Analysis of SOCS-3 Promoter Responses to Interferon γ

Luana Gatto,Chiara Berlato,Valeria Poli,Silvia Tininini,Ichiko Kinjyo,Akihiko Yoshimura,Marco A Cassatella,Flavia Bazzoni

doi:10.1074/jbc.m308999200

Abstract

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.

Highlights

(SBE)) whose specific mutation totally abolished the re- mainly mediated through the regulation of gene expression
Induction of SOCS-3 Gene and Protein Expression by IFN␥—We initially investigated the ability of IFN␥ to induce SOCS-3 protein expression in the mouse macrophage cell line J774 and peritoneal elicited macrophages (PEM)
Following IFN␥ stimulation SOCS-3 mRNA was rapidly induced, reaching maximum levels within 1 h (Fig. 1C). These results extend at the protein level previous observations showing that IFN␥ is able to transiently induce SOCS-3 mRNA in different cell types [17, 18, 20, 37,38,39]

Summary

Introduction

(SBE)) whose specific mutation totally abolished the re- mainly mediated through the regulation of gene expression. Stimulation with IFN␥ caused no further increase of luciferase activity in J774 cells transfected with construct 6 (Ϫ50/ϩ968 nucleotides) as compared with J774 transfected with pGL2Basic alone (1.4 Ϯ 0.08 and 1.3 Ϯ 0.1-fold, respectively) (Fig. 3), indicating that the region from nucleotides Ϫ50 to ϩ968 is likely not involved in promoter activity.

Results

Conclusion