Multiple Signaling Pathways Mediate Interleukin-4-Induced 3ß-Hydroxysteroid Dehydrogenase/
5-
4 Isomerase Type 1 Gene Expression in Human Breast Cancer Cells
Sébastien Gingras,
Stéphanie Côté and
Jacques Simard
Medical Research Council Group in Molecular Endocrinology
CHUL Research Center and Laval University Quebec City, G1V
4G2, Canada
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ABSTRACT
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The 3ß-hydroxysteroid dehydrogenase/
5-
4
isomerase (3ß-HSD) isoenzymes catalyze an essential step in the
formation of all classes of active steroid hormones. We have recently
shown that 3ß-HSD type 1 gene expression is specifically induced by
interleukin (IL)-4 and IL-13 in breast human cancer cell lines and in
normal human mammary epithelial cells in primary culture. There is
evidence that IL-4 stimulates bifurcating signaling pathways in which
the signal transducer and activator of transcription-6
(Stat6)-signal pathway is involved in differentiation and gene
regulation, whereas insulin receptor substrate (IRS) proteins mediate
the mitogenic action of IL-4. In fact, we have shown that Stat6 was
activated by IL-4 in all cell lines studied where IL-4 induced 3ß-HSD
expression, but not in those that failed to respond to IL-4. The
present study was designed to investigate the potential contribution of
IRS proteins and their downstream targets to IL-4-induced 3ß-HSD type
1 gene expression. IL-4 rapidly induced IRS-1 and IRS-2 phosphorylation
in ZR-751 human breast cancer cell lines. Moreover, insulin-like
growth factor (IGF)-I and insulin, which are well known to cause IRS-1
and IRS-2 phosphorylation, increased the stimulatory effect of IL-4 on
3ß-HSD activity. IRS-1 and IRS-2 are adapter molecules that provide
docking sites for different SH2-domain-containing proteins such as the
phosphatidylinositol (PI) 3-kinase. In this light, the inhibition of
IL-4-induced 3ß-HSD expression by wortmannin and LY294002, two potent
PI 3-kinase inhibitors, indicates the probable involvement of the PI
3-kinase signaling molecules in this response to IL-4. Furthermore, it
has been suggested that the IRS proteins are part of the signaling
complexes that lead to activation of the mitogen-activated
protein (MAP) kinase by insulin; thus we investigated the potential
role of the MAP kinase (MAPK) cascade in the IL-4 action. In
ZR-75-1 cells, both the activation of MAPK by IL-4 and the IL-4-induced
3ß-HSD activity were completely blocked by PD98059, an inhibitor of
MAPK activation. Wortmannin also blocked MAPK activation by IL-4,
IGF-I, and insulin, suggesting that the MAPK cascade acts as a
downstream effector of PI 3-kinases. To further understand the
cross-talk between signaling pathways involved in IL-4 action, we
investigated the possible involvement of protein kinase C (PKC). The
potential role of PKC was suggested by the observation that the well
known PKC activator phorbol-12-myristate-13-acetate (PMA) potentiated
the IL-4-induced 3ß-HSD activity. Taken together, these findings
suggest the existence of a novel mechanism of gene regulation by IL-4.
This mechanism would involved the phosphorylation of IRS-1 and IRS-2,
which transduce the IL-4 signal through a PI 3-kinase- and
MAPK-dependent signaling pathway. The inability of IGF-I, insulin, and
PMA to stimulate 3ß-HSD expression by themselves in the absence of
IL-4 makes obvious the absolute requirement of an IL-4-specific
signaling molecule. Our findings thus suggest that the multiple
pathways downstream of IRS-1 and IRS-2 must act in cooperation with the
IL-4-specific transcription factor Stat6 to mediate the induction of
3ß-HSD type 1 gene expression in ZR-751 human breast cancer
cells.
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INTRODUCTION
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The 3ß-hydroxysteroid dehydrogenase/
5-
4 isomerase
(3ß-HSD) isoenzymes catalyze an essential step in the formation of
all classes of active steroid hormones. Two 3ß-HSD isoenzymes exist
in humans: the type 2 gene codes for the 3ß-HSD isoform that is
predominantly expressed in the adrenal gland, ovary, and testis, and
the type 1 gene accounts for the 3ß-HSD activity found in many
peripheral tissues (1). We have recently shown that interleukin (IL)-4
and IL-13 induce 3ß-HSD type 1 gene expression, at the transcription
level, in human ZR-751, T47-D, and MDA-MB-231 breast cancer cells, as
well as in normal human mammary epithelial cells in primary culture (2, 3).
IL-4 is produced by T cells, mast cells, and basophils. IL-4 has
biological effects on many immune cells, including B and T
lymphocytes, mast cells, and macrophages. The most specific effects of
IL-4 are differentiation of T cells to the Th2 phenotype, and in B
cells, immunoglobulin class switching to IgE (reviewed in Ref. 4).
However, the IL-4 receptor (IL-4R) is also expressed by certain
nonimmune cells, thus suggesting that IL-4 may regulate some functions
within those cells. Although it was found that IL-4 stimulates B cell
proliferation, an antiproliferative effect in breast cancer cells has
been documented (5, 6).
Studies of the signal transduction of the interleukin signals have
clarified the mechanism by which IL-4 functions. The signal transducer
and activator of transcription-6 (Stat6) is an IL-4-activated
transcription factor (7, 8, 9) that belongs to the STAT gene family
(reviewed in Ref. 10). Furthermore, recent experiments performed on
Stat6-deficient mice demonstrated that Stat6 plays an essential role in
IL-4 and IL-13 signaling (11, 12, 13). The cytoplasmic protein IRS-1
(insulin receptor substrate-1) was first identified as a major
substrate for the insulin receptor and insulin-like growth factor-I
(IGF-I) receptor, while IRS-2 [also designated 4PS (IL-4
phosphorylated substrate)] was first identified as a substrate for the
IL-4R (reviewed in Ref. 14). However, both proteins share extensive
structural and functional identities and are tyrosine phosphorylated in
response to IL-4, insulin, and IGF-I (15, 16). Moreover, it has been
demonstrated recently that activated IRS-1 and IRS-2 act as critical
mediators of IL-4 mitogenic signaling (16, 17, 18). After tyrosine
phosphorylation these intracellular proteins associate with SH2
domain-containing proteins, such as PI 3-kinase, Grb2, SHP2, fyn, and
nck, to generate downstream signals (14, 19, 20). Among those proteins,
the signal transduction pathways involving PI 3-kinase and Grb2 have
been studied the most extensively.
PI 3-kinase activity plays a central role in a broad range of
biological responses and has also been linked to wide variety of
stimuli (reviewed in Ref. 21). One group of PI 3-kinase isoforms is
activated by receptors with intrinsic or associated tyrosine kinase
activity. PI 3-kinase isoforms from that group are comprised of a
regulatory/adapter subunit (p85) and a catalytic subunit (p110). These
enzymes can use phosphatidylinositol (PtdIns), PtdIns 4-phosphate, and
PtdIns (4, 5)-bisphosphate as substrates to generate
PtdIns(3, 4)P2 and
PtdIns(3, 4, 5)P3. The lipid products of PI
3-kinase act as both membrane anchors and allosteric regulators, thus
serving to localize and activate downstream enzymes, such as PKB/AKT
and p70S6 kinase (22, 23) and different protein kinase C (PKC) isoforms
(24, 25, 26, 27, 28). It was suggested that Grb2 might link Sos to IRS-1 signaling
complexes as part of the mechanism by which insulin activates Ras, thus
leading to activation of the mitogen-activated protein kinase (MAPK)
cascade (20).
We have recently observed that Stat6 DNA-binding activity is activated
within 30 min after exposure to IL-4 in all the cell lines where IL-4
induces 3ß-HSD expression, but not in unresponsive cell lines. We
have also found Stat6 DNA-binding elements in the 3ß-HSD type 1 gene
promoter (2). However, despite extensive attempts, we did not succeed
in observing transactivation of a luciferase reporter gene under the
control of the 3ß-HSD type 1 gene promoter (2). Thus, to gain further
insight into the molecular mechanism of IL-4 action, the present study
was designed to investigate whether or not IRS-1 and IRS-2 and their
downstream targets contribute to the induction of 3ß-HSD type 1 gene
expression by IL-4. The present study reports, that in ZR-751 human
breast cancer cells, IL-4 induces the phosphorylation of IRS-1 and
IRS-2, thus mediating the IL-4 signal that causes the induction of
3ß-HSD activity through a PI3-kinase- and MAPK-dependent pathway.
This pathway should act in concert with the IL-4-specific transcription
factor, Stat6, to mediate the potent induction of the 3ß-HSD activity
by this cytokine.
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RESULTS
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IL-4 Induces IRS-1 and IRS-2 Phosphorylation
To determine whether IRS-1 and IRS-2 could be involved in the
induction of 3ß-HSD type 1 gene expression by IL-4, we first
investigated whether or not IL-4 could induce their tyrosine
phosphorylation in ZR-751 breast cancer cells. As shown in Fig. 1
, neither IRS-1 nor IRS-2 (first
and third panels, respectively) was tyrosine phosphorylated in
untreated cells as revealed by immunoprecipitation followed by Western
blotting with an antiphosphotyrosine antibody. However, incubation with
IL-4 caused rapid tyrosine phosphorylation of both IRS-1 and IRS-2,
which was readily detectable after a 15-min exposure. The effect of
IL-4 on this parameter reached a peak at 30 min and then declined
gradually. To evaluate the efficacy of the immunoprecipitation,
membranes were then stripped and reblotted with either an anti-IRS-1 or
an anti-IRS-2 antibody (second and fourth panel,
respectively).

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Figure 1. IL-4 Induces the Phosphorylation of IRS-1 and IRS-2
in ZR-751 Human Breast Cancer Cells
ZR-751 cells were incubated for the indicated time with IL-4 (10
ng/ml). Immunoprecipitation (IP) was carried out as described in
Materials and Methods with anti-IRS-1 or anti-IRS-2.
Immunoprecipitated proteins were separated on SDS-PAGE and
tyrosine-phosphorylated protein was analyzed by Western blotting (WB)
with the 4G10 antibody. Control blotting was performed by stripping the
membrane and reprobing with either anti-IRS-1 or anti-IRS-2. Detection
was performed by enhanced chemiluminescence.
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IGF-I and Insulin Potentiate the Induction of 3ß-HSD Activity by
IL-4
To further understand the role of IRS-1 and IRS-2 in IL-4-induced
3ß-HSD type 1 gene expression, we next studied the effect of insulin
and IGF-I alone or in combination with IL-4 on 3ß-HSD activity. It is
important to note that both IRS-1 and IRS-2 are also tyrosine
phosphorylated by insulin and IGF-I and that these growth factors have
been shown to induce PI 3-kinase in ZR-751, MCF-7, and T47-D breast
cancer cell lines (29). ZR-751 cells, therefore, were treated for
6 h with increasing concentrations of IGF-I (Fig. 2A
) or insulin (Fig. 2B
) in the presence
or absence of a suboptimal concentration of IL-4 (10 pM).
As illustrated in Fig. 2
, neither IGF-I nor insulin, even at high
concentrations, induced 3ß-HSD activity in the absence of IL-4. The
stimulatory effect of IGF-I and insulin on the IL-4-induced 3ß-HSD
activity was exerted at EC50 values of 0.8
± 0.4 nM and 3 ± 1 nM, respectively.

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Figure 2. IGF-I and Insulin Increase 3ß-HSD Activity Only
in the Presence of IL-4
ZR-751 cells were incubated for 6 h with increasing
concentrations of IGF-I (A) or insulin (B) in the presence or absence
of 10 pM IL-4. Thereafter, cell homogenates equivalent to
100,000 cells were used to measure 3ß-HSD activity for 6 h.
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IL-4-Induced 3ß-HSD Activity Is Blocked by PI 3-Kinase
Inhibitors
After tyrosine phosphorylation IRS-1 and IRS-2 are
recognized to interact with SH2 domain-containing proteins, such as PI
3-kinase, to generate downstream signals. To further address the role
of PI 3-kinase in the induction of 3ß-HSD type 1 expression by IL-4,
ZR-751 cells were incubated for 6 h with 100 pM IL-4
in the presence of increasing concentrations of wortmannin (Fig. 3A
) or LY294002 (Fig. 3B
). Wortmannin, a
well characterized selective inhibitor of PI 3-kinase, is able to
inhibit the following IL-4 effects: 1) induction of germline
gene
expression, 2) induction of the translocation of the PKC
isoform; 3)
prevention of apoptosis in the 32D cells and B cells, and 4) the
regulation of HMG-I(Y) phosphorylation (30, 31, 32). Specific inhibition of
PI 3-kinase occurs in the range of 1 and 100 nM, and at
higher concentrations, it also inhibits some isoforms of
PtdIns-4-kinase and phospholipase A2 (33, 34).
LY294002 is another, structurally unrelated, PI 3-kinase inhibitor,
with a half-maximal inhibitory concentration
(IC50) of 1.4 µM, but it does not
inhibit PtdIns 4-kinase, PKC, protein kinase A (PKA), p70S6 kinase, or
MAPK (35). As illustrated in Fig. 3
, both inhibitors completely blocked
the IL-4-induced 3ß-HSD activity, their half-maximal inhibitory
effect being exerted at IC50 values of 37 ±
6 nM for wortmannin and 1.5 ± 0.1 µM
for LY294002, which are within the range of their known specificity for
PI 3-kinase activity.

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Figure 3. PI 3-Kinase Inhibitors Block IL-4-Induced 3ß-HSD
Activity in ZR-751 Cells
ZR-751 cells were incubated for 6 h with increasing
concentrations of wortmannin or LY294002 in the presence of 100
pM IL-4. Cell homogenates equivalent to 100,000 cells were
used to measure 3ß-HSD activity for 6 h.
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Involvement of MAPK Pathway
Phosphorylated IRS-1 and IRS-2 can activate the MAPK pathway by
binding Grb2 and engaging the Ras/Raf pathway (20, 36). Although it was
reported that IL-4 failed to activate MAPK in lymphohematopoietic cells
(37, 38), MAPK was activated by IL-4 in human keratinocytes and in
breast cancer cell lines (29, 39). To investigate whether the latter
pathway was involved in the IL-4-induced 3ß-HSD activity, we used a
specific inhibitor of MAPK kinase (MEK) activation, PD98059. This
compound selectively prevents the activation of MEK by Raf in
vitro (IC50 27
µM), without inhibiting the activity of
phosphorylated MAPK or other serine/threonine protein kinases,
including Raf-1, PKA, and PKC (40). As shown in Fig. 4
, micromolar concentrations of PD 98059
were able to inhibit the induction of 3ß-HSD expression by IL-4.

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Figure 4. Inhibition of IL-4 Induced 3ß-HSD Activity by an
Inhibitor of MAPK Activation in ZR-751 Cells
ZR-751 cells were incubated for 6 h with increasing
concentrations of PD98058 in the presence of 100 pM IL-4.
Cell homogenates equivalent to 100,000 cells were used to measure
3ß-HSD activity for 6 h.
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To further explore the involvement of the MAPK pathway in
IL-4-induced 3ß-HSD type 1 gene expression, ZR-751 cells were
treated with IL-4, IGF-I, or insulin for 15 min, and MAPK activation
was determined by Western blotting using an antiphospho-MAPK (p42 and
p44)-specific monoclonal antibody (Fig. 5
, top panel). MAPK was found
to be phosphorylated in cells treated with IL-4, IGF-I, or insulin
(Fig. 5
, lanes 24) but not in untreated cells (Fig. 5
, lane 1).
Wortmannin was added to determine whether the activation of MAPK could
be linked to the activation of the PI 3-kinase pathway. This treatment
blocked the phosphorylation of MAPK induced by all three factors (Fig. 5
, lanes 68), suggesting that MAPK activation is a downstream
effector of PI 3-kinase, which is activated by all three factors. As
expected, PD98059 also completely blocked the activation of MAPK (Fig. 5
, lanes 1012). As a control, the membrane was then stripped and
reblotted with an anti-MAPK antibody (Fig. 5
, bottom panel),
which indicates that the levels of total MAPK were equal in all the
samples.

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Figure 5. Activation of MAPK by IL-4, IGF-I, and Insulin in
ZR-751 Cells
ZR-751 cells were stimulated for 15 min with 100 pM IL-4,
5 nM IGF-I, or 15 nM insulin in the presence or
absence of wortmannin (0.5 µM) or PD98059 (50
µM). MAPK activation was determined by the extent of MAPK
phosphorylation by Western blotting using an antiphospho-MAPK (p42 and
p44)-specific mouse monoclonal antibody (top panel).
Total MAPK protein content was determined by stripping and reprobing
the blot using an anti-MAPK (p42 and p44) rabbit immunoaffinity
purified IgG antibody (bottom panel). Proteins were
visualized using enhanced chemiluminescence.
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Stat6 Is Specifically Activated by IL-4, Independently of PI
3-Kinases and MAPKs
To confirm that Stat6 is specifically activated by IL-4 and not by
IGF-I or insulin, ZR-751 cells were incubated with 100 pM
IL-4, 5 nM IGF-I, or 15 nM insulin for 30 min.
As shown in Fig. 6
, Stat6 DNA-binding
activity was only induced by IL-4. To determine whether the inhibitory
effects of wortmannin and PD98059 on the IL-4-induced 3ß-HSD activity
were due to a modulation of the activation of Stat6, ZR-751 cells
were incubated with 100 pM IL-4 in the presence or absence
0.5 µM wortmannin or 50 µM PD98058 for 30
min. Neither wortmannin nor PD98059 blocked the induction of Stat6
DNA-binding activity by IL-4 (Fig. 6
, lanes 5 and 6).

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Figure 6. Stat6 Is Specifically Activated by IL-4,
Independently of the PI 3-Kinases and the MAPKs
ZR-751 cells were incubated with 100 pM IL-4, 5
nM IGF-I, or 15 nM insulin in the presence or
absence of 0.5 µM wortmannin or 50 µM
PD98059 for 30 min. EMSA was performed as described in Materials
and Methods.
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Phorbol-12-Myristate-13-Acetate (PMA) Potentiates the Induction of
3ß-HSD Activity by IL-4
To investigate the potential involvement of PKC activity in
IL-4-induced 3ß-HSD type 1 expression, ZR-751 cells were treated
6 h with increasing concentrations of IL-4 in the presence or
absence of 10 nM PMA, a well recognized activator of PKC
activity. As illustrated in Fig. 7A
, incubation with 10 nM PMA increased the maximal stimulatory
effect of IL-4, which led to almost complete conversion of
DHEA into 4-DIONE. It can also be seen in Fig. 7A
, that the stimulatory effect of IL-4 was exerted at an
EC50 value of 29 ± 1 pM and
7.5 ± 0.4 pM in the absence or presence of PMA,
respectively. To determine whether PMA could induce 3ß-HSD type 1
expression on its own, ZR-751 cells were incubated with increasing
concentrations of PMA (Fig. 7B
) in the presence or absence of a low
concentration of IL-4 (10 pM). It is interesting to note
that PMA, even at high concentrations, was unable to induce 3ß-HSD
activity in the absence of IL-4. Nevertheless, the marked potentiating
action of PMA on the IL-4-induced 3ß-HSD activity was obtained at an
EC50 of 0.5 ± 0.1 nM, which is
well within the previously reported range of concentrations needed to
activate PKC activity (41).

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Figure 7. PMA Increases IL-4-Induced 3ß-HSD Activity in
ZR-751 Cells
ZR-751 cells were incubated for 6 h with increasing
concentrations of IL-4 in the presence or absence of 10 nM
PMA (A), or with increasing concentrations of PMA in the presence or
absence of 10 pM IL-4 (B). Cell homogenates equivalent to
100,000 cells were used to measure 3ß-HSD activity for 6 h.
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To determine whether the potentiating action of PMA was due to
modulation of IL-4-induced activation of Stat6, ZR-751 cells were
incubated with increasing concentrations of IL-4 in the presence or
absence of 10 nM PMA for 30 min. As shown in Fig. 8
, PMA did not change the potency of IL-4
to induce Stat6 DNA-binding activity. Note that an overexposed film
(bottom panel) is provided to demonstrate that even at 1
pM IL-4, Stat6 DNA-binding activity was
detectable and the level of Stat6 activation was not changed by the
simultaneous exposure to PMA.

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Figure 8. PMA Does Not Modify the Potency of IL-4 to Induce
Stat6 DNA-Binding Activity
ZR-751 cells were incubated for 30 min with increasing concentrations
of IL-4 in the presence or absence of 10 nM PMA. EMSA was
performed as described in Materials and Methods. The gel
was exposed overnight (upper panel) or for 6 days
(lower panel).
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DISCUSSION
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The present study demonstrates for the first time that multiple
signal transduction pathways are involved in the induction of 3ß-HSD
type 1 expression by IL-4 in ZR-751 breast cancer cells. We have
previously shown that IL-4-induced expression of the 3ß-HSD type 1
gene required new mRNA synthesis but not new synthesis of proteins,
thus indicating this induction takes place at the transcriptional
level, by activating latent transcription factors (2). We have also
presented data showing that activated Stat6 binds to the two regions in
the 3ß-HSD type 1 gene promoter containing a Stat6 consensus sequence
in human breast cancer cell lines as well as in human mammary
epithelial cells in primary culture (2). The present study describes a
novel mechanism of gene regulation by IL-4. This mechanism includes
phosphorylation of IRS-1 and IRS-2, which transduces the IL-4 signal
via a PI 3-kinase- and MAPK-dependent signaling pathway. However, the
inability of IGF-I, insulin, and PMA to stimulate 3ß-HSD expression
in the absence of IL-4 indicates the absolute requirement of an
IL-4-specific signaling molecule, such as Stat6. Our original findings
thus suggests that the pathways downstream of IRS-1 and IRS-2 should
act in concert with the IL-4-specific transcription factor Stat6 to
mediate the induction of 3ß-HSD type 1 gene expression.
We first observed that treatment with IL-4 induced tyrosine
phosphorylation of both IRS-1 and IRS-2 in ZR-751 human breast cancer
cells. This observation was confirmed by another group during the
progress of our investigation (29). Whether the phosphorylation of
either IRS molecule is physiologically relevant in IL-4 signaling
remains to be established. Both proteins have highly conserved
structures and share many functions, although they are not functionally
interchangeable signaling intermediates for the stimulation of
mitogenesis (42). Moreover, IRS-1 plays a more important role in
mediating growth than metabolic responses because IRS-1-deficient mice
are growth retarded, but mildly insulin resistant (43, 44), while
IRS-2-deficient mice are diabetic as a result of combined insulin
resistance and impaired insulin production (45). Based on the
phenotypes of insulin receptor-, IGF-I receptor-, IRS-1-, and
IRS-2-deficient mice, it was suggested that IRS-1 is predominantly an
IGF-I receptor substrate, and IRS-2 is predominantly an insulin
substrate (46). However, investigators have not yet focused on the
status of IL-4 signaling in those mice.
We have also observed that both IGF-I and insulin are able to
potentiate the effect of IL-4 on 3ß-HSD activity. To our knowledge,
this is the first report of potentiation of an IL-4 response by IGF-I
and/or insulin. The recent observation that IGF-I and insulin activate
IRS-1 and IRS-2, PI 3-kinase, and MAPK in ZR-751 breast cancer cells
(29) further supports the potential involvement of IRS proteins and
their downstream targets in the IL-4 action on the regulation of the
3ß-HSD type 1 gene expression. The relevance of this original finding
also pertains to the observation that most invasive breast tumors
appear to express IGF-I receptor, and their growth is stimulated
in vitro in response to exogenous IGF-I (47). Furthermore,
it is of interest to note that the amplitude of the effect of insulin
or IGF-I on IL-4-induced 3ß-HSD activity was more striking in the
presence of submaximal concentrations of the cytokine. It is thus
tempting to speculate that the action of these growth factors could be
significant under physiological conditions when IL-4 is present at low
concentrations. Finally, because both IGF-I and insulin exerted their
stimulatory action on IL-4-induced 3ß-HSD activity at
EC50 values in the low nanomolar range, and
knowing that there is no significant binding of insulin to the IGF-I
receptor or of IGF-I to insulin receptor at concentrations less than 10
nM (48), our data suggest that both factors exert
their potentiating action through binding to their specific
receptors.
The IRS-1 and IRS-2 possess multiple phosphorylation sites containing
consensus sequences for the binding of SH2/SH3 adapter proteins (14).
In accordance with the activation of IRS-1 and IRS-2, we found that
some of their downstream targets (PI 3-kinases and MAPKs) were involved
in the regulation of 3ß-HSD expression. In this respect, our study
demonstrates that PI 3-kinase plays an important role in the IL-4
signaling pathway leading to the induction of 3ß-HSD expression,
based on the fact that two structurally unrelated inhibitors of PI
3-kinase blocked the induction at IC50 values in
the range known to exert a specific inhibition of PI 3-kinase. The
recruitment of PI 3-kinase by IL-4-phosphorylated IRS proteins as a
part of the mechanism responsible for the IL-4-induced 3ß-HSD type 1
expression is also supported by the observation that the IL-4-induced
PI 3-kinase activity and the IL-4-dependent binding of p85 to IRS-1 was
recently reported in ZR-751 cells (29). The present study also
demonstrates that the MEK inhibitor, PD98059, is able to inhibit
IL-4-induced 3ß-HSD activity. In support of the involvement of the
MAPK pathway in this induction, we have also shown that IL-4 induces
MAPK activity in ZR-751 breast cancer cells. This observation is in
agreement with the observation that PD98059 blocked the activation of
MAPK by IL-4, IGF-I, and insulin in ZR-751 cells. Although IL-4
failed to activate MAPK in lymphohemopoietic cells (37, 38), the
activation of MAPK in ZR-751 cells is in accordance with reports
showing that MAPK is activated by IL-4 in human keratinocytes (39) as
well as in breast cancer cell lines (29). We have also observed that
the activation of MAPK by IL-4, IGF-I, and insulin was blocked by
wortmannin, thus suggesting that MAPK was activated through a PI
3-kinase-dependent pathway. We have also found that wortmannin and
PD98059 failed to inhibit 3ß-HSD activity measured in cells
transfected with a 3ß-HSD type 1 expression vector (data not shown),
thus excluding the possibility that those compounds are able to
directly block the activity of the enzyme rather that its expression.
In accordance with this observation, wortmannin was found to inhibit
the induction of 3ß-HSD type 1 mRNA by IL-4 (data not shown).
Our observation of the potentiating action of PMA suggests that at
least one signaling molecule that is involved in the signal
transduction of the IL-4 action on the expression of 3ß-HSD is also a
substrate for PKC. It is tempting to speculate that PKC
is the
potential link between the PI 3-kinase activity and the activation of
MAPK for the following reasons: 1) IL-4 is able to activate PKC
(32); 2) Raf, a molecule directly downstream of Ras, is an important
physiological substrate of PKC (41); thus, direct Raf activation
provides a potential mechanism for the activation of the MAPK cascade
downstream and independently of Grb2, Sos, and Ras; 3) PtdIns(3, 4, 5)P3, a product of PI 3-kinase, activates
different PKC isoenzymes (24), especially PKC
(25, 26, 27, 28); 4) PKC
is
a PMA-insensitive PKC isoform, and we have observed that
down-regulation of diacylglycerol (DAG)-sensitive PKC by
overnight pretreatment with 1 µM PMA failed to block
IL-4-induced 3ß-HSD activity in ZR-751 cells (data not shown).
The lipid products of PI 3-kinase also serve to localize and activate
the p70S6 kinase (22, 23). Rapamycin selectively inhibits the
phosphorylation and activation of p70S6 kinase. This selective
inhibitor has, in fact, been reported to inhibit the effects of IL-4 on
the following parameters: 1) stimulation of T cell proliferation, 2)
activation of the human germline
gene promoter, 3) IgE production,
and 4) serine phosphorylation of nonhistone chromosomal protein
HMG-I(Y) in B lymphocytes (49, 50, 51). However, it is interesting that
rapamycin (5 ng/ml) had no effect on the induction of 3ß-HSD activity
by IL-4 in ZR-751 cells (data not shown), thus suggesting that the
p70S6 kinase is not involved in IL-4-induced 3ß-HSD type 1
expression.
Taking into consideration these findings and the current knowledge in
the literature, we propose the following working hypothesis for the
induction of 3ß-HSD type 1 expression by IL-4. The binding of IL-4 to
its specific receptor causes dimerization of IL-4R and activation of
Janus kinases (JAKs). The JAKs then phosphorylate the IL-4R on its
tyrosine(s), thus providing docking sites for both Stat6 and IRS
proteins which are, in turn, phosphorylated on their tyrosine residues.
Binding of the regulatory subunit (p85) of PI 3-kinase to
phosphorylated IRS proteins within IRS proteins has been shown to
activate PI 3-kinase and to change its subcellular location. PI
3-kinase generates PtdIns(3, 4)P2 and
PtdIns(3, 4, 5)P3, which, in turn, could activate,
in a PI 3-kinase-dependent manner, a nonclassical, DAG-insensitive-PKC,
such as PKC
. This PKC isoform would activate Raf, leading to the
activation of the MAPK pathway, independently of Ras. In accordance
with this model, IGF-I and insulin increase the pool of receptors able
to phosphorylate IRS proteins and activate MAPK in a PI
3-kinase-dependent manner. Our observation that PMA caused a greater
potentiating effect of IL-4-induced 3ß-HSD expression than that
exerted by IGF-I or insulin may be explained by its capacity to
activate a different set of PKC isoforms, i.e. the
DAG-sensitive PKC isoenzymes (41), while IL-4, IGF-I, and insulin
activate atypical PKC (27, 28). The inability of IGF-I and insulin to
induce 3ß-HSD activity in the absence of IL-4 indicates that IRS-1-
and IRS-2-phosphorylated proteins and their downstream effectors may
cooperate with another IL-4-specific signaling transduction pathway.
Thus, we postulate that IL-4-induced 3ß-HSD type 1 gene expression
requires the independent activation of at least two transcription
factors, i.e. Stat6, an IL-4-specific transcription factor,
and a second unidentified factor, which can be activated not only by
IL-4, but also by IGF-I, insulin, and PMA. Moreover, the inability of
PMA to induce 3ß-HSD activity in the absence of IL-4 also strongly
suggests the absolute requirement of an IL-4-specific signaling
molecule. The fact that IL-4-induced Stat6 DNA-binding activity is not
affected by wortmannin or PD98059, and that PMA does not modify the
potency of IL-4 to induced Stat6 DNA-binding activity, suggests that
Stat6 is activated independently of IRS proteins, as previously
suggested in another model system (52).
Another possibility, is that the PI 3-kinase-MAPK-dependant pathway
described herein might regulate the transcriptional activity of Stat6
directly, especially if one takes into account previous studies
demonstrating a positive role of serine phosphorylation of the
carboxyl-terminal end of Stat1, Stat3, and Stat5 in the maximal
transcriptional activation by those factors (53, 54, 55). MAPK has been
implicated in the phosphorylation of serine 727, which occurred
in a MAPK consensus phosphorylation sites, in both Stat1 and Stat3
(53). Stat5a and Stat5b have been shown to be phosphorylated on
positionally homologous serines (725 and 730, respectively), but those
are not in a MAPK consensus sequence but rather in a P-S-P motif (56).
Furthermore, the nonconserved serine of Stat5a (780) is also the target
of MAPK (57). In addition to those reports, it has also been
demonstrated that Stat3 and Stat5 proteins are serine phosphorylated by
MAPK-independent pathways (54, 58, 59, 60). There is no report showing that
Stat6 is serine phosphorylated, and Stat6 does not contain either MAPK
consensus phosphorylation site (P-X-S-P) or the proline-flanked motif
(P-S-P). Thus, in view of this information, it is difficult to predict
the potential role, if any, of MAPK serine phosphorylation of Stat6.
Moreover, we have not observed any difference in IL-4-induced Stat6
DNA-binding activity when the cells were incubated in the presence of
either IGF-I, insulin, PMA, wortmannin, or PD98059, strongly
suggesting that the PI 3-kinase-MAPK-dependent pathway does not seem to
modulate Stat6 tyrosine phosphorylation and DNA-binding activity.
Finally, neither wortmannin nor PMA modulates IL-4-induced reporter
gene activity from a reporter construct containing Stat6 response
elements linked to the thymidine kinase promoter, which was
transfected with Stat6 in ZR-751 cells (data not shown). Moreover,
the transactivation domain of Stat6, when fused to the GAL4 DNA-binding
domain, has transactivation activity that is independent of tyrosine
phosphorylation (61). When such a construct was transfected into
ZR-751 cells along with a GAL4 reporter construct, wortmannin and PMA
again failed to modulate the activity of this reporter gene (data not
shown). Thus, it is more likely that the PI 3-kinase-MAPK-dependent
pathway induced by IL-4 regulates another transcription factor rather
than Stat6 itself.
Although our data might suggest that other pathways or signaling
molecules might be involved in IL-4-induced 3ß-HSD type 1 gene
expression, none can provide a satisfactory explanation and be in
accordance with all of our data and published research,
especially with the PI 3-kinase-dependent activation of MAPK. For
example, Shc is another adapter molecule that can recruit Grb2 and lead
to the activation of the MAPK cascade. Although the JAKs can
phosphorylate Shc, all the cytokines, which induce the phosphorylation
of Shc by the JAKs, contain docking sites for SHC in one of their
receptor chains. There is no report indicating that Shc can bind to the
IL-4 receptors as is the case for the IGF-I and insulin receptors.
Moreover, there is no evidence suggesting that the Shc can directly
dock to the JAKs. Furthermore, there is no evidence that IL-4 induced
the phosphorylation of a protein with the size of Shc.
There is also a possibility that IL-4 activates the MAPK, at least in
part, via Grb2 binding to IRS proteins. According to the current
literature, if the binding of Grb2 to IRS proteins was involved
in IL-4-induced MAPK activity it would have to act upstream of Ras.
However, the relationship between PI 3-kinase and Ras in the activation
of MAPK by insulin is a matter of debate, and in the case of IL-4 this
question has not yet been addressed. It was reported that PI 3-kinase
can be either downstream or upstream of Ras (20, 42, 62, 63, 64), but this
apparent discrepancy most likely results from the measurement of
different parameters. Furthermore, overexpression of dominant negative
p85 increased the basal state GTP-bound Ras activity, and this
parameter was not further enhanced by insulin, while insulin-induced
MAPK activation was still inhibited (65). This suggests, in fact, that
PI 3-kinase can be required for the activation of MAPK independently or
downstream of Ras. Since there are no data in the literature that could
explain how the PI 3-kinase would regulate the binding of Grb2 to IRS,
the possibility that IL-4 activates the MAPK via the binding of Grb2 to
IRS proteins was excluded. In this regard, it should be mentioned that
LY294002 and wortmannin inhibited insulin-induced activation of Raf-1
and MAPK, whereas these compounds exert no effect on the formation of
the GTP-Ras complex (66). Furthermore, the mechanism proposed to
explain the activation of MAPK by erythropoietin involved sequential
recruitment of PI 3-kinase to the erythropoietin receptor and
activation of MAPK activity, independent of the Shc/Grb2-adapter
pathway and of Stat proteins. In fact, an atypical PKC was also
suggested to be the mediator connecting PI 3-kinase with the MAPK
cascade (67).
The induction of the 3ß-HSD type 1 gene expression by IL-4 provides a
unique model to aid in the identification of one or several
transcription factors involved in PI 3-kinase and MAPK pathways that
cooperate with Stat6 to regulate gene transcription. Thus, elucidation
of the mechanism through which IL-4 induces 3ß-HSD type 1 gene
expression in ZR-751 cells will provide new insights into the
mechanism of action of IL-4 in nonimmune cells. However, the widespread
expression of 3ß-HSD type 1 and IL-4R suggested that the IL-4 action
on 3ß-HSD type 1 gene expression might be relevant in physiological
and pathological conditions in various tissues. In this regard, IL-4
may play a role in the biosynthesis of active sex steroids from the
inactive adrenal steroid DHEA, not only in breast cells
but also in various cell types derived from peripheral target tissues,
such as normal human prostate epithelial cells, immortalized
keratinocytes, as well as colon and cervix cancer cell lines (68).
 |
MATERIALS AND METHODS
|
---|
Cell Culture and Chemicals
All media and supplements for cell culture experiments were
obtained from Sigma (St. Louis, MO), with the exception of
FBS, which was purchased from HyClone Laboratories, Inc.
(Logan, UT). Human IL-4 was purchased from R&D Systems (Minneapolis,
MN), human IGF-I from Genzyme Corp. (Cambridge, MA), and
human insulin from Sigma. Rapamycin was purchased from ICN
Canada Ltd. (Montreal, Quebec, Canada), PMA, wortmannin, and LY294002
were from Calbiochem (San Diego, CA), and PD98059 was from
Research Biochemicals International (Oakville, Ontario,
Canada).
The ZR-751 human breast cancer cells were obtained from the
American Type Culture Collection (Manassas, VA). ZR-751
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, and 10% FBS.
Immunoprecipitation and Western Blotting
For the phosphorylation analysis of IRS proteins, ZR-751 cells
were plated at a density of 1,000,000 cells per 10-cm dish. Three days
after plating, cells were incubated for the indicated time periods with
IL-4 (10 ng/ml). Thereafter, cells were washed with cold PBS and lysed
with 1 ml/dish of lysis buffer (150 mM Tris, pH 8.0, 150
mM NaCl, 1% NP-40) for 30 min at 4 C. All subsequent steps
were performed at 4 C. Cell lysate was cleared by centrifugation at
10,000 x g for 15 min. Cell lysates, equivalent to
500,000 cells, were incubated overnight with 3 µg of either
anti-IRS-1 or anti-IRS-2 (Upstate Biotechnology, Inc. Lake
Placid, NY). The following day, 30 µl of protein G+/A coupled to
agarose beads (Calbiochem) were then added for 3 h,
followed by three washes in lysis buffer. Beads were resuspended in
loading buffer and were separated on an 8% SDS-PAGE. Gels were
transferred onto a nitrocellulose membrane. Analysis of tyrosine
phosphorylation was performed by probing the membranes with the 4G10
monoclonal antibody (Upstate Biotechnology, Inc.) and
antimouse IgG linked to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as
secondary antibody. Control blotting was performed by stripping the
membrane followed by incubating the membrane with either anti-IRS-1 or
anti-IRS-2 and a horseradish peroxidase-linked antirabbit IgG
(Amersham Pharmacia Biotech, Oakville, Ontario,
Canada) was used as a secondary antibody. For the MAPK
activation experiment, ZR-751 cells were directly lysed in
sample buffer after the indicated treatment, after which 15 µg of
each protein extract were loaded on a 12% SDS-PAGE and
transferred onto a nitrocellulose membrane. MAPK activation was
determined with an antiphospho-MAPK (p42 and p44)-specific mouse
monoclonal antibody (Upstate Biotechnology, Inc.) and
antimouse IgG antibody linked to horseradish peroxidase. Thereafter,
the blot was stripped and reprobed with an anti-MAPK (p42 and p44)
rabbit immunoaffinity purified IgG antibody (Upstate Biotechnology, Inc.) and a horseradish peroxidase-linked
antirabbit IgG to control for total MAPK protein content. The
recognized proteins were visualized using enhanced chemiluminescence
(Amersham Pharmacia Biotech).
Assay for 3ß-HSD Activity
Assay for 3ß-HSD activity was performed as previously
described (2). Briefly, cells were plated at 200,000 cells per well in
6-well plates. After the indicated treatment, cells were harvested and
resuspended in 3ß-HSD assay buffer (50 mM
NaH2PO4, pH 7.4, 1
mM EDTA, 20% glycerol), submitted to three freeze-thaw
cycles, and kept frozen at 80 C. Optimal amounts of cell homogenates
were used to measure the conversion of 10 nM
[4-14C(N)]DHEA (55.2 mCi/mmol)
(Mandel Scientific Company Ltd., Guelph, Ontario, Canada) in 3ß-HSD
assay buffer in the presence of 1 mM NAD+, into
[14C]-4-DIONE.
[14C]-labeled steroids were separated on TLC
plates, which were then 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.
Electrophoretic Mobility Shift Assay (EMSA)
Cells were treated as indicated for 30 min and EMSA was
performed using whole- cell extracts as previously described (2, 69).
Complexes were resolved in a 4% polyacrylamide gel in 0.25x
Tris-borate-EDTA buffer. The classical Stat6 double-stranded DNA probe
5'-GTCAACTTCCCAAGAACAGAA-3' derived from the human Ig constant region E
(IgE) promoter was used.
 |
ACKNOWLEDGMENTS
|
---|
We thank doctors Marie-Louise Ricketts and Richard Moriggl for
helpful discussion and advice in the preparation of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jacques Simard, Laboratory of Hereditary Cancers, CHUL Research Center, 2705 Laurier Boulevard, Quebec City, Quebec G1V 4G2, Canada.
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 Senior Scientist from Le Fond de la Recherche
en Santé du Québec (FRSQ).
Received for publication March 23, 1999.
Revision received September 14, 1999.
Accepted for publication October 21, 1999.
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