SKF-82958 Is a Subtype-selective Estrogen Receptor-α (ERα) Agonist That Induces Functional Interactions between ERα and AP-1

Abstract

The transcriptional activity of estrogen receptors (ERs) can be regulated by ligands as well as agents such as dopamine, which stimulate intracellular signaling pathways able to communicate with these receptors. We examined the ability of SKF-82958 (SKF), a previously characterized full dopamine D1 receptor agonist, to stimulate the transcriptional activity of ER␣ and ER␤. Treatment of HeLa cells with SKF-82958 stimulated robust ER␣-dependent transcription from an estrogen-response element-E1b-CAT reporter in the absence of estrogen, and this was accompanied by increased receptor phosphorylation. However, induction of ER␤-directed gene expression under the same conditions was negligible. In our cell model, SKF treatment did not elevate cAMP levels nor enhance transcription from a cAMPresponse element-linked reporter. Control studies revealed that SKF-82958, but not dopamine, competes with 17␤-estradiol for binding to ER␣ or ER␤ with comparable relative binding affinities. Therefore, SKF-82958 is an ER␣-selective agonist. Transcriptional activation of ER␣ by SKF was more potent than expected from its relative binding activity, and further examination revealed that this synthetic compound induced expression of an AP-1 target gene in a tetradecanoylphorbol-13-acetate-response element (TRE)-dependent manner. A putative TRE site upstream of the estrogen-response element and the amino-terminal domain of the receptor contributed to, but were not required for, SKF-induced expression of an ER␣-dependent reporter gene. Overexpression of the AP-1 protein c-Jun, but not c-Fos, strongly enhanced SKF-induced ER␣ target gene expression but only when the TRE was present. These studies provide information on the ability of a ligand that weakly stimulates ER␣ to yield strong stimulation of ER␣-dependent gene expression through cross-talk with other intracellular signaling pathways producing a robust combinatorial response within the cell.The effects of estrogens are mediated by the products of two separate genes, one for estrogen receptor-␣ (ER␣) 1 and another for ER␤. Both are members of the nuclear receptor superfamily of ligand-activated transcription factors. The mechanisms by which ERs activate target gene expression in response to estrogen signaling have been the subject of intense investigation since their respective cDNAs were cloned (1, 2). Because of the relatively recent identification of ER␤, the bulk of our knowledge regarding the genomic effects of estrogens is derived from ER␣ studies. For instance, upon binding to 17␤-estradiol (E 2 ), ER␣ undergoes a series of biochemical alterations including increased phosphorylation and conformational changes as well as homodimerization and binding of the receptor to its target DNA sequence, the estrogen-response element (ERE; see Refs. 3-5). ER␤ also undergoes conformational changes in response to ligand binding (6, 7) and is phosphorylated in vivo (8). With respect to DNA binding, ER␤ binds to the same consensus ERE that ER␣ does, although the latter receptor has an ϳ4-fold ...

Mechanistic Differences in the Activation of Estrogen Receptor-α (ERα)- and ERβ-dependent Gene Expression by cAMP Signaling Pathway(s)

“…Although the sequence (TGACACA) present in our ERE-E1b-CAT reporter diverges from the consensus TRE sequence, it has been shown to bind to in vitro translated Jun and Fos (56), and our gel mobility shift assays reveal that the levels of both c-Jun and c-Fos expressed in HeLa cells are sufficient to bind to this DNA. Moreover, our laboratory demonstrated previously through overexpression studies that c-Jun can enhance ER␣-mediated transcription of ERE-E1b-CAT but not of the corresponding TRE mutant reporter, ERE-E1b-CAT(mTRE) (39). Because we demonstrate a requirement for the ERE and TRE DNA sites, our data suggest that cooperation between ER and AP-1 transcription factors bound to their respective target promoter sequences results in robust forskolin/IBMX activation of target gene expression.…”

“…In ERE-E1b-CAT such a putative TRE is located ϳ255 bp upstream from the ERE. To determine whether this TRE played any role in the ability of forskolin/ IBMX to stimulate the activity of either ER, we examined the ability of forskolin/IBMX to increase CAT gene expression from a reporter plasmid (ERE-E1b-CAT(mTRE)) in which the TRE was mutated to GGACTCA, a mutation previously demonstrated to abolish AP-1 binding (39,57). As shown in Fig.…”

“…demonstrated that amino acids 259 -302 of ER␣ constitute a major interaction site with c-Jun (58), and because the ability of ER␣-179C to mediate forskolin-induced activation of CAT expression was dependent on a TRE site within the reporter gene that has been shown to support c-Jun physical and functional interactions (39,56), we investigated the possibility that cAMP activation of ER␣-179C was caused by a direct interaction between the hinge region of the receptor and c-Jun. To test this hypothesis, the EF domain of ER␣ (amino acids 302-595) and the corresponding ER␤ fragment (amino acids 254 -530) were fused to the Gal4 DNA binding domain (Gal-ER␣ EF and Gal-ER␤ EF) and examined for their abilities to stimulate expression of 17mer-E1b-CAT, which contains four Gal4 binding sites upstream from the TATA box and CAT gene.…”

Runx2 Integrates Estrogen Activity in Osteoblasts

“…Unlike the inhibitory effects of estradiol on TGF-␤-sensitive and bone morphogenetic proteinsensitive Smads that occur in mesangial and breast cancer cells (69,70), estradiol enhances Smad-dependent gene expression in response to TGF-␤ in osteoblasts. Interactions between ERs and AP-1 transcription factors are also complex, because estrogen can either increase or decrease the expression of AP-1-sensitive genes (56,57,71,76). We found that estrogen reduced TGF-␤ activation of the AP-1-sensitive promoter 3TP-Lux.…”

Marinobufagenin interferes with the function of the mineralocorticoid receptor

“…The ability of MBG to bind to MR was determined in vivo essentially as previously described [20]. Briefly, 1 μg of the MR expression plasmid was transfected into Cos-1 cells using Lipofectamine and 24 h thereafter media was aspirated from wells and replaced with DMEM containing 5% sFBS and ∼1.0 nM …”