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Departments of Medicine and Biochemistry and Molecular Biology Albuquerque Veterans Administration Medical Center and University of New Mexico Health Sciences Center Albuquerque, New Mexico 87108
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Data suggest that hypothalamic CRH repression is mediated, in part, through direct effects on hypothalamic CRH-producing neurons (6, 7), which express high levels of classical (type II) glucocorticoid receptor (GR) (8). However, other sites of corticosteroid action may contribute to repression of hypothalamic CRH, as both GR and corticosteroid-responsive mineralocorticoid receptors are expressed in extrahypothalamic central nervous system sites (9, 10). Glucocorticoids appear to regulate CRH through direct inhibition of gene transcription (11). This effect is tissue specific; CRH mRNA levels are unaffected by glucocorticoids at several extrahypothalamic central nervous system sites (12, 13) and are paradoxically up-regulated by glucocorticoids in cultured human placental trophoblasts (14).
In vitro studies suggest that glucocorticoids inhibit CRH gene transcription through specific sites within the CRH promoter (15, 16, 17). Regulatory elements contained within the proximal 1 kb of the CRH promoter are necessary and sufficient to confer cAMP-dependent activation and glucocorticoid-dependent transcriptional regulation to the stably transfected CRH gene (18) or to CRH promoter-reporter constructs in transient transfection studies (15, 16, 17). The cAMP response element (CRE, 5'-TGACGTCA-3') centered at -224 nucleotide (nt) affects basal promoter activity as well as cAMP-dependent, 12-O-tetradecanoylphorbol-13-acetate-dependent, and depolarization-dependent transcriptional activation of the CRH promoter (19, 20, 21). In contrast, localization and characterization of cis-acting elements mediating glucocorticoid-dependent repression within the CRH promoter and potential mechanisms involved in hormonal repression remain uncertain (22).
Glucocorticoid effects are mediated through specific, ligand-dependent
interactions with GR. After corticosteroid binding, GR influences
transcription through distinct mechanisms that can involve direct DNA
binding (23, 24) and/or protein-protein interactions (25, 26, 27). For
example, at the osteocalcin promoter GR binds a GRE that overlaps the
binding site for TATA box-binding protein, and GR-DNA binding reduces
transcription by preventing the binding of a basal transcription factor
(28, 29). Within the model of composite regulation, GR interacts with
other transcription factors at adjacent or overlapping
DNA-regulatory elements (30, 31). Several examples of composite
regulation between GR and AP-1 nucleoproteins (c-jun and
c-fos) have been observed (31, 32). In this setting, the
transcriptional effect of liganded GR may be influenced by
cell-specific factors as well as relative concentrations of
c-jun and c-fos (31). In addition, hormonal
repression may occur through mechanisms that do not require direct DNA
binding of GR at specific target genes, but rather involve soluble
interaction between GR and regulatory proteins such as AP-1 family
members or NF-
B (33, 34). In addition, GR interacts with several
functionally important coadaptors, including CREB-binding protein (CBP)
and GR-interacting protein (GRIP-1) (35, 36, 37).
In an effort to better understand potential mechanisms involved in glucocorticoid-dependent repression of CRH, we have attempted to specifically localize cis-acting region(s) of the CRH promoter critical for hormonal repression. The mouse corticotroph AtT-20 cell line is a useful in vitro model for repression of CRH, since glucocorticoids repress transcription of the endogenous POMC gene as well as exogenous CRH promoter introduced by either stable or transient transfection. Using a series of CRH promoter-luciferase constructs, we have previously reported that nested deletion of the CRH promoter (from -278 to -249 nt) results in complete loss of glucocorticoid-dependent repression of cAMP-stimulated CRH promoter activity (15). Analysis of this highly conserved sequence between -278 and -249 nt demonstrated the presence of potential glucocorticoid and AP-1 response element motifs, suggesting that direct DNA binding of both GR and AP-1 nucleoproteins may play a role in regulation of CRH promoter activity. Our objectives in the present series of studies were 1) to further localize the negative glucocorticoid response element (nGRE); 2) to characterize protein-DNA interactions involving GR and AP-1 nucleoproteins at this site; and 3) to assess the functional role of putative GR and AP-1 binding sites within this element in the context of both intact CRH and heterologous promoter constructs. Our results establish that the nGRE plays an important role in hormonal activation mediated by both cAMP and AP-1 nucleoproteins and also mediates glucocorticoid-dependent repression. Further, promoter mutations that interrupt either AP-1 or GR binding activity to the nGRE lead to abrogation of glucocorticoid-dependent repression, suggesting that interactions between GR and AP-1 or related nucleoproteins may mediate hormonal repression at this regulatory element.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). As previously shown,
glucocorticoid-dependent repression was retained in constructs
containing sequences to -278 nt, while deletion to -249 nt completely
eliminated DEX-dependent repression. Two deletions that disrupt the
region between -278 and -249 nt [CRH(-270)luc and CRH(-260)luc]
retained partial glucocorticoid-dependent repression. As expected,
cAMP responsiveness was retained in all constructs containing the
cAMP-response element (CRE) at -224 nt (data not shown). Loss of
DEX-dependent repression with preservation of cAMP responsiveness
in both CRH(-249)luc and CRH(-235)luc indicates that glucocorticoids
do not repress CRH promoter activity through interference with the CRE
or CRE-binding proteins. This result is in contrast to the findings of
Guardiola-Diaz et al. (16) who reported colocalization of a
glucocorticoid-dependent repression to the CRE (see
Discussion).
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). In contrast to consensus GRE sites
that support GR dimer formation and are in palindromic orientation
(Fig. 2A), the GRE half-sites of the CRH gene are organized as direct
or inverted repeats. In addition, two sites having homology with
consensus AP-1 response elements (Fig. 2C) were identified within the
functionally defined nGRE (Fig. 2B). For subsequent binding and
functional studies, putative GR binding sites 1–3 and putative AP-1
binding sites 1 and 2 refer to upstream and downstream elements
indicated in Fig. 2B (see Materials and Methods).
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). As expected,
GR-DBD specifically bound a positive control probe containing the GRE
from the mouse mammary tumor virus (MMTV) at two sites and, as
previously described, interacted with the CRH nGRE probe (CRH, -278 to
-249 nt) at two sites (15). In contrast, GR-DBD failed to shift the
negative control CRE probe (CRH, -220 to -228 nt). GR-DBD bound MMTV
and CRH probes with similar affinities (see Table 1);
however, at the same protein concentration (20 ng),
GR-DBD interacted with MMTV to yield a protein-DNA complex consistent
with dimeric binding, while GR-DBD interactions at the CRH nGRE
produced a complex consistent with predominantly monomeric GR-DBD
binding. Interestingly, at higher GR-DBD concentrations a second,
higher mol wt band appeared that is consistent with GR-DBD binding at
two sites within the CRH nGRE probe (15).
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, data
summarized in Table 1). Mutation of the middle GRE to an
EcoRI site (Fig. 3, mut2) resulted in a 10-fold reduction in
the affinity (kd) of GR-DBD for the probe and a
commensurate reduction in percentage of two-site GR-DBD binding. In
contrast, mutation of the upstream or downstream GR binding sites
(mut1, mut3) reduced kd by 2-fold and dimer
formation by 4-fold. This identifies the middle GR binding site as the
high-affinity GR binding site within the functionally defined nGRE.
These data are supported by studies of nGRE probes containing mutations
at two GR binding sites. Mutation of GR-sites 1 and 2 (mut1,2) markedly
reduced GR-DBD affinity for the nGRE, while a less dramatic reduction
in affinity was observed when the middle GR binding site was preserved
(mut1, 3). As expected, mutation of all three GR binding sites
(mut1,2,3) eliminated detectable GR-DBD binding. The high-affinity GR
binding site identified in these EMSA experiments corresponds to the
same region of the CRH promoter in which high-affinity GR binding was
identified by deoxyribonuclease I (DNase I) footprinting (16).
AP-1 Interactions within the CRH nGRE
EMSA was also used to evaluate binding of purified,
bacterially expressed AP-1 proteins at putative AP-1 sites within the
CRH nGRE. As shown in Fig. 4A, probes
containing either the consensus AP-1 binding site of the collagenase
promoter (5'-TGACTCA-3') or the CRH nGRE interacted with AP-1 proteins
as jun:jun homodimers and jun:fos heterodimers. Mutation of the
upstream AP-1 binding site to an EcoRI site (AP1-mut1) had
little effect on AP-1 binding, while mutation of the downstream
putative AP-1 binding site (AP1-mut2), markedly reduced both jun:jun
and jun:fos binding in vitro. These findings indicate that
the downstream AP-1 element (Fig. 2, AP1-site 2) represents the
high-affinity AP-1 binding site within the nGRE. AP-1 binding activity
was confirmed by cross-competition studies (data not shown). In these
studies, excess unlabeled CRH, colAP1, or AP1-mut1 probes effectively
competed for AP-1 binding to labeled CRH or colAP1 probes. In contrast,
excess unlabeled AP1-mut2 probe (CRH probe containing a mutated
downstream AP-1 site) failed to compete for AP-1 binding at either of
these probes. Interestingly, mutation of the upstream AP-1 site
(AP1-mut1) somewhat reduced GR-DBD binding to the CRH nGRE, while
mutation of the downstream AP1-site (AP1-mut2) did not (Fig. 4A). Since
the AP1-mut1 mutation also disrupts the distal GRE half-site, decreased
GR-DBD dimer formation suggests that this half-site participates in
GR-DBD dimerization at the nGRE. That AP-1 binding was preserved with
mutation of the high-affinity GR binding site (Fig. 4B) indicates that
GR and AP-1 binding activities can be experimentally distinguished
through selective mutation of binding sites.
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, internal deletion of the entire nGRE
(CRH(-918)[
278–249]luc) significantly reduced, but did not
eliminate, DEX-dependent repression relative to the wild-type CRH
promoter. This confirms an important functional role for sequences
between -278 and -249 nt, but suggests that additional upstream sites
may participate in glucocorticoid-dependent repression. In an effort to
localize secondary glucocorticoid-responsive elements, we extended the
internal deletion to include additional 5'-regulatory sequences from
(CRH(-918)[295–249]luc and CRH(-918)[340–249]luc). Both
of these constructs retained partial glucocorticoid-dependent
repression that was not different from the more limited nGRE internal
deletion construct (CRH(-918) [278–249]luc). Thus, it appears
that CRH promoter sequences between -340 and -918 nt contribute to
glucocorticoid-dependent regulation in a manner independent of the nGRE
between -278 to -249 nt.
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). Mutation
of all three GR binding sites (CRH(-918)[GRsites1,2,3]luc)
reduced glucocorticoid-dependent repression by approximately 50%.
Similar reductions in glucocorticoid-dependent repression were seen
when only the high-affinity GR binding site was mutated
(CRH(-918)[GRsite2]luc, which corresponds to the mut2 probe in
Fig. 3). This confirms the functional importance of the
high-affinity GR binding site defined by EMSA. Interestingly,
mutation of the high-affinity AP-1 binding site (CRH(-918)[AP1
site2]luc, which corresponds to the AP1-mut2 probe in Fig. 3) also
decreased glucocorticoid-dependent repression relative to the wild-type
CRH promoter. Since this mutation does not interfere with GR-DBD
binding (Fig. 4A), the decreased DEX-dependent repression associated
with this mutation suggests a specific role for AP-1 binding in
hormonal regulation.
The possibility of interactions between GR- and CRE-binding proteins or
their cognate coadaptors is also of interest, since GR is capable of
physical interactions with CREB (43) and can repress cAMP-stimulated
promoter activity through interference with mutually required cofactors
(40, 44). Furthermore, within the CRH promoter, Guardiola-Diaz et
al. localized glucocorticoid-dependent repression and cAMP
stimulation to the CRE (16). Experiments summarized in Fig. 6 specifically examine the regulatory
relationship between nGRE and CRE sequences within the CRH promoter. As
previously reported, internal deletion of the CRE
(CRH(-918)[CRE]luc) significantly reduced cAMP-dependent
activation relative to the intact promoter but did not interfere with
hormonal repression (15). Similarly, internal deletion of the CRE in
the context of CRH(-249)luc also reduced cAMP activation relative to
the parent construct. Interestingly, internal deletion of the high
affinity AP-1 site (CRH(-918)[A P1site2]luc) significantly
reduced cAMP responsiveness, suggesting either that the high-affinity
AP-1 site of the nGRE functions as a secondary CRE or that the AP-1 and
CRE interact synergistically. As expected, constructs lacking a defined
CRE, including CRH(-200)luc, CRH(-38)luc, and 
hCG(-100)luc,
demonstrated only mild (
1.5 fold) nonspecific cAMP induction that
was not repressible by glucocorticoids.
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). The
basal Adh promoter demonstrated no hormonal regulation; however, CRH
promoter fragments that included both CRE and nGRE sequences
(CRH[-285 to -90])Adh and CRH[-285 to -160]Adh) conferred both
cAMP-dependent stimulation and DEX-dependent repression to the Adh
promoter. Studies of the CRE and nGRE in isolation confirm the
structural independence of these elements. One or three copies of the
CRE (1x CRH CRE-Adh and 3x CRH CRE-Adh) conferred cAMP-dependent
induction but not DEX-dependent repression to the Adh promoter. In
contrast, the nGRE (1x CRH nGRE-Adh and 3x CRH nGRE-Adh) conferred
mild cAMP responsiveness as well as glucocorticoid-dependent repression
to the Adh promoter, consistent with the function of this element as
both a nGRE and secondary CRE.
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, the
PLF cGRE conferred both cAMP- and glucocorticoid-dependent regulation
to the Adh promoter. Thus, it appears that cAMP is capable of
stimulating transcriptional activation through AP-1 responsive elements
in the context of AtT-20 cells. It is unclear whether this effect is
mediated through changes in AP-1 nucleoprotein levels, phosphorylation
status, or through phosphorylation of nucleoproteins more classically
associated with protein kinase A (PKA)-dependent activation pathway,
such as CREB or ATF-1.
The nGRE and CRE Regulate Basal Promoter Activity
Many hormonally responsive elements influence the rate of basal
transcription. We observed that both the high-affinity AP-1 site within
the nGRE and the consensus CRE influenced basal CRH promoter activity
(Table 2). Mutations that replaced the high-affinity
AP-1 or disrupted the CRE both decreased basal transcription,
suggesting that both of these elements influence transcription in the
absence of cAMP. This conclusion was supported by parallel experiments
using the heterologous Adh promoter. Relative to the minimal Adh
promoter, one or three copies of the CRE dramatically augmented basal
transcription. Similarly, one or three copies of the nGRE (-278 to
-249 nt) placed upstream of the heterologous Adh promoter produced a
3- to 6-fold increase in basal transcription. The cGRE from the
proliferin gene produced a similar increase in basal transcription.
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). Transfection of the c-jun
expression plasmid induced the transcriptional activity of the intact
CRH promoter by 9-fold. This induction was repressible by
glucocorticoids. In contrast, overexpression of c-fos
produced less dramatic induction of CRH that was minimally inhibited by
DEX. A different pattern of AP-1 activation and
glucocorticoid-dependent repression was obtained when the nGRE was
examined in the context of the heterologous Adh promoter. In this
context, c-fos produced a greater magnitude of induction
than c-jun (22-fold vs. 8-fold), and conditions
of jun or fos excess were both sensitive to DEX-dependent repression.
Two control plasmids were also evaluated. The composite element from
the proliferin gene was activated by both jun and fos, and DEX
effectively inhibited activation under conditions of c-jun
excess. In contrast, the CRE was minimally responsive to overexpressed
AP-1 proteins and was not DEX repressible under any conditions
tested.
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, mutation of either the high-affinity GR binding site
or the high-affinity AP-1 site significantly decreased the magnitude of
cAMP-dependent induction relative to the intact nGRE. In addition, both
mutations led to complete loss of glucocorticoid repression of
cAMP-stimulated activity. Stimulation by AP-1 proteins and
glucocorticoid repression of this activity were also examined using
these mutant constructs. As shown in Fig. 9B, mutation of high-affinity
GR or AP-1 sites had no effect on c-jun-mediated induction
but decreased c-fos-mediated induction. Interestingly,
glucocorticoid-dependent repression of AP-1-stimulated activity was
preserved, suggesting that, at this element, repression of both cAMP
activation and AP-1 activation is mediated through distinct
mechanisms.
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). Thus, we studied the role of CREB at the CRH nGRE
in using cotransfection of plasmids that express either full-length
CREB (CREB-FL) or a dominant negative truncated CREB (CREB-BR) capable
of binding DNA but lacking the transactivation domain (47). As shown in
Fig. 10A, CRH nGRE and CRH CRE were
induced by 8-Br-cAMP, and this induction was reduced by cotransfection
of CREB-BR. Furthermore, overexpression of full-length CREB stimulated
activity at each of these elements in a non-cAMP-dependent manner (Fig. 10A). The nGRE was unexpectedly more sensitive to overexpressed CREB
(40-fold induction) than classical CRE (5-fold induction,
P < 0.05). This stimulation appears to be mediated by
the high-affinity AP-1 site, since mutation of this site (3x CRH nGRE
AP-1 site 2) abrogated stimulation by both cAMP and CREB.
Transcriptional activation mediated by CREB and cAMP was clearly due to
enhancer elements contained within the nGRE, as no stimulation was
observed at the basal Adh promoter. Interestingly, CREB-stimulated
activity at the CRH nGRE or CRH CRE was not repressed by
glucocorticoids (Fig. 10B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The present study accomplishes the following: 1) a cis-acting site of the CRH promoter that mediates negative regulation by glucocorticoids was precisely localized; 2) the functional role of this element was defined in the context of both intact CRH and heterologous Adh promoters; 3) the sites and affinities of GR and AP-1 binding within the functionally defined nGRE were determined; and 4) the nGRE was established as a site that can mediate transcriptional activation by both cAMP and AP-1 proteins as well as transcriptional repression by glucocorticoids. Together these results suggest that glucocorticoids inhibit CRH gene transcription through a mechanism that involves direct binding of GR, as well as AP-1 or related nucleoproteins, to be defined cis-acting element of the CRH promoter.
Although the proximal 1 kb of the CRH promoter confers
glucocorticoid-dependent regulation to various reporter genes (16, 50),
the localization of cis-acting elements within the CRH
promoter and potential mechanism of glucocorticoid-dependent
repression remain uncertain. Sequence analysis demonstrates remarkable
conservation between species within the proximal promoter of the CRH
gene (>90% between rat, ovine, and human genes between -300 and +1),
suggesting an important role for functional elements contained within
this region. Although no classical consensus GRE sequences have been
identified within the CRH promoter, Guardiola-Diaz et al.
demonstrated several regions of high-affinity GR binding using rat GR
DNA-binding domain in a DNase I protection assay (16). Indeed, the
functional nGRE localized in our study corresponds precisely to one of
these sites. Interestingly, Guardiola-Diaz et al. observed
that glucocorticoid-dependent repression was maintained in the plasmid
CRH(-249)CAT (16), while we observed loss of repression in an
equivalent luciferase-based construct (CRH(-249)luc, see Fig. 6). In
addition, Guardiola-Diaz et al. reported suppression of
heterologous construct containing the CRE linked to an SV40 promoter
(16). In contrast, we observed no repression when the CRE was linked to
a heterologous Adh promoter and both cAMP-dependent induction and
glucocorticoid-dependent repression when the nGRE (-278 to -249) was
placed upstream of this same promoter (Fig. 7).
The experimental basis for these disparate results is uncertain. Results obtained in the present study, including evaluation of the nGRE through additional nested and internal deletion constructs as well as heterologous contexts, clearly establish the localization and reproducibility of the nGRE under experimental conditions used in our laboratory. A possible explanation for these different results may be related to the presence of a cryptic AP-1 site in the puc18 plasmid backbone used in the aforementioned study (16), which is absent in the pBR-based backbone used in the present series of experiments. The potential for artifactual glucocorticoid repression in the pUC18-derived plasmid containing enhancer elements has been previously reported (51) and would explain the apparent glucocorticoid repression observed in CRH or heterologous promoter constructs containing the CRE reported by Guardiola-Diaz et al. (16). However, unpublished data indicate a similar pattern of glucocorticoid-dependent repression is observed for these constructs when the cryptic AP-1 site is deleted (S. Coon and A. Seasholtz, personal communication). An alternative possibility is that differences in the host AtT-20 cell line may account for different regulatory effects. Since CRH is not expressed in the corticotroph and its regulation in AtT-20 cells is of interest primarily as a model system, mechanisms of glucocorticoid-dependent repression defined by the Seasholtz laboratory (16) and in the present experiments are both of heuristic interest. Clearly, it will be important to determine which potential cis-acting element(s) and mechanisms defined by in vitro studies of the CRH promoter are critical in glucocorticoid repression of hypothalamic CRH expression in situ.
Our observation that internal deletion of the nGRE in the context of
the intact CRH promoter partially reduced glucocorticoid-dependent
repression suggests that upstream sequences within the CRH promoter
also contribute to transcriptional repression. Glucocorticoid
repression in constructs containing internal deletions of the nGRE and
surrounding sequences is specific, since neither constitutive nor
cAMP-responsive control promoters demonstrate DEX-dependent
repression. In an effort to localize potential regions of the CRH
promoter that contribute to hormonal repression independent of the
nGRE, we extended the internal deletion nGRE to include additional
upstream sequences. However, both CRH(-295/-249) and
CRH(-340/-249) demonstrated a magnitude of DEX-dependent
repression comparable to the more limited nGRE deletion
(CRH(-278/-249)). These results suggest that a previously
identified GR binding site between -313 and -301 (16) does not
represent a functional nGRE, and that additional hormonally responsive
element(s) are located upstream of -340 nt.
Mechanisms of glucocorticoid-mediated repression can be broadly divided
into those that require direct GR binding to the target gene promoter
(e.g. occlusion, composite regulation) and those that act
without DNA binding (e.g. soluble interactions,
cross-repression, squelching). Our gel-shift results demonstrating
high-affinity GR binding at the middle GRE half-site confirm the DNase
I protection results of Guardiola-Diaz et al. (16). A
mechanism of glucocorticoid-dependent repression that requires direct
DNA binding is supported by the observation that mutations that disrupt
GR binding activity at this site reduce hormonal repression (Fig. 5).
This is consistent with previous studies of Majzoub and co-workers
(50), who observed that GR mutants lacking DNA-binding activity fail to
repress CRH gene expression.
GR typically interacts as a dimer at "simple" or activating GREs
(52). Within negatively regulated promoters, dimeric GR binding has
been observed at the proliferin composite GRE (31), while trimeric
GR-DNA interactions have been observed at the POMC gene (38). Within
the CRH nGRE, the high-affinity GRE half-site is flanked by two lower
affinity GRE half-sites, suggesting that GR interacts as a dimer or
trimer at this site. However, the classic palindromic GRE organization
that supports dimeric GR binding is not present within the CRH nGRE
(Fig. 2). At higher concentrations of GR-DBD, we have observed GR-DNA
interactions at the lower affinity GRE half-sites (15). However,
because our studies used the DNA-binding domain rather than full-length
GR, it is unclear whether this element would support homodimeric
binding of full-length GR.
Several examples of glucocorticoid-dependent regulation involve interactions between GR and members of the basic leucine zipper superfamily (e.g. AP-1 and CREB/ATF family members (31, 40, 53). Our results establish that the proximal AP-1 sequence within the CRH nGRE is capable of specific and high-affinity interactions with both jun:jun homo- and jun:fos heterodimers. Furthermore, the functional activity mediated through the proximal AP-1 element in response to both cAMP and overexpressed AP-1 proteins establish that AP-1 binding activity is associated with transcriptional activation. The magnitude of transcriptional activation produced by overexpression of c-jun and c-fos, respectively, differed between intact CRH and minimal heterologous promoters. This may be related to the presence of other functional AP-1-responsive elements within the CRH promoter (54) or to endogenous expression of AP-1 family members within AtT-20 corticotrophs.
In addition to mediating activation in response to overexpressed AP-1 nucleoproteins, we found that the nGRE functions as a cAMP-response element independent of the CRE at -224 nt. When examined in a heterologous context, the consensus CRE increased both basal and cAMP-stimulated transcription more than the nGRE. However, within the context of the intact CRH promoter, internal deletion of either element produced a similar decrement in cAMP response. The mechanism of cAMP-dependent activation through the nGRE is uncertain. One possibility is that cAMP acts through a classical PKA-dependent pathway to stimulate phosphorylation of CREB, ATF-1, or related nucleoproteins. CREB and related nucleoproteins are capable of binding some AP-1 response elements and may lead to either transcriptional activation or repression in a cell- and promoter-specific fashion (45, 55, 56). In addition, CREB/ATF and AP-1 family members may heterodimerize at certain CRE and/or AP-1 elements (55, 57, 58). The ability of a dominant negative CREB mutant that lacks the transcriptional activation domain but retains DNA-binding activity to suppress the cAMP response mediated through the nGRE suggests that CREB is capable of interacting at the CRH nGRE. Another possibility is that cAMP acts through PKA-dependent transcriptional effects leading to changes in the absolute or relative concentrations of c-jun or c-fos. Another hypothesis is that synergy exists between nucleoproteins interacting at nGRE (AP-1) and CRE sites. In this model, synergistic nucleoprotein interactions could contribute to cAMP-dependent activation mediated through the nGRE, and liganded GR could disrupt these interactions to produce glucocorticoid-dependent repression. Alternatively, PKA stimulation may influence other signal transduction elements, like mitogen/stress-activated kinases that regulate phosphorylation-dependent activation of CREB, as well as activation of AP-1 response elements in several neuronal and endocrine cell types (59, 60, 61, 62).
The localization of GR and AP-1 binding within a single regulatory element, coupled with the loss of hormonal repression observed after mutation of either site, suggests that the CRH nGRE functions as a composite regulatory element. This conclusion is supported by the observation that hormonal regulation of the CRH nGRE parallels that of the well characterized composite element from the mouse proliferin gene. In addition, the CRH nGRE has a complex structure similar to that of the previously described composite GREs from the mouse proliferin and bovine PRL genes (31, 63). The mechanism by which GR and AP-1 nucleoproteins interact at composite elements to activate or repress gene transcription in a tissue-specific fashion remains uncertain. While we did not examine co-occupancy of GR and AP-1 nucleoproteins at the nGRE, it is possible that GR binding may interfere with AP-1 binding or influence the composition of AP-1 nucleoproteins at this element. In addition, the role of coadaptors in mediating regulatory responses from composite elements has yet to be defined. One interesting possibility is that the host of transcription factors recruited to a composite element then determines the composition of the coadaptor complex and hence the signal transmitted to the basal transcription apparatus. Within this model, the ability of glucocorticoids to effect positive and negative regulation from a composite response element may be mediated through differential recruitment of coadaptors that facilitate regional histone acetylation and deacetylation. The cell specificity observed for directional regulation of composite glucocorticoid response elements may be influenced not only by relative concentrations and phosphorylation status of AP-1 and related nucleoproteins, but also the host cell repertoire of coactivator and corepressor proteins. In any case, it will be crucial to extend the current findings related to glucocorticoid-dependent relation of the CRH promoter to other in vitro and in vivo models to better understand the molecular mechanisms of negative glucocorticoid feedback of the HPA axis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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)dATP, and purified by
chromatography over G50 Sephadex (Pharmacia Biotech,
Uppsala, Sweden) and phenol-chloroform extraction. Probes with the
following target sequences were prepared (consensus sequences shown in
bold): MMTV GRE, containing the palindromic consensus GRE
from mouse mammary tumor virus (sense,
5'-GTTGGGTTACAAACTGTTCT-3'; antisense,
5-'TGGTTAGAACAGTTTGTAAC-3'); CRE, containing the
8-bp CRE present in the hCRH promoter at -224 nt (sense,
5'-GTTGGTGACGTCA-3'; antisense,
5'-TGGTTTGACGTCA-3'); CRH, containing the nGRE from hCRH
(-278 to -249 nt) (sense, 5'-ATTTTTGTCAATGGACAAGTCATA-3'; antisense,
5'-TT-CTTATGACTTGTCCATTGACA-3'); colAP1, containing the consensus
AP-1 binding site from the collagenase promoter (-73 to -66 nt)
(sense, 5'-GTTGGTGAGTCA-3'; antisense,
5'-GGTTGTGACTCA -3').
In addition, CRH nGRE (-278 to -249 nt) probes were created in which
potential GR binding sites (Fig. 2B, GR-sites 1–3) or potential
AP-1-binding sites (Fig. 2B, AP1-sites 1 and 2) were mutated to
EcoRI restriction sites (5'-GAATTC-3'). GR binding site
mutants designated mut1 to mut3 refer to putative GR binding sites 1–3
enumerated in Fig. 2B. AP-1 binding site mutants (AP1 mut1 and AP1
mut2) are designated with respect to putative AP-1 binding sites
illustrated in Fig. 2B. The following mutant CRH probes were prepared
(mutated bases shown in bold): MUT 1, mutation of GR-site 1
(sense, 5'-ATGAATTCCAATGGACAAGTCATA-3'; antisense,
5'-TTCTTATGACTTGTCCATTGGAA-3'); MUT 2, mutation of
GR-site 2 (sense, 5'-ATTTTTGTCAAGAATTCAGTCATA-3';
antisense, 5'-TTCTTATGACTGAATTCTTGACA-3'); MUT 3,
mutation of GR-site 3 (sense,
5'-ATTTTT-GTCAATGGACAAGTCGAA-3'; antisense,
5'-TGAATTCG-ACTTGTCCATTGACA-3'); MUT 1,2, mutation of
GR-sites 1 and 2 (sense,
5'-ATGAATTCCAAGAATTCAGTCATA-3'; antisense,
5'-TTCTTATGACTGAATTCTTGGAA-3'); MUT 1,3, mutation
of GR-sites 1 and 3 (sense,
5'-ATGAATTCCAATGGACAAGTCGAA-3'; antisense,
5'-TGAATTCGACTTGTCCATTGGAA-3'); MUT 1,2,3,
mutation of GR-sites 1–3 (sense,
5'-ATGAATTCCAAGAATTCAGTCGAA-3';
antisense,
5'-TGAATTCGACTGAATTCTTGGAA-3');
AP1-MUT1, mutation of AP1-site 1 (sense,
5'-ATTTGAATTCTTGTCAATGGACAAGTCATA-3'; antisense,
5'-TTCTTATGACTTGTCCATGAATC-3'); AP1-MUT2, mutation of
AP1-site 2 (sense, 5'-ATTTTTGTCAATGGACGAATTCTA-3';
antisense, 5'-TTCT-TAGAATTCGTCCATTGACA-3').
Note that in AP-1 mutants, since a 6-bp EcoRI site
replaced a 7-bp AP1 site, the 5'-base of each AP-1 site (shown in
italics on the sense strand) was not altered. Radiolabeled
probe (0.2 ng) was incubated with 0–20 ng purified rat GR-DNA
binding domain (GR-DBD, amino acids 440–525, kindly provided by L.
Freedman, Sloan-Kettering Institute, New York, NY) or 0–20 ng AP-1
proteins (c-jun and c-fos, kindly provided by K.
Yamamoto, University of California, San Francisco, CA) in 10 µl
binding buffer (20 mM Tris-HCl, pH 7.9, 1 mM
EDTA, 1 mM DTT, 0.1% NP-40, 10% glycerol, 1 µg poly
dIC) for 15 min at room temperature. Bound and unbound species
were separated by either 10% nondenaturing PAGE for reactions
containing GR-DBD or 6% nondenaturing PAGE for reactions containing
AP-1 proteins. Autoradiograms were visualized using a PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA); free
and shifted probes were quantitated using ImageQuant software
(Molecular Dynamics, Inc.). Binding curves for nGRE probes
were created using a constant amount of probe and varying the amount of
protein (GR-DBD) added to each reaction. Affinity (kd) was
calculated using the method described by Freedman and Alroy (64), which
is summarized in the following equation:
 |
Plasmid Constructs
CRH(-918)luc, CRH(-664)luc, CRH(-364)luc,
CRH(-295)luc, CRH(-278)luc, CRH(-249)luc, and
CRH(-918)[CRE]luc have been previously described (15).
CRH(-270)luc, CRH(-260)luc, CRH(-235)luc, and CRH(-200)luc were
prepared by PCR of CRH(-918)luc template using 30 mer sense primers
complementary to the corresponding sites of the hCRH gene containing
5'-HindIII tails and an antisense primer to the 5'-region of
luciferase (PGL2, Promega Corp., Madison, WI,
5'-CTTTATGTTTTTGGCGTCTTCCA-3'). After transitional subcloning into TA
cloning vector (Invitrogen, Carlsbad, CA), the PCR product
was digested with HindIII and ligated into
HindIII-digested pA3luc luciferase vector (65). CRH(-38)luc
was prepared by ligation of the HindIII fragment of
CRH(-918)luc (CRH, -918 to +38 nt) into the HindIII site
of the PxpI luciferase vector [ATCC, Manassas,
VA (66)], which was then digested with SalI and religated.
CRH(-918)[-340 to -249]luc, CRH(-918)[-295 to
-249]luc, and CRH(-918)[-278 to -249]luc were generated by
recombinant PCR of CRH(-918)luc template. The upstream fragment was
generated using a sense primer to the pA3luc backbone (pA3luc sense,
5'-CTGGATCCCCGGGTACC-3') and an antisense primer complementary to the
region 5' of the desired deletion and containing a 5'-tail
complementary to the region directly 3' of the desired deletion. The
downstream fragment was generated using the PGL2 antisense primer and a
sense primer complementary to the region 3' of the desired deletion and
containing a 5' tail complementary to the region 5' of the desired
deletion. After gel purification, first-round PCR products were
denatured, allowed to anneal to each other, and then subjected to a
second round of PCR using the outside primers (PGL2 and pA3luc sense).
After transitional subcloning into TA cloning vector
(Invitrogen, San Diego, CA), the recombinant
HindIII fragment was ligated into
HindIII-digested pA3luc. CRH(-918)[AP1-site 2],
CRH(-918)[GR-site 2], and CRH(-918)[GR-sites 1,2,3], in which
the indicated site(s) were replaced with EcoRI site(s) (see
Fig. 2B), were generated by recombinant PCR as described above, but
with CRH-specific primers containing the desired mutations.
CRH(-918)[GR-site 2], CRH(-918)[GR-sites 1,2,3], and
CRH(-918)[AP1-site 2] correspond to mut2, mut1,2,3, and AP1
mut2 probes used in EMSA experiments. CRH-(-249)[CRE]luc was
created by digestion of CRH(-918)-[CRE]luc with
SmaI and HindIII and ligation of this product
into SmaI/HindIII-digested pA3luc. All plasmid
constructs were confirmed by Sanger sequencing using the Sequenase
2.0 kit (United States Biochemical Corp., Cleveland,
OH).
HCG(-100)luc (67) and expression vector for full-length CRE binding
protein (CREB-FL, amino acids 1–327) were kindly provided by J.
Hoeffler (Invitrogen). CREB-FL was placed under the
control of the CMV promoter by ligation of the
HindIII/XbaI fragment of CREB-FL into
HindIII/XbaI-digested CMV4 vector (kindly
provided by J. Omdahl, Department of Biochemistry and Molecular
Biology, University of New Mexico Health Science Center). CMV-driven
CREB-binding region (CREB-BR, amino acids 254–327) was created by
BglII digestion of CMV-CREB-FL and religation of the
digested product (47). GR expression vector, pRSVhGR (68), was
kindly provided by R. Evans (Salk Institute, La Jolla, CA). Expression
vectors for c-jun and c-fos were kindly provided
by R. Tijan (University of California, Berkley, CA). pODLO
(luciferase vector driven by a minimal Drosophila Adh
promoter (-33 to +53 nt) and PLFG3.1 (containing three copies of the
composite GRE from the proliferin promoter in pODLO(31)) were kindly
provided by K. Yamamoto (University of California, San Francisco, CA).
Regulatory elements were inserted upstream of the minimal Adh promoter
into PstI/SalI-digested pODLO using annealed
pairs of com-plementary oligonucleotides designed to have
PstI/SalI overhangs.
The following plasmids were created: [1x nGRE]Adh, containing the
nGRE of hCRH (-278 to -249 nt); [2x nGRE]Adh, containing two
copies of the nGRE of hCRH separated by a XhoI site; [1x
CRE]Adh, containing the CRE of hCRH (-231 to -217 nt); [3x
CRE]Adh, containing three copies of the CRE of hCRH; [3x
colAP1]Adh, containing three copies of the AP1 site of the collagenase
promoter (-77 to -62 nt); [1x nGRE-GR site 2]Adh, containing
the nGRE of hCRH in which the middle GR binding site has been mutated
to an EcoRI site; [1x nGRE-AP1 site 2], containing the
nGRE of hCRH in which the downstream AP-1 site has been mutated to an
EcoRI site.
[3x nGRE]Adh, containing three copies of the nGRE of hCRH, was
created by inserting an annealed oligonucleotide pair containing the
nGRE of hCRH with XhoI overhangs into the XhoI
site of [2x nGRE]Adh. [3x nGRE-GR site 2]Adh and [3x
nGRE-AP1 site 2] were created by inserting an oligonucleotide pair
containing two copies of the appropriate nGRE mutant with
PstI/XhoI overhangs into the PstI and
(engineered) XhoI sites of the 1x parent construct.
[CRH(-285 to -90)]Adh and [CRH(-285 to -160)]Adh were generated
by PCR of CRH(-918)luc template with 30 mer primers complementary to
the appropriate regions of the CRH gene. The sense primer contained a
5'-PstI tail and the antisense primer contained a
5'-SalI tail. PCR-generated fragments were digested with
PstI/SalI and inserted into
PstI/SalI-digested pODLO.
Cell Culture, Transfection, and Luciferase Assay
AtT-20 cells were maintained and transfected as previously
described (22). Briefly, AtT-20 cells were maintained under standard
conditions in DMEM containing 10% FBS. Cells were transfected with
CsCl2 purified DNA at 70–80% confluence by
calcium-phosphate precipitation with glycerol shock. Each 60-mm plate
received 16 µg DNA, consisting of 2–5 µg luciferase reporter, 0–2
µg GR expression vector, 0–4 µg of expression vector for
c-jun, c-fos, CMV-CREB-FL, or CMV-CREB-BR, and
Bluescript KS+ carrier to 16 µg.
Statistical Analysis
Data are expressed as fold induction over a baseline level of
1.0 or as percentage of 8-Br-cAMP-stimulated promoter activity. Each
experimental condition in all transfection experiments was performed in
duplicate. The full-length CRH promoter construct (CRH[-918]luc) was
included in each independent experiment as a positive control for cAMP
induction and glucocorticoid repression. Pooled data represent the
mean ± SEM of at least three independent experiments.
The number of independent experiments for plasmid CRH(-918)luc was 76;
for other constructs the number of experiments ranged from 3 to 20.
Overall statistical analysis was performed using repeated-measures
ANOVA with post-hoc pairwise comparison. Statistical significance of
induction by 8-Br-cAMP, AP-1 proteins, or CREB-FL, and repression by
DEX, was determined by paired Student’s t test. Differences
in hormonal responses between different reporter/receptor combinations
were assessed by unpaired Student’s t test.
P < 0.05 was considered to be statistically
significant.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
This research was supported by Veterans Administration Research Service.
Received for publication March 2, 1999. Revision received May 20, 1999. Accepted for publication June 23, 1999.
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Indicates significantly less repression than CRH(-918)luc. Nested
deletion constructs containing an intact nGRE (constructs extending
upstream of -278 nt) demonstrated approximately a 2-fold DEX-dependent
repression of cAMP-stimulated promoter activity (
