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Endocrinology Vol. 145, No. 12 5525-5531
Copyright © 2004 by The Endocrine Society

Estrogen Up-Regulates Hepatic Expression of Suppressors of Cytokine Signaling-2 and -3 in Vivo and in Vitro

Gary M. Leong, Sofia Moverare, Jesena Brce, Nathan Doyle, Klara Sjögren, Karin Dahlman-Wright, Jan-Åke Gustafsson, Ken K. Y. Ho, Claes Ohlsson and Kin-Chuen Leung

Pituitary Research Unit (G.M.L., J.B., N.D., K.S., K.K.Y.H., K.-C.L.), Garvan Institute of Medical Research, and Department of Endocrinology (K.K.Y.H.), St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia; Center for Bone Research at the Sahlgrenska Academy (S.M., K.S., C.O.), Division of Endocrinology, Department of Internal Medicine, Göteborg University, Göteborg SE-41345, Sweden; and Departments of Medical Nutrition and Bioscience (K.D.-W., J.-Å.G.), Karolinska Institute, Novum, Huddinge SE-14186, Sweden

Address all correspondence and requests for reprints to: Dr. Kin-Chuen Leung, Pituitary Research Unit, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, Sydney, New South Wales 2010, Australia. E-mail: k.leung{at}garvan.org.au.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Suppressors of cytokine signaling (SOCS) are important negative regulators of cytokine action. We recently reported that estrogen stimulates SOCS-2 expression and inhibits GH signaling in kidney cells. The effects of estrogen on SOCS expression in other tissues are unclear. The aim of this study was to investigate in vivo and in vitro whether estrogen affected SOCS expression in the liver, a major target organ of GH. The in vivo hepatic effects of estrogen on ovariectomized mice lacking estrogen receptor (ER)-{alpha}, ERß, or both and their wild-type littermates were examined by DNA microarray analysis. In vitro, the effects of estrogen on SOCS expression in human hepatoma cells were examined by reverse transcription quantitative PCR. Long-term (3 wk) estrogen treatment induced a 2- to 3-fold increase in hepatic expression of SOCS-2 and -3 in wild-type and ERß knockout mice but not in those lacking ER{alpha} or both ER subtypes. Short-term treatment (at 24 h) increased the mRNA level of SOCS-3 but not SOCS-2. In cultured hepatoma cells, estrogen increased SOCS-2 and -3 mRNA levels by 2-fold in a time- and dose-dependent manner (P < 0.05). Estrogen induced murine SOCS-3 promoter activity by 2-fold (P < 0.05) in constructs containing a region between nucleotides –1862 and –855. Moreover, estrogen and GH had additive effects on the SOCS-3 promoter activity. In summary, estrogen, via ER{alpha}, up-regulated hepatic expression of SOCS-2 and -3, probably through transcriptional activation. This indicates a novel mechanism of estrogen regulation of cytokine action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYTOKINES EXERT BIOLOGICAL effects through the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway (1). On cytokine activation, JAKs phosphorylate STATs, which translocate to the nucleus and bind specific DNA motifs within the promoter of target genes to initiate transcription. JAK/STAT signaling is negatively regulated by several members of the suppressor of cytokine signaling (SOCS) family, which comprises SOCS-1 to -7 and the cytokine-induced SH2-containing protein (CIS) (2). SOCS-1, -2, and -3 and CIS are expressed in response to several cytokines and hormones, including GH, and feedback to inhibit JAK/STAT signaling (2, 3).

The biological actions of estrogens are exerted through specific nuclear estrogen receptor (ER) subtypes, designated as ER{alpha} and ERß, which exhibit overlapping patterns of tissue expression, ligand binding, and function (4, 5, 6, 7). We recently demonstrated that estrogen induces SOCS-2 expression, which inhibits GH activation of JAK/STAT signaling in human embryonic kidney cells (8). The expression of SOCS-1 and -3 were unaffected. Concurrently Jelinsky et al. (9) reported up-regulation of SOCS-2 expression by estrogen in kidneys of wild-type (WT) and ERß knockout mice, suggesting a role of ER{alpha} rather than ERß in mediating this effect.

The selective SOCS-2 induction by estrogen stands in contrast to previous findings that cytokines and hormones, as exemplified by IL-6 and GH, often stimulate expression of multiple SOCS proteins (2). The effects of estrogen on SOCS regulation in tissues other than kidney are not known. In this study, we investigated the effects of estrogen on SOCS expression in the liver, a major target of GH action. Clinical studies have indicated that estrogen administered via the oral route inhibits GH-induced IGF-I production and substrate metabolism (10, 11, 12, 13). It is possible that the inhibitory effect of estrogen on GH-regulated hepatic function is also mediated by SOCS. This possibility is supported by previous studies in rodents showing that impairment of GH signaling in the liver induced by IL-6, chronic renal failure, and sepsis is accompanied by elevated expression of SOCS-2 and -3 (14, 15, 16, 17, 18, 19). Altogether these studies point to a central role in the regulation of GH action by SOCS proteins in the liver.

We investigated the in vivo and in vitro effects of estrogen on hepatic SOCS expression. Female mice lacking ER{alpha} (ER{alpha}–/–), ERß (ERß–/–), or both (ER{alpha}–/–ß–/–) and their WT littermates were ovariectomized and treated with estrogen before the liver was collected for DNA microarray analysis. In vitro, SOCS expression in response to estrogen and GH was studied in human hepatoma cells (HuH7) by RT-quantitative PCR (RT-qPCR). Furthermore, we compared the effects of estrogen and GH on murine SOCS-3 promoter activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
WT (C57B1/6) and ER knockout mice were bred, and the diet, housing, and genetic background have been previously described (7, 20). The female mice were ovariectomized at 2 months of age and after a 4-d recovery period, injected sc with 2.3 µg/d of 17ß-estradiol benzoate (E2) (Sigma, St. Louis, MO) 5 d/wk for 3 wk before being killed for tissue collection. This treatment regimen resulted in serum E2 levels similar to those normally seen during estrus in female mice (21, 22). Control mice received injections of vehicle (olive oil, Apoteksbolaget, Göteborg, Sweden). For the short-term E2 studies, 2-month-old female mice were ovariectomized; received a single injection of 2.3 µg E2 after recovery for 2 wk; and were killed at 10, 24, and 48 h as previously described (23).

DNA microarray analysis
RNA was isolated from liver homogenates (n = 6 for vehicle- or E2-treated WT, ERß–/– and ER{alpha}–/–ß–/– mice; n = 4 for vehicle- or E2-treated ER{alpha} –/– mice) and purified using RNeasy kit (Qiagen, Chatsworth, CA) as described (24). RNA samples were combined into two pools per animal group for microarray assays, whereas RNA from individual animals was analyzed separately in the confirmatory RT-qPCR as previously described (23).

Bioinformatics
Scanned output files were analyzed using Micro Array Suite (version 4.0.1, Affymetrix, Santa Clara, CA) software and globally scaled to an average intensity of 500 for comparison between GeneChips as described (23). Results from the two vehicle- and E2-treated GeneChips were compared, generating a total of four comparisons. The criteria applied to determine the response of a gene to E2 treatment were: 1) the absolute call for the gene had to be present in all GeneChips, and the average difference had to be above 200; 2) the responses in at least two of the four comparisons were increased or decreased; and 3) the average increase or decrease of the four comparisons should be at least 80%, compared with the control samples.

Quantitation of SOCS mRNA
The DNA microarray findings of SOCS-2, SOCS-3, and CIS were verified by RT-qPCR on individual samples using the ABI Prism 7000 sequence detection system (PE Applied Biosystems, Stockholm, Sweden) with specific probes labeled with the reporter fluorescent dye FAM. The sequences for forward and reverse primers and probes for murine SOCS-2 (accession no. MMU88327) were 5'-CGCGTCTGGCGAAAGC (nucleotides 323–338), 5'-TTCATTAACAGTCATACTTCCCCAGTAC (nucleotides 393–366), and 5'-TCCTGTTTGACTGAGCTCGCGCA (nucleotides 363–341), respectively, and those for murine SOCS-3 (accession no. MMU88328) were 5'-CCCGCGGGCACCTT (nucleotides 207–220), 5'-TTGACGCTCAACGTGAAGAAGT (nucleotides 271–250), and 5'-CCGCGACAGCTCGGACCAGC (nucleotides 227–246), respectively. The oligonucleotide primers and probes were supplied by PE Applied Biosystems including for CIS RT-qPCR by assay on demand ID:Mm00515488_ml. The cDNA amplification conditions were adjusted for the expression of 18S rRNA as previously described (23).

The mRNA abundance of SOCS-2 and -3 in HuH7 cells treated with E2 or GH were quantified by RT-qPCR from RNA obtained from cells transfected with expression plasmids for human (h) ER{alpha} (pcDNA3.1-ER{alpha}) (0.1 µg) or human GH receptor (GHR) (pcDNAI/Amp-GHRfl) (0.1 µg), respectively, using Effectene transfection reagent as per the manufacturer (Qiagen). The expression plasmids for hER{alpha} (25) and hGHR (pcDNAI/Amp-GHRfl) (26) were kindly provided by Patrick Matthias (Friedrich Miescher Institute, Basel, Switzerland) and Richard Ross (Sheffield University, Sheffield, UK), respectively. Cells were then treated with 100 nM E2 or 500 ng/ml GH (27) for 0.25, 0.5, 1, 2, and 4 h or in the dose-dependence studies with 1–100 nM E2 or 5–500 ng/ml GH as indicated. At the indicated time, total RNA was extracted with TRIzol (Invitrogen/Gibco, Carlsbad, CA). reverse transcription was performed using the Omniscript RT kit (Qiagen), and RT-qPCR was performed using a Lightcycler (Roche Molecular Biochemicals, Indianapolis, IN) as previously described (8). PCR primers for human SOCS-2 and SOCS-3 (8) were obtained from Sigma Genosys (Sydney, New South Wales, Australia).

Cell cultures and transcription assays
HuH7 cells were cultured as previously described (8). Cells seeded at a density of 5 x 104 cells/cm2 in 12-well plates were transiently transfected for 24 h with expression plasmids for hER{alpha} (0.1 µg), hGHR (0.1 µg), and ß-galactosidase (IEP-gal-CMV) (0.01 µg) and various reporter clones of the murine SOCS-3 promoter (0.25 µg) (see Fig. 4Go). The SOCS-3 promoter luciferase reporter constructs were generous gifts from Shlomo Melmed (Cedars Sinai Research Institute, Los Angeles, CA) (28). The cells were then treated in triplicate with E2 (1, 10, or 100 nM) and/or GH (5, 50, or 500 ng/ml) at 37 C for 18 h before solubilization in lysis buffer and measurement of luciferase and ß-galactosidase activities as previously described (8).



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  		FIG. 4. Murine SOCS-3 promoter deletion analysis. Transactivation of murine SOCS-3 promoter truncation clones (left panel) with 100 nM E2 (middle panel) or 500 ng/ml GH (right panel) in HuH7 cells. Luciferase reporter activity is expressed as fold induction over vehicle-treated control. Results are mean ± SEM of at least three independent experiments each performed in triplicate. *, P < 0.05 between control and treated samples by Student’s t test. The shaded box indicates promoter region containing an SBE. 6D1 clone has mutated nonfunctional SBE indicated by X. Numbers over clones in the left panel indicate nucleotide sequence in the SOCS-3 promoter (28 ).

 
Statistical analysis
Data were analyzed by Student’s paired t test or two-way ANOVA (STATVIEW version 4.5, Abacus Concepts, Berkeley, CA) where appropriate, and significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vivo effects of estrogen in mice
To determine whether estrogen regulated SOCS expression in the liver, ovariectomized mice were treated with E2 or vehicle for 3 wk, and expression of SOCS-1, -2, and -3 and CIS analyzed by DNA microarray (Table 1Go). E2 significantly increased the abundance of SOCS-2 and -3 mRNA, whereas the CIS level was unaffected. SOCS-1 expression was not detectable in the microarray study, and RT-qPCR analysis revealed that the SOCS-1 level was very low and not affected by E2 treatment.


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  		TABLE 1. Estrogen regulation of SOCS expression in the liver

 
To determine the effect of short-term E2 treatment on SOCS expression, mice were killed after a single injection of E2 at 10, 24, and 48 h. This short-term exposure to E2 significantly increased SOCS-3 mRNA abundance only at 24 h (163 ± 9.2%; P < 0.01 by Student’s paired t test). No significant change in SOCS-3 expression was observed at 10 or 48 h of E2 treatment, nor was SOCS-2 expression altered by E2 treatment at any time points (data not shown).

To establish ER subtype specificity for SOCS stimulation by estrogen, the effects of E2 were compared between ER{alpha}+/+ (i.e. combined WT and ERß–/–) and ER{alpha}–/– (i.e. combined ER{alpha}–/– and ER{alpha}–/–ß–/–) mice. RT-qPCR analysis revealed that E2 increased both the SOCS-2 and -3 mRNA levels in ER{alpha}+/+ but not ER{alpha}–/– mice (P < 0.05 by two-way ANOVA; Table 2Go). No effect of E2 on CIS expression was observed in WT, ER{alpha}–/–, and ER{alpha}–/–ß–/– mice. Unexpectedly, CIS expression was up-regulated in ERß–/– mice. Overall, these results suggest that ER{alpha} mediates E2-stimulated expression of SOCS-2 and -3.


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  		TABLE 2. SOCS expression in response to estrogen in WT and ER knockout mice

 
In vitro effects of estrogen in hepatoma cells
In the light of the DNA microarray findings, we examined, using RT-qPCR, whether E2 regulated SOCS-2 and -3 expression in HuH7 cells and compared the effects with those of GH. E2 (100 nM) induced a time-dependent increase in both SOCS-2 and -3 mRNA abundance to a maximal 2-fold response, compared with untreated controls at 2 h (P < 0.05; Fig. 1AGo). GH (500 ng/ml) induced expression of SOCS-2 and -3 maximally by 2-fold (P < 0.05; Fig. 1BGo) at 2 and 0.5 h, respectively.



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  		FIG. 1. SOCS expression in vitro in response to E2 and GH. HuH7 cells were treated with 100 nM E2 (A) (black bars) or 500 ng/ml GH (B) (hatched bars) for indicated time, and SOCS-2 and -3 mRNA was measured by RT-qPCR as per Materials and Methods. Results expressed as percentage of untreated control are mean ± SEM of three independent experiments each performed in triplicate. *, P < 0.05 by two-way ANOVA for both E2 and GH experiments between untreated and treated samples.

 
The dose effect of E2 (1–100 nM) and GH (5–500 ng/ml) on SOCS-2 and -3 mRNA expression were next investigated. E2 increased the abundance of SOCS-2 and -3 mRNA in a dose-dependent manner (P = 0.05 and P < 0.001 by two-way ANOVA, respectively; Fig. 2AGo). Similarly, the mRNA levels of SOCS-2 and -3 significantly increased with increasing concentration of GH (P < 0.01 by two-way ANOVA; Fig. 2BGo). At lower doses of E2 (10 nM), significant increases in SOCS-2 and -3 mRNA expression were observed, whereas GH at doses as low as 5 and 50 ng/ml increased SOCS-2 and SOCS-3 expression above controls, respectively (P < 0.05 by Student’s paired t test).



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  		FIG. 2. E2 and GH dose studies on SOCS expression in vitro. HuH7 cells were treated in triplicate with 1–100 nM E2 for 2 h (A) (black bars) or 5–500 ng/ml GH (B) (hatched bars) for 30 min (SOCS-3) and 2 h (SOCS-2). SOCS-2 and -3 mRNA was measured by RT-qPCR as per Fig. 1Go. P values by ANOVA are indicated.

 
Modulation of SOCS-3 promoter activity by estrogen and GH
To determine whether the stimulation of SOCS by E2 and GH occurred at the transcriptional level, their effects on transactivation of a luciferase reporter construct containing a 3.6-kb region (clone 6, nucleotides –2757 to +929) of the murine SOCS-3 promoter were examined. The SOCS-3 promoter contains a STAT-binding element (SBE) between nucleotides –72 and –64 (28). Both E2 and GH increased reporter activity in a dose-dependent manner, to a maximum of 2- and 1.5-fold of untreated controls, respectively (P < 0.05; Fig. 3AGo).



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  		FIG. 3. Transactivation of murine SOCS-3 promoter by E2 and GH. HuH7 cells transiently transfected with the full-length SOCS-3 promoter (clone 6) reporter and expression plasmids for hER{alpha} and hGHR were treated with E2 (1–100 nM; black bars) or GH (5–500 ng/ml; hatched bars) (A), E2 (1–100 nM) plus or minus GH (50 ng/ml) (B), or GH (5–500 ng/ml) plus or minus E2 (10 nM) (C) for 18 h as described in Materials and Methods. Luciferase reporter activity is expressed as fold induction over vehicle-treated control (white bars). *, P < 0.05 and **, P < 0.001 in A by ANOVA or in B and C by Student’s t test between single and combined treatments. The changes in fold-induction with either increasing doses of GH or E2 alone or in combination were significant (P < 0.05 by ANOVA). Results are mean ± SEM of at least four independent experiments performed in triplicate.

 
To investigate possible interaction between E2 and GH on the SOCS-3 promoter, the effects of combined treatment were examined. The addition of 50 ng/ml GH to E2-treated cells significantly enhanced transcriptional activity by about 25% at all concentrations of E2 (Fig. 3BGo). Similarly, addition of 10 nM E2 to GH-treated cells resulted in a further increase of transcriptional activation by about 30% (Fig. 3CGo). Taken together, E2 and GH exerted additive effects on the SOCS-3 promoter activity.

To investigate the region of the SOCS-3 promoter mediating the stimulatory effects of E2 and GH, studies were performed in promoter mutants lacking the SBE and those with 5' truncation (clones 2, 6D1, 6T1, 6T2, 6T3, and 6T4) (28). The characteristics of each of these clones are shown in Fig. 4Go. Clones lacking the SBE (clones 2, 6D1, and 6T4) failed to respond to GH, whereas those containing this element (clones 6T1, 6T2, and 6T3) were activated by GH to levels similar to that of clone 6. In contrast, clones with 3' truncation (clone 2), a nonfunctional SBE (clone 6D1) or 5' truncation to nucleotide –1862 (clone 6T1) showed a full responsiveness to E2. Further 5' truncation to nucleotide –855 and beyond (clones 6T2, 6T3, and 6T4) rendered the promoter mutants unresponsive to E2. These findings suggest that the SBE between nucleotides –72 and –64 may be responsible for the GH stimulation and that the estrogen-responsive region lies between nucleotides –1862 and –855.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We investigated the effects of estrogen on expression of SOCS-1 to -3 and CIS in hepatic tissue. In the liver of ovariectomized mice, estrogen up-regulated expression of SOCS-2 and -3 but had no detectable effect on SOCS-1 and CIS. Estrogen action was mediated by ER{alpha} because this effect was observed in WT and ERß–/– mice, but not in ER{alpha}–/– and ER{alpha}–/–ß–/– mice. Estrogen stimulation of SOCS-2 and -3 was confirmed in vitro in human hepatoma cells. GH also induced expression of the two SOCS mRNAs in the hepatoma cells. Both estrogen and GH activated SOCS-3 promoter activity, and their effects were additive. Promoter elements between nucleotides –1862 and –855 were required for estrogen activation, whereas the GH effect was dependent on the presence of a SBE in a region between nucleotides –72 and –64. Taken together, these data suggest estrogen stimulation of hepatic SOCS expression to be transcriptionally regulated.

SOCS are up-regulated by several cytokines and hormones (2). We and others (8, 9) have shown that estrogen stimulates expression of SOCS-2 but not other SOCS members in the kidney. These findings led us to investigate the effects of estrogen on SOCS expression in the liver, which is a major metabolic organ responsive to estrogen. In contrast to the kidney, estrogen stimulated hepatic expression of SOCS-2 and -3, suggesting tissue-dependent response of SOCS-3 to estrogen.

Whereas a 3-wk treatment of estrogen up-regulated hepatic expression of both SOCS-2 and -3, a single injection of estrogen increased the mRNA level of SOCS-3 but not SOCS-2 at 24 h. Reasons for the lack of SOCS-2 response to the short-term E2 treatment are not obvious, but this may reflect differences in the tempo of gene expression between SOCS-2 and -3 induced by estrogen. There is also possible existence of estrogen-responsive factors that in some way affect the expression and/or stability of SOCS-2 mRNA.

Using mice with selective deletion of the two ER subtypes, ER{alpha} was shown to be a prerequisite for the estrogen stimulation of hepatic SOCS expression. In ER{alpha}+/+ (i.e. WT and ERß–/–) but not in ER{alpha}–/– (i.e. ER{alpha}–/– and ER{alpha}–/–ß–/–) mice, SOCS-2 and SOCS-3 expressions were up-regulated by estrogen. Taken together, these data support that ER{alpha} mediates the up-regulation of SOCS expression in the liver. However, it should be noted that, whereas ER subtype expression in the mouse liver and kidney has not been characterized, ER{alpha} is the predominant subtype in these two organs in rats (29). The exact role of ERß, if any, in modulation of SOCS expression in other tissues remains to be determined.

Whereas the SOCS-3 promoters in humans and rodents are cloned (28, 30, 31), no studies of the SOCS-2 promoter have been reported. To gain further insights into the mechanisms for SOCS regulation by estrogen, its effects on transactivation of the murine SOCS-3 promoter were compared with those of GH. This promoter contains a classical SBE at nucleotides –72 to –64 and is activated by cytokines, such as leukemia inhibitory factor (28). Whereas both estrogen and GH activated the SOCS-3 promoter, their respective effects were mediated by different regions of the promoter. GH acted through the SBE, whereas the promoter region between nucleotides –1862 and –855 appeared to be estrogen responsive (Fig. 4Go). That the effects of estrogen and GH were additive supports that the two hormones exert independent effects on the SOCS-3 promoter.

Sequence analysis of the estrogen-responsive region revealed the presence of a motif (AGGTCAgaaAGCCCA) at nucleotides –1366 to –1352, which is highly homologous to the canonical estrogen response element (ERE; AGGTCAnnnTGACCT) (32, 33). However, attempts to demonstrate a direct role of this motif in estrogen regulation of the SOCS-3 promoter activity were unsuccessful. Estrogen did not affect the activity of a luciferase reporter containing this incomplete ERE motif alone, nor was ER{alpha} binding to this element detected on an EMSA (data not shown). It is noteworthy that ER also activates gene expression through interaction with transcription factors such as activating protein-1 (AP-1) (34) and specificity protein 1 (Sp1) (35). Because there is an AP-1 site adjacent to the incomplete ERE motif in the murine SOCS-3 promoter (Leung, K.-C., unpublished observation), further studies are required to ascertain whether ER may interact with AP-1 to regulate the promoter activity.

Estrogen induction of SOCS expression may have a significant physiological role in regulation of GH action. Clinical studies have shown that estrogen at pharmacological concentrations suppresses the hepatic function of GH (10, 11, 12, 13). Conceivably, estrogen inhibition of GH action involves the mediation of both SOCS-2 and -3 in the liver. Stimulation of SOCS-3 expression by estrogen may be relevant to the regulation of other members of the cytokine receptor superfamily, such as the IL-6 receptor. Mice with conditional knockout of the hepatic SOCS-3 gene display increased IL-6 signaling (36). Estrogen is a key negative regulator of IL-6-dependent transcription (37), and postmenopausal osteoporosis is secondary to increased IL-6-mediated bone resorption (37, 38, 39, 40, 41). Our present observation suggests SOCS-3 stimulation by estrogen to be a potential novel mechanism for estrogen regulation of IL-6 function.

In conclusion, estrogen stimulates hepatic expression of SOCS-2 and -3 in vivo and in vitro, and the up-regulation likely occurs at the transcriptional level. This action by estrogen may explain in part the sexual dimorphism in GH action on body growth and composition (42) and in autoimmune and inflammatory disorders (43).


   Acknowledgments
 
We thank Drs. Sholmo Melmed, Richard Ross, and Patrick Matthias for the plasmids.


   Footnotes
 
This work was supported by the National Health and Medical Research Council of Australia, the Swedish Medical Research Council, the Swedish Cancer Society, KaroBio AB, the Swedish Foundation for Strategic Research, the European Commission, the Lundberg Foundation, the Torsten and Ragnar Söderberg’s Foundation, the Emil and Vera Cornell Foundation, the Novo Nordisk Foundation and Petrus, and the Augusta Hedlunds Foundation.

Abbreviations: AP-1, Activating protein-1; CIS, cytokine-induced SH2-containing protein; E2, 17ß-estradiol; ER, estrogen receptor; ERE, estrogen response element; GHR, GH receptor; h, human; HuH7, human hepatoma cells; JAK, Janus kinase; RT-qPCR, reverse transcription quantitative PCR; SBE, STAT-binding element; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; WT, wild-type.

Received January 20, 2004.

Accepted for publication August 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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