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First Department of Internal Medicine, Gunma University School of Medicine, Maebashi 371, Japan
Address all correspondence and requests for reprints to: Teturou Satoh, M.D., Ph.D., First Department of Internal Medicine, Gunma University School of Medicine, 3–39-15 Showa-machi, Maebashi 371, Japan.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epidermal growth factor (EGF), a growth factor initially isolated from the mouse salivary gland (10), is distributed throughout the body and elicits a number of biological responses for cell differentiation and growth (11). In addition, EGF has been reported to stimulate the synthesis and release of several hormones in the pituitary gland. It has been established that EGF stimulates pituitary PRL synthesis at the level of gene transcription through the serum response element of the PRL gene promoter (12, 13). EGF has also been reported to stimulate TSH secretion from perfused rat pituitary glands (14). The iv administration of EGF stimulates TSH release in vivo (14). Fan et al. recently reported that cold stress induced the expression of EGF and its receptor mRNA in pituitary thyrotrophs in rats, suggesting that EGF may have autocrine and/or paracrine roles in the maintenance of the TSH response to cold (15). Although these findings indicated that EGF may modulate thyroid function at the pituitary level by stimulating TSH release in response to some stresses such as cold exposure, the possibility that TSH secretion is augmented by EGF stimulation of the synthesis and secretion of hypothalamic TRH in vivo cannot be ruled out.
It is well known that cold exposure increases the blood level of TSH
and thyroid hormone by stimulating the secretion of TRH from the median
eminence in the hypothalamus (16). In addition to its stimulatory
effect on TRH release, acute and chronic cold exposure elevates the
cellular level of TRH mRNA (17, 18). Although several
neurotransmitters, such as catecholamines, serotonin, and
-aminobutyric acid, appear to be involved in the cold
exposure-induced TRH release from the median eminence (16), the factors
directly stimulating TRH gene expression by cold remain to be
determined. Several growth factors, including EGF, have been reported
to be synthesized locally in the hypothalamus (19, 20). In response to
stress, EGF has been postulated to function as a stimulator of the
hypothalamic-pituitary-adrenal axis, and its site of action appears to
be at the levels of both the hypothalamus and the pituitary (21, 22).
It has been reported that transforming growth factor-
, a member of
the EGF family, stimulates the release of hypothalamic LHRH (23). Taken
together, these results prompted us to evaluate the possible regulatory
roles of EGF regarding the action of hypothalamic TRH. In the present
study we found that EGF stimulated ppTRH gene expression both in
vivo and in vitro. Transfection studies using
GH4C1 cells revealed that the stimulatory
effects may be mediated by direct activation of gene transcription
through two separate DNA regions in the TRH gene promoter. We also
found that a transcription factor, Sp1, in cooperation with other
transcription factors might mediate the stimulatory effect of EGF on
mouse TRH gene transcription.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RNA isolation and Northern blot analysis
Total RNA was isolated from individual hypothalami, and Northern
blot analysis was performed using a 32P-labeled rat ppTRH
complementary RNA probe as described previously (24). The density of
the hybridization signals was quantitated by a laser densitometer.
Cell culture
GH4C1, GH3, CV-1, HeLa, and
I-10 cells were grown in DMEM supplemented with 10% (vol/vol) FBS,
penicillin, streptomycin, and amphotericin B as described previously
(6).
Plasmid construction
Plasmids -1893/+127 Luc and -254/+127 Luc were described
previously (6), in which -1893/+127 and -254/+127 fragments of the
5'-flanking region of the mouse ppTRH gene, respectively, were ligated
to a reporter gene, pA3Luc, possessing the firefly luciferase
complementary DNA as a reporter (25, 26) (provided by Dr. William M.
Wood). DNA fragments containing various lengths of the 5'-flanking
region of the mouse ppTRH gene were amplified by PCR using the
-1893/+127 fragment in pGEM4Z (Promega, Madison, WI) as a template.
All sense primers for PCR were designed from the nucleotide sequence of
the mouse ppTRH gene reported by us (6) and contained a
HindIII site in the middle of the primers by changing 1 or 2
bp (bp) from the original sequence to facilitate subsequent subcloning.
A common antisense primer (PCR2) corresponding to the region between
+68 and +91 was used for PCR, which also contained a HindIII
site by changing the C residue at +82 to T. All of the PCR-amplified
5'-flanking sequences, therefore, ended at the same position. The
amplified DNA fragments were cut by HindIII and subcloned
into pGEM4Z, and the nucleotide sequences were verified by the dideoxy
chain termination method (27) using Sequenase version 2 (U.S.
Biochemical Corp., Cleveland, OH). Sequence-confirmed DNA fragments
were subcloned into a unique HindIII site of the
pA3Luc, and the orientation of the inserts was confirmed by
sequencing.
Oligonucleotides
The nucleotide sequences of the upper strand of
oligonucleotides used for the construction of heterologous promoters
and the gel mobility shift assays were as follows (lowercase letters
indicate HindIII linker sequences, and
underlining indicates putative Sp1-binding sites and point
mutations introduced); EGF response element-1 (EGFRE1),
5'-agcTTCCCGGGACGGTCTCTCTCCCTCCCTTTGTTCCCTAGTCAGAGTGTa-3';
EGFRE2, 5'-agcttGGTTTCCGGAAAGCGGGCGGGTCCCCCGGCTCTGCC
GTCAGCGCCCCTa-3'; consensus Sp1,
AGTCGATCGGGGCGGGGCGA-3'; mutant Sp1,
5'-AGTCGATCGGTTCGGGGCGA-3'; and palindromic thyroid
hor-mone response element (TREPAL),
5'-AGTCATCAGGTCATGA-CCTGCGA-3'.
Heterologous promoters
A heterologous promoter (EGFRE1 and -2-TK-Luc) was constructed
by insertion of the SmaI/XhoI fragment (-254 to
-37) of the mouse ppTRH gene into pT109Luc, which possesses the
minimal promoter of the herpes simplex virus thymidine kinase (TK) gene
fused to the luciferase gene (28). EGFRE1-TK- and EGFRE2-TK-Luc were
constructed by the insertion of a single copy of synthetic
oligonucleotide corresponding to the region from -258 to -213
(EGFRE1) or from -130 to -84 (EGFRE2), respectively, into a unique
HindIII site of pT109Luc.
PCR-generated site-directed mutagenesis
Point mutations were introduced into the Sp1-binding site at
-117 by sequential PCR steps as described previously (29). Two PCR
fragments encompassing the mutation were amplified, using the
-1893/+127 fragment as the template by the combination of a sense
primer (PCR1; 5'-CAAGCACAGGTGCCACTAGAT-3'; residues -381 to -359) and
a mutant antisense primer (5'-CAGAGCCGGGGGACCCGAACGCTT-3';
residues -121 to -97) and by the combination of a mutant sense primer
(5'-CGGAAAGCGTTCGGGTCCCCCGGCT-3'; residues -125 to -101)
and an antisense primer (PCR2; 5'-GACTCTGCAAAGCTTTCCAAGATG-3'; residues
+68 to +91). The mutant sense and antisense primers overlapped one
another by 20 bp. The two PCR products of the expected lengths were gel
purified, heat denatured, and then annealed to each other. The 3'-ends
were extended by a Taq DNA polymerase and amplified using
the PCR1 and PCR2 primers. The final PCR product was digested by
SmaI and HindIII and subcloned into pGEM4Z. The
introduced mutation was confirmed by dideoxy sequencing, and the
SmaI/HindIII fragment (-254/+83) was subcloned
into the SmaI/HindIII sites of pA3Luc.
Transient transfection and luciferase assay
Transient transfection into GH4C1 cells
was performed by a calcium phosphate precipitation method with 3 µg
reporter constructs as reported previously (5). Glycerol shock was
performed for 2 min, 16 h after transfection. The cells were
incubated for an additional 48 h in the presence or absence of EGF
(100 nM; Collaborative Biomedical Products, Waltham, MA;
culture grade). The luciferase assay was carried out as described
previously (5). The protein concentrations were measured by Bradford’s
method (30), and the luciferase activities were normalized by protein
concentration and expressed as light units per µg protein.
Nuclear extracts
Nuclear extracts from HeLa cells were purchased from Stratagene
(La Jolla, CA). Nuclear extracts from GH4C1
cells were prepared according to the method of Andrews et
al. (31). In brief, confluent GH4C1 cells
in a 100-mm culture dish were rinsed twice with ice-cold PBS and then
scraped into 0.75 ml PBS. The cells were spun down; resuspended in 200
µl buffer A containing 10 mM HEPES-KOH (pH 7.9), 1.5
mM MgCl2, 10 mM KCl, 0.5
mM dithiothreitol, and 0.2 mM
phenylmethylsulfonylfluoride (Sigma Chemical Co., St. Louis, MO); and
allowed to swell on ice for 10 min. After centrifugation, nuclear
pellets were resuspended in 50 µl buffer C containing 20
mM HEPES-KOH (pH 7.9), 25% glycerol, 420 mM
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA,
0.5 mM dithiothreitol, and 0.2 mM
phenylmethylsulfonylfluoride. After incubation on ice for 20 min
followed by a quick spin, the supernatant was used as the nuclear
extract. The protein concentration was determined as described above,
using BSA as a standard.
Gel mobility shift assays
The double strand oligonucleotides described above were
radiolabeled by fill-in reaction with [-32P]deoxy-CTP
(New England Nuclear, Boston, MA) and were separated from
unincorporated isotopes using Sephadex G-25 columns (Boehringer
Mannheim, Mannheim, Germany). Binding reactions were performed with
50,000 cpm purified probes and 1 µg nuclear extract for 20 min under
conditions described previously (6). For competition experiments, a
200-fold molar excess of cold oligonucleotides was included unless
otherwise indicated. For supershift experiments, either 2 µl of a
specific rabbit polyclonal antibody for the human Sp1 that cross-reacts
to the rat Sp1 (Santa Cruz Biotechnology, Santa Cruz, CA) or normal
rabbit serum (NRS) was incubated for an additional 30 min at room
temperature. Gel electrophoresis and autoradiography were performed as
previously described (6).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
densitometric analysis revealed that EGF significantly stimulated TRH
mRNA levels by 2-fold (Fig. 1B). Although we did not measure EGF levels
in the hypothalamus, EGF has been suggested to be transported to the
central nervous system from the peripheral circulation through the
choloid plexuses, which constitute the blood-cerebrospinal fluid
barrier (32).
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, EGF stimulated TRH gene transcription
in a dose-dependent manner. Significant stimulation was observed at
10-10 M EGF. Approximately 4-fold stimulation
compared with the control value was observed at 10-9
M, but no additional stimulation was observed at
10-8 M EGF. We next studied the time course of
EGF stimulation of TRH gene transcription (Fig. 2B). The luciferase
activities in EGF-treated and control groups at 6 and 12 h did not
differ from those at time zero. TRH gene transcription was
significantly stimulated after 24 h until 48 h by the
addition of 10-8 M EGF, and it decreased
thereafter.
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, EGF stimulated the promoter activity
of the longest construct (-1893/+127) by approximately 5-fold. In
contrast, no significant stimulation was observed in the herpes simplex
virus TK gene promoter. The deletion from -1893 to -255 (-254/+127)
did not significantly influence EGF stimulation. In contrast, EGF
activation was significantly decreased from 5- to 2-fold by deletion of
the sequence between -254 and -218 (-217/+83). The further deletion
of fragments between -217 and -178 (-177/+83) and between -177 and
-131 (-130/+83) did not affect EGF stimulation. Finally, deletion of
the element between -130 and -84 (-83/+83) completely abolished the
EGF stimulatory effect. No significant stimulation was observed in the
-36/+127 construct. These deletion analyses indicated that the DNA
elements mediating EGF stimulation of the mouse ppTRH gene were located
at two separate regions, i.e. between -254 and -218 and
between -130 and -84. We designated the upstream region EGFRE1 and
the downstream region EGFRE2.
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shows the nucleotide
sequences of the two EGF response elements identified by deletion
analysis. The nucleotide sequence of EGFRE1 is TC rich, and contains a
sequence (TCCCTCCC) homologous to a novel Sp1-binding site identified
in the acetylcholine receptor 3 gene promoter (CCCCTCCC) (35). It
also possesses a sequence (GGGACG) similar to the GA box (GGGAGG) found
in the glial-specific JC virus early promoter (36) that binds Sp1. In
contrast, the EGFRE2 sequence is GC rich and possesses a consensus core
hexamer of the Sp1-binding site (GGGCGG) (37).
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, EGF stimulated EGFRE1 and -2-TK-Luc
significantly, by 1.8-fold. In contrast, neither EGFRE1-TK-Luc nor
EGFRE2-TK-Luc was stimulated by EGF. These results indicated that the
two separate EGF response elements in the ppTRH gene promoter may
function in a cooperative manner. Moreover, the fold stimulation of the
EGFRE1 and -2-TK-Luc by EGF was significantly lower than that of the
wild-type promoter, suggesting that the positioning of the two EGFREs
relative to the transcription start site affects the magnitude of EGF
stimulation.
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, lane 2 in both panels). With the
EGFRE1 probe, an intense, fast migrating complex B was observed in
addition to a weaker, slowly migrating complex A (lanes 3 and 4 in the
left panel). In contrast, with the EGFRE2 probe, one intense
DNA-protein complex C, migrating in a position similar to that of
complex A on EGFRE1, was observed with three additional faster
migrating complexes, D, E, and F (lanes 3 and 4 in the right
panel). Complex F was present in a position similar to that of
complex B on EGFRE1. No new protein-DNA complexes were observed on
either probe with nuclear extracts from EGF-treated
GH4C1 cells (lanes 5 and 6
in both panels). DNA-protein complexes identical to those detected with
HeLa cells were found with
GH4C1 cell nuclear extracts
on both probes (data not shown).
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, four similar DNA-protein
complexes formed on EGFRE2 were detected with the consensus Sp1 site
(compare lanes 2 in the two panels). Complexes C, D, E, and F formed on
the Sp1 oligonucleotide were diminished by incubation with an excess of
cold EGFRE2 (lane 5 in the left panel). Conversely, the
formation of these four DNA-complexes on EGFRE2 was completely
abolished by incubation with unlabeled Sp1 oligonucleotides (lane 5 in
the right panel). Unlabeled Sp1 oligonucleotide more
effectively inhibited formation of protein-DNA complexes on EGFRE2 than
did cold EGFRE2 itself (Fig. 6C). Unlabeled EGFRE1 did not affect the
four DNA-protein complexes on the Sp1 probe (lane 4 in the left
panel), but slightly reduced the complex formation on the EGFRE2
probe (lane 3 in the right panel). A mutant Sp1
oligonucleotide in which the second and third G residues in the GGGCGG
motif were substituted by T residues failed to prevent formation of the
four DNA-protein complexes (Fig. 6C). Furthermore, a TREPAL
oligonucleotide, with no relation to the Sp1-binding site, did not
inhibit the formation of these complexes (Fig. 6C). These results
suggest that the formation of all four complexes on the GGGCGG motif in
EGFRE2 was specific.
To assess whether the nuclear protein bound to EGFRE2 was bona
fide Sp1, a specific antibody for Sp1 was added to the reaction
mixtures. As shown in Fig. 6D, an anti-Sp1 antibody, but not NRS,
supershifted complex C on the EGFRE2 and Sp1 probes, confirming that
the major nuclear protein binding to EGFRE2 was the transcription
factor Sp1. In contrast, complexes D, E, and F on both probes were not
supershifted by the Sp1 antibody.
Two distinct classes of nuclear proteins bind to EGFRE1
The binding of nuclear proteins to the EGFRE1 probe was also
reduced by the consensus Sp1 oligonucleotide in a manner similar to
that by cold EGFRE1 itself (Fig. 7).
However, higher concentrations of unlabeled Sp1 oligonucleotide were
required to compete with the binding on the EGFRE1 probe compared with
the competition of binding to the EGFRE2 probe (Figs. 6C and 7). The
major protein-DNA complex B on EGFRE1 migrated faster than did complex
C on EGFRE2 (Fig. 6A), and complex A was retarded in a position similar
to that of complex C on EGFRE2 (Fig. 6A). The two complexes, A and B,
on EGFRE1 were not supershifted by an Sp1 antibody (data not shown).
These results imply that the EGFRE1-binding proteins were able to bind
to the consensus Sp1 sequence with low affinity and were
distinguishable from the authentic Sp1. We next examined whether the
EGFRE1-binding proteins were the Sp1-related protein that also
possesses zinc finger motifs (38, 39). The zinc finger motif requires
zinc as a cofactor and can be eliminated by the chelation of zinc ions
by adding EDTA to the binding buffer (40). As shown in Fig. 8A, the addition of EDTA at a dose as low
as 5 mM diminished the complexes C and E on the EGFRE2
probe. In contrast, complexes D and F on EGFRE2 were not affected, even
by 50 mM EDTA. These data suggest that complex C on the
EGFRE2 probe has a nature compatible with that of Sp1. Complex A on the
EGFRE1 probe was obviously sensitive to ETDA, as were complexes C and E
on the EGFRE2 probe (Fig. 8B), but the formation of complex B was not
prevented by 50 mM EDTA. Addition of increasing
concentrations of CaCl2 in the presence of 50
mM EDTA did not restore binding of all EDTA-sensitive
protein-DNA complexes (data not shown). These results indicate that two
distinctive classes of nuclear proteins could bind to EGFRE1, and that
the protein-forming complex A might be an Sp1-related zinc finger
protein.
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, EGF stimulation was
significantly reduced in the mutant -254/+83 Luc to approximately 40%
of that in the wild-type promoter. The result further confirmed that
the GGGCGG motif in EGFRE2 was necessary for the binding of a nuclear
protein such as Sp1 to confer the maximal EGF stimulatory action on the
mouse ppTRH gene promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three additional protein-DNA complexes (complexes D, E, and F) were observed on EGFRE2, as were seen on the 10-bp consensus Sp1 sequence. The formation of multiple complexes between nuclear extracts containing the Sp1 and a consensus Sp1 probe has been reported by several groups (38, 39, 42). However, binding factors other than Sp1 have not been fully characterized. In the present study, the consensus Sp1 sequence inhibited the formation of these three complexes on the EGFRE2 probe. Moreover, mutant Sp1 oligonucleotides failed to inhibit the formation of these complexes, and an Sp1 antibody did not supershift the three complexes. These results indicate that nuclear proteins other than Sp1 can bind to the GGGCGG motif in EGFRE2. As the formation of one of these complexes was sensitive to EDTA, the protein forming complex E may be an Sp1-related protein that bound to the GC box by zinc finger motifs but was different in structure from the authentic Sp1. Alternatively, complex E may represent a partially degraded Sp1-DNA complex that was not recognized by an Sp-1 antibody, as the antibody used in the present study was raised against amino acid residues 436–454 of the human Sp1 protein, and these amino acids were not involved in DNA binding (43).
Homologous sequences to Sp1-binding sites identified in other genes (35, 36) were found in the EGFRE1 sequence. However, our competition and supershift analyses demonstrated that the GH4C1 cell nuclear proteins binding to EGFRE1 were distinct from authentic Sp1. As the formation of two complexes on EGFRE1 was differentially affected by EDTA, it was unlikely that the slower migrating band represented oligomerization of the faster migrating one. It is plausible that two different classes of nuclear proteins in GH4C1 cells, which could bind to the Sp1 sequence with lower affinities, may bind to EGFRE1. The protein forming the slower migrating complex on EGFRE1 might also be an Sp1-related zinc finger protein because of its sensitivity to EDTA.
Our transfection studies with heterologous promoters demonstrated that the two EGFREs did not confer EGF stimulation when an individual element was linked to the TK promoter. No stimulatory effect on heterologous promoter activities by EGF has been reported in the EGFREs of other genes fused to the TK gene promoter (44). However, when the DNA fragment containing both EGFREs of the mouse TRH gene was ligated to the TK promoter, the heterologous promoter was significantly stimulated by EGF. These results suggest that the two EGFREs of the mouse TRH gene may mediate EGF stimulation in a cooperative manner. In contrast, a point mutation introduced into EGFRE2 reduced EGF stimulation by approximately 40%, indicating that EGFRE1 can partially mediate EGF stimulation when positioned properly in a homologous promoter. Taken together, these results suggest that the spatial constraint between the two EGFREs and the transcription start site are important factors in the exertion of full EGF stimulation. The requirement for other DNA-binding proteins in addition to Sp1 for full EGF stimulation of the mouse ppTRH gene might be reinforced by the finding that the TK gene promoter, in which Sp1 plays an important role for basal promoter function (37, 41), was not activated by EGF. A cooperative interaction between EGFRE1- and EGFRE2-binding proteins might be necessary to exert maximal EGF stimulation of the mouse ppTRH gene promoter.
The transcription factor Sp1, initially isolated from HeLa cells, binds to a GC-rich sequence in the promoter region and activates a wide variety of cellular genes as well as many viral genes (37, 40). However, whether Sp1 plays a direct role in any signal transduction pathways remains to be clarified. In this context, there are some reports showing an interaction between Sp1 and other transcription factors for the control of gene expression (45, 46). An interesting result was reported by Merchant et al., who showed that stimulation of the gastrin promoter by EGF in GH4 cells was mediated by Sp1 in conjunction with two additional uncharacterized transcription factors binding to the overlapping half-sites of the GC-rich gastrin EGF response element (47). Taken together with these observations, the present results are interpreted to support the possibility that an interaction between different sets of transcription factors and Sp1 is one of the nuclear pathways for the EGF stimulation of specific genes.
In conclusion, the present results suggest that EGF serves as a possible stimulator of ppTRH gene expression. EGF or related growth factors produced locally or from the systemic circulation might be involved in regulation of the level of hypothalamic TRH mRNA. It is known that TRH can be produced by anterior pituitary cells (48, 49). Because the transfection experiments in this study were performed using a clonal pituitary tumor cell line, it is possible that EGF stimulates ppTRH gene expression in subsets of cell types also in the pituitary gland. Further study is required to elucidate the physiological significance of EGF in the regulation of TRH function in vivo.
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Acknowledgments |
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Received July 21, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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