Induction of 3ß-Hydroxysteroid Dehydrogenase/{Delta}5-{Delta}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 l’Université 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-75–1 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-75–1 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{alpha}-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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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{alpha}-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-{gamma} (IFN-{gamma}) and IL-4 (19). The potential role of cytokines in breast cancer cells is supported by the observation that interleukin-1{alpha} (IL-1{alpha}), IL-4, IL-6, and IL-13 inhibit the proliferation of ZR-75–1, 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-75–1 cells (26). ZR-75–1 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-75–1 cells under basal growth condition, as indicated by the absence of detectable conversion of [3H]DHEA. However, incubation with IL-4 (Fig. 1AGo) or IL-13 (Fig. 1BGo) 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{alpha} (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-{gamma} (15 U/ml), and epidermal growth factor (EGF) (30 µg/ml) all failed to induce 3ß-HSD activity in ZR-75–1 cells (data not shown).



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Figure 1. Induction of 3ß-HSD Activity by IL-4 in ZR-75–1 Human Breast Cancer Cells

Cells were plated at a density of 20,000 cells per well; 3 days after plating, ZR-75–1 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.

 
In untreated ZR-75–1 cells, [3H]-5-androstene-3ß,17ß-diol (5-DIOL) was only converted into [3H]DHEA (Fig. 1Go, 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. 2Go), which is consistent with the data shown in Fig. 1Go, C and D. On the other hand, in IL-4-treated ZR-75–1 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{alpha}-reductase activity. This latter activity was also detectable in ZR-75–1 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{alpha}-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-75–1 Human Breast Cancer Cells

Cells were plated at a density of 20,000 cells per well; 3 days after plating, ZR-75–1 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.

 
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. 3AGo), indicating that the IL-4 action on 3ß-HSD activity was not restricted to ER+ ZR-75–1 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. 3BGo).



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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.

 
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. 4AGo, 5Go-DIOL (10 nM) increased by 12-fold the luciferase activity from the androgen-sensitive 4XARE-luciferase reporter construct. Pretreatment of ZR-75–1 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. 4BGo 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-75–1 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 (A–C, hAR; D–F, hER{alpha}) and 0.65 µg of the reporter construct (A–C, 4XARE-LUC; D–F, 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-75–1 Human Breast Cancer Cells

ZR-75–1 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 7–10). The position of primers P1 and P2 used for RT-PCR amplification of 3ß-HSD transcripts is also illustrated.

 
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. 4CGo). 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. 4DGo, 5Go-DIOL increased by 3-fold the luciferase activity from the estrogen-sensitive PS2-luciferase reporter construct, while pretreatment of ZR-75–1 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. 4EGo, 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. 4FGo). 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-75–1 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-75–1 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. 5Go (lane 1), 3ß-HSD transcripts are not detectable in control ZR-75–1 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. 5Go, 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. 5Go, 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. 5Go, lane 6). The PCR product amplified using a plasmid containing the 3ß-HSD type 2 cDNA was not digested by HpaI (Fig. 5Go, lane 7). This experiment indicates that 3ß-HSD type 1 transcript levels are induced in ZR-75–1 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. 6AGo), 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-75–1 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. 6BGo, upper panel). GAPDH mRNA levels were unchanged by IL-4 treatment (Fig. 6BGo, 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-75–1 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-75–1 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).

 
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 7Go shows that in extracts from IL-4-treated HeLa cells transfected with both human Stat6 and human IL-4 receptor {alpha}-chain expression vectors, activated-Stat6 binds to the I{epsilon} 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. 7Go, 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. 7Go, lanes 5 and 11). These complexes have the same mobility as the one formed by activated Stat6 on the I{epsilon} probe. These complexes were also completely supershifted by the anti-Stat6 antibody (Fig. 7Go, lanes 6 and 12). No such complexes were formed on the mutant Stat6#1 and mutant Stat6#2 probes (Fig. 7Go, lanes 7–9 and 13–15). 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{alpha} 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{epsilon} probe: lanes 1–3) or from the 3ß-HSD type 1 promoter Stat6#1 (lanes 4–6), Stat6#1 mutant (lanes 7–9), Stat6#2 (lanes 10–12), and Stat6#2 mutant (lanes 13–15). Stat6-containing complexes were supershifted with anti-Stat6 antibody (lanes 3, 6, 9, 12, and 15).

 
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-75–1 cells (Fig. 8Go). The binding of Stat6 to the 3ß-HSD type 1 Stat6#1 and Stat6#2 probes was competed by an excess of nonradioactive I{epsilon} oligonucleotide (Fig. 8Go, lanes 3–4, 9–10, respectively), but it was not affected by a 300-fold excess of an unrelated (SP1) cold oligonucleotide (Fig. 8Go, lanes 5–6, 11–12, respectively). Stat6 binding to the I{epsilon} probe was competed by an excess of nonradioactive 3ß-HSD type 1 Stat6#1 or Stat6#2 oligonucleotides (Fig. 8Go, lanes 16 and 18), whereas a 300-fold excess of cold SP1 oligonucleotide failed to displace the Stat6-binding to I{epsilon} 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. 8Go, 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-75–1 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 1–6), 3ß-HSD type 1 Stat6#2 (lanes 7–12), and I{epsilon} (lanes 13–20) 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{epsilon} (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-75–1, but also in T-47D and MDA-MB-231 cells as well as in HMECs. As illustrated in Fig. 9Go, Stat6 DNA-binding activity was induced by IL-4 and IL-13 in these cells as revealed by EMSA using the I{epsilon} 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-75–1 (lanes 1–5), T-47D (lanes 6–10), MDA-MB-231 (lanes 11–15), and HMEC (lanes 16–20) 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{epsilon} 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-75–1 cells with staurosporine for 1 h blocked the IL-4 induction of both the 3ß-HSD activity (Fig. 10AGo) and the 3ß-HSD type 1 transcripts (Fig. 10Go B) and concomitantly abolished the activation of the DNA-binding activity of Stat6 by IL-4 (Fig. 10CGo).



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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-75–1 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{epsilon}-labeled probes.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our study demonstrates that IL-4 and IL-13 induce 3ß-HSD type 1 gene expression in ZR-75–1 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-75–1 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-75–1 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. 2Go clearly show that in ZR-75–1 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. 4Go), the induction of the 3ß-HSD expression by IL-4 may modulate the balance between estrogenic and androgenic biological responses when ZR-75–1 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 {alpha}-chain (IL-4R{alpha}) and the common {gamma}-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 {alpha}1-chain (54, 55) and {alpha}2-chain (56). Those two proteins can heterodimerize with IL-4R{alpha} 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 20–80 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-75–1 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{epsilon}. Moreover, Stat6 binds as efficiently to the 3ß-HSD type 1 STAT6#1 and STAT6#2 probes as to I{epsilon} 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 {epsilon} 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-{kappa}B site in the promoter of mouse Ig heavy-chain germ line {epsilon} 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-{alpha}/{gamma}-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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-75–1, 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-75–1 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-{gamma}, and EGF were purchased from R&D Systems (Minneapolis, MN). IL-1{alpha} 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-75–1 cells were transfected in OPTI-MEM (GIBCO BRL, Burlington, Ontario, Canada) with the lipofectin reagent (GIBCO BRL) according to the manufacturer’s 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{alpha} 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{alpha} cDNAs (76) (10 µg each) according to the manufacturer’s 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-75–1 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-75–1 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 manufacturer’s 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 {alpha}-[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 supplier’s 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{epsilon}, 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 {gamma}-[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 637–847 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 l’Université 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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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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