Analysis of SOCS-3 promoter responses to interferon gamma.
SOCS-3 (suppressor of cytokine signaling 3) is an intracellular protein that is selectively and rapidly induced by appropriate agonists and that modulates responses of immune cells to cytokines by interfering with the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. On the basis of the observations that interferon gamma (IFNgamma) up-regulates SOCS-3 gene and protein expression in primary mouse macrophages, J774 macrophage cell line and embryonal fibroblasts, we investigated which sequences of the 5' SOCS-3 gene are responsive to IFNgamma. By promoter deletion analysis we identified a functional IFNgamma-responsive element, located at nucleotides -72/-64 upstream from the transcription initiation, whose presence and integrity is necessary to ensure responsiveness to IFNgamma. This element contains a STAT consensus binding sequence (SOCS-3/STAT-binding element (SBE)) whose specific mutation totally abolished the responsiveness to IFNgamma. In contrast, discrete deletion of other 5' regions of the SOCS-3 promoter did not substantially modify the inducibility by IFNgamma. Electromobility shift assay analyses revealed that IFNgamma promotes specific DNA binding activities to an oligonucleotide probe containing the SOCS-3/SBE sequence. Even though IFNgamma triggered tyrosine phosphorylation of both STAT1 and STAT3 in macrophages and J774 cells, only STAT1 was appropriately activated and thus found to specifically bind to the SOCS-3/SBE oligonucleotide probe. Accordingly, IFNgamma-induced SOCS-3 protein expression was not impaired in STAT3-deficient embryonal fibroblasts. Taken together, these results demonstrate that the induction of SOCS-3 by IFNgamma depends upon the presence of a STAT-binding element in the SOCS-3 promoter that is specifically activated by STAT1.
Gatto
Luana
L
Department of Pathology, University of Verona, Strada le Grazie 4, 37134 Verona, Italy.
Berlato
Chiara
C
Poli
Valeria
V
Tininini
Silvia
S
Kinjyo
Ichiko
I
Yoshimura
Akihiko
A
Cassatella
Marco A
MA
Bazzoni
Flavia
F
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000276:immunology; D007371:Interferon-gamma / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics*
D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; D050794:STAT1 Transcription Factor; D050796:STAT3 Transcription Factor; C491411:Socs3 protein, mouse; C494080:Stat1 protein, mouse; C494087:Stat3 protein, mouse; D000071223:Suppressor of Cytokine Signaling 3 Protein; D050826:Suppressor of Cytokine Signaling Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D007371:Interferon-gamma