Interaction and Functional Interference of Glucocorticoid Receptor and SOCS1

Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNγ and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNγ treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling. Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNγ and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNγ treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling. Key principles determining the cellular response to cytokine signaling are duration of stimulus, prevalence of inhibitory feedback mechanisms, and operation of cross-talk with other cellular pathways. All of these factors interact to influence the strength and the quality of the physiological response. For cytokines operating via the Janus kinase (JAK) 2The abbreviations used are: JAKJanus kinaseSTATsignal transducers and activators of transcriptionSOCSsuppressor of cytokine signalingGRglucocorticoid receptorFCSfetal calf serumPBSphosphate-buffered salineIFNγinterferon γwtwild typeDexdexamethasoneGCglucocorticoidLBDligand binding domain. /signal transducers and activators of transcription (STAT) pathway, both suppressor of cytokine signaling (SOCS) proteins and the glucocorticoid receptor (GR) have been identified as important intracellular regulators in this respect. Janus kinase signal transducers and activators of transcription suppressor of cytokine signaling glucocorticoid receptor fetal calf serum phosphate-buffered saline interferon γ wild type dexamethasone glucocorticoid ligand binding domain. SOCS proteins have been shown to serve both as feedback inhibitors as well as mediators of cross-talk with other signaling pathways. They comprise a family of 8 members, initially described as negative feedback regulators of the JAK/STAT pathway. All SOCS proteins share two functional domains: an SH2 domain, which primarily enables binding of phosphorylated tyrosine residues, and a C-terminal SOCS box, which serves as a recruiting site for ubiquitin ligases, thereby combining specific inhibition of JAK catalytic activity with generic mechanisms such as competition over binding sites and targeting of associated proteins to proteasomal degradation (1Yoshimura A. Naka T. Kubo M. Nat. Rev. Immunol. 2007; 7: 454-465Crossref PubMed Scopus (1193) Google Scholar, 2Alexander W.S. Hilton D.J. Annu. Rev. Immunol. 2004; 22: 503-529Crossref PubMed Scopus (607) Google Scholar). Recent evidence suggests that the negative regulatory function of SOCS is not restricted to the JAK/STAT pathway. Through interaction with other signaling intermediates SOCS proteins can interfere with crucial signaling pathways such as the NF-κB and insulin receptor signaling pathways (3Ilangumaran S. Ramanathan S. Rottapel R. Semin. Immunol. 2004; 16: 351-365Crossref PubMed Scopus (117) Google Scholar). Furthermore glucocorticoids (GC) can also influence cytokine signaling. They act together with cytokines in an integrated fashion to control inflammation, immune response, and other more diverse physiological functions in mammals (4Rogatsky I. Ivashkiv L.B. Tissue Antigens. 2006; 68: 1-12Crossref PubMed Scopus (113) Google Scholar). The effect of GCs is mediated by the GR, a member of the nuclear receptor superfamily (5Webster J.I. Tonelli L. Sternberg E.M. Annu. Rev. Immunol. 2002; 20: 125-163Crossref PubMed Scopus (739) Google Scholar), which can serve as a transactivator or repress gene expression by interfering with other transcription factors (6De Bosscher K. Van den Berghe W. Haegeman G. Endocr. Rev. 2003; 24: 488-522Crossref PubMed Scopus (735) Google Scholar). In this respect, the GR has been described to interact directly or indirectly with essential transcription factors in cytokine signaling, namely AP-1, NF-κB, and STAT proteins. Interactions of the GR with AP-1 and NF-κB are involved in the negative cross-talk between cytokine and GC signaling (7Kassel O. Herrlich P. Mol. Cell. Endocrinol. 2007; 275: 13-29Crossref PubMed Scopus (223) Google Scholar), whereas the interaction between GR and STATs can be either synergistic or antagonistic, depending on the cell type, duration of stimulus, and STAT factor involved in the interaction (4Rogatsky I. Ivashkiv L.B. Tissue Antigens. 2006; 68: 1-12Crossref PubMed Scopus (113) Google Scholar, 8Engblom D. Kornfeld J.W. Schwake L. Tronche F. Reimann A. Beug H. Hennighausen L. Moriggl R. Schutz G. Genes Dev. 2007; 21: 1157-1162Crossref PubMed Scopus (91) Google Scholar). Here we establish a novel mode of interaction between cytokine and GC signaling, which was apparent in the early phase of GC stimulation. We provide evidence that SOCS1 is associated with the GR, and forms an intracellular complex that is disassembled after long term stimulation with GC. Furthermore, SOCS1 nuclear levels were found to increase in GC-treated cells. We tested the hypothesis that the interaction between GR and SOCS1 is influencing GC signaling. Our results led us to propose that SOCS1 is able to inhibit the transactivation activity of the GR and thereby to attenuate the transcriptional activation of GC-regulated genes. The study establishes a new link between cytokine and GC signaling with important implications for the regulation of inflammation as well as other more diverse physiological processes, where interactions of GC with JAK/STAT signaling are involved. Cell Culture—COS7 monkey kidney cells, were propagated in MEM medium supplemented with 10% heat-inactivated FCS and 50 μg/ml gentamycin; MEFs and NIH3T3 cells were propagated in Dulbecco's modified Eagle's medium with 10% FCS and gentamycin (50 μg/ml); P388D1 murine lymphoma cells were kept in RPMI with 10% FCS and gentamycin (50 μg/ml). Murine BMDM were prepared as described recently (9Tuckermann J.P. Kleiman A. Moriggl R. Spanbroek R. Neumann A. Illing A. Clausen B.E. Stride B. Forster I. Habenicht A.J. Reichhardt H.M. Tronche F. Schmid W. Schütz G. J. Clin. Investig. 2007; 117: 1381-1390Crossref PubMed Scopus (213) Google Scholar). All cells were cultivated in 5% CO2 at 37 °C and subcultured every 3–4 days. To eliminate basal activation of the glucocorticoid receptor by serum glucocorticoids, all cells were washed prior to experiments three times with prewarmed serum-free F-12/DMEM supplemented with 50 μg/ml gentamycin and 200 μg/ml Albumax II (Invitrogen). Hormones, Cytokines, and Plasmids—Murine IFNγ was purchased from PeproTech (Rocky Hill, NJ) and used at a concentration of 20 ng/ml. Dexamethasone (Sigma) was diluted in 70% ethanol and applied in concentrations ranging from 100 nm to 1 μm. RU486 was kindly provided by Dr. E. Baulieu and used at a final concentration of 1 μm. Myc-tagged wild-type SOCS1 and SOCS1 deletion mutants were described previously (10Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (602) Google Scholar). Rat GR mutants (11Starr D.B. Matsui W. Thomas J.R. Yamamoto K.R. Genes Dev. 1996; 10: 1271-1283Crossref PubMed Scopus (109) Google Scholar) were kindly provided by Dr. Starr. Transient Transfections—Transient transfections were carried out using Transfast transfection reagent (Promega, Madison, WI). For a 10-cm cell culture dish, 3 μg of DNA were mixed with 9 μl of Transfast in 1200 μl of serum-free Opti-Mem I (Invitrogen). After 3 h, medium supplemented with 10% FCS was added. Antibodies—Rabbit polyclonal anti-GR antibody (M-20, Santa Cruz Biotechnologies) was used at a dilution of 1:350 for immunoblotting and 1:150 for immunofluorescence microscopy. Anti-c-Myc mouse monoclonal antibody (9E10, Santa Cruz Biotechnology) was used at 1:1000 and 1:500 for immunoblotting and immunofluorescence microscopy, respectively. Anti-GAPDH (MAB374, Chemicon Int.) was used at a 1:1500 dilution, anti-SOCS1 (4H1, MBL International, Woburn, MA) at 1:200, and anti-GR (MAI-510, ABR, Golden, CO) at 1:350. Immunoprecipitation and Immunoblotting—For total cell lysates (TCL), cells were washed three times with ice-cold PBS and then lysed in coIP lysis buffer (50 mm HEPES, pH 7.5, 1 mm EGTA, 2 mm EDTA, 12.5 mm β-glycerophosphate, 3.2 mm MgCl2, 10% glycerol, 1% Triton X-100, 5 μg/ml aprotinin, 5 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 1.19 mm Na3VO4, 2.5 mm NaF) for 30 min at 4 °C on a rocking platform. Lysates were centrifuged (21,000 × g) at 4 °C for 30 min to remove insoluble fractions and precleared with Cruz Biotechnology) for 1 To SOCS1 GR interaction, were with 5 μg of All experiments were in with the and have been by the Tissue were prepared from or of to and lysed in 1200 μl of coIP lysis buffer supplemented with μg/ml by two and by Lysates were then to × g) at 4 °C for 30 were precleared as described and with specific as To immune was added. were by were washed three times with lysis in lysis and in buffer by and were prepared as described previously Mol. Cell. PubMed Google Scholar). were in or by depending on and to were with reagent for for 1 h, and were applied at the in reagent at 4 and were used at were using a were using provided by was carried out using for cells were on and after cells were washed with with in PBS for min on and by for 5 to antibody cells were with serum in PBS for 1 and were diluted in PBS with serum and for 1 were washed three times with 20 in PBS after with the and were in medium and by using a with a was with of SOCS1 and the genes and FKBP5 was by using the was from cells using the of prepared was by of and in an μg of total was with to the All were carried out on an as described previously B. J.P. F. E. G. W. 2007; 7: PubMed Scopus Google Scholar). To control for in quality and expression of the gene of was to the expression of the expression levels were to the where was as of and as of and used in the study can be found as SOCS1 with the SOCS1 with the GR in we Myc-tagged SOCS1 and GR in cells. Cell lysates were to using either the of SOCS1 or the GR. proteins were by and for SOCS1 and GR SOCS1 was in the GR immune complex and GR was with the that both proteins form a of SOCS1 has been to be and has been for the or from and required the of and immunoblotting S. J. W.S. Hilton D.J. J. 2006; PubMed Scopus Google Scholar, A. S. A. J. 275: PubMed Scopus Google Scholar). To the of SOCS1 and GR we experiments with prepared from of described previously S. J. W.S. Hilton D.J. J. 2006; PubMed Scopus Google Scholar), mouse SOCS1 expression and as a negative control for SOCS1 SOCS1 be in anti-GR in lysates from that SOCS1 and GR are also associated in a complex at physiological lysates from and lysates with an anti-SOCS1 antibody as a control for of the SOCS1 to the GR after GC GR is a transcription factor and has been shown to interact with and in a fashion Endocr. Rev. 18: PubMed Scopus Google Scholar). We which domain of the GR is required for SOCS1 interaction and ligand binding has implications on the interaction of the receptor with SOCS1. cells were with SOCS1 and GR or GR deletion We used wild-type GR, which we have shown to SOCS1 the C-terminal deletion the ligand binding domain and the deletion the (11Starr D.B. Matsui W. Thomas J.R. Yamamoto K.R. Genes Dev. 1996; 10: 1271-1283Crossref PubMed Scopus (109) Google Scholar). Cell lysates were with an antibody the of SOCS1 and by We observed that the the of GR did not with that the interaction of SOCS and GR the of the in suggesting an of this domain. deletion of the DNA binding domain W. M. J. J. S. T. J. S. H. D.B. H. D. S. Mol. Cell. 21: PubMed Scopus Google Scholar) did not interfere with the of the GR to to SOCS1 not To ligand binding has an effect on the of the cells were with SOCS1 and GR Cell lysates were prepared and after GC stimulation, and anti-GR were for SOCS1 the of SOCS1 after stimulation with the GR dexamethasone After a SOCS1 binding to the GR was We this in binding of SOCS1 to the GR was the of transcriptional activation by the or also with the shown in the two of both and in the of SOCS1 that the effect not on a GR. The was by experiments with a GR previously described by and S. PubMed Scopus Google Scholar), that is able to RU486 not and is of transcriptional RU486 for in binding of SOCS1 to the GR whereas did not effect on SOCS1 GR interaction as We observed that the SOCS1 which the C-terminal the SOCS box, as the that proteasomal degradation of other proteins by SOCS1 for the observed in binding after GC stimulation not together these results suggest that long term ligand binding the interaction of GR and SOCS1 and not on GR transcriptional activity and SOCS proteasomal The SH2 of SOCS1 for GR the site on SOCS1 for the interaction with GR, wild-type and of SOCS1 were tested in the SOCS1 a in the SH2 domain which binding to and the which the SOCS the GR. of the of the also did not in a binding binding was abolished in the of a deletion the and the SH2 domain these results that the SH2 domain is required for GR SOCS1 interaction, not the of the SH2 domain to to which of SOCS1. the SH2 domain is the SOCS we other of this family share the to to the GR. shown in and were with the GR, with as by the of in from in the of SOCS1 to and in the of and respectively. observed for long term GC stimulation in binding of and to the GR. results suggest that the GR is able to interact with the SH2 domain of other SOCS proteins by a as with GC of observed in experiments the ligand nuclear of the GR can influence the nuclear concentration of SOCS1 in cells. the of GR and SOCS1 in nuclear and prepared from cells for 3 with or from cells, was by stimulation in nuclear of the GR. also a in the nuclear to SOCS1 suggesting that SOCS1 is the in a cells with SOCS1 expression did not in nuclear stimulation. We the of GR and SOCS1 by in cells with expression for both proteins or with were with for 3 or and by microscopy. In cells with GR a nuclear of the GR was observed stimulation as in cells with did not an effect on the of SOCS1 both proteins were the of SOCS1 and both proteins a In cells, SOCS1 was in the and in nuclear of that the GR is of influencing the of SOCS1 and results from experiments SOCS1 GR the interaction of SOCS1 and GR an on the transactivation activity of the GR, the effect of SOCS1 expression on the transcription of GC-regulated genes was in lymphoma cells, murine and NIH3T3 SOCS1 expression was by IFNγ for 2.5 h, by activation of the GR with for FKBP5 and were as GC genes, because have been shown to be by GC H. B. M. J. Clin. Endocrinol. 2003; PubMed Scopus Google Scholar, T. Kleiman A. P. M. J. J. J. 2006; PubMed Scopus Google Scholar). FKBP5 for a which forms of the receptor complex H. B. M. J. Clin. Endocrinol. 2003; PubMed Scopus Google Scholar). kinase 1 as is a essential for inhibition of kinase and and thereby to the response of GCs T. Kleiman A. P. M. J. J. J. 2006; PubMed Scopus Google Scholar). levels were by and in levels were In all three cell with IFNγ in a of levels IFNγ and with suggests an inhibitory effect of IFNγ on GR because IFNγ GR function via mechanisms, we experiments with SOCS1-deficient MEFs and compared with control MEFs were with IFNγ to SOCS1 expression in control MEFs and then treated with for the expression levels of FKBP5 or were not by IFNγ and were in and control MEFs For both GC-regulated genes MEFs levels stimulation as compared with control were 8 and 4 after stimulation for FKBP5 and MKP1, in the of between and control MEFs was observed in the of FKBP5 and by results hypothesis of a negative effect of SOCS1 on GR After stimulation, the FKBP5 and levels in to be from control for an early phase effect of SOCS1 on GR transactivation which is after long term stimulation. The is in with the of the interaction after GC stimulation as observed in experiments Interactions of signaling pathways an important role in the and of the cellular response. inflammation, a between and signaling has to be to and GCs are for and cytokine signaling, through the JAK/STAT pathway, Here we propose a new mode of cross-talk between GC and cytokine signaling mediated by an interaction between the GR and the SOCS1 which is after long term with GC. of and for for the of an intracellular complex between SOCS1 and GR was from experiments with of cells as well as of lysates of the interaction is that the GR the of SOCS1. shown in cells GR and of cells with GC SOCS1 nuclear that ligand SOCS1 the together with GR is that the of has also been described to be by the GR B. Mol. Cell. 18: PubMed Scopus Google Scholar). the of cells with GCs in nuclear of suggesting that is with GR nuclear we have not the between GR and we that physiological GR is in and that a of intracellular SOCS1 associated and the together with the GR. The of a is by the of SOCS1 with a GR antibody from of murine with GR mutants the C-terminal of the GR to the DNA binding domain to be required for the interaction with SOCS1 of the GR the binding sites as well as transcription activation Endocr. Rev. 18: PubMed Scopus Google Scholar, S. Yamamoto K.R. EMBO J. PubMed Scopus Google Scholar, Cell. PubMed Scopus Google Scholar). All these functional are and on the of the GR. is that an of SOCS1 with the interfere with the of proteins such as receptor and transcriptional factor 2 P. H. EMBO J. 1996; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar), which the negative regulatory effect of SOCS1 on GR transactivation observed in study The binding of SOCS1 to the GR after of the transactivation domain to the role of The of SOCS1 and GR in the have implications on GR function by interfering with the of transcriptional and to genes, which has to be in The GR interaction domain of SOCS1 was to the SH2 domain domain has previously been shown to the binding of a of proteins in a and fashion (10Yasukawa H. Misawa H. Sakamoto H. Masuhara M. Sasaki A. Wakioka T. Ohtsuka S. Imaizumi T. Matsuda T. Ihle J.N. Yoshimura A. EMBO J. 1999; 18: 1309-1320Crossref PubMed Scopus (602) Google Scholar, P. S. Rottapel R. J. 275: PubMed Scopus Google Scholar, M. T. H. T. M. M. Yoshimura A. 2004; PubMed Scopus Google Scholar, S. E. S. D. P. S. A. 2002; PubMed Scopus Google Scholar). with mutants be required to the interaction of the GR and SOCS1 in more the interaction of the GR was not restricted to SOCS1 was also observed with the SH2 proteins and that the binding to the GR be a function of SOCS proteins and a level of for cross-talk between GR and cytokine signaling. the of the other proteins was compared with the physiological of these interactions to be SOCS proteins have been shown to and degradation of proteins via SOCS (3Ilangumaran S. Ramanathan S. Rottapel R. Semin. Immunol. 2004; 16: 351-365Crossref PubMed Scopus (117) Google Scholar, D. S. M. R. W. W.S. Hilton D.J. S. A. PubMed Scopus Google Scholar), suggesting the that the negative effect of SOCS1 on GR GR in experiments with and cells to GR in the or of we not an influence of SOCS1 on GR Furthermore, we did not a in the level of GR in these cells in the or of an important role of SOCS1 in GR SOCS1 also not to be for the of GR and because of either SOCS1 or the SOCS deletion in suggest that the observed effect of SOCS1 on GR transactivation is not a of degradation of the GR. of the Transient of of the intracellular complex of GR and SOCS1 was by GR ligand binding with effect of ligand well after the effect of ligand on the early of receptor which of nuclear and The effect did not require transactivation via the GR, because it was observed with a GR in transcriptional activity as well as after binding of the GR The interaction between SOCS1 and GR to the more of the GR on two other cytokine signaling AP-1 and NF-κB (6De Bosscher K. Van den Berghe W. Haegeman G. Endocr. Rev. 2003; 24: 488-522Crossref PubMed Scopus (735) Google Scholar, O. Herrlich P. Mol. Cell. Endocrinol. 2007; 275: 13-29Crossref PubMed Scopus (223) Google Scholar). mechanisms for the functional of GC signaling with the JAK/STAT have been which comprise long term of the GR via activation of GR-regulated genes (4Rogatsky I. Ivashkiv L.B. Tissue Antigens. 2006; 68: 1-12Crossref PubMed Scopus (113) Google Scholar, Ivashkiv L.B. J. Immunol. 2003; PubMed Scopus Google Scholar, S. M. B. O. J. Immunol. PubMed Scopus Google Scholar). GR was shown to with a of inflammation via the IFNγ JAK/STAT S. M. B. O. J. Immunol. PubMed Scopus Google Scholar). In this of transcriptional activity was observed to enhanced transcription of the receptor and this was to the of transcriptional by the GR. In long term GC negatively the of IFNγ signaling by expression at the level Ivashkiv L.B. J. Immunol. 2003; PubMed Scopus Google Scholar). the novel mechanisms for negative between GC and the SOCS1 described by was prominent in the early phase of GC action. shown in the interaction of GR with SOCS1 inhibited GR and this effect was apparent early after GC stimulation and not observed after long term in with the of the GR SOCS1 complex these of in of on the negative effect of SOCS1 on GR function is that expression levels of SOCS1 as observed in P. T. P. A. M. T. T. O. 2007; PubMed Scopus Google Scholar) in to GC and to of GR function in inflammation has been to the of which can the of the negative of GR and thereby GC S. A. PubMed Scopus Google Scholar), or to a negative effect of NF-κB on GR transactivation S. A. PubMed Scopus Google Scholar, Mol. Endocrinol. PubMed Scopus Google Scholar). the GR T. J. PubMed Scopus Google Scholar) and SOCS1 (1Yoshimura A. Naka T. Kubo M. Nat. Rev. Immunol. 2007; 7: 454-465Crossref PubMed Scopus (1193) Google Scholar) have been for a long as intracellular regulators of the response. have been to act from Our provide a novel link by which the two proteins can interact with is that the negative cross-talk between GR and SOCS1 a to a effect of a of these two inhibitors of cytokine signaling. To this hypothesis and to the of the interaction in the of the cellular response, it be important to mutants of the GR or SOCS1 that are in negative cross-talk. We for for of and and for in BMDM and for the of and and for and on the with