Induction of 3ß-Hydroxysteroid Dehydrogenase/
5-
4 Isomerase Type 1 Gene Transcription in Human Breast Cancer Cell Lines and in Normal Mammary Epithelial Cells by Interleukin-4 and Interleukin-13
Sébastien Gingras,
Richard Moriggl,
Bernd Groner and
Jacques Simard
Medical Research Council Group in Molecular
Endocrinology (S.G., J.S.) Centre Hospitalier de
lUniversité Laval Research Center and Laval
University Quebec City, Quebec, G1V 4G2, Canada
Institute
for Experimental Cancer Research (R.M., B.G.) Tumor Biology
Center D-79106 Freiburg, Germany
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ABSTRACT
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Sex steroids play a crucial role in the
development and differentiation of normal mammary gland as well as in
the regulation of breast cancer growth. Local intracrine formation of
sex steroids from inactive precursors secreted by the adrenals, namely,
dehydroepiandrosterone and its sulfate, may regulate growth and
function of peripheral target tissues, including the breast. Both
endocrine and paracrine influences on the proliferation of human breast
cancer cells are well recognized. Breast tumors harbor tumor-associated
macrophages and tumor-infiltrating lymphocytes that secrete a wide
spectrum of cytokines. These factors may also contribute to neoplastic
cell activity. The present study was designed to investigate the action
of cytokines on 3ß-hydroxysteroid dehydrogenase (3ß-HSD) activity,
which is an essential step in the biosynthesis of active estrogens and
androgens in human breast cancer cell lines and in normal human mammary
epithelial cells in primary culture. 3ß-HSD activity was undetectable
in ZR-751 and T-47D estrogen receptor-positive
(ER)+ cells under basal growth conditions. This
activity was markedly induced after exposure to picomolar
concentrations of interleukin (IL)-4 or IL-13. The potent stimulatory
effect of these cytokines on 3ß-HSD activity was also observed in the
ER- MDA-MB-231 human breast cancer cell line
and in normal human mammary epithelial cells (HMECs) in primary
culture. The stimulation of 3ß-HSD activity by IL-4 and IL-13
results from a rapid increase in 3ß-HSD type 1 mRNA levels as
measured by RT-PCR and Northern blot analyses. Such an induction of the
3ß-HSD activity may modulate androgenic and estrogenic biological
responses as demonstrated using ZR-751 cells transfected with
androgen- or estrogen-sensitive reporter constructs and treated with
the adrenal steroid 5-androstene-3ß,17ß-diol. The DNA-binding
activity of Stat6, a member of the signal transducers and activators of
transcription gene family, is activated 30 min after exposure to IL-4
and IL-13 in human breast cancer cell lines as well as in HMECs in
primary culture. In these cells, Stat6 activated by IL-4 or IL-13 binds
to two regions of the 3ß-HSD type 1 gene promoter, containing Stat6
consensus sequences. IL-4 induction of 3ß-HSD mRNA and activity is
sensitive to staurosporine. This protein kinase inhibitor also inhibits
IL-4-induced Stat6 DNA-binding activity. Our data demonstrate for the
first time that IL-4 and IL-13 induce 3ß-HSD type 1 gene expression,
thus suggesting their involvement in the fine control of sex steroid
biosynthesis from adrenal steroid precursors in normal and tumoral
human mammary cells. Furthermore, aromatase and/or 5
-reductase(s)
are expressed in the mammary gland and in a large proportion of human
breast tumors. An increase in the formation of their substrates,
namely, 4-androstenedione and testosterone, may well have a significant
impact on the synthesis of active estrogens and androgens in these
tissues.
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INTRODUCTION
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Sex steroids play a predominant role in the regulation of cell
growth and differentiation of normal mammary gland as well as in breast
carcinomas. Estrogens stimulate cell growth of hormone-sensitive breast
cancer cells (1, 2), while androgens exert an antiproliferative effect
on breast cancer cells (2, 3, 4). Local intracrine formation of active
estrogens and androgens from adrenal steroid precursors secreted by the
adrenals, namely, dehydroepiandrosterone (DHEA) and its sulfate,
may regulate growth and function of peripheral target tissues,
including the breast (5, 6, 7, 8). Various types of human enzymes catalyzing
17ß-hydroxysteroid dehydrogenase (17ß-HSD), 3ß-hydroxysteroid
dehydrogenase/isomerase (3ß-HSD), 5
-reductase activities, and the
alternative promoter usage of the aromatase gene, because of their
tissue- and/or cell-specific expression and substrate specificity,
provide each cell with the necessary mechanisms to control the level of
intracellular active estrogens and androgens (6, 9, 10, 11).
The 3ß-HSD isoenzymes are responsible for the oxidation and
isomerization of 5-ene-3ß-hydroxysteroid precursors into
4-ene-ketosteroids. These 3ß-HSD isoenzymes catalyze an essential
step in the formation of all classes of active steroid hormones
(reviewed in Ref. 10). In humans, there are two 3ß-HSD isoenzymes,
which were chronologically designated type 1 and 2 (10). Type 1 gene is
the almost exclusive 3ß-HSD expressed in the placenta and peripheral
tissues, including the mammary gland and the skin, whereas the type 2
gene is the predominant 3ß-HSD expressed in the human adrenal gland,
ovary, and testis. Deficiency in type 2 3ß-HSD is responsible for a
form of congenital adrenal hyperplasia (10, 12, 13). In mammals,
3ß-HSD activity is also found in many other peripheral tissues
including adipose tissue, lung, endometrium, prostate, liver, kidney,
epididymis, and brain (10). Although 3ß-HSD gene expression has been
demonstrated in human mammary gland, several human breast cancer cell
lines failed to possess detectable 3ß-HSD activity. It thus appears
relevant to find factors that regulate 3ß-HSD gene expression in
breast cells, since DHEA levels in normal and neoplastic breast tissues
are much higher than in serum (14).
Both endocrine and paracrine influences on the proliferation of human
breast cancer cells are well recognized, thus supporting that breast
tumors can modulate their hormonal environment (15, 16). Considerable
numbers of tumor-associated macrophages and tumor-infiltrating
lymphocytes are present in breast tumors. They secrete a wide spectra
of cytokines that might play a key role in neoplastic cell activity
(17, 18). It has also been reported that natural killer (NK) cells
isolated from ductal invasive breast tumor secreted interferon-
(IFN-
) and IL-4 (19). The potential role of cytokines in breast
cancer cells is supported by the observation that interleukin-1
(IL-1
), IL-4, IL-6, and IL-13 inhibit the proliferation of ZR-751,
T-47D, and/or MCF-7 human breast cancer cells (20, 21, 22, 23, 24, 25, 26, 27). Cytokines can
also regulate the expression of several enzymes involved in formation
and inactivation of sex steroids in breast cells. For example, IL-6
regulates the expression of 17ß-HSD (15, 28, 29), the estrone
sulfatase (30), and p450 aromatase (31), whereas IL-4 also regulates
17ß-HSD activity (29).
These data prompted us to investigate the potential regulatory effect
of cytokines on 3ß-HSD expression in human breast cancer cells. The
present study demonstrates for the first time the potent and rapid
induction of the 3ß-HSD type 1 gene expression by both IL-4 and IL-13
in several estrogen receptor-positive (ER+) and
ER- human breast cancer cell lines as well as in normal
human mammary epithelial cells (HMECs) in primary culture.
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RESULTS
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IL-4 and IL-13 Induce 3ß-HSD Activity in Normal and Tumoral
Mammary Cells
The potential action of IL-4 and IL-13 in breast cancer cells is
based on the presence of high-affinity IL-4 receptors in human breast
carcinomas (32), their inhibitory effect on the estrogen-induced breast
cancer cell proliferation (22, 25, 27, 29), and their potent
stimulatory effect on GCDFP-15/PIP (gross cystic disease fluid
protein-15/PRL-inducible protein) gene expression (25) as well
as spermidine transport in ZR-751 cells (26). ZR-751 human breast
cancer cells were exposed to increasing concentrations of IL-4 or IL-13
for 6 days. There is no detectable 3ß-HSD activity in ZR-751 cells
under basal growth condition, as indicated by the absence of detectable
conversion of [3H]DHEA. However, incubation with IL-4
(Fig. 1A
) or IL-13 (Fig. 1B
) induced a
marked conversion of [3H]DHEA into
[3H]-4-androstenedione (4-DIONE), indicating an induction
of 3ß-HSD activity in these cells. This potent up-regulation of
3ß-HSD activity induced by IL-4 and IL-13 was observed at
EC50 values of 48 ± 3 pM and 10 ± 5
pM, respectively. IL-4 and IL-13 also stimulate 3ß-HSD
activity in T-47D, another ER+ breast cancer cell line
(data not showed). It should be noted that IL-1
(5 U/ml), IL-2 (5
U/ml), IL-3 (5 U/ml), IL-6 (100 U/ml), IL-8 (2 ng/ml), IL-10 (5 U/ml),
IFN-
(15 U/ml), and epidermal growth factor (EGF) (30 µg/ml) all
failed to induce 3ß-HSD activity in ZR-751 cells (data not
shown).

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Figure 1. Induction of 3ß-HSD Activity by IL-4 in ZR-751
Human Breast Cancer Cells
Cells were plated at a density of 20,000 cells per well; 3 days after
plating, ZR-751 cells were incubated for 6 days with the indicated
concentrations of IL-4 (A and C) or IL-13 (B and D). Media were changed
every 2 days. Thereafter, 3ß-HSD activity was assayed for 16 h
using 10 nM [3H]DHEA (A and B) or
[3H]-5-DIOL (C and D). Data are expressed as the
mean ± SEM of triplicate dishes. When SEM
overlaps with the symbol, only the symbol is shown.
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In untreated ZR-751 cells,
[3H]-5-androstene-3ß,17ß-diol (5-DIOL) was only
converted into [3H]DHEA (Fig. 1
, C and D), which is
most likely due to the endogenous expression of 17ß-HSD type 2 in
these cells (29). The induction of the 3ß-HSD activity by IL-4 and
IL-13 was responsible for the increased conversion of
[3H]-5-DIOL into the 4-ene-ketosteroids,
[3H]testosterone (TESTO) and
[3H]-4-DIONE.
To further characterize the predominant [3H]-5-DIOL
metabolic pathway(s) and thus measure the relative contribution of both
3ß-HSD and 17ß-HSD activities, we have performed a time course
experiment after a 6-day incubation in the presence or absence of 140
pM IL-4. In the absence of IL-4, [3H]-5-DIOL
is only converted into [3H]DHEA (Fig. 2
), which is consistent with the data
shown in Fig. 1
, C and D. On the other hand, in IL-4-treated ZR-751
cells, there was no [3H]DHEA formed, but rather a rapid
conversion into [3H]TESTO, which was thereafter converted
into [3H]-4-DIONE by the 17ß-HSD type 2 activity and
finally into [3H]androstanedione (A-DIONE) by the
endogenous 5
-reductase activity. This latter activity was also
detectable in ZR-751 cells in the absence of IL-4 (data not shown).
When using [3H]-4-DIONE as starting substrate, IL-4
failed to increase its conversion into [3H]-A-DIONE. This
indicate that the 5
-reductase activity is not regulated by IL-4 in
this cell line (data not shown).

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Figure 2. Time Course of the IL-4 Action on the Conversion of
5-DIOL in ZR-751 Human Breast Cancer Cells
Cells were plated at a density of 20,000 cells per well; 3 days after
plating, ZR-751 cells were incubated for 6 days in the presence
(right) or absence (left) of 140
pM (2 ng/ml) IL-4. Media were changed every 2 days.
Thereafter, [3H]-5-DIOL was added to the medium and
incubated for the indicated time intervals. Data are expressed as the
mean ± SEM of triplicate dishes. When SEM
overlaps with the symbol, only the symbol is shown.
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In the ER- MDA-MB-231 cells, IL-4 increased this enzymatic
activity by 3.3-fold as measured by the conversion of
[3H]-DHEA in both [3H]-4-DIONE and
[3H]-A-DIONE (Fig. 3A
),
indicating that the IL-4 action on 3ß-HSD activity was not restricted
to ER+ ZR-751 and T-47D (data not shown) human breast
cancer cells. Furthermore, the physiological relevance of our finding
was strengthened by the observation of a 5.5-fold increase in 3ß-HSD
activity in normal HMECs in primary culture after exposure to IL-4
(Fig. 3B
).

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Figure 3. Stimulatory Effect of IL-4 on 3ß-HSD Activity in
ER-Negative MDA-MB-231 Human Breast Cancer Cells and in Normal HMECs
MDA-MB-231 cells (A) were plated at a density of 10,000 cells per well;
HMECs (B) were plated at a density of 2,500 cells per well. Cells were
allowed to adhere for 72 h and then incubated for 48 h with
the indicated concentrations of IL-4. At the end of incubation period,
3ß-HSD activity was assayed for 16 h using
[3H]DHEA as substrate. Data are expressed as the
mean ± SEM of triplicate dishes.
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IL-4-Induced Up-Regulation of 3ß-HSD Expression May Modulate
Estrogenic and Androgenic Responses of 5-DIOL
The C19 adrenal steroid 5-DIOL has the structure
of androgens and binds the androgen receptor, but it is also well
recognized to possess an intrinsic potent estrogenic activity due to
its relatively high affinity for ERs (2, 4, 5, 33). Transient
transfections of androgen- or estrogen-sensitive reporter constructs
were used to measure androgenic and estrogenic responses of 5-DIOL in
the presence or absence of IL-4 (500 pM). As illustrated in
Fig. 4A
, 5
-DIOL (10 nM)
increased by 12-fold the luciferase activity from the
androgen-sensitive 4XARE-luciferase reporter construct. Pretreatment of
ZR-751 cells with increasing concentrations of IL-4 further enhances
the luciferase activity up to 30-fold. This effect could be explained
by the conversion of 5-DIOL into TESTO. To investigate whether such an
effect would change the amount of 5-DIOL needed to stimulate this
androgenic responses, we have compared the effect of increasing
concentrations of 5-DIOL on cells transfected with 4XARE-luciferase
reporter construct and pretreated in the presence or absence of IL-4
(500 pM). As shown in Fig. 4B
the maximal androgenic
response in control cells was achieved with 10 nM 5-DIOL.
On the other hand, in cells pretreated with IL-4, the same activation
could be achieved with only 0.1 nM 5-DIOL. However, 5-DIOL
at this latter concentration failed to stimulate this parameter in
control cells.

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Figure 4. Effect of the IL-4 Stimulation of 3ß-HSD Activity
on the Transcriptional Activation of Androgen-Sensitive 4XARE-LUC and
Estrogen-Sensitive PS2-LUC Reporter Constructs
ZR-751 cells were plated at a density of 70,000 cells per well; the
day after plating, cells were transfected with 0.35 µg receptor
expression vector (AC, hAR; DF, hER ) and 0.65 µg of the
reporter construct (AC, 4XARE-LUC; DF, PS2-LUC) as well as 0.1 µg
of cytomegalovirus-ß-galactosidase and 4 µl of the lipofectin
reagent in OPTI-MEM media. Thereafter, media were replaced by control
RPMI media or media containing indicated concentrations of IL-4 for
24 h. Then the cells were incubated for 16 h with new media
containing the indicated concentrations of 5-DIOL, TESTO or
E2, in the presence or absence of the 3ß-HSD inhibitor,
epostane (1 µM), the pure antiandrogen OH-FLU (1
µM), or the pure antiestrogen EM-800 (10 nM).
Cells were then lysed and luciferase activity was measured. Data are
expressed as the mean ± SEM of triplicate dishes.
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Figure 5. Induction of 3ß-HSD Type 1 Gene Expression by
IL-4 and IL-13 in ZR-751 Human Breast Cancer Cells
ZR-751 cells were plated in T-75 flasks and allowed to adhere for
72 h. Thereafter, cells were incubated for 6 days with fresh
medium (medium changed every 2 days) containing either no
interleukin or 140 pM IL-4 or 150 pM IL-13.
RT-PCR was performed as described in Materials and
Methods. RT-PCR amplification of 3ß-HSD transcripts were done
with total RNA from untreated cells (lane 1) or from IL-4-or
IL-13-treated cells (lane 2 and 3, respectively). RT-PCR
products from IL-4 (lane 4)- or IL-13 (lane 5)-treated cells and from
control amplification of 3ß-HSD type 1 cDNA (lane 6) and type 2 cDNA
(lane 7) were digested with Hpa1. Control
amplification of GAPDH was performed as control for each reverse
transcriptase reaction (lanes 710). The position of primers P1 and P2
used for RT-PCR amplification of 3ß-HSD transcripts is also
illustrated.
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To demonstrate that the increase of androgenic response induced
by IL-4 in cells incubated with 5-DIOL is primarily due to the
induction of 3ß-HSD expression, we next investigated whether this
IL-4 effect would be inhibited by the potent 3ß-HSD inhibitor
epostane (Fig. 4C
). The complete blockade of the stimulatory effect of
IL-4 on this parameter in cells incubated with 5-DIOL, but not in those
incubated with TESTO, supports this hypothesis. The specificity of this
androgenic response is confirmed by the observation that the pure
antiandrogen OH-Flutamide (OH-FLU), but not the pure antiestrogen
EM-800 (34), was able to block the stimulatory effects of both 5-DIOL
and TESTO.
Since 5-DIOL also has estrogenic activity (2, 5, 33), the effect of
IL-4 on the estrogenic responses of 5-DIOL was also tested. As
illustrated in Fig. 4D
, 5
-DIOL increased by 3-fold the luciferase
activity from the estrogen-sensitive PS2-luciferase reporter construct,
while pretreatment of ZR-751 cells with increasing concentrations of
IL-4 reduced this stimulation by 75%. The effect of increasing
concentration of 5-DIOL in the cells pretreated in the presence or
absence of IL-4 (500 pM) was then studied to determine
whether such pretreatment would decrease the estrogenic potency of
5-DIOL. As shown in Fig. 4E
, an estrogenic response was observed at 1
nM, and the maximal estrogenic response was achieved with
10 nM of 5-DIOL in control cells. Whereas in cells
pretreated with 500 pM of IL-4, no stimulation of this
parameter was observed with 1 nM, and even at 10
nM 5-DIOL the maximal stimulation was not attained. Thus,
the concentration of 5-DIOL needed to achieve the same estrogenic
response in IL-4-treated cells is approximately 10-fold higher than in
control cells. Finally, to investigate whether the IL-4-induced
decrease in the estrogenic response of 5-DIOL resulted from an
induction of 3ß-HSD expression, we have also studied the effect of
epostane on this inhibitory effect of IL-4. Epostane was able to block
this effect of IL-4 in 5-DIOL-treated cells but exerted no significant
effect in E2-treated cells (Fig. 4F
). As expected, OH-FLU
had no effect on this parameter, whereas the pure antiestrogen EM-800
completely blocked the estrogenic action of 5-DIOL and E2.
It should also be mentioned that the small, but significant,
IL-4-induced decrease of the PS2-luciferase reporter activity in
E2-treated cells is likely due to the stimulatory effect of
IL-4 on oxidative 17ß-HSD in ZR-751 cells as recently described
(29). These findings are also consistent with the IL-4-induced
conversion of 5-DIOL into TESTO resulting from an induction of 3ß-HSD
activity.
IL-4 and IL-13 Induce 3ß-HSD Type 1 Transcripts Selectively
To determine whether IL-4 and IL-13 cause the induction of type 1
and/or type 2 3ß-HSD gene expression, we have investigated the effect
of these cytokines on 3ß-HSD transcripts by the analysis of the
RT-PCR products amplified from total RNA isolated from ZR-751 cells
incubated for 6 days in the presence or absence of IL-4 or IL-13.
Because the primers selected for this analysis do not discriminate
between type 1 and type 2 3ß-HSD transcripts, the RT-PCR products
were then digested with HpaI, which selectively cut type 1
3ß-HSD, since this restriction site is absent in the type 2 3ß-HSD
products. Moreover, these primers spanned three introns of the 3ß-HSD
genes, thus excluding the possibility that the RT-PCR products arose
from contaminating genomic DNA. As illustrated in Fig. 5
(lane 1), 3ß-HSD transcripts are not
detectable in control ZR-751 cells, although the internal control
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was
amplified, as revealed by the presence of the expected 545-bp band
(Fig. 5
, lanes 8). The 3ß-HSD transcripts were detected after RT-PCR
when total RNA isolated from cells incubated with IL-4 (lane 2) or
IL-13 (lane 3) was used. Digestion of the RT-PCR products generated
from cytokine-treated cells produced two fragments of 652 bp and 128 bp
(Fig. 5
, lanes 4 and 5), which have the same size as the fragment from
the digestion of PCR amplification product amplified from a control
plasmid containing the 3ß-HSD type 1 cDNA insert (Fig. 5
, lane 6).
The PCR product amplified using a plasmid containing the 3ß-HSD type
2 cDNA was not digested by HpaI (Fig. 5
, lane 7). This
experiment indicates that 3ß-HSD type 1 transcript levels are induced
in ZR-751 after IL-4 or IL-13 treatment. This observation was
confirmed by sequencing the products obtained after a 5'-RACE
experiment (data not shown).
IL-4 Induces 3ß-HSD Type 1 Gene Expression
We characterized the kinetics of IL-4 induction of 3ß-HSD
activity and mRNA levels. Significant 3ß-HSD activity is already
detectable after a short incubation period of 6 h with IL-4 and it
continues to increase for at least 48 h (Fig. 6A
), thus showing a very rapid induction
of 3ß-HSD activity by IL-4. To further characterize the time course
of IL-4 action on 3ß-HSD expression, we measured the 3ß-HSD mRNA
levels after short-term exposure. 3ß-HSD type 1 transcripts were
undetectable in untreated ZR-751 cells, which is consistent with the
absence of 3ß-HSD activity in these cells. IL-4 induces 3ß-HSD gene
expression as early as 2 h after exposure (Fig. 6B
, upper
panel). GAPDH mRNA levels were unchanged by IL-4 treatment (Fig. 6B
, lower panel). The IL-4 induction of 3ß-HSD mRNA levels
was completely abolished by actinomycin-D, whereas cycloheximide did
not block its effect. These findings suggest that mRNA synthesis is
needed for the increase of 3ß-HSD mRNA levels induced by IL-4, and
that this takes place without the need of ongoing protein
synthesis.

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Figure 6. Rapid Induction of 3ß-HSD Activity and 3ß-HSD
Type 1 Transcripts by IL-4 in ZR-751 Human Breast Cancer Cells
A, Induction of 3ß-HSD activity. Cells were plated at a density of
40,000 cells per well; 3 days after plating (time zero), ZR-751 cells
were incubated for the indicated time periods with or without 140
pM IL-4. During the last 2 h of each incubation
period, medium was replaced with fresh medium containing
[3H]DHEA in the presence or absence of IL-4. Data are
expressed as the mean ± SEM of triplicate dishes. B,
Increase of 3ß-HSD type 1 mRNA levels. Cells were treated with 140
pM IL-4 for the indicated time periods. Northern analysis
was performed as described in Materials and Methods.
Membrane probed with 3ß-HSD type 1 cDNA was exposed for 5 days, and
then stripped and reprobed with GAPDH followed by an overnight exposure
period. C, Inhibition of the IL-4-induced 3ß-HSD type 1 mRNA by
actinomycin-D but not by cycloheximide. Cells were treated for 2 h
with 140 pM IL-4 in the presence or absence of
actimonycin-D (4 µg/ml) or cycloheximide (10 µg/ml).
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Stat6 Binds to Consensus Sequences in the 3ß-HSD Type 1
Promoter
The action of several cytokines is mediated through the Stat
pathway (signal transducers and activators of transcription) (reviewed
in Refs. 35, 36). IL-4 and IL-13 mediate their biological effects by
activating Stat6 (37, 38, 39, 40). Recent studies performed with
Stat6-deficient mice have demonstrated that Stat6 plays an essential
role in IL-4 and IL-13 signaling (41, 42, 43, 44, 45). Computer analysis of the
sequences upstream of the 3ß-HSD type 1 gene revealed the presence of
two sequences matching the Stat6 consensus sequence (TTCNNNNGAA). These
two putative Stat6 sequence 5'-TTCCACTGAA-3' and 5'-TTCCTGGGAA-3' are
located at position -847 to -838 and -156 to -137,
respectively.
To investigate whether Stat6 was able to bind to these sites, two
probes containing these consensus sequences, designated 3ß-HSD type 1
Stat6#1, for the distal site and 3ß-HSD type 1 Stat6#2 for the
proximal site, were synthesized. Figure 7
shows that in extracts from IL-4-treated HeLa cells transfected with
both human Stat6 and human IL-4 receptor
-chain expression vectors,
activated-Stat6 binds to the I
probe, a well recognized
Stat6-binding sequence found in the IgE promoter. Incubation of the
complex with an anti-Stat6 antibody caused a supershift and confirmed
the specificity of the binding (Fig. 7
, lane 3). Electrophoretic
mobility shift assay (EMSA) performed with those cell extracts produced
complexes on the 3ß-HSD type 1 Stat6#1 and Stat6#2 probes (Fig. 7
, lanes 5 and 11). These complexes have the same mobility as the one
formed by activated Stat6 on the I
probe. These complexes were also
completely supershifted by the anti-Stat6 antibody (Fig. 7
, lanes 6 and
12). No such complexes were formed on the mutant Stat6#1 and mutant
Stat6#2 probes (Fig. 7
, lanes 79 and 1315). These mutant probes
contained two single-base pair substitutions in the Stat6 consensus
sequence as described in Material and Methods. Similar
substitutions have already been showed to disrupt Stat6-DNA binding
(46).

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Figure 7. Recombinant Stat6 Binds to Consensus Stat6 Sequence
Sites in the 3ß-HSD Type 1 Gene Promoter
Analysis of Stat6 activation using an EMSA was performed as described
in Materials and Methods. HeLa cells were transfected
with hStat6 and hIL-4R cDNAs and were incubated in the absence
(lanes 1, 4, 7, 10, and 13) or presence of 10 ng/ml of IL-4 for 30 min
(lanes 2, 3, 5, 6, 8, 9, 11, 12, 14, and 15). Labeled probes were
derived from the IgE-promoter (I probe: lanes 13) or from the
3ß-HSD type 1 promoter Stat6#1 (lanes 46), Stat6#1 mutant (lanes
79), Stat6#2 (lanes 1012), and Stat6#2 mutant (lanes 1315).
Stat6-containing complexes were supershifted with anti-Stat6 antibody
(lanes 3, 6, 9, 12, and 15).
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To provide further evidence that the Stat6-binding to the 3ß-HSD type
1 Stat6#1 and Stat6#2 probe was specific, a competition assay was
performed with activated endogenous Stat6 from ZR-751 cells (Fig. 8
). The binding of Stat6 to the 3ß-HSD
type 1 Stat6#1 and Stat6#2 probes was competed by an excess of
nonradioactive I
oligonucleotide (Fig. 8
, lanes 34, 910,
respectively), but it was not affected by a 300-fold excess of an
unrelated (SP1) cold oligonucleotide (Fig. 8
, lanes 56, 1112,
respectively). Stat6 binding to the I
probe was competed by an
excess of nonradioactive 3ß-HSD type 1 Stat6#1 or Stat6#2
oligonucleotides (Fig. 8
, lanes 16 and 18), whereas a 300-fold excess
of cold SP1 oligonucleotide failed to displace the Stat6-binding to
I
probe. These findings support the hypothesis that the two
sequences, upstream of 3ß-HSD type 1 gene promoter, matching the
Stat6 consensus, are indeed competent Stat6-binding sites. On the other
hand, the faster migrating complex was competed by an excess of an
unrelated SP1 oligonucleotide (Fig. 8
, lanes 6, 12 and 20), suggesting
that the formation of this complex does not require a specific
sequence.

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Figure 8. Putative Stat6-Binding Sites from the 3ß-HSD Type
1 Gene Promoter Can Compete a High-Affinity Binding Site from the IgE
Constant Region Gene Promoter
Competition analysis of the binding of activated Stat6 to Stat6-binding
probes from 3ß-HSD type 1 gene promoter. Band shift experiments were
performed with whole-cell extracts from ZR-751 cells treated with
IL-4 (10 ng/ml/30 min) except in lanes 1, 7, and 13 EMSAs were
performed with untreated cellular extracts. Stat6 binding on labeled
probes was as follows. 3ß-HSD type 1 Stat6#1 (lanes 16), 3ß-HSD
type 1 Stat6#2 (lanes 712), and I (lanes 1320) were competed by
using the indicated excess of unlabeled oligonucleotides: 3ß-HSD type
1 Stat6#1 (lanes 15 and 16), 3ß-HSD type 1 Stat6#2 (lanes 17 and 18),
I (lanes 3, 4, 9 and 10), and SP1 (lanes 5, 6, 11, 12, 19 and 20).
|
|
IL-4 and IL-13 Induce Stat6 DNA-Binding Activity in Normal and
Tumoral Mammary Cells
Since Stat6 binds consensus sequences in the 3ß-HSD type 1
promoter, we determined whether Stat6 DNA-binding activity was induced
by IL-4 and IL-13 not only in ZR-751, but also in T-47D and
MDA-MB-231 cells as well as in HMECs. As illustrated in Fig. 9
, Stat6 DNA-binding activity was induced
by IL-4 and IL-13 in these cells as revealed by EMSA using the I
probe (upper panel), the 3ß-HSD type 1 Stat#1 probe
(middle panel) 3ß-HSD type 1 Stat#2 probe (lower
panel). The addition of anti-Stat6 antibody to the binding
reaction completely abrogated the formation of the IL-4- and
IL-13-induced complexes, causing the formation of a slower migrating
complex and confirming that Stat6 is induced in those cells by
both IL-4 and IL-13.

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Figure 9. IL-4 Induction of Stat6 DNA-Binding in Normal and
Tumoral Mammary Cells
ZR-751 (lanes 15), T-47D (lanes 610), MDA-MB-231 (lanes 1115),
and HMEC (lanes 1620) cell lines were treated with 10 ng/ml IL-4
(lanes 2, 3, 7, 8, 12, 13, 17, and 18) or 10 ng/ml IL-13 (lanes 4, 5,
9, 10, 14, 15, 19, and 20) for 30 min. Stat6 DNA-binding activity was
assayed on labeled probes derived from the IgE promoter (I probe,
upper panel) or from the 3ß-HSD type 1 promoter
(Stat6#1, middle panel, and Stat6#2, bottom
panel). Stat6-containing complexes were supershifted with
anti-Stat6 antibody (lane 3, 5, 8, 10, 13, 15, 18, and 20).
|
|
Involvement of a Staurosporine-Sensitive Pathway in the IL-4 Action
on 3ß-HSD Type 1 Gene Expression and Stat6 DNA-Binding Activity
Tyrosine phosphorylation is known to be involved in
multiple-signal transduction events of transmembrane receptors and in
the activation of the Jak-Stat pathway. We took advantage of the
ability of staurosporine to inhibit tyrosine phosphorylation, to
further characterize the action of IL-4 stimulation on 3ß-HSD type 1
expression. Preincubation of ZR-751 cells with staurosporine for
1 h blocked the IL-4 induction of both the 3ß-HSD activity (Fig. 10A
) and the 3ß-HSD type 1
transcripts (Fig. 10
B) and concomitantly abolished the activation of
the DNA-binding activity of Stat6 by IL-4 (Fig. 10C
).

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|
Figure 10. Involvement of a Staurosporine-Sensitive Pathway
in the IL-4 Action on 3ß-HSD Type 1 Gene Expression and Stat6
DNA-Binding Activity
In all panels, ZR-751 cells were preincubated with 250 ng/ml (0.5
µM) staurosporine where indicated for 1 h before
induction with IL-4 (2 ng/ml, 140 pM) for the indicated
times. A, 3ß-HSD activity: after cells were stimulated with IL-4 for
6 h, 3ß-HSD activity was measured, for 6 h, in cell
homogenate equivalent to 50,000 cells, in the presence of 1
mM NAD+ using 10 nM [14C]DHEA. B,
Northern analysis: cells were treated with IL-4 for 2 h. Northern
analysis was performed as described above. Membrane probed with
3ß-HSD cDNA was exposed for 5 days, and then stripped and
reprobed with GAPDH followed with an overnight exposition for GAPDH. C,
Activation of Stat6: cells were treated with IL-4 for 30 min. Band
shift analysis was performed as described above using I -labeled
probes.
|
|
 |
DISCUSSION
|
---|
Our study demonstrates that IL-4 and IL-13 induce 3ß-HSD type 1
gene expression in ZR-751 cells. This is in agreement with our
previous demonstration that 3ß-HSD type 1 is the predominant type of
3ß-HSD transcript detectable in peripheral tissues such as the
mammary gland, whereas the 3ß-HSD type 2 is almost exclusively
expressed in the adrenals and gonads (10). The potent action of IL-4
was not restricted to ER+ human breast cancer cell lines,
but was also observed in ER- MDA-MB-431 as well as in
normal HMECs. This marked stimulatory effect is coupled with an
activation of Stat6, a member of the Stat (signal transducers and
activators of transcription) protein family. The present study shows
that IL-4 and IL-13 may modulate not only the human breast cancer cell
proliferation (22, 25, 27, 28), but also the bioavailability of sex
steroids in these cells by regulating their formation and/or
inactivation. In support of this notion, we have recently reported that
in ZR-751 cells, both IL-4 and IL-6 increased the oxidative 17ß-HSD
activity and decreased the reductive pathway, resulting most likely
from an increase of 17ß-HSD type 2 expression. Such a regulatory
effect favoring the inactivation of active sex steroids was observed
with both estrogenic and androgenic substrates (29). On the other hand,
in T-47D cells, IL-6 failed to regulate the 17ß-HSD activity, whereas
IL-4 caused a marked increase in the reductive estrogenic 17ß-HSD
activity, thus suggesting an increase of the 17ß-HSD type 1
expression (29). These cytokines may exert a finely tuned control on
these enzymatic activities and provide an additional mechanism by which
the intracellular levels of estrogens and androgens are regulated in
breast cancer cells.
Because aromatase is expressed in a large proportion of human breast
carcinomas (15, 47), the induction of 3ß-HSD activity by IL-4 and
IL-13 would markedly increase the formation of its substrates, 4-DIONE
and TESTO, and this may well have a significant impact on the estrogen
synthesis in breast tumors. However, it should be taken into
consideration that androgens are well recognized to exert an
antiproliferative action in breast cancer cells (2, 3, 4). Thus, the
IL-4-induced 3ß-HSD activity would increase the intracellular levels
of TESTO, which in return would inhibit breast cancer cell growth. In
support of this hypothesis, DHEA exerts an inhibitory effect on the
development of ZR-751 human breast cancer cell xenografts in
ovarariectomized nude mice (10). DHEA has also been shown to exert a
potent inhibitory effect on the development of
7,12-dimethylbenz[a]anthracene-induced mammary carcinomas in
the rat (48). Finally DHEA exerts an almost exclusive androgenic effect
in the rat mammary gland (49).
The data presented in Fig. 2
clearly show that in ZR-751 cells, the
induction of 3ß-HSD activity by IL-4 caused the transient conversion
of 5-DIOL into TESTO. Furthermore, IL-4-induced 3ß-HSD activity has a
greater impact on the metabolism of 5-DIOL when compared with the
17ß-HSD activity. This is suggested by the absence of transformation
into DHEA. The C19 adrenal steroid 5-DIOL has the structure
of an androgen and binds the androgen receptor, but it is also well
recognized to exert a potent estrogenic action by itself due to its
relatively high affinity for ERs (2, 4, 5, 33). Thus, as demonstrated
by the experiment using both estrogen- and androgen-sensitive reporter
constructs (Fig. 4
), the induction of the 3ß-HSD expression by IL-4
may modulate the balance between estrogenic and androgenic biological
responses when ZR-751 cells are incubated with 5-DIOL.
The induction of 3ß-HSD type 1 gene expression by IL-4 is also
relevant to the presence in solid breast tumors of considerable numbers
of infiltrating stromal/immune cells secreting cytokines (17, 18). The
potential activity of IL-4 in normal and tumoral breast tissues is
supported by the findings that NK cells isolated directly from breast
tumor site secrete more IL-4 than NK cells from peripheral blood of the
same patients (19). It is also of interest to note that IL-4 secretion
by NK cells derived from healthy donors was stimulated after exposure
to conditioned medium by MCF-7 cells (19). Early studies have reported
that the presence or absence of lymphocytes within tumors is related to
increased survival and represent host resistance (Ref. 18 and
references therein). This could simply be explained by immune responses
against the tumor, although our data suggest that the cytokines
secreted might in turn also regulate enzymatic activity of both normal
and neoplastic cells.
The redundant effects of IL-4 and IL-13 observed in the present study
are in agreement with the proposed pleiotropic action of these two
cytokines (50). The similar effects of IL-4 and IL-13 in breast cancer
cells are also consistent with the observation that IL-4 and IL-13
receptors share at least one subunit (51). One type of IL-4 receptor is
composed of two subunits, the IL-4 receptor
-chain (IL-4R
) and
the common
-chain from the IL-2 receptor (52). Neither of these two
proteins binds IL-13 (53), and this type is specific for IL-4. On the
other hand, two IL-13-binding proteins have been cloned, the IL-13
receptor
1-chain (54, 55) and
2-chain (56). Those two proteins
can heterodimerize with IL-4R
to form receptors for both IL-4
and IL-13 (57). The stimulatory effect of IL-4 and IL-13 on the
3ß-HSD expression was observed at concentrations lower than 100
pM. Because the dissociation constant (Kd) for
the binding of IL-4 to the IL-4R is about 2080 pM (22, 58), the effects of IL-4 and IL-13 are likely mediated through their
specific receptor. Furthermore, it has been reported that both IL-4 and
IL-13 inhibit IL-1ß-induced procoagulant activity of tissue factor in
human monocytes with half-maximal inhibitory effect (IC50)
of 30 and 98 pM, respectively (59).
During the past few years a general mechanism has been proposed to
explain the intracellular signaling pathway involved in the modulation
of transcription of target genes in response to extracellular
stimulation by various cytokines. These agents induce tyrosine
phosphorylation of latent cytoplasmic transcription factors, now
designated Stat (35, 36). IL-4 and IL-13 have been shown to induced
tyrosine phosphorylation of Stat6 (37, 38, 39, 40, 60, 61). Moreover,
experiments carried out with Stat6-deficient mice indicated that Stat6
plays an essential role in IL-4 and IL-13 signaling (41, 42, 43, 44, 45). However,
IL-4 and IL-13 may also induce the rapid phosphorylation of the 170-kDa
substrate termed 4PS (63). 4PS is related to the major insulin-induced
phosphorylated substrate, insulin receptor substrate 1 (IRS-1) (64),
and was thus designated IRS-2 (65). Phosphorylated IRS-2 associates
with phosphatidylinositol 3-kinase as well as with other
SH2-domain-containing proteins and mediates important signals for
proliferation. It was suggested that IL-4 stimulates bifurcating
signaling pathways in which the Stat-signaling pathway is involved in
differentiation or gene regulation whereas IRS-2 would be involved in
mitogenic signal (66, 67).
Since IL-4 and IL-13 exert an antiproliferative effect in breast cancer
cell lines (22, 25, 27, 29), it is more likely that Stat6 is the
transcription factor involved in the induction of 3ß-HSD type 1 gene
expression by these cytokines. This hypothesis is supported by the
following findings: 1) the Stat6 DNA-binding activity is induced by
IL-4 and IL-13 in all breast cancer cell lines tested and even in
normal mammary epithelial cells; 2) although one report describes the
induction of Stat6 by platelet-derived growth factor (PDGF) in NIH 3T3
fibroblasts (68), and another describes the induction of Stat6 by IL-3
in murine Da-3 cells expressing erythropoietin receptor (39),
IL-4 and IL-13 are the only cytokines known to induce gene
transcription through the induction of Stat6; 3) all other cytokines
tested that were reported to induce other Stats failed to induce
3ß-HSD activity in the ZR-751 cells; 4) Stat6 is the only Stat able
to bind inverted Stat-binding site TTC spaced by 4 bp (69) like the two
consensus sequences found in the promoter region of 3ß-HSD type 1
gene. These sites bind Stat6 and can compete the binding of Stat6 to a
well recognized Stat6-binding sequence I
. Moreover, Stat6 binds as
efficiently to the 3ß-HSD type 1 STAT6#1 and STAT6#2 probes as to
I
probe; 5) The 3ß-HSD type 1 gene expression is rapidly activated
by a latent transcription factor, and this parameter, as well as
the activation of Stat6, is sensitive to the inhibitor of tyrosine
phosphorylation, staurosporine.
However, after extensive efforts using several alternative experimental
procedures, we failed to observe transactivation of luciferase reporter
gene constructs under the control of several DNA fragments
corresponding to different 5'- and 3'-deletions of the upstream
promoter region (-1082 to +182) of the 3ß-HSD type 1 gene. Thus, the
possibility that another pathway is also involved in the IL-4 induction
of 3ß-HSD type 1 gene expression cannot be excluded. It has been
reported that a reporter construct carrying multiple copies of the IL-4
response element derived from the human Ig heavy-chain germ line
promoter required both C/EBP and Stat6 binding sites for IL-4-induced
gene expression (70). It was also shown that mutation in the NF-
B
site in the promoter of mouse Ig heavy-chain germ line
reduces IL-4
responsiveness (62). Furthermore, it was reported that the
glucocorticoid receptor can act as a transcriptional coactivator for
Stat3 and Stat5 (71, 72) and CBP/p300 interacts with Stat2 and Stat1 to
increase IFN-
/
-activated transactivation, respectively (73, 74, 75).
Thus, it might be possible that the upstream region (-1082 to +182) of
the 3ß-HSD type 1 gene used in our experiments is missing DNA
recognition sequence(s) for a cofactor that remains to be characterized
or it might be necessary to overexpress some additional transcriptional
factor(s) to clearly observe an IL-4 responsiveness (61). We are thus
unable, given the present state of our knowledge, to demonstrate
whether either or both of these consensus sequences are essential for
the induction by IL-4 of the 3ß-HSD type 1 gene transcription.
The present study shows, for the first time, that IL-4 and IL-13 can
regulate 3ß-HSD type 1 gene expression in several human breast cancer
lines as well as in normal HMECs. The present findings strongly support
the relevance of the action of IL-4 and IL-13 in the intracrine
biosynthesis of active sex steroids from adrenal precursors (DHEA and
5-DIOL) in both human normal and tumoral breast tissues.
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Transfection
All media and supplements for cell culture were obtained from
Sigma Chemical Co. (St. Louis, MO), except for FBS, which was purchased
from Hyclone (Logan, UT). The ZR-751, T-47D, and MDA-MB-231 human
breast cancer cells and HeLa human cervix cancer cells were obtained
from the American Type Culture Collection (Manassas, VA. Normal HMECs,
media, and supplements for their culture were purchased from Clonetics
Corp. (San Diego, CA). ZR-751 and T-47D cells were routinely grown in
phenol red-free RPMI-1640 medium supplemented with 1 nM
E2, 2 mM L-glutamine, 1
mM sodium pyruvate, 15 mM HEPES, 100 IU/ml
penicillin, 50 µg/ml streptomycin sulfate, 0.5 µg/ml bovine
insulin, and 10% FBS. MDA-MB-231 cells were cultured in MEM
supplemented with MEM nonessential amino acids, 100 IU/ml penicillin,
50 µg/ml streptomycin sulfate, and 5% FBS. HMECs were cultured in
phenol red-free defined MEBM (mammary epithelial cell basal medium)
supplemented with 26 mg/500 ml bovine pituitary extract, 5 µg/ml
insulin, 10 ng/ml recombinant human (h)EGF, 0.5 µg/ml hydrocortisone,
50 µg/ml gentamicin sulfate, and 50 µg/ml amphotericin-B. IL-4 was
kindly provided by Drs T. L. Nagabhushan and S. Narula
(Schering-Plough Research Institute, Kenilworth, NJ). IL-13, IFN-
,
and EGF were purchased from R&D Systems (Minneapolis, MN). IL-1
was purchased from Genzyme (Cambridge, MA). IL-6 was from Boehringer
Mannaheim (Laval, Quebec, Canada). IL-2, IL-3, IL-8, and IL-10 were
from Sigma Chemical.
ZR-75-1 cells were plated at 70,000 cells per well in 24-well plates.
The following day ZR-751 cells were transfected in OPTI-MEM (GIBCO
BRL, Burlington, Ontario, Canada) with the lipofectin reagent (GIBCO
BRL) according to the manufacturers instructions for 6 h. Cells
were transfected, with 0.35 µg of the indicated receptor and
0.65 µg of the reporter construct, 0.1 µg
cytomegalovirus-ß-galactosidase, and 4 µl of the lipofectin reagent
for each well. The PS2-LUC reporter and ER
expression vector were
described previously; the human AR (M. V. Govindan) and the
4XARE-LUC reporter construct containing four copies of an androgen
response element in front of a thimidine kinase minimal promoter was a
kind gift of E. Lesvesque and A. Bélanger (Laboratory of
Molecular Endocrinology, Quebec City) and will be described
elsewhere.
HeLa cells were cultured in MEM supplemented with nonessential amino
acids, 100 IU/ml penicillin, 50 µg/ml streptomycin sulfate, and 5%
FBS. Cells were plated at a density of 3,000,000 cells per 10-cm dish.
Cells were transfected for 3 h with the ExGen 500 reagent (MBI
Fermentas, Inc., Amherst, NY) with hStat6 and hIL-4R
cDNAs (76) (10
µg each) according to the manufacturers instructions. Two days
after transfection, cells were treated with or without 10 ng
IL-4/ml for 30 min.
Assay for 3ß-HSD Activity
The following radioactive steroids were purchased from Mandel
Scientific Company Ltd. (Guelph, Ontario, Canada):
[1,2,6,7-3H (N)]DHEA (83.2 Ci/mmol);
[1,2,3-3H (N)]androstenediol (5-DIOL) (52.2 Ci/mmol), and
[4-14C(N)]-DHEA (55.2 mCi/mmol).
In Intact Cells
ZR-751 and T-47D cells were seeded in 24-well plates in RPMI-1640
plus 5% dextran-coated charcoal-treated FBS and allowed to adhere for
72 h, while other cells were seeded in the same medium as they
were routinely cultured. Cells were then treated for the indicated time
intervals; thereafter, medium was replaced with 1 ml of medium
containing 10 nM of the indicated
[3H]-labeled steroids. After incubation, medium was
harvested and steroids were extracted by adding 5 vol of diethyl ether.
Organic fractions were analyzed by TLC. TLC plates of
[3H]-labeled substrates were analyzed using a Berthold
model 440E scanner, while TLC plates from [14C]DHEA were
analyzed by PhosphorImager imaging system (Molecular Dynamics, Inc.,
Sunnyvale, CA). EC50 (half-maximal stimulatory effect)
values were calculated using a weighted iterative nonlinear
least-squares regression. All results were expressed as means ±
SEM of triplicate dishes.
In Cell Homogenates ZR-751 cells were plated in six-well
plates at a density of 250,000 cells per well. After the indicated
treatment, cells were harvested by enzymatic digestion with 0.1%
pancreatin. Cell pellets were then resuspended in 3ß-HSD assay buffer
(50 mM NaH2PO4, 1 mM
EDTA, 20% glycerol, pH 7.4), submitted to three freeze/thaw cycles,
and kept frozen at -80 C. Cell homogenates equivalent to 50,000 cells
were used to measure 3ß-HSD activity for 6 h in the presence of
1 mM NAD+ using [14C]DHEA as substrate.
Northern Analysis and RT-PCR
After the indicated treatment, cells were harvested by enzymatic
digestion with 0.1% pancreatin. Cell pellets were resuspended in
Tri-Reagent (Molecular Research Center, Inc. Cincinnati, OH). RNA was
then extracted according to the manufacturers instructions. Total RNA
was solubilized in FORMAzol (Molecular Research Center, Inc.) and
stored at -80 C. For RT-PCR, 5 µg of total RNA were precipitated
with 4 vol of methanol and resuspended in sterile water. This RNA was
reverse transcribed using SuperScript II (GIBCO BRL), with 2.5 µg of
an oligonucleotide T12VN and 0.5 mM
deoxynucleoside triphosphate using the supplied buffer. Reaction
was carried out at 37 C for 1 h, and then purified with
Quiax II (Qiagen Inc., Santa Clarita, CA) and eluted in 100 µl of
sterile water. PCR reactions were carried out with 1/20 vol of
RT reaction product using the following primers: for
3ß-HSD; P1, 5'-TGGAGCTGCCTTGTGACAGGA-3'; P2,
5'-TATCATAGCTTTGGTGAGGCG-3'; and for GAPDH,
5'-ATTGACCTCAACTACATGGT-3', 5'-CTTGCCCACAGCC-TTGGCAG-3'.
For Northern blot analysis 20 µg or total RNA was loaded in each lane
and subjected to electrophoresis in 1.2% agarose gel containing 2%
formaldehyde in 1x 3-[N-morpholino]propanesulfonic acid
(MOPS) buffer. Gel was then transferred by capillarity to
GeneScreen Plus positively charged nylon membranes (Mandel Scientific
Co. Ltd), and RNA was immobilized by UV-cross-linking. Hybridization
with
-[32P]dCTP-labeled cDNA probe corresponding to
the whole human 3ß-HSD type 1 cDNA was performed in 50%
formamide-containing buffer at 42 C, according to the membrane
suppliers protocol. Washes were 2x saline sodium citrate
(SSC) at room temperature for 10 min (twice), once in 2x SSC, 1% SDS
at room temperature for 10 min, and once in 2x SSC, 1% SDS at 52 C
for 10 min. Control hybridization was performed using a human GAPDH
cDNA HindIII-XbaI fragment of 548 bp. For GAPDH,
washes were twice 2x SSC, 0.1% SDS at room temperature for 30 min,
and once in 0.1x SSC, 0.1% SDS at 50 C for 30 min. Membranes were
exposed to Hyperfilm-MP x-ray film at -80 C.
Electrophoretic Mobility Shift Assay
After treatment with IL-4 or IL-13 for 30 min, cells were washed
twice in cold PBS and then scraped off the plates in cold PBS.
Whole-cell extract were prepared by resuspension of pellet in the
following buffer: 20 mM HEPES, pH 7.9, 400 mM
NaCl, 1 mM EDTA, 20% glycerol, 1 mM
dithiothreitol, 0.2 mM phenylmethylsulfonyl fluoride, and
100 µM sodium orthovanadate. Lysates were made by three
freeze-thaw cycles, and cleared lysates were obtained by centrifugation
at 14,000 rpm at 4 C. Supernatants equivalent to 100,000 cells were
used for band shift assays as previously described (77). Briefly,
complex formation was carried in 20 µl of binding buffer (10
mM Tris-HCl, pH 7.5, 50 mM NaCl, 1
mM EDTA, 1 mM dithiothreitol, 5% glycerol,
0.1% Nonidet P-40, and 0.2 mM phenylmethylsulfonyl
fluoride) containing 1 mg/ml BSA, 0.1 µg of poly (dI-dC) and 100,000
cpm (
10 fmol) of labeled double-stranded oligonucleotide. Complex
were resolved in a 4% polyacrylamid gel in 0.25x
Tris-borate-EDTA as buffer. The following double-stranded DNA
probes were used: I
, 5'-GTCAACTTCCCAAGAACAGAA-3' from the human Ig
constant region E promoter; Stat6#1,
5'-CTGTCAAGTTCCACTGAACTGAACAC-3'; Stat6#2,
5'-TCTTCCTGTTCCTGGGAAGAATTAGA-3'; Stat6#1
mutant, 5'-CTGTCAAGTgCCACTGcACTGAACAC-3'; and
Stat6#2 mutant,
5'-TCTTCCTGTgCCTGGGcAGAATTAGA-3' from the
3ß-HSD type 1 genes. The mutant probes each contained two 1-bp
substitutions in the Stat6 consensus sequence, which has already been
shown to disrupt Stat6-DNA binding (46). Nucleotides critical for the
binding of Stat6 are underlined, and mutated nucleotides are
in lower case.
Stat6#1 and Stat6#2 were positioned from -855 to -830 and from -164
to -129 bp from the cap site, respectively. Probes were end-labeled
with
-[32P]dATP by T4 polynucleotide kinase (Amersham
Pharmacia Biotech, Inc., Arlington Heights, IL) to a specific
activity of approximately 10,000 cpm/fmol. Supershifting of Stat6
complex was achieved by concomitant addition to the binding reaction of
1 µg of a chicken antibody directed to amino acids 637847 of murine
Stat6 (76). Similarly, competition experiments were performed by
concomitantly adding the indicated amounts of cold oligonucleotides to
the binding reaction. We used 5'-ATATCTGCGGGGCGGGGCAGACACAG-3'
containing an SP1 site as an unrelated oligonucleotide.
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs T. L. Nagabhushan and S. Narula
(Schering-Plough Research Institute, Kenilworth, NJ) who kindly
provided us with IL-4.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jacques Simard, Laboratory of Hereditary Cancers, Centre Hospitalier de lUniversité Laval Research Center, 2705 Laurier Boulevard, Quebec, Quebec G1V 4G2, Canada. E-mail: Jacques.Simard{at}crchul.ulaval.ca
Financial support was provided by the Medical Research Council of
Canada (MRC Group in Molecular Endocrinology). S.G. holds a studentship
from MRC and J.S. is a MRC Scholar.
Received for publication July 6, 1998.
Revision received September 18, 1998.
Accepted for publication September 28, 1998.
 |
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