help button home button Endocrine Society Molecular Endocrinology ENDO 08 Sessions Library
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wissink, S.
Right arrow Articles by van der Saag, P. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wissink, S.
Right arrow Articles by van der Saag, P. T.
Molecular Endocrinology 15 (4): 543-552
Copyright © 2001 by The Endocrine Society

Synergistic Activation of the Serotonin-1A Receptor by Nuclear Factor-{kappa}B and Estrogen

Sacha Wissink, Bart van der Burg, Benita S. Katzenellenbogen and Paul T. van der Saag

Hubrecht Laboratory (S.W., B.v.d.B., P.T.v.d.S.) Netherlands Institute for Developmental Biology 3584 CT Utrecht, The Netherlands Departments of Molecular and Integrative Physiology (B.S.K.) University of Illinois Urbana, Illinois 61801-3704


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen exerts profound effects on mood and mental state. The ability of estrogen to modulate serotonergic function raises the possibility that it may play a role in the mechanism associated with depression and its treatment. A cellular mechanism for estrogen to influence mood might be through the regulation of genes involved at various levels of the serotonin system. Here we report that estrogen can up-regulate the expression of the serotonin-1A receptor via a new mechanism involving synergistic activation by nuclear factor-{kappa}B (NF-{kappa}B) with estrogen receptor {alpha}. Interestingly, we observed that only estrogen receptor-{alpha}, and not -ß, was able to mediate this effect of estrogens. The partial antiestrogen, 4-hydroxytamoxifen, had the same effect as estrogen. In addition, mutation analysis showed that both the transactivation function of p65 and activation function 1 of estrogen receptor-{alpha} were essential for this synergistic regulation. Therefore, we propose that NF-{kappa}B complexes cooperate with estrogen receptor-{alpha} to recruit cofactors into the complex and thereby synergistically activate the serotonin-1A receptor promoter through nonclassical estrogen response elements by a mechanism that does not involve direct receptor binding to DNA.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen and other gonadal steroids have profound effects on the central nervous system (1). Specifically, the ability of estrogen to modulate the brain serotonin system suggests that estrogens may play a role in the mechanism associated with depression and its treatment (2, 3). Serotonin (5-hydroxytryptamine, 5-HT) is involved in the control of a variety of behavioral processes (4). Dysregulation of the serotonin system is thought to play an important role in neuropsychiatric disorders, such as depression and anxiety (5, 6). The complex action of serotonin is mediated by a large family of related receptors (7). Particular attention has focused on the 5-HT1A receptor, which is a G protein-coupled receptor that negatively regulates adenylate cyclase (8). The 5-HT1A receptor is expressed in a restricted pattern in the brain, and high levels of receptors were observed in the limbic areas, cerebral cortex, and raphe nuclei of the brain (9, 10, 11). Studies in rats have shown that ovariectomy caused decreases in 5-HT binding and 5-HT transporter binding sites and that estrogen replacement reversed this decline (12, 13, 14), suggesting possible estrogen regulation of serotonin receptor expression.

The effects of estrogen are now known to be mediated by two estrogen receptors (ER{alpha} and ß), that belong to the superfamily of nuclear hormone receptors (15, 16, 17, 18). The two ERs share a well conserved modular structure. While the DNA-binding domain is highly conserved between ER{alpha} and ß (96% identity) and the ligand-binding domain is relatively well conserved (58% identity), the A/B region is poorly conserved between the two receptors (20% identity). Upon ligand binding, the activated receptor dimerizes and interacts with specific DNA sequences, termed estrogen response elements (EREs), located in the regulatory region of target genes. The DNA-bound receptor can then regulate transcription either positively or negatively. It is known for ER{alpha} that the regulation of transcription is mediated by two transactivation regions: AF-1 located in the A/B domain and AF-2 located in the ligand-binding domain. The two transactivation regions may function independently or cooperate, depending on cell and promoter context (19, 20). Several other mechanisms have been discovered recently by which estrogen regulates target genes. These include genes that utilize nonclassical EREs as the target sequence of ER action (21) or genes that are regulated by ER through interaction with other transcription factors bound to their respective DNA-binding sites, such as AP-1, Sp 1, and nuclear factor-{kappa}B (NF-{kappa}B) (22, 23, 24).

To explore the molecular mechanism by which estrogen modulates the serotonin system, we have investigated the effect of estrogen on the 5-HT1A receptor gene. In the present study, we show that ER{alpha} acts synergistically with NF-{kappa}B to activate the 5-HT1A receptor promoter. This activation already occurred in the absence of hormone and could be further induced by the addition of either 17ß-estradiol (E2) or 4- hydroxytamoxifen (OH-T), but not by ICI 164384 (ICI). In contrast to ER{alpha}, ERß was hardly able to mediate this effect, suggesting different roles in gene regulation for the two receptors. We also found that this synergistic activation was dependent both on the transactivation domains of the p65 subunit of NF-{kappa}B and the A/B domain of ER{alpha}, containing AF-1. Our findings show that estrogens may regulate the expression of the 5-HT1A receptor via a new mechanism involving synergistic activation by NF-{kappa}B with ER{alpha}.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Synergistic Activation of the 5-HT1A Receptor Promoter by NF-{kappa}B and ER{alpha}
To determine the effect of estrogens on 5-HT1A receptor promoter activity, we transiently transfected COS-1 cells with a reporter construct containing the 5-HT1A receptor promoter together with an expression vector encoding ER{alpha} or ERß. As shown in Fig. 1AGo, cotransfection of ER{alpha} or ERß and treatment of the cells with E2 had a minimal effect on 5-HT1A promoter activity. However, in addition to direct regulation, ER target genes can also be regulated indirectly through interaction of ER with other transcription factors. Putative NF-{kappa}B binding sites were shown to be present in the -901luc 5-HT1A receptor promoter construct (see Fig. 3AGo), and transfection of this reporter construct with expression vectors encoding the p50 and p65 subunit of NF-{kappa}B resulted in an 10-fold induction of the reporter. Interestingly, cotransfection of ER{alpha} in combination with NF-{kappa}B now resulted in a very strong induction of promoter activity, which could be further increased by the addition of E2 (Fig. 1AGo). In contrast to ER{alpha}, ERß showed only minimal induction when cotransfected with NF-{kappa}B, and no effect of E2 could be observed (Fig. 1BGo). Similar results were obtained in 293 cells (results not shown), although the level of activation by ER{alpha} was less high compared with COS-1 cells. These results indicate that the 5-HT1A receptor promoter can be synergistically activated by NF-{kappa}B and ER{alpha}.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 1. Synergistic Activation of the 5-HT1A Receptor Promoter by NF-{kappa}B and Human ER{alpha} (A) or ERß (B)

COS-1 cells were transiently transfected with the -901luc reporter construct together with empty expression vector or expression vectors encoding the p50 and p65 subunits of NF-{kappa}B (white bars) in combination with expression vectors encoding ER{alpha} or ERß (hatched bars) and treated with 10-8 M E2 for 24 h (black bars). Depicted is the induction of luciferase activity evoked by NF-{kappa}B over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD.

 


View larger version (17K):
[in this window]
[in a new window]
  		Figure 3. Importance of {kappa}B Elements in 5-HT1A Receptor Promoter Regulation by NF-{kappa}B and Human ER{alpha}

A, Schematic representation of luciferase (luc) reporter constructs used, containing rat 5-HT1A receptor (5-HT1AR) promoter deletions. The two open circles (O) represent NF-{kappa}B binding sites. The 5-HT1A receptor promoter contains no estrogen response element. B, COS-1 cells were transiently transfected with the different -901 reporter constructs as indicated, together with empty expression vector or expression vectors encoding the p50 and p65 subunits of NF-{kappa}B (white bars) in combination with expression vectors encoding ER{alpha} (hatched bars) and treated with 10-8 M E2 for 24 h (black bars). Depicted is the induction of luciferase activity evoked by NF-{kappa}B over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD. C, COS-1 cells were transiently transfected with the different -81 reporter constructs as described in panel B.

 
In the past, several groups have reported an inhibitory effect of estrogens on NF-{kappa}B activity (33, 34, 35). Therefore, we also studied the effect of estrogen on a reporter construct containing four NF-{kappa}B elements from the human immunodeficiency virus-long terminal repeat (HIV-LTR) in front of the thymidine kinase promoter coupled to luciferase in combination with expression constructs encoding ER{alpha} or ERß and the p50 and p65 subunits of NF-{kappa}B. On this reporter construct, cotransfection of ER{alpha} resulted in repression of the transcriptional activity of NF-{kappa}B already in the absence of hormone, while addition of hormone resulted in a further repression (Fig. 2BGo). Cotransfection of ERß also showed some repression of NF-{kappa}B activity. Similar results were obtained in 293 cells (results not shown). These results indicate that while ER{alpha} acts as a transcriptional repressor of NF-{kappa}B on an artificial NF-{kappa}B reporter construct, ER{alpha} acts as a transcriptional activator with NF-{kappa}B on the 5-HT1A receptor promoter.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. COS-1 Cells Were Transiently Transfected with 4xNF-{kappa}B(HIV)tkluc in Combination with Empty Expression Vector or Expression Vectors Encoding the p50 and p65 Subunits of NF-{kappa}B and Expression Vectors Encoding ER{alpha} or ERß

Cells were either untreated (white bars) or treated with 10-8 M E2 (black bars) for 24 h. Depicted is the induction of luciferase activity evoked by NF-{kappa}B over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD.

 
Involvement of NF-{kappa}B Elements in 5-HT1A Receptor Promoter Regulation by NF-{kappa}B and ER{alpha}
To localize the effect of ER{alpha} on the 5-HT1A receptor promoter, several promoter deletion constructs were used (Fig. 3AGo). Mutation of both NF-{kappa}B elements (-901 365/64Mluc) completely abolished the effect of NF-{kappa}B on the 5-HT1A receptor promoter (Fig. 3BGo). However, ER{alpha}, only in combination with NF-{kappa}B, was still able to induce promoter activity as efficient as on the wild-type promoter (-901luc). Likewise, the promoter construct -81luc could not be induced by NF-{kappa}B, although it still contained one NF-{kappa}B element. However, also on this promoter construct, the effect of ER{alpha} with NF-{kappa}B was maintained. When the single NF-{kappa}B element present in the -81luc construct was mutated (-81 64Mluc), the ER{alpha} effect was almost completely abolished (Fig. 3CGo). Thus, synergistic activation of the 5-HT1A receptor promoter involves NF-{kappa}B binding sites, although activation of the promoter by NF-{kappa}B itself appears not to be required for the effect of ER{alpha}. These results suggest that this synergistic promoter activation by ER{alpha} is independent of DNA binding and involves protein-protein interactions.

Effects of Antiestrogens on the 5-HT1A Receptor Promoter
Antiestrogens have been described to have differential effects depending on promoter context and receptor subtype. In transactivation experiments, tamoxifen inhibited transcription of genes regulated by a classical ERE, while, like E2, it activated transcription of genes that are under the control of an AP-1 element with ER{alpha} (22). Moreover, only antiestrogens were transcriptional activators with ERß at an AP-1 site (36). We examined the effect of antiestrogens on the 5-HT1A receptor promoter using the partial antagonist OH-T, which blocks AF-2, and the pure antagonist ICI, which blocks AF-1 and AF-2. As shown in Fig. 4Go, ICI treatment did not enhance the activity of the 5-HT1A receptor promoter by ER{alpha} and NF-{kappa}B, while OH-T was as potent as E2 in transcriptional activation. These data indicate that the partial antagonist OH-T, still able to activate AF-1, is as potent as E2 in synergistic activation of the 5-HT1A receptor promoter by ER{alpha}.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 4. OH-T, but Not ICI, Can Enhance Activity of the 5-HT1A Receptor Promoter by NF-{kappa}B and Human ER{alpha}

COS-1 cells were transiently transfected with the -901luc reporter construct together with empty expression vector or expression vectors encoding the p50 and p65 subunits of NF-{kappa}B in combination with expression vectors encoding ER{alpha} and treated with E2, OH-T, or ICI for 24 h as indicated. Depicted is the induction of luciferase activity evoked by NF-{kappa}B over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD.

 
Domains of NF-{kappa}B and ER{alpha} Involved in the Synergistic Activation of the 5-HT1A Receptor Promoter
To determine the importance of the transactivation function of NF-{kappa}B, we examined the effect of deleting the transactivation domains, or impairing the DNA-binding function of the p65 subunit of NF-{kappa}B, on its ability to synergistically activate the 5-HT1A receptor promoter with ER{alpha} in a transient transfection assay. While cotransfection of p50 and p65 or p65 alone strongly activated the promoter in combination with ER{alpha} and E2, cotransfection of p50 alone, which has no transactivation function, had almost no effect (Fig. 5Go). Deletion of the transactivation domains of p65 resulted in a construct containing only the Rel homology domain (p65RHD). P65RHD was still able to bind to DNA (27), but was unable to activate the promoter both in the absence or presence of ER{alpha} and E2. The DNA-binding defective mutant (p65Nsi) still contained intact transactivation domains, but was also unable to synergistically activate the promoter. Taken together, these data show that both the transactivation function as well as the DNA binding function of p65 are essential for synergistic activation of the 5-HT1A receptor promoter by ER{alpha}.



View larger version (18K):
[in this window]
[in a new window]
  		Figure 5. The Transactivation Function of p65 Is Required for Synergistic Activation of the 5-HT1A Receptor Promoter by NF-{kappa}B and Human ER{alpha}

COS-1 cells were transiently transfected with the -901luc reporter construct together with empty expression vector or expression vectors encoding p50, p65, p65RHD, or p65Nsi as indicated (white bars) in combination with expression vectors encoding ER{alpha} (hatched bars) and treated with 10-8 M E2 for 24 h (black bars). Depicted is the induction of luciferase activity evoked by the NF-{kappa}B subunits over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD.

 
To identify the regions of ER{alpha} involved in activation of the 5-HT1A receptor promoter, deletion constructs of mouse ER{alpha} that lack part of the A/B region containing AF-1, or that lack part of the ligand binding region containing AF-2, were used. While deletion of the A/B region (ER{alpha} 121–599) inhibited the synergistic activation of the promoter, deletion of the ligand-binding domain (ER{alpha} 1–339) resulted in a receptor that was at least as active as wild-type ER{alpha} (Fig. 6AGo). The DNA-binding defective mutant of ER{alpha} (ER{alpha} C241/244A) was unable to activate the 5-HT1A receptor promoter, possibly because a functional DNA-binding domain is needed for interaction with NF-{kappa}B (34). Note that in contrast to human ER{alpha}, no ligand dependency can be observed for mouse ER{alpha} in synergistic activation of the 5-HT1A receptor promoter. This synergistic activation of the promoter by ER{alpha} could be observed only in combination with NF-{kappa}B, although in the absence of NF-{kappa}B a small activation of the promoter could be found with ER{alpha} 1–339 (results not shown). In a control experiment, ER{alpha}, ERß, and the deletion mutants were cotransfected with a reporter construct containing three copies of a consensus ERE and a TATA box coupled to luciferase to determine their ability to activate transcription from a classical ERE. As shown in Fig. 6BGo, both ER{alpha} and ERß stimulate transcription from 3xERE-TATA-luc, although the transcriptional activity of ERß was significantly less than that of ER{alpha}, a phenomenon that has been described previously (28). Both deletion mutants, lacking either AF-1 or AF-2, stimulated transcription although much less efficiently than wild-type ER{alpha}, indicating that the transactivation domains are able to synergize on this promoter construct. Furthermore, it was shown that ER{alpha} 1–339 was already maximally activated in the absence of ligand, clearly demonstrating the ligand-independent activity of AF-1. As expected, the DNA-binding defective mutant, ER{alpha} C241/244A, was unable to activate this reporter construct.



View larger version (16K):
[in this window]
[in a new window]
  		Figure 6. The Synergistic Activation of the 5-HT1A Receptor Promoter by Mouse ER{alpha} Occurs in an AF-2-Independent Fashion

A, COS-1 cells were transiently transfected with the -901luc reporter construct together with empty expression vector or expression vectors encoding the p50 and p65 subunits of NF-{kappa}B in combination with expression vectors encoding mouse ER{alpha}, ERß, ER{alpha} 121–599, ER{alpha} 1–339, or ER{alpha} C241/244A. Cells were either untreated (hatched bars) or treated with 10-8 M E2 (black bars) for 24 h. Depicted is the induction of luciferase activity evoked by NF-{kappa}B over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD. B, COS-1 cells were transiently transfected with 3xERE-TATAluc in combination with empty expression vector or expression vectors encoding mouse ER{alpha}, ERß, ER{alpha} 121–599, ER{alpha} 1–339, or ER{alpha} C241/244A. Cells were either untreated (hatched bars) or treated with 10-8 M E2 (black bars) for 24 h. Depicted is the induction of luciferase activity evoked by ER over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD.

 
Since ER{alpha} and not ERß was able to synergistically activate the 5-HT1A receptor promoter with NF-{kappa}B, chimeric constructs with ER{alpha} and ERß were used to further determine the region of ER{alpha} involved in this activation. Replacement of the A/B region of ERß with the A/B region of ER{alpha} (ER{alpha}/ß) resulted in a chimeric receptor that was even more potent than wild-type ER{alpha} in activation of the 5-HT1A receptor promoter (Fig. 7AGo). However, replacement of the A/B region of ER{alpha} with the A/B region of ERß (ERß/{alpha}) totally abolished the ability of the receptor to synergistically activate the promoter. Again this synergistic activation of the promoter by ER{alpha} and ER{alpha}/ß could only be observed in combination with NF-{kappa}B, although in the absence of NF-{kappa}B a small ligand-independent activation of the promoter could be seen with ER{alpha}/ß (results not shown). In a control experiment, both chimeric constructs were able to activate transcription from 3xERE-TATA-luc as efficiently as wild-type ER{alpha}, while some ligand-independent activity could be observed only with ER{alpha}/ß (Fig. 7BGo). These results suggest that the synergistic activation of the 5-HT1A receptor promoter by ER{alpha} and NF-{kappa}B is dependent on the DNA binding domain and the A/B region of ER{alpha} containing AF-1.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 7. The A/B domain of ER{alpha} Is Essential for the Synergistic Activation of the 5-HT1A Receptor Promoter

A, COS-1 cells were transiently transfected with the -901luc reporter construct together with empty expression vector or expression vectors encoding the p50 and p65 subunits of NF-{kappa}B in combination with expression vectors encoding human ER{alpha}, ERß, ER{alpha}/ß, or ERß/{alpha}. Cells were either untreated (hatched bars) or treated with 10-8 M E2 (black bars) for 24 h. Depicted is the induction of luciferase activity evoked by NF-{kappa}B over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD. B, COS-1 cells were transiently transfected with 3xERE-TATAluc in combination with empty expression vector or expression vectors encoding human ER{alpha}, ERß, ER{alpha}/ß, or ERß/{alpha}. Cells were either untreated (hatched bars) or treated with 10-8 M E2 (black bars) for 24 h. Depicted is the induction of luciferase activity evoked by ER over cells transfected with empty expression vector. Bars represent the mean of at least three independent experiments ± SD.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we show that estrogen may regulate the 5-HT1A receptor promoter via a new mechanism involving synergistic activation of NF-{kappa}B with ER{alpha}. The cis-regulatory region of the 5-HT1A receptor contains two putative NF-{kappa}B binding sites, and the presence of NF-{kappa}B proteins is critical for synergistic induction by ER{alpha}. This suggests that NF-{kappa}B complexes cooperate with ER{alpha} to synergistically regulate 5-HT1A receptor gene expression.

In most systems that have been examined the estrogen and NF-{kappa}B signaling pathways are mutually antagonistic. For instance, regulation of the IL-6 promoter has been extensively studied, and NF-{kappa}B- induced activation of this gene could clearly be inhibited by estrogen (33, 34). Consistent with these findings, we have shown that on an artificial NF-{kappa}B reporter construct, estrogen inhibits NF-{kappa}B activity. However, on the 5-HT1A receptor promoter, estrogen further enhanced NF-{kappa}B-induced activity, indicating that positive or negative regulation by estrogen is dependent on the promoter context. Similar results have been described for both negative and positive regulation of AP-1-dependent promoters by estrogens (36) and glucocorticoids (37, 38).

Based on several different approaches, synergistic activation of the 5-HT1A receptor promoter by NF-{kappa}B and ER{alpha} was found to be dependent on the N-terminal region of ER{alpha}, containing AF-1. First, in contrast to ER{alpha}, ERß was unable to mediate this synergistic effect. The two receptors show a high degree of homology in the DNA-binding domain and moderate homology in the ligand-binding domain; however, the A/B region is poorly conserved between the two receptors. This already suggested the importance of the A/B region of ER{alpha} in the synergistic activation. Second, synergistic activation by ER{alpha} 1–339, an AF-2 defective mutant, was comparable to wild-type ER{alpha}, while ER{alpha} 121–599, an AF-1-defective mutant, was unable to mediate this effect, comparable to wild-type ERß. Third, replacement of the A/B region of ERß with the A/B region of ER{alpha} (ER{alpha}/ß) resulted in a chimeric receptor that was even more potent than wild-type ER{alpha} in activation of the 5-HT1A receptor promoter. However, replacement of the A/B region of ER{alpha} with the A/B region of ERß (ERß/{alpha}) totally abolished the ability of the receptor to synergistically activate the promoter. Fourth, the partial antiestrogen OH-T, which blocks only AF-2, was as potent as E2 in activation of the 5-HT1A receptor promoter. The AF-1-mediated agonistic effect of antiestrogens has recently been reported to be mediated via the A/B region of ER{alpha} but not by the A/B region of ERß (28). These differences between ER{alpha} and ERß suggest different regulatory functions for the two ER subtypes. Finally, the fact that the ER{alpha} effect is mostly estrogen-independent also indicates the involvement of AF-1, which is the hormone-independent activation function that resides in the N terminus of ER{alpha}. Taken together, these data clearly show the involvement of ER{alpha} AF-1 in 5-HT1A receptor promoter regulation.

Although the regulation by ER of AP-1-dependent promoters and the NF-{kappa}B-dependent 5-HT1A receptor promoter shares several features, clear differences are also present. Estrogen-induced transcription from an AP-1-dependent promoter requires both ER{alpha} and AP-1 transcription factors (22). Similarly, estrogen- induced transcription from an NF-{kappa}B-dependent promoter requires ER{alpha} and NF-{kappa}B. Both pathways appear to require the amino terminus of ER{alpha}, containing AF-1. While both tamoxifen and ICI activate transcription via AP-1 sites, only tamoxifen induces synergistic activation of the 5-HT1A receptor promoter with NF-{kappa}B. This lack of activity of ICI and its apparent capacity to decrease the ability of ER{alpha} to activate the 5-HT1A receptor promoter could be explained by differences in receptor conformation, but may also be due to enhanced receptor turnover (39, 40). Furthermore, while with ERß, E2 inhibited AP-1-dependent transcription, antiestrogens stimulated AP-1-dependent transcription (36). In contrast to this, E2 was not a very potent activator of the 5-HT1A receptor promoter with ERß. These findings highlight the unique pharmacology of estrogen receptor-regulated transcription at different gene sites.

Our findings clearly show a crucial role for NF-{kappa}B complexes and NF-{kappa}B binding sites in the synergistic activation of the 5-HT1A receptor promoter by ER{alpha}. However, mutation of the two NF-{kappa}B elements in the -901luc construct abolished the NF-{kappa}B effect while the ER{alpha} effect was maintained. One explanation could be that NF-{kappa}B proteins bind as monomers to these mutated {kappa}B elements. This NF-{kappa}B-DNA complex, unable to activate transcription in this conformation, might be stabilized by ER{alpha} and consequently result in activation. Furthermore, it is evident from the use of ER{alpha} mutants and chimeras that, although a functional DNA binding domain of ER{alpha} is required, there is no clear correlation between ER{alpha} activity on the ERE reporter and the 5-HT1A receptor promoter. Therefore, the most likely explanation for the synergistic activation of the 5-HT1A receptor promoter is that ER{alpha} activates this promoter not via direct binding to DNA but via protein-protein interactions. This model is supported by the fact that both the DNA-binding domain of ER{alpha} and an intact RHD of p65 are required for the synergistic activation, since ER{alpha} has been described to directly interact with p65 involving the DNA-binding domain of ER{alpha} and the RHD of p65 (34). In addition, ER{alpha} could also interact with other transcription factors present in COS-1 cells or with other components of the transcription machinery involved in promoter regulation. Furthermore, the fact that both the transactivation domains of p65 and AF-1 of ER{alpha} are essential for this response clearly indicates the involvement of cofactors. Therefore, we propose that NF-{kappa}B complexes cooperate with ER{alpha} to recruit coactivators into the complex via AF-1 and thereby synergistically activate the 5-HT1A receptor promoter. An alternative explanation could be that the 5-HT1A receptor promoter contains a cryptic site that directly binds ER{alpha} but requires functional cooperation with NF-{kappa}B, bound to nearby DNA binding sites. Since our data cannot rule out direct binding of ER{alpha} to this promoter, it could be possible that the 5-HT1A receptor promoter contains a composite element that simultaneously binds ER{alpha} and NF-{kappa}B.

In addition to the classical hormone activation pathway, other signal transduction pathways have been described to regulate a number of steroid receptors, including ER{alpha}, independently of hormonal ligands. Nuclear receptors have been shown to be activated by nonsteroidal agents, such as dopamine, growth factors, and PKA activators, via phosphorylation (41). Phosphorylation of ER{alpha} was shown to enhance receptor activity and major phosphorylation sites are located in the A/B region of the receptor (42, 43). Recently it was demonstrated that phosphorylation of ERß AF-1 regulates cofactor recruitment and gene activation by nonsteroidal activators (44), whereas phosphorylation of the A/B domain of peroxisome proliferator-activated receptor {gamma} decreased its transcriptional activity (45). The presence of several kinase sites within the A/B region of ER{alpha} and ERß suggests that differential phosphorylation of the AF-1 domain may result in diverse responses of the receptors by different activators. The existence of this additional pathway emphasizes the importance of AF-1 in hormone-independent receptor activation.

These studies were all performed in nonneuronal cells, and it would be interesting to determine whether the same effects can be observed in a serotonergic neuronal environment. Additional neuronal-specific transcription factors might regulate the 5-HT1A receptor promoter in the same way or different from nonneuronal cells. However, several lines of evidence suggest that estrogen also regulates 5-HT1A receptor expression in the central nervous system (CNS). For instance, the decline in estrogen before parturition and at the onset of menopause has been correlated with negative affect (46), while estrogen replacement therapy can, in some cases, alleviate depression or anxiety in women (47, 48). Moreover, ovariectomy caused decreases in 5-HT binding and 5-HT transporter binding sites (12, 13, 14), while replacement of estrogen to ovariectomized rats reversed this decline. Both ER{alpha} and ERß have been identified in multiple regions of the brain, including the cortex, hippocampus, and raphe nuclei (49). In addition, NF-{kappa}B has also been described to be active in the brain, particularly in the cortex and hippocampus (50). At the same time evidence is emerging that NF-{kappa}B not only functions in immune cells, but also has unique roles in processes such as neuronal plasticity, neurodegeneration, and neuronal development (51). Thus, these transcription factors and pathways may play an important role in regulation of 5-HT1A receptor gene expression in the brain. Furthermore, in addition to the direct mechanism described above, estrogen may also have an indirect effect in the CNS. Estrogen may induce the formation of an intermediate protein which might be able to further induce 5-HT1A receptor expression. In conclusion, the ability of estrogen to modulate serotonergic receptor function may underlie, at least in part, the profound effects of this hormone on mood and mental state.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Special Reagents
E2 was obtained from Sigma (St Louis, MO). 4-Hydroxytamoxifen and ICI 164384 were kind gifts from Dr. A. Wakeling (Zeneca Pharmaceuticals, Macclesfield, UK).

Cell Culture
Monkey COS-1 cells and human 293 embryonal kidney cells were obtained from American Type Culture Collection (ATCC; Manassas, VA) and were cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium (DF; Life Technologies, Inc., Gaithersburg, MD), buffered with bicarbonate and supplemented with 7.5% FCS. Dextran-coated charcoal (DCC)-FCS was prepared by treatment of FCS with DCC to remove steroids, as described previously (25).

Plasmids
-901luc was created by partial digestion of -1,588luc (a kind gift from Dr. O. Meijer, Leiden, The Netherlands), with StyI, filling-in and ligation into pGL3 digested with SmaI, redigestion with HindIII, and religation; -81luc was created by digestion of -1,588luc with StyI, filling-in and digestion with BglII, and ligation into pGL3 digested with SmaI/BglII; -901 365Mluc and -901 64Mluc were constructed by introducing point mutations into the original promoter constructs by site-directed mutagenesis using the oligonucleotides 5'-gagccgaattctacagactaa-3' and 5'-aactgcaaggagatctacatcgcccctcg-3', respectively. -901 365/64Mluc was created by digestion of -901 64Mluc with SacII/HindIII and ligation into -901 365Mluc digested with SacII/HindIII; -81 64Mluc was made by partial digestion of -901 64Mluc with StyI and religation. The CMV4 expression vectors containing full-length cDNAs encoding human p65 (RelA), p50 (NF-{kappa}B1) and p65RHD (1–305), and p65Nsi (1–551E39I) have been described previously (26, 27). The expression vectors encoding human ER{alpha} (pSG5-HEGO) and human ERß (pSG5- ERß530) were kind gifts of Dr. Chambon (Strasbourg, France) and Dr. Gustafsson (Stockholm, Sweden), respectively. Chimeric human ER{alpha}/ERß and ERß/ER{alpha} receptors were described previously (28) and contained the A/B domain of ER{alpha} and domain C, D, E, and F of ERß in the ER{alpha}/ERß chimera and the A/B domain of ERß and domain C, D, E, and F of ER{alpha} in the ERß/ER{alpha} chimera. Mouse ER{alpha} (pMT2MOR), ER{alpha} 1–339, ER{alpha} 121–599, and ER{alpha} C241/244A (29) were kindly provided by Dr. Parker (London, UK). The reporter plasmids used, 4xNF-{kappa}B(HIV)tkluc and 3xERE-TATA-luc, have been described previously (30, 31).

Transient Transfections
For transient transfections, COS-1 cells and 293 cells were cultured in 24-well plates in phenol red-free DF supplemented with 5% DCC-FCS. Cells were transfected using calcium-phosphate coprecipitation with 0.4 µg of luciferase reporter, 0.6 µg of PDMlacZ, and 0.2 µg of the indicated expression plasmids. pBluescript SK- was added to obtain a total amount of 1.8 µg of DNA/well. After 16 h, the medium was refreshed and when indicated hormone was added. Cells were harvested 24 h later and assayed for luciferase activity using the Luclite luciferase reporter gene assay kit (Packard Instruments, Meriden, CT) according to the manufacturer’s protocol and the Topcount liquid scintillation counter (Packard Instruments). Values were corrected for transfection efficiency by measuring ß-galactosidase activity (32).


   ACKNOWLEDGMENTS
 
We thank Drs. M. Parker, P. Chambon, and J.-Å Gustafsson for ER cDNAs. We thank Dr. A. Wakeling for providing us with 4-hydroxytamoxifen and ICI 164384. We thank J. Heinen and F. Vervoordeldonk for photographic reproductions.


   FOOTNOTES
 
Address requests for reprints to: Paul T. van der Saag, Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. E-mail: paul{at}niob.knaw.nl

This work was supported by grants from the Netherlands Organization for Scientific Research (STIGO project no. 014–80-005) and NV Organon, Oss, The Netherlands (to S.W.) and NIH Grant CA-18119 (to B.S.K.).

Received for publication April 28, 2000. Revision received December 4, 2000. Accepted for publication January 10, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. McEwen BS, Alves SE 1999 Estrogen actions in the central nervous system. Endocr Rev 20:279–307[Abstract/Free Full Text]
  2. Bethea CL, Pecins-Thompson M, Schutzer WE, Gundlah C, Lu ZN 1998 Ovarian steroids and serotonin neural function. Mol Neurobiol 18:87–123[Medline]
  3. Joffe H, Cohen LS 1998 Estrogen serotonin and mood disturbance: where is the therapeutic bridge? Biol Psychiatry 44:798–811[CrossRef][Medline]
  4. Buhot MC 1997 Serotonin receptors in cognitive behaviors Curr Opin Neurobiol 7:243–254[CrossRef][Medline]
  5. Murphy DL 1990 Neuropsychiatric disorders and the multiple human brain serotonin receptor subtypes and subsystems. Neuropsychopharmacology 3:457–471[Medline]
  6. Maes M, Meltzer H 1995 In: Bloom F, Kupfer D (eds) The Serotonin Hypothesis of Depression. Raven, New York, pp 933–944
  7. Julius D 1991 Molecular biology of serotonin receptors. Annu Rev Neurosci 14:335–360[CrossRef][Medline]
  8. Fargin A, Raymond JR, Regan JW, Cotecchia S, Lefkowitz RJ, Caron MG 1989 Effector coupling mechanisms of the cloned-5-HT1A receptor. J Biol Chem 264:14848–14852[Abstract/Free Full Text]
  9. Albert PR, Zhou Q-Y, Van Tol HHM, Bunzow JR, Civelli O 1990 Cloning functional expression and messenger RNA tissue distribution of the rat 5-hydroxytryptamine1a receptor gene. J Biol Chem 265:5825–5832[Abstract/Free Full Text]
  10. Chalmers DT, Watson SJ 1991 Comparative anatomical distribution of 5-HT1A receptor messenger rna and 5-HT1A binding in rat brain - a combined in situ hybridisation/in vitro receptor autoradiographic study. Brain Res 561:51–60[CrossRef][Medline]
  11. Pompeiano M, Palacios JM, Mengod G 1992 Distribution and cellular localization of mRNA coding for 5-HT1A receptor in the rat brain; correlation with receptor binding. J Neurosci 12:440–453[Abstract]
  12. Biegon A, McEwen BS 1982 Modulation by estradiol of serotonin receptors in brain. J Neurosci 2:199–205[Abstract]
  13. Fink G, Sumner BE, McQueen JK, Wilson H, Rosie R 1998 Sex steroid control of mood mental state and memory. Clin Exp Pharmacol Physiol 25:764–775[Medline]
  14. McQueen JK, Wilson H, Fink G 1997 Estradiol-17 ß increases serotonin transporter (sert) mRNA levels and the density of sert-binding sites in female rat brain. Brain Res Mol Brain Res 45:13–23[Medline]
  15. Green S, Walter P, Kumar V, Krust A, Bornet JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence expression and homology to v-erb-A. Nature 320:134–139[CrossRef][Medline]
  16. Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J 1986 Sequence and expression of human estrogen receptor complementary DNA. Science 231:1150–1154[Abstract/Free Full Text]
  17. Kuiper GJM, Enmark E, Peto-Huikko M, Nilsson S, Gustaffson JA 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5935–5930
  18. Mosselman S, Polman J, Dijkema R 1996 ERß: identification and characterization of a novel human estrogen receptor. FEBS Lett 392:49–53[CrossRef][Medline]
  19. Tora L, White J, Brou C, Tasset D, Webster N, Scheer E, Chambon P 1989 The human estrogen receptor has two independent nonacidic transcriptional activation functions. Cell 59:477–478[CrossRef][Medline]
  20. Tzukerman MT, Esty A, Santiso-Mere D, Danielian P, Parker MG, Stein RB, Pike JW, McDonnell DP 1994 Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions. Mol Endocrinol 8:21–30[Abstract]
  21. Elgort MG, Zou A, Marschke KB, Allegretto EA 1996 Estrogen and estrogen receptor antagonists stimulate transcription from the human retinoic acid receptor-{alpha} 1 promoter via a novel sequence. Mol Endocrinol 10:477–487[Abstract]
  22. Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation of the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9:443–456[Abstract]
  23. Krishnan V, Wang X, Safe S 1994 Estrogen receptor-Sp1 complexes mediate estrogen-induced cathepsin D gene expression in MCF-7 human breast cancer cells. J Biol Chem 269:15912–15917[Abstract/Free Full Text]
  24. Stein B, Yang MX 1995 Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-{kappa} B and C/EBP ß. Mol Cell Biol 15:4971–4979[Abstract]
  25. Van der Burg B, Rutteman GR, Blankenstein MA, de Laat SW, van Zoelen EJ 1988 Mitogenic stimulation of human breast cancer cells in a growth factor-defined medium: synergistic action of insulin and estrogen. J Cell Physiol 134:101–108[CrossRef][Medline]
  26. 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 antiinflammatory action of glucocorticoids. Mol Endocrinol 9:401–412[Abstract]
  27. 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-{kappa}B subunit are required for negative cross-talk and direct interaction with the glucocorticoid receptor. J Biol Chem 272:22278–22284[Abstract/Free Full Text]
  28. McInerney EM, Weis KE, Sun J, Mosselman S, Katzenellenbogen BS 1998 Transcription activation by the human estrogen receptor subtype ß (ER ß) studied with ER ß and ER {alpha} receptor chimeras. Endocrinology 139:4513–4522[Abstract/Free Full Text]
  29. Danielian PS, White R, Lees JA, Parker MG 1992 Identification of a conserved region required for hormone dependent transcriptional activation by steroid hormone receptors. EMBO J 1992 11:1025–1033
  30. Wissink S, van de Stolpe A, Caldenhoven E, Koenderman L, van der Saag PT 1997 NF-{kappa} B/Rel family members regulating the ICAM-1 promoter in monocytic THP-1 cells. Immunobiology 198:50–64[Medline]
  31. Legler J, van den Brink CE, Brouwer A, Murk AJ, van der Saag PT, Vethaak AD, van der Burg B 1991 Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line. Toxicol Sci 48:55–66[Abstract/Free Full Text]
  32. Pfahl M, Tzukerman M, Zhang XK, Lehmann JM, Hermann T, Wills KN, Graupner G 1990 Nuclear retinoic acid receptors: cloning analysis and function. Methods Enzymol 189:256–270[Medline]
  33. Ray A, Prefontaine KE, Ray P 1994 Down-modulation of interleukin-6 gene expression by 17 ß-estradiol in the absence of high affinity DNA binding by the estrogen receptor. J Biol Chem 269:12940–12946[Abstract/Free Full Text]
  34. Stein B, Yang MX 1995 Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-{kappa} B and C/EBP ß. Mol Cell Biol 15:4971–4979
  35. Galien R, Evans HF, Garcia T 1996 Involvement of CCAAT/enhancer-binding protein and nuclear factor-{kappa} B binding sites in interleukin-6 promoter inhibition by estrogens. Mol Endocrinol 10:713–722[Abstract]
  36. Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson J-Å, Kushner PJ, Scanlan T 1997 Differential ligand activation of estrogen receptors ER{alpha} and ERß at AP1 sites. Science 277:1508–1510[Abstract/Free Full Text]
  37. Jonat C, Rahmsdorf HJ, Park KK, Cato ACB, Gebel S, Ponta H, Herrlich P 1990 Antitumor promotion and antiinflammation: down-modulation of AP1 (Fos/Jun) activity by glucocorticoid hormone. Cell 62:1189–1204[CrossRef][Medline]
  38. Harrison RJ, McNeil GP, Dobner PR 1995 Synergistic activation of neurotensin/neuromedin N gene expression by c-Jun and glucocorticoids: novel effects of Fos family proteins. Mol Endocrinol 9:981–993[Abstract]
  39. Van den Bemd GJCM, Kuiper GGJM, Pols HAP, Van Leeuwen JPTM 1999 Distinct effects on the conformation of estrogen receptor {alpha} and ß by both the antiestrogens ICI-164,384 and ICI 182,780 leading to opposite effects on receptor stability. Biochem Biophys Res Commun 261:1–5[CrossRef][Medline]
  40. Gibson MK, Nemmers LA, Beckman WC, Davis VL, Curtis SW, Korach KS 1991 The mechanism of ICI-164,384 antiestrogenicity involves rapid loss of estrogen receptor in uterine tissue. Endocrinology 129:2000–2010[Abstract]
  41. Weigel NL, Zhang Y 1998 Ligand-independent activation of steroid hormone receptors. J Mol Med 76:469–479[CrossRef][Medline]
  42. Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor. Identification of hormone-regulated sites and examination of their influence on transcriptional activity. J Biol Chem 11:4458–4466
  43. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 270:1491–1494[Abstract/Free Full Text]
  44. Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß through phosphorylation of activation function AF-1. Mol Cell 3:513–519[CrossRef][Medline]
  45. Hu E, Kim JB, Sarraf P, Spiegelman BM 1996 Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPAR {gamma}. Science 274:2100–2103[Abstract/Free Full Text]
  46. Gitlin MJ, Pasnau RO 1989 Psychiatric disorders associated with specific periods in women’s lives. Am J Psychiatry 146:1413–1422[Abstract/Free Full Text]
  47. Oppenheim G 1983 Estrogen in the treatment of depression: neuropharmacological mechanisms. Biol Psychiatry 18:721–725[Medline]
  48. Gregoire AJ, Kumar R, Everitt B, Henderson AF, Studd JW 1996 Transdermal oestrogen for treatment of severe postnatal depression. Lancet 347:930–933[CrossRef][Medline]
  49. Shughrue PJ, Lane MV, Merchenthaler I 1997 Comparative distribution of estrogen receptor-{alpha} and -ß mRNA in the rat central nervous system. J Comp Neurol 4:507–525
  50. Schmidt-Ullrich R, Memet S, Lilienbaum A, Feuillard J, Raphael M, Israel A 1996 NF-{kappa} B activity in transgenic mice: developmental regulation and tissue specificity. Development 122:2117–2128[Abstract]
  51. O’Neill LAJ, Kaltschmidt C 1997 NF-{kappa} B: a crucial transcription factor for glial and neuronal cell function. Trends Neurosci 20:252–258[CrossRef][Medline]



This article has been cited by other articles:


Home page
 EndocrinologyHome page
A. D. Adamson, S. Friedrichsen, S. Semprini, C. V. Harper, J. J. Mullins, M. R. H. White, and J. R. E. Davis
Human Prolactin Gene Promoter Regulation by Estrogen: Convergence with Tumor Necrosis Factor-{alpha} Signaling
Endocrinology, February 1, 2008; 149(2): 687 - 694.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
S. Friedrichsen, C. V. Harper, S. Semprini, M. Wilding, A. D. Adamson, D. G. Spiller, G. Nelson, J. J. Mullins, M. R. H. White, and J. R. E. Davis
Tumor Necrosis Factor-{alpha} Activates the Human Prolactin Gene Promoter via Nuclear Factor-{kappa}B Signaling
Endocrinology, February 1, 2006; 147(2): 773 - 781.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
G. Soucy, G. Boivin, F. Labrie, and S. Rivest
Estradiol Is Required for a Proper Immune Response to Bacterial and Viral Pathogens in the Female Brain
J. Immunol., May 15, 2005; 174(10): 6391 - 6398.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
M. H. Herynk and S. A. W. Fuqua
Estrogen Receptor Mutations in Human Disease
Endocr. Rev., December 1, 2004; 25(6): 869 - 898.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
A. van de Stolpe, A. J. Slycke, M. O. Reinders, A. W. M. Zomer, S. Goodenough, C. Behl, A. F. Seasholtz, and P. T. van der Saag
Estrogen Receptor (ER)-Mediated Transcriptional Regulation of the Human Corticotropin-Releasing Hormone-Binding Protein Promoter: Differential Effects of ER{alpha} and ER{beta}
Mol. Endocrinol., December 1, 2004; 18(12): 2908 - 2923.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
I. Bjorn, I. Sundstrom-Poromaa, M. Bixo, S. Nyberg, G. Backstrom, and T. Backstrom
Increase of Estrogen Dose Deteriorates Mood during Progestin Phase in Sequential Hormonal Therapy
J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2026 - 2030.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
M. E. Marin-Castano, S. J. Elliot, M. Potier, M. Karl, L. J. Striker, G. E. Striker, K. G. Csaky, and S. W. Cousins
Regulation of Estrogen Receptors and MMP-2 Expression by Estrogens in Human Retinal Pigment Epithelium
Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 50 - 59.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
K. Kawashima, K. Yamakawa, W. Takahashi, S. Takizawa, P. Yin, N. Sugiyama, S. Kanba, and J. Arita
The Estrogen-Occupied Estrogen Receptor Functions as a Negative Regulator to Inhibit Cell Proliferation Induced by Insulin/IGF-1: A Cell Context-Specific Antimitogenic Action of Estradiol on Rat Lactotrophs in Culture
Endocrinology, July 1, 2002; 143(7): 2750 - 2758.
[Abstract] [Full Text] [PDF]

Home page
 Endocr. Rev.Home page
J. Pfeilschifter, R. Koditz, M. Pfohl, and H. Schatz
Changes in Proinflammatory Cytokine Activity after Menopause
Endocr. Rev., February 1, 2002; 23(1): 90 - 119.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services