissue. Expression of GHRH-R in the pituitary is under the control of the transcription factor Pit-1; neither GHRH-R nor GH is expressed in the dwarf mouse bearing a putative null mutation in the Pit-1 transcription factor (13).

A mutation of the GHRH-R gene has been defined in the lit/lit dwarf mouse, which is characterized by anterior pituitary hypoplasia, a marked decrease in pituitary GH mRNA and protein, and some decrease of PRL. Sequencing of the GHRH-R gene revealed a single nucleotide alteration (A->G) in the second nucleotide of codon 60, predicting an encoded glycine residue rather than an aspartic acid (19, 20). The Asp 60->Gly GHRH-R was functionally defective and unable to regulate intracellular cAMP levels. In situ hybridization analysis revealed no apparent effect during early anterior pituitary development. In contrast, the defective GHRH-R did not allow the continued replication of the somatotrophs in the mature pituitary, leading to a 10-fold decrease in somatotroph cells (20). Therefore, it has been suggested that initial somatotroph stem cell proliferation is not under control of a cAMP-dependent signal transduction system. As somatotrophs proliferate centrally in the mature anterior pituitary, GHRH is required for continous cell replicaton. Similiar to the lit/lit dwarf mouse, a mutation of the GHRH-R has been identified in humans with severe GH deficiency (21). The Glu72Stop mutation described would be expected to produce a severely truncated GHRH-R protein lacking any of the membrane-spanning regions or the G-protein-binding site.

Mutations in the GHRH-R may also play a role in GH excess and pituitary tumorigenesis. The sequence of events leading to pituitary adenoma formation is not completely understood. Several studies have shown that most pituitary tumors are monoclonal in origin and may therefore result from an intrinsic defect that results in either activation of a cell stimulator or inactivation of an inhibitor of cell proliferation. In 30–40% of somatotroph adenomas, dominant somatic mutations of the Gs {alpha} gene (gsp), which cause an activated GTP-bound state leading to constitutive adenylate cyclase induction, have been identified (22). The mutated Gs {alpha} gene mimicks the effects of GHRH on the hormone signaling at the cell membrane. In contrast, the role of exogenic factors is unclear. In transgenic mice, chronic GHRH-R stimulation by GHRH overexpression leads to pituitary tumors (23, 24). Therefore, changes in specific ligand-receptor complexes could lead to hormone hypersecretion and pituitary cell proliferation. Interestingly, in somatotrophic tumors, an up-regulation of GHRH-R mRNA has been demonstrated (25). Recently, activating mutations in related G-protein-coupled receptors have been demonstrated as for the LH receptor in male precocious puberty (26), the TSH receptor in hyperthyroidism (27), and the PTH receptor in metaphyseal chondrodysplasia (28). These data suggest the possibility that the GHRH-R could function as a protooncogene subject to activating mutations in some pituitary adenomas.

In order to facilitate screening for mutations of the GHRH-R in different diseases and to understand regulation of the GHRH-R, we isolated a genomic clone of the GHRH-R and investigated stucture and regulation of the GHRH-R gene.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of a Genomic Clone of the Human (h)GHRH-R Gene
Screening of ~1,000,000-phage clones of a human genomic library with a 5'-hGHRH-R cDNA probe resulted in the isolation of one positive clone. Phage DNA from this positive clone was prepared and digested with NotI restriction enzyme to release the insert. Southern analysis confirmed hybridization of the 5'-hGHRH-R cDNA probe to an approximate 20-kb insert, which could not be separated from an approximate 20-kb {lambda}-arm by gel electrophoresis. Restriction digestion of the genomic clone with NotI and EcoRI released three fragments of approximate 15 kb, 1.6 kb, and 3.5 kb. The genomic fragments were individually subcloned into a pBluescript SKII+ vector and designated p15/SKII, p1.6/SKII, and p3.5/SKII, respectively. After identification with three different cDNA probes (fragments 2–313, 419-1067, 1068–1599) and sequencing part of the subclones, a restriction map of the genomic clone was constructed, as shown in Fig. 1AGo. Probe 2–313 hybridized only to the 15-kb fragment, whereas probe 419-1067 hybridized to all three fragments. Probe 1068–1599 did not hybridize to any of the fragments (data not shown).



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Figure 1. Genomic Organization of the Human GHRH Receptor

Schematic representation of a Not-1/EcoRI restriction map of the isolated genomic clone (a), the deliniated 5'-flanking region and exon-intron structure of the human GHRH-R gene (b), and comparison with the GHRH-R cDNA (c). Drawn in scale. In panel b, the black boxes represent the 10 exons identified between nucleotides +1 and +1025 and are numbered with roman numerals. Introns are represented by a double line, the 5'-flanking sequence by a hatched box. Dashed lines represent genomic sequences that have not been analyzed. In panel c, the white box represents the 5'- untranslated region, and the hatched box represents the coding region of the GHRH-R gene. The open boxes describe the transmembrane regions between nucleotides +1 and +1025 (as described in Ref. 14) and are numbered with roman numerals.

 
Structure of the hGHRH-R Gene
The nucleotide sequence of the coding region was determined on both DNA strands. Intron-exon junctions were determined by DNA sequence comparison with hGHRH-R cDNA. The exons have been numbered, beginning with exon number I containing the 5'-untranslated region. The 15-kb NotI-EcoRI fragment p15/SKII was shown to contain the first five exons. The EcoRI fragment p1.6/SKII contained exon VI; the EcoRI-NotI fragment contained p3.5/SKII exon VI to X (Fig. 1BGo). The 10 exons contained residues 1–1025 of the cDNA of the hGHRH-R (numbering of residues as in Ref.14). Residues 1026–1617 are not included in the isolated genomic clone, as already suggested by Southern blotting. The exons were fully sequenced. The introns were amplified by PCR, and their sizes were determined by agarose gel electrophoresis, except for introns II, VII, and VIII, which were sequenced. The 10 exons defined range in size from 61–154 bp, and introns range from 126 bp to 1.8 kb (Table 1Go). Altogether, the hGHRH-R gene spans more than 8 kb and contains more than 10 exons. Partial nucleotide sequences of the introns revealed no deviations from the consensus sequences for splice site in the 5'-donor and 3'-acceptor splice site (Table 1Go), and all intron-exon splice junctions followed the GT/AG rule (29). Exon I is composed of the 5'-untranslated region as well as the first 19 amino acids. hGHRH-R genomic DNA matched the cDNA sequences (as shown in Refs. 14 and 16) except for the first few nucleotides of the 5'-untranslated region and two nucleotides (CG->GC) at +583/584 (14), encoding alanine instead of arginine at position 178. The locations of the coding regions for the first five transmembrane regions of the hGHRH-R, in comparision to the intron-exon boundaries, are indicated in Fig. 1CGo.


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Table 1. Intron-Exon Junctional Sequences of the Human GHRH-R Gene

 
Analysis of the 5'-Flanking Sequence and Transcriptional Start Site
p15/SKII contains approximately 12 kb of 5'-flanking region of the hGHRH-R gene. The nucleotide sequence of 2 kb of the 5'-flanking region was determined on both DNA strands (Fig. 2Go). The hGHRH-R transcription start site was determined by ribonuclease (RNase) protection analysis using total RNA extracted from human somatotroph pituitary tumors. We generated a 32P-labeled probe consisting of exon I and the immediate 5'-flanking genomic sequence. Figure 3Go shows a major 101-nucleotide protected fragment that maps to a cytosine residue located 40 bp upstream of the translation start site (as determined by comparison with the juxtaposed sequence). In the following, this position is defined as the transcription initiation site, position (1) (Fig. 2Go). It represents pos.12 of the cDNA as defined by Mayo (14). Several other bands of less intensity were also detected, which might represent alternative transcription start sites. Furthermore, the design of the probe does not exclude additional major transcription start sites upstream of nucleotide -108. Yeast RNA, used as a negative control, did not yield any protected fragments.



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Figure 2. Nucleotide Sequence of the 2-kb 5'-Flanking Region and 5'-Untranscribed Region

The transcription start site is indicated by an arrow and defined as +1. Nucleotide markers are marked on the left side. Upstream nucleotides have negative numbers. Potential transcriptional regulatory sequences identified by a computer-assisted analysis are underlined. The translation start site ATG is printed in bold.

 


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Figure 3. Determination of the Transcription Start Site of the Human GHRH-R Gene by RNase Protection Analysis

A 209-bp fragment containing the immediate 5'-flanking region of the GHRH-R gene and the first exon was amplified by PCR using S2 and A2 as primers and subcloned into pCRII. The antisense probe was generated by in vitro transcription driven by T7 RNA polymerase in the presence of [{alpha}-32P]CTP after linerization of the construct with Spe1. The 32P-labeled cRNA was hybridized with 4 µg human pituitary total RNA (lane 1) or 10 µg yeast RNA (lane 2 + lane 3) and then digested with RNases A/T1 (lane 1 + 2). Lane 3 shows an undigested probe sample. The protected fragments were resolved juxtaposed to a DNA-sequencing reaction (GATC) of the same probe using primer A2 in a 6% polyacrylamide gel. The autoradiogram shows a major band of 101 bp marked by the lower arrow in lane 1 that maps the transcription start site to position -40 bp with respect to the translation start site. Some minor bands appeared after longer exposure time, which may describe alternative transcription start sites. Control yeast RNA in lane 2 did not generate any protected bands. The purified probe in lane 3 is denoted by the upper arrow. Lane 4 was loaded with a 100-fold dilution of sample 1.

 
No potential element that is required for accurate initiation of transcription, including the TATA box, CCAAT box, or an initiator sequence, is evident in the appropriate location in the sequence of the 5'-flanking region (Fig. 2Go). One GC box motif is located around nucleotide -210.

The 5'-flanking region contains a number of other putative response elements (Fig. 2Go). These include two putative binding sites for the enhancer factor AP-1 (activator protein-1) at bp -991 and -609 and one for AP-4 at bp -28, the consensus sequences for the nuclear factor NF-1 at bp -355, -294, and -107, and a binding site for the upstream stimulatory factor USF at bp -1406. Furthermore, several binding sites for tissue-specific transcription factors, such as the POU-domain factors Pit-1 at bp -1009, -799, and -127, and Brn-2 at bp -700, were identified. In addition, the promoter region contains consensus motifs corresponding to inducible promoter elements that are known to bind transcription factors induced by exogenous stimuli. These include binding sites for the transcription factors CREB [cAMP-response element (CRE)-binding protein] at bp -483, estrogen receptor at bp -888 and -317, and NF-{kappa}B at bp -429.

Transient Expression Analysis of the 5'-Flanking Region
To determine whether the hGHRH-R 5'-flanking region can direct cell-specific expression, the proximal 108 bp and 1456 bp were inserted into a transient expression vector, pGL2-Basic, which contains luciferase as the reporter gene, and the resulting plasmids (-108 hGHRHR/luc and -1456 hGHRHR/luc) were transiently transfected into various cultured cell lines. Gene transfer studies were done by calcium-phosphate transfection, and luciferase enzyme activity was measured in light units as an indication of promoter activity. Cells were cotransfected with pSV-ß-GAL as an internal control for transfection efficiency. As shown in Fig. 4Go, 1456 bp of the hGHRH-R promoter directed high levels of luciferase expression in GH4 rat pituitary cells as compared with the promoterless pGL2-Basic luciferase vector. In contrast, we observed no significant activity of -1456 hGHRH-R/luc in chorion carcinoma cells JEG3 or monkey kidney cells COS-7. One hundred eight base pairs of the hGHRH-R promoter directed no significant activity in any of the cell lines tested. Relative activity of pGL2-Control in COS-7, JEG3, and GH4 cells was 1217-fold, 370-fold, and 59-fold, respectively. The finding that hGHRH-R promoter activity is restricted to pituitary cells demonstrates that 1456 bp of the hGHRH-R 5'-flanking region are sufficient to direct appropiate cell-selective expression in transient transfection analysis.



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Figure 4. Promoter Activity of the GHRH-R 5'-Flanking Region in Various Cell Lines

The -108 hGHRHR/luc (open bars) and -1456 hGHRHR/luc (hatched bars) constructs were transfected in parallel with pGL2-Basic, which lacks promoter activity, into GH4 rat pituitary cells, COS-7 monkey kidney cells, and JEG3 chorion carcinoma cells. Cotransfection with CMV-ß-galactosidase was used to control for transfection efficiency. The luciferase activity of each construct was normalized with the ß-galactosidase activity, and values were expressed as fold induction relative to the activity of the promoterless construct pGL2-Basic. Values represent the mean ± SEM of at least three determinations.

 
Elements Required for hGHRH-R Promoter Activity Are Located within 202 bp of the Transcription Initiation Site
To further analyze the 5'-flanking region of the hGHRH-R gene for constitutive promoter activity in GH4 rat pituitary cells, varying lengths of 5'-flanking regions created by PCR were placed upstream of the luciferase reporter gene (left panel of Fig. 5Go). Only background activity was obtained with the construct containing 108 nucleotides 5' to the major transcription initiation site (right panel of Fig. 5Go). A 13-fold increase in activity was observed with the construct containing 202 bp of 5'-flanking region. Another increase in activity was observed with the construct containing 276 bp. With constructs between -276 bp and -1013 bp, the level of luciferase activity was nearly constant, about 27- to 36-fold. The construct containing 1181 bp produced a 58-fold increase in activity compared with the promoterless control; 1456 bp of promoter produced a similiar activity of 50-fold. These results suggest that the regions between -108 and -202, between -202 and -276, and between -1013 and -1181 contain important positive regulator of hGHRH-R expression in GH4 rat pituitary cells. The negative control, pGL2-Basic without any 5'-flanking region, caused very low luciferase activity.



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Figure 5. Promoter Activity of Various Deletion Constructs of the GHRH-R 5'-Flanking Region

The schematic diagram on the left represents a series of hGHRH-R promoter-luciferase gene chimeric plasmids with variable 5'-ends (from -1456 to -108) and the same 3'-end (+24). Each construct was transiently transfected into GH4 rat pituitary cells. Promoter activity is normalized for transfection efficiency by the ß-galactosidase activity and is expressed relative to the activity of the pGL2-Basic control (right panel). Data are the means ± SEM of at least three independent experiments performed in triplicate.

 
The hGHRH-R Promoter Is under Control of the Pituitary-Specific Transcription Factor Pit-1
Selective expression of the hGHRH-R 5'-flanking region in GH4 pituitary cells suggest that an interaction between pituitary-specific factors and the hGHRH-R promoter is required for expression of the hGHRH-R. The expression of POU-domain transcription factor Pit-1 is strictly pituitary-specific and absolutely required for transcription of the hGHRH-R gene in vivo (13). COS-7 monkey kidney cells do not produce any significant amount of Pit-1. hGHRH-R 5'-promoter deletion constructs transiently transfected into COS-7 cells were used to determine the effect of Pit-1 cotransfection and the approximate location of the Pit-1-responsive element(s) in the hGHRH-R promoter. As shown in Fig. 6Go, cotransfection of Pit-1 did not enhance the activity of the construct containing 108 bp of 5'-flanking region compared with the mock cotransfected cells. In contrast, cotransfection of Pit-1 to any of the larger constructs ranging from 202-1456 bp of 5'-flanking region resulted in markedly higher promoter activity. These results suggest that a Pit-1-responsive region is localized upstream of position -108.



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Figure 6. Effect of Pit-1 Cotransfection on the Activity of Various Deletion Constructs of the GHRH-R 5'-Flanking Region

The indicated series of GHRH-R promoter deletions were cotransfected with (hatched bars) or without (open bars) pCMV-hpit1 transiently into COS-7 monkey kidney cells, as described in Materials and Methods. Promoter activity is normalized for transfection efficiency by the ß-galactosidase activity and is expressed relative to the activity of the pGL2-Basic control, respectively. Results are the mean ± SEM of three transfections.

 
Activity of the hGHRH-R Promoter Is Inversely Regulated by Glucocorticoids and Estrogen
To investigate the hormonal regulation of the hGHRH-R 5'-flanking region, we also analyzed the effect of various agents on 1456 bp of hGHRH-R promoter in GH4 rat pituitary cells. As shown in Fig. 7Go, treatment with 10-6 M forskolin, 10-7 M phorbol-myristate-acetate (TPA), and 10-9 M thyroid hormone (T3) did not significantly influence activity of the hGHRH-R promoter. In contrast, treatment with 10-7 M hydrocortisone significantly enhanced activity of 1456 bp of hGHRH-R 5'-flanking region whereas we did not observe any significant effect on 108 bp of promoter. In initial studies we did not observe any significant effect of estrogen on a control promoter that included several estrogen-responsive elements (data not shown), suggesting that GH4 pituitary cells do not express significant amounts of intact estrogen receptor. Therefore, cotransfection studies using a vector encoding the human estrogen receptor were performed. As shown in Fig. 7Go, treatment with 10-9 M ß-estradiol significantly inhibited 1456 bp of hGHRH-R promoter in GH4 rat pituitary cells, whereas 108 bp were not significantly influenced.



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Figure 7. Hormonal Regulation of the Human GHRH-R Promoter

Two different GHRH-R deletion constructs, -108 hGHRH-R/luc (open bars) and -1456 hGHRH-R/luc (hatched bars), and the promoterless vector pGL2-Basic (closed bars) were transiently transfected into GH4 rat pituitary cells. Regulation by various agents was tested by treatment with 10-6 M forskolin (For), 10-7 M TPA, 10-7 M hydrocortisone (HC), 10-9 M triiodothyronine (T3), and 10-9 M ß-estradiol (E2). To determine the ß-estradiol effect, 1.5 µg pER were cotransfected to the indicated GHRH-R constructs. Activity is expressed as fold induction relative to that driven by each construct transfected alone in the absence of treatment and represents the mean ± SEM of three independent experiments.

 
We next performed 5'-deletion analysis to identify specific sequences that regulate hormone-induced promoter activity. Plasmids containing progressively decreasing amounts of hGHRH-R 5'-flanking region upstream of the luciferase gene were transiently transfected into GH4 rat pituitay cells, with or without hormonal treatment, and promoter activity was measured by luciferase assay. As shown in Fig. 8AGo, deletion of sequences from bp -1456 to -1181 significantly impaired induction of the GHRH-R promoter by glucocorticoids. These results suggest that the GHRH-R promoter contains a glucocorticoid-responsive element between bp -1456 and -1181. Progressive deletion of sequences from bp -1456 to -1181, from bp -1181 to -1013, from bp -1013 to -809, from bp -809 to -276, and from bp -276 to -202 did not significantly impair estrogen-inhibited promoter activity (Fig. 8BGo). Further deletion from bp -202 to -108 considerably diminished inhibition of the promoter by estrogen. These results indicate that the region between bp -202 and -108 contains an estrogen-responsive element.



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Figure 8. Mapping of the cis Elements Required for Activation of the Human GHRH-R Promoter by Dexamethasone (A) and Inhibition of the Human GHRH-R Promoter by Estrogen (B)

A, Glucocorticoid regulation was mapped by transfecting 3 µg of the indicated GHRH-R promoter deletion constructs or the pGL2-Basic vector into GH4 pituitary cells followed by treatment with 10-7 M dexamethasone. B, Sex steroid regulation was mapped by cotransfecting 3 µg of the indicated GHRH-R promoter deletion constructs or the pGL2-Basic vector with 1.5 µg pER into GH4 pituitary cells followed by treatment with 10-9 M ß-estradiol. The total DNA transfected was fixed using nonspecific DNA. Activity is expressed as fold induction relative to that driven by each construct transfected alone in the absence of treatment and represents the mean ± SEM of three independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To gain insight into the developmental and differential regulation of the GHRH-R, characterization of the 5'-promoter elements is essential. We now present data regarding the structure and regulation of the human GHRH-R gene. Abstracts demonstrating the genomic structure have been presented (30, 31, 32, 33, 34), but a systematic study of the regulation of the GHRH-R gene has not yet been described.

In contrast to other G protein-coupled receptors, such as the receptors for somatostatin, the GHRH-R gene is of complex genomic structure including more than 10 exons. It spans more than 8 kb; the average size of its exons in the region investigated is approximately 100 bp. The intron/exon structure of the human gene reported here is very similiar to the structure of the murine gene as reported in Ref. 20. Consensus sequences for splice donor and acceptor sites are present at all intron-exon boundaries. Unfortunately, screening of ~1,000,000-phage clones of a human genomic library with a 5'-hGHRH-R cDNA probe resulted in the isolation of only one positive clone. Screening with a probe specific for the 3'-region has been unsuccessful so far. As the isolated clone misses the 3'-end of the GHRH-R gene, we cannot comment on the exon structure encoding the residues +1026 to +1617 of the GHRH-R cDNA. In an abstract presented recently (30) the hGHRH-R gene was shown to span at least 12 kb and to consist of 13 exons. Some data about the gene structure of the 3'-region can be delineated from reports describing splicing variants of the GHRH-R. These have been found in the rodent (14), mouse (13), swine (15), and humans (35, 36). Three variant forms of the GHRH-R, which originate through different splicing of the intron X located at +1025/1026, have been demonstrated (36). The first transcript is generated by complete splicing of this intron; the second transcript contains the entire unspliced intron of 561 bp; and the third transcript results from utilization of the normal donor and an alternative acceptor signal in the intron, which removes a 123-bp intronic sequence. The alternatively processed intron at +1025/1026 possesses numerous stop codons. Therefore, the second transcript results in a protein coded out of frame from the 326th amino acid for 12 additional residues, and the third transcript gives rise to a protein truncated at the 325th amino acid. The authors have not yet reported on the functional significance of these transcripts. The second splicing variant has also been demonstrated by another group in some somatotrophic adenomas that were derived from patients who did not respond to GHRH (35). By cell transfection studies, these authors showed that the truncated receptor could not transduce the signal stimulated by GHRH. The genomic clone identified by us includes approximately 200 bp of the 561-bp insert described (data not shown), which confirms the notion of an alternative splicing mechanism for generation of this fragment. The locations and sizes of exon VIII to exon X reported by Tang et al. (36) by amplification of genomic DNA are in agreement with our data.

The isolation of a genomic human GHRH-R clone has allowed for the characterization of its transcription start site and identification of the proximal promoter sequence. In eukaryotes, the key step in gene regulation is the process of transcriptional initiation mediated by site-specific DNA-binding proteins. Neither TATA nor other initiator motifs were evident in the 2-kb 5'-flanking region upstream of the translation start site. In TATA-less genes, the mechanism of transcriptional initiation and its regulation are not uniformally established. In general, GC-rich domains and initiator elements have been proposed to act cooperatively to direct gene transcription (37). GC-rich promoters, found primarily in housekeeping genes, usually contain several trancription start sites spread over a fairly large region and several potential binding sites for the transcription factor Sp1. Other TATA-less promoters are not GC-rich and initiate transcription at only one or a few tightly clustered start sites (38). Many of these latter type of promoters, including the promoter for the terminal deoxynucleotidyl transferase gene, are regulated during differentiation or development (39). Due to the low GC content and the absence of a TATA element in the upstream region, the GHRH-R promoter most likely belongs to the same promoter class as the terminal deoxynucleotidyl transferase promoter, which would fit with the lineage-restricted and developmentally regulated expression pattern of the GHRH-R.

Putative binding sites for several transcription factors that are known to bind on the GH promoter and are responsible for its basal activity were also identified on the GHRH-R promoter. Similiar to the GHRH-R, GH is specifically expressed in somatotroph pituitary cells. The upstream stimulatory factor (USF) has been found in pituitary cells and shown to bind on the GH promoter (40). Binding of Sp1 on the GH promoter has been demonstrated in GC and HeLa cells, but is inhibited by Pit-1 under in vitro conditions (40). Nuclear Factor 1 (NF-1) was originally identified from HeLa cells as a DNA-binding protein required for efficient adenovirus replication (41). It is capable of binding to the GH promoter in a pituitary cell line (42). AP-2 is a transcription factor that binds in a mutually exclusive character on the NF-1 binding site of the GH promoter (42). Activator proteins are involved in the basal control of promoters, e.g. the metallothionein-IIA gene mediating transcriptional activation by phorbol esters and cAMP (43).

Several sequences related to consensus binding sites for the POU-domain transcription factors, Pit-1, Oct-1, and Brn-2, may play an important role in the tissue-specific expression of the GHRH-R gene. The transcription factor Pit-1 is specifically expressed in the pituitary and capable of activating both PRL and GH promoter in nonpituitary cells (44, 45). The octamer-binding protein Oct-1 differs in its ubiquitous tissue distribution and its ability to activate certain eukaryotic promoters that lack a TATA box (46), but is also coexpressed in cells of the anterior pituitary. The neuron-specific transcription factor Brn-2 is expressed in a distinct spatial and temporal pattern in the brain (47). Pit-1 can bind as a heterodimer with the widely expressed Oct-1 protein to critical tissue-specific cis-active elements in the rat PRL gene (48). It has been suggested by the authors that a combinatorial pattern of heterodimeric interactions between different members of the POU-domain gene family potentially regulates differential developmental gene regulation. The functional significance of these promoter elements remains to be determined.

RNase protection analysis indicate that a major transcription start site begins 40 nucleotides upstream from the ATG initiating codon. The sequence of the cDNA reported previously (14) contains 51 nucleotides upstream of the ATG codon. The reason for a nucleotide difference between the RNA analysis experiment and cDNA cloning is unknown. It might reflect alternative transcription start sites, as indicated by several less intense bands in the RNase protection assay, or may be due to electrophoresis conditions. The design of the probe for the RNase protection assay allowed only for detection of transcription start sites located up to 148 bp 5' to the translation start site. Therefore, we cannot exclude additional transcription start sites further upstream. Primer extension analysis did not give any results regarding determination of the transcription start site, possibly indicating low abundance of the receptor or secondary structures hindering extension. In 15/SK a few nucleotides of the 5'-untranslated region and two nucleotides (CG->GC) at +583/584 (numbering as in Ref.14) are different from those found in the GHRH-R cDNA as reported in Ref. 14 but match closely the sequence reported in Ref. 16. These nucleotide differences are probably due to alteration by processing of the GHRH-R gene and sequencing error, respectively.

GHRH acts on the GHRH-R in the anterior pituitary to stimulate the synthesis and secretion of GH. Furthermore, GHRH has been implicated in control of appetite (49), sleep (50, 51), and medullary oxygenation of the kidney (52). High expression of GHRH-R was demonstrated in the pituitary by Northern blot analysis and RNase protection assay, whereas no expression was found in liver, stomach, intestine, brain, ovary, testis, kidney, muscle, heart, or placenta (13, 14, 16). Matsubara et al. (52) detected expression of GHRH-R in the renal medulla of the rat by Northern blot analysis; however, no expression was found in renal cortex, renal pelvis, and liver by the same technique. Using a more sensitive RT-PCR technique, the same investigators decribe low-level expression of GHRH and GHRH-R in a variety of rat tissues, including heart, lung, duodenum, small intestine, spleen, adrenal gland, epididymis, and skeletal muscle. Therefore, GHRH may act as a paracrine or autocrine factor in these tissues. Only GHRH was detected in ovary, testis, and placenta; only GHRH-R was detected in the cerebellum, thyroid gland, colon, renal cortex, and ureter. Neither GHRH nor GHRH-R mRNA was detected in stomach, liver, and adipose tissue (52). Interestingly, Takahashi et al. (53) describe expression of the GHRH-R in the hypothalamus, as analyzed by RNase protection assay, which could indicate an ultrashort feedback mechanism by GHRH, and explain the effect of GHRH on feeding behavior. In our studies we did not find any significant activity of 1.4 kb of 5'-flanking region of the GHRH-R gene in a chorion carcinoma cell line and a monkey kidney cell line. Similiarly, the endometrium cell line Skut-1B did not allow for sufficient activity of the GHRH-R promoter (data not shown). In contrast, we found high activity of the hGHRH-R promoter in the pituitary cell line GH4. These findings suggest a strong tissue-specific regulation of the GHRH-R gene, but we cannot exclude that elements in introns or 5' of the investigated promoter region may allow for expression in other tissues. Alternatively, the transient transfection assay used may not be sensitive enough to detect significant activity in the nonpituitary cell lines examined.

Our studies show that 202 bp of 5'-flanking region contain element(s) that support gene expression preferentially in a pituitary cell line, whereas a minimal 108-bp promoter did not allow for considerable transcription. Expression of the GHRH-R gene has been shown to depend on the presence of Pit-1 (13). Putative binding sites for Pit-1 have been identified in the promoter of the human GHRH-R at position -1009, -799, and -127 (Fig. 2Go). In contrast to GH4 cells, COS-7 cells do not show any detectable presence of Pit-1. The transcriptional activity of the GHRH-R promoter in COS-7 cells is approximately 20-fold lower than in GH4 cells. Cotransfection of a Pit-1 expression vector did not change activity of the -108GHRHR/luc construct in COS-7 cells, but the activity of constructs containing at least 202 bp of 5'-flanking region was enhanced by Pit-1. The Pit-1-binding site at location -127 may therefore be important for the pituitary-specific expression of the GHRH-R; further experiments must verify this hypothesis. Lin et al. (13) demonstrated a 5- to 10-fold stimulation of 1.4 kb of the mouse GHRH-R promoter by rat Pit-1 in CV-1 cells without further comment on the promoter sequence (13). The lower stimulation in our studies may demonstrate species differences or result from the experimental design (e.g. different reporter vectors).

Results from several systems suggest that the GHRH-R gene may be under regulatory control. Therefore, the hormonal regulation of the GHRH-R promoter was studied. Regulation of the GHRH-R by GHRH itself has been demonstrated. Passive immunization to GHRH induced a marked reduction in pituitary GHRH-R mRNA in neonatal rats (54); therefore, GHRH might be essential during a critical period in the ontogeny of the GHRH-R. In contrast, in primary rat pituitary cells a dose-dependent decrease in GHRH-R mRNA was observed after treatment with GHRH, as assayed by quantitive RT-PCR (55). Bilezikjian et al. (56) reported a 50% reduction in GHRH-binding sites after incubation with GHRH in vitro. Miki et al. (57) report that GHRH-R mRNA levels were significantly increased by immunoneutralization of endogenous GHRH with its specific antiserum. These authors suggest that GHRH inhibits the production of its receptor by a receptor-mediated, cAMP-dependent reduction of GHRH-R mRNA accumulation. The promoter of the GHRH-R contains a putative CRE at position -483 for binding of the transcription factor CREB (Fig. 2Go), which could transduce the signaling cascade induced by GHRH to the GHRH-R promoter. GH4 pituitay cells do not possess any endogenous GHRH-R; therefore we could not test the direct effects of GHRH on GHRH-R promoter activity. Forskolin is an activator of protein kinase A, which is an essential element of the signaling cascade regulated by the GHRH-R. Aleppo et al. (55) describe inhibition of GHRH-R mRNA accumulation by forskolin similiar to GHRH. We did not find any significant regulation of 1.4 kb of 5'-flanking region of the GHRH-R gene by forskolin. We cannot exclude that physiologically important CREs may be located 5' of the analyzed promoter region or in any of the numerous introns. Alternatively, GHRH might regulate GHRH-R mRNA levels by controlling degradation of the mRNA. Interestingly, a reduced responsiveness of the GHRH-R, both in terms of GH secretion and activation of the adenylyl cyclase, is observed in aging (58). Qing et al. (59) found similar changes after passive immunization of male rats with supramaximal doses of GHRH antiserum and suggest that reduction of endogenous GHRH priming of its receptors induces a lower responsiveness of the same receptors, and that these alterations present in aging are related to a deficiency of the endogenous neurohormone secretion.

An alternative signal transduction pathway in somatotrophic pituitary cells controlling GHRH-R expression may involve activation of protein kinase C. Activation of protein kinase C by diacylglycerol analogs and phorbol esters stimulated the release of GH from anterior pituitary cells (60). In contrast, French et al. (61) did not find any significant alteration of protein kinase C activity and translocation in somatotrophs by GHRH, but describe the presence of protein kinase C in somatotrophs. To investigate the effect of protein kinase C activation, we used TPA as an activator of the protein kinase C pathway. In our cell transfection studies we did not observe any significant changes of GHRH-R promoter activity by TPA using the -1456GHRHR/luc construct in GH4 pituitary cells.

Glucocorticoids act at multiple levels of the GH pathway (62). GHRH-binding sites in dispersed pituitary cells from adrenalectomized rats are down-regulated, and hormone replacement with dexamethasone restores GHRH binding (63). Miller and Mayo (64) describe decreased GHRH-R mRNA levels in adrenalectomized rats that increase after corticosterone treatment. GHRH-R expression is elevated in anterior pituitary cells after treatment with dexamethasone (64, 65), suggesting a direct effect of glucocorticoids on GHRH-R expression at the level of the pituitary. Lam et al. (66) demonstrated increased GHRH-R mRNA levels in dexamethasone-treated rats. A search of 2 kb of 5'-flanking region of the GHRH-R vs. TFMATRIX did not identify any putative glucocorticoid receptor response elements by homology comparison. In contrast, our studies of a transient expression system indicate a positive glucocorticoid responsive element located between bp -1456 and -1181. We therefore suggest an interaction of the glucocorticoid receptor with the GHRH-R gene to regulate its expression, possibly explaining the ability of glucocorticoids to enhance pituitary responsiveness to GHRH. NF-1 is known to be involved in the glucocorticoid-dependent stimulation of transcription of the mouse mammary tumor virus (MMTV) promoter (67), and similiar interactions of Sp1 with the glucocorticoid receptor have been demonstrated (68). Synergism of the glucocorticoid receptor with jun homodimers has been demonstrated to activate AP-1-regulated promoter lacking glucocorticoid-responsive elements (69). Several AP-1-binding sites have been identified on the GHRH-R promoter at location bp -991 and -609. Further analysis will identify the glucocorticoid-responsive elements within the GHRH-R gene. Physiologically, a single administration of dexamethasone causes an early stimulatory effect on GH secretion in normal subjects, followed by a late inhibitory effect (70). Long time exposure to glucocorticoids exerts a growth-suppressive effect (71). Children chronically treated with glucocorticoids show decreased growth rates; patients with hypercortisolism have almost lost the ability to secrete GH. The overall growth-suppressive effect of glucocorticoids in contrast to its positive actions on both GHRH-R and GH within the pituitary is indicative of its complex actions at multiple levels of the GH axis.

Thyroid hormones are essential for growth in mammals. Like glucocorticoids, thyroid hormone affects the GH axis at different levels (72). Thyroid hormone enhanced somatotrope sensitivity to GHRH in vitro (73). Thyroidectomy in rats decreased pituitary GHRH-R mRNA by 60%, this decrease could be partially reversed by T4 replacement therapy (74). We did not observe any significant regulation of 1456 bp of 5'-flanking region of the GHRH-R gene by thyroid hormone. The intactness of thyroid hormone receptors in GH4 cells was demonstrated by significant stimulation of the GH promoter by thyroid hormone (data not shown). A search of 2 kb of 5'-flanking region of the GHRH-R vs. TFMATRIX did not identify any putative thyroid hormone receptor-response elements. The mechanisms of GHRH-R mRNA regulation by thyroid hormone remain to be further investigated.

GH secretion exhibits marked sexual dimorphism in many species (75), apparently depending on the presence of GHRH (76). The growth rate in rats is gender-specific, with male rats growing faster and bigger than female rats. Sex differences are observed at various levels of the GH axis, but the greatest gender difference has been reported for the levels of pituitary GHRH-R mRNA: the female level is only 15% of the male value in rats (77). In a transient expression system we observed significant inhibition of the GHRH-R promoter by ß-estradiol. Our studies suggest an negative estrogen-responsive element located between bp -202 and -108. Down-regulation of GHRH-R expression in females by estrogen could explain the observed gender differences in GHRH-R mRNA levels and may play a role in the sex differences in growth. A search of the 5'-flanking region of the GHRH-R gene vs. TFMATRIX identified putative estrogen receptor-response elements that are located at bp -317 and -888 and therefore cannot transduce the estrogen effect to the minimal responsive 202-bp promoter. A number of different mechanisms for estrogen-receptor mediated gene regulation in the absence of a classic hormone-responsive element have been described (78, 79). Interactions between nuclear factors and the transcription factor Sp1 can modulate promoter activity (80). Widely spaced, directly repeated PuGGTCA elements (81) and a new subclass of Alu DNA repeats (82) can function as estrogen receptor-dependent enhancers. The precise estrogen-responsive elements within the GHRH-R gene must be identified by further analysis.

In summary, our initial characterization of the promoter region of the human GHRH-R gene demonstrates that this gene contains a TATA-less promoter region with specific activity in a pituitary cell line. A number of transcription factor-binding sites were identified by sequence homology. Regulation of the GHRH-R promoter by glucocorticoids and estrogen was shown. Further study is necessary and will provide insights into the mechanism that regulates the expression of the GHRH-R gene. Analysis of structure and regulation of the GHRH-R gene provides tools to investigate the relevance of the GHRH-R in diseases with GH excess or GH deficiency.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Material
Forskolin, TPA, hydrocortisone, T3, and ß-estradiol were purchased from Sigma Chemical Co. (St. Louis, MO).

Plasmids
The plasmid construct pCMV-hpit1 contains the full-length human Pit-1 cDNA under control of the cytomegalovirus (CMV)-1 promoter. Plasmid pER contains the human estrogen receptor-coding region under control of the herpes simplex virus thymidine kinase (HSV TK) promoter and a CMV enhancer (83). Plasmid pGL2-Basic is a luciferase vector lacking eukaryotic promoter and enhancer sequences (Promega Corp., Madison, WI). pGL2-Control contains an SV40 promoter and an SV40 enhancer inserted into the structure of pGL2-Basic (Promega Corp.). pSV-ß-GAL contains an SV40 promoter and an SV40 enhancer. Both promoter and enhancer drive transcription of the lacZ gene, which encodes the ß-galactosidase enzyme (Promega Corp.).

Isolation of GHRH-R cDNA Probe and Screening of Genomic DNA Library
Total RNA was extracted from a human somatotrophic pituitary tumor. Total RNA (0.5 µg) was reverse transcribed and amplified by the polymerase chain reaction (PCR) using a GeneAmp RNA PCR Kit (Perkin Elmer, Norwalk, CT). The DNA fragment containing residues 2–1599 of the hGHRH-R cDNA (numbering of residues as in Ref.14) was amplified (95 C 20 sec, 65 C 30 sec, 72 C 30 sec, 40 cycles) using S1 (5'-GCA-GCC-AAG-GCT-TAC-TGA-GGC-TGG-TGG-AGG-3') and A1 (5'-CAG-GTT-TAT-TGG-CTC-CTC-TGA-GCC-TTG-GGC-3') as primers. The PCR product was fractionated on a 0.8% agarose gel and subsequently cloned into pCRII (phGHRHR/pCRII) using the TA-Cloning Kit (Invitrogen, San Diego, CA). Sequencing analysis confirmed the identity of the amplified DNA. A DNA fragment containing residues 2–313 was isolated by restriction digestion with EcoRI and HinfI, purified and labeled with [{alpha}-32P]dCTP (Amersham International, Buckinghamshire, U.K.) by the random primer method. The probe was used to screen a human placenta {lambda} FIXRII genomic DNA library (Stratagene Corp, La Jolla, CA). Approximately 1 x 106 recombinant phage plaques were screened with 1 x 107 cpm/ml of the probe. Prehybridization was performed for 4 h at 42 C in 50% formamide, 2x piperazine-N,N'-bis[2-ethane-sulfonic acid] (PIPES) (10x PIPES being 0.8 M NaCl, 0.02 M PIPES, pH 6.5), 0.5% SDS, and 100 µg/ml denatured sonicated salmon sperm DNA. The prehybidization solution was exchanged against fresh solution for hybridization. The probe was denatured at 100 C for 4 min before being added to the hybridization solution at 42 C overnight. The filters were then washed twice in 0.1x SSC, 0.1% SDS at 60 C and exposed to Kodak XAR-5 film. Positive recombinant plaques were purified by replating twice and grown in liquid culture. Phage DNA was prepared with a QIAGEN Lambda Midi Kit (QIAGEN GmbH, Hilden, Germany).

Subcloning of Phage DNA and Mapping by Southern Blot
The phage DNA was digested with various restriction enzymes and separated on a 0.7% agarose gel. cDNA fragments containing residues 2–313, residues 419-1067, and residues 1068–1599 were isolated by restriction digestion with EcoRI and HinfI from the phGHRHR/pCRII plasmid, purified and labeled with [{alpha}-32P]dCTP (Amersham International) by the random primer method. Genomic fragments were mapped by hybridization with different probes (hybridization as above, Southern blotting as described in Ref.84). Subsequentely, genomic fragments were purified by QIAEX Gel Extraction Kit (QIAGEN GmbH) and subcloned into Bluescript SKII+ Vector (Stratagene Corp). Plasmids were prepared by QIAGEN Plasmid Maxi Kits (QIAGEN GmbH).

Nucleotide Sequence Determination
Double-stranded plasmid DNA was sequenced by fluorescent sequencing using dye-labeled terminators (ABI PRISM Dye Terminator Cycle Seqencing Ready Reaction Kit, PE Applied Biosystems, Warrington, U.K.) and Applied Bioystems instrumentation. Sequences were assembled using Lasergene computer software (DNASTAR, Madison, WI). To avoid errors, all sequences were determined by sequencing both strands of the DNA. The nucleotide sequence data reported in this paper have been submitted to Genbank and assigned the accession number AF029342. Transcription factor-binding sites were identified using TFSEARCH on the internet, which searches sequence fragments vs. TFMATRIX, the transcription factor-binding site profile database, by E. Wingender, R. Knueppel, P. Dietze, and H. Karas (GBF-Braunschweig).

Determination of Exon/Intron Gene Structure
Individual exon/intron boundaries were determined by a loss of identity between the genomic and cDNA nucleotide sequence and also by the presence of consensus donor and acceptor signals at the point of divergence. The size of introns was determined by PCR amplification using exon-derived oligonucleotide primers with the exception of introns II, VII, and VIII, which were determined by sequencing.

Ribonuclease Protection Assay
A DNA fragment spanning 209 nucleotides 5' of position 112 (numbering of residues as in Ref.14) was obtained by PCR amplification (95 C 20 sec, 58 C 30 sec, 72 C 30 sec, 30 cycles) of the genomic clone using a GeneAmp PCR Kit (Perkin Elmer, Norwalk, CT) and primers S2 (5'-CCC-TTG-GCT-AGC-TCC-TGC-CTA-TG-3') and A2 (5'-ATA-CGG-TCG-GTA-ACG-GGC-TC-3'). The PCR product was fractionated on a 0.8% agarose gel and subsequently cloned into pCRII using the TA-Cloning Kit (Invitrogen). Sequencing analysis using T7 primer determined the identity and orientation of the amplified DNA. The recombinant plasmid was linearized with SpeI, and transcribed with [{alpha}-32P]CTP (Amersham International) to generate 32P-labeled cRNA probes, using T7 RNA polymerase of the MAXIscript Transcription Kit (Ambion Inc., Austin, TX). Riboprobes were purified by electrophoresis through an 5% denaturing polyacrylamide gel. The full-length band was excised, and the riboprobes were eluted in probe elution buffer (0.5 M ammonium acetate, 1 mM EDTA, 0.2% SDS) by shaking at 37 C overnight. Approximately 4 µg of human pituitary total RNA were hybridized to 1 x 105 cpm of the probe in hybridization puffer (80% formamide, 100 mM sodium citrate, pH 6.4, 300 mM sodium acetate, pH 6.4, 1 mM EDTA) overnight and then treated with RNase A/RNase TI (5 µg/ml RNase A/20 U/ml RNase TI) for 30 min at 37 C, following the protocol for the Ambion RPA II Kit (Ambion Inc.). After RNase inactivation and precipitation of the protected probe fragments, the samples were analyzed on a 6% denaturing polyacrylamide gel. A sequence ladder of the riboprobe template was obtained using the A2 primer. Double-stranded DNA sequencing was performed by the dideoxy chain termination method using [{alpha}-35S]dATP and the Sequenase 2.0 DNA Sequencing Kit (USB Corp., Cleveland, OH). Autoradiography was performed at -80 C using Kodak XAR-5 films.

Construction of Luciferase-Expression Vectors Containing Upstream Sequence
Upstream sequences were obtained by amplification of the genomic clone using A3 (5'-GCT-CCC-TCC-ACC-AGC-CTC-AGT-AAG-3', starting at position +24) as antisense primer and S3 (5'-GGT-TCT-AGC-TTT-CCC-TTC-A-3', starting at position -1456), S4 (5'-AAC-CCC-TGC-TGA-TGT-CAA-AAT-AAG-3', starting at position -1181), S5 (5'-TGG-GAT-ATT-CAG-GTC-TTT-CA-3', starting at position -1013), S6 (5'-TGT-CCT-CCC-CCT-ATT-CAA-GA-3', starting at position -809), S7 (5'-TGG-GCC-CTT-GCT-ATC-AGG-ACA-GA-3', starting at position -276), S8 (5'-GCT-CCT-GCC-TGC-TGG-AAA-CAG-AG-3', starting at position -202), and S9 (5'-CCC-TTG-GCT-AGC-TCC-TGC-CTA-TG-3', starting at position -108) as sense primers (numbering of residues, as shown in this paper). The PCR products were fractionated on a 1.0% agarose gel. Fragments of correct size were subsequently cloned into pCRII using the TA-Cloning Kit (Invitrogen). Sequencing analysis confirmed the identity and orientation of the amplified DNA. DNA fragments were isolated by restriction digestion with KpnI and NotI, purified by QIAEX Gel Extraction Kit (QIAGEN GmbH), and inserted upstream of the luciferase reporter gene into pGL2-Basic mammalian expression vector (Promega Corp.). Plasmids were prepared by QIAGEN Plasmid Maxi Kits (QIAGEN GmbH).

Cell Culture, Transient Transfection, Luciferase Assay, and ß-Galactosidase Assay
Rat pituitary GH4, monkey kidney cells COS-7, and human chorion carcinoma JEG3 cells were grown in DMEM (GIBCO BRL, Grand Island, NY) containing 10% FCS (Serva, Heidelberg, Germany). Cells were maintained at 37 C in 5% CO2. Cells (5 x 105)/well were seeded in six-well plates for transfection. The medium was changed 3 h before transfection. Experimental and control plasmids were mixed and transfected in triplicates by CaPO4-DNA coprecipitation. Transfections included 3 µg reporter gene construct and 2 µg pSV-ß-GAL as an internal control of transfection efficiency. For cotransfection studies, 1.5 µg pCMV-hPit1 or 1.5 µg pER were added, respectively. The total amount of DNA was maintained constant with nonspecific DNA. After 16 h in the presence of DNA, cells were shocked for 2 min at room temperature with 15% glycerol in PBS, and then serum-free DMEM containing 3% BSA was replaced. Cells were harvested 64 h after transfection in lysis buffer (Promega Corp.). The luciferase assay was performed in a final volume of 120 µl, containing 20 µl cell extract, following the protocol for the Promega Luciferase assay system (Promega Corp.). Luciferin was added just before measurement of light units, which were measured during the first seconds of the reaction at 25 C in a Luminometer. The ß-galactosidase assay was performed following the protocol for the Promega ß-galactosidase assay system (Promega Corp.). Cell extract (50 µl) was incubated with 50 µl assay buffer until color developed (30–120 min), and the reaction was stopped by adding 150 µl 1 M Na2CO3. Absorbance was then read at 405 nM. Luciferase light units were normalized to the activity of ß-galactosidase. Data are expressed as the mean ± SEM. All experiments were repeated at least three times.


    ACKNOWLEDGMENTS
 
We wish to thank Dr. Gabriel DiMattia for the Pit-1 expression vector pCMV-hpit1; Dr. R. J. Miksicek for the estrogen receptor expression vector pER; and Dr. B. Gellersen for helpful discussion.


    FOOTNOTES
 
Address requests for reprints to: Dr. S.Petersenn, IHF Institute for Hormone and Fertility Research, University of Hamburg, Grandweg 64, 22529 Hamburg, Germany.

This work was presented in part at the 10th International Congress of Endocrinology, San Francisco, CA, June 12–15, 1996.

This work is based in part on the doctoral study by A.R. performed at the Faculty of Biology, University of Hamburg, and was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG) Schu 669/5–1.

Dedicated to Prof. Dr. F. A. Leidenberger on the occasion of his 60th birthday.

Received for publication September 8, 1997. Revision received October 16, 1997. Accepted for publication October 29, 1997.


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 DISCUSSION
 MATERIALS AND METHODS
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