17β-Estradiol (E2) induces c-fosprotooncogene expression in MCF-7 human breast cancer cells, and deletion analysis of the c-fos promoter showed that the serum response element (SRE) at −325 to −296 ...was E2-responsive. The mechanism of ligand-activated estrogen receptor α (ERα)-dependent activation of gene expression through the SRE was determined by mutational analysis of the promoter, analysis of mitogen-activated protein kinase (MAPK) pathway activation by E2, and transforming growth factor α (TGF-α) as a positive control. In addition, ERα-negative MDA-MB-231 breast cancer and Chinese hamster ovary cells were used as reference cell lines. The results showed that transcriptional activation of the SRE by E2 was due to ERα activation of the MAPK pathway and increased binding of the serum response factor and Elk-1 to the SRE. Subsequent studies with dominant negative Elk-1, wild type, and variant GAL4-Elk-1 fusion proteins confirmed that phosphorylation of Elk-1 at serines 383 and 389 in the C-terminal region of Elk-1 is an important downstream target associated with activation of an SRE by E2. Both E2 (ERα-dependent) and growth factors (ERα-independent) activated the SRE in breast cancer cells via the Ras/MAPK pathway; however, in ER-negative CHO cells that do not express a receptor for TGF-α, only hormone-induced activation was observed in cells transfected with ERα.
17β-Estradiol (E2) induces proliferation and c-
fos gene expression in MCF-7 cells and both responses are partially blocked by wortmannin and LY294002 which are inhibitors of ...phosphatidylinositol-3-kinase (PI3-K). Analysis of the c-
fos gene promoter shows that the effects of wortmannin and LY294002 are associated with inhibition of E2-induced activation through the serum response factor (SRF) motif within the proximal serum response element at −325 and −296. E2 activates constructs containing multiple copies of the SRF (pSRF) and a GAL4–SRF fusion protein; these responses are accompanied by PI3-K-dependent phosphorylation of Akt and inhibited by wortmannin/LY294002, the antiestrogen ICI 182780, but not by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD98059. Using a series of kinase inhibitors and dominant negative kinase expression plasmids, it was shown that the non-genomic activation of SRF by E2 was associated with src–ras–PI3-K pathway, thus, demonstrating hormonal activation of the SRE through src–ras activation of both PI3-K- and MAPK-dependent signaling pathways.
Prolactin (Prl) and progesterone (P) cooperate synergistically during mammary gland development and tumorigenesis. We hypothesized that one mechanism for these effects may be through mutual induction ...of receptors (R). EpH4 mouse mammary epithelial cells stably transfected with PR-A express elevated levels of PrlR mRNA and protein compared to control EpH4 cells that lack the PR. Likewise, T47D human breast cancer cells treated with P overexpress the PrlR and activate PrlR promoter III. PrlR promoter III does not contain a classical P response element but contains several binding sites for transcription proteins, including C/EBP, Sp1 and AP1, which may also interact with the PR. Using promoter deletion and site directed mutagenesis analyses as well as gel shift assays, cooperative activation of the C/EBP and adjacent Sp1A, but not the Sp1B or AP1, sites by P is shown to confer P responsiveness leading to increased PrlR transcription.
Human prolactin (hPRL) is a pleiotropic and versatile hormone that exercises more than 300 biological activities through binding to its cognate receptors. Recently, multiple studies have implicated ...hPRL in the development of human breast cancer. As a target of hPRL, both normal and neoplastic human breast cells also synthesize and secrete hPRL, which therefore establishes an autocrine/paracrine action loop in the mammary gland. In contrast to the extensive studies of regulation of hPRL expression in the pituitary gland, regulation of hPRL in mammary tissue and human breast cancer cells has not been extensively addressed. Extrapituitary PRL expression is primarily regulated by a distal promoter located 5.8
kb upstream to the pituitary promoter. As a result of alternative promoter usage, extrapituitary PRL is regulated by different signalling pathways and different hormones, cytokines or neuropeptides compared to regulation in the pituitary. Here, we present evidence that shows estrogen directly induces hPRL gene expression in T47D human breast cancer cells. We have identified a functional, non-canonical estrogen responsive element (ERE) and an AP1 site located in the hPRL distal promoter. Gel shift and chromatin immunoprecipitation assays demonstrated that both estrogen receptor (ER)α and ERβ directly bind to the ERE. However, only ERα interacts with AP1 proteins that bind to the AP1 site in the hPRL distal promoter. Promoter–reporter gene studies demonstrate that both ERE and AP1 sites are required for full induction of the promoter activity by estradiol. Our studies suggest that the interactions between estrogens, ERs, the ERE and AP1 transcription factors in regulation of autocrine/paracrine PRL in the human breast may be critical for oncogenesis and may contribute to progression of breast cancer.
17β-Estradiol (E2)
induced c-fos protooncogene mRNA levels in MCF-7 human
breast cancer cells, and maximal induction was observed within 1 h
after treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin
...(TCDD) inhibited the E2-induced response within
2 h. The molecular mechanism of this response was further
investigated using pFC2-CAT, a construct containing a −1400 to +41
sequence from the human c-fos protooncogene linked to a
bacterial chloramphenicol acetyltransferase (CAT) reporter gene. In
MCF-7 cells transiently transfected with pFC2-CAT, 10
nm E2 induced an
8.5-fold increase of CAT activity, and cotreatment with 10
nm TCDD decreased this response by more than
45%. α-Naphthoflavone, an aryl hydrocarbon receptor (AhR)
antagonist, blocked the inhibitory effects of TCDD; moreover, the
inhibitory response was not observed in variant Ah-nonresponsive MCF-7
cells, suggesting that the AhR complex was required for estrogen
receptor cross-talk. The E2-responsive sequence
(−1220 to −1155) in the c-fos gene promoter contains two
putative core pentanucleotide dioxin-responsive elements (DREs) at−
1206 to −1202 and −1163 to −1159. In transient transfection assays
using wild-type and core DRE mutant constructs, the downstream core DRE
(at −1163 to −1159) was identified as a functional inhibitory DRE.
The results of photo-induced cross-linking, gel mobility shift, and
in vitro DNA footprinting assays showed that the AhR
complex interacted with the core DRE that also overlapped the
E2-responsive GC-rich site (−1168 to −1161),
suggesting that the mechanism for AhR-mediated inhibitory effects may
be due to quenching or masking at the Sp1-binding site.
Treatment of MCF-7 human breast cancer cells with 10 nM 17 beta-estradiol (E2) resulted in a 2-fold induction of heat shock protein (Hsp) 27 mRNA levels, and this response persisted for up to 24 h. ...The 5'-promoter region of the gene was further investigated to identify genomic sequences associated with E2 responsiveness. An Sp1 and half-palindromic estrogen response element (ERE) separated by 10 nucleotides, GGGCGGG(N)10GGTCA, were identified at -105 to -84, and formation of the Sp1/estrogen receptor (ER) complex was investigated by in vitro assays using synthetic Hsp 27-32PSp1/ERE oligonucleotides in a gel mobility shift assay and transient transfection studies using short (-108/-84) and long (-108/+23) 5'-promoter sequences linked to a thymidine kinase promoter and the bacterial chloramphenicol acetyl transferase (CAT) reporter gene (Hsp-CATs and Hsp-CATl, respectively). Incubation of nuclear extracts from MCF-7 cells with an Hsp 27-32PSp1/ERE oligonucleotide results in formation of an Sp1/ER complex. The formation of this complex was inhibited by coincubation with unlabeled Sp1/ERE, ERE, and Sp1 oligonucleotides and by preincubation with ER or Sp1 antibodies (immunodepletion). In addition, the complex was supershifted by coincubation with ER antibodies. Mutation of either Sp1 or ERE sites also decreases formation of the retarded band. E2 induced CAT activity in MCF-7 cells transiently transfected with either Hsp-CATs or Hsp-CATl plasmids. It was also demonstrated that E2 did not significantly induce CAT activity in MCF-7 cells transiently transfected with Hsp-CATl-containing mutations in both the Sp1 and ERE sites. The results of this study demonstrate that an Sp1/ER complex is involved in E2-induced Hsp 27 gene expression.
Adenosine deaminase (ADA) gene expression is induced by 17β-estradiol
(E2) in MCF-7 human breast cancer cells, whereas the
antiestrogens 4′-hydroxytamoxifen and ICI 182,780 exhibit partial
estrogen ...receptor (ER) agonist/antagonist and antagonist activities,
respectively. Previous studies have shown that the −211 to +11 region
of the ADA gene promoter contains six GC-rich sites (I–VI) that bind
Sp1 protein, and these elements are required for high basal expression.
In transient transfection studies with pADA211, which contains the−
211 to +11 ADA gene promoter linked to a bacterial chloramphenicol
acetyl transferase (CAT) reporter gene, E2 and tamoxifen
(but not ICI 182,780) induced CAT activity. Ligand-induced
transactivation was observed only in cells cotransfected with
expression plasmids for wild-type ER or HE11, which does not contain
the DNA-binding domain of the ER. Cotransfection with HE15 and HE19,
which contain the DNA-binding domain and activation function-1 (AF-1)
and AF-2 of the ER, respectively, did not result in
E2-induced activity. Subsequent deletion analysis of the
ADA gene promoter showed that Sp1 binding site IV (−79 to −73) was
primarily responsible for hormone responsiveness. ER activation of ADA
gene expression is another example of an E2-induced gene
that is dependent on ER/Sp1 interactions with a site-specific GC-rich
motif.
17β-Estradiol (E2) induces c-fos
protooncogene expression in MCF-7 human breast cancer cells, and
previous studies in HeLa cells identified an imperfect palindromic
estrogen-responsive element (−1212 ...to −1200) that was required for
trans-activation. In contrast, the estrogen-responsive
element was not required for E2 responsiveness in MCF-7
cells, and using a series of constructs containing wild-type (pF1) and
mutant 5′-flanking sequences (−1220 to −1155) from the
c-fos protooncogene promoter in transient transfection
assays, it was shown that a GC-rich motif (5′-GGGGCGTGG) containing an
imperfect Sp1-binding site was required for hormone-induced activity.
This sequence also bound Sp1 protein in gel mobility shift assays, and
coincubation with the estrogen receptor (ER) enhanced Sp1-DNA binding.
E2 and 4′-hydroxytamoxifen, but not ICI 164,384, induced
reporter gene activity in cells transiently transfected with pF1.
E2 induced reporter gene activity in MDA-MB-231 breast
cancer cells transiently cotransfected with pF1 and wild-type ER or
variant ER in which the DNA-binding domain was deleted (HE11); plasmids
expressing N-terminal or C-terminal domains of the ER containing
activator function-1 or -2, respectively, were inactive in these
assays. In contrast, only wild-type ER mediated
4′-hydroxytamoxifen-induced activity. Induction of
c-fos protooncogene expression by E2 in
MCF-7 cells is dependent on the formation of a transcriptionally active
ER/Sp1 complex that binds to a GC-rich enhancer element.
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