title: SOCS-1 and SOCS-3 inhibit IL-4 and IL-13 induced activation of Eotaxin-3/CCL26 gene expression in HEK293 cells.
abstract: Secretion of various chemokines including Eotaxin-3/CCL26 results in the attraction of eosinophils to sites of allergic inflammation. IL-4/IL-13-induced activation of the Eotaxin-3/CCL26 gene in human dermal fibroblasts was shown to be a STAT6-dependent process mediated by a single STAT6 binding motif located upstream of the transcription initiation site. The suppressors of cytokine signaling 1-3 (SOCS 1-3) are members of a recently discovered family of proteins acting as negative regulators of cytokine signaling. We show here, that transfection of SOCS-1 and SOCS-3 but not SOCS-2 expression vectors inhibited IL-4/IL-13 induced secretion of Eotaxin-3/CCL26. Further, using Eotaxin-3/CCL26 promoter reporter gene constructs, we could show that, upon cotransfection of SOCS-1 and SOCS-3 expression vectors, IL-4 and IL-13 induced luciferase activity was strongly reduced. This effect was not seen when SOCS-2 was cotransfected. Further, EMSA studies with nuclear extracts prepared from IL-4/IL-13 induced HEK293 cells were conducted. The nuclear extracts of cells transfected with SOCS-1 or SOCS-3 did not form complexes with oligonucleotide probes corresponding to the STAT6 binding site in the Eotaxin-3/CCL26 promoter. In contrast, complex formation upon SOCS-2-transfection was comparable to mock-transfected cells. Further, the levels of phosphorylated STAT6 in IL-4 and IL-13 treated cells were markedly reduced when the cells had been transfected with SOCS-1 or SOCS-3, confirming the role of these negative regulators for the IL-4 and IL-13 induced activation of Eotaxin-3/CCL26 gene expression. The insertion of amino acid exchanges into the kinase inhibitory regions of SOCS-1 and SOCS-3 demonstrated a requirement of these domains for a proper inhibitory function.
authors.affiliation: Department of Molecular Biology, University of Salzburg, Hellbrunner Str. 34, 5020 Salzburg, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Hebenstreit
authors.forename: Petra
authors.initials: P
authors.lastname: Luft
authors.forename: Angela
authors.initials: A
authors.lastname: Schmiedlechner
authors.forename: Albert
authors.initials: A
authors.lastname: Duschl
authors.forename: Jutta
authors.initials: J
authors.lastname: Horejs-Hoeck
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D002460:Cell Line; D000075906:Chemokine CCL26; D019742:Chemokines, CC / Q000096:biosynthesis / Q000235:genetics*; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression; D006801:Humans; D018793:Interleukin-13 / Q000037:antagonists & inhibitors*; D015847:Interleukin-4 / Q000037:antagonists & inhibitors*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics; D050800:STAT6 Transcription Factor; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C120429:CCL26 protein, human; D000075906:Chemokine CCL26; D019742:Chemokines, CC; D018793:Interleukin-13; D047908:Intracellular Signaling Peptides and Proteins; D012097:Repressor Proteins; C108944:SOCS1 protein, human; C106644:SOCS3 protein, human; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D000071222:Suppressor of Cytokine Signaling 1 Protein; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D015847:Interleukin-4