Multiple Signaling Pathways Mediate Interleukin-4-Induced 3ß-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}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-75–1 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-75–1 human breast cancer cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}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-75–1, 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-75–1 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-75–1 breast cancer cells. As shown in Fig. 1Go, 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-75–1 Human Breast Cancer Cells

ZR-75–1 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.

 
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-75–1, MCF-7, and T47-D breast cancer cell lines (29). ZR-75–1 cells, therefore, were treated for 6 h with increasing concentrations of IGF-I (Fig. 2AGo) or insulin (Fig. 2BGo) in the presence or absence of a suboptimal concentration of IL-4 (10 pM). As illustrated in Fig. 2Go, 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-75–1 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.

 
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-75–1 cells were incubated for 6 h with 100 pM IL-4 in the presence of increasing concentrations of wortmannin (Fig. 3AGo) or LY294002 (Fig. 3BGo). Wortmannin, a well characterized selective inhibitor of PI 3-kinase, is able to inhibit the following IL-4 effects: 1) induction of germline {epsilon} gene expression, 2) induction of the translocation of the PKC{zeta} 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. 3Go, 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-75–1 Cells

ZR-75–1 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.

 
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 2–7 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. 4Go, 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-75–1 Cells

ZR-75–1 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.

 
To further explore the involvement of the MAPK pathway in IL-4-induced 3ß-HSD type 1 gene expression, ZR-75–1 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. 5Go, top panel). MAPK was found to be phosphorylated in cells treated with IL-4, IGF-I, or insulin (Fig. 5Go, lanes 2–4) but not in untreated cells (Fig. 5Go, 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. 5Go, lanes 6–8), 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. 5Go, lanes 10–12). As a control, the membrane was then stripped and reblotted with an anti-MAPK antibody (Fig. 5Go, 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-75–1 Cells

ZR-75–1 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.

 
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-75–1 cells were incubated with 100 pM IL-4, 5 nM IGF-I, or 15 nM insulin for 30 min. As shown in Fig. 6Go, 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-75–1 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. 6Go, 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-75–1 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.

 
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-75–1 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. 7AGo, 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. 7AGo, 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-75–1 cells were incubated with increasing concentrations of PMA (Fig. 7BGo) 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-75–1 Cells

ZR-75–1 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.

 
To determine whether the potentiating action of PMA was due to modulation of IL-4-induced activation of Stat6, ZR-75–1 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. 8Go, 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-75–1 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).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-75–1 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-75–1 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-75–1 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-75–1 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-75–1 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-75–1 cells. Although IL-4 failed to activate MAPK in lymphohemopoietic cells (37, 38), the activation of MAPK in ZR-75–1 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{zeta} 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{zeta} (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{zeta} (25, 26, 27, 28); 4) PKC{zeta} 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-75–1 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 {epsilon} 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-75–1 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{zeta}. 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-75–1 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-75–1 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-75–1 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-75–1 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA). ZR-75–1 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-75–1 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-75–1 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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