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Endocrinology Vol. 143, No. 9 3284-3294
Copyright © 2002 by The Endocrine Society

ARTICLE

Stimulation of Hepatic Signal Transducer and Activator of Transcription 5b by GH Is Not Altered by 3-Methylcholanthrene

Yoav E. Timsit and David S. Riddick

Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8

Address all correspondence and requests for reprints to: David S. Riddick, Ph.D., Department of Pharmacology, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. E-mail: david.riddick{at}utoronto.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We are investigating the mechanisms by which aromatic hydrocarbons, such as 3-methylcholanthrene (MC), suppress hepatic cytochrome P450 2C11 (CYP2C11) gene expression. CYP2C11 is an enzyme expressed in the liver of male rats and is regulated by a pulsatile pattern of GH secretion. We have previously shown that MC attenuates the stimulatory effect of GH on CYP2C11 expression in hypophysectomized male rats. In follow-up studies we evaluated the effect of MC on GH-stimulated signal transducer and activator of transcription 5b (STAT5b) phosphorylation, nuclear translocation, and DNA-binding activity. GH-stimulated increases in hepatic nuclear STAT5b and phospho-STAT5b levels were not different between groups of hypophysectomized rats receiving MC or vehicle. This observation was corroborated at the DNA-binding level by EMSA. We also measured GH-induced STAT5b activation in the H4IIE rat hepatoma cell line. STAT5b DNA-binding activity detected in GH-treated cells was not affected by MC. Immunocytochemistry experiments revealed no effect of MC on GH-stimulated STAT5b nuclear translocation in H4IIE cells. These in vivo and in vitro data suggest that interference with GH-stimulated STAT5b activation does not constitute a mechanism by which MC attenuates the stimulatory effect of GH on CYP2C11 gene expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HALOGENATED aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, and polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are ubiquitous environmental pollutants whose endocrine-disrupting effects are well documented (1, 2, 3, 4, 5). Most adaptive and toxic responses to aromatic hydrocarbons are mediated by a ligand-activated transcription factor known as the aromatic hydrocarbon receptor (AHR) (1, 2, 3, 4). Endocrine effects of these compounds include modulation of the expression of many genes, such as the estrogen receptor, glucocorticoid receptor, and progesterone receptor (6, 7, 8); growth factors and related receptors (9, 10); genes that play a role in androgen, estrogen, and thyroid hormone metabolism (1, 3); and numerous hormone-regulated genes, such as pS2 and cathepsin D (5). Due to the diversity of the genes involved in homeostatic processes that are regulated by endocrine factors, endocrine disruption plays a significant role in the etiology of toxic responses elicited by aromatic hydrocarbons. Hence, changes in tissue function at the cellular level in this context can often be viewed as a response secondary to endocrine disruption.

In addition to endocrine disruption caused by alterations in hormone levels or hormone receptors, xenobiotics may also directly interfere with hormone-stimulated signal transduction pathways. Studies of substances belonging to a variety of chemical classes provide useful examples of this type of endocrine disruption. These include the attenuating effect of dexamethasone on estrogen-stimulated rat uterine IGF-I mRNA expression (11), the suppressive effect of insulin on T3-stimulated GH mRNA levels in rat pituitary cells (12), and the suppressive effect of ethanol on rat hepatic cytochrome P450 2C11 (CYP2C11) expression causally related to altered GH secretion (13). This type of interaction has also been implicated in the suppression of CYP2C11 expression caused by aromatic hydrocarbons. CYP2C11, the predominant CYP enzyme expressed constitutively in the liver of adult male rats (14), is down-regulated by aromatic hydrocarbons, including the prototypical model compound 3-methylcholanthrene (MC) (15, 16, 17). Our in vivo studies indicate that MC down-regulates hepatic CYP2C11 gene expression at least partially via a transcriptional mechanism (17, 18), and there is some evidence for a role of the AHR in this process (19). An interaction with an endocrine process is inferred from studies that demonstrated a loss in the ability of MC and small aromatic hydrocarbons such as ethylbenzene to suppress CYP2C11 expression in hypophysectomized (hypx) male rats (20, 21). These studies suggest that aromatic hydrocarbons may modulate hepatic gene expression by disrupting pituitary-dependent signaling, and our recent demonstration of an attenuating effect of MC on GH-stimulated hepatic CYP2C11 expression in hypx male rats (22) provides additional support for such a mechanism.

The goal of the present study was to investigate a potential mechanism by which MC may disrupt hepatic GH signaling. In view of the role played by GH-activated signal transducer and activator of transcription 5b (STAT5b) in the regulation of expression of CYP2C11 (23) and other male-specific CYPs (24), we hypothesized that MC attenuates GH-stimulated CYP2C11 expression by altering the activation and DNA-binding activity of STAT5b. Such a mechanism is not without precedence, as it has been shown that STAT activation is inhibited by substances such as endotoxin (STAT5) (25), ethanol (STAT3) (26, 27), and insulin (STAT5) (28). In addition, inhibitory cross-talk has been observed between GH-activated STAT5b and the peroxisome proliferator-activated receptor-{alpha} pathway (29), and 2,4,4'-trichlorobiphenyl has recently been shown to modulate STAT5 activity (30). Therefore, in this study we measured STAT5b and phosphorylated STAT5b levels in hepatic nuclear extracts prepared from hypx rats treated with GH in the presence or absence of MC cotreatment. GH-induced STAT5b DNA-binding activity was also assessed in these extracts. Furthermore, we have developed an H4IIE rat hepatoma cell culture model to investigate the effect of MC on GH-stimulated STAT5b activation. In this cell culture model, we evaluated the effect of MC on GH-inducible STAT5b DNA-binding activity; STAT5b nuclear translocation was also assessed by immunocytochemistry. Thus, we have employed a combination of in vivo- and in vitro-based strategies to examine the effect of a representative aromatic hydrocarbon on GH-stimulated STAT5b activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Source of chemicals
Rat GH (rGH-B-13, BIO) was supplied by the NIDDK’s National Hormone and Pituitary Program and Dr. Albert Parlow. [{gamma}-32P]ATP (specific activity, >5000 Ci/mmol; radiochemical purity, >95%) and T4 polynucleotide kinase were purchased from Amersham Pharmacia Biotech (Baie d’Urfé, Canada). MC (chemical purity, 98%) was purchased from Sigma-Aldrich (St. Louis, MO). Electrophoresis and blotting equipment and reagents were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Poly[d(I-C)] was obtained from Roche (Laval, Canada). {alpha}MEM was purchased from Media Preparation Services, University of Toronto (Toronto, Canada). FBS was obtained from Life Technologies, Inc. (Gaithersburg, MD). Plasmid purification kits were purchased from QIAGEN (Valencia, CA). Transfast transfection reagent, the pRL-TK Renilla luciferase vector, and luciferase assay reagents were obtained from Promega Corp. (Madison, WI).

Animals and treatment
Male Fischer 344 rats were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Hypophysectomy operations were performed at 8 wk of age by the supplier. Rats received 5% glucose in the drinking water for 1 wk after surgery. Rats were acclimatized to living and handling conditions for a total of 13 d after arrival in the Division of Comparative Medicine, University of Toronto. Body weights were monitored during this time and during the treatment schedule to establish the effectiveness of hypophysectomy. Rats were fed Purina Rodent Laboratory Chow (no. 5001, Ralston Purina, St. Louis, MO) and water ad libitum and were housed under controlled conditions (two per cage; 12-h light, 12-h dark cycle, with lights on at 0700 h). All animals were cared for in accordance with the principles of the Canadian Council on Animal Care, and all animal experimentation was approved by the University of Toronto animal care committee. Treatments were performed as described previously (22) with some modifications. Groups of four or five hypx rats received the following treatments: vehicle (-MC/-GH), GH alone (-MC/+GH), MC alone (+MC/-GH), and both MC and GH (+MC/+GH). Rats received either MC (20 mg/kg) or corn oil by gavage on d 1, 3, and 5. A single ip injection of GH (125 µg/kg) or saline was administered on d 5, 1 h after the final gavage treatment. Rats were killed by decapitation on d 5, 1 h after the GH or saline injection. Livers were immediately removed and weighed, and nuclear extracts were prepared by standard differential centrifugation methods (31, 32, 33). Nuclear extracts were dialyzed twice for 2 h at 4 C in nuclear extract buffer [25 mM HEPES, 40 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 0.5 mM phenylmethylsulfonylfluoride (PMSF), 0.5 mM NaF, and 1 mM sodium orthovanadate, pH 7.6]. Nuclear extracts were frozen and stored in liquid nitrogen until use. Protein concentrations were determined by the method described by Bradford (34).

Cell culture
The rat hepatoma cell line H4IIE was obtained from the American Type Culture Collection (Manassas, VA). Cells were grown as monolayer cultures in antibiotic-free {alpha}MEM supplemented with 10% FBS and were maintained in an atmosphere of 5% CO2 and 95% air at 37 C. For treatments, cells were seeded into 60-mm culture dishes and maintained in complete medium. When cell growth reached approximately 50% confluence, cells were washed twice with PBS and maintained in serum-free {alpha}MEM for 24 h. The medium was then replaced with serum-free {alpha}MEM containing the appropriate combination of MC (10 nM, 100 nM, or 1 µM), or dimethylsulfoxide (DMSO) vehicle and GH (500 ng/ml), or saline vehicle in accordance with the treatment conditions of the experiment. At the end of treatment, cells were harvested, and whole cell extracts were prepared as previously described (28). Briefly, cells were quickly washed with PBS and then scraped into 1 ml ice-cold wash buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 50 mM NaF, 0.5 mM sodium orthovanadate, and 1 mM PMSF]. Cells were collected by centrifugation at 800 x g for 2 min and resuspended in lysis buffer [50 mM Tris (pH 8.0), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1.5 mM MgCl2, 1 mM EDTA, 10 mM sodium pyrophosphate, 50 mM NaF, 1 mM sodium orthovanadate, 1 mM PMSF, 10 mM benzamidine, and 2 µg/ml aprotinin]. Cell lysis was carried out for 1 h at 4 C with gentle rocking, and extracts were centrifuged at 15,000 x g for 15 min. Supernatants were frozen and stored in liquid nitrogen until use. Whole cell extract protein concentrations were determined by the method of Bradford (34).

Transient transfection and luciferase assay
Two GH-responsive luciferase constructs were provided by Dr. Bernd Groner (Institute of Biomedical Research, Frankfurt, Germany): 1) ß-casein-luciferase (ß-cas-luc), in which luciferase is under control of the ß-casein 5'-flank and promoter; and 2) (STAT5)6-thymidine kinase-luc, in which luciferase is under control of six copies of a consensus STAT5 site fused upstream of the thymidine kinase promoter. H4IIE cells were cultured in six-well plates to approximately 80% confluence, at which time transfection was performed. Cells were overlaid with 1 ml serum-free medium containing 3.3 µg luciferase, 0.5 µg Renilla plasmid DNA, and 22.5 µl Transfast reagent. Cells were incubated with the transfection mixture for 1 h at 37 C, after which 4 ml complete medium were added to the wells. Cells were allowed to recover for 24 h before treatment with GH (500 ng/ml) or saline vehicle in serum-free {alpha}MEM. The following GH treatment protocols were used: continuous GH for 8, 16, or 24 h; a single 20-min GH pulse, followed by wash and culture in serum-free {alpha}MEM for 8 h; and one, two, or three 20-min GH pulses, each followed by wash and culture in serum-free {alpha}MEM for 8 h. At the end of treatment, cells were harvested, and cell extract (40 µl) was used for the luciferase/Renilla assays, which were performed according to the manufacturer’s instructions using the Dual Luciferase Reporter Assay System (Promega Corp.). Luciferase activity was normalized to Renilla activity. Cell extract protein concentrations were determined by the method of Bradford (34).

Immunoblot analysis
Antibodies were obtained from the following sources: rabbit polyclonal anti-STAT5b antibody from Dr. Li-Yuan Yu-Lee (Baylor College of Medicine, Houston, TX), and rabbit polyclonal anti-phospho-STAT5a/b (pSTAT5) antibody from Dr. David Frank (Dana-Farber Cancer Institute, Boston, MA). Antibody conditions for immunoblotting were as follows. For primary incubation, anti-STAT5b was used at a 1:3,000 dilution in TNT1 [20 mM Tris (pH 7.4), 150 mM NaCl, and 0.1% Tween 20] containing 1% skim milk powder or anti-pSTAT5 was used at a 1:10,000 dilution in TNT2 [10 mM Tris (pH 8.0), 150-mM NaCl, and 0.5% Tween 20]. For secondary incubation, donkey antirabbit Ig-horseradish peroxidase conjugate (Amersham Pharmacia Biotech) was used at a 1:5,000 dilution in TNT1 containing 1% skim milk powder for STAT5b immunoblots or in TNT2 for pSTAT5 immunoblots. Nuclear extract protein from each rat liver sample (3 µg for STAT5b detection and 5 µg for pSTAT5 detection) was resolved by SDS-PAGE (35) and transferred to nitrocellulose (Hybond-ECL, Amersham Pharmacia Biotech). An enhanced chemiluminescence system (Amersham Pharmacia Biotech) was used for protein detection, films were scanned on a SuperVista S-12 scanner (UMAX Technologies, Inc., Fremont, CA), and relative quantitation was performed using IPLab Gel for the Power Macintosh version 1.5e (Signal Analytics, Vienna, CA). STAT5b and pSTAT5 quantitative analyses were performed under conditions that yielded a linear relationship between the amount of nuclear extract protein and immunoreactive signal intensity.

Immunoprecipitation
Rat liver nuclear extracts (10 µg) were incubated with anti-STAT5b antibody (1:500 dilution) in a 0.5-ml volume of buffer A [50 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA, 0.25% gelatin, and 0.02% sodium azide] for 2 h at 4 C on an orbital shaker. Immune complexes were collected after an additional overnight incubation at 4 C with 60 µl of a 1:1 (v/v) mixture of protein A-agarose (Repligen Corp., Needham, MA) and buffer B [50 mM Tris (pH 7.5), 4% BSA, and 0.02% sodium azide]. Immunoprecipitated protein was eluted by boiling in 1x sample loading buffer [1x = 62.5 mM Tris (pH 6.8), 10% glycerol, 2% SDS, 5% ß-mercaptoethanol, and 0.001% bromophenol blue], and proteins were separated by SDS-PAGE. Proteins were transferred to nitrocellulose, followed by immunoblotting using anti-STAT5b antibody. The membranes were subsequently stripped and reprobed with anti-pSTAT5 antibody. Protein was visualized by enhanced chemiluminescence detection, and quantitation was performed as described above.

EMSA
The following pairs of cDNA oligonucleotides were synthesized and purified by ACGT Corp. (Toronto, Canada) and used in EMSA experiments, with the consensus STAT5-binding site underlined: ß-casein probe [wild-type (wt) ß-casein] containing bases -100 to -80 from the rat ß-casein gene (5'-GATCCCCCTTAATTCCAAGAAGTCCA-3' and 5'-GATCTGGACTTCTTGGAATTAAGGGG-3'), mutant ß-casein probe (mutated ß-casein) containing five mutations in the consensus STAT5 binding site (in bold; 5'-GATCCCCCTTAATTAGTTTAAGTCCA-3' and 5'-GATCTGGACTTAAACTAATTAAGGGG-3'), and CYP2C11 STAT5 probe (2C11 STAT) containing bases -1186 to -1167 of the CYP2C11 gene relative to the transcription start site (5'-GATCCAAACATTTTCCATGAAAAAAA-3' and 5'-GATCTTTTTTTCATGGAAAATGTTTG-3'). Each oligonucleotide was designed as a 26-mer with creation of BamHI and BglII sticky ends at the 5' and 3' ends, respectively, after annealing. Complementary pairs of oligonucleotides were annealed and used as unlabeled competitor probes, or some annealed oligonucleotides (wt ß-casein and 2C11 STAT) were end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. Nuclear extract (1 µg from rat liver) or whole cell extract (5 µg from H4IIE cells) was incubated with poly[d(I-C)] (0.5 µg) in the absence or presence of a specific concentration of unlabeled competitor probe for 15 min. The 32P-labeled probe was added (~100,000 cpm, 0.25–1 ng), and 15 min later the anti-STAT5b antibody (0.5 µl of a 1:10 dilution) was added. Binding reactions were carried out in 15-µl volumes in binding buffer [10 mM Tris (pH 7.4), 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, and NaCl (33.3 mM for liver nuclear extracts and 50 mM for H4IIE cell extracts)]. All incubations were performed at room temperature. Equal amounts of protein from each binding reaction were loaded, and protein-DNA complexes were analyzed by electrophoresis on nondenaturing 4% polyacrylamide gels and autoradiography. Relative quantitation was performed by PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA) using IPLab Gel for the Power Macintosh version 1.5e. Quantitative analysis of STAT5-DNA binding was performed under conditions that yielded a linear relationship between the amount of extract protein and the PhosphorImager signal.

Immunocytochemistry (ICC)
H4IIE cells were cultured in complete medium on sterile poly-D-lysine-coated cover slips placed in individual wells of six-well plates. Upon reaching approximately 50% confluence, cells were treated in serum-free {alpha}MEM with the appropriate combination of MC (1 µM) or DMSO vehicle and GH (500 ng/ml) or saline vehicle in accordance with the treatment conditions of the experiment. At the end of treatment, cells were washed with PBS and fixed with a 0.2 M phosphate buffer solution containing 4% paraformaldehyde for 30 min at room temperature. After three washes with PBS, cells were subjected to immunostaining. Briefly, cells were blocked with PBST (PBS plus 0.01% Triton X-100) containing 10% horse serum for 1 h with gentle rocking. Fixed cells were then incubated with primary antibody solution (PBST containing 2% horse serum and anti-STAT5b antibody at a 1:2000 dilution) for 48 h at 4 C. After three 10-min washes with PBST, cells were reblocked for 1 h at room temperature, followed by incubation with a 1:500 dilution of secondary antibody (biotinylated goat antirabbit IgG, Vector Laboratories, Inc., Burlingame, CA) in PBST containing 2% horse serum for 1 h at room temperature. Cells were washed twice with PBST, followed by quenching of endogenous peroxidase with 0.3% hydrogen peroxide in PBS, incubation with avidin-biotin-peroxidase conjugate (ABC Elite standard kit, Vector Laboratories, Inc.), and finally, diaminobenzidine staining with nickel ion enhancement (diaminobenzidine kit, Vector Laboratories, Inc.). Cells on coverslips were then dehydrated by sequential immersion in increasing concentrations of ethanol. After immersion in xylene, coverslips were mounted on slides, and stained cells were visualized by microscopy using a Zeiss Axioscop microscope (Carl Zeiss, New York, NY) at x100 magnification.

Statistical analysis
Where appropriate, data are expressed as the mean ± SD of multiple determinations. All statistical analyses were performed on the original raw data and not on the percent control data presented in the figures. For group comparisons, a randomized design one-way ANOVA was performed, followed by a post hoc Newman-Keuls test. A result was considered statistically significant if P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of MC on GH-stimulated STAT5b activation in vivo
A single ip dose of GH leads to rapid, high level tyrosine phosphorylation and nuclear accumulation of a 93-kDa liver protein identified as STAT5 in hypx male rats, and intermittent GH administration, mimicking the male GH secretion pattern, triggers repeated phosphorylation of this protein (33, 36). STAT5 activation by GH in male rats requires Janus kinase 2 (JAK2), a GH receptor-associated tyrosine kinase that phosphorylates STAT5 after hormone-induced dimerization of the GH receptor (37, 38). Intermittent GH administration also leads to STAT5 nuclear translocation and elevated DNA-binding activity (33, 36), resulting in transcriptional activation (39).

Consistent with these hepatic signal transduction events after GH stimulation in hypx male rats, we detected an approximately 93-kDa protein corresponding to STAT5b in nuclear extracts prepared 1 h after a single GH injection (Fig. 1AGo). Although it has been shown that hepatic nuclear STAT5b can be detected in hypx male rats as early as 15 min after a single GH injection, maximal elevation of the level of nuclear STAT5b is seen at about 45 min, and nuclear STAT5b levels return to background levels by 4 h (33). Treatment of hypx rats with MC alone did not result in STAT5b nuclear accumulation, whereas treatment with GH and MC resulted in a strong nuclear STAT5b signal (Fig. 1AGo). Quantitation of immunoreactive STAT5b in nuclear extracts under conditions that yielded a linear relationship between immunoreactive signal and the amount of loaded protein (Fig. 1BGo) revealed that the level of nuclear STAT5b was not different between hypx rats treated with both MC and GH and those treated with GH alone (Fig. 1CGo).



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  		Figure 1. Effect of MC on GH-stimulated STAT5b nuclear accumulation in vivo. A, Immunoblot analysis of hepatic nuclear extract protein (3 µg) using a STAT5b polyclonal antibody. Numbers on the left indicate the size of molecular mass markers in kilodaltons. Results for one representative hypx rat from each treatment group are shown. B, Standard curve showing a linear relationship between the immunoreactive band intensity and the amount of nuclear extract protein loaded for GH-treated hypx male rats. The equation of the line of best fit was generated by least squares linear regression analysis. C, Quantitative analysis of nuclear levels of STAT5b. Results are presented as a percentage of the mean for the -MC/+GH treatment group. All data are expressed as the mean ± SD of determinations from five rats. *, Significantly different (P < 0.01) from -MC/-GH and +MC/-GH groups based on randomized design, one-way ANOVA and post hoc Newman-Keuls test.

 
Similar results were obtained when levels of phosphorylated STAT5a/b were assessed in nuclear extracts of hypx rats from each of the four treatment groups using an anti-pSTAT5 polyclonal antibody (Fig. 2Go). However, this does not provide specific information on phosphorylation of the b isoform of STAT5, since the anti-pSTAT5 antibody recognizes both phosphorylated STAT5a and -5b. Therefore, to measure levels of STAT5b phosphorylation, nuclear extracts from hypx rats were subjected to immunoprecipitation using the STAT5b polyclonal antibody, followed by sequential immunoblotting with anti-STAT5b and then anti-pSTAT5 antibody. No difference was detected in the levels of hepatic nuclear pSTAT5b between hypx rats treated with both MC and GH and those treated with GH alone (data not shown).



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  		Figure 2. Effect of MC on GH-stimulated nuclear accumulation of phosphorylated STAT5a/b in vivo. A, Immunoblot analysis of hepatic nuclear extract protein (5 µg) using a pSTAT5 polyclonal antibody. Numbers on the left indicate the size of molecular mass markers in kilodaltons. Results for one representative hypx rat from each treatment group are shown. B, Standard curve showing a linear relationship between the immunoreactive band intensity and the amount of nuclear extract protein loaded for GH-treated hypx male rats. The equation of the line of best fit was generated by least squares linear regression analysis. C, Quantitative analysis of nuclear levels of pSTAT5. Results are presented as a percentage of the mean for the -MC/+GH treatment group. All data are expressed as the mean ± SD of determinations from four or five rats. *, Significantly different (P < 0.001) from -MC/-GH and +MC/-GH groups based on randomized design, one-way ANOVA and post hoc Newman-Keuls test.

 
GH-stimulated STAT5b DNA-binding activity was also assessed in nuclear extracts of hypx rats by EMSA using labeled oligonucleotide probes containing consensus STAT5 sites. In most experiments we used the well characterized STAT5-binding sequence from the rat ß-casein gene 5'-flank (40, 41, 42), which contains a perfect interferon-{gamma}-activated site (GAS; 5'-TTCNNNGAA-3') known to bind STAT5 homo- or heterodimers (43). Consistent with a recent report (23), we also identified a similar GAS sequence within the 5'-flank of the CYP2C11 gene (bases -1186 to -1167), and an EMSA probe was designed based on this sequence. As shown in Fig. 3AGo, a single ip GH injection dramatically increased the formation of a protein-DNA complex (lane 3), using the wt ß-casein radiolabeled probe. DNA sequence specificity for this interaction was confirmed by demonstrating effective competition by unlabeled wt ß-casein sequence (lanes 6 and 7), partial competition by the 2C11 STAT sequence (lanes 10 and 11), and lack of competition by the mutated ß-casein sequence (lanes 8 and 9). Additional confirmation that the protein-DNA complex contains STAT5b was provided by a supershift caused by the addition of anti-STAT5b polyclonal antibody (lane 13). Quantitative EMSA analysis revealed that GH strongly stimulated STAT5b DNA binding to the wt ß-casein probe, and this stimulatory effect was not altered by MC treatment (Fig. 3Go, A and B).



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  		Figure 3. Effect of MC on GH-stimulated STAT5b DNA-binding activity in vivo. A, Rat liver nuclear extract (1 µg) was analyzed by EMSA using a 32P-labeled wt ß-casein probe. Binding reactions were performed with no nuclear extract (lane 1) or extract from a representative hypx rat from each treatment group: -MC/-GH (lane 2), -MC/+GH (lanes 3 and 6–13), +MC/-GH (lane 4), and +MC/+GH (lane 5). Binding reactions contained either no competitor oligonucleotide (lanes 1–5, 12, and 13), a 10- or 100-fold molar excess of unlabeled wt ß-casein probe (lanes 6 and 7), a 10- or 100-fold molar excess of unlabeled mutated ß-casein probe (lanes 8 and 9), or a 10- or 100-fold molar excess of unlabeled 2C11 STAT probe (lanes 10 and 11). Binding reactions contained no antibody (lanes 1–11), nonimmune serum (NIS; lane 12), or anti-STAT5b antibody (lane 13). The positions of the STAT5b-DNA complex and the antibody-supershifted complex (SS) are shown. B, Quantitative analysis of STAT5b DNA-binding activity in hepatic nuclear extracts. Results are presented as a percentage of the mean for the -MC/+GH treatment group. All data are expressed as the mean ± SD of determinations from four or five rats. *, Significantly different (P < 0.001) from -MC/-GH and +MC/-GH groups based on randomized design, one-way ANOVA and post hoc Newman-Keuls test.

 
To confirm that the 2C11 STAT site represents a potentially important STAT5b-binding site, direct binding of STAT5b was examined using 32P-labeled 2C11 STAT as the probe (Fig. 4Go). Once again, GH strongly increased the formation of a protein-DNA complex (lane 2), and competition and antibody supershift analyses were consistent with a STAT5b-DNA interaction. This analysis confirmed that the CYP2C11 STAT5 consensus site has a lower affinity for STAT5b than the ß-casein sequence. Consistent with results obtained with the wt ß-casein radiolabeled probe, use of the 2C11 STAT radiolabeled probe revealed no effect of MC on GH-stimulated STAT5b DNA binding (data not shown).



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  		Figure 4. EMSA analysis demonstrating direct binding of rat liver nuclear STAT5b to the 2C11 STAT DNA probe. Rat liver nuclear extract (1 µg) was analyzed by EMSA using a 32P-labeled 2C11 STAT probe. Binding reactions were performed with nuclear extract from a representative rat from the -MC/-GH (lane 1) and -MC/+GH (lanes 2–13) groups. Binding reactions contained either no competitor oligonucleotide (lanes 1, 2, 12, and 13); a 1-, 10-, or 100-fold molar excess of unlabeled wt ß-casein probe (lanes 3–5); a 1-, 10-, or 100-fold molar excess of unlabeled mutated ß-casein probe (lanes 6–8); or a 1-, 10-, or 100-fold molar excess of unlabeled 2C11 STAT probe (lanes 9–11). Binding reactions contained no antibody (lanes 1–11), nonimmune serum (NIS; lane 12), or anti-STAT5b antibody (lane 13). The positions of the STAT5b-DNA complex and the antibody-supershifted complex (SS) are shown. Similar results were obtained in at least one additional experiment with an independent set of nuclear extracts.

 
Using whole liver extracts prepared from the same rats used to assess STAT5b activation, we confirmed that all MC-treated rats showed a dramatic induction of hepatic CYP1A1-immunoreactive protein, a classic positive control response for AHR activation (data not shown). Taken together, our in vivo data suggest that a MC dosing regimen that activates the AHR and causes CYP1A1 induction (22) does not inhibit GH-stimulated STAT5b phosphorylation, nuclear uptake, or DNA binding.

Effect of MC on GH-stimulated STAT5b activation in vitro
We were also interested in evaluating the effect of MC on GH-stimulated STAT5b activation in a cell culture system where it is more convenient to alter the MC concentration and the duration and timing of MC exposure relative to GH stimulation. Although many studies have shown GH activation of the JAK2-STAT5b pathway in cell lines after transfection of pathway components, we were interested in using a liver-derived cell line that possesses an intact JAK2-STAT5b pathway stimulated by GH. GH-stimulated STAT5b activation in the H4IIE rat hepatoma cell line was reported in one study examining the inhibitory effect of insulin on STAT5b activation (28). In addition, the H4IIE cell line has an intact and well characterized AHR signaling pathway (44). We therefore used this cell line to examine the effect of MC on STAT5b activation by GH.

Although we could not measure STAT5b or pSTAT5 by immunoblot analysis in our H4IIE whole cell extracts, we report the first demonstration of GH-stimulated STAT5b DNA-binding activity in H4IIE cells without requiring transfection of any components of the GH receptor-JAK2-STAT5b pathway (Fig. 5Go). EMSA experiments using whole cell extracts prepared from H4IIE cells showed that GH (500 ng/ml) stimulated STAT5b DNA binding; for the time points that we assessed, the highest DNA binding was observed at 20 min (Fig. 5AGo). The results of competitive EMSA experiments such as those performed with rat nuclear extracts confirmed that the GH-inducible protein-DNA complex represents STAT5b binding, and a similar difference in the affinity of STAT5b for the 2C11-STAT vs. wt ß-casein probe was found (Fig. 5BGo).



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  		Figure 5. EMSA analysis of GH-stimulated STAT5b DNA-binding activity in H4IIE whole cell extracts. Cell extract (5 µg) was analyzed by EMSA using a 32P-labeled wt ß-casein probe. A, Time course for activation of STAT5b DNA binding in response to GH treatment (500 ng/ml). Binding reactions were performed with no extract (lane 1), extract prepared from saline-treated cells (lanes 2, 4, 6, and 8), or extract prepared from GH-treated cells (lanes 3, 5, 7, and 9). B, Demonstration of DNA sequence specificity of the GH-inducible complex. Binding reactions were performed with no extract (lane 12), extract prepared from saline-treated cells (lane 1), or extract prepared from cells exposed to GH for 20 min (lanes 2–11). Binding reactions contained no competitor oligonucleotide (lanes 1 and 2); a 1-, 10-, or 100-fold molar excess of unlabeled wt ß-casein probe (lanes 3–5); a 1-, 10-, or 100-fold molar excess of unlabeled mutated ß-casein probe (lanes 6–8); or a 1-, 10-, or 100-fold molar excess of unlabeled 2C11 STAT probe (lanes 9–11). For A and B, similar results were obtained in at least one additional experiment with an independent set of cell extracts.

 
To evaluate the effect of MC on GH-stimulated STAT5b activation in vitro, H4IIE cells were preincubated with MC (10 nM, 100 nM, or 1 µM) or the vehicle DMSO for 30 min, 2 h, 3.5 h, or 16 h, then treated with GH (500 ng/ml) or saline vehicle for 20 min in the continuing presence of the appropriate MC concentration. Figure 6AGo shows a representative EMSA experiment in which cells were pretreated for 2 h with DMSO or MC, and then GH-inducible STAT5b DNA binding was assessed. At all time points and all MC concentrations examined, no significant effect of MC on GH-stimulated STAT5b activation was observed (Fig. 6BGo).



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  		Figure 6. Effect of MC on GH-stimulated STAT5b DNA-binding activity in H4IIE whole cell extracts. A, Cell extract (5 µg) was analyzed by EMSA using a 32P-labeled wt ß-casein probe. Binding reactions were performed with no extract (lane 1) or extract from one representative sample for each treatment group (lanes 2–9). Cells were exposed to either DMSO (lanes 2 and 3), or MC at 10 nM (lanes 4 and 5), 100 nM (lanes 6 and 7), or 1 µM (lanes 8 and 9) for 2 h, followed by a 20-min exposure to saline (lanes 2, 4, 6, and 8) or GH (lanes 3, 5, 7, and 9) in the continued presence of DMSO or MC. B, Quantitative analysis of STAT5b DNA-binding activity in H4IIE whole cell extracts. Experiments were performed as described for A, except that cells were pretreated with DMSO or MC for 30 min, 2 h, 3.5 h, or 16 h. The average intensity for the -MC/-GH group was subtracted from the intensity of individual samples in all other groups. Results are presented as a percentage of the mean for the -MC/+GH treatment group. All data are expressed as the mean ± SD of triplicate determinations in a single experiment. *, Significantly different (P < 0.01) from all -GH groups based on randomized design one-way ANOVA and post hoc Newman-Keuls test. Similar results were obtained in at least one additional independent experiment.

 
We used ICC analysis to assess STAT5b nuclear accumulation in H4IIE cells in response to GH. There was minimal staining when primary anti-STAT5b antibody was not included in the ICC procedure, for either saline- or GH-treated cells (Fig. 7AGo, a and b). In saline-treated cells probed with the anti-STAT5b antibody (Fig. 7AGo, c), cell staining was diffuse and generally cytoplasmic, with visibly weaker staining in the nucleus. In cells treated with GH (500 ng/ml) for 20 min (Fig. 7AGo, d), there was a dramatic increase in nuclear staining that is indicative of STAT5b nuclear translocation. These results confirm that the GH-inducible STAT5b DNA binding observed in whole cell extracts is accompanied by pronounced STAT5b nuclear translocation. Pretreatment of H4IIE cells with MC (1 µM) for the time periods described previously had no observable effect on GH-stimulated STAT5b nuclear translocation (Fig. 7BGo). These data are consistent with the lack of effect of MC on STAT5b DNA-binding activity observed under identical treatment conditions.



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  		Figure 7. ICC analysis of STAT5b nuclear translocation in H4IIE cells. A, Assay optimization experiments: a, saline-treated cells, no primary antibody; b, GH-treated cells, no primary antibody; c, saline-treated cells, with primary antibody; d, GH-treated cells, with primary antibody. B, Representative ICC results with H4IIE cells pretreated for 30 min with DMSO or MC (1 µM) before exposure to saline or GH (500 ng/ml) for 20 min in the continued presence of DMSO or MC. STAT5b staining is shown for each of the treatment groups: a, -MC/-GH; b, -MC/+GH; c, +MC/-GH; d, +MC/+GH. For A and B, magnification is x100.

 
Lastly, we attempted to examine the effect of MC on GH-inducible, STAT5b-mediated transcriptional activation in H4IIE cells; however, neither of the reporter plasmids containing functional GAS elements (45, 46, 47) responded to GH after transfection in H4IIE cells (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
GH is known to regulate CYP2C11 gene expression at the transcriptional level (48, 49), and the CYP2C11 5'-flanking region has been shown to contain sequences that interact in a sex-dependent and GH-regulated manner with putative transcription factors (49, 50). Further characterization of GH signal transduction pathways involved in the masculinization of liver CYP expression by GH pulses led to the identification of STAT5b as a key intracellular mediator of this process (24, 39, 51). STAT5b is activated at each GH pulse by GH receptor-associated JAK2, which becomes activated after GH-induced dimerization of the GH receptor (37, 52). The process of deactivation of this pathway has recently been characterized and is crucial for resetting the pathway to a GH-responsive state in time for the next GH pulse (43). In light of 1) the role of pulsatile GH in regulating CYP2C11 expression, 2) the binding of GH-dependent transcription factors to the CYP2C11 5'-flank, and 3) the importance of STAT5b in masculinizing liver CYP expression, STAT5b is likely to be a key transcription factor in the regulation of CYP2C11. Recent evidence supports that this is indeed the case (23), although it appears that STAT5b may be but one component in a complex cascade of liver-enriched transcription factors.

Several reports have demonstrated an ability of aromatic hydrocarbons to modulate various kinase pathways (53, 54, 55, 56, 57) as well as a pathway involving a 91-kDa STAT (58) and nuclear factor-{kappa}B (NF-{kappa}B) (59) signaling pathways, with all effects associated with AHR activation. These findings establish precedence for the ability of aromatic hydrocarbons to interact with components of various signaling pathways in an AHR-dependent fashion. The AHR is a ligand-activated transcription factor that binds aromatic hydrocarbons such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and MC, resulting in altered expression of a diverse array of genes, including those involved in drug and hormone metabolism, cell growth, and cell differentiation. Therefore, taking into consideration the role of STAT5b in the regulation of CYP2C11, the fact that aromatic hydrocarbons are able to modulate kinase-mediated signaling pathways, and our previous observation of the attenuating effect of MC on GH-stimulated CYP2C11 expression (22), we explored whether MC modulates hepatic GH signaling via the STAT5b pathway.

We measured the effect of MC on STAT5b activation by GH both in vivo and in vitro. Biochemical analysis of STAT5b both in vivo and in vitro demonstrated that GH-inducible STAT5b phosphorylation, nuclear translocation, and DNA binding were not affected by MC. The information derived from the EMSA data is particularly important. Under most circumstances, DNA-binding activity represents a culmination of all signaling events upstream of STAT5b DNA binding. These events include 1) GH receptor dimerization upon ligand binding, 2) JAK2 activation, 3) JAK2-catalyzed STAT5b phosphorylation, 4) STAT5b dimerization, and finally 5) STAT5b nuclear translocation. Defects in any of these events would probably be reflected in changes in DNA binding; however, no effects of MC were observed at the DNA-binding level. We, therefore, conclude that MC has no direct effect on GH signal transduction at any level up to STAT5b DNA binding, and that direct interference with GH signal transduction via the STAT5b pathway is not a likely mechanism underlying the attenuation of GH-stimulated CYP2C11 expression by MC.

Nevertheless, it remains to be explored whether MC attenuates GH-stimulated CYP2C11 expression at the level of GH-induced, STAT5b-mediated transcriptional activation. We speculate that the lack of GH-inducible luciferase expression in the H4IIE cell line with two reporter constructs known to be activated by GH in a STAT5-dependent fashion may be due to 1) a high expression level of transcriptional repressors that inhibit STAT5b-mediated trans-activation, or 2) a lack of transcriptional coactivators required for STAT5b trans-activation. Nevertheless, the H4IIE cell line in our hands is presently unsuitable for addressing whether MC interferes with GH-stimulated gene expression at the level of transcriptional activation. In the absence of changes in STAT5b DNA binding, it remains possible that MC could alter transcriptional activation by modulating the interaction of STAT5b with transcriptional coactivators or repressors. This problem could be studied in the CWSV-1 immortalized rat hepatocyte cell line that displays intact GH receptor-JAK2-STAT5b signaling (60) or in cell lines transfected with the necessary components of the signaling pathway (23).

Various transcription factors have been shown to interact with STAT5b. Studies of the regulation of hepatic female-specific CYP2C12 gene expression suggest an interaction between STAT5b and hepatocyte nuclear factors (HNFs). STAT5b was shown to negatively regulate the activity of HNF-3ß and -6 (61) and to synergize with HNF-4 (62) by mechanisms that probably involve protein-protein interactions; further evidence for functional antagonism between STAT5b and HNF-3ß was obtained recently (23). Other transcription factors found to interact with STAT5a/b include members of the steroid receptor family (63, 64, 65), the thyroid hormone receptor (66), CCAAT enhancer-binding protein ß (indirectly via the glucocorticoid receptor) (67), cAMP response element-binding protein-binding protein/p300 (68), yin-yang 1 (69), and nuclear factor-Y (70). However, whether MC changes any transcription factor’s interaction with STAT5b with resulting alterations in gene expression is speculative at this point. This could also be explored in an appropriate cell system that responds to GH with a STAT5b-dependent transcriptional response.

An alternate hypothesis for MC interference with GH signaling is one that takes into consideration the mechanisms by which the JAK-STAT pathway is negatively regulated. There is a growing list of proteins that function as negative regulators of the JAK-STAT pathway (71, 72). These include the phosphotyrosine phosphatases SHP-1 (73, 74) and PTP1B (75), signal regulatory protein {alpha} (76), and members of the suppressors of cytokine signaling family (77, 78, 79). It is conceivable that aromatic hydrocarbons such as MC interfere with STAT5b-mediated signaling by increasing the activity of these inhibitory proteins, although we would have expected such effects to be manifest in our studies of STAT5b DNA binding. With respect to negative regulators at a gene promoter level, a potential candidate that may mediate an inhibitory effect of MC on STAT5b-mediated trans-activation is NF-{kappa}B. Four pieces of evidence support this suggestion: 1) NF-{kappa}B has been shown to interact with and inhibit STAT5b (80), although it has been found that the inhibition can also occur in the reverse direction (81); 2) the AHR has been found to interact with NF-{kappa}B (59); 3) DNA elements located within the CYP2C11 5'-flank have been found to bind both STAT5b (Ref. 23 and this study) and the AHR (82); and 4) NF-{kappa}B can mediate the suppression of CYP2C11 caused by IL-1ß (83). It would be very interesting to determine whether the attenuating effect of MC on the ability of GH to stimulate CYP2C11 expression can be explained by an AHR-directed recruitment of NF-{kappa}B to the CYP2C11 5'-flank and thereby inhibition of STAT5b-mediated transcriptional activation.

The present study examined the effect of MC on the JAK2-STAT5b pathway; however, it is well known that GH activates multiple signaling pathways, including those involving MAPKs, phosphatidylinositol 3-kinase, and protein kinase C (43). In preliminary experiments we attempted to assess the effect of MC on two of these additional GH signaling pathways: 1) as a marker for activation of a mitogen-activated protein kinase pathway, we monitored GH-stimulated phosphorylation of extracellular signal-regulated kinase 1/2; and 2) as a marker for activation of the phosphatidylinositol 3-kinase pathway, we monitored GH-stimulated phosphorylation of protein kinase B. Using whole liver extracts prepared from the same rats used to assess STAT5b activation in vivo and the same H4IIE cell extracts used for in vitro studies, GH did not consistently and reproducibly stimulate phosphorylation of extracellular signal-regulated kinase 1/2 or protein kinase B (data not shown). Thus, it remains to be determined whether aromatic hydrocarbons disrupt any of these GH signaling pathways.

In summary, we have shown that MC does not interfere with GH-stimulated STAT5b activation, thus eliminating this as a likely mechanism to explain the attenuating effect of MC on the ability of GH to stimulate CYP2C11 expression in hypx male rats. This was demonstrated by an assessment of STAT5b activation by GH in the presence or absence of MC using a combination of both in vivo- and in vitro-based strategies. No effect of MC was observed on GH-inducible STAT5b phosphorylation, nuclear accumulation, and DNA-binding activity. Nevertheless, we have developed H4IIE cells as a simple cell culture system in which STAT5b nuclear accumulation and DNA binding can be readily assessed. We also demonstrated that STAT5b is able to bind a STAT5 consensus site located within the CYP2C11 5'-flank. These findings set the stage for future studies aimed at understanding the molecular mechanisms by which aromatic hydrocarbons interfere with the ability of GH to stimulate the expression of hepatic CYP2C11, whose gene product plays an important role in the metabolism of both endogenous and foreign compounds.


   Acknowledgments
 
We thank the following individuals for their contributions to this work: Drs. David Waxman, Denis Grant, Allan Okey, and Rachel Tyndale for valuable discussions; Dr. Dwayne Barber for helpful suggestions and for sharing protocols and valuable reagents; Dr. Albert Parlow for providing rat GH; Drs. Li-Yuan Yu-Lee and David Frank for generous gifts of antibodies; Dr. Bernd Groner for the generous gift of luciferase reporter plasmids; Drs. Sharon Miksys and A. José Lança for assistance with ICC experiments; Anahita Bhathena for assistance with H4IIE transfections; and Chunja Lee for skilled technical assistance.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research (MOP-42399).

Abbreviations: AHR, Aromatic hydrocarbon receptor; ß-cas-luc, ß-casein-luciferase; DMSO, dimethylsulfoxide; GAS, interferon-{gamma}-activated site; HNF, hepatocyte nuclear factor; hypx, hypophysectomized; ICC, immunocytochemistry; JAK2, Janus kinase 2; MC, 3-methylcholanthrene; NF-{kappa}B, nuclear factor-{kappa}B; PBST, PBS plus 0.01% Triton X-100; PMSF, phenylmethylsulfonylfluoride; STAT, signal transducer and activator of transcription; wt, wild-type.

Received February 21, 2002.

Accepted for publication May 29, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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