Homeobox Protein Gsh-1-Dependent Regulation of the Rat GHRH Gene Promoter
Noriko Mutsuga,
Yasumasa Iwasaki,
Minako Morishita,
Atsushi Nomura,
Etsuko Yamamori,
Masanori Yoshida,
Masato Asai,
Nobuaki Ozaki,
Fukushi Kambe,
Hisao Seo,
Yutaka Oiso and
Hidehiko Saito
First Department of Internal Medicine (N.M., M.M., A.N., E.Y.,
M.Y., M.A., N.O., Y.O., H.S.), Department of Clinical Laboratory
Medicine (Y.I.), Nagoya University School of Medicine and Hospital,
Nagoya, Japan 466-8560; and Research Institute of Environmental
Medicine (F.K., H.S.), Nagoya University, Nagoya, Japan 464-8601
Address all correspondence and requests for reprints to: Yasumasa Iwasaki, M.D., Ph.D., Department of Clinical Laboratory Medicine, Nagoya University School of Medicine and Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan, E-mail: iwasakiy{at}med.nagoya-u.ac.jp
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ABSTRACT
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Although GHRH is known to play a pivotal role in the
regulation of the GHRH-GH-IGF-I axis, the molecular mechanism of GHRH
gene expression has not yet been examined. Here we studied the
transcriptional regulation of the GHRH gene 5'promoter using an
in vitro experimental model system. We especially
focused on the role of homeobox transcriptional factor Gsh-1, because a
dwarf phenotype and abolished GHRH expression was observed in Gsh-1
knockout mice. First, we cloned human Gsh-1, which showed 87.3%
homology with mouse Gsh-1 at the nucleotide level. When the 5'-promoter
region of the rat GHRH gene was introduced into the human placental
cell line JEG-3, in which we found the endogenous expression of Gsh-1
as well as GHRH mRNA, substantial transcriptional activity of the
promoter was recognized. Promoter activity was further enhanced by
overexpression of Gsh-1 protein, whereas it was substantially reduced
by elimination of Gsh-1 binding sites. EMSA confirmed the actual
binding of Gsh-1 on the multiple binding sites of GHRH gene promoter.
Finally, coexpression of CREB-binding protein significantly enhanced
the Gsh-1-induced GHRH gene expression, suggesting the cooperative role
of the coactivator protein. Because Gsh-1 is found to be expressed in
the hypothalamus of the adult rat, our data provide evidence that the
Gsh-1 homeobox protein plays a key role in the expression of the GHRH
gene.
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INTRODUCTION
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SOMATIC GROWTH IN the mammalian organism is
controlled by a complex regulatory system that originates in the
hypothalamus, from which two peptides important for the regulation of
pituitary somatotroph cells are released: GHRH and somatostatin
(1). Of these, GHRH facilitates GH secretion from the
pituitary gland, which in turn enhances the production of IGF-I in the
liver or other tissues, mediating a variety of somatic and/or metabolic
effects of GH (2). Thus, GHRH is a key regulatory factor
in the positive control of the function of GH-IGF-I axis. GHRH also
plays an important role in the proper proliferation and differentiation
of the somatotroph through the development of the pituitary gland
(3).
GHRH was first isolated from human pancreatic tumors (4, 5), and since then intensive physiological studies regarding the
hormone have been carried out. Its cDNA and gene in various species
have been cloned as well (6, 7, 8). Nevertheless, there has
been virtually no study attempting to elucidate the transcriptional
regulation of GHRH gene expression. This may be due, in part, to the
lack of availability of appropriate host cells that would allow the
expression of the GHRH gene promoter. Recently, however, it has been
reported that genetic ablation of a homeobox gene Gsh-1 showed a dwarf
phenotype in the mouse, in which no expression of the GHRH gene in the
arcuate nucleus of the hypothalamus was reported (9, 10).
This raises the possibility that Gsh-1 is an indispensable
transcriptional factor for the tissue-specific expression of the GHRH
gene, and those cells expressing Gsh-1 may be adequate for studying the
GRH gene expression.
In this study, we first investigated the transcriptional regulation of
the GHRH gene, using the JEG-3 human placental cell line. We found that
endogenous Gsh-1 as well as GHRH mRNA was expressed in the cells, and
that a substantial level of GHRH gene promoter activity was observed
when the GHRH 5'-promoter-luciferase fusion gene was introduced.
Furthermore, we obtained evidence showing that Gsh-1 is indeed a key
transcription factor for maintaining the expression of the GHRH
gene.
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RESULTS
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Cloning of the Human Gsh-1 cDNA
We first cloned the human homolog of Gsh-1 cDNA. By screening the
human fetal brain cDNA library with mouse Gsh-1 gene as a probe, we
isolated the cDNA clone of human Gsh-1. When the obtained sequences
were compared with the mouse cDNA, 87.3% and 96.6% homology were
observed at the nucleotide and protein levels, respectively (Fig. 1
, A and B; GenBank accession nos.
AB044157 and AB044158 for human Gsh-1 cDNA and gene). The gene
structures (two exons and one intron) were also similar between the
mouse and human (data not shown), indicating that the homeobox protein
Gsh-1 is a highly conserved transcriptional factor among the
species.

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Figure 1. Comparison of the Sequences of Mouse and Human
Gsh-1 cDNA and Proteins
A, Alignment of the human and mouse Gsh-1 cDNA. Numbers in the
right column correspond to nucleotide residues.
Asterisks indicate residues identical to human Gsh-1. B,
Alignment of the predicted protein sequences of human and mouse Gsh-1.
Numbers in the right column correspond to amino acid
residues. Asterisks indicate residues identical to human
Gsh-1.
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Analyses of GHRH and Gsh-1 mRNA Expression
We then screened the expression of Gsh-1 and GHRH mRNA by RT-PCR
in various endocrine cell lines and adult rat tissues. We found that
both mRNAs were coexpressed in the human placental cell line JEG-3
(Fig. 2A
) and in the hypothalamus (Fig. 2B
) and the testis (not shown in the figure). Although Gsh-1 expression
was detected in some other cell lines and tissues without GHRH
expression, we could not see any GHRH mRNA without Gsh-1 expression
(Table 1
). These results suggest that the
homeobox gene Gsh-1 is expressed in adult as well as fetal tissues, and
that the presence of Gsh-1 is necessary, although not sufficient, for
GHRH expression. In addition, the intrinsic expression of both Gsh-1
and GHRH in JEG-3 cells suggests that the cell line is suitable for
studying the transcriptional regulation of the GHRH gene.

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Figure 2. Expression of Gsh-1 and GHRH mRNA in Human
Placental Cell Line JEG-3 and the Rat Hypothalamus Analyzed by RT-PCR
A, The amplified DNA fragments with the predicted lengths (381 bp and
331 bp for the human Gsh-1 and GHRH mRNA, respectively) derived from
JEG-3 cells using specific primer sets for the human Gsh-1 and GHRH. B,
The amplified DNA fragments with the predicted lengths (381 bp and 214
bp for the rat Gsh-1 and GHRH mRNA, respectively) derived from the
adult rat hypothalamic tissues using specific primer sets for the rat
Gsh-1 and GHRH. M refers to DNA markers. Control reactions
without RT showed no amplifications (data not shown).
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Transcriptional Activity of GHRH Gene 5'-Promoter in JEG-3 or
Neuronal Cell Lines Expressing Gsh-1
When the rat GHRH gene 5'-promoter (-472 to +71; +1 designates
the hypothalamic transcription start site)-luciferase fusion gene was
introduced into the JEG-3 cells, substantial promoter activity was
observed compared with that of the promoterless plasmid (Fig. 3A
). To confirm whether the promoter
activity may be attributable to the presence of Gsh-1, we examined the
expression of the promoter in mouse hypothalamic progenitor cell lines
with or without Gsh-1 (Gsh+/+ and Gsh-/-; kindly provided by Dr.
Potter) (14). The Gsh+/+ cell line, which intrinsically
expresses Gsh-1, was derived from the hypothalamus of Gsh-1-SV40
T-antigen transgenic mice, and the Gsh-/- cell line, which lacks
Gsh-1, was derived from the hypothalamus of Gsh-1 knockout mice
(14). Again, the GHRH gene promoter activity was more than
4-fold higher in Gsh+/+ cells than in Gsh-/- cells (Fig. 3B
).
Furthermore, when Gsh-1 protein was coexpressed with GHRH in JEG-3
cells, the promoter activity was further enhanced in a dose-dependent
manner (Fig. 4
). Altogether, our data
strongly suggest that the homeobox protein Gsh-1 plays a key role in
the expression of the GHRH gene.

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Figure 3. Gsh-1-Dependent Expression of the GHRH Gene
5'-Promoter
A, JEG-3 cells were transfected with GRH472Luc or a
promoterless plasmid (pA3Luc), and the promoter activities were
determined by luciferase assay. *, P < 0.01
vs. the promoterless plasmid. B, Gsh-1+/+ and Gsh-1-/-
cells were transfected with GRH472Luc, and the promoter
activities were determined by luciferase assay. *,
P < 0.001 vs. Gsh-1-/- cells. In
both experiments, ß-galactosidase expression vector was used as an
internal control.
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Figure 4. Dose-Dependent Effect of Gsh-1 Overexpression on
the GHRH Gene 5'-Promoter in JEG-3 Cells
JEG-3 cells were transfected with GRH472Luc and various
amounts of the Gsh-1 expression vector, and the GHRH 5'-promoter
activities were determined by luciferase assay. *,
P < 0.05 vs. the control group (C)
without Gsh-1 coexpression. In both experiments ß-galactosidase
expression vector was used as an internal control.
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Binding of Gsh-1 Protein to the 5'-Promoter Region of GHRH
An analysis of the nucleotide sequences of the GHRH gene
5'-promoter region revealed five putative binding sites of Gsh-1
[consensus sequence:
GCT/CA/C
ATTA G/A] (9)
(see legend of Fig. 5
). To see whether
the Gsh-1 protein binds to some or all of the sequences, we carried out
an EMSA using glutathione-S-transferase (GST)-Gsh-1 fusion
protein. As shown in Fig. 5
, a DNA-protein complex was observed when
Gsh-1 protein was treated with the DNA fragments containing four
(G2G5) of the five sequences analyzed (lanes 3, 6, 9, and 14). No
binding was obtained with GST protein without Gsh-1 (lanes 2, 5, 8, and
13). All of the gel shift entities formed by GST-Gsh-1 fusion protein
were antagonized when incubated with 100 molar excess of each unlabeled
DNA fragment, but not with a mutated sequence (M). Furthermore,
32P-labeled probe M failed to bind to GST-Gsh-1
(lane 12). Addition of antibody to Myc-tag caused a supershift and/or
displacement, confirming the specificity. Altogether, these data
suggest that Gsh-1 indeed binds to at least four sites in the
5'-promoter region of the GHRH gene.

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Figure 5. Binding Analysis of Gsh-1 Protein to the
5'-Promoter Region of the Rat GHRH Gene
A, Localization of the Gsh-1 binding sites in the rat GHRH 5'-promoter.
The boxed sequences indicate the putative binding
elements based on the Gsh-1 consensus sequence
(GCT/CA/C ATTA
G/A) (9 ). Sequences below
each box are the mutated one used in EMSA or in promoter
analysis (Fig. 6 ). B, EMSA of Gsh-1 binding sites. In the left
panel, the 32P-labeled fragments spanning
approximately 20 bp of each Gsh-1 binding site were incubated with GST
(lanes 2, 5, 8, and 13) or GST-Gsh-1 fusion protein (lanes 1, 3, 4, 6,
7, 912, and 1423). The mutated probe of G4 (M) was used in lanes 11
and 12. Nonlabeled fragments were used as a competitor (lanes 4, 7, 10,
and 15). In the right panel, each reaction mixture was
incubated for another 60 min with anti-Myc tag antibody (lanes 17, 19,
21, and 23).
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Deletion/Mutation Analyses of the Gsh-1 Binding Sites of the GHRH
Gene Promoter
To confirm that some or all of the Gsh-1 binding sites are
functional, transcriptional activity of deleted or mutated promoter was
analyzed in JEG-3 cells. As shown in Fig. 6A
, the GHRH promoter activity
significantly decreased when all the Gsh-1 binding sites were
eliminated (GRH47Luc), whereas deletion of G1 to
G4 had no effect. Furthermore, introduction of mutation in an
individual binding site (mG1mG5) caused no effect (Fig. 6B
), whereas
the promoter activity dramatically decreased when all binding sites
were mutated (mG15) (Fig. 6C
). These results suggest that alternative
binding of one or more Gsh-1 proteins contribute to maintain the
transcriptional activity of the GHRH gene.

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Figure 6. Deletion/Mutation Analyses of the GHRH Gene
Promoter
A, JEG-3 cells were transfected with full-length
(GRH472Luc) or deleted promoter constructs
(GRH304Luc, GRH247Luc, GRH187Luc,
GRH133Luc, GRH47Luc) with a constant amount of
Gsh-1 expression vector (RSV-Gsh-1). G1G5 represents putative Gsh-1
binding sites. *, P < 0.05 vs.
GRH472Luc. B, JEG-3 cells were transfected with a
full-length construct (GRH472Luc) or constructs mutated in
each of the Gsh-1 binding sites (mG1, mG2, mG3, mG4, and mG5) with a
constant amount of RSV-Gsh-1. X represents the mutated sites. C, JEG-3
cells were transfected with wild-type (GRH472Luc) or the
mutant construct in which all of the putative Gsh-1 binding sites were
mutated (mG15). *, P < 0.001 vs.
GRH472Luc. In all experiments, ß-galactosidase expression
vector was used as an internal control.
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Functional Cooperation of Gsh-1 with CREB-Binding Protein in the
Regulation of the GHRH Gene
Finally, we examined whether Gsh-1 functions by interacting with
some other transcription factors or cofactor(s). Specifically, we
focused on the coactivator protein CBP, because some of the
transcription factors determining tissue-specific expression (such as
Pit-1) have been shown to interact with CBP (15). We found
that again, expression of Gsh-1 markedly enhanced the promoter activity
of GHRH. Furthermore, coexpression of CBP significantly enhanced the
effect of Gsh-1, although expression of CBP alone had no effect (Fig. 7A
). In the mG15 construct in which all
of the Gsh-1 binding sites are mutated, CBP had no enhancing effect
either, suggesting that the binding of Gsh-1 is necessary for Gsh-1/CBP
synergism (Fig. 7B
). In addition, when each individual Gsh-1 binding
site (G2G5) was located in a heterologous promoter (thymidine kinase
minimal promoter), no synergistic effect of Gsh-1 and CBP was observed
(Fig. 8
).

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Figure 7. Synergistic Activation of Gsh-1 and CBP on the GHRH
Gene Promoter
A, JEG-3 cells were cotransfected with GRH472Luc and the
Gsh-1 and/or CBP expression vectors, and the GHRH 5'-promoter
activities were determined by luciferase assay. B, JEG-3 cells were
cotransfected with the mG15 construct and the Gsh-1 and/or CBP
expression vectors, and the GHRH 5'-promoter activities were determined
by luciferase assay. ß-Galactosidase expression vector was used as an
internal control. *, P < 0.01 vs.
GRH472Luc alone. **, P < 0.05
vs. GRH472Luc+Gsh-1.
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Figure 8. Effects of Gsh-1/CBP Coexpression in Gsh-TK-Luc
Constructs
A, Structure of the Gsh-TK-Luc construct. Each Gsh-1 binding site
(G2G5) in the GHRH 5'-promoter was isolated and located in front of
the TK minimal promoter-luciferase gene. B, JEG-3 cells were
cotransfected with each Gsh-TK-Luc construct and the Gsh-1 and/or CBP
expression vectors, and the promoter activities were determined by
luciferase assay. ß-Galactosidase expression vector was used as an
internal control. *, P < 0.05 vs.
each Gsh-TK-Luc activity without CBP/Gsh-1.
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DISCUSSION
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Although more than 10 yr have passed since the GHRH gene with its
promoter region was cloned (6, 7), virtually no report has
emerged dealing with its transcriptional regulation of GHRH. In fact,
previous efforts have been hampered mainly by the lack of a homologous
cell line that would be appropriate for studying the tissue-specific
regulation of the GHRH gene. Recent findings of the Gsh-1 transcription
factor of the homeobox type (9, 10), however, prompted us
to search for appropriate host cells expressing the homeobox gene, and
we finally found the GHRH gene promoter to be well expressed in some
placental and neuronal cell lines in which Gsh-1 is endogenously
expressed.
Tissue specificity of the expression of the hypothalamic or pituitary
hormone genes is, in many cases, determined by the existence of
specific transcription factors. For example, the expression of GH, PRL,
and TSH genes are largely dependent on the POU domain
transcription factor Pit-1 (16, 17, 18), and CRH and
vasopressin by homeobox protein Brain-2 (19, 20).
Regarding GHRH, the work by Potter and associates (9, 10)
using homologous recombination techniques strongly suggests that Gsh-1
is the key determinant of GHRH gene expression. Our data obtained in
this study support the notion, showing that Gsh-1 is a prerequisite for
maintaining the transcriptional activity of the GHRH gene. Indeed, the
promoter activity was well maintained in the JEG-3 and Gsh+/+
hypothalamic cell lines, both of which express Gsh-1 (14),
whereas much less activity was observed in the Gsh-/- cell line.
Overexpression of Gsh-1 in JEG-3 further enhanced the promoter
activity, indicating the dependency of GHRH gene expression on the
homeobox protein. Moreover, RT-PCR analysis showed Gsh-1 mRNA
expression to be present in any cell line or tissue that expressed GHRH
mRNA. On the other hand, expression of Gsh-1 is not always accompanied
by that of GHRH, and, in fact, short-term expression of Gsh-1 alone did
not rescue the promoter activity of GHRH gene in Gsh-/- or other cell
lines (data not shown). We assume that some additional Gsh-1-dependent
transcriptional factor(s) is also needed to maintain the expression of
the GHRH gene.
In the 5'-promoter region of the GHRH gene we examined, five putative
Gsh-1 binding sites were recognized through the homology search
technique. The results from the EMSA method showed that Gsh-1 actually
binds to most of the consensus sequences in the promoter. The binding
characteristics of each site may be somewhat different, because a clear
supershift was observed in G2 and G3 whereas the binding was disrupted
in G4 and G5. In any event, the functional significance of the binding
was supported by the deletion analysis, which showed a marked decrease
in the basal transcriptional activity when all the binding sites were
eliminated. Induction of mutation into each binding site, however, did
not affect the promoter activity, whereas induction into all sites
markedly decreased the promoter activity of the GHRH gene, suggesting
that alternative Gsh-1 binding is necessary and sufficient to maintain
the promoter activity. A similar regulatory pattern is reported in the
rat PRL gene promoter, in which four Pit-1 binding sites exist and
function to maintain the transcriptional activity (21, 22).
In this study, we also showed the positive role of CBP, a coactivator
protein that is ubiquitously expressed in the nucleus of most cells.
Specifically, the promoter activity of the GHRH gene was found to be
maximal when CBP and Gsh-1 were coexpressed, suggesting the positive
cooperation between the two factors. Cohen et al.
(15) reported the direct interaction between Pit-1 and CBP
in the regulation of the GH gene promoter, and that a similar mode of
regulation may be occurring in the promoter of GHRH. Our efforts,
however, have so far failed to demonstrate the protein-protein
interaction between the two factors using GST pull-down assay or EMSA
(using full-length, in vitro translated CBP and GST-Gsh-1
fusion protein), or the yeast two-hybrid system (using fragments of CBP
and Gsh-1) (data not shown). Thus, some additional unknown factor(s)
may be necessary to reconstitute the CBP interaction to Gsh-1 in
vitro. This hypothesis is supported by our data showing that
Gsh-1/CBP synergy was not observed when each Gsh-1 binding site alone
was located in the heterologous promoter.
Homeobox proteins usually play an important role in development and
organogenesis during the embryonic stage. In fact, Gsh-1-deficient mice
show agenesis of GHRH neurons in the arcuate nucleus with the dwarf
phenotype (10). However, our results showing the
expression of Gsh-1 mRNA in the hypothalamus of the 7-month-old rat
strongly suggest that Gsh-1 protein exists and plays an important role
in the brain of mature animals.
We carried out most of the experiments in this study using the JEG-3
placental cell line. Although the cells have endogenous GHRH mRNA and
peptide, expression of GHRH is believed to be regulated mainly
by placenta-specific alternative promoter (23). However,
the construct we used in our experiments includes only the hypothalamic
promoter region and does not contain the placenta-specific region,
indicating that the regulation observed does not reflect the
placenta-specific promoter. Instead, the results are likely to show the
hypothalamic mode of regulation because of the Gsh-1-dependent
regulation occurring in the hypothalamic promoter region.
Finally, the dwarf phenotype of Gsh-1 knockout mice suggests that some
human familial dwarfism may be caused by mutation of the Gsh-1 gene.
Since we cloned the human homolog of the Gsh-1 gene, the nucleotide
sequence of which is now available, it is an additional possible
candidate for disease-causing genes in dwarf patients in which
no mutation is found in GHRH, GHRH receptor, or GH genes. Further
research may add a novel type of genetic dwarfism in the future.
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MATERIALS AND METHODS
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RT-PCR
Expression of Gsh-1 and GHRH in various cell lines and adult rat
tissues (see Table 1
) was examined by the RT-PCR method. Total RNA was
isolated from the monolayer culture of the cells or tissues of the
adult Sprague Dawley (Chubu Science Materials, Nagoya, Japan)
rat using TRIzol reagent (Life Technologies, Inc.,
Gaithersburg, MD), and 1 µg each was applied for each reaction with
specific primer sets: sense, 5'-GACAAGAAGGCTCCGGAGGG-3', and
antisense, 5'-CAGCTGGTTCGAGCTGCTGT-3' for the human/rat Gsh-1 mRNA;
sense, 5'-GGGTGTTCTTCTTTGTGATCC-3', and antisense,
5'-TCACAGGAGGAATCTTCATC-3' for the human GHRH mRNA; sense,
5'-TATGCAGATGCCATCTTCACC-3', and antisense,
5'-TCAAGCCTCCGCTGAAAGCT-3' for the rat and mouse GHRH mRNA. The
primer sets for GHRH span exons 35 and thus amplify generic GHRH
transcripts. An RNA sample without reverse transcriptase was used as a
negative control.
Plasmid Constructions
The 5'-flanking region of the rat GHRH gene (-472 to +71; +1
designates the transcription start site), kindly provided by Dr. Kelly
E. Mayo (Northwestern University, Evanston, IL) (7), was
fused with the luciferase reporter gene in the pA3Luc plasmid
(GRH472Luc). Deletion mutants of various lengths
of the promoter were constructed as well
(GRH304Luc, GRH247Luc,
GRH187Luc, GRH133Luc,
GRH47Luc). The mutated
GRH472Luc constructs (mG1, mG2, mG3, mG4, mG5,
and mG15) (see Fig. 5
for details) were constructed by the PCR-based
site directed mutagenesis technique (24). The Gsh-TK-Luc
constructs (G2-TK-, G3-TK-, G4-TK-, and G5-TK-Luc) were constructed by
insertion of annealed oligonucleotides in the pFlashII plasmid
(SynapSys Co., Burlington, MA) containing thymidine kinase (TK) minimal
promoter: sense, 5'-GGCCGAAATATAATTACTAGG-3', and antisense,
5'-GATCCCTAGTAATTATATTTC-3' for G2; sense, 5'-GGCCGATTCAACATTATATTG-3',
and antisense, 5'-GATCCAATATAATGTTGAATC-3' for G3; sense,
5'-GGCCGGGTCTAAATTAGTGGG-3', and antisense, 5'-GATCCCCACTAATTTAGACCC-3'
for G4; sense, 5'-GGCCGTTTCCCCATTACTTTG-3', and antisense,
5'-GATCCAAACTAATGGGGAAAC-3' for G5. Mouse Gsh-1 cDNA was isolated
from mouse genomic DNA and subcloned into pRC/RSV (Rous sarcoma virus)
mammalian expression vector (Invitrogen, Groningen, The
Netherlands). Sequence analyses were performed for all constructs made
by PCR. Dr. Toshihiro Nakashima (University of Tsukuba, Tsukuba,
Ibaragi, Japan) kindly provided the CBP expression vector
(RSV-CBP).
Cell Culture and Transfection
The human placental cell line JEG-3 and the mouse
hypothalamic neuronal cell lines (Gsh+/+, Gsh-/-) were grown in DMEM
supplemented with 10% FBS. Cultures were maintained at 37 C in a humid
atmosphere of 5% CO2. In each experiment, cells
were plated in 3.5-cm dishes with 70% confluency (
2 x
106 cells per dish), and the plasmids used (3
µg of test plasmid(s) and 0.3 µg of ß-galactosidase expression
vector as an internal control per dish) were transfected with TransIT
LT-100 (Mirus, Madison, WI). In Gsh-1 or CBP coexpression studies,
0.30.6 µg of Gsh-1/CBP expression plasmids or empty vector
(promoter alone) was used so that the total amount of expression vector
was equal.
Forty-eight hours after transfection, cells were harvested with
lysis buffer containing 1% Triton X-100 and centrifuged, and the
supernates were used for luciferase and ß-galactosidase assays.
Luciferase assay was performed as previously described
(25). Briefly, 100 µl of each supernatant were added to
400 µl of reaction buffer, and the reactions were started by the
injection of 200 µl of luciferin solution containing 0.2
mM D-luciferin (Sigma, St. Louis,
MO). Light output was measured for 20 sec at room temperature using a
luminometer (Lumat LB 9501, EG&G Berthold, Oak Ridge, TN).
ß-Galactosidase assay was determined using commercially available
assay kits (Galacto-Light Plus Kit, Tropix, Inc., Bedford, MA) based on
the manufacturers instructions. Luciferase activities were divided by
the ß-galactosidase activities to correct for the transfection
efficiency.
Preparation of Fusion Protein
Mouse Gsh-1 cDNA was incorporated into pcDNA 3.1/myc-His (+) A
plasmid (Invitrogen). After the myc-tagged cDNA was
subcloned into pGEX 4T-2 expression vector (Amersham Pharmacia Biotech, Piscataway, NJ) to produce GST- and myc-tagged fusion
protein, the protein product was subsequently purified using
glutathione-sepharose 4B beads (Amersham Pharmacia Biotech). Western blot of the tagged protein using anti-Myc
antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
showed a single band in the appropriate size. The purified fusion
protein was used in EMSA as shown below.
EMSA
To assay protein binding, synthetic double-stranded
oligonucleotides encompassing the Gsh-1 binding sequences of the rat
GHRH 5'-promoter region (G1: sense, 5'-TGAACAAAACAGAATTATAGGG-3',
antisense, 5'-AGTTCCCTATAATTCTGTTTTG-3'; G2: sense,
5'-TGGTAAATATAATTACTAGA-3', antisense, 5'-AGAGTCTAGTAATTATATTT-3'; G3:
sense, 5'-TAGTCATTCAACATTATATTC-3', antisense,
5'-TGAACAGAATATAATGTTGAATG-3'; G4: sense,
5'-GTATGGGTCTAAATTAGTGGG-3', antisense,
5'-TGCCCCCACTAATTTAGACCC-3'; G5: sense, 5'-CGACTTTTCCCCATTAGTTTG-3',
antisense, 5'-CACGCAAACTAATGGGGAAAA-3') were labeled with
[
32P] deoxy-CTP (Amersham Pharmacia Biotech) and DNA polymerase (Klenow enzyme, New England Biolabs, Inc., Beverly, MA). The labeled fragment (25 fmol,
40,000 cpm) was then incubated with 12 µg of GST gsh-1-Myc fusion
proteins in the EMSA binding buffer (40 mM HEPES, pH 7.9,
75 mM KCl, 0.2 mM EDTA, 10% glycerol, 1
mM dithiothreitol) and 2 µg poly dI-dC (Amersham Pharmacia Biotech) for 15 min on ice. Pure GST protein
was used as a control. For competition assays, a 100-fold molar excess
of nonlabeled probe was added to the reaction solution. For supershift
analysis, anti-Myc antibody (Santa Cruz Biotechnology, Inc.) was added to the reaction and incubated for additional 60
min on ice. The mixture was then subjected to 4% nondenaturing
polyacrylamide gel, and the electrophoresis was run at 160 V with
cooling for 4 h. The gels were dried and subjected to
autoradiography by exposing the gel to Kodak X-AR film
(Eastman Kodak Co., Rochester, NY).
Data Analysis
Samples in each group of the experiments were in triplicate. All
experiments were repeated three times at independent times, and the
data are presented as mean ± SE of the averaged data
from the three experiments. When statistical analyses were performed,
data were compared by one-way ANOVA with Fishers protected least
significant difference test, and P values of <0.05 were
considered significant.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Drs. Kelly E. Mayo (Northwestern University,
Evanston, IL) and Steven S. Potter (University of Cincinnati,
Cincinnati, OH) for providing the rat GHRH gene and Gsh+/+, -/-
cells, respectively. We also thank Dr. Toshihiro Nakashima (Tsukuba
University, Tsukuba, Japan) for providing the CBP expression
vector.
 |
FOOTNOTES
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This work was supported in part by the Novo Nordisk Growth Award (to
Y.I.), Japan.
Abbreviations: CBP, CREB-binding protein; GST,
glutathione-S-transferase; RSV, Rous sarcoma virus; TK,
thymidine kinase.
Received for publication December 29, 2000.
Accepted for publication August 29, 2001.
 |
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