Activities of estrogen receptor alpha- and beta-selective ligands at diverse estrogen responsive gene sites mediating transactivation or transrepression

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Abstract

Estrogens exert their regulatory transcriptional effects, which can be stimulatory or repressive, at diverse gene sites via two estrogen receptors, ERα and ERβ. Since these two ERs have different tissue distributions, ligands that have the capacity to selectively activate or inhibit these two ERs would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity. We have developed several ligands that showed ERα or ERβ selectivity at promoter-gene sites containing consensus estrogen response elements (EREs): ERα-selective agonist (propyl-pyrazole-triol (PPT)), ERα-selective antagonist (methyl-piperidino-pyrazole (MPP)), ERβ-potency selective agonist (diarylpropionitrile (DPN)) and ERβ-selective antagonist/ERα-agonist (R,R-tetrahydrochrysene (R,R-THC)). In this study, we have examined the activity of these compounds at a range of gene sites where ER stimulates gene expression through non-consensus EREs (complement C3), or multiple half-EREs (NHE-RF/EBP50), or by tethering to DNA via other proteins (TGF β3 and progesterone receptor A/AP-1), and at gene sites where ER represses gene transcription (interleukin-6). At all of these genes, PPT showed full stimulation through ERα while displaying no agonism through ERβ. MPP antagonized estradiol actions on gene transactivation and transrepression through ERα, with little or no effect on transcription mediated through ERβ. DPN displayed subtype-selective agonism, being ca. 30-fold more potent through ERβ. R,R-THC was a complete antagonist through ERβ and displayed agonism through ERα, the level of which was promoter dependent. Because these ligands maintain their agonist or antagonist character and ER subtype-selectivity at gene sites of diverse nature, where estradiol is either stimulatory or inhibitory, these compounds should prove useful in elucidating the biological functions of ERα and ERβ.

Introduction

Estrogens play important roles in a number of physiological processes, including the growth and development of reproductive tissues and maintenance of bone mineral density, and in pathologic conditions, including cancers that develop in estrogen-responsive tissues (Katzenellenbogen et al., 2000). The estrogen receptor (ER), a ligand-inducible transcription factor responsible for mediating the effects of estrogens, exists as two distinct subtypes encoded by different genes. These are ERα, the first characterized ER, and the more recently identified ERβ (Katzenellenbogen and Korach, 1997, Enmark and Gustafsson, 1999, Dechering et al., 2000, Pettersson and Gustafsson, 2001). Although these two subtypes of ER have high identity in their DNA binding domains (∼96% amino acid identity), there are significant differences between the ligand binding domains of the two subtypes (∼59% amino acid identity). Hence, although estradiol (E2) binds with relatively similar affinity to ERα and ERβ, due to this divergence in ligand binding domain sequences, the two ER subtypes bind some ligands with quite different affinities (Kuiper et al., 1997). In addition, while the expression of the two ERs may overlap in some target tissues, they are differentially expressed in others. Therefore, ligands that have the capacity to selectively bind to and activate or inhibit these ER subtypes would be useful in elucidating the biology of these two receptors and might assist in the development of estrogen pharmaceuticals with improved tissue selectivity.

The estradiol-occupied ER complex stimulates or represses the transcription of many target genes. Although some estrogen responsive genes contain palindromic estrogen responsive elements (EREs) in their promoter regions to which the liganded ER binds directly, much recent information has highlighted the complexity of ER transactivation and transrepression (Hall et al., 2001). In addition to binding to promoter elements that contain a consensus ERE, ligand-bound ER can also regulate transcription at genes that contain non-consensus EREs or ERE half-sites. ER is also able to alter transcription at particular sites without directly binding to DNA. At these sites, ER exerts its effects by tethering to another transcription factor, such as AP-1 or Sp1 (Paech et al., 1997, Webb et al., 1999, Kushner et al., 2000, Safe, 2001). Likewise, ER can transrepress gene expression by inhibiting the DNA interaction or activity of other factors such as NFkB, which appears to underlie its inhibition of the interleukin-6 (IL-6) gene (Pottratz et al., 1994, Ray et al., 1994, Stein and Yang, 1995, Galien et al., 1996, Galien and Garcia, 1997, Kurebayashi et al., 1997, Ray et al., 1997). Interestingly, the pharmacology of ER and other nuclear receptors at a given target gene can vary, being determined by the ER subtype or nature of the ligand (McDonnell, 1999, Kushner et al., 2000, Katzenellenbogen and Katzenellenbogen, 2002).

We have developed a number of non-steroidal compounds that we characterized as selective agonists or antagonists for ERα or ERβ based on their transactivation effects on gene constructs containing consensus EREs. Propylpryazole–triol (PPT) is a triarylpyrazole that was found to be approximately 1000-fold more potent as an agonist on ERα than on ERβ (Kraichely et al., 2000, Stauffer et al., 2000). Methyl-piperidino-pyrazole (MPP) is a pyrazole compound containing a basic side-chain substituent that converts the pyrazole into an antagonist that retains high preferential binding affinity (approximately 200-fold) for ERα (Sun et al., 2002). Diarylpropionitrile (DPN) is an agonist that displayed potency selectivity for ERβ (Meyers et al., 2001). R,R-tetrahydrochrysene (R,R-THC) showed partial agonist activity through ERα, but was a pure antagonist through ERβ (Meyers et al., 1999, Sun et al., 1999).

In this study, we have investigated the pharmacological character of these ligands by evaluating their transcriptional activities at a range of estrogen-responsive promoters containing diverse response elements where the ER acts to up-regulate or down-regulate gene expression via mechanisms that involve direct DNA binding or indirect DNA interaction via tethering of the ER to other DNA-binding transcription factors. Hence, the activities of these ligands with ERα or ERβ were studied at two promoters where ER directly binds to the promoter-gene DNA, namely at the complement component 3 (C3) promoter, which contains three EREs, one consensus and two nonconsensus, and the sodium–hydrogen exchanger regulatory factor ezrin–radixin–moesin binding protein 50 (NHE-RF/EBP50) promoter, where ER acts through multiple ERE-half sites (Yang et al., 1996, Petz et al., 2002). In addition, we examined ligand activity at promoters where ER acts through a protein–protein tethering interaction to regulate transcription (TGFβ3 promoter, or AP1 site in the progesterone receptor A promoter) (Petz et al., 2002) or where ER acts as a transrepressor (IL-6 promoter) by inhibiting activity of NFkB (Pottratz et al., 1994, Ray et al., 1994, Galien et al., 1996, Galien and Garcia, 1997, Kurebayashi et al., 1997, Ray et al., 1997). Our findings reveal that these ligands retain their ERα- or ERβ-selective character at a range of estrogen-responsive genes where the receptor mediates transactivation or transrepression. Intriguingly, the agonist or antagonist pharmacology of the ligands is retained at all the genes examined, however, the extent of ERα agonism of the ERα agonist/ERβ antagonist ligand R,R-THC is found to vary, dependent on the nature of the particular promoter. The findings are discussed in terms of our understanding of the structural basis for the ERα or ERβ selectively of these ligands.

Section snippets

Chemicals, materials, and plasmid constructions

Cell culture media were purchased from Life Technologies, Inc. (Grand Island, NY). Calf serum was obtained from HyClone Laboratories (Logan, UT), and fetal calf serum was purchased from Atlanta Biologicals (Atlanta, GA). [14C]Chloramphenicol (50–60 Ci/mmol) was obtained from DuPont, NEN Life Science Products (Boston, MA). The luciferase assay system was from Promega Corp. (Madison, WI). The expression vectors for human ERα (pCMV5-hERα) and human ERβ (pCMV5-ERβ, 530 residues) were constructed as

Results

Fig. 1 shows the structures of the ligands used in these studies, PPT, DPN, R,R-THC and MPP. The ligands are all non-steroidal, and the MPP compound contains a piperidino side-chain found in the antiestrogen raloxifene. Characterization of these ligands using promoter-gene constructs containing consensus estrogen response elements (EREs) indicated PPT to be an ERα-selective agonist, MPP to be an ERα-selective antagonist, DPN to be an ERβ-potency selective agonist and R,R-THC to be an

Discussion

The ER acts at estrogen-responsive target genes to either transactivate or transrepress gene expression (Valentine et al., 2000). We were interested, in this study, in determining the effects of ERα or ERβ selective ligands on diverse estrogen-responsive gene sites. In particular, would an ERα antagonist (MPP) reverse E2-repression on an inhibited promoter such as that in IL-6 only through ERα and not through ERβ? How would a ligand, namely R,R-THC, with known ERα agonist and ERβ antagonist

Acknowledgements

This work was supported by NIH grants CA18119 and DK15556, NIH training grants T32HD07028 and T32ES07326, and The Breast Cancer Research Foundation.

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