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Molecular Endocrinology, doi:10.1210/me.2007-0030
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Molecular Endocrinology 21 (10): 2516-2528
Copyright © 2007 by The Endocrine Society

Characterization of the SOCS3 Promoter Response to Prostaglandin E2 in T47D Cells

Johanna L. Barclay, Stephen T. Anderson, Michael J. Waters and Jon D. Curlewis

School of Biomedical Sciences (J.L.B., S.T.A., M.J.W., J.D.C.) and Institute for Molecular Bioscience (M.J.W.), University of Queensland, Queensland 4072, Australia

Address all correspondence to: Dr. Jon D. Curlewis, School of Biomedical Sciences, University of Queensland, Queensland 4072, Australia. E-mail: j.curlewis{at}uq.edu.au.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Suppressor of cytokine signaling 3 (SOCS3), a negative regulator of cytokine signaling, is expressed in breast cancer cells where it can modify sensitivity and responsiveness to cytokine signaling through the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. Although it is widely accepted that SOCS3 expression is in itself regulated by STATs, we and others have shown that prostaglandins can also up-regulate SOCS3 expression. Here we used T47D breast cancer cells treated with prostaglandin E2 (PGE2) to examine this pathway. T47D cells responded to PGE2 stimulation with a significant increase in SOCS3 mRNA that was independent of de novo protein synthesis. PGE2 stimulation resulted in STAT3 serine and tyrosine phosphorylation, although mutation of either of the two previously characterized STAT response elements on the SOCS3 promoter did not affect SOCS3 promoter activation by PGE2. In addition, overexpression of STAT3 wild-type, constitutively active or dominant-negative constructs did not affect PGE2-induced SOCS3 promoter activation, indicating that STATs are unlikely mediators of this pathway in these cells. PGE2 is a known activator of the cAMP/protein kinase A (PKA) pathway, and in T47D cells, up-regulation of SOCS3 mRNA by PGE2 was abolished by pretreatment with H89, a PKA inhibitor and increased by cAMP and forskolin treatment. Consistent with this, PGE2 treatment increased cAMP response element (CRE)-binding protein serine phosphorylation. However, mutation of the activator protein 1/CRE on the promoter did not affect basal or PGE2-stimulated activation, suggesting a role for cAMP/PKA that is independent of CRE-binding protein binding. Mutation of the GC-rich region of the SOCS3 promoter, a putative Sp1/Sp3 binding site, abolished both basal and PGE2-stimulated activation. Gel-shift assays showed increased complex formation after treatment, and this was inhibited by the addition of an Sp1 antibody or pretreatment with PKA inhibitor. Chromatin immunoprecipitation assay verified Sp1 binding to the promoter in response to PGE2. Sp1 overexpression increased SOCS3 promoter activation, and both basal and PGE2-induced SOCS3 mRNA expression was prevented by mithramycin, an inhibitor of Sp1 DNA binding. Finally, a physiological role for PGE2 was demonstrated with PGE2 pretreatment reducing lipopolysaccharide-induced STAT3 activation. Collectively, this study details a novel mechanism of SOCS3 up-regulation by PGE2 in breast cancer cells that appears to be STAT independent and involve Sp1 binding to the promoter. This process has possible implications for cytokine responsiveness and tumor progression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN THE MAMMARY GLAND, cytokines including prolactin (PRL) and GH as well as TGF-ß, IGF-I, and colony-stimulating factor 1 (CSF-1), signal through their cognate receptors to activate a range of signaling cascades, thereby regulating many cellular processes including proliferation, differentiation, and apoptosis. The role of cytokines in breast cancer formation and progression remains controversial, but increasing evidence suggests that PRL (1) and GH (2, 3), key regulators of mammary development and function, as well as other type 1 cytokines, potentially promote mammary gland tumorigenesis through uncontrolled signaling.

One of the key pathways employed after cytokine stimulation is the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. Cytokine binding causes receptor activation, which allows autophosphorylation of receptor-associated JAK, and subsequent receptor phosphorylation. This creates docking sites for STATs, which are in turn phosphorylated by JAK. Activated STATs form homo- or heterodimers and translocate to the nucleus where they bind to STAT response elements (SREs) on target genes and activate transcription (4). In the mammary gland, PRL receptor signaling through STAT5a is crucial for lactation, whereas in contrast, STAT3 activation occurs in association with involution at the end of lactation. The role of STAT1 in mammary gland development and function is not so clear, although it is expressed and regulated in a manner distinct from that of STAT3 and -5, indicating that it does contribute in some way (5).

One of the many target genes of activated STATs is suppressor of cytokine signaling 3 (SOCS3), which is up-regulated in response to cytokines. Increased SOCS3 protein then blocks the phosphorylation of STATs by JAK, effectively deactivating cytokine signaling. In this way, SOCS3 can modulate cytokine stimulation and prevent overstimulation in cytokine-responsive cells (6, 7). In the mammary gland, SOCS3 is induced by a number of cytokines, in particular PRL (8, 9), GH (10), and epidermal growth factor (11). SOCS3 expression is increased during involution of the mammary gland and functions to modulate the apoptotic effects of STAT3 during this process (12, 13, 14). SOCS3 expression is also increased in breast cancer, both in human breast carcinomas in vivo and in breast cancer cell lines (15), although its specific role in tumor progression is controversial. Although this increased SOCS3 expression could be due directly to cytokines, we and others have shown that prostaglandins are capable of inducing SOCS expression (16, 17, 18, 19), although no previous study has examined this action in a breast cancer model. T47D cells are a differentiated epithelial substrate from an infiltrating ductal carcinoma and were shown by Raccurt et al. (15) to express SOCS3. Using T47D cells, we have reported that prostaglandin E2 (PGE2) treatment stimulates expression of SOCS3 and SOCS1 (20). Breast tumors and breast cancer cells, including T47D cells (21), express high levels of cyclooxygenase 2 (COX-2), which results in increased PGE2 synthesis in these cells. This results in increased intracellular cAMP and subsequent estrogen synthesis (22), which is strongly implicated in breast cancer progression (23). In addition to this well-known effect, PGE2-induced SOCS3 expression may be important in regulating responsiveness to cytokines and could be involved in tumor progression.

The transcriptional regulation of SOCS3 expression has been studied in a number of cell systems in response to various stimuli, and the minimal functional SOCS3 promoter has been shown to contain two SREs, one proximal (–72/–64) and one distal (–95/–87) (24), as well as an activator protein 1 (AP1) element (–105/–99) (25), a GC-rich region (–58/–52) (26), and an A/T-rich region (–37/–31) (27, 28). Activation of this promoter in response to cytokines appears to involve the proximal SRE and one or more of the STATs, although there is some debate as to whether these SREs are more suitable for STAT1/3 or STAT5 binding (28, 29). In addition to the proximal SRE, it was shown that SOCS3 promoter activation by IL-6 in RAW 264-7 cells required binding of Sp3, but not Sp1, to the GC-rich region of the promoter (26). In hepatocytes, GH and IL-6 stimulation of SOCS3 transcription shows a requirement for both the GC- and A/T-rich regions (27). Stimulation of the SOCS3 promoter by cAMP-elevating agents in AtT-20 cells was shown to also be independent of SRE binding, rather relying on c-fos and JunB binding to the AP1 element on the promoter (25). In the present study of T47D breast cancer cells, we show that PGE2 uses the E prostanoid-4 (EP4) receptor to stimulate SOCS3 mRNA expression, and this action is independent of STATs. Rather, we show that PGE2-induced SOCS3 expression relies on protein kinase A (PKA) activation and subsequent Sp1 binding to the promoter, outlining a unique model of SOCS3 transcriptional regulation that is of potential importance for cytokine responsiveness and tumorigenesis in breast cancer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PGE2 Treatment Induces SOCS3 Expression
After treatment of T47D cells with PGE2, SOCS3 mRNA expression was significantly up-regulated at 30 and 60 min, returning to basal levels by 120 min (Fig. 1AGo). This induction was not inhibited by pretreatment of cells with the protein synthesis inhibitor cycloheximide (CHX) (Fig. 1BGo), indicating that PGE2-induced expression of SOCS3 does not require the synthesis of protein intermediates. In fact, CHX alone increased basal levels of SOCS3 mRNA, suggesting that a repressor of SOCS3 expression may be synthesized in these cells. However, the fold induction in response to PGE2 was similar in vehicle- and CHX-treated cells. To our knowledge, only one repressor of SOCS3 transcription has been identified, GFI-1B, which binds to specific motifs on the promoter (30). Finally, the effect of PGE2 treatment on SOCS3 protein was established by Western blot, with increased SOCS3 protein detected by 120 min (Fig. 1CGo).


Figure 1
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  		Fig. 1. PGE2 Stimulation of SOCS3 mRNA Expression

A, T47D cells were treated with PGE2 (250 ng/ml) for up to 120 min, and SOCS3 mRNA was measured by QPCR. Results are expressed as fold induction relative to vehicle-treated cells, and different superscripts denote significant differences (P < 0.01). B, T47D cells were pretreated with CHX (10 µM) for 1 h followed by treatment with PGE2 (250 ng/ml) for 1 h, and SOCS3 mRNA was measured by QPCR. Results are expressed as fold induction relative to vehicle-treated cells, and different superscripts denote significant differences (P < 0.01). C, T47D cells were treated with PGE2 (250 ng/ml) for up to 120 min and lysed in TK+ buffer. SOCS3 protein was detected by Western blot, and membrane was stripped and reprobed with an antibody to ß-actin to establish loading. D, RT-PCR was used to examine the expression of prostanoid receptors in T47D cells. E, T47D cells were treated with prostaglandin agonists butaprost (But), latanoprost (Lat), PGE2 (PGE), or sulprostone (Sulp) (250 ng/ml) for 1 h, and SOCS3 mRNA measured by QPCR. F, T47D cells were pretreated with L-161982 (10 µM) for 30 min and then treated with PGE2 for 1 h, and SOCS3 mRNA was measured by QPCR. Results are expressed as fold induction relative to vehicle-treated cells, and different superscripts denote significant differences (P < 0.01).

 
The expression of prostaglandin-EP and F-prostanoid (FP) receptors in T47D cells was examined using RT-PCR (Fig. 1DGo). PCR products were seen for EP2, EP3, and EP4 receptors but not EP1 and FP receptors. A series of prostaglandin agonists were then used to treat T47D cells, and SOCS3 mRNA expression was measured by quantitative real-time PCR (QPCR) (Fig. 1EGo). Although PGE2 increased SOCS3 expression, latanoprost (FP receptor agonist), butaprost (EP2 receptor agonist), and sulprostone (EP3 receptor agonist) had no effect. PGE2 can signal through all four EP receptors but has much higher affinity for EP3 and EP4 compared with either EP1 or EP2 (31). Because T47D cells do not express EP1 receptor (Fig. 1CGo), these results suggest that PGE2 up-regulates SOCS3 expression through the EP4 receptor. This was confirmed using a specific EP4 antagonist, L-161982, to pretreat cells before PGE2 treatment. Antagonism of the EP4 receptor abolished PGE2-induced SOCS3 mRNA expression (Fig. 1FGo).

SOCS3 Promoter-Luciferase Constructs: Response to PGE2
To study the SOCS3 promoter, a deletion series of the murine promoter cloned into the promoterless luciferase expression vector (pGL3) (Fig. 2AGo) was transfected into T47D cells, and luciferase activity was measured in response to PGE2 treatment. All constructs were responsive to treatment. Truncation at both the 3' (clone 4) and 5' (6T3) ends increased basal luciferase activity compared with that of clone 6, without diminishing overall responsiveness (Fig. 2BGo). 6T3 (–159/+959), representing the minimal functional promoter, was used for all additional experiments. It is important to note that 6T4, which is truncated at the 5' end to remove the AP1/cAMP response element (CRE) and both the SREs, still responded to PGE2 treatment.


Figure 2
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  		Fig. 2. SOCS3 Promoter Deletion Series

A, A murine SOCS3 promoter deletion series in the pGL3 luciferase reporter construct (24 ). B, Luciferase activity of these constructs was measured in response to PGE2 stimulation. T47D cells were transfected with each of the constructs and activity measured after treatment with PGE2 (250 ng/ml) for 6 h. Results are expressed as RLU, and different superscripts denote significant differences (P < 0.01). C, A schematic illustration of transcriptional response elements on the 6T3 minimal promoter, showing a distal SRE (–95/–87), a proximal SRE (–71/–64), an AP1 element (–105/–99), and a GC-rich region (–58/–52).

 
Analysis of the 6T3 promoter, in accordance with previous studies, showed the presence of two SREs, one distal (–95/–87) and one proximal (–72/–64) (24), an AP1 element (–105/–99) (25), and a GC-rich region (–58/–52) (27) (Fig. 2CGo). The AP1 element has also been described as a potential CRE (25), and the GC-rich region has been reported as capable of binding Sp3 (26).

SOCS3 Response to PGE2 Is Not Mediated by STATs
Involvement of the JAK/STAT pathway in cytokine-induced SOCS3 expression is well established (24). To examine whether PGE2 signals through this pathway, T47D cells were treated with PGE2, and subsequent STAT activation was determined by immunoprecipitation (IP)/Western blot. T47D cells showed readily detectable basal levels of STAT3-Y705 (STAT3-Y) and STAT3-S727 (STAT3-S) phosphorylation, which was increased by PGE2 treatment (Fig. 3AGo). Phosphorylation of Y705 is necessary for STAT3 dimerization and nuclear translocation, whereas phosphorylation of S727 conveys enhanced transcription activation (32). There was little detectable STAT5-Y694/699 or STAT1-Y701 phosphorylation, and further activation was not seen after PGE2 treatment (data not shown). To examine the importance of STAT3 activation in this system, we studied the SREs on the SOCS3 promoter by using site-directed mutagenesis to create distal and proximal mutants (dSREm and pSREm, respectively). These mutant promoters were compared with the parent 6T3 promoter for their ability to respond to PGE2 in a luciferase reporter assay. Mutation of either the dSRE or pSRE did not prevent PGE2-stimulated promoter activation (Fig. 3BGo), suggesting that there is no role for either SRE in this signaling pathway. Interestingly, mutation of the pSRE did reduce basal promoter activation and resulted in a greater fold increase in luciferase activity with PGE2 treatment. This suggests that although basal SOCS3 expression in these cells may be dependent on the integrity of this response element, SOCS3 expression in response to PGE2 is not. To address the possibility of redundancy between these response elements, a promoter was generated in which both SREs were mutated (d+pSREm). This promoter was still responsive to PGE2 treatment (Fig. 3CGo).


Figure 3
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  		Fig. 3. Involvement of STAT3 in SOCS3 Expression

A, Western blot analysis of STAT3 activation in response to PGE2. T47D cells were treated with PGE2 (250 ng/ml) for 15 and 30 min, and protein extracts were immunoprecipitated with STAT3 antibody. STAT3-S and STAT3-Y phosphospecific antibodies were used for Western blots, and STAT3 antibody was used to establish loading. B, Luciferase activity of WT promoter and SRE mutants in response to PGE2 treatment was measured. The 6T3 minimal promoter was mutated to generate distal (d)SRE (WT, ttacaagaa; mutant, tCCcaGgaa) and proximal (p)SRE mutants (WT, ttccaggaa; mutant, AtcGaCgaT). T47D cells were transfected with these constructs, and luciferase activity was measured after treatment with PGE2 (250 ng/ml) for 6 h. Results are expressed as percent RLU relative to vehicle-treated cells expressing 6T3, and different superscripts denote significant differences (P < 0.05). C, The 6T3 promoter was mutated to generate a dSRE and pSRE mutant promoter (d+pSREm). T47D cells were transfected with this construct, and luciferase activity was measured after treatment with PGE2 (250 ng/ml) for 6 h. Results are expressed as RLU relative to vehicle-treated cells expressing 6T3, and different superscripts denote significant differences (P < 0.05). D, Luciferase activity in response to PGE2 stimulation was measured in the presence of STAT3 overexpression. T47D cells were cotransfected with the 6T3 minimal promoter and pCDNA3 empty vector, STAT3 WT, STAT3 CA, or STAT3 DN expression constructs. Cells were treated with PGE2 (250 ng/ml) for 6 h. Results are expressed as RLU. Different superscripts denote significant differences (P < 0.05).

 
Finally, the involvement of STAT3 in the SOCS3 response to PGE2 was studied by coexpression of the 6T3 promoter-luciferase construct with either STAT3 wild-type (WT), constitutively active (CA), or dominant-negative (DN) constructs (Fig. 3DGo). Overexpression of STAT3 WT or CA did not affect basal promoter activity, whereas the DN construct significantly reduced basal promoter activation. Nevertheless, in all cases, there was a significant increase in promoter activation in response to PGE2 (P < 0.001), which was of similar magnitude when compared with the respective vehicle control (analysis not shown). Collectively, these results suggest that there is no involvement of STATs in PGE2-induced SOCS3 expression in these cells.

PGE2-Stimulated SOCS3 Expression Is PKA Dependent and Requires the GC-Rich Region of the Promoter But Does Not Involve CRE-Binding Protein (CREB) Activation or the AP1 Response Element
PGE2 is a known activator of the cAMP signaling pathway, and because SOCS3 expression can be stimulated by cAMP-elevating agents (25), it was necessary to investigate the possible involvement of cAMP in the PGE2 response. First we treated T47D cells with cAMP (Fig. 4AGo) or forskolin (Fig. 4BGo), a potent inducer of cAMP synthesis, and examined whether these could stimulate SOCS3 mRNA expression. Both treatments induced SOCS3 mRNA expression. Protein kinase A (PKA) is a downstream component of the cAMP signaling cascade. To assess the involvement of PKA, cells were pretreated with H89, a PKA inhibitor, followed by PGE2 treatment. PGE2-induced SOCS3 expression was significantly reduced by H89 pretreatment (Fig. 4CGo), suggesting the involvement of PKA in this signaling pathway. PKA activates CREB, which can bind directly to AP1/CRE motifs on promoters, alone or in conjunction with AP1 complexes. We therefore used IP/Western blot to determine whether PGE2 treatment caused CREB activation, and found that CREB-S phosphorylation was induced at 15 min (2.6-fold, P < 0.001) but not at 30 min (Fig. 4DGo). Finally, we tested the involvement of the CRE/AP1 element of the SOCS3 promoter by generating a mutant (designated AP1m) of the 6T3 promoter. This was compared with the parent 6T3 promoter in a luciferase reporter assay for its ability to respond to PGE2. Mutation of the AP1 element had no effect on basal or PGE2-induced SOCS3 promoter activation (Fig. 4EGo), suggesting that although CREB is activated by PGE2, subsequent binding to the promoter is not required for increased SOCS3 expression.


Figure 4
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  		Fig. 4. Involvement of cAMP and PKA Inhibitor H89 on SOCS3 mRNA Expression

A and B, Effect of cAMP (A) and forskolin (B) on SOCS3 mRNA expression. T47D cells were treated with cAMP (10 mM) for 1 h or forskolin (1 or 10 µM) for 1 h. SOCS3 mRNA was measured by QPCR, and results are expressed as fold induction relative to vehicle-treated cells. Different superscripts denote significant differences (P < 0.05). C, T47D cells were pretreated with H89 (10 µM) for 1 h, followed by PGE2 (250 ng/ml) for 1 h. SOCS3 mRNA was measured by QPCR, and results are expressed as fold induction relative to vehicle-treated cells. Different superscripts denote significant differences (P < 0.01). D, Western blots were used to examine CREB activation after PGE2. T47D cells were treated with PGE2 (250 ng/ml) for indicated time periods, and protein extracts were immunoprecipitated with CREB antibody. Western blot analysis using CREB-S antibody and CREB antibody to establish loading was performed. E, Luciferase activity of WT 6T3, AP1 mutant (AP1m), and GC mutant (GCm) promoters. The 6T3 minimal promoter was mutated to generate AP1m (WT, gtgactaa; mutant, AAgCTtaa) and GCm (WT, gggcgg; mutant, gTTcgg) promoters. T47D cells were transfected with the constructs and treated with PGE2 (250 ng/ml) for 6 h. Results are expressed as RLU. Different superscripts denote significant differences (P < 0.001). F, Luciferase activity of the Sp1 mutant promoter coexpressed with a STAT5 CA construct. T47D cells were cotransfected with the GCm promoter and either empty vector or STAT5 CA. Results are expressed as RLU.

 
Having eliminated a role for both SREs and the AP1 response element in the stimulation of the minimal SOCS3 promoter by PGE2, and considering previous studies showing the importance of the GC-rich region in PGE2 signaling (33) and SOCS3 expression (26, 27), a mutant of the GC-rich region (designated GCm) was generated. Mutation of the GC-rich region of the promoter completely abolished both basal and PGE2-induced promoter activation when compared with the 6T3 promoter construct (Fig. 4EGo), indicating that the integrity of this region is required for SOCS3 expression in these cells. The GC-rich region has been shown to be important for SOCS3 expression in other systems (26, 27), although not without the involvement of transcription factor binding other cis-regulator elements. Due to the complete loss of basal activity with this promoter, it is important to question the relevance of this site in PGE2-stimulated promoter activity, because this mutation may render the promoter completely inactive. However, the functional integrity of this mutant promoter was verified by overexpression of STAT5 CA, which caused activation of this promoter (Fig. 4FGo). Furthermore, the mutant promoter has been verified in pro-B cells, where it had no effect on basal activation and remained responsive to STAT5b activation (60).

PGE2 Treatment Causes Increased Sp1 Binding to the GC-Rich Element of the Promoter
Having established the importance of the GC-rich element, it was necessary to confirm transcription factor binding in response to treatment. Using EMSA with a probe encompassing the GC-rich region of the SOCS3 promoter, we saw that PGE2 treatment stimulated increased binding of nuclear protein, which was reduced by pretreatment with H89 (Fig. 5AGo). Interestingly, this nuclear protein was also detected on the promoter in vehicle-treated cells albeit in lower amounts, and the formation of this complex was prevented by the addition of a specific Sp1 antibody to either vehicle or PGE2-treated extracts (Fig. 5BGo). The absence of an upper supershift band suggests that the Sp1 antibody interacts with the DNA-binding region of the protein, preventing interaction with the labeled oligonucleotide, although this could not be confirmed with the manufacturer. Increased Sp1 binding to the SOCS3 promoter after PGE2 treatment was further verified using chromatin IP (ChIP) assay with an antibody against Sp1 (Fig. 5CGo).


Figure 5
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  		Fig. 5. Binding of Nuclear Proteins to GC-Rich Region of the SOCS3 Promoter

A, EMSA was used to examine nuclear protein binding to the GC-rich region of the SOCS3 promoter. Nuclear extracts from cells treated with PGE2 (250 ng/ml) for 1 h, or cells pretreated with H89 (10 µM) for 1 h followed by PGE2 (250 ng/ml) for 1 h, were combined with a labeled SOCS3 promoter probe (5'-cggggggcggggcgcggcggcc-3') encompassing the putative Sp1 response element (in bold type). B, EMSA was performed as above and exposed to film for a longer time. The addition of a specific Sp1 antibody was used to establish specificity of nuclear protein in the complex. C, ChIP assay was performed using an antibody to Sp1 and PCR primers to the human SOCS3 promoter. A no-antibody control (NC) and a total input control (PC) have been included.

 
SOCS3 Expression Is Dependent on Sp1 Binding and Is Augmented by Sp1 Overexpression
To assess the importance of Sp1 as a transcriptional activator of the SOCS3 promoter, Sp1 was overexpressed with the 6T3 promoter, and luciferase activity was measured (Fig. 6AGo). Sp1 overexpression increased basal promoter activation, which was further increased by PGE2 treatment, indicating that Sp1 was capable of driving SOCS3 transcription. Finally, to assess the importance of Sp1 in the SOCS3 mRNA response to PGE2, the Sp1 DNA binding inhibitor mithramycin was used. T47D cells were pretreated with or without mithramycin, followed by PGE2, and SOCS3 mRNA expression was determined using QPCR (Fig. 6BGo). Mithramycin reduced basal SOCS3 mRNA expression and prevented further expression after PGE2 treatment.


Figure 6
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  		Fig. 6. Sp1 Overexpression Causes SOCS3 Promoter Activation

A, Luciferase activity was measured in T47D cells that were transfected with 6T3 and either Sp1 or empty vector and treated with PGE2 (250 ng/ml) for 6 h. Results are expressed as RLU relative to vehicle-treated cells expressing 6T3, and different superscripts denote significant differences (P < 0.05). B, T47D cells were pretreated with mithramycin (10 µM) for 24 h followed by PGE2 (250 ng/ml) for 1 h. SOCS3 mRNA was measured by QPCR and expressed relative to vehicle-treated cells. Different superscripts denote significant differences (P < 0.05).

 
Pretreatment of T47D Cells with PGE2 Reduces LPS-Induced STAT3 Activation
Given that SOCS3 is an important negative regulator of STAT3 activation, the ability of PGE2-induced SOCS3 to affect STAT3 activation in response to lipopolysaccharide (LPS) was examined. The ability of LPS to activate STAT3 has been documented previously in a number of systems (34, 35), and here T47D cells were treated with LPS to induce STAT3 phosphorylation after PGE2 pretreatment for 2 h (to give optimal SOCS3 expression, Fig. 1CGo) (Fig. 7Go). Pretreatment with PGE2 significantly inhibited STAT3 tyrosine phosphorylation in LPS-treated cells (1.0 ± 0.2 and 0.65 ± 0.2 for LPS alone and PGE2 plus LPS, respectively, P < 0.05), indicating a physiological role of PGE2 on STAT3 activation, potentially through the up-regulation of SOCS3.


Figure 7
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  		Fig. 7. PGE2 Pretreatment Reduces LPS-Induced STAT3 Activation

Western blots were used to analyze STAT3 activation in T47D cells pretreated with or without PGE2 (250 ng/ml) for 2 h, followed by LPS (100 ng/ml) for 1 h. Phosphospecific STAT3-Y antibody was used to detect STAT3 activation, and STAT3 antibody was used to establish loading. Results of three experiments were analyzed by densitometry and show a 45% reduction in STAT3 tyrosine phosphorylation after PGE2 pretreatment (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
SOCS3 has the potential to modify responsiveness of breast cancer cells to important cytokines including PRL and GH as well as ILs. SOCS3 is expressed in breast cancer cells, and although cytokines themselves are likely to contribute to this, here we show that PGE2, which is produced in breast cancers and breast cancer cell lines (36), can also stimulate SOCS3 expression. The current study shows that in T47D breast cancer cells, this process depends on Sp1, most likely activated by PKA, binding to the SOCS3 promoter. We have also shown that this process is independent of STAT activation in these cells.

Although not previously studied in a breast cancer cell line, SOCS3 promoter activation by cytokines predominantly requires STAT activation and promoter binding (24, 26, 28). However transcriptional regulation of SOCS3 by noncytokine ligands, such as PGE2, does not appear to involve STAT activation and association with the SREs. For example, in pituitary tumor cells, cAMP-elevating agents induce SOCS3 expression via PKA activation and increase binding of c-fos and junB to the AP1 element on the promoter (25). Our results indicate that neither SRE is required for PGE2-stimulated SOCS3 expression in T47D cells, despite increased activation of STAT3 after PGE2 treatment, because mutation of these elements did not prevent PGE2 stimulation of the promoter (Fig. 3Go). In addition, the SOCS3 promoter was not regulated by binding of AP1 transcription factors to the AP1/CRE motif (Fig. 4Go). Rather, in T47D cells, PGE2 increased SOCS3 transcriptional activation by increased binding of Sp1 to the GC-rich region of the promoter.

Differential regulation of SOCS3 expression by distinct stimuli creates a situation whereby potentiation of SOCS3 expression is possible. For example, although cAMP-stimulating agents in isolation induce SOCS3 expression, costimulation with leukemia inhibitory factor (LIF) has an additive effect (25), and stimulation of PGE2-pretreated cells with IL-10 and IL-6 has, respectively, an additive and synergistic effect on SOCS3 expression (18). In contrast, treatment of erythroid cells with PGE2 alone was unable to induce expression of SOCS3, but pretreatment with PGE2 augmented erythropoietin-mediated SOCS3 expression (19). These studies highlight the potential cross talk between cytokine- and cAMP-mediated signaling pathways as well as the plasticity of the SOCS3 promoter, resulting in potentiation of SOCS3 expression.

The importance of the GC-rich region of the SOCS3 promoter in some systems has been established (26, 27), but no previous study has linked PGE2 stimulation to this cis-element or the involvement of Sp1. However, in another model, PGE2 treatment of smooth muscle cells stimulated VEGF expression via activation of Sp1. In the current study, the involvement of Sp1 in SOCS3 transcription was evaluated by its DNA binding in response to stimulation. Our results in T47D cells show that PGE2 stimulation caused increased nuclear protein binding to an oligonucleotide encompassing the GC-rich region of the SOCS3 promoter, and this protein was identified as Sp1 using a specific antibody (Fig. 5Go). Furthermore, ChIP analysis showed increased Sp1 association with the SOCS3 promoter after PGE2 treatment (Fig. 5Go). Sp1 is a tissue-specific transcription factor that regulates gene expression in response to various cell stimuli (33). It is widely expressed and binds to GC-rich motifs on promoters, influencing many cellular processes such as inflammation, apoptosis, cell cycle regulation, and cytokine responses (37). Sp1 can be phosphorylated on at least five confirmed residues by a number of kinases, such as PKA and PKC (38). It is also subject to further modification by glycosylation and sumoylation, and each of these posttranslational modifications has subsequent effects on the efficiency of Sp1 nuclear translocation, DNA binding, and transcriptional activity (39, 40). Although the current study does not identify such posttranslational modifications, this is worthy of further investigation.

Due to the marked loss of basal activity with the GCm promoter (Fig. 4Go), it is important to question the relevance of this site in PGE2-stimulated promoter activity, because this mutation may render the promoter completely inactive. However, this promoter was still activated by coexpression of STAT5 CA in T47D cells (Fig. 4Go), and in a model of pro-B cells, mutation of this element had no effect on basal or GH-stimulated activity (60). Furthermore, the shortest promoter in the deletion series, which is truncated immediately distal to the GC-rich cis-element and contains only 61 nucleotides before the ATG start codon, is still responsive to PGE2 treatment (Fig. 2BGo). Although it is feasible that this short region of promoter may contain additional response elements necessary for PGE2-induced activation, extensive promoter analysis did not reveal any likely candidates. The reason for this reduction in basal GCm promoter activity could be consequent to additional loss of STAT-dependent SOCS3 expression. STAT3 is directly associated with Sp1 in T47D cells, and STAT-dependent transcriptional activity of SOCS3 is dependent on both the pSRE and the GC-rich region of the promoter in these cells (our unpublished results). This is supported by the reduction in basal promoter activity seen with the overexpression of STAT3 DN and mutation of the pSRE (Fig. 3Go).

This study presents a model of SOCS3 expression in breast cancer cells in response to stimulation by PGE2, a ligand that is synthesized and secreted by these cells (21), and has a strong correlation with tumor growth (41). PGE2 can bind to EP receptors 1–4, each of which is associated with different G proteins and downstream signaling pathways, although it shows highest affinity for EP3 and EP4 (31). In T47D cells, it appears that EP4, which couples to G{alpha}s and stimulates cAMP-mediated pathways, is used by PGE2 to stimulate SOCS3 expression, as we show here (Fig. 1Go). Interestingly, a recent study of PGE2 signaling in murine breast cancer cells showed that antagonism of the EP4 receptor decreased metastatic potential of these cells (42).

The role of SOCS3 in breast cancer is controversial. SOCS3 is highly expressed in breast carcinomas and some breast cancer cell lines when compared with normal breast tissue or mammary cell lines (15). As a negative regulator of cytokine signaling via inhibition of the JAK/STAT pathway, SOCS3 has the potential to influence breast cancer development and progression through interference with these signaling cascades. For example PRL and GH, the key mammotropic hormones, rely on JAK/STAT signaling pathways to exert their effects on proliferation and differentiation, in particular the activation of STAT5a. There is evidence suggesting that aberrant activation by these ligands may be implicated in rodent models of breast cancer (4), although evidence in human breast cancer is inconsistent, with numerous studies showing conflicting results (43, 44, 45, 46, 47). However, overexpression of STAT5 DN constructs causes a reduction in tumor size in mice with T47D-derived tumors, strongly implicating STAT5 in tumor progression. We observed low levels of active STAT5 (and STAT1) in T47D cells. Active STAT3, on the other hand, was detectable in these cells and further inducible after PGE2 treatment.

STAT3 regulates apoptosis during involution of the mammary gland because STAT3-null mammary glands show delayed involution (48), and SOCS3 appears to modulate STAT3 because loss of SOCS3 in the mammary gland results in aberrant STAT3 activation (13). The proapoptotic role of STAT3 appears to be lost during tumorigenesis, because STAT3 activation is increased in mammary carcinomas (49) and gene silencing of STAT3 prevents tumor formation in immunodeficient mice injected with 4T1 cells (50). In addition, overexpression of STAT3 DN constructs in breast cancer cells leads to inhibition of colony formation (51), and chemical inhibition of STAT3 by indirubin (52) or resveratrol (53) results in cell cycle arrest in breast cancer cell lines. In the current study, we confirm that T47D cells express readily detectable levels of basal and PGE2-induced serine- and tyrosine-phosphorylated STAT3 but show that induction of SOCS3 expression by PGE2 is not dependent on this activated STAT3 (Fig. 3Go). That is not to say that this active STAT3 is not stimulating basal SOCS3 expression in these cells, as discussed above. The ability of SOCS3 to inhibit STAT3 activation has been previously demonstrated in many cancers other than breast cancer (54, 55, 56). Here we show in T47D breast cancer cells that treatment with PGE2 to induce maximal SOCS3 expression reduces LPS-stimulated STAT3 activation, confirming a physiological effect of PGE2 on STAT3 in these cells, which is potentially mediated by SOCS3. Furthermore, studies in our laboratory have shown that SOCS3 overexpression severely reduces STAT3 expression in T47D cells (our unpublished results). SOCS3 overexpression has been effectively used for the treatment of inflammatory disorders in rodents (57, 58, 59) and could be equally valuable for the treatment of breast cancer.

In the current study, PGE2 treatment of T47D cells resulted in a modest increase in SOCS3 promoter activity (Fig. 2Go) in comparison with the much larger increase in SOCS3 mRNA expression (Fig. 1Go). The physiological relevance of this response is indicated by the elevated SOCS3 protein in these cells after PGE2 treatment (Fig. 1Go). However, the modest response of the SOCS3 promoter to PGE2 treatment suggests that there may be additional mechanisms regulating the levels of SOCS3 protein in these cells. Alternatively, it is feasible that the minimal SOCS3 promoter used in this study does not contain all key elements and that other cis-regulatory sequences present further upstream or, in downstream introns, may contribute to PGE2-induced SOCS3 expression.

In conclusion, this study has described a novel pathway of SOCS3 up-regulation by PGE2 in breast cancer cells, involving Sp1 binding to the GC-rich region of the promoter. This pathway occurs via PGE2 signaling through the EP4 receptor and is independent of STAT activation. This has particular relevance in the case of STAT3, which is strongly implicated as a pro-oncogene in the mammary gland and is negatively regulated by SOCS3. The implications of such a signaling cascade in a breast cancer model are potentially significant, both for a better understanding of breast cancer signaling pathways and for the development of therapeutic drugs for treatment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
PhosphoPlus CREB (Ser133) Antibody Kit, phospho-STAT5 (Tyr694), and phospho-Stat3 (Ser727 and Tyr705) antibodies were purchased from Cell Signaling Technology (Beverly, MA); STAT3 (C-20), STAT1 (E-23), STAT5 (C-17), and Sp1 (PEP2) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); and SOCS3 antibody was from Zymed Laboratories (San Francisco, CA). ECL plus Western Blotting Detection System, Protein A Sepharose 6MB, and Nick columns were from Amersham Biosciences (Little Chalfont, UK). DNA-free kit was purchased from Geneworks (Adelaide, Australia), and Trizol reagent, SuperScript first-strand kit, and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA). TaqMan 2x PCR MasterMix was obtained from Applied Biosystems (Foster City, CA). Complete Inhibitors were purchased from Roche Diagnostics (Mannheim, Germany), and Steady-Glo Luciferase Assay System, Glo-Lysis buffer, Pfu DNA polymerase, and JM105 competent cells were from Promega (Madison, WI). The PKA inhibitor H89 and cAMP were from Calbiochem (La Jolla, CA), and forskolin, CHX, mithramycin, and dIdC were from Sigma (Victoria, Australia). dNTPs and DpnI were from New England Biolabs (NEB, Boston, MA), and QIAprep Spin miniprep kit was from QIAGEN (Victoria, Australia). Bicinchoninic acid (BCA) protein assay kit and Klenow fragment were obtained from Quantum Scientific (Queensland, Australia). All custom oligos were supplied by Sigma. The EP4 antagonist L-161982 was generously supplied by Merck Frosst (Quebec, Canada).

Expression Plasmids
The SOCS3 promoter deletion series in the luciferase expression vector pGL3 were generously donated by Shlomo Melmed (University of California, Los Angeles, Los Angeles, CA) (24). STAT3 and STAT1 WT, CA, and DN forms in the pCDNA3.1 expression vector were kindly donated by Dr. Robert Arceci (Johns Hopkins University, Baltimore MD). The STAT5 CA construct was obtained from Toshio Kitamura (University of Tokyo, Tokyo, Japan). The Sp1 expression plasmid was obtained from Robert Tjian (Howard Hughes Medical Institute, Berkeley, CA) and Levon Khachigian (University of New South Wales, Sydney, Australia).

Cell Culture
T47D cells were maintained in HEPES-modified RPMI with 10% fetal bovine serum, 10 µg/ml human insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37 C and 5% CO2. Cells were starved for 18 h before experiments in serum-free culture media unless stated otherwise.

Cell Stimulation and PCR
Cells were starved in 12-well dishes for 18 h before treatment with inhibitors (concentration and time dependent on the inhibitor) with or without PGE2 (250 ng/ml for 1 h). Cells were then washed in cold PBS and resuspended in Trizol reagent. RNA was extracted according to the Trizol reagent protocol and resuspended in a minimal volume of water. RNA was quantitated using a spectrophotometer and visualized on a 1.2% agarose gel. Two micrograms of RNA were DNase treated followed by reverse transcription, according to kit instructions. For QPCR, cDNA was diluted 1:20 in water, and PCR was performed using 4.5 µl cDNA, 3 µM and 2 µM TaqMan probes for ß-actin and SOCS3, respectively, 2 µM primers, and 13 µl TaqMan MasterMix in a 25-µl final volume. The following primers and probes were used: SOCS3 forward primer 5'-gaccagcgccacttcttcac-3', SOCS3 reverse primer 5'-ctggatgcgcagttcttg-3', SOCS3 probe, 6-FAM-5'-ctcagcgtcaagacccagtctggga-3'-TAMRA, ß-actin forward primer 5'-ctggcacccagcacaatg-3', ß-actin reverse primer 5'-gccgatccacacggagtact-3', and ß-actin probe 6-FAM-5'-atcaagatcattgctcctcctgagcgc-3'-TAMRA. Experiments were repeated at least twice, with all treatments done in triplicate, and each sample was analyzed in triplicate by QPCR using the ABI Prism 7000 TaqMan Real-Time PCR machine. Analysis was performed by calculating the relative change in target sequence between actin and SOCS3 and expressed as fold change relative to vehicle-treated cells (comparative CT method). For RT-PCR of prostaglandin receptor subtypes, cDNA was diluted 1:4. and 2 µl was used in a 50-µl reaction with 2 U Taq polymerase, 1x ThermoPol buffer, 400 µM dNTP, 500 µM primers, and 2.5% dimethylsulfoxide. Cycling conditions for EP1–EP4 receptor PCR were as follows: 94 C for 2 min; 35 cycles of 94 C for 30 sec, 60 C for 30 sec, and 72 C for 30 sec; and 72 C for 2 min. Cycling conditions for FP receptor PCR were 94 C for 2 min; 35 cycles of 94 C for 30 sec, 50 C for 30 sec, and 72 C for 30 sec; and 72 C for 2 min. Human prostanoid receptor primers were as follows: EP1 forward 5'-tcaacctgagcctggcgg-3', EP1 reverse 5'-cggcgtggagcagcggc-3', EP2 forward 5'-gcctccaatgactcccagtc-3', EP2 reverse 5'-ggctgaagaaggtcatggcg-3', EP3 forward 5'-tggcgaggattgcggatc-3', EP3 reverse 5'-acgctgcttggacaggtaca-3', EP4 forward 5'-cctccttgagccccgacc-3', EP4 reverse 5'-agaaataggcatgtttgatggc-3', FP forward 5'-atgaacaattccaaacagct-3', and FP reverse 5'-gtgactccaatacaccgc-3'.

Cell Transfection and Luciferase Reporter Assays
Cells were cultured in 24-well dishes to 70–80% confluency without antibiotics. They were then transfected using Lipofectamine 2000 reagent according to the manufacturer’s instructions, with 1 µg reporter construct with or without 1 µg expression plasmid or empty vector to standardize DNA content, and rested for 18 h. Cells were then starved in insulin- and serum-free culture media for 24 h and treated with PGE2 for 6 h. Cells were then lysed in 100 µl Glo-Lysis buffer, and 50 µl/well was added to 96-well plates along with 50 µl/well Steady-Glo Luciferase Reagent. Luciferase activity was measured using a BMG FLUOstar OPTIMA microplate reader. Results were normalized to protein concentration determined by BCA assay and expressed as relative luciferase units (RLU). Periodically, cells were cotransfected with green fluorescent protein to ensure transfection efficiency was not altered by different expression plasmids.

Immunoprecipitation and Western Blot
Treated cells were washed in cold PBS and resuspended in TK lysis buffer: 50 mM HEPES (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM Na3VO4, 30 mM NaF, 10 mM Na4P2O7, 10 mM EDTA with (TK+) or without (TK–) Complete Inhibitors. Protein A beads were washed twice in PBS and blocked in 1% BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature with rotation. Beads were then washed in PBS and TK– and incubated overnight with 2 µg antibody in TK– at 4 C with rotation. Beads were then washed in PBS and twice in TK– and incubated with 500 µg to 1 mg protein in TK+ buffer for 2 h at 4 C with rotation. Beads were washed three times in TK+ and then eluted with sample buffer [15 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 100 mM dithiothreitol (DTT)] at 100 C for 5 min. Samples were then electrophoresed in 8% SDS-PAGE gels, transferred onto nitrocellulose membranes, blocked in 2% BSA in TBST for 1 h at room temperature, and incubated with primary antibody in blocking buffer over night at 4 C with rocking. Membranes were then washed three times for 5 min each in TBST, followed by secondary antibody for 2 h at room temperature with rocking. Finally, membranes were washed, and ECL plus reagent used according to the manufacturer’s instructions to develop signal. For Western blots not requiring IP, cells were lysed into TK+ buffer, or 2% SDS for nonreducing conditions, before electrophoresis.

Site-Directed Mutagenesis
Mutant primers were used at 10 µM in a 50-µl PCR with Pfu DNA polymerase, 10 mM dNTPs, and 40 ng template (6T3 promoter construct). PCR was performed at 95 C for 30 sec, followed by 16–20 cycles of 96 C for 30 sec, 55–65 C for 60 sec, and 68 C for 10 min, followed by a 4 C hold. NEB reaction buffer 4 (5.5 µl) and 1 µl DpnI was added to each PCR, and samples were digested at 37 C for 2 h. Two microliters of this digestion were transformed into chemically competent JM105 cells. Colonies were picked from plates, and DNA was prepared by miniprep and sequenced by standard procedures.

EMSA
Confluent 10-cm dishes of T47D cells were starved for 18 h before treatment. Cells were washed in cold PBS, scraped, and centrifuged at 13,000 rpm for 1 min at 4 C. Pellets were resuspended in 400 µl buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM sodium vanadate, and Complete Inhibitors] and incubated on ice for 30 min. Cells were vortexed and centrifuged at 13,000 rpm for 1 min at 4 C, and the pellet was resuspended in 100 µl cold buffer C [20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 1 mM sodium vanadate, and Complete Inhibitors]. Nuclei were incubated on ice for 30 min, vortexed, and centrifuged at 13,000 rpm for 5 min at 4 C. Protein concentration of the supernatant was determined by BCA assay, and 5–10 µg nuclear extracts were used in each reaction. Synthetic oligonucleotides were annealed using 1 µg of each strand with 4 µl NEB buffer 2 in a 40-µl reaction, boiled for 2 min in a water bath, and cooled slowly with stirring. Four microliters annealed oligos with 3 µl each of 5 µM dGTP, dATP, and dTTP; 5 µl NEB buffer 2; and 1 µl Klenow fragment were labeled with 4 µl [32P]dCTP (250 µCi, 9.25-MBq stock) in a 50-µl reaction at room temperature for 1 h. Reactions were purified on Nick columns, counted, and diluted to 25,000–35,000 cpm/µl. Binding reaction was prepared with 5x binding buffer [50 mM Tris (pH 8.0), 200 mM KCl, 30% glycerol, 5 mM DTT, 0.25% NP40, to 10 ml with dH2O], 4 µg poly dIdC, 100 ng denatured salmon sperm DNA, 100 ng nonspecific single-stranded oligo, 10 µg BSA, 5–10 µg nuclear extract, with or without antibody, and incubated on ice for 20 min. Three microliters (~100,000 cpm) labeled probe were added and incubated for 10 min at room temperature. The reaction was loaded onto a 5% 0.5 x TBE nondenaturing polyacrylamide gel and run for 1.5–2 h at 150 V at room temperature. The gel was dried onto filter paper, and exposed to film for 18–48 h.

ChIP
Treated cells were cross-linked using 1% formaldehyde on a rocking platform for 10 min at room temperature. Cross-linking was stopped by the addition of 0.125 M glycine for 5 min, and cells were washed twice in cold PBS and lysed in 1 ml lysis buffer [1% SDS, 10 mM EDTA, 50 mM Tris (pH 8.0)]. Chromatin was sonicated on ice to an average length of 600 bp using four 30-sec pulses. Chromatin was centrifuged at 14,000 rpm for 10 min at 4 C, supernatant collected and diluted [0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), 167 mM NaCl]. Chromatin was precleared with 40 µl protein A-Sepharose (50% slurry), 2 µg sonicated salmon sperm DNA, and 20 µl preimmune rabbit serum for 1 h at 4 C. After gentle centrifugation to pellet beads, antibodies (2–4 µg) were added and bound at 4 C overnight with rotation, including a no-antibody negative control. Forty-five microliters protein A-Sepharose were added and further rotated for 1 h, and beads were washed for 10 min in each of TSI [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl, protease inhibitors], TSII [0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl, protease inhibitors], and TSIII [0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1), protease inhibitors]. Beads were washed twice in Tris-EDTA buffer and eluted three times for 15 min each on a vortex in elution buffer (1% SDS, 0.1 M NaHCO3) followed by centrifugation at 12,000 rpm for 3 min. Eluates were pooled and, after 8 µl 5 M NaCl was added, heated to 65 C for 5 h to reverse cross-linking. An additional sample of chromatin, for a total input positive control, was included. Samples were purified using QIAGEN PCR Purification Kit, eluted in 30 µl water, and used for PCR. PCR primers were designed specifically for the human SOCS3 promoter (forward 5'-gttccaggaatcggggggcggg-3', reverse 5'-cggctggctgcgtgcggggc-3'), and PCR (2 U Taq polymerase, 1x ThermoPol buffer, 400 µM dNTP, and 500 µM primers) was performed as follows: 94 C for 2 min; 35 cycles of 94 C for 30 sec, 70 C for 30 sec, and 72 C for 30 sec; and 72 C for 2 min.

Analysis of Results
Data were log transformed and one-way ANOVA performed to identify significant main events. Newman-Keuls multiple comparison test was then performed post hoc where significant.


   ACKNOWLEDGMENTS
 
We thank Shlomo Melmed for generously providing the SOCS3 promoter deletion series, Robert Arceci for kindly providing the STAT1 and 3 expression plasmids, Toshio Kitamura for the STAT5 expression plasmids and Robert Tjian and Levon Khachigian for the Sp1 expression plasmid.


   FOOTNOTES
 
Address requests for reprints to: Johanna Barclay, School of Biomedical Sciences, University of Queensland, Queensland 4072, Australia.

This work was supported by the Australian Research Council and a National Health and Medical Research Council Dora Lush Postgraduate Scholarship.

Disclosure: The authors have nothing to disclose.

First Published Online July 17, 2007

Abbreviations: AP1, Activator protein 1; BCA, bicinchoninic acid; CA, constitutively active; ChIP, chromatin immunoprecipitation; CHX, cycloheximide; CRE, cAMP response element; CREB, CRE-binding protein; DN, dominant-negative; DTT, dithiothreitol; dSREm, distal SRE mutant; EP4, E prostanoid-4; FP, F-prostanoid; JAK, Janus kinase; LPS, lipopolysaccharide; PGE2, prostaglandin E2; PKA, protein kinase A; PRL, prolactin; pSREm, proximal SRE mutant; QPCR, quantitative real-time PCR; RLU, relative luciferase units; SOCS3, suppressor of cytokine signaling 3; SRE, STAT response element; STAT, signal transducers and activators of transcription; TBST, Tris-buffered saline containing 0.1% Tween 20.

Received for publication January 17, 2007. Accepted for publication July 9, 2007.


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J. L. Barclay, S. T. Anderson, M. J. Waters, and J. D. Curlewis
Regulation of Suppressor of Cytokine Signaling 3 (SOC3) by Growth Hormone in Pro-B Cells
Mol. Endocrinol., October 1, 2007; 21(10): 2503 - 2515.
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