| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
and ERß
Hubrecht Laboratory (A.v.d.S., A.J.S., M.O.R., A.W.M.Z., P.T.v.d.S.) Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands; Institute of Physiological Chemistry and Pathobiochemistry (S.G., C.B.), Johannes Gutenberg University Mainz, 55099 Mainz, Germany; and Mental Health Research Institute (A.F.S.), Ann Arbor, Michigan 48109-0720
Address all correspondence and requests for reprints to: Anja van de Stolpe, M.D., Ph.D., Hubrecht Laboratory, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. E-mail: anja{at}niob.knaw.nl.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
induced and ERß repressed promoter activity in a dose-dependent manner. TNF inhibited promoter induction by ER in the absence and presence of estradiol. Three ERE half-sites in the CRH-BP promoter bound ER and ERß in an EMSA, and disruption of ERE half-sites by site-directed mutagenesis abolished ligand-independent induction by ER and ERß and promoter enhancement by estradiol-activated ER. Repression by estradiol/ERß was unaffected by disruption of ERE half-sites, activating protein 1, cAMP response element, GATA, or nuclear factor 
B sites, and reversed to promoter induction by estrogen antagonists, tamoxifen and ICI 182,780, suggesting corepressor involvement. In hypothalamic GT1–7 cells, Western blotting demonstrated rapid induction of endogenous CRH-BP expression by estradiol-bound ER, which was inhibited by TNF. We propose a model in which ERs maintain basal CRH-BP expression in pituitary and neurosecretory cells, whereas in the presence of ER estrogen enhances CRH-BP transcription, causing down-regulation of the HPA axis, and nuclear factor B-activating cytokines activate the HPA axis by inhibiting ER. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Estrogens are well known positive modifiers of mood, although the exact molecular mechanism of action remains to be determined (13). Estrogens act through binding to two related estrogen receptors (ERs), ER and ERß (14, 15). Ligand-bound ERs homo- or heterodimerize and function as transcription factors that recognize and transactivate estrogen response elements (EREs). Alternatively, multiple so called ERE half-sites, or incomplete EREs, have been demonstrated to confer estrogen responsiveness upon a subset of ER-target genes (16, 17). In addition, ERs have been reported to cross-talk with members of different transcription factor families, resulting in modulation of each others transcriptional activity (18, 19, 20, 21, 22 ). Notably, transcriptional activity of ERs can be negatively modified by interaction with the nuclear factor B (NFB) transcription factor (21). NFB is an important transcription factor in inflammatory processes, and activation of NFB is associated with stimulation of the HPA axis (3).
Recently, it was shown in vivo that mouse pituitary CRH-BP protein expression is positively regulated by estrogens (2), which suggests that CRH-BP may be a target gene for estrogens. As such, it may be involved in mediating positive effects of estrogen on mood. The human CRH-BP (hCRH-BP) promoter has been cloned but not yet functionally characterized (23). Comparison between human, mouse, and rat CRH-BP (rCRH-BP) promoters reveals that all three contain ERE half-site sequences (Refs.1 and 23 , and Fig. 1
), providing a potential molecular basis for CRH-BP as a novel estrogen target gene.
|
and ERß constitutively activate the hCRH-BP promoter. In the presence of the estrogenic hormone estradiol, the CRH-BP promoter is differentially regulated, i.e. further enhanced by ER and repressed by ERß. In addition, TNF, as a typical inflammatory cytokine, markedly inhibited the ER-mediated induction in CRH-BP promoter activity. We have extended these results to the endogenous CRH-BP gene using a murine hypothalamic neurosecretory cell line, in which estradiol rapidly up-regulates CRH-BP protein expression, whereas this effect is dramatically inhibited by TNF.
We propose a model in which estrogens play a role in regulation of the neuroendocrine HPA axis by up-regulating CRH-BP expression in hypothalamic neurosecretory and pituitary cells, whereas inflammatory cytokines such as TNF activate the HPA axis, at least in part, by blocking the estrogen effect on CRH-BP transcription.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
). Percentage homology between the three species was highest in the proximal 300 bp with a mean of 82%, which was reduced to 56% over the next 500 bp. Further upstream the percentage homology varied around 35% (only shown up to –1500 in Fig. 1A). This indicated that the –841/+46 region, relative to the transcription start site in the human promoter, was likely to contain most important regulatory elements and to represent the functional promoter region of the hCRH-BP gene.
ER and ERß Mediate Opposite Effects of Estradiol on Transactivation of the hCRH-BP Promoter
To investigate the effect of estrogen on CRH-BP expression, human osteosarcoma U-2 OS cells were transiently transfected with the –841 hCRH-BP promoter coupled to luciferase. The sequence of the –841/+46 hCRH-BP promoter region obtained by PCR was 100% homologous with the human genome sequence containing the CRH-BP gene (Ref|NT_006713.13|Hs5_6870, nucleotide 5640160-5641047). This sequence varied two nucleotides (GA instead of AG) from the published sequence GI|300094 at –774 (23).
U-2 OS do not express endogenous ER (data not shown), and ER and ERß, contained in the same expression vectors, were cotransfected. In the absence of ligand, both ER and ERß induced the hCRH-BP promoter (Fig. 2, A and B), whereas estradiol (10–8 M) induced the promoter an additional three times in the presence of ER (Fig. 2A), but repressed transcription by more than 50% in the presence of ERß (Fig. 2B and Fig. 3B). A 10-fold reduction in the amount of transfected ERß did not change ERß-mediated promoter repression (Fig. 2C), which makes squelching as a cause of the repressive effect very unlikely. Dose-response experiments showed that the minimal effective estradiol concentration was 10–10 M for both ER and ERß (Fig. 2, D and E). These results demonstrate that in U-2 OS cells unliganded ER and ERß were both capable of inducing the hCRH-BP promoter, whereas estradiol modified this ligand-independent effect in opposite ways.
|
|
(Fig. 3A), but repressed in the presence of cotransfected ERß (Fig. 3B). Taken together, the response of the CRH-BP promoter to estrogen appears to be an evolutionary conserved phenomenon.
ERE Half-Sites in the hCRH-BP Promoter Mediate Estradiol-Dependent and -Independent Induction by ER, and Estradiol-Independent Induction, But Not Estradiol-Dependent Repression, by ERß
To clarify the mechanism by which estrogen regulates transcription from the CRH-BP promoter, we proceeded to identify potential estrogen-sensitive regulatory elements in the promoter region contained in the –841 CRH-BP-luciferase construct: 935 bp upstream from the ATG start codon of the human (Ref|NT_ 006713.13|Hs5_6870, nucleotide 5640160–5641094), mouse (ref|NT 039590.1|Mm13 39630 30, nucleotide 3454740–3453121), and rat (Ref|NW_043493.1|Rn2_ 796, nucleotide 220494–219560) CRH-BP genes were analyzed for transcription factor binding sites using MatInspector and regulatory sequence analysis (RSA) Tools. This part of the human 5'-regulatory region appeared to contain three ERE half-sites (AGGTCA at –267, –682, –735, relative to the transcription start site, consensus half-site sequence in bold), the mouse contained two (AGGTCA at –303 and –457 relative to the ATG start codon), and the rat contained one (AGGTCA at –303 relative to the ATG) (Fig. 1B). In addition, the rat 5'-upstream region contains four additional ERE half-sites in the 3500 bp which make up the promoter construct that was used for the transfection experiments [AGGTCA at –1491, GGTCA at –1657, GGTCA at –2585, and GGTCA on the reverse strand at –2767 (data not shown)].
The three ERE half-sites in the hCRH-BP promoter are located at variable distance from each other (Fig. 1B): 53 bp between the two most distal sites (–682 and –735) and 415 bp between the –682 and the most proximal site (–267). Multiple ERE half-sites have been reported to be able to cooperate in estrogen-induced transcription activation (17). To determine which of these ERE half-sites in the hCRH-BP promoter might mediate the effects of ER on CRH-BP expression, we either mutated or selectively deleted each of the three ERE half-sites in the human promoter.
The hormone-independent promoter induction observed with ER was significantly reduced upon disruption of either the –682 or –735 ERE half-site (but not the –267 half-site) or all three ERE half-sites (C-267/–682/–735) when only background luciferase levels remained (Fig. 4A). Similarly, basal transcription in the presence of unliganded ERß was reduced to half by eliminating all three half-sites, however, inactivation of a single site had no evident effect (Fig. 4B). These results indicate an important role for the ERE half-sites in constitutive transcriptional activity of this promoter, which is mediated through ER and ERß.
|
failed to reach significance upon disruption of the –682 and 735 ERE half-sites, whereas disruption of the most proximal –267 half-site was without effect (Fig. 4A). With the triple mutant, induction by estradiol was completely abolished (Fig. 4A), indicating that all three intact ERE half-sites are required for maximal enhancement of the hCRH-BP promoter by ligand-activated ER. In contrast to ER, estradiol-dependent repression of the promoter in the presence of ERß persisted upon disruption of all three half-sites (Fig. 4B), suggesting that the observed estradiol/ERß-effect occurred independently of intact ERE half-sites.
ER and ERß Directly Bind to the ERE Half-Sites in the hCRH-BP Promoter
To investigate whether ERE half-sites would be capable of directly binding ER and ERß, fluorescent EMSAs were performed, using FAM-labeled oligo duplexes containing either a consensus ERE (FAM-ERE), each of the three ERE half-sites in the hCRH-BP promoter (FAM-267, FAM-682, FAM-735), the mutated –267 half-site (as present in the mutant promoter construct) (FAM-267m), or a random nucleotide sequence (FAM-EREm) of the same length. Human recombinant ER or ERß was used to assess binding (Figs. 5 and 6). Both ER (Fig. 5) and ERß (Fig. 6) specifically retarded all three of the ERE half-site oligo duplexes, indicating specific binding of both receptors to these half-sites with their respective flanking sequences (Fig. 5A, lanes 6–10; and Fig. 6, lanes 6–10). The mobility of the ERE half-site-ER complexes was similar to the ERE-ER complexes, suggesting that both ER and ERß bound as dimers to the half-sites. ER seemed to bind stronger to the –682 half-site than to the –735 and –267 half-sites (Fig. 5A, lanes 7–9), and strongest to the palindromic ERE (Fig. 5A and Fig. 6; compare lane 6 and lane 8). An additional EMSA performed on the –682 half-site, an important contributor to ER-mediated promoter induction, showed that the amount of ER bound to the ERE half-site was dependent on protein concentration (Fig. 5B, lanes 7–9). Receptor bound to the ERE or ERE half-site could be specifically competed away with excess unlabeled ERE oligo duplex (Fig. 5C). As expected, based on the transfection experiments, neither ER nor ERß bound to the mutated –267 ERE half-site (Fig. 7). Cell extract from COS-1 cells overexpressing another transcription factor, i.e. the p65 subunit of the NFB transcription factor, did not bind to the –267 half-site (Fig. 7, lanes 4 and 8), whereas cell extract from U2OS cells showed only aspecific binding both to the –267 and to the mutant –267 oligo (Fig. 7, lanes 3 and 7), thus lending further credit to the specificity of ER binding to the half-sites. Together, these results provide strong evidence that, at least in vitro, direct and specific binding of ER and ERß occurs to the three independent ERE half-sites in the human –841 CRH-BP promoter.
|
|
|
and ERß to all three ERE half-sites is required for significant induction of the hCRH-BP promoter. For ERß, binding requirements seem to be less stringent, as binding to either combination of two half-sites appeared to be sufficient for ligand-independent promoter activation, whereas estradiol-dependent repression occurred in the absence of functional ERE half-sites and apparently represented an indirect effect.
In Contrast to Estradiol, Estrogen Antagonists Induce the CRH-BP Promoter in the Presence of ERß
Our finding, that promoter repression by estradiol-activated ERß was independent of ERE half-sites, prompted us to consider an alternative mechanism, either negative cross-talk between estradiol-bound ERß and another transcription factor involved in constitutive activation of the hCRH-BP promoter, or estradiol-induced binding of a corepressor to ERß (18, 24).
With respect to the first option, estrogen-activated ERß has been demonstrated previously to directly interact with, and inhibit, specific transcription factors, e.g. activating protein 1 (AP1) and GATA-1 (18, 20, 22). The observation that both the human and rat CRH-BP promoters were similarly repressed by estradiol-bound ERß (Fig. 3B) suggested a conserved mechanism, e.g. involvement of a response element that both promoter regions have in common. Promoter alignment and analysis of conserved regions for transcription factor binding sites revealed an AP1 (–195, TGACTGA), a cAMP response element (CRE) (–127, core sequence CGTCA), and a GATA site (–590, GATAAAG), which were conserved with respect to both sequence and location (Fig. 1B). Two putative NFB response elements (GGAAAATTCCC at –315, and GGGCTTTCC on the complementary strand at +37) were partly conserved, as indicated in Fig. 1B. Using site-directed mutagenesis, each of these regulatory elements was disrupted in separate constructs. Subsequent transfection experiments demonstrated that basal activity of the hCRH-BP promoter was reduced upon disruption of the CRE, GATA, or –315 NFB (Fig. 8A) site, indicating their involvement in constitutive transcriptional activity of the promoter. However, neither of these mutations (Fig. 8, B–D), nor mutation of the +37 NFB (Fig. 8E) or AP1 (Fig. 8F) sequence, abolished estradiol/ERß-induced promoter repression, thus excluding negative cross-talk with the corresponding transcription factors as an explanation for the repressive effect (Fig. 8, B–F).
|
). This result indicates that the repressive effect of ERß is specific for estradiol as ligand, which is suggestive of corepressor binding.
|
Represses ER-Mediated Induction of the hCRH-BP Promoter Both in the Absence and Presence of Estradiol, which exert their effects through activation of the NFB signaling pathway (3). Notably, the p65 subunit of NFB is known to modify effects of estradiol through cross-talk with estrogen receptors (21). Therefore, we investigated whether TNF might modify transcriptional effects of ER or ERß on the CRH-BP promoter (Fig. 10). TNF did not affect transcription of the hCRH-BP promoter in U-2 OS cells in the absence of ER (Fig. 10A). However, transcription in the presence of unliganded ER was reduced to half, whereas estradiol/ER-induced transcription was inhibited by more than 70% by simultaneous treatment with TNF (Fig. 10B). In contrast, no effect was observed on estradiol/ERß-mediated repression (Fig. 10C). Interestingly, TNF significantly induced the hCRH-BP promoter in the presence of unliganded ERß, suggesting a potential synergistic effect between the two transcription factors.
|
B may interfere with estradiol-induced CRH-BP expression, especially in cells predominantly expressing ER. However, the ERE half-site-independent repressive effect of estradiol/ERß was not affected by TNF, again emphasizing differences between the molecular mechanisms by which ER and ERß regulate CRH-BP transcription.
Estradiol Increases Endogenous Expression of CRH-BP Protein in Hypothalamic Neuronal GTI-7 Cells, and TNF Inhibits the Effect of Estradiol
The endogenous CRH-BP gene has been shown to be expressed in pituitary and in several brain regions, including hypothalamus (5). Our finding that the CRH-BP promoter is a target for estradiol-bound ER implies that the endogenous CRH-BP gene may similarly be under estrogen control. However, to our knowledge no human hypothalamic or pituitary cell line that expresses both CRH-BP and ER is currently available to investigate this issue. We therefore used the murine GT1–7 hypothalamic secretory neuronal cell line, which has been reported to express both ER and ERß (25). Expression of both ER and ERß was confirmed by Western blotting under our experimental conditions (data not shown). Western blot analysis further demonstrated that treatment with estradiol increased CRH-BP protein expression within 12 h, and this increase was completely prevented by pretreatment of the cells with the estrogen antagonist ICI (Fig. 11A). Because ICI competes specifically with estradiol for ER, these results demonstrate that the effect of estradiol is mediated by binding to the receptor, whereas the rapid increase in CRH-BP protein expression is at least supportive of a direct transcriptional response. Interestingly, TNF did not change CRH-BP expression by itself, but instead inhibited the estradiol-induced increase in protein expression (Fig. 11B).
|
. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and ERß Exert Opposing Effects on the hCRH-BP Promoter, requiring multiple intact ERE half-sites to induce the promoter, whereas estradiol-bound ERß repressed transcription in a ERE half-site-independent manner. ERs classically enhance promoter activity by binding to an ERE consisting of two well-defined palindromic half-sites separated by three nucleotides. However, cooperation between multiple ERE half-sites at considerable distance from each other may also confer estrogen/ER inducibility upon gene promoters, probably involving direct binding of an ER dimer to separated direct repeat half-sites (17, 26, 27, 28). ER has been documented to bind to some, but not all, ERE half-sites, e.g. in promoter regions of the prolactin, the human Na+/H+ exchanger regulatory factor/Ezrin-Radixin-Moesin binding protein 50 (NHE-RF/EBP50), and the high-density lipoprotein receptor genes (15, 17, 27, 28). Whether or not ER binds to a half-site may depend on the flanking sequence and the ER subtype, i.e. ER or ERß (17, 27). The half-sites in the CRH-BP promoter contained 5'-flanking nucleotides (underlined) which have been shown to provide additional support for ER binding to an ERE: –267 CAGGTCA, –682 CAGGTCA, and –735 AGGTCA (29), and both ER and ERß bound specifically to all three half-sites in an EMSA, but not to the mutated –267 half-site. The identical mobility patterns for full ERE/ER and ERE half-site/ER complexes further support the concept that both ERs bind as dimers, and not monomers, to two direct repeat half-sites (16, 27).
Whereas disruption of the –682 or –735 ERE half-site in the hCRH-BP promoter effectively abolished the significant estrogen/ER response, disruption of the –682 ERE half-site, which in the EMSA demonstrated strong binding to ER, had the most profound effect on the estrogen/ER response. Interestingly this half-site with its 3'-flanking sequence closely resembles an ERE with one mismatch (A instead of C) and two nucleotides instead of the obligatory three separating the palindromic half-sites: atcCAGGTCAagTGACAgag. Palindromic EREs mediate stronger estrogen responses than multiple ERE half-sites, and we speculate that this DNA sequence may bend in the right way to allow an ER homodimer to bind and transactivate, as has been suggested for the ERE-like sequence (GGTCATGACC) in the NHE-RF/EBP50 promoter (17, 30). Alternatively, because direct repeats are more flexible in binding ER than a palindromic ERE, the –735 half-site may function in a direct repeat ERE with the –682 half-site, which at close distance is probably preferred over the –267 half-site. Such a direct repeat may allow the ER homodimer to bind effectively to both half-sites with looping DNA in between (16). A similar DNA looping mechanism has been proposed to explain cooperative activity of widely spaced ERE sequences (31) and of ERE half-sites in the rat prolactin promoter (28). Interestingly, the distance of 53 nucleotides between the –735 and –682 site represents five helical turns, which has been shown to be the optimal distance between EREs to act synergistically (31).
Remarkably, in the presence of estrogenic hormone, ER and ERß exerted opposite effects on CRH-BP transcription. Whereas ligand-activated ERß has been proposed to negatively control enhancing effects of ER on an ERE (32, 33), estradiol-bound ERß repressed the hCRH-BP promoter independently of ERE half-sites or the presence of ER. This repressive effect of estradiol-bound ERß persisted when a 10-fold lower amount of ERß expression vector was transfected, excluding aspecific squelching of essential transcriptional coactivators as a cause for the observed promoter repression. The observation that estradiol/ERß-mediated repression was also evident in the rat promoter led us to search for an evolutionary conserved mechanism. The CRH-BP promoter contains several conserved response elements, i.e. AP1, CRE, GATA, and NFB sequences, of which the corresponding transcription factors can both contribute to constitutive transcription and interact with ERß (18, 19, 20, 21, 34, 35, 36, 37). However, disruption of individual response elements did not abolish the repressive effect of estradiol-bound ERß, eliminating the possibility of negative interaction between ERß and these transcription factors. Despite this, mutation of the CRE, GATA, or –315 NFB site did reduce basal activity of the CRH-BP promoter, indicating that CRE binding protein (CREB), GATA, and NFB transcription factors indeed contributed to constitutive activity of the CRH-BP promoter.
An intriguing alternative explanation for ERß-mediated repression is a ligand-specific change in ERß conformation, leading to differential binding of coregulator proteins (for review, see Ref.38). Interestingly, the corepressor N-CoR has been shown recently to fit this concept and bind specifically to the ligand-binding domain of estradiol-bound ERß, but not to antagonist-bound ERß (24). In support of such a mechanism, we observed that the estrogen antagonists tamoxifen and ICI significantly induced, instead of repressed, the hCRH-BP promoter in the presence of ERß. At present, we can only speculate with regard to the nature of a putative corepressor molecule, which is the subject of current investigations.
ERE Half-Sites Are Important for Constitutive Transactivation of the hCRH-BP Promoter
In the absence of hormone, both ER and ERß induced the hCRH-BP promoter. Although ERß has been observed to bind to its response element in the absence of ligand, both unliganded ERs must be activated by kinases such as protein kinase A or MAPK to be able to transactivate a palindromic ERE (19, 39, 40, 41, 42). To our knowledge no evidence is available on a similar mechanism to account for unliganded transactivation through ERE half-sites. Alternatively, unliganded ERs may form transcriptionally active heterodimers with components of transcription factors that are known to participate in constitutive promoter activity (e.g. AP1, GATA, NFB, or CREB), or otherwise interact on the hCRH-BP promoter with specific components of the basal transcription machinery (e.g. TATA-binding protein, transcription factor IIB, and TATA-binding protein-associated factor) to enhance basal transcription rate (18, 36, 37, 43, 44, 45, 46). Our results indicated that CREB, NFB, and GATA transcription factors were indeed involved in basal transactivation; however, upon disruption of their corresponding response elements, unliganded ER and ERß could still induce the hCRH-BP promoter (Fig. 8 and our unpublished results). This excludes important collaboration between these transcription factors and ER. Although the mechanism thus remains to be defined, it is an important finding that, even in the absence of estrogenic hormones, ER and ERß may play a role in the regulation of basal levels of CRH-BP.
TNF Inhibits the Enhancing Effect of Estradiol/ER on the hCRH-BP Promoter
Whereas an estradiol-induced increase in CRH-BP expression is expected to limit activation of the HPA axis by scavenging CRH, factors that inhibit CRH-BP expression are likely to stimulate this important neuroendocrine axis. Well-known stimulators of the HPA axis, and therefore candidates to repress CRH-BP transcription, are inflammatory cytokines such as TNF (3). Interestingly, we found that TNF inhibited the inducing effect of ER on the hCRH-BP promoter, both in the presence and absence of estradiol. TNF is a prototypical activator of the NFB transcription factor complex, which consists of a homo- or heterodimer of two members of the NFB family, classically a p50/p65 heterodimer, and enhances transcription by binding to NFB response elements (47). The p65 subunit of NFB also binds to members of the nuclear receptor family to generally repress transcription of their target genes independently of DNA binding (21, 48). Such a cross-talk between p65 and ER may explain the inhibitory effect of TNF on the CRH-BP promoter. On the other hand, NFB has also been described to functionally interact with ERß (49), and this may provide a tentative explanation for the observed small synergistic effect of TNF and unliganded ERß. We propose that inhibition of CRH-BP expression by interaction between activated NFB and ER contributes to the stimulatory effect of inflammatory cytokines on activity of the HPA axis.
The Physiological Significance of CRH-BP Regulation by Estrogen and NFB
Our observation that the rCRH-BP promoter responded in a similar fashion to estradiol, despite the fact that individual ERE half-sites were not conserved at corresponding positions, suggests that in rodents as well as in humans, the CRH-BP promoter is likely to be under estrogen control. The larger induction by estradiol/ER observed with the rat promoter is probably due to the larger 5'-upstream region in the rat luciferase construct (–3500). This sequence contains an additional four ERE half-sites, of which two half-sites resemble an imperfect ERE (at –2585 with respect to the ATG: GGTCAtagaTGACa, and at –1491: AGGTCAtggTatgC), and may conceivably contribute to the stronger estrogen response.
The U-2 OS cell line was well suited for analyzing transcriptional mechanisms involved in regulation of the hCRH-BP promoter, because it does not express endogenous ERs, allowing controlled expression of transfected ER or ERß. However, results thus obtained may not always represent transcriptional regulation of the corresponding endogenous gene, which would require a direct assay on the endogenous hCRH-BP gene promoter, e.g. a chromatin immunoprecipitation assay. Unfortunately, no human cell line has been identified that expresses CRH-BP and ERs, precluding such an experiment. Instead, we decided to use a nonhuman cell line uniquely expressing CRH-BP and both ER and ERß (25) to test estrogen responsiveness of the endogenous CRH-BP gene. In these murine hypothalamic neuronal GT1–7 cells, estradiol increased CRH-BP protein expression, and this increase was blocked by the estrogen antagonist ICI, demonstrating that estradiol induced the endogenous gene through binding to ER. Moreover, the rapid increase in protein expression within 12 h suggested direct transcriptional regulation of the murine CRH-BP gene and provided an explanation for the in vivo increase in CRH-BP mRNA and protein expression reported in the mouse pituitary during proestrus (2). Despite the fact that both GT1–7 cells and the pituitary express ERß in addition to ER (Refs.25 and 50 , and our own results), the net effect of exposure to estrogen was in each case an increase in CRH-BP expression. This suggests that ER may be the dominant ER in these cells. However, the exact ratio between ER and ERß expression levels in pituitary and GT1–7 cells remains to be established.
The finding that TNF strongly inhibited the estradiol-induced increase in CRH-BP expression in GT1–7 cells perfectly matched with our transfection data on the human promoter and again emphasizes the apparently conserved nature of CRH-BP regulation. It strongly suggests that TNF, and/or other NFB-activating cytokines, play a role in regulation of the HPA axis by reducing local estrogen-stimulated CRH-BP expression. Interestingly, in the absence of estrogen, TNF had no effect on endogenous CRH-BP expression, whereas our transfection results clearly demonstrated that TNF inhibited ER-, and enhanced ERß-, mediated hCRH-BP promoter induction. Because GT1–7 cells express both ERs, we propose that these opposite effects of TNF effectively cancelled each other out. These results do not preclude a role for TNF to differentially regulate CRH-BP protein expression in the absence of estrogen, depending on the ER subtype expressed.
Interestingly, GT1–7 cells are GnRH-releasing neurosecretory cells derived from the hypothalamus (25). Using in situ hybridization, CRH-BP expression has been reported to be present in a few secretory cells in the male rat hypothalamus (5); however, CRH-BP expression in hypothalamic secretory neurons may be more prominent in female animals due to the presence of estrogens. We propose that estrogens may also up-regulate CRH-BP expression in neurosecretory cells, where it may similarly play a role in blocking actions of CRH.
The results presented in this paper, taken together with the previously published results on in vivo CRH-BP protein expression in the mouse (2), provide compelling evidence that estrogen responsiveness of the CRH-BP promoter is conserved between rodents and humans and is likely to play a role in the physiological regulation of CRH-BP expression in pituitary and possibly also in hypothalamus. In addition, the observed inhibition of CRH-BP expression by TNF may play a role in activation of the HPA axis as a normal response to inflammation, e.g. during systemic infection, but also as a pathological mechanism in certain affective disorders characterized by increased activated NFB and abnormal activation of the HPA axis (3, 11).
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
was from Roche Molecular Biochemicals (Mannheim, Germany), and baculovirus-expressed human recombinant ER and ERß were from PanVera (Madison, WI). The goat antimouse CRH-BP and actin polyclonal antibodies were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Cell Culture
Human osteosarcoma U-2 OS cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium (DF; Life Technologies, Inc., Gaithersburg, MD), buffered with 44 mM NaHCO3 and supplemented with 7.5% fetal calf serum (FCS) (Bodinco, Alkmaar, The Netherlands). Cells were cultured at 37 C in a 7.5% CO2 humidified atmosphere. GT1–7 cells were kindly supplied by Dr. R. Weiner (University of California, San Francisco, CA) and have been described previously (25). Cells were cultured in DMEM, supplemented with 10% FCS under standard conditions (37 C, 5% CO2). COS-1 cells were cultured as described previously (51).
Plasmids
All primers were purchased from Isogen Bioscience BV (Maarsen, The Netherlands). The expression vector encoding human ER (pSG5-hER) and human ERß (pSG5-hERß) were kind gifts of Dr. P. Chambon (Strasbourg, France) and Dr. J.-Å. Gustafsson (Huddinge, Sweden), respectively. The –841 hCRH-BP-luciferase promoter construct (–841 to +46, relative to the transcription initiation site), containing all three ERE half-sites (located at –267, –682, and –735), was amplified with Pfu polymerase from human genomic DNA with the following primers: forward primer, 5'-CCGTCGACAGCTTCCTGGCAGTTCCTTCTA-3'; reverse primer, 5'-GCGGTACCGTCCTGGGCTTTCCTTCCTACACC-3'. A SalI restriction site was added to the forward primer, and a KpnI site was added to the reverse primer. The PCR product was cloned in the pCRII-TOPO vector (Invitrogen), cut out with SalI/KpnI, and subsequently ligated into p19-luc (52). Mutant or deletion promoter constructs were created by using the QuikChange Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) using the following forward primers (mutated nucleotides indicated in lower case). Reverse primers were complementary to the forward primers. To mutate the –267 ERE half-site (CRHBP-267), the forward primer 5'-GTAGCATTTAGCAGtTCcACAAAATGCACTCCC-3' was used. The –682 and –735 ERE half-sites were selectively deleted (CRHBP-682 and CRHBP-735) using, respectively, the forward primers 5'-GTGTACATCATGTAAACATCCAGTGACAGAGCTCAGCTAAAATCC-3' and 5'-GGTTCAGATGGATGCGATGGCTACAGCTACCTGGTCTAGG-3'. To obtain the triple mutant with all three ERE half-sites mutated/deleted (C-267/–682/–735), we consecutively used the same primers. To create the AP1m-CRHBP promoter construct, the –195 TGACTGA sequence was mutated using the forward primer 5'-GTGCACTAAGTTGTAGTGgCgGAAATCATTCTTGGTG-3'; for the CREm-CRHBP construct the CGTCA sequence at –127 was mutated using the forward primer 5'-GTGGCTGAGAGCTGGACCCTCGgCcTCGCCACGTACTCTG-3'; for the GATAm-CRHBP construct the sequence –590 GATAAAG was mutated using the forward primer 5'-GAAACTGGACCAGGtTtAAGCAAAGAGTAAGGGG-3'; for the –315NFBm-CRHBP construct the sequence –315 GGAAAATTCCC was mutated using the forward primer 5'-GCAAAATGGAAAATggCCTGAATGCCCACAG; and for the +37NFBm-CRHBP construct the +37 GGAAAGCCC sequence was mutated using the forward primer 5'-GGGTGTAGGAAGGAtAAGCCCAGGAC-3'. All promoter constructs were sequenced completely in both directions to avoid amplification errors. The –3500/+66 promoter region of the rCRH-BP promoter was cloned in the PXP2 luciferase vector and has been described previously (1). The p65 expression plasmid has been described elsewhere (51).
Transient Transfections and Luciferase Assay
For transfection purposes cells were cultured in 24-well plates in phenol red-free DF medium (Life Technologies, Inc.), supplemented with 5% dextran-coated charcoal FCS (53). For transient transfections 25 x 103 U-2 OS cells were added per well, grown to 50% confluency, and transfected using the calcium-phosphate coprecipitation method. Cells were transfected with a total amount of 2 µg DNA/well, consisting of a mixture of 0.4 µg luciferase reporter plasmid, 0.2 µg ER or ERß expression plasmid (unless otherwise indicated), or empty pSG5 vector, and pBluescript SK–. In all experiments 0.2 µg of estradiol/ER- and TNF-insensitive ß-galactosidase reporter construct (under control of a PGK- or SV2 promoter) was cotransfected to correct for differences in transfection efficiency. The medium was refreshed 16 h after transfection and, when indicated, estradiol (10–13–10–7 M), tamoxifen (10–6 M), ICI (10–6 M), or TNF (1 ng/ml) (or corresponding vehicle) was added to the medium. Cells were harvested 24 h later and assayed for luciferase activity in a scintillation counter (Top Count, Packard-Instruments, Meriden, CT) using the Luclite luciferase reporter gene assay kit (Packard-Instruments) and for ß-galactosidase (54). Within independent experiments, all experimental conditions were performed in triplicate, and the mean was used to calculate induction values of the promoter reporter construct. For each transfection mix, relative transfection efficiency was calculated based on ß-galactosidase activity. Subsequently, luciferase values were corrected according to transfection efficiency of the different transfection mixes and normalized (55).
EMSA
p65-transfected COS-1 cells and the preparation of whole-cell extract has been described previously (52). All oligonucleotides were purchased from Biolegio (Malden, The Netherlands). The following HPLC-purified FAM-labeled double-stranded oligonucleotides were used: the consensus ERE from the vitellogenin promoter (29) (FAM-ERE: 5'-FAM-CGCGAGATATCAGGTCAGAGTGACCTGGAGATTCCT-3'), a nonsense sequence (FAM-EREm: 5'-CGCGAGATATCACATCGCGATGAGATGGAGATTCCT 3'), the –267 ERE half-site with flanking sequence (FAM-267: 5'-AAGTATAATGTAGCATTTAGCAGGTCAACAAAATGC-3'), the mutant –267 ERE half-site (FAM-267m: 5'-AAGTATAATGTAGCATTTAGCAGtTCcACAAAATGC-3'), the –682 ERE half-site (FAM-682: 5'-CATGTCAACATCCAGGTCAAGTGACAGAGCTCAGCT-3'), the –735 ERE half-site (FAM-735: 5'-ATGGATGCGAAGGTCATGGCTACAGCTACCTGGTCT-3'). Only the forward sequence is given; both forward and reverse sequence were 5'-FAM labeled. For competition experiments a nonlabeled oligonucleotide duplex containing the ERE sequence 5'-AGGTCACTGTGACCT-3' was used. The fluorescent EMSA was performed based on the on-line published protocol from Amersham Biosciences (Sunnyvale, CA) with several modifications (56). All steps were carried out in the dark. Forward and reverse FAM-labeled oligo [15 pmol (or 150 pmol in case of nonlabeled competitor oligo)] were incubated for 10 min at 95 C in a total volume of 50 µl annealing buffer (final concentration: 20 mM Tris-HCl, 5 mM MgCl2, 0.1 mM dithiothreitol, 0.01 mM EDTA, pH 7.6), followed by slowly cooling down to room temperature. FAM-oligo duplex (1.5 pmol) and 50–400 ng human recombinant ER, ERß (see special reagents), or 5 µg of whole-cell extract were incubated for 20 min at room temperature in a total volume of 15 µl binding buffer [final concentration 10% (vol/vol) glycerol, 50 mM KCl, 15 mM Tris, 0.2 mM EDTA, 1 mM MgCl2, 0.4 mM dithiothreitol, 33 µg/ml BSA, pH 7.9], subsequently kept on ice, and loaded on a 5% polyacrylamide (29:1), 2.5% glycerol, TAE (Tris-acetate EDTA, pH 8) gel. Tracking dye (15 µl) was loaded in a separate lane. 1xTAE supplemented with 1 mM MgCl2 was used as running buffer. For competition experiments nonlabeled oligo duplex was added in molar excess to the binding mix as indicated. The gel was run at 4 C in the dark at 150 V for 1.5 h. The fluorescent signal was immediately analyzed at 488 nm using a FluorImager 595 (Molecular Dynamics, Inc., Sunnyvale, CA) and ImageQuant software (ImageQuant 5.2, Amersham Biosciences).
Western Blotting
GTI-7 cells were seeded onto 6-cm culture dishes at a density of approximately 50% confluency and allowed to adhere overnight under normal serum conditions. The cells were then starved for a further 24 h by replacing the medium with serum-free, phenol red-free DMEM containing low glucose (1000 mg/liter). Estradiol was added to the medium in a final concentration of 10–8 M for 12 h. Where indicated, ICI was added in a concentration of 10–5 M, 2 h before estrogen treatment, or TNF was added at a final concentration of 1 ng/ml, simultaneously with estradiol. The appropriate vehicle was added to the control cells. After exposure, the medium was removed, and cells were harvested and stored at –80 C. The procedure for Western blotting has been described previously (57). In short, cellular proteins (10 µg) were denatured with 4% (wt/vol) sodium dodecyl sulfate, electrophoresed on a 7.5% (wt/vol) SDS-PAGE gel, and transferred onto nitrocellulose membrane. After preincubation with 5% milk powder, primary antibodies at a dilution of 1:200 for the CRH-BP and 1:1000 for the actin antibody were added to the membranes and allowed to hybridize overnight at 4 C. The antibody was removed and the membrane was washed three times for 10 min, followed by incubation with horseradish peroxidase-conjugated secondary antibody (diluted in 1% milk powder) for 2 h at room temperature. The membrane was washed again as described above and developed using an enhanced chemiluminescence detection system (Amersham Biosciences). Results were quantitated using Gelpro software (Image Processing Solutions, North Reading, MA), normalized, and expressed as "fold induction" over control values.
Bioinformatics and Software
Genomic sequences of the human (Ref|NT_006713.13| Hs5_6870, nucleotide 5637095–5641094), mouse (gi|28527136, nucleotide 3453121–3458121 and ref|NT 039590.1|Mm13 39630 30, nucleotide 3454740–3453121), and rat (Ref|NW_043493.1| Rn2_796, nucleotide 219560–224559) CRH-BP genes were retrieved from the National Center for Biotechnology Information GenBank database. Vector NTI software (Informax, Invitrogen Life Science Software) was used to align sequences; RSA Tools software (accessible at: http://rsat.ulb.ac.be/rsat/) and MatInspector (accessible at http://www.genomatix.de/) were used to analyze promoter sequences for regulatory elements.
Statistical Analysis
Unless otherwise stated, statistical analysis was performed using a one-way ANOVA followed by a multiple comparison Bonferroni posttest for selected pairs, where all selected groups were analyzed simultaneously. Analysis was performed using GraphPad Prism Statistics software (San Diego, CA). P values < 0.05 were considered significant (95% confidence interval) and are indicated by asterisks. For Fig. 4, A and B, Bonferroni posttest on selected pairs was performed to compare estradiol-treated groups with the untreated group for each promoter construct separately, and to compare untreated mutant promoter constructs with the untreated wild-type promoter construct.
|
FOOTNOTES |
|---|
Abbreviations: AP1, Activating protein 1; CRE, cAMP response element; CREB, CRE-binding protein; CRH-BP, CRH-binding protein; ER, estrogen receptor; ERE, estrogen response element; FCS, fetal calf serum; HPA, hypothalamus-pituitary-adrenal; ICI, ICI 182,780; NFB, nuclear factor B; RSA, regulatory sequence analysis.
Received for publication November 17, 2003. Accepted for publication August 23, 2004.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and ERß at AP1 sites. Science 277:1508–1510B and C/EBPß. Mol Cell Biol 15:4971–4979[Abstract]
(ER)- and ERß-expressing GT1–7 GnRH neurons. Endocrinology 140:5045–5053 gene. Gene 294:279–290[CrossRef][Medline]
interaction with estrogen response element half sites from the rat prolactin gene. Biochemistry 39:3842–3847[CrossRef][Medline]
-regulated gene transcription, supporting a "ying yang" relationship between ER and ERß in mice. Mol Endocrinol 17:203–208 in the uterus. Proc Natl Acad Sci USA 97:5936–5941 and JUN proteins. J Biol Chem 276:36361–36369 regulates expression of the orphan receptor small heterodimer partner. J Biol Chem 278:36418–36429 transcriptional activity and is a key regulator of the cellular response to estrogens and antiestrogens. Endocrinology 140:5566–5578 at serine 118 by two distinct signal transduction pathways by phosphorylation-specific antisera. Oncogene 21:4921–4931[CrossRef][Medline]
(ER)- and ERß-dependent gene expression by cAMP signalling pathway(s). J Biol Chem 278:12834–12845 and ERß. Mol Endocrinol 16:674–693B puzzle. Cell 109:S81–S96
B and estrogen. Mol Endocrinol 15:543–552 and ß in the rat pituitary gland. Endocrinology 139:3976–3983B/Rel family members regulating the ICAM-1 promoter in monocytic THP-1 cells. Immunobiology 198:50–64[Medline]
NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  P. Haeger, M. E. Andres, M. I. Forray, C. Daza, S. Araneda, and K. Gysling Estrogen receptors alpha and beta differentially regulate the transcriptional activity of the Urocortin gene. J. Neurosci., May 3, 2006; 26(18): 4908 - 4916. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-M. O'Carroll, S. J Lolait, and G. M Howell Transcriptional regulation of the rat apelin receptor gene: promoter cloning and identification of an Sp1 site necessary for promoter activity J. Mol. Endocrinol., February 1, 2006; 36(1): 221 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. J. Westphal and A. F. Seasholtz Gonadotropin-Releasing Hormone (GnRH) Positively Regulates Corticotropin-Releasing Hormone-Binding Protein Expression via Multiple Intracellular Signaling Pathways and a Multipartite GnRH Response Element in {alpha}T3-1 Cells Mol. Endocrinol., November 1, 2005; 19(11): 2780 - 2797. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S K Nair, T J Thomas, N J Greenfield, A Chen, H He, and T Thomas Conformational dynamics of estrogen receptors {alpha} and {beta} as revealed by intrinsic tryptophan fluorescence and circular dichroism J. Mol. Endocrinol., October 1, 2005; 35(2): 211 - 223. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
