Human Insulin Receptor Substrate-2 (IRS-2) Is a Primary Progesterone Response Gene
Lothar Vaßen,
Wojciech Wegrzyn and
Ludger Klein-Hitpass
Institut für Zellbiologie (Tumorforschung)
Universitätsklinikum Essen D-45122 Essen, Germany
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ABSTRACT
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Elevated cAMP has been shown to unmask agonist
activity of antiprogestin/antiglucocorticoid RU486. In our search for
cellular target genes induced through this cross-talk mechanism, we
identified human insulin receptor substrate-2 (IRS-2), a cytoplasmic
signaling molecule that mediates effects of insulin, insulin-like
growth factor-1 (IGF-I), and other cytokines by acting as a molecular
adaptor between diverse receptor tyrosine kinases and downstream
effectors. Our analysis of the regulation of IRS-2 in HeLa cell models
shows that synergistic induction of IRS-2 by cAMP and RU486 can be
mediated by progesterone receptors (PR) and glucocorticoid receptors
(GR) and occurs through a relative slow mechanism that requires ongoing
protein synthesis. Importantly, we demonstrate that IRS-2 mRNA is also
inducible by progesterone, while glucocorticoid effects are only
observed in the presence of cAMP. Up-regulation of IRS-2 by
progesterone depends strictly on the presence of PR and occurs through
a rapid mechanism, suggesting that it represents a primary
transcriptional response. Furthermore, we show that expression of
IRS-1, which also binds to receptors of insulin, IGF-I, and cytokines,
is unaffected by progesterone. Thus, our results demonstrate that
progesterone alters the ratio of IRS-1 and IRS-2 in PR-positive cells
and implicate a mechanism through which progesterone can modulate the
effects of insulin, IGF-I, and cytokines on cell proliferation,
differentiation, and homeostasis.
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INTRODUCTION
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Progesterone and glucocorticoids regulate complex events in
development, growth, differentiation, and cellular homeostasis. The
underlying mechanisms are thought to be triggered through rapid
transcriptional regulation of a set of primary response genes, some of
which might have the potential to induce secondary responses of gene
networks that contribute to the observed complex changes in gene
expression. However, particularly in the case of progesterone, searches
for primary cellular targets have resulted in only a very
limited number of genes, most of which appear not to represent key
factors in signaling cascades (1). The primary transcriptional
responses to progesterone or glucocorticoids are mediated by the
progesterone and glucocorticoid receptors (PR and GR, respectively),
which belong to the nuclear hormone receptor superfamily (2). Upon
activation by binding of the cognate agonist, these receptors can both
bind to the same regulatory element, termed GRE/PRE (glucocorticoid
response element/progesterone response element) (3, 4), located
in target gene promoters or enhancers to stimulate transcription by RNA
polymerase II.
In many cases the hormone-induced activation of target genes by
agonist-loaded PR and GR can be completely antagonized by excess of
RU486, a synthetic steroid that displays both antiprogestin and
antiglucocorticoid activities (5). RU486 is used as an abortive drug
and may also be useful to antagonize the growth-promoting effects of
progesterone on breast tumors (6, 7). However, recent studies showed
that the antagonist RU486 can be rendered into a pure agonist, if the
protein kinase A (PKA) pathway is simultaneously activated by elevated
cAMP levels (8, 9, 10, 11, 12). This cAMP-induced switching of RU486
antagonist/agonist activities is regarded as a potential mechanism
through which antihormone-resistant cell populations might be selected
during long-term therapy (13). The induction of GRE/PRE-containing
reporter genes through cAMP and RU486 occurs in a clearly synergistic
fashion and depends on the presence of GRE/PREs and on PR or GR,
suggesting that the mechanism involves specific binding of RU486-loaded
receptors to GRE/PREs (9, 11). Thus, gene induction by progesterone and
glucocorticoids or by the combined action of RU486 and PKA activators
share common regulatory elements and trans-acting factors.
Yet the underlying mechanisms appear to be different, since gene
induction by progesterone or cAMP/RU486 exhibit different kinetics and
differential sensitivity to partial inhibition of protein synthesis
(9).
To identify cellular genes synergistically induced by activated PKA and
progesterone antagonist RU486, we have performed differential display
using cDNA from appropriately treated HeLa3B2 cells, which stably
express the PR isoform B (9). Since all cAMP/RU486-inducible reporter
genes analyzed so far contain GRE/PREs and therefore are also
progesterone and glucocorticoid responsive, we reasoned that a search
for cAMP/RU486-inducible cellular genes could also lead us to potential
progesterone and glucocorticoid target genes. By cloning of the
corresponding full-length cDNA, we show here that a cDNA probe, which
proved to be inducible by cAMP and RU486 in a synergistic manner, is
derived from the human insulin receptor substrate-2 gene (hIRS-2), a
key molecule in insulin-, insulin-like growth factor I (IGF-I)-, and
cytokine-signaling pathways (14, 15, 16). In the current study we analyzed
the regulation of IRS-2 by cAMP, RU486, progesterone, and
dexamethasone. We demonstrate that IRS-2 is inducible by progesterone
through a direct PR-mediated mechanism, while the related IRS-1 is
unaffected. The progesterone-induced alteration of the relative
abundance of IRS-1 and IRS-2 proteins provides a new cross-talk
mechanism through which progesterone may modulate gene networks
regulated by many growth factors, including insulin and
IGF-1.
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RESULTS
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Cloning of hIRS-2 cDNA
To identify genes potentially responding to cAMP and RU486 in a
synergistic manner, we employed the differential display RT-PCR
(DDRT-PCR) method by comparing RNA from PR-positive HeLa cells
(HeLa3B2) treated with either 8-Br-cAMP alone or 8-Br-cAMP plus RU486.
By ribonuclease (RNase) protection analysis, we could confirm that a
273-bp DDRT-PCR probe indeed detected a transcript that was regulated
by cAMP and RU486 in a synergistic fashion (see below). Sequencing of
the 373-bp fragment revealed no extended open reading frame (ORF) and
no homology to any known gene. To identify the corresponding gene, we
used the isolated cDNA fragment to screen cDNA libraries from human
fetal brain and R5020-induced HeLa3B2 cells. Overlapping cDNA clones
isolated during multiple rounds of screening were used to assemble the
6996-bp cDNA sequence, which contains an ORF of 4014 bp and 5'- and
3'-untranslated regions of 516 bp and 2466 bp, respectively (Fig. 1
, A and B). The
3'-untranslated region contains four consensus polyadenylation signals
(AATAAA). Analysis of various cDNA clones and a number of expressed
sequence tags (EST database, NCBI) showed that the two signals
at positions 6891 and 6965 are functional and used at about equal
frequency.

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Figure 1. A cAMP/RU486-Regulated DDRT-PCR Probe Is Derived
from the Human IRS-2 Gene
A, Molecular cloning of hIRS-2 cDNA. Top, a schematic
representation of the hIRS-2 cDNA is given. The location of the ORF as
well as a number of restriction sites are indicated.
Bottom, the partial cDNA probe (1.6) obtained by
DDRT-PCR and overlapping cDNAs used to assemble the complete cDNA
sequence are aligned. X, XhoI; K, KpnI;
Bg, BglII; Ks, Ksp632I; S,
SalI; RV, EcoRV. B, cDNA sequence and
deduced protein sequence of hIRS-2. The putative Kozac start site at
position 517 is shown in bold and the in-frame
stop codon 5' of the translation start site is
underlined. IH-1PH and IH-2PTB
homology domains are shaded in gray and potential
tyrosine phosphorylation motifs are highlighted as white
characters on black background. Consensus polyadenylation
signals within the 3'-untranslated region are in bold,
and the part of the cDNA as isolated by DDRT-PCR is
underlined. C, Schematic diagram of hIRS-2, showing
regions of homology with mouse IRS-2 (mIRS-2) and human IRS-1 (hIRS-1).
Numbers within boxes represent percentages of amino acid
identity between hIRS-2 and the corresponding domains of mIRS-2 (18 )
and hIRS-1 (31 ) after pairwise alignment using Multalin version
5.3.3 (32 ). IH-1PH and IH-2PTB homology domains
of IRS proteins are indicated as black boxes.
Small numbers refer to amino acids bordering the
homology regions.
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The putative initiator methionine at nucleotide position 517 is
preceded by an in-frame stop codon at position 397. The ORF encodes a
protein of 1338 amino acids and a molecular mass of 138 kDa. A homology
search revealed that this ORF showed similarities to various members of
the insulin receptor substrate (IRS) gene family. IRS are cytoplasmic
signal transduction proteins, which after binding to activated insulin
and IGF-I receptors become phoshorylated at multiple tyrosine residues
(14, 15, 16). Tyrosine-phosphorylated IRS proteins then serve as docking
sites for a number of enzymes and effector proteins, which are linked
to pathways regulating metabolism, differentiation, and cell growth. As
indicated in Fig. 1C
, the greatest homology was found with IRS-2 of the
mouse (mIRS-2, Refs. 17, 18). The IRS-2 pleckstrin-homology
[IH-1PH, amino acids (aa) 32143] and the
phosphotyrosine-binding (IH-2PTB, aa 195353) domains,
which are both important for binding and coupling to the
phosphotyrosine kinase domains of insulin and IGF-I receptors (19),
showed high amino acid sequence identities of 94% and 99%,
respectively. A high level of amino acid sequence identity (83%) with
mIRS-2 was also found within the C-terminal part of the protein (aa
355-1338), whereas the N-terminal domain (74% identity) and the region
between IH-1PH and IH-2PTB (63% identity) were
less conserved. Moreover, all potential tyrosine phosphorylation motifs
identified previously in the mIRS-2 protein are well conserved in the
predicted protein sequence (Fig. 1B
). Within all domains, similarities
with human IRS-1 (20) were clearly lower than those observed with
mIRS-2, and homology with rat IRS-3 (21) and human IRS-4 (22) was only
detected within the IH-1PH and IH-2PTB domains
(Fig. 1C
and data not shown). Together, these data indicate that the
cloned cDNA represents the human homolog of mIRS-2.
Alignment of our sequence with a partial hIRS-2 cDNA sequence published
during preparation of this manuscript (GeneBank accession no. AB000732)
revealed a number of point mutations, which might represent
polymorphisms or sequencing errors. However, compared with our hIRS-2
cDNA sequence, the nucleotide sequence published by Ogihara et
al. (23) contains four 1-bp insertions within the last 80
bp of the coding region, which result in a C-terminal end of the
protein that shows no extended homology to mIRS-2 any more (data not
shown). We are positive that our nucleotide sequence and translation of
the C-terminal end of the ORF represents the correct sequence, as our
cDNA sequence is identical with human EST clone AA375248 in the NCBI
database. Moreover, sequencing of the corresponding part of a genomic
clone isolated from a phage library confirmed our cDNA sequence (data
not shown).
Expression of hIRS-2 in Human Tissues
To determine the size and the expression profile of hIRS-2 mRNA, a
Northern analysis of multiple tissue blots was performed. On a 2-day
exposure, a single mRNA species of approximately 7.5 kb in size was
detected in prostate, testis, ovary, small intestine, colon, peripheral
blood, heart, brain, placenta, and skeletal muscle (Fig. 2
). Longer exposure revealed also
expression in spleen, thymus, kidney, liver, and pancreas (data not
shown). Thus, in accordance with a Northern analysis of IRS-2
expression in mice (18), hIRS-2 appears to be expressed at low levels
in all tissues analyzed. Assuming a poly(A) tail of 150200
nucleotides in length, we conclude that the 6996-bp long hIRS-2 cDNA
sequence represents full-length or nearly full-length transcripts.

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Figure 2. Expression of IRS-2 in Human Tissues
Premade Northern blots of poly A+ RNA (2 µg each) from
human tissues were hybridized at high stringency with an IRS-2 cDNA
probe derived from the 3'-untranslated region. The position of the
7.5-kb mol wt marker is indicated on the right.
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Regulation of hIRS-2 mRNA by cAMP and Steroids
To study the regulation of hIRS-2 in detail, we analyzed RNA from
PR-positive HeLa cells (HeLa3B2) treated with cAMP and various steroids
by RNAse protection (Fig. 3A
). The RNA
antisense probe used was synthesized from a pBluescript plasmid
containing the 273-bp IRS-2 cDNA fragment isolated by DDRT-PCR, which
is derived from the most 3'-end of the hIRS-2 mRNA (see Fig. 1B
). This
probe generates two distinct protected bands since it covers the two
functional polyadenylation sites at positions 6891 and 6965, which are
used at about equal frequency. PR and
-actin mRNA, which did not
respond to hormone treatment, were measured as internal controls.
hIRS-2 mRNA was almost unaffected by RU486 alone (compare lane 1 with
lane 3), while cAMP treatment (lane 4) resulted in about 2-fold
induction. In contrast, combined treatment with 8-Br-cAMP and RU486
induced hIRS-2 mRNA levels 7.5-fold (lane 5). Thus, hIRS-2 responds to
cAMP and RU486 in a synergistic fashion. As the synergistic induction
of genes by cAMP and RU486 is a characteristic property of PR target
genes, it was of interest to determine whether hIRS-2 mRNA could also
be induced by PR agonists. Indeed, treatment with the synthetic
progestin R5020 led to a 7-fold increase of hIRS-2 mRNA (Fig. 3A
, lane 2). Thus, our study identifies hIRS-2 as a novel progesterone
target gene, which can also be induced by RU486 upon costimulation of
the PKA-signaling pathway.

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Figure 3. Regulation of hIRS-2 mRNA by cAMP and Steroids
A, Analysis of IRS-2 mRNA expression in HeLa3B2 (PR-positive) by RNase
protection. Cells were treated for 20 h with R5020 (R; 10
nM), RU486 (RU; 10 nM), 8-Br-cAMP (100
µM), and cycloheximide (Chx, 40 µM) as
indicated above the lanes. Protected bands resulting
from hybridization of total RNA (20 µg each) with the IRS-2, PR, and
-actin probes after digestion with RNases T1 and A are indicated on
the left. The IRS-2 probe covers two alternative
polyadenylation sites that are used at about equal frequency, resulting
in two protected bands. Left and right panels are
derived from the same experiment and represent identical film exposure
times. In the bar diagram below, absolute IRS-2 mRNA
levels as determined by laser densitometer analysis of x-ray films are
given in arbitrary units for each lane. Numbers above the
bars represent induction factors normalized to -actin mRNA.
Induction factors of untreated controls in the absence (lane 1) or
presence of cycloheximide (lane 6) were set to be 1. Similar results
were obtained in three independent experiments. B, Hormonal regulation
of IRS-2 mRNA in PR-negative HeLa cells. Total RNA (20 µg each) from
HeLa3B2 cells treated for 20 h with hormones as indicated was
analyzed by RNase protection analysis. Dexamethasone (D) was used at a
concentration of 10 nM. Note the complete absence of PR
transcripts. Lane 7 shows a control hybridization with 20 µg of yeast
tRNA. Lane 8 contains pBR322 HpaII mol wt marker. C,
Graphic representation of a representative RNase protection experiment
analyzing induction of IRS-2 mRNA in HeLa3B2 cells after 4 (open
bars) or 24 h (filled bars) of hormone
treatment. Treatment of cells and RNA analysis was done as described in
panel A. IRS-2 signals were quantified by laser densitometry of
appropriate autoradiographs, normalized to -actin signals, and are
expressed relative to untreated controls. Qualitatively similar results
were obtained in three independent experiments.
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To confirm the role of PR in induction of hIRS-2 by progestins
and cAMP/RU486, we investigated the regulation of IRS-2 in the parental
HeLa cells, which do not express PR (Fig. 3B
). In these cells, both in
the absence and presence of 8-Br-cAMP, hIRS-2 mRNA levels could not be
stimulated by R5020 (compare lane 1 with lanes 2 and 6) proving that
progestin induction of hIRS-2 indeed requires the PR. RU486 (lane 3) or
cAMP (lane 5) alone had no effect, but when applied together (lane 9),
an increase of hIRS-2 mRNA became evident. Most likely, this induction
is mediated by the GR, which is known to bind RU486 with high affinity
and can bind to the same regulatory DNA element as PR.
Because high concentrations of dexamethasone can also activate
the PR (L. Klein-Hitpass, unpublished observation), potential
regulation of hIRS-2 mRNA levels by the synthetic glucocorticoid was
also investigated in HeLa cells to rule out a possible interference of
PR. Dexamethasone treatment alone did not significantly increase hIRS-2
mRNA (Fig. 3B
, lane 4). However, upon costimulation with cAMP,
dexamethasone increased hIRS-2 mRNA about 3-fold (lane 10). This result
suggests that, at least in the presence of elevated cAMP, the hIRS-2
gene is also a target for GR.
Induction of hIRS-2 mRNA by Progesterone or cAMP/RU486 Occurs
through Different Mechanisms
To investigate whether R5020 and 8-Br-cAMP/RU486 induction of
hIRS-2 mRNA are primary responses, we analyzed the effect of
cycloheximide in HeLa3B2 cells by RNase protection. As shown in Fig. 3A
, cycloheximide treatment reproducibly elevated basal expression
levels of IRS-2 about 3-fold (compare lane 1 with 6). However, this
effect of the inhibitor on basal IRS-2 mRNA levels appears to be
nonspecific, since PR mRNA levels were also about 3-fold stimulated by
cycloheximide. Partial inhibition of protein synthesis by cycloheximide
did not inhibit induction of hIRS-2 mRNA by progestin R5020 (compare
lane 6 with lane 7), suggesting that R5020 induction of hIRS-2 occurs
through a direct PR-mediated mechanism. In contrast, induction of
hIRS-2 mRNA by cAMP/RU486 was largely prevented in the presence of the
inhibitor (compare lane 6 with lane 10), indicating that cAMP/RU486
induction occurs through a distinct mechanism that requires ongoing
protein synthesis.
To compare the kinetics of progesterone and cAMP/RU486 induction of
hIRS-2 in the presence and absence of cAMP, we performed RNAse
protection analysis with RNA from HeLa3B2 cells that were incubated
with hormones for 4 or 24 h. As summarized in Fig. 3C
, induction
of hIRS-2 mRNA by progestin R5020 was already 12-fold after 4 h of
treatment and decreased to about 8-fold at 24 h. In contrast,
cotreatment with cAMP and RU486 had very little effect after 4 h,
but resulted in about 20-fold induction after 24 h. cAMP or RU486
alone caused at most 2-fold induction of hIRS-2 mRNA both at 4- and
24-h time points. Taken together, our results show that progesterone
induction of hIRS-2 mRNA is a rapid and direct transcriptional
response, while induction by cAMP/RU486 occurs through a relatively
slow mechanism that requires ongoing protein synthesis.
Differential Regulation of IRS-1 and IRS-2 by Steroids
To verify the induction of hIRS-2 by R5020 or cAMP/RU486 and their
differential kinetics at the protein level, we performed Western blot
analysis on whole cell extracts prepared from HeLa3B2 cells treated for
248 h. As shown in Fig. 4
, A and B, by
immunoblotting with an
hIRS-2 antibody, R5020 treatment resulted in
a detectable induction of IRS-2 after as early as 4 h. Longer
treatment led up to a 17-fold enhancement of IRS-2 expression at the
48-h time point. In agreement with the RNase protection data (Fig. 3
),
8-Br-cAMP or RU486 alone had only minor effects on IRS-2 levels, while
combined treatment with 8-Br-cAMP and RU486 resulted in about 8-fold
induction after 48 h. Thus, both progesterone and cAMP/RU486
induction of IRS-2 are also clearly detectable at the protein level.
Again, consistent with the RNA data, induction of hIRS-2 by cAMP/RU486
occurred much more slowly than by R5020 (Fig. 4B
), supporting our
conclusion that regulation of IRS-2 by R5020 and cAMP/RU486 occurs
through different mechanisms. In the absence of cAMP, the induction of
IRS-2 by R5020 observed in HeLa3B2 cells could be antagonized by a
100-fold molar excess of RU486, confirming the involvement of PR (Fig. 5
). It should be noted that the level of
IRS-2 protein induced by cAMP/RU486 remains lower than the
R5020-induced IRS-2 level at all time points investigated (Fig. 4B
),
although cAMP/RU486-induced IRS-2 mRNA levels are at least as high as
the R5020-induced mRNA at the 20 and 24 h time points (Fig. 3
, A
and C). We assume that this apparent discrepancy is due to a presumably
long half-life time of the IRS-2 protein and the faster onset of the
R5020 induction, which would allow a greater accumulation of the IRS-2
protein in the presence of R5020.

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Figure 4. Time Course of hIRS-2 Induction by Steroids and
8-Br-cAMP
A, Western blot analysis. HeLa3B2 cells were treated with steroids as
indicated on the left (R5020, 10 nM; RU486,
10 nM) or 8-Br-cAMP (cAMP, 0.2 mM). Whole-cell
extracts were harvested at the indicated time points after addition of
hormones and analyzed by Western blot using antibody directed against
IRS-2. Similar results were obtained in three to four independent
experiments. B, Quantitative representation of the effects of steroids
and cAMP on hIRS-2 expression after hormone addition. ECL signals were
quantified using a CCD video camera (Raytest).
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Figure 5. Differential Regulation of IRS-1 and IRS-2 Proteins
by Progestin R5020
Whole-cell extracts (20 µg/lane) were isolated from HeLa3B2
(left panel) and HeLa cells (right
panel), which were incubated with the indicated hormones (cAMP,
0.2 mM; RU486, 10 nM; R5020, 10 nM;
R5020/100xRU; 10 nM R5020 plus 1 µM RU486;
Dex, 10 nM) for 10 h and analyzed by Western blot
using antibodies directed against IRS-2 or IRS-1. The positions of
IRS-2 and IRS-1 are indicated on the left. Similar
results were obtained in two independent experiments.
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Next we compared the expression of IRS-2 and IRS-1 in HeLa3B2 and HeLa
cells, which were treated with cAMP, R5020, RU486, and dexamethasone
for 10 h (Fig. 5
). Confirming the result shown in Fig. 4
, expression of IRS-2 was clearly induced by progestin R5020 in HeLa3B2
cells, whereas no effect was seen in PR-negative HeLa cells.
Dexamethasone treatment for 10 h did not result in a comparable
induction of IRS-2 either in HeLa 3B2 or in HeLa cells, although both
cell lines contain functional GR. Similarly, cAMP/dexamethasone
cotreatment for 10 h had very little or no effect on the level of
IRS-2 protein in HeLa3B2 and HeLa cells (Fig. 5
and data not shown),
whereas about 3-fold induction of IRS-2 mRNA levels was observed in a
20-h experiment (Fig. 3B
). This result suggests that induction of IRS-2
mRNA levels by cAMP/dexamethasone treatment follows a slow kinetic,
which is similar to the kinetic of the cAMP/RU486 induction. Expression
of the IRS-1 gene, which is homologous to IRS-2, was completely
unaffected by treatment with cAMP, RU486, dexamethasone, and
cotreatment with cAMP/dexamethasone or cAMP/RU486 both in HeLa3B2 and
HeLa cells (Fig. 5
and data not shown). In striking contrast to IRS-2,
IRS-1 could not be induced by progestin treatment in the
PR-positive cell line (Fig. 5
). Together, these results demonstrate
that progesterone, through a PR-mediated mechanism, specifically
increases IRS-2 expression without altering IRS-1 levels, resulting in
an increased ratio of IRS-2 to IRS-1 signaling proteins.
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DISCUSSION
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Induction of hIRS-2 by cAMP/RU486 Represents a Delayed PR-Mediated
Response
Using a differential display technique and cDNA cloning, we found
that the endogenous IRS-2 gene is synergistically induced by elevated
cAMP and the antiprogestin RU486 in a HeLa-derived cell line that
expresses human PRB. Consistent with a previous study in
which we analyzed well defined transfected reporter genes (9), we show
here that cAMP/RU486 induction of IRS-2 is prevented through partial
inhibition of protein synthesis (Fig. 3A
), suggesting that intermediate
synthesis of a yet unknown cofactor is required. Moreover, we showed,
both at the mRNA and protein level, that cAMP/RU486 induction of hIRS-2
occurs much slower than progesterone induction (Figs. 3C
and 4
),
underlining our previous conclusion that induction of
progesterone-responsive genes by cAMP/RU486 is not a direct
transcriptional response mediated by PR (9). Whether
cAMP/RU486-mediated induction of IRS-2 could represent a step of a
mechanism through which antihormone-resistant tumor cell populations
might be selected during therapy remains to be elucidated.
IRS-2 Is a Primary PR Target Gene but Responds Poorly to
Glucocorticoid
Like the well defined cAMP/RU486-responsive reporter genes used in
previous transfection studies (8, 9, 10, 11, 12), the endogenous IRS-2 gene proved
to be strongly inducible by progesterone in the PR-expressing HeLa3B2
cell line. We showed that progesterone induction of hIRS-2 is dependent
on the presence of PR, is largely prevented by excess of RU486 (in the
absence of cAMP), is detectable as early as after 4 h, and is
insensitive to cycloheximide (
Figs. 35

). Together, these findings
clearly suggest that progesterone induction of hIRS-2 occurs at the
transcriptional level through a direct PR-mediated mechanism that most
likely involves the binding of PR to one or more GRE/PREs located in
the promoter or enhancer(s) of the hIRS-2 gene. In contrast to IRS-2,
the endogenous IRS-1 gene proved to be unresponsive to progesterone in
our experimental system, suggesting that the regulatory element(s)
conferring inducibility have not been conserved throughout evolution of
the IRS gene family.
Surprisingly, although HeLa cell lines contain functional GR that can
bind to consensus GRE/PREs, treatment with dexamethasone alone did not
result in a rapid induction of hIRS-2 mRNA and protein (Figs. 3B
and 5
). Thus, it seems possible that the hIRS-2 gene contains a special PRE
that is not recognized by GR. However, this possibility seems unlikely,
since both GR-mediated dexamethasone and RU486 effects were detectable
upon cotreatment with cAMP for 20 h (Fig. 3B
). Alternatively, in
the absence of the potentiating action of cAMP, the level of GR present
in our cell lines may be insufficient to ensure sufficient occupancy of
the responsible elements of the IRS-2 gene. Clearly, identification of
the elements that confer progesterone regulation to the IRS-2 gene in
HeLa3B2 cells and an analysis of their interactions with PR and GR are
required to gain further insight into the regulatory mechanism
responsible for the differential inducibility of IRS-2 in response to
progesterone and glucocorticoid treatment.
Possible Implications
IRS-1 knockout mice display a mild form of insulin resistance of
peripheral tissues that can be overcome by increased insulin secretion,
while IRS-2 knockout mice exhibit all characteristics of a type II
diabetes disorder, including reduced ß-cell mass that prevents
compensation through increased insulin secretion (24, 25, 26). Moreover,
analysis of IRS-dependent signaling pathways in cells derived from
IRS-1 knockout mice showed that IRS-2 overexpression cannot completely
restore the impaired effect of IGF-I on cell cycle progression and that
IRS-2 may not be necessary for activation of ERK1 and ERK2 (27).
Together, these results suggest that IRS-1 and IRS-2, despite their
many functional and structural similarities, are not completely
interchangable. The molecular basis for the different signaling
capabilities of IRS-1 and IRS-2, which seem to be coexpressed in many
cell types (17, 28) and bind both to activated insulin and IGF-I
receptors (19, 29), is not fully understood, but these differences
could be due in part to different affinities for downstream SH2 domain
interaction partners (17). We show here that progesterone treatment
results in a up-regulation of IRS-2, whereas there is no effect of
progesterone on IRS-1 expression (Fig. 5
). This specific effect of
progesterone on IRS-2 might affect the ability of cells to respond to
signals, e.g. insulin and IGF-I, which are transduced via
the various IRS proteins. We envision that the induction of IRS-2,
which would occur only in PR-positive cells, could have both positive
and negative effects on insulin and IGF-I signaling, depending on the
progesterone level, the precise equipment with the various IRS
proteins, and their relative affinities and concentrations. For
example, in cells expressing limiting amounts of IRS proteins, modest
induction of IRS-2 could simply enhance IRS-2-dependent signaling
pathways without interfering with IRS-1-dependent signaling. In
contrast, in cells expressing relatively high amounts of IRS-1,
progesterone- induced overexpression of IRS-2 might interfere with
IRS-1-specific signaling pathways, as an excess of IRS-2 might compete
with IRS-1 for binding to activated phosphotyrosine receptors and
downstream effector molecules. Further studies will be required to
confirm whether progesterone-mediated induction of IRS-2 elicits
secondary progesterone responses through IRS-2-dependent gene networks
and to determine the importance of this potential cross-talk mechanism
between progesterone and insulin, IGF-1, or cytokine pathways in the
regulation of growth, differentiation, reproduction, and
homeostasis.
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MATERIALS AND METHODS
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Cell Culture
The generation of HeLa3B2 cells stably expressing PR, cell
culture, and hormone treatment of HeLa and HeLa3B2 cells have been
described by Kahmann et al. (9).
Northern Analysis
Premade human multiple-tissue Northern blots (CLONTECH, Palo
Alto, CA) containing approximately 2 µg of polyA+ mRNA per lane were
hybridized with an hIRS-2 cDNA probe (nucleotides 54125873) labeled
with [
-32P]dCTP using the RediPrime system (Amersham,
Arlington Heights, IL) according to the manufacturers instructions.
The blots were washed at high stringency (0.1x saline sodium
citrate, 0.1% SDS at 64 C) and exposed to x-ray film.
SDS-PAGE and Western Blotting
Preparation of high-salt whole-cell extracts (0.5 M
NaCl) of HeLa and HeLa3B2 cells, separation by SDS-PAGE, and transfer
to nitrocellulose membranes were done as described by Kahmann et
al. (9). After blocking, blots were probed with rabbit
IRS-2 or
IRS-1 antibodies. Blots were then incubated with a horseradish
peroxidase-linked second antibody followed by chemiluminescence
detection using SuperSignal (Pierce, Rockford, IL) as substrate.
IRS-1 antibodies directed against the 14 C-terminal residues of rat
IRS-1 were purchased from Upstate Biotechnology, Inc. (Lake Placid,
NY).
IRS-2 antibodies were prepared in rabbits (Eurogentec,
Belgium) against a glutathione-S-transferase (GST) fusion
protein containing the 76 C-terminal amino acids of human IRS-2, and
specific antibodies were enriched by immunopurification on GST-IRS-2
cross-linked to tresyl-agarose. Signals were quantified by using a
charge-coupled device camera system (Raytest,
Straubenhardt, Germany).
DDRT-PCR
DDRT-PCR, gel electrophoresis on denaturing gels, and
reamplification of bands were performed essentially as described by
Bauer et al. (30). Reamplified cDNA probes were
phosphorylated with T4 polynucleotide kinase and ligated into
pBluescriptIISK+ (Stratagene, La Jolla, CA), which had been
restricted with EcoRV and dephosphorylated with calf
intestinal phosphatase. Plasmid sequencing of cloned fragments was done
using an automated laser fluorescence sequencer (Pharmacia,
Piscataway, NJ).
RNA Extraction and RNase Protection
Total RNA for DDRT-PCR was extracted from HeLa3B2 cells by using
the Optiprep1 RNA isolation kit (Biometra, Göttingen, Germany).
RNA for RNase protections was prepared using Trizol reagent (GIBCO-BRL,
Eggenstein, Germany). 32P-labeled hIRS-2 antisense probe
was synthesized with T7 RNA polymerase from a linearized pBluescript
plasmid containing nucleotides 6751- 6987 of hIRS-2 cDNA (see Fig. 1B
).
An antisense PR probe was prepared by T7 RNA polymerase from a
linearized pBluescript plasmid containing a 249-bp long cDNA fragment
corresponding to aa 556638 of human PR (isoform B), which was
generated by PCR. The
-actin probe was as described by Kahmann
et al. (9). Hybridization of cellular RNA and antisense
probes, RNase digestion, and denaturing gel electrophoresis were done
as described previously (9).
Isolation of cDNA Clones
A human fetal brain cDNA library in
gt10 from CLONTECH
(HL3003a) and two different
gt11 libraries prepared from R5020
induced (6 h) HeLa3B2 cells by oligo dT priming or by priming with an
hIRS-2 specific primer (Superscript
system for cDNA synthesis and
cloning, GIBCO-BRL) were used during multiple rounds of screening to
isolate an overlapping series of partial hIRS-2 cDNAs by plaque filter
screening with 32P-labeled cDNA probes. Phage inserts were
subcloned into pBluescriptIISK+ (Stratagene) or pZErO-1
(Invitrogen, San Diego, CA) and sequenced using T3, T7, and SP6 as well
as gene- specific primers and an automated sequencer
(Pharmacia).
Nucleotide Sequence Accession Numbers
The nucleotide sequence described here has been submitted to the
NCBI nucleotide sequence database under accession number AF073310.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to H. Gronemeyer (Strasbourg, France) and Ian
Kerr (London, U.K.) for providing plasmids, and Roussel-Uclaf
(France) for providing RU486. We thank G. U. Ryffel and S.
Kahmann for a critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Ludger Klein-Hitpass, Institut für Zellbiologie (Tumorforschung), Universitätsklinikum Essen, Virchowstrasse 173, D-45122 Essen, Germany. E-mail:
ludger.klein-hitpass{at}uni-essen.de
This work was supported by BMBF Grant 0310780 to L.K.-H. L.V. is a
postdoctoral fellow of the Schering Research Foundation (Berlin).
Received for publication September 11, 1998.
Revision received November 13, 1998.
Accepted for publication December 8, 1998.
 |
REFERENCES
|
---|
-
Kester HA, van der Leede BM, van der Saag PT, van den
Burg B 1997 Novel progesterone target genes identified by an improved
differential display technique suggest that progestin-induced growth
inhibition of breast cancer cells coincides with enhancement of
differentiation. J Biol Chem 272:1663716643[Abstract/Free Full Text]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kästner P, Mark M, Chambon P 1995 The
nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Strähle U, Klock G, Schütz G 1987 A DNA sequence
of 15 base pairs is sufficient to mediate both glucocorticoid and
progesterone induction of gene expression. Proc Natl Acad Sci USA 84:78717875[Abstract]
-
von der Ahe D, Janich S, Scheidereit C, Renkawitz R,
Schütz G, Beato M 1985 Glucocorticoid and progesterone receptors
bind to the same sites in two hormonally regulated promoters. Nature 313:706709[Medline]
-
Gaillard RC, Riondel A, Muller AF, Herrmann W, Baulieu EE 1984 RU486: a steroid with antiglucocorticosteroid activity that only
disinhibits the human pituitary-adrenal system at a specific time of
day. Proc Natl Acad Sci USA 81:38793882[Abstract]
-
Bakker GH, Setyono-Han B, Portengen H, De JF, Foekens JA,
Klijn JG 1990 Treatment of breast cancer with different antiprogestins:
preclinical and clinical studies. J Steroid Biochem Mol Biol 37:789794[CrossRef][Medline]
-
Klijn JG, De JF, Bakker GH, Lamberts SW, Rodenburg CJ,
Alexieva-Figusch J 1989 Antiprogestins, a new form of endocrine therapy
for human breast cancer. Cancer Res 49:28512856[Abstract]
-
Beck CA, Weigel NL, Moyer ML, Nordeen SK, Edwards DP 1993 The
progesterone antagonist RU486 acquires agonist activity upon
stimulation of cAMP signaling pathways. Proc Natl Acad Sci USA 90:44414445[Abstract]
-
Kahmann S, Vassen L, Klein-Hitpass L 1998 Synergistic
enhancement of PRB-mediated RU486 and R5020 agonist activities through
cyclic adenosine 3',5'-monophosphate represents a delayed primary
response. Mol Endocrinol 12:278289[Abstract/Free Full Text]
-
Nordeen SK, Bona BJ, Beck CA, Edwards DP, Borror KC, DeFranco
DB 1995 The two faces of a steroid antagonist: when an antagonist
isnt. Steroids 60:97104[CrossRef][Medline]
-
Sartorius CA, Tung L, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone receptors bound to DNA are
functionally switched to transcriptional agonists by cAMP. J Biol
Chem 268:92629266[Abstract/Free Full Text]
-
Tung L, Mohamed MK, Hoeffler JP, Takimoto GS, Horwitz KB 1993 Antagonist-occupied human progesterone B-receptors activate
transcription without binding to progesterone response elements and are
dominantly inhibited by A-receptors. Mol Endocrinol 7:12561265[Abstract]
-
Horwitz KB, Tung L, Takimoto GS 1995 Novel mechanisms of
antiprogestin action. J Steroid Biochem Mol Biol 53:917[CrossRef][Medline]
-
Waters SB, Pessin JE 1997 Insulin receptor substrate 1 and 2
IRS1 and IRS 2: what a tangled web we weave. Trends Cell Biol 6:14
-
White MF 1997 The insulin signalling system and the IRS
proteins. Diabetologia 40:S217
-
Yenush L, White MF 1997 The IRS-signalling system during
insulin and cytokine action. Bioessays 19:491500[Medline]
-
Sun XJ, Pons S, Wang LM, Zhang Y, Yenush L, Burks D, Myers Jr
MG, Glasheen E, Copeland NG, Jenkins NA, Pierce JH, White MF 1997 The
IRS-2 gene on murine chromosome 8 encodes a unique signaling adapter
for insulin and cytokine action. Mol Endocrinol 11:251262[Abstract/Free Full Text]
-
Sun XJ, Wang LM, Zhang Y, Yenush L, Myers Jr MG, Glasheen E,
Lane WS, Pierce JH, White MF 1995 Role of IRS-2 in insulin and cytokine
signalling. Nature 377:173177[CrossRef][Medline]
-
He W, Craparo A, Zhu Y, ONeill TJ, Wang LM, Pierce JH,
Gustafson TA 1996 Interaction of insulin receptor substrate-2 IRS-2
with the insulin and insulin-like growth factor I receptors. Evidence
for two distinct phosphotyrosine-dependent interaction domains within
IRS-2. J Biol Chem 271:1164111645[Abstract/Free Full Text]
-
Sun XJ, Rothenberg P, Kahn CR, Backer JM, Araki E, Wilden PA,
Cahill DA, Goldstein BJ, White MF 1991 Structure of the insulin
receptor substrate IRS-1 defines a unique signal transduction protein.
Nature 352:7377[CrossRef][Medline]
-
Lavan BE, Lane WS, Lienhard GE 1997 The 60-kDa phosphotyrosine
protein in insulin-treated adipocytes is a new member of the insulin
receptor substrate family. J Biol Chem 272:1143911443[Abstract/Free Full Text]
-
Lavan BE, Fantin VR, Chang ET, Lane WS, Keller SR, Lienhard GE 1997 A novel 160-kDa phosphotyrosine protein in insulin-treated
embryonic kidney cells is a new member of the insulin receptor
substrate family. J Biol Chem 272:2140321407[Abstract/Free Full Text]
-
Ogihara T, Isobe T, Ichimura T, Taoka M, Funaki M, Sakoda H,
Onishi Y, Inukai K, Anai M, Fukushima Y, Kikuchi M, Yazaki Y, Oka Y,
Asano T 1997 143-3 protein binds to insulin receptor substrate-1, one
of the binding sites of which is in the phosphotyrosine binding domain.
J Biol Chem 272:2526725274[Abstract/Free Full Text]
-
Araki E, Lipes MA, Patti ME, Brüning JC, Haag BLI II,
Johnson RS, Kahn CR 1994 Alternative pathway of insulin signalling in
mice with targeted disruption of the IRS-1 gene. Nature 372:186190[CrossRef][Medline]
-
Kadowaki T, Tamemoto H, Tobe K, Terauchi Y, Ueki K, Kaburagi
Y, Yamauchi T, Satoh S, Sekihara H, Aizawa S, Yazaki Y 1996 Insulin
resistance and growth retardation in mice lacking insulin receptor
substrate-1 and identification of insulin receptor substrate-2. Diabet
Med 13:1031088
-
Withers DJ, Gutierrez JS, Towery H, Burks DJ, Ren JM, Previs
S, Zhang Y, Bernal D, Pons S, Shulman GI, Bonner-Weir S, White MF 1998 Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391:900904[CrossRef][Medline]
-
Brüning JC, Winnay J, Cheatham B, Kahn CR 1997 Differential signaling by insulin receptor substrate 1 IRS-1 and IRS- 2
in IRS-1-deficient cells. Mol Cell Biol 17:15131521[Abstract]
-
Bernal D, Almind K, Yenush L, Ayoub M, Zhang Y, Rosshani L,
Larsson C, Pedersen O, White MF 1998 Insulin receptor substrate-2 amino
acid polymorphisms are not associated with random type 2 diabetes among
Caucasians. Diabetes 47:976979[Free Full Text]
-
Dey BR, Frick K, Lopaczynski W, Nissley SP, Furlanetto RW 1996 Evidence for the direct interaction of the insulin-like growth factor I
receptor with IRS-1, Shc, and Grb10. Mol Endocrinol 10:631641[Abstract]
-
Bauer D, Müller H, Reich J, Riedel H, Ahrenkiel V,
Warthoe P, Strauss M 1993 Identification of differentially expressed
mRNA species by an improved display technique DDRT-PCR. Nucleic Acids
Res 21:42724280[Abstract]
-
Araki E, Sun XJ, Haag BL, Chuang LM, Zhang Y, Yang-Feng TL,
White MF, Kahn CR 1993 Human skeletal muscle insulin receptor
substrate-1. Characterization of the cDNA, gene, and chromosomal
localization. Diabetes 42:10411054[Abstract]
-
Corpet F 1988 Multiple sequence alignment with hierarchical
clustering. Nucleic Acids Res 16:1088110890[Abstract]