Two Separate Mechanisms for Ligand-Independent Activation of the Estrogen Receptor
Mohammed K. K. El-Tanani and
Chris D. Green
School of Biological Sciences, University of Liverpool,
Liverpool, L69 3BX, United Kingdom
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ABSTRACT
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Transient transfection experiments in which three
different estrogen response element-containing reporter genes were
cotransfected into HeLa cells, together with constitutively expressed
estrogen receptor (ER) constructs, demonstrate that activation of the
transcription of the reporter genes by epidermal growth factor (EGF)
and by cholera toxin with 3-isobutyl-1-methyl-xanthine, which elevate
cellular cAMP, is dependent upon the presence of functional ER.
Cotransfection of the reporter genes with truncated versions of the ER
shows that the two non-ligand activators of ER require different
regions of the receptor to produce their effects on transcription. EGF
acts primarily by means of transactivation domain AF-1, whereas cAMP
acts via transactivation domain AF-2 of the ER. A point mutation that
removes a major site of inducible phosphorylation within the AF-1
domain of the ER abolishes the response to EGF, but the response to
estradiol and cAMP is retained. Specific inhibition of cAMP-activated
protein kinase (protein kinase A) prevents the response to elevated
cAMP but does not affect EGF or estradiol responses. Overexpression of
the protein kinase A catalytic subunit in HeLa cells results in an
amplified response to estradiol, similar to that induced by cholera
toxin with 3-isobutyl-1-methyl-xanthine. Comparable experiments
performed using COS-1 cells produce similar results but also reveal
cell type- and promoter-specific aspects of the activation mechanisms.
Apparently, the ER may be activated by three different signal
molecules, estradiol, EGF, and cAMP, each using a mechanism that is
distinguishable from that of the others.
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INTRODUCTION
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The estrogen receptor (ER) is a member of a superfamily of nuclear
receptors that function as ligand-activated transcription factors
(1, 2, 3). Binding the hormone activates the receptor protein so that it
may bind to discrete estrogen response element (ERE) sequences in the
genomic DNA and stimulate the transcription of specific structural
genes. The ER, in common with other members of the nuclear receptor
superfamily, possesses a modular structure in which various aspects of
receptor function are associated with specific regions of the peptide
sequence (4, 5). In addition to well defined DNA-binding and
ligand-binding domains, nuclear receptors possess two separate regions
that are required for optimal transcriptional activation. An
amino-terminal transcription activation function (AF-1) operates in a
manner that is independent of ligand binding (6, 7). A second
activation function (AF-2) is located in the ligand-binding domain of
the receptor toward the carboxy terminus of the molecule, and its
activity is dependent upon the binding of an agonistic ligand (6, 7).
Both AF-1 and AF-2 are required for optimal stimulation of
transcription, but the relative contributions of the two varies in a
promoter- and cell type-specific manner (6, 7, 8).
However, more recent evidence has suggested that the ER, in particular,
may be activated in a ligand-independent manner by a variety of agents
that include several growth factors (9, 10, 11), the neurotransmitter
dopamine (12), and cAMP (13, 14). The expression of a variety of
estrogen-responsive genes has been shown to be stimulated by these
agents by a mechanism that is inhibited by the presence of the pure
antiestrogen, ICI 164384 (9, 11, 13). Because ICI 164384 acts by
binding to the ER (15), this is taken as evidence that transcriptional
activation by these alternative agents involves the ER. Stimulation by
epidermal growth factor (EGF) of the expression of estrogen-responsive
reporter gene constructs in HeLa cells, which do not express endogenous
ER, has been shown to be dependent upon coexpression of ER (9). In
addition, it has been shown that the estrogen-like effects of EGF upon
the mouse uterus do not occur in ER-deficient transgenic mice (10).
We now present evidence that the activation of the human ER by EGF and
by cAMP involves mechanisms distinct from that of estradiol and
distinguishable from each other. Furthermore, the transactivation
effect of EGF is not dependent upon estradiol effects upon receptor
activity.
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RESULTS
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HeLa cells from human cervical carcinoma do not contain ERs, but
their growth is stimulated by EGF (16). We transfected eukaryotic
expression vectors containing cDNAs derived from the human ER (4) into
HeLa cells. HEG0 contains an insert coding for the full-length wild
type human ER (amino acids 1595) (17). HE0 codes for a full-length ER
that contains a single point mutation resulting in an amino acid
substitution, Gly400, which is replaced by a Val residue
(17). HE457 codes for a full-length ER containing a point mutation
resulting in the replacement of Ser118 by an Ala residue
(18). HE15 contains an insert coding for a carboxyl-terminal truncated
receptor (amino acids 1282). HEG19 codes for an amino-terminal
truncated receptor (amino acids 179595). As indicated in Fig. 1a
, the product of HE15 consists of the A/B and C
domains and therefore retains AF-1, but not AF-2, transactivation
function. The HEG19 product consists of domains C, D, and E/F and
therefore has ligand-binding activity and retains AF-2 but not AF-1
transactivation function. We investigated the ability of these ER
constructs to stimulate the expression of chloramphenicol
acetyltransferase (CAT) reporter gene constructs, in response to
estradiol, EGF, and elevated cAMP. The relative contributions of AF-1
and AF-2 to overall transcriptional activation are reported to be
promoter specific (6, 7, 8), and we therefore used three reporter
constructs, each with a different promoter structure (Fig. 1b
).

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Figure 1. DNA Constructs Transfected into HeLa and COS-1
Cells
a, ER derivatives. Amino acid positions are numbered from the amino
terminus of the ER and indicate the boundaries of the functional
domains of the receptor and of the truncated receptors. Positions of
point mutations (amino acid substitutions) in HE0 and HE457 are
indicated. b, Reporter constructs (not drawn to scale). Solid
squares indicate consensus EREs. ERE.VIT contains two
contiguous (nonconsensus) endogenous EREs together with an additional
consensus ERE inserted upstream of the endogenous EREs (31). VA, An
NF1-related vitellogenin activator element (33). 2ERE.TATA is a
synthetic enhancer/promotor sequence containing the elements indicated
(31). ERE.TK consists of a consensus ERE linked to the promotor
sequence (nucleotides -150 to +51) of the herpes simplex virus
thymidine kinase gene (6).
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In the absence of ER, the expression, in HeLa cells, of an
estrogen-responsive reporter gene is not stimulated by estradiol, EGF,
or cholera toxin + 3-isobutyl-1-methylxanthine (CT/IBMX) (Fig. 2
). CT causes irreversible stimulation of adenylate
cyclase, and IBMX inhibits cAMP phosphodiesterase; together they cause
a major increase in intracellular cAMP (13). Cotransfection of the full
length ER (HEG0) with the reporter gene enables all three agents to
stimulate CAT expression (Fig. 2
). EGF alone causes an increase in CAT
expression that is less than that seen with estradiol; however,
simultaneous treatment of the cells with EGF and estradiol results in a
response that is the sum of the two separate responses. CT/IBMX alone
causes only a weak stimulation of reporter gene activity but has a
synergistic effect when combined with estradiol. The Gly->Val mutation
in HE0 has been reported to prevent the ligand-independent activation
of the ER by dopamine (12). However, we found that HE0 is able to
support a pattern of response to estradiol, EGF, and CT/IBMX, alone and
in combination, similar to that seen in the presence of the wild type
receptor. The pattern of response to the inducers is similar for the
different reporter gene constructs, although the scale of the increase
varies.

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Figure 2. ER Dependence of Reporter Gene Response to
Estradiol, EGF, and Elevated cAMP in HeLa Cells
HeLa cells were withdrawn from estradiol and transferred to serum-free
medium before being transfected with reporter plasmid DNA with or
without ER plasmid (HEG0 or HE0) DNA as indicated. All cells were
cotransfected with pSV-ß-galactosidase plasmid DNA. After 24 h,
cells were transferred to serum-free medium with additions (E, EGF,
CT/IBMX) or without addition (C) as indicated. Cells were harvested
after 24 h of treatment and assayed for CAT and ß-galactosidase
activity. CAT activity was normalized relative to thr ß-galactosidase
activity and is expressed as a percentage of the activity in
HEGO-transfected, estradiol-treated cells. Results are the means
± SD of three experiments.
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Cotransfection of the truncated receptors, HE15 and HEG19, with the
reporter genes reveals differences between the three inducers and their
relationships with each other. EGF is able to induce an increase in CAT
expression in the presence of HE15 but not of HEG19 (Fig. 3
). The scale of increase produced by EGF with HE15
(e.g. 2ERE.TATA, 5.6 ± 0.8-fold increase) is
equivalent to that seen with the complete receptor (2ERE.TATA, 4.8
± 0.7-fold increase). The product of HE15 does not possess a
hormone-binding site and is therefore unresponsive to both estradiol
and ICI 164384. HEG19 can respond to estradiol, but there is no further
increase seen when EGF is combined with the hormone.

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Figure 3. The Ability of Carboxyl-Terminal, but not
Amino-Terminal, Truncated ER to Support EGF Stimulation of Reporter
Gene Expression in HeLa Cells
HeLa cells were transfected with the indicated reporter plasmid DNA
plus expression vector DNA containing either HE15- or HEG19-truncated
ER cDNA. Experiments were conducted as described for Fig. 2 (ICI,
antiestrogen ICI 164384). CAT activity is expressed as a percentage of
the activity measured in HEG0-transfected, estradiol-treated cells.
Results are the mean ± SD of three separate
experiments.
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In contrast, CT/IBMX is able to stimulate reporter gene activity in the
presence of HEG19 but not of HE15 (Fig. 4
). The pattern
of response to CT/IBMX, alone or in combination with estradiol or with
ICI 164384, in the presence of HEG19 is similar to that seen with HEG0.
The response to estradiol in the presence of HEG19 is reduced
(e.g. ERE.VIT, 6.1-fold increase) compared with that seen
with HEG0 (11.1-fold increase), but CT/IBMX in combination with
estradiol still produces a synergistic response with ERE.VIT and
2ERE.TATA reporters, although the ERE.TK response is merely
additive (data not presented). Cells transfected with HEG19 were
rigorously depleted of estrogen before they were treated with
CT/IBMX, making it unlikely that the response seen in the absence of
exogenous estradiol involves an interaction between cAMP and
ligand-occupied receptor.

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Figure 4. The Ability of Amino-Terminal, but not
Carboxyl-Terminal, Truncated ER to Support cAMP Stimulation of Reporter
Gene Expression in HeLa Cells
HeLa cells were transfected with the indicated reporter plasmid DNA
plus expression vector DNA containing either HE15- or HEG19-truncated
ER cDNA. Experiments were conducted as described for Fig. 2 . CAT
activity is expressed as a percentage of the activity measured in
HEG0-transfected, estradiol-treated cells. Results are the means
± SD of three separate experiments.
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The simultaneous presence of HE15 and HEG19 enables the ERE.VIT and
ERE.TATA reporter genes to respond to both estradiol and EGF (Fig. 5a
). In addition, the response to EGF is now inhibited
by ICI 164384, indicating that the two truncated receptors interact
with each other. Phosphorylation of Ser118 has been shown
to be necessary for full activation of AF-1 (18) and to occur via
mitogen-activated protein kinase in EGF-stimulated cells (19, 20). The
HE457 mutant ER cannot be phosphorylated at this residue and does not
support stimulation of the ERE.VIT or 2ERE.TATA reporter genes by EGF,
although their responsiveness to estradiol and to increased cAMP is
retained (Fig. 5b
).

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Figure 5. Interaction between Amino-Terminal and
Carboxyl-Terminal Truncated ERs in HeLa Cells and the Effect of a Point
Mutation in AF-1 of the ER upon the Stimulation of Reporter Gene
Expression
a, HeLa cells were transfected with both HE15- and HEG19-truncated ER
plasmid DNA plus the indicated reporter plasmid DNA. b, HeLa cells were
transfected with HE457-mutated ER plasmid DNA plus the indicated
reporter plasmid DNA. Experiments were conducted as described for Fig. 2 . CAT activity is expressed as a percentage of the activity measured
in estradiol-treated cells. Results are the mean ± SD
of three separate experiments.
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The differences in their responses to mutations in the ER indicate that
the two ligand-independent activators of the ER, EGF and cAMP, achieve
their effects by different mechanisms. We investigated further the
involvement of protein kinases in these two mechanisms. The compound
N-\[2-((p-bromocinnamyl)amino)ethyl\]-5-isoquinolinesulfonamide,
2 HCl (H-89) selectively inhibits cAMP-activated protein kinase
[protein kinase A (PKA)] (21) whereas bisindolylmaleimide I (BIMD) is
a highly selective inhibitor of protein kinase C (22). The response to
cAMP of the ERE.VIT reporter gene, cotransfected into HeLa cells with
the HEG0 expression vector, is abolished by H-89 but is unaffected by
the presence of BIMD (Fig. 6
). Interestingly, the
response of the reporter gene to estradiol or to EGF is unaffected by
either of these protein kinase inhibitors. The involvement of PKA in
the activation of the ER by cAMP is supported by the results shown in
Fig. 7
. HeLa cells were cotransfected with the ERE.VIT
reporter gene, with the HEG0 ER expression vector and, as indicated,
with an expression vector coding for either the
- or the ß-isoform
of the human PKA catalytic subunit (23). Overexpression of PKA alone
had no detectable effect upon reporter gene expression but, in the
presence of estradiol, there was a nearly 4-fold increase in the
response above that seen with estradiol alone. Both PKA
and PKAß
produced a synergistic increase, equivalent to that seen upon elevation
of cellular cAMP levels with CT/IBMX. The effects of PKA upon ERE.VIT
expression were abolished by the presence of ICI 164384, indicating
their requirement for ER.

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Figure 6. The Ability of Inhibitors of Protein Kinases to
Influence the Induction of Reporter Gene Expression in HeLa Cells
HeLa cells were transfected with HEG0 ER plasmid DNA, ERE.VIT reporter
plasmid DNA, and pSV-ß-galactosidase plasmid DNA. Experiments were
conducted as described for Fig. 2 . Cells were treated for 24 h
with the indicated agents, including H-89 (20 µM, PKA
inhibitor) or BIMD 1 (10 µM, protein kinase C inhibitor).
CAT activity is expressed as a percentage of the activity measured in
estradiol-treated cells. Results are the mean ± SD of
three separate experiments.
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Figure 7. The Influence of Overexpression of PKA Catalytic
Subunit upon the Induction of Reporter Gene Expression in HeLa Cells
HeLa cells were transfected with HEG0 ER plasmid DNA, ERE.VIT reporter
plasmid DNA, pSVß-galactosidase plasmid DNA and, where indicated,
with either pC EV (PKAa) or pCßEV (PKAb) expression plasmid DNA.
Experiments were conducted as described for Fig. 2 . Cells were treated
for 24 h with the indicated agents in medium containing 80
µM ZnSO4. CAT activity is expressed as a
percentage of the activity measured in estradiol-treated cells. Results
are the mean ± SD of three separate experiments.
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Because responses of transfected estrogen-induced reporter genes have
been reported to be cell-specific (6, 7, 8), we repeated some of our
experiments using COS-1 cells from monkey kidney. The full-length ER
(HEG0) supported a response to estradiol comparable to that seen in
HeLa cells (Fig. 8
). Neither reporter gene showed a
significant response to EGF alone, although the CAT activity with EGF +
estradiol (ERE.VIT, 121 ± 4%; ERE.TK, 125 ± 5%) was
marginally higher than that with estradiol alone. Both reporter genes
responded to CT/IBMX, but only ERE.VIT showed a synergistic response
when CT/IBMX was combined with estradiol (CT/IBMX, 2-fold increase;
estradiol, 14-fold increase; estradiol + CT/IBMX 36-fold increase).

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Figure 8. The Ability of Estradiol, EGF, and cAMP to
Stimulate Reporter Gene Expression in the Presence of ER in COS-1 Cells
COS-1 cells were withdrawn from estradiol and transferred to serum-free
medium before being transfected with reporter plasmid DNA and ER
plasmid (HEG0) DNA as indicated. Experiments were conducted as
described for Fig. 2 . CAT activity is expressed as a percentage of the
activity measured in estradiol-treated cells. Results are the mean
± SD of three separate experiments.
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In the presence of HE15, ERE.VIT showed a pattern of responses similar
to that seen in HeLa cells (Fig. 9
), i.e.
induction by EGF but not by estradiol or CT/IBMX. However, ERE.TK
responded to EGF and failed to respond to estradiol, but also showed a
clear response to CT/IBMX. ERE.VIT and ERE.TK, in the presence of
HEG19, reproduced the pattern of responses to the three inducers that
was seen in HeLa cells, i.e. induction by estradiol and by
CT/IBMX, but only ERE.TK showed a synergistic response to their
simultaneous presence.

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Figure 9. The Ability of Truncated ERs to Support the
Stimulation of Reporter Gene Expression by Estradiol, EGF, and cAMP in
COS-1 Cells
COS-1 cells were withdrawn from estradiol and transferred to serum-free
medium before being transfected with reporter plasmid DNA and either
HE15- or HEG19-truncated ER plasmid DNA, as indicated. Experiments were
conducted as described for Fig. 2 . CAT activity is expressed as a
percentage of the activity measured in HEG0-transfected,
estradiol-treated cells. Results are the mean ± SD of
three separate experiments.
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DISCUSSION
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Our results demonstrate that the expression of estrogen-responsive
reporter genes may also be stimulated by a polypeptide growth factor,
EGF, and by elevated cAMP, in the absence of exogenous estradiol and in
a manner that is dependent upon the presence of the ER. The
simultaneous presence of an optimally inducing concentration of
estradiol (24) and either EGF or CT/IBMX results in a level of reporter
gene expression that is higher than that seen with estradiol alone.
This suggests that the mechanism of ER activation by EGF or cAMP
differs from that used by estradiol. Furthermore, the fact that EGF +
estradiol causes an additive increase whereas CT/IBMX + estradiol
causes a synergistic increase suggests that the two non-ligand
activators differ from each other in their mechanisms of action.
This latter conclusion is supported by our experiments using truncated
versions of the ER, which demonstrate that EGF and cAMP require
different regions of the receptor to achieve their effects upon
transcription. The fact that EGF can cause stimulation of transcription
in the absence of the carboxyl-terminal AF-2 transactivation domain
suggests that its effects are mediated via AF-1. EGF can stimulate
reporter gene transcription via HE15, a truncated version of the ER
that completely lacks the ligand-binding domain of the receptor,
clearly establishing that EGF does not simply enhance the action of
estradiol, but is able to activate the ER de novo. On the
other hand, elevated cellular cAMP levels, produced by treating cells
with CT/IBMX, appear to act by increasing the transactivation activity
of AF-2, located in the carboxyl-terminal half (E domain) of the
receptor. In this connection it is interesting to note that the only
difference we observed between the activity of the wild type receptor
(HEG0) and the receptor with a point mutation in the E domain (HE0) is
that the latter showed a diminished response to CT/IBMX (Fig. 2
).
Because the HEG19 construct retains the ligand-binding domain of the
receptor, we cannot determine whether or not the cAMP effect requires
the occupation of the LBD by an agonistic ligand. The fact that
stimulation of transcription by CT/IBMX can be seen in cells that have
been rigorously depleted of estrogen makes this unlikely.
The role played by phosphorylation of the ER in the ability of the
receptor to stimulate transcription is unclear. The ER protein is
phosphorylated at a basal level in the absence of estradiol, and
numerous reports (e.g. Refs. 18, 25, and 26) have shown that
the level of phosphorylation is increased upon hormone binding. The
identity of the amino acids phosphorylated, their relative levels of
phosphorylation, and the functional consequences of their
phosphorylation are, however, the subjects of conflicting reports (18, 25, 26, 27). Nevertheless, the fact that ER phosphorylation is also
increased, at apparently identical sites, by antiestrogen binding (18, 25) indicates that ligand-induced transactivation activity of the
receptor is not determined solely by its level of phosphorylation. The
evidence we present here supports the idea that ligand-independent
activation of the ER requires phosphorylation of the receptor protein
at sites that differ with the identity of the activator.
Ser118, which is situated within the AF-1 domain (28), has
been identified as a major site of ligand-stimulated phosphorylation
(18, 26) and has also been shown to be phosphorylated by
mitogen-activated protein kinase in response to EGF stimulation of
cells (19, 20). Conversion of this residue to a nonphosphorylatable
alanine (HE457) abolishes activation of the ER by EGF (Fig. 5b
).
However, HE457 is still activated, albeit to a reduced extent, by
estradiol and by elevated cAMP. Elevation of cellular cAMP has been
reported to stimulate ER phosphorylation at a site(s) outside the A/B
domain and distinct from those responsive to ligand binding (25). We
show that inhibition of cAMP-stimulated protein kinase (PKA), by
treating cells with the specific inhibitor H-89, prevents stimulation
of reporter gene activity by CT/IBMX and, most strikingly, prevents the
synergism between estradiol and CT/IBMX (Fig. 6
). The involvement of
PKA in the activation of ER by elevated cAMP is also supported by our
observation that increasing kinase activity by overexpression of the
PKA catalytic subunit duplicates the synergistic response to estradiol
seen with CT/IBMX treatment (Fig. 7
). This stimulation of reporter gene
expression by elevated PKA level was prevented by the antiestrogen, ICI
164384, indicating the requirement for ER.
ICI 164384 is termed a "pure" antiestrogen (15) because, unlike
non-steroid antiestrogens such as tamoxifen, it does not exhibit any
agonist activity but inhibits both the AF-1 and the AF-2
transactivation functions of the receptor (29). The mechanism by which
ICI 164384 occupation of the ligand-binding domain, in the
carboxy-terminal half of the receptor, may interfere with the activity
of the amino-terminal AF-1 function is unclear. Our experiments (Fig. 5a
), in which both truncated versions of the ER (HE15+HEG19) were
simultaneously expressed in HeLa cells, show that the AF-1 domain and
the ligand-binding domain do not need to be situated in the same
molecule for inhibition to occur. ICI 164384 was able to inhibit the
action of EGF, previously shown to operate by stimulating AF-1 but not
AF-2, indicating that the two truncated receptors interacted with each
other rather than acting independently. The mechanism of this
intermolecular interaction is unclear. Both HE15 and HEG19 possess
DNA-binding domains so that they may be capable of binding to the ERE
as a heterodimer, and the association of HEG19, in an inactive
configuration, with HE15 may inactivate HE15 also.
Both AF-1 and AF-2 contribute to the overall transactivation activity
of the ligand-occupied ER, but the nature of their contributions
differs. Truncated receptors have shown that AF-1 can exhibit
transactivation in the absence of estrogen binding to the ER whereas
AF-2 activity is dependent upon hormone binding (6, 7). We have now
demonstrated that AF-1 activity, although ligand-independent, is not
constitutive but may be indirectly regulated by signal molecules that
bind to plasma membrane receptors. The relative contributions of AF-1
and AF-2 to gene activation are markedly influenced both by the
structure of the target gene promoter and by the cell type containing
the target gene (6, 7, 8). Differences in their extent of response were
observed between the reporter genes, although the pattern of response
to the inducers was similar for all three reporter gene constructs.
However, it is difficult to detect any consistent pattern in the
relative responsiveness of the reporter genes. In general, the ERE.VIT
reporter gene, which has the largest number of copies of the ERE and
the most complex promoter, gave the largest response, but there were
exceptions to this, e.g. with HEG19 in COS-1 cells (Fig. 9
).
We assume that these differences between promoters arise from
differences in requirements for possible intermediary factors and
differences in the availability of these molecules in different cell
types (30). We compared the responses of the ERE.VIT and ERE.TK
reporter genes in COS-1 cells with those in HeLa cells. The only
significant difference between the results with the two cell lines was
that ERE.TK was able to respond to CT/IBMX in the presence of HE15, in
COS-1 cells. Apparently, in the presence of certain promoters and in a
cooperative cellular context, cAMP may also work through AF-1. A
potential PKA phosphorylation site does exist within the HE15 sequence
at Ser236 (25).
Our results show that the ER can be stimulated to activate
transcription by EGF and cAMP as well as by binding estradiol.
Transcriptional activation by estradiol is believed to involve both the
AF-1 and AF-2 transactivation functions. Our experiments indicate that,
at least in certain promoter/cell type contexts, EGF and cAMP effects
each involve only one transactivation function: AF-1 for EGF and AF-2
for cAMP. The fact that addition of either EGF or CT/IBMX in the
presence of a saturating concentration of estradiol results in a
further increase in reporter gene expression indicates that there are
basic differences between the mechanisms of ligand-independent and
ligand-dependent activation. We have presented evidence that specific
protein kinases are involved in the mechanisms triggered by the two
non-ligand activators of the ER. It is increasingly recognized that the
many intracellular signal transduction pathways in eukaryotic cells do
not operate independently of each other but that cross-talk between
pathways is possible and potentially important. It appears that the ER
provides a site for the integration of three well recognized signal
transduction pathways, i.e. those of steroid hormones, cAMP,
and tyrosine kinase receptors.
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MATERIALS AND METHODS
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Chemicals and Materials
Tissue culture medium, newborn calf serum (NBCS), FCS,
antibiotics, and trypsin-EDTA were purchased from GIBCO BRL (Paisley,
Scotland). 17ß-Estradiol, insulin, CT, IBMX, EGF (human,
recombinant), proteinase K, and vanadyl complex were obtained from
Sigma (Poole, England). ICI 164384 was kindly provided by Dr. A. E.
Wakeling, Zeneca Pharmaceuticals (Macclesfield, England). H-89 and BIMD
were obtained from Calbiochem-Novabiochem (U.K.) Ltd (Nottingham,
England). Other reagents were obtained from Boehringer Mannheim UK
(Lewes, England).
The ER plasmids, reporter constructs, and PKA plasmids were obtained
from the laboratories in which they were constructed. ER derivatives,
HE0 (4), HEG0, HEG19 (17), and HE457 (18) are contained within the
eukaryotic expression vector pSG5, and HE15 is contained in pKCR2 (4).
pC
EV and pCßEV consist of cDNAs coding for the
- and
ß-isoforms of the PKA catalytic subunit, contained within the Zem3
vector in which transcription is driven by the mouse metallothionein
promoter (23). ERE.VIT is derived from the Xenopus laevis
vitellogenin B1 gene (31). 2ERE.TATA is an entirely synthetic
enhancer/promoter sequence (31). ERE.TK (pERE BLCAT) consists of a
consensus ERE linked to the promoter sequence (nucleotides -105 to
+51) of the herpes simplex virus, thymidine kinase gene (6).
Cell Culture and Transient Transfections
HeLa cells and COS-1 cells were obtained from the European
Collection of Animal Cell Culture (Porton Down, U.K). Both cell lines
were cultured in MEM medium (GIBCO BRL) containing 100 µg/ml
penicillin (GIBCO), 100 µg/ml streptomycin (GIBCO), and 10%
(vol/vol) FCS (GIBCO). Cells were depleted of estrogen by culture for 6
days in phenol red-free RPMI 1640 medium (GIBCO), supplemented with 5%
(vol/vol) dextran/charcoal-treated NBCS (32). Cells were further
cultured for 1 day in medium supplemented with 1% (vol/vol)
dextran/charcoal-treated NBCS followed by 1 day in serum-free medium
[phenol red-free RPMI 1640 supplemented with: 10 µg/ml transferin;
10 ng/ml sodium selenite; 1% (wt/vol) glutamine; 0.2% (wt/vol) BSA].
Cells were then harvested and seeded, in multiwell plates, at a density
of 2 x 105 cells per 35-mm well, in serum-free
medium. After 24 h, cells were transfected with DNA \[300 ng ER
plasmid (HEG0, HE0, HE15, HEG19 or HE457), 1000 ng each of 2ERE.TATA,
ERE.VIT, or ERE.TK plasmid, 20 ng pC
EV or pCßEV plasmid, 700 ng
pSV-ß-galactosidase plasmid (Promega, Madison, WI)\] using the
LipofectAMINE reagent (Life Technologies, Gaithersburg, MD) according
to the manufacturers protocol. After 5 h an equal volume of
serum-free medium was added to each well. After a further 19 h the
medium was changed for fresh serum-free medium containing the inducing
agents: 10-8 M estradiol (E); 100 ng/ml EGF; 1
µg/ml CT, 10-4 M IBMX; 10-6
M ICI 164384 (ICI); or without addition (C). For
transfections involving the pC
EV and pCßEV vectors, the culture
medium was supplemented with 80 µM ZnSO4.
Cells were incubated for 24 h and then were harvested and CAT
(liquid scintillation method) and ß-galactosidase activities were
assayed in whole cell extracts using assay kits (Promega) according to
the manufacturers protocols. CAT activity results were normalized
relative to the ß-galactosidase activity, to correct for differences
in efficiency of transfection, and are expressed as a percentage of the
estradiol-induced level, as indicated. Results are the mean ±
SD of at least three separate experiments.
 |
ACKNOWLEDGMENTS
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We wish to thank P. Chambon for the gift of HEG0, HE0, HE15,
HEG19, and HE457; G. S. McKnight for the gift of pC
EV and pCßEV;
D. J. Shapiro for the gift of ERE.VIT and 2ERE.TATA; M. G. Parker for
the gift of ERE.TK; and A. E. Wakeling for the gift of ICI 164384.
 |
FOOTNOTES
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Address requests for reprints to: Dr. C. D. Green, School of Biological Sciences, Life Sciences Building, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX, U.K.
This work was supported by the Association for International Cancer
Research.
Received for publication July 19, 1996.
Accepted for publication March 11, 1997.
 |
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