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
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ABSTRACT |
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INTRODUCTION |
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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 (AG) 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
3040% of somatotroph adenomas, dominant somatic mutations of the
Gs gene (gsp), which cause an activated
GTP-bound state leading to constitutive adenylate cyclase induction,
have been identified (22). The mutated Gs
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.
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RESULTS |
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The 5'-flanking region contains a number of other putative response
elements (Fig. 2). 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-
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. 4, 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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DISCUSSION |
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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 (CGGC) 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. 2). 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. 2), 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.
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MATERIALS AND METHODS |
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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 21599 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 2313 was isolated by
restriction digestion with EcoRI and HinfI,
purified and labeled with [-32P]dCTP (Amersham
International, Buckinghamshire, U.K.) by the random primer method. The
probe was used to screen a human placenta
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
2313, residues 419-1067, and residues 10681599 were isolated by
restriction digestion with EcoRI and HinfI from
the phGHRHR/pCRII plasmid, purified and labeled with
[-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
[-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
[
-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 (30120 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was presented in part at the 10th International Congress of Endocrinology, San Francisco, CA, June 1215, 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 669/51.
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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REFERENCES |
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