title: Characterization of the SOCS3 promoter response to prostaglandin E2 in T47D cells.
abstract: 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.
authors.affiliation: School of Biomedical Sciences, University of Queensland, Queensland 4072, Australia.
authors.forename: Johanna L
authors.initials: JL
authors.lastname: Barclay
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Anderson
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Waters
authors.forename: Jon D
authors.initials: JD
authors.lastname: Curlewis
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology*; D020862:GC Rich Sequence; D006801:Humans; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000187:drug effects; D020218:Response Elements; D050796:STAT3 Transcription Factor / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D016329:Sp1 Transcription Factor / Q000378:metabolism; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics* / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D008070:Lipopolysaccharides; C106644:SOCS3 protein, human; D050796:STAT3 Transcription Factor; D016329:Sp1 Transcription Factor; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D012694:Serine; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone