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Endocrinology Vol. 140, No. 10 4573-4584
Copyright © 1999 by The Endocrine Society

ARTICLES

Induction of 3ß-Hydroxysteroid Dehydrogenase/ Isomerase Type 1 Expression by Interleukin-4 in Human Normal Prostate Epithelial Cells, Immortalized Keratinocytes, Colon, and Cervix Cancer Cell Lines1

Sébastien Gingras2 and Jacques Simard3

Medical Research Council Group in Molecular Endocrinology, CHUL Research Center and Laval University, Québec City, G1V 4G2, Canada

Address all correspondence and requests for reprints to: Jacques Simard, Laboratory of Hereditary Cancers, T3–57, CHUL Research Center, 2705 Laurier Boulevard, Sainte-Foy, Québec, G1V 4G2, Canada. E-mail: Jacques.Simard{at}crchul.ulaval.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD) isoenzymes catalyze an essential step in the formation of all classes of active steroid hormones. In humans there are two 3ß-HSD isoenzymes, the type 1 gene being predominantly expressed in the placenta and peripheral tissues, whereas the type 2 gene is the predominant 3ß-HSD expressed in the adrenal glands and gonads. We have recently showed that interleukin (IL)-4 and IL-13 induce 3ß-HSD type 1 gene expression in human breast cancer cell lines as well as in normal human mammary epithelial cells. The present study was designed to investigate whether such a cytokine-induced 3ß-HSD type 1 expression would also be observed in cell types derived from other peripheral sex steroid target tissues. To gain further knowledge about the molecular mechanism of IL-4 action, we have studied whether the induction of 3ß-HSD type 1 expression in IL-4-responsive cell types would always be associated with the activation of Stat6, a member of the Signal Transducers and Activators of Transcription (STAT) gene family. Stat6 is recognized as the principal transcription factor mediating the effects of IL-4. In normal human prostate epithelial cells (PrEC), no 3ß-HSD activity was detectable under basal culture conditions, while exposure to IL-4 or IL-13 caused a potent induction of this activity. This effect results from a rapid induction of 3ß-HSD type 1 messenger RNA levels as determined by Northern blot and RT-PCR analyses. Furthermore, IL-4 and IL-13 also increased 3ß-HSD type 1 gene expression in human HaCaT immortalized keratinocytes, ME-180 cervix cancer cells, HT-29 colon cancer cells as well as in BT-20 and ZR-75–1 breast cancer cells. However, IL-4 and IL-13 failed to modulate the 3ß-HSD type 1 expression in human LnCAP and PC-3 prostate cancer cells, Caco-2 colon cancer cells as well as in JAR and JEG-3 choriocarcinoma cell lines. The DNA-binding activity of Stat6 was activated after a 30-min exposure to IL-4 in PrEC and in all the cell types where IL-4 induced 3ß-HSD expression, but not in those that failed to respond to IL-4. Our data therefore suggest that IL-4 and IL-13 may play a role in the biosynthesis of active sex steroids from the inactive adrenal steroid dehydroepiandrosterone, 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. Our data also demonstrates that the stimulatory effect of IL-4 was always associated with the activation of Stat6, thus supporting the essential role of Stat6 in this induction of 3ß-HSD type 1 gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS WELL RECOGNIZED that humans and certain other primates are unique among animal species in having adrenals that secrete large amounts of the inactive steroid precursors, dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) (1). These androgens of adrenal origin do not bind to the androgen receptor (2) but exert androgenic action after their conversion into active androgens in target tissues. Indeed, plasma DHEA-S levels are 100 to 500 times higher than those of testosterone (TESTO) (3, 4), thus providing the high levels of the substrate required for conversion into active sex steroids in target tissues. The tissue- and cell-specific expression, as well as the substrate specificity of the various types of human enzymes catalyzing 17ß-hydroxysteroid dehydrogenase (17ß-HSD), 3ß-HSD and 5{alpha}-reductase activities, provide each cell with the necessary mechanisms to control the level of intracellular active sex steroids (1, 5, 6, 7).

The 3ß-HSD isoenzymes are responsible for the oxidation and isomerization of 5-ene-3ß-hydroxysteroid precursors into 4-ene-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones (6). In humans there are two 3ß-HSD isoenzymes, which were chronologically designated type 1 and 2 (6). Type 2 gene is the predominant 3ß-HSD expressed in the human adrenal gland, ovary and testis and its deficiency is responsible for a rare form of congenital adrenal hyperplasia (6, 8, 9, 10). In contrast, the expression of the type 1 is responsible for the 3ß-HSD activity found in many peripheral tissues such as placenta, mammary gland, and skin (6).

We have recently shown that IL-4 and IL-13 cause a rapid and potent induction of 3ß-HSD type 1 gene transcription in human breast cancer cell lines as well as in normal human mammary epithelial cells (11). This induction was associated with the activation of the DNA-binding activity of Stat6, a member of the signal transducers and activators of transcription gene family. We have also characterized two Stat6 DNA-binding elements in the 3ß-HSD type 1 gene promoter (11). The similar effects exerted by IL-4 and IL-13 are explained by the fact that their receptors share at least one subunit (12, 13). One type of IL-4 receptor (IL-4R) is composed of the IL-4R{alpha} chain and the common {gamma} chain from the IL-2 receptor. While the IL-13 receptor {alpha}1 chain can heterodimerize with the IL-4R{alpha} chain to form receptors for both IL-4 and IL-13 (14).

The primary function for both IL-4 and IL-13 is recognized to be promotion of humoral responses and suppression of inflammation, those effects are mediated through regulation of T-helper cells and B cells (12, 15, 16). However, the expression of their receptors is not restricted to hematopoietic cells but was also found in a large number of normal and tumoral tissues or cells lines such as breast, prostate, colon, stomach, liver, lung, adrenals, keratinocytes, chorionic trophoblast, and amnion epithelial cells (17, 18, 19, 20, 21, 22, 23, 24).

Knowing that several peripheral tissues possess 3ß-HSD activity, and that the receptors for IL-4 and IL-13 are expressed in many of those tissues, the present study was first designed to investigate whether the stimulatory effect IL-4 and IL-13 on 3ß-HSD type 1 would be restricted to breast cells. Another objective of this work was to gain further knowledge about the molecular mechanism of IL-4 action on this parameter and to verify the hypothesis that the induction of 3ß-HSD type 1 gene expression in IL-4-responsive cells would always be associated with the activation of Stat6, and that the activation would not be observed in cell lines where IL-4 failed to increase 3ß-HSD activity. To address those questions we studied the effects of IL-4 and IL-13 on 3ß-HSD type 1 gene expression and on Stat6 DNA-binding activity in several cell types derived from peripheral tissues that are known to have 3ß-HSD activity or in cell lines that are known to be IL-4-responsive.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
All media and supplements for cell culture were obtained from Sigma (St. Louis, MO), except FBS, which was purchased from HyClone Laboratories, Inc. (Logan, UT). IL-4 and IL-13 were purchased from R&D Systems (Manassas, VA). The estrogen receptor (ER) positive ZR-75–1 and ER negative BT-20 human breast cancer cells, LnCAP and PC-3 human prostate cancer cells, HT-29 and Caco-2 human colon cancer cells, ME-180 human cervix cancer cells, JAR and JEG-3 human choriocarcinoma cells were obtained from the American Type Culture Collection (Rockville, MD). The immortalized human keratinocyte HaCaT cells were obtained from Dr. N. E. Fusening (German Cancer Research Center, Heiderberg, Germany). Normal human prostate epithelial cells (PrEC), media and supplements for their culture were purchased from Clonetics Corp. (San Diego, CA). PrEC were cultured in PrEBM (prostate epithelial cell basal medium), which was supplemented with bovine pituitary extract, hydrocortisone, epidermal growth factor, epinephrine, transferrin, insulin, retinoic acid, triiodothytonin, gentamicin sulfate, and amphotericin-B according to the supplier’s instructions. LnCAP, PC-3, JAR, HaCaT and ZR-75–1 cells were routinely grown in phenol red-free RPMI-1640 medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 15 mM HEPES, and 10% FBS. For ZR-75–1 cells, 1 nM 17ß-estradiol was added to the growth medium. BT-20 and Caco-2 cells were cultured in MEM supplemented with MEM nonessential amino acids and 5% FBS. JEG-3 cells were cultured in DMEM low glucose supplemented with 10% FBS and 2 mM L-glutamine. ME-180 and HT-29 cells were cultured in McCoy’5A supplemented with 10% FBS and 2 mM L-glutamine. 100 IU/ml penicillin and 50 µg/ml streptomycin sulfate were added to the culture media of all the cell lines.

Assay for 3ß-HSD activity
The following radioactive steroids were purchased from Mandel Scientific Company Ltd. (Guelph, Ontario, Canada): [4-14C(N)]DHEA (55.2 mCi/mol) and [1,2,3-3H(N)]-androstenediol (5-DIOL) (52.2 Ci/mmol). Assays for 3ß-HSD activity in intact cells were performed as previously described (11). TLC plates of [3H]-substrates were analyzed using a Berthold model 440E scanner, while TLC plates from [14C]DHEA were analyzed using a PhosphorImager imaging system (Molecular Dynamics, Inc.). Half-maximal stimulatory effect (EC50) values were calculated using a weighed iterative nonlinear least-squares regression. Assays for 3ß-HSD activity in cell homogenates were performed as previously described (11). Briefly, cells were plated at 200,000 cells per well in six-well plates, following the indicated treatment, cells were harvested and resuspended in 3ß-HSD assay buffer (50 mM NaH2PO4 pH 7.4, 1 mM EDTA, 20% glycerol), the samples were then submitted to three freeze/thaw cycles and frozen at -80 C. Different amounts of cell homogenates were used for each cell line to obtain optimal conversion of the substrate for both treated and untreated cells. The protein content in each homogenate was measured using the Bio-Rad Laboratories, Inc. Protein Assay Kit (Bio-Rad Laboratories, Inc. Mississauga, Ontario, Canada), and enzymatic activity was expressed as pmol of product formed per mg of proteins per h.

Western blot analysis
Ten micrograms of total cell extracts were separated on an 8% SDS-PAGE gel. The gel was transferred onto a nitrocellulose membrane, and equal sample loading and transfer efficiency was confirmed by staining of the membrane with Ponceau Red. The membrane was probed with a Stat6 antibody (Santa Cruz Biotechnology, Inc., Santa Clara, CA, catalog no. SC-621X). Antirabbit IgG linked to horseradish peroxidase (Amersham Pharmacia Biotech, Oakville, Ontario, Canada) was used as secondary antibody and proteins were visualized using ECL (enhanced chemiluminescence, Amersham Pharmacia Biotech).

Northern blot analysis and RT-PCR
After the indicated treatments, RNA was extracted using Tri-Reagent (Molecular Research Center, Inc. Cincinnati, OH) according to the manufacturer’s instructions. Total RNA was solubilized in FORMAzol (Molecular Research Center, Inc.) and stored at -80 C. Northern blot analysis was performed as previously described (11) using 20 µg of total RNA in each lane. Hybridization with {alpha}-[32P]dCTP-labeled complementary DNA (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. Control hybridization was performed using a 548 bp human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) cDNA HindIII-XbaI fragment.

RT-PCR experiments were performed as previously described (11). Briefly, 5 µg of total RNA was reverse transcribed using SuperScript II (Life Technologies, Inc., Burlington, Ontario, Canada) with 2.5 µg of an oligonucleotide T12VN and 0.5 mM deoxynucleoside triphosphate using the supplied buffer. Reaction was carried out at 42 C for 1 h, and then purified with QIAquick PCR Purification Kit (QIAGEN Inc., Santa Clarita, CA). 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'-CTTGCCCACAGCCTTGGCAG-3'.

Electrophoretic mobility shift assay (EMSA)
Cells were treated for 30 min with 10 ng/ml of IL-4 and EMSA was performed using whole cell extracts as previously described (11, 25). Complexes were resolved in a 4% polyacrylamide gel in 0.25 x Tris-borate-EDTA buffer. The following double stranded DNA probes were used: the well established Stat6 responsive element I{epsilon}, 5'-GTCAACTTCCCAAGAACAGAA-3' from the human Ig constant region E (IgE) promoter, Stat6 #1, 5'-CTGTCAAGTTCCACTGAACTGAACAC-3' and Stat6 #2, 5'-TCTTCCTGTTCCTGGGAAGAATTAGA-3' positioned from -855 to -830 and from -164 to -129 bp from the cap site of the 3ß-HSD type 1 genes, respectively (11). The same Stat6 antibody used for Western blot analysis was included in the binding reaction where indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-4 and IL-13 induce 3ß-HSD activity in normal prostate epithelial cells in primary culture
It is now well recognized that in the prostate, the amounts of TESTO and DHT synthesized locally from inactive adrenal steroid precursors (DHEA and 4-androstenedione (4-DIONE)) are estimated to be comparable to the quantity of TESTO and DHT of testicular origin (1, 4). This is supported by the observation that, after castration, the intraprostatic concentration of DHT remains at 40–50% of the level measured in the prostate of intact control subjects (3, 4). Because the 3ß-HSD is the first step in the conversion of DHEA into active androgens, we have investigated the potential action of IL-4 and IL-13 on PrEC in primary culture.

PrEC were exposed to increasing concentrations of IL-4 or IL-13 for 2 days. The 3ß-HSD activity was not detectable in PrEC under basal growth conditions, 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-DIONE, indicating an induction of 3ß-HSD activity in these cells. This potent up-regulation in 3ß-HSD activity, induced by IL-4 and IL-13, was observed at EC50 values of 20 ± 2 pM and 170 ± 12 pM, respectively. In control PrEC, [3H]-5-androstene-3ß, 17ß-diol (5-DIOL) was only converted into [3H]DHEA (Fig. 1Go, C and D), resulting from the endogenous oxidative 17ß-HSD activity in these cells. It can also be seen in Fig. 1Go, C and D, that the induction of 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]TESTO and [3H]-4-DIONE.



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  		Figure 1. Induction of 3ß-HSD activity by IL-4 and IL-13 in normal human prostate epithelial cells. Normal human prostate epithelial cells (PrEC) in primary culture were plated at 10,000 cells per well in 24-well plates. Two days after plating, PrEC were incubated for 2 days with the indicated concentrations of IL-4 (A and C) or IL-13 (B and D). Thereafter, 3ß-HSD activity was assayed for 8 h using 10 nM [14C]DHEA (A and B) or [3H]-5-DIOL (C and D). Data are expressed as the mean ± SEM of triplicate dishes.

 
To further characterize the predominant [3H]-5-DIOL metabolic pathway(s) and to measure the relative contribution of both 3ß-HSD and 17ß-HSD activities, we performed a time course experiment after a 2-day incubation in the presence or absence of 100 pM IL-4. In the absence of IL-4, [3H]-5-DIOL is only converted into [3H]DHEA by oxidative 17ß-HSD activity (Fig. 2AGo), which is consistent with the data shown in Fig. 1Go, C and D. On the other hand, in IL-4-treated PrEC (Fig. 2BGo), [3H]-5-DIOL was converted into [3H]DHEA and [3H]TESTO by 17ß-HSD and 3ß-HSD activities, respectively. Thereafter, these products were both converted to [3H]-4-DIONE, which was finally converted into [3H]androstanedione (A-DIONE) by an endogenous 5{alpha}-reductase activity. This latter activity was also detectable in PrEC cells in the absence of IL-4 and was not regulated by IL-4 in these cells (data not shown).



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  		Figure 2. Time course of the IL-4 action on the conversion of 5-DIOL in normal human prostate epithelial cells. Normal human prostate epithelial cells (PrEC) in primary culture were plated at 10,000 cells per well in 24-well plates. Two days after plating, cells were incubated for 2 days in the presence (B) or absence (A) of 100 pM IL-4. Thereafter, medium was changed for medium containing [3H]-5-DIOL and incubated for the indicated time intervals. Data are expressed as the mean ± SEM of triplicate dishes.

 
Rapid induction of 3ß-HSD expression by IL-4 in PrEC
We have characterized the kinetics of IL-4 induction of 3ß-HSD messenger RNA (mRNA) levels. The PrEC were treated for up to 24 h with 100 pM IL-4 (Fig. 3Go). 3ß-HSD transcripts were undetectable in untreated PrEC, which is in accordance with the absence of 3ß-HSD activity in these cells. However, IL-4 induced 3ß-HSD gene expression as early as 3 h after exposure (Fig., 3 lane 3, upper panel). GAPDH mRNA levels were unchanged by IL-4 treatment (Fig. 3Go, lower panel). To characterize the kinetics of the IL-4 induction of 3ß-HSD activity, PrEC were treated for increasing times with 100 pM IL-4 (Fig. 4AGo). The 3ß-HSD activity was detectable after a short incubation period of 8 h with IL-4, and it continued to increase for at least 48 h, thus showing a rapid induction of 3ß-HSD activity by IL-4. It should be noted that IL-4 induces 3ß-HSD activity with the same kinetics in both PrEC and ZR-75–1 cells (Fig. 4DGo).



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  		Figure 3. Rapid induction of 3ß-HSD mRNA by IL-4 in normal human prostate epithelial cells. Normal human prostate epithelial cells (PrEC) in primary culture were treated with 140 pM IL-4 for the indicated periods. Northern blot analysis was performed as described in Materials and Methods. The membrane probed with 3ß-HSD cDNA (upper panel) was exposed for 5 days. It was then stripped and reprobed with GAPDH cDNA (lower panel), followed by an overnight exposure.

 


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  		Figure 4. Rapid induction of 3ß-HSD activity by IL-4 in a variety of cell types derived from peripheral tissues. PrEC, Normal human prostate epithelial cells in primary culture (A); LnCAP (B) and PC-3 (C) prostate cancer cells; ZR-75–1 (D) and BT-20 (E) breast cancer cells; HaCaT (F) human immortalized keratinocytes; HT-29 (G) and Caco-2 (H) human colon cancer cells; ME-180 (I) human cervix cancer cells or JAR (J) and JEG-3 (K) human choriocarcinoma cells were plated at 200,000 cells per well in six-well plates. Two days after plating, cells were incubated for the indicated periods with 100 pM IL-4. Thereafter, cells were harvested and 3ß-HSD activity was measured as described in Materials and Methods with 10 nM [14C]DHEA in the presence of 1 mM NAD+. Data are expressed as the mean ± SEM of triplicate dishes.

 
Effect of IL-4 on 3ß-HSD activity in cell types derived from peripheral tissues
The 3ß-HSD is expressed in many intracrine peripheral tissues (6), and the IL-4R is expressed in most of those tissues (17, 18, 19, 20, 21, 22, 23, 24), the effect of IL-4 was thus investigated in human immortalized keratinocytes and in several human cancer cell lines. In contrast to the induction of 3ß-HSD activity in PrEC in primary culture, IL-4 failed to regulate 3ß-HSD activity in LnCAP and PC-3 human prostate cancer cells (Fig. 4Go, B and C, respectively). While LnCAP cells expressed low but detectable levels of 3ß-HSD activity under basal culture conditions, this activity was undetectable in PC-3 cells. As illustrated in Fig. 4EGo, IL-4 increased 3ß-HSD activity in ER-negative BT-20 breast cancer cells, which is consistent with our previous findings showing that IL-4 also increased 3ß-HSD activity in MDA-MB-231, another ER-negative breast cancer cell line. BT-20 cells had significant 3ß-HSD activity under basal growth conditions, and a 48-h exposure to IL-4 caused a 28-fold increase in 3ß-HSD activity over the basal level.

It was demonstrated that human skin converted DHEA to testosterone, which is consistent with the expression 3ß-HSD type 1 in this tissue (8, 26, 27). On the other hand, IL-4 was detected in human skin in certain pathological conditions, such as atopic dermatitis (21), and in murine skin during the elicitation phase of contact sensitivity (28, 29). Moreover, human keratinocytes constitutively express IL-4R and its expression is also increased in some epidermal proliferative diseases (21, 22). Therefore, the effect of IL-4 on 3ß-HSD expression was investigated in HaCaT human immortalized keratinocytes. No 3ß-HSD activity was detectable in these cells under basal culture conditions, but incubation with IL-4 caused a rapid induction of this activity (Fig. 4FGo). The kinetics of the induction was similar to that observed in PrEC and ZR-75–1 cells.

The IL-4R is known to be expressed in more than 85% of colon carcinomas in addition to the normal colon mucosa of these patients (18). Thus, the effect of IL-4 was studied in HT-29 and Caco-2 human colon cancer cells (Fig. 4Go, G and H). Both cells lines possess relatively high levels of 3ß-HSD activity under basal culture conditions. However, a 48-h exposure to IL-4 caused a 19-fold stimulation in HT-29 cells, whereas it failed to change 3ß-HSD expression in Caco-2 cells.

The ME-180 human cervix cancer cells are IL-4-responsive cells, and these cells have previously been used to measure reporter gene activity in response to IL-4 (30). Consequently, we have investigated the potential action of this cytokine on 3ß-HSD expression. There is no 3ß-HSD activity under basal culture conditions in these cells; however, when the cells were incubated with IL-4, a rapid induction of 3ß-HSD was observed (Fig. 4IGo).

The expression of IL-4R was also reported in JAR and JEG-3 human choriocarcinoma cell lines (24). Furthermore, it was suggested that colocalization of IL-4 and IL-4R within gestational tissues may indicate auto- and/or paracrine mechanisms of action for these cytokines within the uteroplacental unit (23, 31). The production of progesterone is essential for the maintenance of human pregnancy and the high levels of progesterone produced by the placenta indicate that the placenta is an important site for 3ß-HSD activity. Thus, the effect of IL-4 on 3ß-HSD activity was investigated in JAR and JEG-3 cells (Fig. 4Go, J and K). Both cell lines express relatively high levels of 3ß-HSD activity under basal culture conditions; however, incubation with IL-4 for up to 48 h did not produce any significant change in the level of activity in these cells.

Stimulatory effect of IL-13 on 3ß-HSD activity in BT-20, HaCaT, HT-29 and ME-180 cell lines
Knowing that IL-4 induces 3ß-HSD activity in BT-20 breast cancer cells, HaCaT immortalized keratinocytes, HT-29 colon cancer cells, and ME-180 cervix cancer cells; the effect of IL-13 was investigated in these cells. The cells were grown for 48 h in the presence or absence of 100 pM IL-13. As shown in Fig. 5Go, IL-13 increases 3ß-HSD activity in all these cell lines and the amplitude of the stimulation was similar to that observed with IL-4. It should be noted that all the other cell lines that failed to respond to IL-4 also failed to response to IL-13 (data not shown). These data support the hypothesis that the effects of IL-4 and IL-13 are mediated through their common receptor.



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  		Figure 5. Induction of 3ß-HSD activity by IL-13 in cell lines derived from peripheral tissues. BT-20 breast cancer cells (A), HaCaT human immortalized keratinocytes (B), HT-29 human colon cancer cells (C) and ME-180 human cervix cancer cells (D) were plated at a density of between 150,000 and 200,000 cells per well in six-well plates. Two days after plating, cells were incubated in presence or absence of 100 pM IL-13 for 48 h. Thereafter, cells were harvested and 3ß-HSD activity was measured as described in Materials and Methods with 10 nM [14C]DHEA in the presence of 1 mM NAD+. Data are expressed as the mean ± SEM of triplicate dishes.

 
IL-4 selectively induces 3ß-HSD type 1 transcripts
To determine whether IL-4 induced type 1 and/or type 2 3ß-HSD gene expression, we performed a RT-PCR assay that discriminates between these two transcripts. This method has been previously used to demonstrate the selective induction of 3ß-HSD type 1 mRNA by IL-4 in ZR-75–1 cells (11). Because the primers selected spanned three introns of the 3ß-HSD genes, it excludes the possibility that the RT-PCR products arose from contaminating genomic DNA. In accordance with the data obtained from the measurement of 3ß-HSD activity, 3ß-HSD transcripts were not detectable in total RNA of untreated PrEC, ZR-75–1, HaCaT, ME-180 and PC-3 cells (Fig. 6Go, top panel, lanes 3, 5, 9, 13, and 16). However, the 3ß-HSD transcripts were detected following RT-PCR performed with total RNA from IL-4-treated PrEC, ZR-75–1, BT-20, HaCaT, HT-29, and ME-180 cells (Fig. 6Go, top panel, lanes 4, 6, 8, 10, 12, and 14) as well as in untreated BT-20, HT-29, LnCAP, Caco-2, JAR and JEG-3 cells (Fig. 6Go, top panel, lanes 7, 11, 15, and 17–19). Because the primers selected for this analysis amplified both the type 1 and type 2 3ß-HSD transcripts (Fig. 6Go, top panel, lanes 1 and 2), the RT-PCR products were then digested with HpaI, which selectively cut 3ß-HSD type 1 transcript. Digestion of the amplicons produced a fragment of 652 bp (Fig. 6Go, middle panel), 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. 6Go, middle panel, lane 1). On the other hand, the PCR product amplified using a plasmid containing the 3ß-HSD type 2 cDNA was not digested by HpaI (Fig. 6Go, middle panel, lane 2). This experiment indicates that 3ß-HSD type 1 gene is responsible for the 3ß-HSD activity detected in all these cells. As a control, the internal control GAPDH transcript was amplified from all the RT reactions, as revealed by the presence of the expected 545 bp band (Fig. 6Go, bottom panel).



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  		Figure 6. Induction of 3ß-HSD type 1 gene expression by IL-4 in cell types derived from peripheral tissues. The IL-4 responsive, normal human prostate epithelial cells (PrEC), ZR-75–1 and BT-20 breast cancer cells, HaCaT human immortalized keratinocytes, HT-29 human colon cancer cells; ME-180 human cervix cancer cells were incubated for 24 h in the presence or absence of 100 pM IL-4, whereas the IL-4 unresponsive LnCAP and PC-3 prostate cancer cells, Caco-2 colon cancer cells, as well as JAR and JEG-3 human choriocarcinoma cells were incubated only with control medium. Selective RT-PCR analysis for 3ß-HSD transcripts (top panel) was performed as described in Materials and Methods. Amplification of 3ß-HSD type 1 cDNA and type 2 cDNA were performed as control (top panel, lane 1 and 2). RT-PCR products and control amplifications were digested with HpaI (middle panel). Control amplification of GAPDH was performed as control for each RT reaction (bottom panel). The position of primers P1 and P2 used for RT-PCR amplification of 3ß-HSD transcripts is also illustrated.

 
Stat6 expression and activation in different cell types
The action of several cytokines is mediated through the signal transducers and activators of transcription (Stat) pathway (reviewed in Refs. 32, 33, 34). IL-4 and IL-13 mediate their biological effects by activating Stat6 (35, 36, 37). Recent studies performed with Stat6-deficient mice have demonstrated that Stat6 plays an essential role in IL-4 and IL-13 signaling (38, 39, 40). Therefore, the expression of Stat6 was investigated by Western analysis to find out whether the absence of its expression could explain the failure of IL-4 to regulate 3ß-HSD expression in some of the cell lines tested. As shown in Fig. 7Go, with the exception of LnCAP prostate cancer cells, all the cell lines studied express Stat6. We have also used extract from human embryonic kidney 293 cells as a control; these cells have been previously shown to not express Stat6 (41). Although there are some differences in the level of Stat6 expression, this could not be responsible for the failure of IL-4 to modulate 3ß-HSD expression in the PC-3, Caco-2, JAR and JEG-3 cell lines.



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  		Figure 7. Stat6 expression in cell types derived from peripheral tissues. Ten micrograms of total cell extract from ZR-75–1 and BT-20 breast cancer cells, normal human prostate epithelial cells (PrEC) in primary culture, LnCAP and PC-3 prostate cancer cells, HaCaT human immortalized keratinocytes; HT-29and Caco-2 human colon cancer cells, ME-180 human cervix cancer cells, JAR and JEG-3 human choriocarcinoma and 293 human embryonic kidney cells were separated on SDS-PAGE. Western blot analysis was performed as described in Materials and Methods. Equal sample loading and transfer efficiency was confirmed by staining of the membrane with Ponceau Red.

 
IL-4-binding to its receptors at the cell surface induces receptors dimerization and activation of the cytoplasmic receptor associated Janus kinases (JAK). The JAK then phosphorylate a specific tyrosine residue at position 641 in Stat6 (42), causing Stat6 dimerization and translocation to the nucleus and leading to specific DNA-binding to promoter sequences. Stat6 phophorylation is an absolute prerequisite for its activation and its DNA-binding activity (42). Hence, EMSA were performed to detect Stat6 DNA-binding activity and to determine whether IL-4 activated Stat6 in the different cell lines. EMSA were performed with the classical Stat6-binding probe derived from the IgE promoter. As shown in Fig. 8Go, a slower migrating complex was formed in PrEC, BT-20, HaCaT, HT-29, and ME-180 when these cells were treated with IL-4 for 30 min, ZR-75–1 cells were added as a positive control, because Stat6 is activated by IL-4 in this cell line (11). The identity of Stat6 in that complex was confirmed by the addition of an antibody against Stat6 to the binding reaction, which completely supershifted the complex. On the other hand, this Stat6-containing complex was not formed in response to IL-4 in LnCAP, PC-3, Caco-2, JAR, and JEG-3 cells. The only complex detectable in those cell lines was the nonspecific (NS) complexes described previously (11). Although this complex is nonspecific, it provides a useful control for sample loading. This experiment demonstrates that the IL-4-induced 3ß-HSD expression is always associated with the activation of Stat6.



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  		Figure 8. Stat6 activation by IL-4. Normal human prostate epithelial cells (PrEC) in primary culture, LnCAP and PC-3 prostate cancer cells, ZR-75–1 and BT-20 breast cancer cells, HaCaT human immortalized keratinocytes, HT-29 and Caco-2 human colon cancer cells, ME-180 human cervix cancer cells or JAR and JEG-3 human choriocarcinoma cells were incubated in the presence of absence of IL-4 (10 ng/ml for 30 min). Analysis of Stat6 activation using EMSA was performed as described in Materials and Methods using a well established Stat6 responsive element derived from the IgE-promoter. A Stat6 antibody was included in the binding reaction where indicated.

 
We have previously demonstrated the presence of two Stat6 DNA-binding elements in the 3ß-HSD type 1 gene promoter located at position -847 to -838 and -156 to -137, respectively (11). To confirm that activated Stat6 was also able to bind to these sites, EMSA were performed with the 3ß-HSD type 1 Stat6#1 and Stat6#2 probes. Figure 9Go shows that in IL-4 treated PrEC cells extracts, activated-Stat6 binds to the 3ß-HSD type 1 Stat6#1 and Stat6#2 probes. Similar results were obtained with ZR-75–1, BT-20, HaCaT, HT-29 and ME-180 cell extracts (data not shown).



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  		Figure 9. IL-4-activated Stat6 binds to consensus Stat6 sequence sites in the 3ß-HSD type 1 gene promoter. Normal human prostate epithelial cells (PrEC) in primary culture were incubated in the presence of absence of IL-4 (10 ng/ml for 30 min). Analysis of Stat6 activation using EMSA was performed as described in Materials and Methods using the 3ß-HSD type 1 Stat6#1 and Stat6#2 probes from the 3ß-HSD type gene promoter. A Stat6 antibody was included in the binding reaction where indicated.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study shows the rapid induction of the 3ß-HSD expression by IL-4 and IL-13, in normal human prostate epithelial cells in primary culture, in HaCaT immortalized human keratinocytes, in HT-29 human colon cancer cells and in ME-180 human cervix cancer cells. To the best of our knowledge, this is the first report describing the regulation of an enzyme involved in sex steroid formation in these cells. Our data therefore suggest that IL-4 and IL-13 may play a role in the biosynthesis of active sex steroids from inactive adrenal steroid DHEA not only in normal or tumoral breast cells but also in various cell types derived from peripheral target tissues. The increase of 3ß-HSD activity by IL-4 results from an induction of 3ß-HSD type 1 gene expression. This finding is in accordance with our recent observation that both IL-4 and IL-13 caused a rapid and specific induction of 3ß-HSD type 1 gene transcription in ZR-75–1 human breast cancer cells (11). It is also in accordance with the observation that 3ß-HSD type 1 gene is the predominant, if not the sole, type of 3ß-HSD transcript detectable in peripheral tissues such as the prostate, breast, skin, placenta, whereas 3ß-HSD type 2 gene is almost exclusively expressed in the adrenals and gonads (6, 8).

Our study also indicates that the cell specificity of IL-4 action on 3ß-HSD expression is not related to levels in 3ß-HSD activity under basal cell growth conditions. Indeed, the stimulation of 3ß-HSD expression by IL-4 was observed in cells that do not have any basal 3ß-HSD activity (PrEC, ZR-75–1, HaCaT and ME-180) as well as in cell lines having significant basal levels of activity (BT-20 and HT-29). On the other hand, the absence of IL-4 action in cell lines with (LnCAP, Caco-2, JAR, and JEG-3) or without (PC-3) 3ß-HSD activity under basal culture conditions further supports our conclusion.

Furthermore, the lack of a stimulatory effect of IL-4 on 3ß-HSD type 1 expression in some cell lines cannot be explained by the absence of Stat6 expression because this protein was expressed in the IL-4 nonresponsive PC-3, Caco-2, JAR, and JEG-3 cell lines. Rather, our study shows that the cell-specific action of IL-4 correlates with the activation of Stat6. Indeed, Stat6 was activated in all the cell types where the stimulatory effect of IL-4 on 3ß-HSD type 1 expression was observed, but not in those where IL-4 failed to regulate this parameter. However, it is of interest to note that IL-4 and IL-13 had the capacity to down regulate the production of the chemokine MCP-1 in Caco-2 cells (43) and to up-regulate the expression of the CD44 transmembrane adhesion receptor in HT-29 and Caco-2 (44). Thus, even if Caco-2 is an IL-4 responsive cell line, IL-4 failed to activate Stat6 and to regulate 3ß-HSD type 1 expression in this cell line, thus further supporting our hypothesis.

We have previously characterized two Stat6 DNA-binding elements in the 3ß-HSD type 1 gene promoter (11). We found that two 1-bp substitutions in the Stat6 consensus sequence of the 3ß-HSD type 1 Stat6#1 and Stat6#2 probes, disrupt Stat6 DNA-binding to these probes. We also observed that the 3ß-HSD type 1 Stat6#1 and Stat6#2 probes competed the binding to the classical Stat6 DNA-binding probe derived from the IgE constant region gene promoter. However, we failed after extensive efforts using several alternative experimental procedures to observe transactivation of a luciferase reporter gene under the control of DNA fragment from the upstream region (-1082 to +182) of the 3ß-HSD type 1 gene promoter (11). The present data thus further support our previous hypothesis that the activation of Stat6 is essential for the stimulation of 3ß-HSD type 1 gene transcription by IL-4 (11), because Stat6 was activated by IL-4 in all the cells where IL-4 induces 3ß-HSD type 1 expression, but not in any of the unresponsive cells.

The IL-4-induced regulation of 3ß-HSD activity, which catalyzes an essential step in the formation of active androgens and estrogens from DHEA, in a large number of peripheral tissues, could play an important role in both physiological and pathological conditions. In this regard, it is now well recognized that the amounts of TESTO and DHT synthesized locally in the prostate from inactive adrenal precursors are estimated to be comparable to the quantity of TESTO and DHT of testicular origin. Indeed, after elimination of testicular androgens by medical or surgical castration the intraprostatic concentration of DHT, which is the most meaningful parameter of androgenic action in prostatic tissue, remains at approximately 40% of that measured in the prostate, in intact 65-year-old men, thus leaving important amounts of free androgen to continue stimulating growth of the prostate cancer (3, 45, 46). In IL-4-treated PrEC, the major product formed from 5-DIOL is 4-DIONE. It arises from a transient formation of TESTO (catalyzed by the IL-4-induced 3ß-HSD type 1 activity) and DHEA (catalyzed by the oxidative 17ß-HSD activity). It has recently been shown that 17ß-HSD type 5 is expressed in the human prostate (47, 48) and this enzyme has a reductive activity (5, 49). Thus, the induction of 3ß-HSD activity by IL-4 and IL-13 would markedly increase the formation, from DHEA, of the 17ß-HSD type 5 substrates 4-DIONE and A-DIONE, which would lead to the synthesis of TESTO and DHT, respectively. This may well have a significant impact on the development of prostate cancer because the effect of prolonged presence of androgens that stimulate prostate cancer growth is well established (4, 50). The relevance of the IL-4 action in prostate cells also pertains to the observation that in an immortalized human prostate cell line derived from the primary cultures, the gene expression of the tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-2 (MMP-2) was regulated by IL-4 (51). It was suggested that IL-4 may control the molar ratio of TIMP-1 and MMP-2 to influence the level of protease activity and perhaps the invasive behavior of malignant cells in vivo (51).

The present study also shows for the first time the induction of 3ß-HSD type 1 gene expression by IL-4 in HaCaT human immortalized keratinocytes. The physiological relevance of this finding is well supported by the observation that 1) human skin can convert DHEA into TESTO (26); 2) DHEA can stimulate sebaceous gland secretion (52, 53); 3) the in vivo sebum secretion rate in humans is closely correlated with 3ß-HSD activity (54); and 4) 3ß-HSD type 1 gene is expressed in human skin (8, 27). Moreover, it is interesting that the rat 3ß-HSD type 4, which is the exclusive 3ß-HSD gene expressed in the skin, is also regulated by cytokines in rat skin (55, 56). It has been reported that IL-4 and IL-13 stimulate IL-6 expression in normal keratinocytes and keratinocyte cell lines of human origin (57), and that IL-4 induces proliferation of normal human keratinocytes, which is associated with c-myc gene expression (58). Therefore, it is likely that these cytokines play a role in the regulation of inflammation at both systemic and local levels. Finally, IL-4 can be detected in the human skin in pathological conditions, such as atopic dermatitis and during the elicitation phase of contact sensitivity (21, 28). Thus, in view of the observation that IL-4 induced 3ß-HSD type 1 gene expression in HaCaT immortalized keratinocytes, it would be relevant to investigate the potential effect of androgens or estrogens in those pathologies as well as in the physiological functions of keratinocytes.

Our original information showing the marked stimulation of the 3ß-HSD activity by IL-4 in the HT-29 human colon cancer cells suggested a role for IL-4 and sex steroids, at least in a subset of colon cancers. In support of this hypothesis, it has been reported that IL-4 inhibited the HT-29 cell proliferation (59) and IL-4 is commonly expressed by colon carcinoma tumor-infiltrating lymphocytes and is associated with improved survival (60). There is also increasing amount of data indicating that the colon is a sex steroids target tissue. First, sex steroid hormone receptors are known to be expressed in normal and tumoral colon specimens (61, 62), and it was postulated that sex steroids may influence the function of colonocytes indirectly through stromal-epithelial interactions (63). Furthermore, higher levels of the AR was detected in normal adjacent mucosa compared with colorectal adenomas suggesting a protective role of androgens in colonic mucosa (64). Estrogens also have a protective action of sex steroids on colorectal cancers and expression of the exogenous ER in cultured colon carcinoma cells resulted in marked growth suppression (65). Moreover, epidemiogical analyses suggested that the risk for colorectal cancer was decreased significantly among women who used postmenopausal hormonal replacement therapy (66, 67, 68). However, it should be noted that other studies using several colon cancer cell lines demonstrated that the effect of estrogens and androgens on cell growth may be different (69, 70, 71). Finally, the induction of 3ß-HSD in HT-29 cells is interesting, especially knowing that long-term administration of DHEA to Balb/c mice significantly inhibits the rate of appearance of 1,2-dimethylhydrazine-induced colon tumors (72). Most likely DHEA, is not exerting that effect on its own, but its effect would be exerted by its conversion into active androgens or estrogens, which first required 3ß-HSD activity.

It has been reported that both JAR and JEG-3 human choriocarcinoma cell lines produce IL-4 and express the IL-4R (24). Moreover, IL-4 and IL-13 were detected in human placental samples. It has also been suggested that colocalization of IL-4 and IL-4R within gestational tissues may indicate autocrine and/or paracrine mechanisms of action for these cytokines within the uteroplacental unit (23, 31). Because 3ß-HSD type 1 activity is responsible for the formation of progesterone, which is critical for placental function, it is tempting to speculate that IL-4 might act as an autocrine factor regulating the expression of 3ß-HSD in the placenta. However, we have failed to observe an increase in 3ß-HSD gene expression after treatment with IL-4 in JAR and JEG-3 cell lines. In addition, we were unable to detect, by ELISA, either IL-4 or IL-13 in the supernatant of both cell lines (data not shown). Therefore the effect of IL-4 on 3ß-HSD expression in the normal placenta remains to be determined.

The present findings provide further evidence for the role of Stat6 in the induction of 3ß-HSD type 1 gene expression by IL-4. Our data also strongly suggest that IL-4 and IL-13 may play a crucial role in the biosynthesis of active sex steroids from inactive adrenal precursors in most of the peripheral tissues where 3ß-HSD type 1 is expressed. Finally, the wide spread 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.


   Footnotes
 
1 Financial support was provided by the Medical Research Council of Canada (MRC Group in Molecular Endocrinology) and Endorecherche. Back

2 Holds a studentship from MRC. Back

3 Senior Scientist from Le Fonds de la Recherche en Santé du Québec (FRSQ). Back

Received January 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Labrie F 1991 Intracrinology. Mol Cell Endocrinol 78:C113–C118
  2. Grover PK, Odell WD 1975 Correlation of in vivo and in vitro activities of some naturally occurring androgens using a radioreceptor assay for 5{alpha}-dihydrotestosterone with rat prostate cytosol receptor protein. J Steroid Biochem 6:1373–1379[CrossRef][Medline]
  3. Labrie F, Dupont A, Bélanger A 1985 Complete androgen blockade for the treatment of prostate cancer. In: de Vita S, Hellman S, Rosenberg SA, (eds) Important Advances in Oncology. Lippincott, Philadelphia, pp 193–217
  4. Labrie F, Belanger A, Cusan L, Labrie C, Simard J, Luu-The V, Diamond P, Gomez JL, Candas B 1996 History of LHRH agonist and combination therapy in prostate cancer. Endocrine-Related Cancer 3:243–278
  5. Labrie F, Luu The V, Lin SX, Labrie C, Simard J, Breton R, Belanger A 1997 The key role of 17ß-hydroxysteroid dehydrogenases in sex steroid biology. Steroids 62:148–158[CrossRef][Medline]
  6. Simard J, Durocher F, Mebarki F, Turgeon C, Sanchez R, Labrie Y, Couet J, Trudel C, Rheaume E, Morel Y, Luu The V, Labrie F 1996 Molecular biology and genetics of the 3ß-hydroxysteroid dehydrogenase//{Delta}5-{Delta}4 isomerase gene family. J Endocrinol [Suppl] 150:S189–S207
  7. Russell DW, Wilson JD 1994 Steroid 5{alpha}-reductase: two genes/two enzymes. Annu Rev Biochem 63:25–61[Medline]
  8. Rheaume E, Lachance Y, Zhao HF, Breton N, Dumont M, de Launoit Y, Trudel C, Luu The V, Simard J, Labrie F 1991 Structure and expression of a new complementary DNA encoding the almost exclusive 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase in human adrenals and gonads. Mol Endocrinol 5:1147–1157[Abstract]
  9. Simard J, Rheaume E, Sanchez R, Laflamme N, de Launoit Y, Luu The V, van Seters AP, Gordon RD, Bettendorf M, Heinrich U, Moshang T, New M, Labrie F 1993 Molecular basis of congenital adrenal hyperplasia due to 3ß-hydroxysteroid dehydrogenase deficiency. Mol Endocrinol 7:716–728[Abstract]
  10. Rheaume E, Simard J, Morel Y, Mebarki F, Zachmann M, Forest MG, New MI, Labrie F 1992 Congenital adrenal hyperplasia due to point mutations in the type II 3ß-hydroxysteroid dehydrogenase gene. Nat Genet 1:239–245[CrossRef][Medline]
  11. Gingras S, Moriggl R, Groner B, Simard J 1999 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. Mol Endocrinol 13:66–81[Abstract/Free Full Text]
  12. Zurawski G, de Vries JE 1994 Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells. Immunol Today 15:19–26[CrossRef][Medline]
  13. Zurawski SM, Vega Jr F, Huyghe B, Zurawski G 1993 Receptors for interleukin-13 and interleukin-4 are complex and share a novel component that functions in signal transduction. EMBO J 12:2663–2670[Medline]
  14. Murata T, Taguchi J, Puri R 1998 Interleukin-13 receptor {alpha}' but not {alpha} chain: a functional component of interleukin-4 receptors. Blood 91:3884–3891[Abstract/Free Full Text]
  15. Paul WE 1991 Interleukin-4: a prototypic immunoregulatory lymphokine. Blood 77:1859–1870[Free Full Text]
  16. Zurawski G, de Vries JE 1994 Interleukin 13 elicits a subset of the activities of its close relative interleukin 4. Stem Cells 12:169–174[Abstract]
  17. Debinski W, Puri RK, Kreitman RJ, Pastan I 1993 A wide range of human cancers express interleukin-4 (IL-4) receptors that can be targeted with chimeric toxin composed of IL-4 and pseudomonas exotoxin. J Biol Chem 268:14065–14070[Abstract/Free Full Text]
  18. Kaklamanis L, Gatter KC, Mortensen N, Harris AL 1992 Interleukin-4 receptor and epidermal growth factor receptor expression in colorectal cancer. Br J Cancer 66:712–716[Medline]
  19. Murata T, Noguchi PD, Puri RK 1996 IL-13 induces phosphorylation and activation of JAK2 Janus kinase in human colon carcinoma cell lines: similarities between IL-4 and IL-13 signaling. J Immunol 156:2972–2978[Abstract]
  20. Topp MS, Papadimitriou CA, Eitelbach F, Oelmann E, Koehler B, Oberberg D, Von Marschall Z, Reufi B, Stein H, Thiel E, Winton EF, Berdel WE 1995 Antiproliferative effect of human interleukin-4 in human cancer cell lines: studies on the mechanism. Leuk Lymphoma 19:319–328[Medline]
  21. Junghans V, Jung T, Neumann C 1996 Human keratinocytes constitutively express IL-4 receptor molecules and respond to IL-4 with an increase in B7/BB1 expression. Exp Dermatol 5:316–324[CrossRef][Medline]
  22. Prens E, Hegmans J, Lien RC, Debets R, Troost R, van Joost T, Benner R 1996 Increased expression of interleukin-4 receptors on psoriatic epidermal cells. Am J Pathol 148:1493–1502[Abstract]
  23. Henriques CU, Rice GE, Wong MH, Bendtzen K 1998 Immunolocalisation of interleukin-4 and interleukin-4 receptor in placenta and fetal membranes in association with pre-term labour and pre-eclampsia. Gynecol Obstet Invest 46:172–177[CrossRef][Medline]
  24. de Moraes Pinto MI, Vince GS, Flanagan BF, Hart CA, Johnson PM 1997 Localization of IL-4 and IL-4 receptors in the human term placenta, decidua and amniochorionic membranes. Immunology 90:87–94[CrossRef][Medline]
  25. Schmitt Ney M, Doppler W, Ball RK, Groner B 1991 ß-casein gene promoter activity is regulated by the hormone-mediated relief of transcriptional repression and a mammary-gland-specific nuclear factor. Mol Cell Biol 11:3745–3755[Abstract/Free Full Text]
  26. Thomas JP, Oake RJ 1974 Androgen metabolism in the skin of hirsute women. J Clin Endocrinol Metab 38:19–22[Medline]
  27. Dumont M, Luu The V, Dupont E, Pelletier G, Labrie F 1992 Characterization, expression, and immunohistochemical localization of 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase in human skin. J Invest Dermatol 99:415–421[CrossRef][Medline]
  28. Asada H, Linton J, Katz SI 1997 Cytokine gene expression during the elicitation phase of contact sensitivity: regulation by endogenous IL-4. J Invest Dermatol 108:406–411[CrossRef][Medline]
  29. Salerno A, Dieli F, Sireci G, Bellavia A, Asherson GL 1995 Interleukin-4 is a critical cytokine in contact sensitivity. Immunology 84:404–409[Medline]
  30. Seidel HM, Milocco LH, Lamb P, Darnell JE, Jr, Stein RB, Rosen J 1995 Spacing of palindromic half sites as a determinant of selective STAT (signal transducers and activators of transcription) DNA binding and transcriptional activity. Proc Natl Acad Sci USA 92:3041–3045[Abstract/Free Full Text]
  31. Dealtry G, Clark D, Sharkey A, CharnockJones D, Smith S 1998 Expression and localization of the Th2-type cytokine interleukin-13 and its receptor in the placenta during human pregnancy. Am J Reprod Immunol 40:283–290
  32. Ihle JN 1996 STATs: signal transducers and activators of transcription. Cell 84:331–334[CrossRef][Medline]
  33. Leonard W, O’Shea J 1998 JAKS and STATS: biological implications. Annu Rev Immunol 16:293–322[CrossRef][Medline]
  34. Darnell Jr JE 1997 STATs, and gene regulation. Science 277:1630–1635[Abstract/Free Full Text]
  35. Quelle FW, Shimoda K, Thierfelder W, Fischer C, Kim A, Ruben SM, Cleveland JL, Pierce JH, Keegan AD, Nelms K, Paul WE, Ihle JN 1995 Cloning of murine Stat6 and human Stat6, Stat proteins that are tyrosine phosphorylated in responses to IL-4 and IL-3 but are not required for mitogenesis. Mol Cell Biol 15:3336–3343[Abstract]
  36. Hou J, Schindler U, Henzel WJ, Ho TC, Brasseur M, McKnight SL 1994 An interleukin-4-induced transcription factor: IL-4 Stat. Science 265:1701–1706[Abstract/Free Full Text]
  37. Kohler I, Alliger P, Minty A, Caput D, Ferrara P, Holl Neugebauer B, Rank G, Rieber EP 1994 Human interleukin-13 activates the interleukin-4-dependent transcription factor NF-IL4 sharing a DNA binding motif with an interferon-{gamma}-induced nuclear binding factor. FEBS Lett 345:187–192[CrossRef][Medline]
  38. Shimoda K, van Deursen J, Sangster MY, Sarawar SR, Carson RT, Tripp RA, Chu C, Quelle FW, Nosaka T, Vignali DA, Doherty PC, Grosveld G, Paul WE, Ihle JN 1996 Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 380:630–633[CrossRef][Medline]
  39. Kaplan MH, Schindler U, Smiley ST, Grusby MJ 1996 Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. Immunity 4:313–319[CrossRef][Medline]
  40. Takeda K, Kishimoto T, Akira S 1997 STAT6: its role in interleukin-4-mediated biological functions. J Mol Med 75:317–326[CrossRef][Medline]
  41. Mikita T, Campbell D, Wu P, Williamson K, Schindler U 1996 Requirements for interleukin-4-induced gene expression and functional characterization of Stat6. Mol Cell Biol 16:5811–5820[Abstract]
  42. Kotanides H, Reich NC 1993 Requirement of tyrosine phosphorylation for rapid activation of a DNA binding factor by IL-4. Science 262:1265–1267[Abstract/Free Full Text]
  43. Kucharzik T, Lugering N, Pauels HG, Domschke W, Stoll R 1998 IL-4, IL-10 and IL-13 down-regulate monocyte-chemoattracting protein-1 (MCP-1) production in activated intestinal epithelial cells. Clin Exp Immunol 111:152–157[CrossRef][Medline]
  44. Trejdosiewicz LK, Morton R, Yang Y, Banks RE, Selby PJ, Southgate J 1998 Interleukins 4 and 13 upregulate expression of CD44 in human colonic epithelial cell lines. Cytokine 10:756–765[CrossRef][Medline]
  45. Labrie F, Simard J, Luu The V, Pelletier G, Belghmi K, Belanger A 1994 Structure, regulation and role of 3ß-hydroxysteroid dehydrogenase, 17ß-hydroxysteroid dehydrogenase and aromatase enzymes in the formation of sex steroids in classical and peripheral intracrine tissues. Baillieres Clin Endocrinol Metab 8:451–474[CrossRef][Medline]
  46. Labrie F, Belanger A, Cusan L, Candas B 1997 Physiological changes in dehydroepiandrosterone are not reflected by serum levels of active androgens and estrogens but of their metabolites: intracrinology. J Clin Endocrinol Metab 82:2403–2409[Abstract/Free Full Text]
  47. Lin HK, Jez JM, Schlegel BP, Peehl DM, Pachter JA, Penning TM 1997 Expression and characterization of recombinant type 2 3{alpha}-hydroxysteroid dehydrogenase (HSD) from human prostate: demonstration of bifunctional 3{alpha}/17{alpha}-HSD activity and cellular distribution. Mol Endocrinol 11:1971–1984[Abstract/Free Full Text]
  48. El-Alfy M, Luu-The V, Huang X-F, Berger L, Labrie F, Pelletier G 1999 Localization of type 5 17ß-hydroxysteroid dehydrogenase, 3ß-hydroxysteroid dehydrogenase and androgen receptor in the human prostate by in situ hybridization and immunocytochemistry. Endocrinology 140:1481–1491[Abstract/Free Full Text]
  49. Dufort F, Rheault P, Huang X-F, Soucy P, Luu-The V 1999 Characteristics of a highly labile human type 5 17ß-hydroxysteroid dehydrogenase. Endocrinology 140:568–574[Abstract/Free Full Text]
  50. Wilding G 1992 The importance of steroid hormones in prostate cancer. Cancer Surv 14:113–130[Medline]
  51. Wang M, Fudge K, Rhim JS, Stearns ME 1996 Cytokine regulation of the matrix metalloproteinases and their inhibitors in human papillomavirus-18 transformed human prostatic tumor cell lines. Oncol Res 8:303–315[Medline]
  52. Pochi PE, Strauss JS 1969 Sebaceous gland response in man to the administration of testosterone, {Delta}-4-androstenedione, and dehydroisoandrosterone. J Invest Dermatol 52:32–36[Medline]
  53. Drucker WD, Blumberg JM, Gandy HM, David RR, Verde AL 1972 Biologic activity of dehydroepiandrosterone sulfate in man. J Clin Endocrinol Metab 35:48–54[Medline]
  54. Simpton NB, Cunliffe WJ, Hodgins MB 1983 The relationship between the vitro activity of 3ß-hydroxysteroid dehydrogenase {Delta}5-{Delta}4-isomerase in human sebaceous glands and their secretory activity in vivo. J Invest Dermatol 81:139–144[CrossRef][Medline]
  55. Simard J, Couet J, Durocher F, Labrie Y, Sanchez R, Breton N, Turgeon C, Labrie F 1993 Structure and tissue-specific expression of a novel member of the rat 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase (3ß-HSD) family. The exclusive 3ß-HSD gene expression in the skin. J Biol Chem 268:19659–19668[Abstract/Free Full Text]
  56. Couet J, Martel C, Labrie Y, Luo S, Simard J, Labrie F 1994 Opposite effects of prolactin and corticosterone on the expression and activity of 3ß-hydroxysteroid dehydrogenase/{Delta}5-{Delta}4 isomerase in rat skin. J Invest Dermatol 103:60–64[CrossRef][Medline]
  57. Derocq JM, Segui M, Poinot-Chazel C, Minty A, Caput D, Ferrara P, Casellas P 1994 Interleukin-13 stimulates interleukin-6 production by human keratinocytes. Similarity with interleukin-4. FEBS Lett 343:32–36[CrossRef][Medline]
  58. Yang Y, Yoo HM, Choi I, Pyun KH, Byun SM, Ha H 1996 Interleukin 4-induced proliferation in normal human keratinocytes is associated with c-myc gene expression and inhibited by genistein. J Invest Dermatol 107:367–372[CrossRef][Medline]
  59. Toi M, Bicknell R, Harris AL 1992 Inhibition of colon and breast carcinoma cell growth by interleukin-4. Cancer Res 52:275–279[Abstract/Free Full Text]
  60. Barth Jr RJ, Camp BJ, Martuscello TA, Dain BJ, Memoli VA 1996 The cytokine microenvironment of human colon carcinoma. Lymphocyte expression of tumor necrosis factor-{alpha} and interleukin-4 predicts improved survival. Cancer 78:1168–1178[CrossRef][Medline]
  61. Alford TC, Do HM, Geelhoed GW, Tsangaris NT, Lippman ME 1979 Steroid hormone receptors in human colon cancers. Cancer 43:980–984[CrossRef][Medline]
  62. Wilson CM, McPhaul MJ 1996 A and B forms of the androgen receptor are expressed in a variety of human tissues. Mol Cell Endocrinol 120:51–57[CrossRef][Medline]
  63. Waliszewski P, Blaszczyk M, Wolinska-Witort E, Drews M, Snochowski M, Hurst RE 1997 Molecular study of sex steroid receptor gene expression in human colon and in colorectal carcinomas. J Surg Oncol 64:3–11[CrossRef][Medline]
  64. Marugo M, Aste H, Bernasconi D, Fazzuoli L, Conio M, Cuva A, Meozzi M 1992 Cytosolic and nuclear androgen receptors in colorectal adenomas. Anticancer Res 12:705–708[Medline]
  65. Issa JP, Ottaviano YL, Celano P, Hamilton SR, Davidson NE, Baylin SB 1994 Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nat Genet 7:536–540[CrossRef][Medline]
  66. Grodstein F, Martinez ME, Platz EA, Giovannucci E, Colditz GA, Kautzky M, Fuchs C, Stampfer MJ 1998 Postmenopausal hormone use and risk for colorectal cancer and adenoma. Ann Intern Med 128:705–712[Abstract/Free Full Text]
  67. Fernandez E, La Vecchia C, Braga C, Talamini R, Negri E, Parazzini F, Franceschi S 1998 Hormone replacement therapy and risk of colon and rectal cancer. Cancer Epidemiol Biomarkers Prev 7:329–333[Abstract]
  68. Hebert-Croteau N 1998 A meta-analysis of hormone replacement therapy and colon cancer in women. Cancer Epidemiol Biomarkers Prev 7:653–659[Abstract]
  69. Xu X, Thomas ML 1994 Estrogen receptor-mediated direct stimulation of colon cancer cell growth in vitro. Mol Cell Endocrinol 105:197–201[CrossRef][Medline]
  70. Lointier P, Wildrick DM, Boman BM 1992 The effects of steroid hormones on a human colon cancer cell line in vitro. Anticancer Res 12:1327–1330[Medline]
  71. Harrison JD, Watson S, Morris DL 1989 The effect of sex hormones and tamoxifen on the growth of human gastric and colorectal cancer cell lines. Cancer 63:2148–2151[CrossRef][Medline]
  72. Nyce JW, Magee PN, Hard GC, Schwartz AG 1984 Inhibition of 1,2-dimethylhydrazine-induced colon tumorigenesis in Balb/c mice by dehydroepiandrosterone. Carcinogenesis 5:57–62[Abstract/Free Full Text]



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