Composite Glucocorticoid Regulation at a Functionally Defined Negative Glucocorticoid Response Element of the Human Corticotropin-Releasing Hormone Gene

Stephen P. Malkoski and Richard I. Dorin

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid-dependent negative feedback of the hypothalamic-pituitary-adrenal axis is mediated in part through direct inhibition of hypothalamic CRH gene transcription. In the present study, we sought to further localize and characterize glucocorticoid receptor (GR) and AP-1 interactions at a functionally defined negative glucocorticoid response element (nGRE) of the CRH promoter. Transient transfection studies in mouse corticotroph AtT-20 cells demonstrated that internal deletion of the nGRE (-278 to -249 nucleotides) within the context of 1 kb of the intact CRH promoter resulted in decreased 8-Br-cAMP stimulation and glucocorticoid-dependent repression of CRH promoter activity. The nGRE conferred transcriptional activation by both cAMP and overexpressed c-jun or c-fos AP-1 nucleoproteins as well as specific glucocorticoid-dependent repression to a heterologous promoter. A similar profile of regulation was observed for the composite GRE derived from mouse proliferin promoter. The CRH nGRE was clearly distinct from the consensus cAMP response element (CRE) at -224 nucleotides, which increased basal activity and cAMP responsiveness of a heterologous promoter but did not confer glucocorticoid-dependent repression. High-affinity binding sites for both GR and AP-1 nucleoproteins were identified at adjacent elements within the nGRE. Mutations that disrupted either GR or AP-1 binding activity were associated with loss of glucocorticoid-dependent repression. These results are consistent with a composite mechanism of glucocorticoid-dependent repression involving direct DNA binding of GR and AP-1 nucleoproteins at discrete adjacent sites within the CRH promoter.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hypothalamic CRH-producing neurons integrate afferent central nervous system signals related to stress responsiveness and circadian rhythmicity as well as negative feedback inhibition by glucocorticoids (1, 2). The primacy of CRH in central regulation of the hypothalamic-pituitary-adrenal (HPA) axis is established in animal and human models of CRH deficiency and excess. The CRH knockout mouse demonstrates low levels of ACTH and corticosterone and a blunted HPA response. Thus, even in the setting of reduced corticosteroid feedback, other ACTH secretagogues, such as vasopressin, are unable to compensate for CRH deficiency (3, 4). Within the clinical model of Cushing’s syndrome, the prolonged suppression of ACTH and cortisol secretion that remains after chronic corticosteroid excess can be reversed by administration of exogenous CRH, indicating that repression of central signals limits recovery of the HPA axis (5).

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-{kappa}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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nested Deletions of the hCRH Promoter Localize a nGRE between -278 and -249 nt
In previous studies, nested deletions to -278 nt retained cAMP-dependent activation and dexamethasone (DEX)-dependent repression while DEX-dependent repression was eliminated when CRH promoter constructs were shortened to -249 nt (15). Additional CRH promoter deletion constructs were used to confirm and further localize putative nGRE site(s) mediating hormonal repression (Fig. 1Go). 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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Figure 1. Nested Deletions of the CRH Promoter Confirm the Localization of the nGRE between -278 and -249 nt

AtT-20 cells were transfected with hCRH promoter-luciferase reporter constructs by calcium phosphate precipitation as described in Materials and Methods. Data (mean ± SEM of at least three independent experiments) are expressed as the percentage of 8-Br-cAMP-stimulated promoter activity observed with 8-Br-cAMP and DEX cotreatment; therefore, no repression is 100%. Nested deletions are designated by the most 5' nt of the hCRH promoter retained. * Indicates significant DEX-dependent repression. {diamondsuit} 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 (~50% of 8-Br-cAMP stimulated). Nested deletions that eliminated the entire nGRE (CRH(-249)luc, CRH(-235)luc, CRH(-200)luc) demonstrated no DEX-dependent repression. Nested deletions that disrupted the nGRE (CRH(-270)luc, CRH(-260)luc) demonstrated significant, but reduced, glucocorticoid repression of cAMP-stimulated CRH promoter activity.

 
The region between -278 and -249 nt is highly conserved, with 100% homology between rat, ovine, and human genes. Analysis of this sequence identified three sites with GRE half-site homology (Fig. 2BGo). In contrast to consensus GRE sites that support GR dimer formation and are in palindromic orientation (Fig. 2AGo), 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. 2CGo) were identified within the functionally defined nGRE (Fig. 2BGo). 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. 2BGo (see Materials and Methods).



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Figure 2. Sequence and Putative Binding Sites in the Human CRH Promoter nGRE (-278 to -249 nt)

This region of the hCRH promoter has 100% homology with rat and ovine CRH genes. Sites with GRE half-site homology are shown on the top (sense) strand and have either 4/6 (GR-sites 1 and 3) or 5/6 (GR-site 2) similarity with the GRE half-site consensus (5'-TGTACA-3') sequence. Sites with AP-1 site homology are shown on the bottom (antisense) strand; AP1-site 1 has 6/7 similarity with the nonclassical AP-1 site of the composite GRE of the mouse proliferin gene (30 ), while AP1-site 2 has 5/7 similarity with the consensus AP-1 element (5'-TGACTCA-3'). For mutant probes used in EMSA and mutant constructs used in transient transfection assays, putative transcription factor binding sites were mutated to EcoRI restriction sites (5'-GAATTC-3'). Specifically, GR binding site mutants were designated mut1 to mut3 with respect to GR sites 1–3 shown in panel B (e.g. mut2 indicates mutation of GR-site2). Mutation of GR binding sites in functional constructs was similarly designated (e.g. {Delta}GRsite2 indicates mutation of GR-site2).

 
Localization of GR Binding to the CRH nGRE
Ligand-bound GR can influence transcription by direct DNA binding to regulatory sequences (28, 31, 38, 39) or through protein-protein interactions involving GR and other transcription factors and/or coadaptors (31, 33, 40, 41). To better define the role of GR-DNA binding, we sought to characterize and localize high-affinity GR interactions with the CRH nGRE sequence. Electrophoretic mobility shift assays (EMSAs) were performed using bacterially expressed rat GR DNA-binding domain (GR-DBD, kindly provided by L. Freedman, Sloan-Kettering Institute, New York, NY) (42) and wild-type or mutant CRH nGRE probe (Fig. 3Go). 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 1Go); 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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Figure 3. GR-DBD Binding to CRH nGRE Probes Containing Mutations in Potential GR Binding Sites

Labeled probes were incubated in the absence or presence of 5 ng of rat GR-DBD (amino acids 440–525) and separated by nondenaturing PAGE. MMTV probe contains a classical consensus GRE sequence (5'-GTTACAnnnTGTTCT-3'). CRE probe contains the palindromic cAMP response element (5'-GTCATGAC-3'). CRH probe contains the nGRE of hCRH (-278 to -249 nt) shown in Fig. 2Go. mut 1 to mut 1,2,3 are CRH nGRE probes containing EcoRI restriction sites in place of putative GR binding sites designated in Fig. 2Go. Protein-DNA complexes consistent with GR-DBD monomeric and dimeric binding are designated on the basis of migration patterns observed in previously described gel-shift studies of the MMTV GRE (64 ). Mutation of the middle GR binding site (mut2) reduced affinity and dimer formation more than mutation of either or both outside GR binding sites (mut1, mut3, and mut1, 3). Negative control probes, mut1,2,3, and CRE did not demonstrate a band shift in the presence of GR-DBD. Affinity and dimer formation data are summarized in Table 1Go.

 

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Table 1. Affinities and Dimer Formation for hCRH nGRE Probes Containing Mutations of Potential GR Binding Sites

 
To determine which potential GRE half-sites participate in high-affinity GR-DBD binding, we introduced mutations into upstream, middle, or downstream GRE sequences (designated mut1, mut2, and mut 3, respectively) and examined GR binding using EMSA (Fig. 3Go, data summarized in Table 1Go). Mutation of the middle GRE to an EcoRI site (Fig. 3Go, 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. 4AGo, 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. 2Go, 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. 4AGo). 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. 4BGo) indicates that GR and AP-1 binding activities can be experimentally distinguished through selective mutation of binding sites.



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Figure 4. The 3'-Putative AP-1 Site within the hCRH nGRE Specifically Binds AP-1 Proteins in Vitro

A, Labeled probes were incubated with either 10 ng c-jun and 10 ng c-fos (JF) or 20 ng c-jun (J) or 20 ng GR-DBD (GR), separated by 6% PAGE, and visualized using a PhosphorImager (Molecular Dynamics, Inc.). The colAP-1 probe contains the consensus AP-1 site of the collagenase promoter (-73 to -66 nt, 5'-TGACTCA-3'). The CRH probe contains the hCRH nGRE (-278 to -249 nt) shown in Fig. 2BGo. The AP1-mut1 and AP1-mut2 are CRH nGRE probes containing EcoRI restriction sites at the putative AP-1 sites designated in Fig. 2BGo. GR-DBD monomer and dimer bands are designated with respect to intact CRH probe. AP-1 hetero- and homodimer bands are designated with respect to the colAP1-positive control. Mutation of the 3' AP-1 binding site (AP1-mut2), but not the 5' AP-1 site (AP1-mut1) decreased AP-1 binding to the CRH nGRE. B, A CRH probe containing a mutation of the high-affinity GR binding site (GR-mut2, see Fig. 2Go) retains the ability to bind AP-1 proteins. Reactions are as described for panel A.

 
Internal Deletions and Discrete Mutations within the Intact CRH Promoter
Analysis of nested deletions may be complicated by the serial removal of upstream sequences that influence defined regulatory elements through additive or synergistic interactions. Thus, additional transfection studies using constructs containing internal deletions or discrete mutations of the nGRE (-278 to -249 nt) were performed to further define the functional role of the nGRE within the context of the intact CRH promoter (to -918 nt). As shown in Fig. 5Go, internal deletion of the entire nGRE (CRH(-918)[{Delta}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)[{Delta}295–249]luc and CRH(-918)[{Delta}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) [{Delta}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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Figure 5. Internal Deletions of the CRH Promoter Confirm the Localization of the nGRE and a Functional Role of Putative Transcription Factor Binding Sites

AtT-20 cells were transfected with CRH promoter-luciferase reporter constructs by calcium phosphate precipitation as described in Materials and Methods. Data (mean ± SEM of at least three independent experiments) are expressed as the percentage of 8-Br-cAMP stimulated promoter activity observed with 8-Br-cAMP and DEX cotreatment; therefore, no repression is 100%. Internal deletion constructs are designated by the internally deleted nucleotides. Constructs containing EcoRI substitutions of putative transcription factor binding sites within the nGRE are designated by the mutated binding site(s) as delineated in Fig. 2BGo. * Indicates significant DEX-dependent repression. {diamondsuit} Indicates significantly less repression than CRH(-918)luc. Internal deletion of the nGRE (CRH(-918)[{Delta}340–249]luc, CRH(-918)[{Delta}295–249]luc, CRH(-918)[{Delta}278–249]luc) or mutation of specific binding site(s) within the nGRE (CRH(-918)[{Delta}GRsites1,2,3]luc, CRH(-918)[{Delta}GRsite2]luc, CRH(-918)[AP1 site2]luc) reduces but does not eliminate glucocorticoid repression of cAMP-stimulated CRH promoter activity.

 
We also examined the effects of specific mutations of GR- and AP-1 binding sites within the context of the CRH promoter (Fig. 5Go). Mutation of all three GR binding sites (CRH(-918)[{Delta}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)[{Delta}GRsite2]luc, which corresponds to the mut2 probe in Fig. 3Go). 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)[{Delta}AP1 site2]luc, which corresponds to the AP1-mut2 probe in Fig. 3Go) also decreased glucocorticoid-dependent repression relative to the wild-type CRH promoter. Since this mutation does not interfere with GR-DBD binding (Fig. 4AGo), 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. 6Go specifically examine the regulatory relationship between nGRE and CRE sequences within the CRH promoter. As previously reported, internal deletion of the CRE (CRH(-918)[{Delta}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)[{Delta}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 {alpha}hCG(-100)luc, demonstrated only mild (~1.5 fold) nonspecific cAMP induction that was not repressible by glucocorticoids.



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Figure 6. The Activities of the CRE and nGRE Are Separable

AtT-20 cells were transfected as described in Materials and Methods, except reporter concentration was increased to 5 µg/plate to facilitate analysis of basal promoter activity (see Table 2A). Data (mean ± SEM of at least three independent experiments) are expressed as fold induction over a baseline level of 1.0. DEX-dependent repression as a percentage of 8-Br-cAMP-stimulated promoter activity is shown on the right. Nested and internal deletion constructs are designated as described for Figs. 1Go and 5Go. CRH(-918)[{Delta}CRE]luc and CRH(-249)[{Delta}CRE]luc contain deletions of the CRE at -224 nt. {alpha}HCG(-100)luc is a basal, constitutive, eukaryotic control consisting of the human CG promoter extended to -100 nt. All constructs demonstrated induction with 8-Br-cAMP treatment (see text); however, compared with CRH(-918)luc, induction was reduced in all deletion and control constructs. * Indicates significant DEX-dependent repression. {diamondsuit} Indicates reduced DEX-dependent compared with CRH(-918)luc. ** Indicates reduced cAMP induction compared with CRH(-249)luc. {diamondsuit}{diamondsuit} Indicates reduced cAMP induction compared with CRH(-235)luc.

 
The nGRE and CRE Confer Transcriptional Regulation to a Heterologous Promoter
Many enhancer and repressor elements confer signal-dependent regulation to a heterologous promoter. To examine regulation in a heterologous context, defined regulatory elements were placed upstream of the minimal Drosophila alcohol dehydrogenase (Adh) promoter, and hormonal regulation of these constructs was examined in transfected AtT-20 cells (Fig. 7Go). 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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Figure 7. The CRE and nGRE Regulate a Heterologous Promoter

Hormonal regulation of various regulatory elements was examined in transfected AtT-20 cells. Data are expressed as in Fig. 6Go. Regulatory elements were inserted into the PstI/SalI sites upstream of the Adh promoter as described in Materials and Methods. All constructs except the basal Adh promoter demonstrated significant cAMP stimulation. CRH(-285 to -90)-Adh and CRH(-285 to -160)-Adh contain both the nGRE and CRE sequences and thus conveyed both cAMP and DEX responsiveness to Adh. 1x CRH CRE-Adh and 3x CRH CRE-Adh contain one and three copies, respectively, of the CRH CRE (-231 to -219 nt) and conveyed only cAMP responsiveness to Adh. 1x CRH nGRE-Adh and 3x CRH nGRE-Adh contain one and three copies, respectively, of the CRH nGRE (-278 to -249 nt) and conveyed DEX-dependent repression and mild cAMP responsiveness to Adh. 3x PLF cGRE-Adh contains three copies of the proliferin promoter cGRE [-254 to -230 nt, PLFG3.1 (31 )] and also conveyed mild cAMP induction and DEX-dependent repression to Adh. * Indicates significant DEX-dependent repression. {diamondsuit} Indicates reduced DEX-dependent repression compared with 3x CRH nGRE-Adh. {diamondsuit}{diamondsuit} Indicates increased cAMP induction compared with 1x CRH CRE-Adh.

 
In addition, we examined cAMP and glucocorticoid effects on an additional construct containing three copies of composite GRE from the mouse proliferin promoter [3x PLF cGRE-Adh also called PLFG3.1 (31)]. This element contains both GR and AP-1 responsive elements upstream of the heterologous Adh promoter. As indicated in Fig. 7Go, 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 2Go). 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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Table 2. The CRE and nGRE Regulate Basal Promoter Activity

 
The CRH nGRE Represents a Functional AP-1 Site
To assess the functionality of AP-1 sites identified by sequence analysis and in vitro binding studies, we performed transient expression studies in which relative levels of c-jun and c-fos were manipulated by cotransfection of AP-1 expression plasmids (Fig. 8Go). 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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Figure 8. The nGRE Responds to Overexpressed AP-1 Proteins and Glucocorticoids Repress AP-1-Stimulated Activity of the CRH nGRE

AtT-20 cells were cotransfected with the indicated reporter (2 µg), expression vectors for c-jun or c-fos (4 µg), and expression vector for GR{alpha} (2 µg). Cells were then treated, harvested, and assayed as described in Materials and Methods; data (mean ± SEM of at least three independent experiments) are expressed as fold induction. All constructs demonstrated significant (P < 0.05) induction with overexpressed c-jun or c-fos except 3x CRH CRE-Adh, which did not induce with c-jun. {diamondsuit} Indicates significant DEX-dependent repression of c-jun- or c-fos-stimulated promoter activity.

 
Role of High-Affinity GR- and AP-1 Binding Sites in Stimulation and Repression
High-affinity GR- and AP-1 binding sites were mutated to EcoRI sites in the context of the 3x CRH nGRE-Adh construct to specifically assess the role of these sites in cAMP-dependent induction, AP-1 dependent induction, and DEX-dependent repression. As shown in Fig. 9AGo, 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. 9BGo, 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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Figure 9. Mutation of High-Affinity GR or AP-1 Binding Sites Reduces cAMP Stimulation, DEX-Dependent Repression, and c-fos Stimulation of the CRH nGRE

AtT-20 cells were transfected and treated as described in Materials and Methods (panel A) or Fig. 8Go (panel B). Data (mean ± SEM of at least three independent experiments) are expressed as fold induction. * Indicates DEX-dependent repression. {diamondsuit} Indicates reduced activation or repression vs. 3x CRH nGRE-Adh.

 
CREB Interactions at the CRH nGRE
AP-1 elements can mediate induction by CREB family members (45, 46), and cAMP modestly activates the CRH nGRE (Fig. 9AGo). 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. 10AGo, 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. 10AGo). 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 {Delta}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. 10BGo).



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Figure 10. CREB Interactions at the CRH nGRE

A, AtT-20 cells cotransfected with the indicated reporter and expression vector for full-length CREB (CREB-FL) or a dominant negative CREB mutant (CREB-BR), were treated, harvested, and assayed as described in Materials and Methods. Data (mean ± SEM of at least three independent experiments) are expressed as fold induction. * Indicates significant cAMP- or CREB-FL- dependent induction. {diamondsuit} Indicates reduced cAMP induction with CREB-BR overexpression. {diamondsuit}{diamondsuit} Indicates reduced cAMP- or CREB-FL-mediated induction vs. 3x CRH nGRE-Adh. B, AtT-20 cells were transfected with the indicated reporter and expression plasmids for full-length CREB and GR were then treated with DEX as described in Materials and Methods. Data (mean ± SEM of at least three independent experiments) are expressed as the percentage of CREB-stimulated promoter activity observed with DEX treatment.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
While intracellular signaling events leading to activation of gene transcription by glucocorticoids are relatively well understood, many important biological effects of corticosteroids are mediated through repression of specific gene transcription and involve complex, distinct mechanisms of transcriptional regulation that are cell- and promoter-specific (48). The CRH gene is of particular interest as a model for glucocorticoid-dependent repression, since it is a physiologically relevant target of corticosteroids and a critical component of long-loop feedback regulation of the HPA axis (3, 22). Although the corticotroph-derived AtT-20 cell line used in this series of transient transfection experiments is not a site of endogenous CRH expression (49), it represents a parallel limb of physiological corticosteroid feedback regulation and has been used in several independent laboratories to investigate glucocorticoid-dependent repression of CRH.

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. 6Go). 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. 7Go).

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{Delta}(-295/-249) and CRH{Delta}(-340/-249) demonstrated a magnitude of DEX-dependent repression comparable to the more limited nGRE deletion (CRH{Delta}(-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. 5Go). 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. 2Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EMSA
EMSA was performed as previously described (15, 64). Briefly, DNA probes were created by annealing complementary oligonucleotides designed with 5'-overhangs, labeled using the Klenow fragment of DNA polymerase, dNTPs, and [32P]({alpha})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. 2BGo, GR-sites 1–3) or potential AP-1-binding sites (Fig. 2BGo, 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. 2BGo. AP-1 binding site mutants (AP1 mut1 and AP1 mut2) are designated with respect to putative AP-1 binding sites illustrated in Fig. 2BGo. 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)[{Delta}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)[{Delta}-340 to -249]luc, CRH(-918)[{Delta}-295 to -249]luc, and CRH(-918)[{Delta}-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)[{Delta}AP1-site 2], CRH(-918)[{Delta}GR-site 2], and CRH(-918)[{Delta}GR-sites 1,2,3], in which the indicated site(s) were replaced with EcoRI site(s) (see Fig. 2BGo), were generated by recombinant PCR as described above, but with CRH-specific primers containing the desired mutations. CRH(-918)[{Delta}GR-site 2], CRH(-918)[{Delta}GR-sites 1,2,3], and CRH(-918)[{Delta}AP1-site 2] correspond to mut2, mut1,2,3, and AP1 mut2 probes used in EMSA experiments. CRH-(-249)[{Delta}CRE]luc was created by digestion of CRH(-918)-[{Delta}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).

{alpha}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{alpha} (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). p{Delta}ODLO (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 p{Delta}ODLO(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 p{Delta}ODLO 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-{Delta}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-{Delta}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-{Delta}GR site 2]Adh and [3x nGRE-{Delta}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 p{Delta}ODLO.

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
 
The authors gratefully acknowledge the technical support of Kathy Kilpatrick and the thoughtful review of this work by Drs. J. Omdahl, T. Williams, and T. Graham.


    FOOTNOTES
 
Address requests for reprints to: Richard I. Dorin, M.D., Chief, Endocrinology and Metabolism, Albuquerque Veterans Administration Medical Center, Medical Service (111), 1501 San Pedro Drive SE, Albuquerque, New Mexico 87108.

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Harbuz MS, Lightman SL 1989 Responses of hypothalamic and pituitary mRNA to physical and psychological stress in the rat. J Endocrinol 122:705–711[Abstract]
  2. Lightman SL, Harbuz MS, Knight RA, Chowdrey HS 1993 CRF mRNA in normal and stress conditions. Ann NY Acad Sci 697:28–38[Medline]
  3. Muglia L, Jacobson L, Dikkes P, Majzoub JA 1995 Corticotropin-releasing hormone deficiency reveals major fetal but not adult glucocorticoid need. Nature 373:427–432[CrossRef][Medline]
  4. Majzoub JA, Muglia LJ, Martinez C, Jacobson L 1995 Molecular and transgenic studies of the corticotropin-releasing hormone gene. Ann NY Acad Sci 771:293–300[Medline]
  5. Gomez MT, Magiakou MA, Mastorakos G, Chrousos GP 1993 The pituitary corticotroph is not the rate limiting step in the postoperative recovery of the hypothalamic-pituitary-adrenal axis in patients with Cushing syndrome. J Clin Endocrinol Metab 77:173–177[Abstract]
  6. Kovacs KJ, Mezey E 1987 Dexamethasone inhibits corticotropin-releasing factor gene expression in the rat paraventricular nucleus. Neuroendocrinology 46:365–368[Medline]
  7. Kovacs K, Kiss JZ, Makara GB 1986 Glucocorticoid implants around the hypothalamic paraventricular nucleus prevent the increase of corticotropin-releasing factor and arginine vasopressin immunostaining induced by adrenalectomy. Neuroendocrinology 44:229–234[Medline]
  8. Agnati LF, Fuxe K, Yu ZY, Harfstrand A, Okret S, Wikstrom AC, Goldstein M, Zoli M, Vale W, Gustafsson JA 1985 Morphometrical analysis of the distribution of corticotrophin releasing factor, glucocorticoid receptor and phenylethanolamine-N-methyltransferase immunoreactive structures in the paraventricular hypothalamic nucleus of the rat. Neurosci Lett 54:147–152[Medline]
  9. Alexis MN, Kitraki E, Spanou K, Stylianopoulou F, Sekeris CE 1990 Ontogeny of the glucocorticoid receptor in the rat brain. Adv Exp Med Biol 265:269–276[Medline]
  10. Whitfield Jr HJ, Brady LS, Smith MA, Mamalaki E, Fox RJ, Herkenham M 1990 Optimization of cRNA probe in situ hybridization methodology for localization of glucocorticoid receptor mRNA in rat brain: a detailed protocol. Cell Mol Neurobiol 10:145–157[Medline]
  11. Herman JP, Schafer MK, Thompson RC, Watson SJ 1992 Rapid regulation of corticotropin-releasing hormone gene transcription in vivo. Mol Endocrinol 6:1061–1069[Abstract]
  12. Imaki T, Nahan JL, Rivier C, Sawchenko PE, Vale W 1991 Differential regulation of corticotropin-releasing factor mRNA in rat brain regions by glucocorticoids and stress. J Neurosci 11:585–599[Abstract]
  13. Frim DM, Robinson BG, Pasieka KB, Majzoub JA 1990 Differential regulation of corticotropin-releasing hormone mRNA in rat brain. Am J Physiol 258:E686–692
  14. Robinson BG, Emanuel RL, Frim DM, Majzoub JA 1988 Glucocorticoid stimulates expression of corticotropin-releasing hormone gene in human placenta. Proc Natl Acad Sci USA 85:5244–5248[Abstract]
  15. Malkoski SM, Handanos CM, Dorin RI 1997 Localization of a negative glucocorticoid response element of the human corticotropin releasing hormone gene. Mol Cell Endocrinol 127:189–199[CrossRef][Medline]
  16. Guardiola-Diaz HB, Kolinske JS, Gates LH, Seasholtz AF 1996 Negative glucocorticoid regulation of cyclic adenosine 3',5'-monophosphate-stimulated corticotropin-releasing hormone-reporter expression in AtT-20 cells. Mol Endocrinol 10:317–329[Abstract]
  17. Van LP, Spengler DH, Holsboer F 1990 Glucocorticoid repression of 3',5'-cyclic-adenosine monophosphate-dependent human corticotropin-releasing-hormone gene promoter activity in a transfected mouse anterior pituitary cell line. Endocrinology 127:1412–1418[Abstract]
  18. Adler GK, Smas CM, Majzoub JA 1988 Expression and dexamethasone regulation of the human corticotropin-releasing hormone gene in a mouse anterior pituitary cell line. J Biol Chem 263:5846–5852[Abstract/Free Full Text]
  19. Spengler D, Rupprecht R, Van LP, Holsboer F 1992 Identification and characterization of a 3',5'-cyclic adenosine monophosphate-responsive element in the human corticotropin-releasing hormone gene promoter. Mol Endocrinol 6:1931–1941[Abstract]
  20. Mugele K, Kugler H, Spiess J 1993 Immortalization of a fetal rat brain cell line that expresses corticotropin-releasing factor mRNA. DNA Cell Biol 12:119–126[Medline]
  21. Guardiola-Diaz HM, Boswell C, Seasholtz AF 1994 The cAMP-responsive element in the corticotropin-releasing hormone gene mediates transcriptional regulation by depolarization. J Biol Chem 269:14784–14791[Abstract/Free Full Text]
  22. Vamvakopoulos NC, Chrousos GP 1993 Structural organization of the 5' flanking region of the human corticotropin releasing hormone gene. DNA Sequence 4:197–206[Medline]
  23. Dahlman-Wright K, Wright A, Carlstedt-Duke J, Gustafsson JA 1992 DNA-binding by the glucocorticoid receptor: a structural and functional analysis. J Steroid Biochem Mol Biol 41:249–272[CrossRef][Medline]
  24. Zilliacus J, Wright AP, Carlstedt-Duke J, Gustafsson JA 1995 Structural determinants of DNA-binding specificity by steroid receptors. Mol Endocrinol 9:389–400[Medline]
  25. Chen H, Srinivasan G, Thompson EB 1997 Protein-protein interactions are implied in glucocorticoid receptor mutant 465*-mediated cell death. J Biol Chem 272:25873–25880[Abstract/Free Full Text]
  26. Wissink S, van Heerde EC, Schmitz ML, Kalkhoven E, van der Burg B, Baeuerle PA, van der Saag PT 1997 Distinct domains of the RelA NF-kappaB subunit are required for negative cross-talk and direct interaction with the glucocorticoid receptor. J Biol Chem 272:22278–22284[Abstract/Free Full Text]
  27. Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin Jr AS 1995 Characterization of mechanisms involved in transrepression of NF-kappa B by activated glucocorticoid receptors. Mol Cell Biol 15:943–953[Abstract]
  28. Stromstedt PE, Poellinger L, Gustafsson JA, Carlstedt-Duke J 1991 The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation. Mol Cell Biol 11:3379–3383[Medline]
  29. Ray A, LaForge KS, Sehgal PB 1990 On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion. Mol Cell Biol 10:5736–5746[Medline]
  30. Hoeppner MA, Mordacq JC, Linzer DI 1995 Role of the composite glucocorticoid response element in proliferin gene expression. Gene Expression 5:133–141[Medline]
  31. Diamond MI, Miner JN, Yoshinaga SK, Yamamoto KR 1990 Transcription factor interactions: selectors of positive or negative regulation from a single DNA element. Science 249:1266–1272[Medline]
  32. Zhang XK, Dong JM, Chiu JF 1991 Regulation of {alpha}-fetoprotein gene expression by antagonism between AP-1 and the glucocorticoid receptor at their overlapping binding site. J Biol Chem 266:8248–8254[Abstract/Free Full Text]
  33. Liden J, Delaunay F, Rafter I, Gustafsson J, Okret S 1997 A new function for the C-terminal zinc finger of the glucocorticoid receptor. Repression of RelA transactivation. J Biol Chem 272:21467–21472[Abstract/Free Full Text]
  34. Caldenhoven E, Liden J, Wissink S, Van de Stolpe A, Raaijmakers J, Koenderman L, Okret S, Gustafsson JA, van der Saag PT 1995 Negative cross-talk between RelA and the glucocorticoid receptor: a possible mechanism for the anti-inflammatory action of glucocorticoids. Mol Endocrinol 9:401–412[Abstract]
  35. Shibata H, Spencer TE, Onate SA, Jenster G, Tsai SY, Tsai MJ, O’Malley BW 1997 Role of co-activators and co-repressors in the mechanism of steroid/thyroid receptor action. Recent Prog Horm Res 52:141–64[Medline]
  36. Janknecht R, Hunter T 1996 Transcription. A growing coactivator network. Nature 383:22–23[CrossRef][Medline]
  37. Hong H, Kohli K, Garabedian MJ, Stallcup MR 1997 GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors. Mol Cell Biol 17:2735–2744[Abstract]
  38. Drouin J, Sun YL, Chamberland M, Gauthier Y, De Lean A, Nemer M, Schmidt TJ 1993 Novel glucocorticoid receptor complex with DNA element of the hormone-repressed POMC gene. EMBO J 12:145–156[Abstract]
  39. Sakai DD, Helms S, Carlstedt-Duke J, Gustafsson JA, Rottman FM, Yamamoto KR 1988 Hormone-mediated repression: a negative glucocorticoid response element from the bovine prolactin gene. Genes Dev 2:1144–1154[Abstract]
  40. Stauber C, Altschmied J, Akerblom IE, Marron JL, Mellon PL 1992 Mutual cross-interference between glucocorticoid receptor and CREB inhibits transactivation in placental cells. New Biol 4:527–540[Medline]
  41. Konig H, Ponta H, Rahmsdorf HJ, Herrlich P 1992 Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo. EMBO J 11:2241–2246[Abstract]
  42. Hard T, Kellenbach E, Boelens R, Maler BA, Dahlman K, Freedman LP, Carlstedt-Duke J, Yamamoto KR, Gustafsson JA, Kaptein R 1990 Solution structure of the glucocorticoid receptor DNA-binding domain. Science 249:157–160[Medline]
  43. Imai E, Miner JN, Mitchell JA, Yamamoto KR, Granner DK 1993 Glucocorticoid receptor-cAMP response element-binding protein interaction and the response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids. J Biol Chem 268:5353–5356[Abstract/Free Full Text]
  44. Akerblom IE, Slater EP, Beato M, Baxter JD, Mellon PL 1988 Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer. Science 241:350–353[Medline]
  45. Deutsch PJ, Hoeffler JP, Jameson JL, Habener JF 1988 Cyclic AMP and phorbol ester-stimulated transcription mediated by similar DNA elements that bind distinct proteins. Proc Natl Acad Sci USA 85:7922–7926[Abstract]
  46. Sassone-Corsi P, Ransone LJ, Verma IM 1990 Cross-talk in signal transduction: TPA-inducible factor jun/AP-1 activates cAMP-responsive enhancer elements. Oncogene 5:427–431[Medline]
  47. Hoeffler JP 1992 Structure/function relationships of CREB/ATF proteins. J Invest Dermatol 98:21S–28S
  48. Karin M 1990 Too many transcription factors: positive and negative interactions. New Biol 2:126–131[Medline]
  49. Dorin RI, Zlock DW, Kilpatrick K 1993 Transcriptional regulation of human corticotropin releasing factor gene expression by cyclic adenosine 3',5'-monophosphate: differential effects at proximal and distal promoter elements. Mol Cell Endocrinol 96:99–111[CrossRef][Medline]
  50. Majzoub JA, Emanuel R, Adler G, Martinez C, Robinson B, Wittert G 1993 Second messenger regulation of mRNA for corticotropin-releasing factor. Ciba Found Symp 172:30–43; discussion 43–58[Medline]
  51. Kushner PJ, Baxter JD, Duncan KG, Lopez GN, Schaufele F, Uht RM, Webb P, West BL 1994 Eukaryotic regulatory elements linking in plasmid DNA: the activator protein-1 site in pUC. Mol Endocrinol 8:405–407[Medline]
  52. Chalepakis G, Schauer M, Cao XA, Beato M 1990 Efficient binding of glucocorticoid receptor to its responsive element requires a dimer and DNA flanking sequences. DNA Cell Biol 9:355–368[Medline]
  53. Yang-Yen HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin J, Karin M 1990 Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. Cell 62:1205–1215[Medline]
  54. Vamvakopoulos NC, Chrousos GP 1994 Hormonal regulation of human corticotropin-releasing hormone gene expression: implications for the stress response and immune/inflammatory reaction. Endocr Rev 15:409–420[Medline]
  55. Masquilier D, Sassone-Corsi P 1992 Transcriptional cross-talk: nuclear factors CREM and CREB bind to AP-1 sites and inhibit activation by Jun. J Biol Chem 267:22460–22466[Abstract/Free Full Text]
  56. Hoeffler JP, Deutsch PJ, Lin J, Habener JF 1989 Distinct adenosine 3',5'-monophosphate and phorbol ester-responsive signal transduction pathways converge at the level of transcriptional activation by the interactions of DNA-binding proteins. Mol Endocrinol 3:868–880[Abstract]
  57. Lamph WW, Dwarki VJ, Ofir R, Montminy M, Verma IM 1990 Negative and positive regulation by transcription factor cAMP response element-binding protein is modulated by phosphorylation. Proc Natl Acad Sci USA 87:4320–4324[Abstract]
  58. Benbrook DM, Jones NC 1990 Heterodimer formation between CREB and JUN proteins. Oncogene 5:295–302[Medline]
  59. Iordanov M, Bender K, Ade T, Schmid W, Sachsenmaier C, Engel K, Gaestel M, Rahmsdorf HJ, Herrlich P 1997 CREB is activated by UVC through a p38/HOG-1-dependent protein kinase. EMBO J 16:1009–1022[Abstract/Free Full Text]
  60. Pierrat B, Correia JS, Mary JL, Tomas-Zuber M, Lesslauer W 1998 RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38{alpha} mitogen-activated protein kinase (p38{alpha}MAPK). J Biol Chem 273:29661–29671[Abstract/Free Full Text]
  61. Zhen X, Uryu K, Wang HY, Friedman E 1998 D1 dopamine receptor agonists mediate activation of p38 mitogen-activated protein kinase and c-Jun amino-terminal kinase by a protein kinase A-dependent mechanism in SK-N-MC human neuroblastoma cells. Mol Pharmacol 54:453–458[Abstract/Free Full Text]
  62. Deak M, Clifton AD, Lucocq LM, Alessi DR 1998 Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J 17:4426–4441[Abstract/Free Full Text]
  63. Subramaniam N, Cairns W, Okret S 1997 Studies on the mechanism of glucocorticoid-mediated repression from a negative glucocorticoid response element from the bovine prolactin gene. DNA Cell Biol 16:153–163[Medline]
  64. Freedman LP, Alroy I 1993 Gel mobility shift assay to study nuclear hormone receptor-DNA interactions. Methods Mol Genet 1:280–299
  65. Maxwell IH, Harrison GS, Wood WM, Maxwell F 1989 A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription. Biotechniques 7:276–280[Medline]
  66. Nordeen SK 1988 Luciferase reporter gene vectors for analysis of promoters and enhancers. Biotechniques 6:454–457[Medline]
  67. Kay TWH, Jameson JL 1992 Identification of a gonadotropin-releasing hormone-responsive region in the glycoprotein hormone a-subunit promoter. Mol Endocrinol 6:1767–1773[Abstract]
  68. Giguere V, Hollenberg SM, Rosenfeld MG, Evans RM 1986 Functional domains of the human glucocorticoid receptor. Cell 46:645–652[Medline]