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Molecular Endocrinology, doi:10.1210/me.2005-0301
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Molecular Endocrinology 20 (7): 1587-1596
Copyright © 2006 by The Endocrine Society

Suppressor of Cytokine Signaling-3 Inhibits Interleukin-1 Signaling by Targeting the TRAF-6/TAK1 Complex

Helle Frobøse, Sif Groth Rønn, Peter E. Heding, Heidi Mendoza, Philip Cohen, Thomas Mandrup-Poulsen and Nils Billestrup

Steno Diabetes Center (H.F., S.G.R., P.E.H., T.M.-P., N.B.), 2820 Gentofte, Denmark; Medical Research Council Protein Phosphorylation Unit (H.M., P.C.), School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom; and Department of Molecular Medicine (T.M.-P.), Karolinska Institute, Stockholm, Sweden

Address all correspondence to: Nils Billestrup, Steno Diabetes Center, Niels Steensens Vej 2, 2820 Gentofte, Denmark. E-mail: NBil{at}steno.dk.Address reprint requests to: Helle Frobøse, Steno Diabetes Center, Niels Steensens Vej 2, 2820 Gentofte, Denmark.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IL-1 plays a major role in inflammation and autoimmunity through activation of nuclear factor {kappa} B (NF{kappa}B) and MAPKs. Although a great deal is known about the mechanism of activation of NF{kappa}B and MAPKs by IL-1, much less is known about the down-regulation of this pathway. Suppressor of cytokine signaling (SOCS)-3 was shown to inhibit IL-1-induced transcription and activation of NF{kappa}B and the MAPKs JNK and p38, but the mechanism is unknown. We show here that SOCS-3 inhibits NF{kappa}B-dependent transcription induced by overexpression of the upstream IL-1 signaling molecules MyD88, IL-1R-activated kinase 1, TNF receptor-associated factor (TRAF)6, and TGFß-activated kinase (TAK)1, but not when the MAP3K MAPK/ERK kinase kinase-1 is used instead of TAK1, indicating that the target for SOCS-3 is the TRAF6/TAK1 signaling complex. By coimmunoprecipitation, it was shown that SOCS-3 inhibited the association between TRAF6 and TAK1 and that SOCS-3 coimmunoprecipitated with TAK1 and TRAF6. Furthermore, SOCS-3 inhibited the IL-1-induced catalytic activity of TAK1. Because ubiquitination of TRAF6 is required for activation of TAK1, we analyzed the role of SOCS-3 on TRAF6 ubiquitination and found that SOCS-3 inhibited ubiquitin modification of TRAF6. These results indicate that SOCS-3 inhibits IL-1 signal transduction by inhibiting ubiquitination of TRAF6, thus preventing association and activation of TAK1.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IL-1 IS A PLEIOTROPIC proinflammatory cytokine affecting many cell types. It plays a critical role in immune and inflammatory responses, and IL-1-stimulated cells show an inflamed phenotype, expressing proteins involved in immune reactivity and inflammation (1, 2). The IL-1 family consists of three members; IL-1{alpha}, IL-1ß, and IL-1 receptor antagonist (IL-1Ra), with IL-1ß being the most abundantly expressed in humans (3). Binding of IL-1 to the extracellular part of the IL-1 receptor leads to conformational changes that allow docking of IL-1 receptor accessory protein (IL-1RacP) to the IL-1/IL-1RI complex (Fig. 1Go, step 1). This induces binding and activation of the adaptor protein MyD88, which subsequently recruits IL-1R-activated kinase 4 (IRAK4) to the receptor complex. Preformed Tollip/IRAK1 complexes that reside in the cytoplasm are recruited to the receptor complex where IRAK1 binds to MyD88 through a death domain and is released from Tollip (4). In this way, IRAK4 and IRAK1 are brought into close proximity allowing IRAK4 to phosphorylate IRAK1, triggering multiple autophosphorylations of IRAK1 (Fig. 1Go, step 2). Phosphorylated IRAK1 interacts with TRAF6, thereby bringing about a transient association of TRAF6 with the receptor complex. TRAF6 and IRAK1 dissociate from the receptor and interact at the membrane with a preformed complex consisting of TGF-ß-activated kinase 1 (TAK1) and two TAK1 binding proteins called TAK1 binding protein 1 and 2 (TAB1 and TAB2) (5, 6) (Fig. 1Go, step 3). The TRAF6/TAK1/TAB1/TAB2 complex translocates to the cytosol, where it binds to the ubiquitin ligases Ubc13 and Uev1A (Fig. 1Go, step 4). This leads to the ubiquitination of TRAF6, triggering activation of TAK1 (7), which then phosphorylates the I{kappa}B kinase (IKK) complex (Fig. 1Go, step 5). IKK phosphorylates I{kappa}B, a cytosolic inhibitory protein that retains the transcription factor NF{kappa}B in the cytoplasm (Fig. 1Go, step 6). Once phosphorylated, I{kappa}B dissociates from its complex with NF{kappa}B (Fig. 1Go, step 7) and is rapidly degraded by the proteasome (3, 8). The dissociation allows NF{kappa}B to translocate to the nucleus and bind to various promoters whereby transcription is initiated (Fig. 1Go, step 8) (3, 8, 9). TAK1 also activates MAPK kinases (MKK) 1/3/6/7 (Fig. 1Go, step 9), which subsequently phosphorylate the MAPKs ERK, p38, and JNK (Fig. 1Go, step 10). The activated MAPKs exert their effect through serine/threonine phosphorylation of their substrates, including the transcription factors Elk-1, c-Jun, and ATF2 (Fig. 1Go, step 11).


Figure 1
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  		Fig. 1. Schematic Presentation of the IL-1 Signaling Cascade

When activated by IL-1 binding, the receptor complex starts a cascade of numerous steps. MyD88, IRAK4, IRAK1, and Tollip are recruited to the receptor and upon activation of IRAK1, TRAF6 is activated. A complex consisting of TRAF6, TAK1, TAB1, TAB2, and the two ubiquitin ligases Uev1A and Ubc13 is formed, phosphorylating TRAF6 and subsequently TAK1. TAK1 activation leads to activation of both NF{kappa}B and the MAPKs, followed by gene transcription. The different steps are described in detail in the text and numbered accordingly. DD, Death domain; Ub,ubiquitin.

 
Characterization of the mechanisms of negative regulation of IL-1 signaling is crucial for the understanding of how IL-1 signaling is controlled and how dysregulation might contribute to various conditions such as autoimmunity and inflammation. Cytokine signaling is negatively regulated by decoy and soluble cytokine receptors, the process of receptor internalization and degradation, and cytokine-induced intracellular feedback inhibitors of the cytokine response, including the suppressors of cytokine signaling (SOCS) proteins.

The SOCS family consists of eight intracellular proteins; cytokine-inducible SH2 protein (CIS) and SOCS-1 through 7, all containing a central SH2 domain and a conserved carboxy-terminal region called the SOCS box (10, 11, 12). The expression of CIS, SOCS-1, SOCS-2, and SOCS-3 is induced in response to stimulation by a wide variety of cytokines, and as transcription of the Socs genes is induced by cytokine-induced signal transducer and activator of transcription (STAT) and NF{kappa}B transcription factors, the corresponding SOCS proteins inhibit the same pathway initiating their production, indicating that SOCS proteins form part of a classical negative feedback loop (11, 12).

The most studied signaling pathway regulated by SOCS proteins is the Janus kinase (JAK) and signal transducer and activator of transcription pathway (13, 14). SOCS-1 and -3 inhibit JAK activity through binding to the phosphorylated cytokine-receptor or JAK directly. CIS and SOCS-2 seem to inhibit signaling by binding to phospho-tyrosines in the activated cytokine receptors, thus preventing recruitment of STAT factors to the receptor complex. Finally, SOCS proteins can mediate proteosomal degradation of key signaling molecules by binding to Elongin B and C through the SOCS-box. Elongin B and C associate with cullin-2 to form part of a putative E3 ubiquitin ligase facilitating the ubiquitination and subsequent proteasomal degradation of associated signaling proteins (15). However, the mechanism by which SOCS-3 inhibits IL-1 signaling is unknown.

We have previously shown that expression of SOCS-3 in the insulin-producing cell line INS-1, reduces IL-1-stimulated nitric oxide production and prevents apoptosis induced by IL-1 (16) and have recently characterized the gene expression profile in these cells. The results revealed several IL-1-induced proapoptotic and NF{kappa}B-dependent genes to be inhibited by SOCS-3, and SOCS-3 was found to reduce NF{kappa}B DNA binding induced by IL-1 (17), suggesting that SOCS-3 inhibits IL-1 signaling at the level of NF{kappa}B activation or at a step upstream.

Therefore, we investigated the mechanisms whereby SOCS-3 exerts its inhibitory effect on IL-1-induced signaling in ß-cells, by identification of the site of inhibition in the IL-1 signaling pathway and characterization of the molecular basis for the inhibitory action of SOCS-3.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SOCS-3 Inhibits IL-1-Induced MAPK Phosphorylation, I{kappa}B Degradation, and NF{kappa}B DNA Binding
To investigate the level at which SOCS-3 inhibits IL-1 signaling, we analyzed both the MAPK and NF{kappa}B pathways of the IL-1 signaling cascade for their putative regulation by SOCS-3. When using the doxycycline inducible ß-cell line INSr3#2, SOCS-3 expression levels are induced to physiologically relevant levels similar to those observed in cytokine-stimulated ß-cells (16). As can be seen in Fig. 2Go, IL-1 induced the phosphorylation of JNK and p38 after 15 and 30 min of stimulation. Phosphorylation of ERK in response to IL-1 was not observed. After induction of SOCS-3 expression by doxycycline, the IL-1-induced JNK phosphorylation was significantly reduced, by 63% after 15 min and completely inhibited after 30 min (P < 0.05 and P < 0.05, respectively). The IL-1-induced phosphorylation of p38 was inhibited by 20% and 28% by SOCS-3 after 15 and 30 min, respectively (P < 0.05 and P < 0.05). SOCS-3 did not affect the low level of MAPK phosphorylation observed in unstimulated cells. Expression of SOCS-3 did not affect the levels of JNK, p38, or ERK proteins, indicating that degradation of these proteins could not explain the reduced phosphorylation.


Figure 2
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  		Fig. 2. SOCS-3 Inhibits IL-1-Induced JNK, p38, and ERK Phosphorylation in INS-1 Cells

Western blots were performed on lysates from INSr3#2 cells preincubated for 20 h with or without doxycycline (Dox 1 µg/ml), in the presence or absence of IL-1 (75 pg/ml) for 15 or 30 min as indicated. Specific antibodies directed against phospho-JNK, phospho-p38 or phospho-ERK, were used to determine MAPK activation. Western blot using antibodies against JNK, p38, or ERK were used to determine the total amount of MAPK in the lysates. SOCS-3 expression was confirmed by use of an anti-SOCS-3 antibody. The results shown are representative of three independent experiments.

 
Using the same lysates as in Fig. 2Go, the NF{kappa}B pathway was analyzed by measuring the level of I{kappa}B{alpha} protein after IL-1 stimulation in the absence or presence of SOCS-3. As can be seen in Fig. 3AGo, exposure to IL-1 for 15 min resulted in a degradation of I{kappa}B{alpha} by 68% (P < 0.05), implying activation of NF{kappa}B. However, in the presence of SOCS-3, significantly less I{kappa}B{alpha} degradation in response to IL-1 was observed, 33% degraded (P < 0.05 compared with IL-1 alone). Also, we investigated the effect of SOCS-3 on NF{kappa}B DNA binding in response to different durations of IL-1 stimulation. We found that 15 and 30 min of IL-1 stimulation resulted in induction of NF{kappa}B DNA binding, which was significantly reduced after doxycycline-induced SOCS-3 expression. We observed a 34% and 24% degradation by SOCS-3 after 15 and 30 min, respectively (P < 0.05 for both observations) (Fig. 3BGo).


Figure 3
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  		Fig. 3. SOCS-3 Prevents IL-1-Induced I{kappa}B Degradation and NF{kappa}B Activation in INS-1 Cells

A, INSr3#2 cells were preincubated with or without doxycycline (Dox 1 µg/ml) for 20 h followed by stimulation for 15 min with IL-1 as indicated. Total cell lysates were analyzed by Western blotting using anti-I{kappa}B{alpha} antibody. B, INSr3#2 cells were preincubated with or without doxycycline for 20 h followed by stimulation with IL-1 (75 pg/ml) for the times indicated. Nuclear extracts were prepared and analyzed by EMSA using an NF{kappa}B binding probe. A 100-fold competition is shown in lane 11 and supershift using anti-NFkB, p65 is shown in lane 12. Results are representative of three independent experiments.

 
SOCS-3 Dose-Dependently Inhibits Inducible Nitric Oxide Synthase (iNOS) Activation Induced by IL-1 Signaling Components at or Upstream of the TAK1/TAB1 Level
The fact that SOCS-3 inhibits both the MAPK and NF{kappa}B pathways indicates that the site of action of SOCS-3 is at a level upstream of the IKK/MAPKKs branching point in the IL-1 signal transduction pathway. To identify the level of inhibition by SOCS-3, we carried out transfection studies of several components in the signaling pathway and analyzed the ability of SOCS-3 to inhibit the transcriptional activity initiated by overexpression of each signaling molecule. It has previously been shown that overexpression of MyD88, IRAK1, TRAF6, or TAK1/TAB1 by themselves is sufficient to induce transcription of IL-1-responsive promoters. We used an iNOS promoter-luciferase reporter transfected into the SOCS-3 inducible INS-1 cell line to measure transcription because this promoter is known to be regulated by IL-1 in an NF{kappa}B-dependent manner. When expression plasmids encoding MyD88, IRAK1, TRAF6, TAK1/TAB1, or MAPK/ERK kinase kinase-1 (MEKK1) were transfected into the INS-1 cells, a significant induction of iNOS promoter activity was observed (P < 0.05 for MEKK1 and P < 0.01 for all other plasmids) (Fig. 4AGo). Induction of SOCS-3 expression by doxycycline reduced the iNOS promoter activity stimulated by overexpression of MyD88, IRAK1, TRAF6, or TAK1/TAB1 (P < 0.05 for IRAK1 and TRAF6, and P < 0.01 for IL-1, MyD88 and TAK1/TAB1), but not the activity induced by MEKK1 (P = 0.26) (Fig. 4BGo). To characterize the inhibitory effect of SOCS-3 in more detail, we cotransfected cells with TAK1 and TAB1 because these are the most distal components and exposed the cells to different concentrations of doxycycline. We found the induction of the iNOS promoter by TAK1/TAB1 to be inhibited by SOCS-3 in a dose-dependent manner (Fig. 4CGo). These results indicate that SOCS-3 inhibits IL-1-induced signaling at a level at, or downstream of the TAK1/TAB1 component but upstream MEKK1.


Figure 4
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  		Fig. 4. Inhibition by SOCS-3 of iNOS Promoter Activity Induced by Overexpression of MyD88, IRAK1, TRAF6, TAK1/TAB1

After preincubation for 20 h with or without doxycycline (Dox 1 µg/ml), INSr3#2 cells were transiently transfected with the different IL-1-signaling components as indicated for 4 h. The next day, cells were stimulated with or without IL-1 (75 pg/ml) for 6 h. Control cells and IL-1-stimulated cells were transfected with empty vector. Cells transfected with the IL-1 signaling components were not IL-1 stimulated. Cell lysates were analyzed for luciferase activity. A, iNOS promoter activity in response to the IL-1 signaling components compared with control. B, Effect of SOCS-3 expression (gray bars) on iNOS promoter activity, showed as percentage of the individual iNOS activities without SOCS-3 expression. C, Dose-dependent effect of doxycycline on TAK1/TAB1-induced iNOS promoter activity. Each set of data is presented as the ratio of Luciferase/Renilla, and as means ± SD of at least three individual experiments. Statistical analyses were made by the paired Student’s t test.

 
IL-1-Induced TAK1/TRAF6 Interaction Is Inhibited by SOCS-3
Because TAK1 is able to phosphorylate both upstream MAPK and IKK, this kinase is considered to be at the branching point of the two pathways. Having identified the level of inhibition likely to be at the level of the TAK1/TAB1 complex, we next analyzed whether SOCS-3 was able to inhibit the formation of the active TRAF6/TAK1/TAB complex. Because binding of TAK1 to TRAF6 is a crucial step in the activation of TAK1 catalytic activity, we analyzed the binding of TAK1 to TRAF6 by coimmunoprecipitation. We found that transfection of human embryonic kidney (HEK) 293 cells with TRAF6, TAK1, and TAB1 resulted in TRAF6 and TAK1 interaction revealed by specific coimmunoprecipitation of TRAF6 and TAK1 (Fig. 5Go, middle panel, lane 7). When cotransfected with SOCS-3, the amount of TAK1 coimmunoprecipitated with TRAF-6 was reduced (Fig. 5Go, middle panel, compare lanes 7 and 8). SOCS-3 expression did not affect the level of TRAF6 or TAK1 protein (Fig. 5Go, upper panel). No immunoprecipitation of TAK1 was observed using a control antibody (anti-STAT1) (Fig. 5Go, lower panel).


Figure 5
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  		Fig. 5. TAK1/TRAF6 Interaction Is Inhibited by SOCS-3

HEK 293 cells were transiently transfected for 4 h with the IL-1 signaling components as indicated in each lane. Upper panel, Whole-cell lysates of each condition subjected to Western blotting against TAK1 and TRAF6. Cell lysates were split in two and immunoprecipitated with antibodies against TRAF-6 (middle panel) or STAT1 (lower panel), before Western blotting against TAK1 and TRAF6. Results are representative of three independent experiments.

 
SOCS-3 Interacts with TAK1 and TRAF6
To investigate whether SOCS-3 prevented TAK1/TRAF6 interaction by binding to the complex, lysates from cells transfected with the three components in different combinations were immunoprecipitated with a SOCS-3 antibody, followed by Western blotting against TRAF6 and TAK1 (Fig. 6Go). When cotransfecting cells with SOCS-3 and TAK1 and/or TRAF6 in different combinations, binding of SOCS-3 to TAK-1 as well as TRAF6 was observed. This interaction was specific because no immunoprecipitation was observed using normal rabbit serum instead of SOCS-3 antibody (data not shown). These results indicate that SOCS-3 associates with both TAK1 and TRAF6; however, these interactions might be indirect and mediated by other signaling components.


Figure 6
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  		Fig. 6. SOCS-3 Associates with TAK1 and TRAF6 in HEK 293 Cells

HEK 293 cells were transiently transfected for 4 h with the IL-1 signaling components, as indicated in each lane. Upper panel, Whole-cell lysates of each condition subjected to Western blotting against flag-tagged TAK1 and flag-tagged TRAF6. Lower panel, Cell lysates were immunoprecipitated with SOCS-3 antibodies before Western blotting against flag-tagged TAK1 and flag-tagged TRAF6. Results are representative of three independent experiments.

 
SOCS-3 Inhibits IL-1-Induced TAK1 Kinase Activity
Because TAK1 is able to activate both the NF{kappa}B and MAPK pathway and because SOCS-3 was able to inhibit TAK1 binding to TRAF6, we next analyzed whether SOCS-3 could inhibit IL-1-induced TAK1 kinase activity. IL-1 was found to stimulate TAK1 kinase activity in INS-1 cells (Fig. 7AGo), and this stimulation was reduced by 39% in cells expressing SOCS-3. Kinase activity, i.e. phosphoryation, revealed in a kinase assay has previously been shown to correlate with a reduced electrophoretic mobility when cell lysates are subjected to SDS-PAGE on a 7% gel (5, 18, 19). The reduced electrophoretic mobility is verified to be the result of phosphorylation because it can be reversed by treatment with protein-phosphatase-1 (19). Accordingly, reductions in electrophoretic mobility of both TAB2 and TAB3 have been shown in response to IL-1, lipopolysaccharide (LPS), and osmotic shock (19). Similarly, we show that the adapter protein TAB2, which links TAK1 to TRAF6, is phosphorylated in response to IL-1, and find SOCS-3 to inhibit this phosphorylation, visualized by reversing the reduced electrophoretic mobility (Fig. 7BGo). We analyzed the electrophoretic mobility of TAB2 in cells stimulated with IL-1 and found that IL-1 induced a shift in mobility and that this shift was reduced in cells expressing SOCS-3 (Fig. 7BGo). Recombinant SOCS-3 did not directly affect the catalytic activity of purified TAK1/TAB1 (Fig. 7CGo), indicating that SOCS-3 inhibits pathways used in the activation of TAK1 rather than inhibiting the catalytic activity of TAK1 itself.


Figure 7
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  		Fig. 7. SOCS-3 Inhibits IL-1-Induced Activation of TAK1

INSr3#2 cells were preincubated in the absence or presence of doxycycline (Dox 1 µg/ml) for 20 h followed by stimulation with IL-1 (1 ng/ml) for 15 min. A, Cell lysates were immunoprecipitated using TAK1 antibodies, and the immunoprecipitate was analyzed for TAK1 kinase activity as described in Materials and Methods. Data are presented as means ± SD of four individual experiments. The induction of TAK1 activity by IL-1 is set to 100%. B, Total cell lysates were subjected to Western blotting using TAB2 antibodies. The arrow indicates the mobility of unmodified TAB2. Results are representative of three to five independent experiments. C, GST-TAK1/TAB1 fusion proteins were analyzed for TAK1 kinase activity in the presence or absence of GST-SOCS-3 in two concentrations. As a control TAK1/TAB1 was incubated with the same two concentrations of GST alone; bars 4 and 5. In the control condition (–), the kinase assay was performed without addition of GST-SOCS-3 or GST alone. Data are presented as means ± SD of three individual experiments. *, P < 0.05.

 
SOCS-3 Inhibits TRAF6 Ubiquitination
Having found that SOCS-3 reduces the association between TRAF6 and TAK1 and inhibits IL-1-induced TAK1 activity, we hypothesized that SOCS-3 inhibits steps in the physiological activation of TAK1 by TRAF6. Because ubiquitination of TRAF6 (by lysine-63 ligation) is known to be required for the activation of TAK1 and because SOCS-3 is able to bind Elongin B and C, which are components of cullin-RING E3 ubiquitin ligases, we investigated whether SOCS-3 had an effect on TRAF6 ubiquitination. As shown in Fig. 8Go, ubiquitination of TRAF6 was found in cells overexpressing hemagluttinin (HA)-tagged ubiquitin and TRAF6 at different concentrations. When SOCS-3 was cotransfected with TRAF6, the ubiquitination of TRAF6 was reduced, indicating that SOCS-3 inhibits E3 ubiquitin ligase activity. SOCS-3 reduced the TRAF6 ubiquitination by 36% in the presence of 0.4 µg TRAF6, P < 0.05 and by 18% when using 0.2 µg TRAF6 expressing plasmid (P < 0.05). As can be seen in the lower panel, the total amount of TRAF6 in the immunoprecipitates was not affected by the presence of SOCS-3.


Figure 8
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  		Fig. 8. SOCS-3 Inhibits Ubiquitination of TRAF6

HEK 293 cells were transfected with HA-ubiquitin, SOCS-3, and different amounts of TRAF6, alone or in combination, as indicated, and cell lysates were immunoprecipitated using TRAF6 antibodies. The immunoprecipitates were subjected to SDS-PAGE and Western blotting. HA antibodies were used to detect the heterogeneous population of mono- and polyubiquitinated TRAF6, indicated by the bracket, and total TRAF6 was detected using TRAF6 antibodies. The results shown are representative of three independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Cytokines are known to regulate proliferation, differentiation, and many other important cellular responses (20). Some cytokines, including IL-1, also have cytostatic or cytotoxic actions, primarily contributing to host defense against tumor cells and microorganisms, but also to destruction of normal cells (21), as is the case for ß-cells. Little is known about the mechanisms negatively controlling the IL-1 pathway. Having previously shown the ability of SOCS-3 to inhibit IL-1 signaling in ß-cells, we here investigated how SOCS-3 exerts its protective effect against IL-1-induced ß-cell death and tried to identify the exact site of inhibition in the IL-1 signaling pathway. We found SOCS-3 to inhibit both the MAPK and NF{kappa}B branch of the IL-1 signaling pathway and that SOCS-3 most likely exerts its inhibitory effect by inhibiting ubiquitination of TRAF6, thereby preventing binding to and activation of TAK1.

Recently, SOCS-1 was reported to interact directly with IRAK1, thereby inhibiting LPS/TNF signaling and accordingly the activation of NF{kappa}B and the MAPKs (22, 23). Furthermore, SOCS-1 was shown by others to bind the p65 subunit of NF{kappa}B and mediate its degradation (24). Because SOCS-1 is closely related to SOCS-3, one could imagine that IRAK1 or p65 also could be targets for SOCS-3 action. However, the finding that SOCS-3 inhibited TAK1-induced transcription indicates that IRAK1 cannot be the only target for SOCS-3. The ability of SOCS-3 to inhibit both I{kappa}B degradation and MAPK activation furthermore shows that p65 cannot be the sole target for SOCS-3. The apparent differences in mechanism of action between SOCS-1 and -3 could be due to differences in their ability to interact with various signaling components, or it could be due to cell-specific differences. Our findings were substantiated by results from primary ß-cells, showing the inhibitory effect of SOCS-3 on IL-1-induced MAPK activation and I{kappa}B degradation (Heding, P. E., unpublished data).

Activation of the Toll-like receptors (TLRs) triggers antimicrobial responses and cytokine production (25). SOCS proteins can be induced by stimulation of TLRs and the IL-1R and TLR family are known to use many of the same signaling pathways. It is interesting to note that others recently reported no inhibitory effects of SOCS-3 on TLR-induced NFkB activation (26). Moreover, when analyzing the effect of SOCS-3 overexpression on TLR signaling, they observe no effect on LPS- or CpG-DNA-induced activation of the MAPKs or IkB degradation. However, they find that SOCS-3 induced by TLR stimulation is able to inhibit type I IFN signaling and abolish TLR mediated STAT-1 activation, indicating that the mode of inhibition must be indirect.

In overexpression studies of different components in the signaling pathway, we observed that SOCS-3 dose-dependently inhibited MyD88, IRAK1, TRAF6, and TAK1/TAB1 but not MEKK1-induced iNOS promoter activity (Fig. 4Go). The fact that both pathways of the IL-1 signaling cascade were inhibited indicated a common point of inhibition of both pathways, namely the branching point, or above (see Fig. 1Go). This was verified because we showed that TRAF6 interaction with TAK1 was reduced by SOCS-3 (Fig. 5Go). This reduced interaction could be the result of direct competition between SOCS-3 and TRAF6 and/or TAK1, thereby inhibiting the interaction between TRAF6 and TAK1. The interaction between TAK1 and TRAF6 in HEK 293 cells is supported by other studies (5, 27, 28). In addition, coimmunoprecipitation studies showed SOCS-3 to bind to both TAK1 and TRAF6 (Fig. 6Go). The interactions between TAK1 and SOCS-3 and TRAF6 and SOCS-3 can be the result of a direct binding of SOCS-3 to one of these proteins. However, it can also be an indirect binding via one or more other proteins present in the complex.

Furthermore, we found that SOCS-3 was able to inhibit the IL-1-induced activity of TAK1 (Fig. 7AGo). However, when using recombinant proteins in the TAK1 assays instead of whole-cell lysates, no inhibition of TAK1 activity by SOCS-3 was seen (Fig. 7CGo).

Receptor interaction with SOCS-3 is necessary for its suppression of gp130 (29), GH (30), leptin (31), and erythropoietin (32) signaling, indicating that SOCS-3 binding to phosphorylated tyrosine residues of the receptor is critical for SOCS-3 mediated inhibition of the activity of some cytokines. Once recruited to the activated cytokine receptor SOCS-3 is thought to inhibit JAK activity through its kinase inhibitory region (32). However, TAK1 is not activated by tyrosine phosphorylation, indicating another mechanism of SOCS-3 in the case of IL-1 signaling.

The SOCS proteins are known to mediate protein degradation via the proteosomal machinery through interacting with elongin B/C complexes (33, 34, 35, 36). Together with a ubiquitin-activating enzyme and a ubiquitin-conjugating enzyme, the E3 complex acts to tag proteins with ubiquitin chains assembled via lysine-48, which leads to proteosomal degradation. In this way, the SOCS proteins might function as adapters for a ubiquitin ligase complex mediating the degradation of target proteins bound to the SH2 domain of SOCS. SOCS-3-mediated degradation of proteins via ubiquitination could also be speculated to be the mechanism behind inhibition of IL-1 signaling. However, we did not observe a reduction in TAK1 or TRAF6 protein levels, in the presence of SOCS-3, indicating that the inhibition of TRAF6 and TAK1 interaction by SOCS-3 (Fig. 5Go), and the interaction of SOCS-3 and TAK1, and SOCS-3 and TRAF6 (Fig. 6Go) do not involve degradation of TAK1 or TRAF6.

The ubiquitin ligase activity of the TRAF6, TAK1, TAB1, and TAB2 complex, ubiquitinate TRAF6 in a unique manner through lysine-63 conjugation. Rather than leading to degradation, this ubiquitination results in activation of TRAF6 and binding of TAK1 to TRAF6 as shown in Fig. 1Go. Our observation, that SOCS-3 inhibited this ubiquitin modification of TRAF6, revealed to us a possible explanation for the mode of action of IL-1 inhibition by SOCS-3, which is consistent with the observed results. Inhibiting ubiquitination of TRAF6 results in lack of TAK1 binding, explaining the inhibited kinase activity of TAK1 and thus inhibition of signaling to the MAPKs and NF{kappa}B. This is in contrast to what has been reported for SOCS-3 inhibition of other signaling pathways in which SOCS proteins inhibit signaling by either inhibition of tyrosine kinase activity or by inducing ubiquitination through lysine-48 leading to proteosomal degradation. This would indicate that SOCS-3 inhibits IL-1 signaling by a novel mechanism involving inhibition of ubiquitination through lysine-63. The exact molecular mechanism responsible for this activity remains to be elucidated.

In summary, our results indicate that SOCS-3 inhibits IL-1 signaling to both MAPKs and NF{kappa}B by inhibiting ubiquitination of TRAF6, thus preventing activation of TAK1 and thereby further signaling, as shown in Fig. 1Go, steps 5 and 9. Additional understanding of how IL-1 signaling can be down-regulated may be essential in many circumstances because the effects of IL-1 are numerous. By elucidating how SOCS-3 protects against the inflammatory effects of IL-1, SOCS-3 could represent a therapeutic target that may alter the course of inflammatory diseases, including type 1 diabetes mellitus.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell Culture
INS-r3#2 cells (INS-1 cells with a doxycycline-inducible SOCS-3 expression) were cultured in RPMI-1640 with glutamax-I (Invitrogen, Paisley, Scotland, UK) supplemented with 10% heat-inactivated TeT System Approved fetal calf serum (FCS) (CLONTECH, Palo Alto, CA), penicillin, streptomycin, 50 µM ß-mercaptoethanol (Sigma Aldrich, St. Louis, MO), hygromycin (100 µg/ml), and geneticin (100 µg/ml) (Invitrogen).

For analysis of the effect of SOCS-3, 1 µg/ml doxycycline (Sigma Aldrich) was added and the cells cultured for 20 h to allow SOCS-3 expression. As we have previously shown, doxycycline induces SOCS-3 protein in a dose-dependent manner (16).

HEK 293 cells were cultured in DMEM (4500 mg/liter glucose, + GlutaMAX) supplemented with 10% heat-inactivated FCS, penicillin, and streptomycin. Both cell lines were cultured at 37 C in a humidified atmosphere containing 5% CO2.

Antibodies and Cytokines
The following primary antibodies were used: rabbit-anti-phospho-SAPK/JNK (Cell Signaling, Danvers, MA; no. 9251s), rabbit-anti-SAPK/JNK (Cell Signaling, Carlsbad, CA; no. 9252), rabbit-anti-phospho-ERK (phospho-p44/42) (Cell Signaling; no. 9101s), rabbit-anti-ERK (p44/42) (Cell Signaling, Carlsbad, CA; no. 9102), rabbit-anti-phospho-p38 (Cell Signaling; no. 9211s), rabbit-anti-p38 (Cell Signaling; no. 9212), mouse-anti-I{kappa}B{alpha} (Active Motif; no. 40903), rabbit-anti-TRAF-6 (Santa Cruz, Santa Cruz, CA; no. Sc-7221), mouse-anti-TAK1 (C9) (Santa Cruz; no. Sc-7967), mouse-anti-Flag M2 (Sigma; no. F 3165), goat-anti-TAB2 (Santa Cruz; no. Sc-11851), mouse-anti-HA (Roche), rabbit-anti-Stat1 (Santa Cruz; no. Sc-591), and rabbit-anti-NF{kappa}B, p65 (Santa Cruz; no. Sc-372x). Rabbit-anti-SOCS-3 antibody for the purpose of immunoprecipitation and mouse-anti-SOCS-3 antibody for Western blotting was kindly supplied by Douglas Hilton (The Walter and Eliza Hall Institute for Medical Research, Melbourne, Australia). TAB1 antibodies for the purpose of immunoprecipitating TAK1 in the kinase assay was produced by the Medical Research Council protein phosphorylation unit as described (18). The following secondary antibodies were used: goat antirabbit (Cell Signaling; no. 7074), horse antimouse (Cell Signaling; no. 7076), and mouse antigoat (Santa Cruz; no. Sc-2354), all conjugated to horseradish peroxidase.

Recombinant mouse IL-1 was purchased from BD Biosciences PharMingen (San Jose, CA).

Western Blotting
INSr3#2 cells were cultured for 24 h in complete medium followed by culture for 20 h in medium with or without doxycycline (1µg/ml). The cells were then incubated with or without IL-1 (75 pg/ml, 150 pg/ml, or 1 ng/ml, as indicated) for 15 or 30 min. The cells were washed in Hanks’ buffered salt solution (Invitrogen) and cell lysates were prepared by adding lysis buffer [20 mM Tris(base)acetate (pH 7), 0.27 mM sucrose, 1 mM EGTA, 1 mM EDTA, 50 mM NaF, 1% Triton-X, 5 mM Na-pyrophosphate, 10 mM Na-glycerophosphate, 1 mM benzamidine, 4 µg/ml leupeptin, 1 mM dithiothreitol, 1 mM Na3VO4]. Lysates were adjusted for protein concentration and subjected to SDS-PAGE and transferred to a nitrocellulose membrane according to the NuPAGE Western blotting protocol (Invitrogen, Carlsbad, CA). Protein expression was detected by use of the antibodies described above and horseradish peroxidase-conjugated antibodies were visualized by the chemiluminescence detection system, LumiGLO (Cell Signaling) and visualized by use of an imaging system (LAS3000; Fuji Film, Tokyo, Japan), according to manufacturer’s instructions. Quantifications of the Western blots were made by use of the Fuji Film Multi Gauge software program. For analysis of ubiquitinated TRAF6, the bands within the bracket shown in Fig. 8Go were quantitated.

For analysis of phosphorylation-dependent electrophoretic mobility shift, the lysates were run on 6% Tris-glycine gels, and for analysis of ubiquitination of TRAF6 lysates were run on 7% Tris-acetate gels, as opposed to 10% bis-Tris gels, which were used for all other Western blots (Invitrogen).

EMSA
Time-dependent IL-1-induced NF{kappa}B DNA binding and inhibition by SOCS-3 was investigated in INSr3#2 cells. Cells were grown in normal media for 2 d, followed by 20 h incubation in medium containing 0.5% FCS in the presence or absence of 1 µg/ml doxycycline. This was followed by IL-1 stimulation (75 pg/ml) for the times indicated in Fig. 3BGo, nuclear extraction and EMSA as described (37). Specificity was evaluated by competition using 100-fold molar excess of nonlabeled oligonucleotide. The identity of the DNA-protein complex was verified by supershift experiments using anti-NFkB, p65.

iNOS Promoter Assay/Activity
Two hundred thousand INSr3#2 cells/well were seeded in duplicates in 24-well dishes. After preculturing for 24 h, cells were cultured with or without doxycycline (1 µg/ml) for 20 h before transfection. The transient transfection was performed using SuperFect (QIAGEN, Hilden, Germany) for 4 h with a total of 2 µg plasmid DNA according to the manufacturer’s description. The cells were cultured in the presence or absence of doxycycline overnight, stimulated with IL-1 (150 pg/ml) for 6 h, and lysed in Passive lysis buffer (Promega, Madison, WI) for 30 min while shaking. The promoter activity in the lysates was analyzed by use of the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s description.

Immunoprecipitation
HEK 293 cells were seeded in six-well dishes and transfected the next day. The transfection was performed with FUGENE 6 transfection reagent (Roche, Basel, Switzerland) according to the manufacturer’s description, each condition being transfected with 2 µg DNA for 24 h. After transfection, the cells were grown in complete medium for 24 h and lysates were prepared by adding 500 µl IP buffer [20 mM HEPES, 1.5 mM MgCl2, 2 mM dithiothreitol, 1 mM Na3VO4, 2 mM EGTA, 0.5% Triton-X, 10 mM NaF, 150 mM NaCl, 12.5 mM ß-glycerophosphate, and Complete Mini (Roche)] according to manufacturers prescription). One microgram of the respective antibody was added to the cell lysate to be immunoprecipitated and incubated for 2 h at 4 C followed by addition of 30 µl Protein G-Sepharose (Amersham Biosciences, Pistcataway, NJ) and subsequent incubation at 4 C for 1 h. Samples were washed four times in IP buffer and analyzed by SDS-PAGE and Western blotting. Ten percent of cell lysate was not immunoprecipitated but used as a control and reference to the immunoprecipitates, i.e. input.

TAK-1 Assay (18)
INS-r3#2 cells were seeded in 100-mm dishes, and after 24 h 1 µg/ml doxycycline was added to the relevant samples. After an additional 20 h, the cells were stimulated with 1 ng/ml IL-1 for 15 min and cell lysates were prepared. Ten microliters of Protein G-Sepharose were incubated with 2.5 µg anti-TAB1 antibody for 5 min (incubation with TAB1 antibody results in immunoprecipitation of TAK1, which is bound to TAB1), subsequently washed in lysis buffer [50 mM TRIS-HCl (pH 7.5), 0.1 mM EGTA, 1 mM EDTA, 1% Triton-X, 50 mM NaF, 5 mM tetrasodium pyrophosphate, 0.27 M sucrose, 1 mM Na3VO4, 0.1% ß-mercaptoethanol, complete mini inhibitor cocktail (Roche)] and incubated with 150 µg lysate for 90 min. For each lysate, three samples were analyzed. Samples were washed twice in 0.5 M NaCl, 0.1% ß-mercaptoethanol lysis buffer and twice in 50 mM Tris (pH 7.5), 0.27 M sucrose, 0.1% ß-mercaptoethanol. 5 µl 0.1 M MgAcetate was added to the wall of the Eppendorf (Hamburg, Germany) tube and 35 µl kinase buffer 1 [50 mM Tris (pH 7.5), 0.1% ß-mercaptoethanol, 0.1 mM sodium orthovanadate, 0.1 mM EGTA, 0.1 mg/ml BSA, 2.5 µg MKK6, 5 µg SAPK2a, 0.1 mM cold ATP] prewarmed to 30 C was added and samples incubated for 30 min at 30 C. A control without MKK6 was included for each lysate. The reactions were started with 20-sec intervals. A 45-µl kinase buffer 2 [50 mM Tris (pH 7.5)/0.1% ß-mercaptoethanol, 0.1 mM sodium orthovanadate, 0.1 mM EGTA, 0.1 mg/ml BSA, 0.33 mg/ml myelin basic protein, 0.1 mM hot ATP, 10 mM MgAcetate] prewarmed to 30 C was added to new Eppendorf tubes, and 5 µl of the first kinase reaction was added to kinase buffer 2 and incubated for 10 min at 30 C. Then 40 µl of the kinase reaction were spotted on P81 Whatman paper and washed three times in 75 mM phosphoric acid, once in acetone and allowed to dry before counting in a ß-counter. A sample containing beads but no lysate was included in each experiment and subtracted as background.

Statistical analysis
Results are presented as means ± SD. Statistical differences were determined by use of a paired Student’s t test with significance levels at P < 0.05.


   ACKNOWLEDGMENTS
 
We thank technicians Anette Hellgren and Susanne Munch for excellent technical assistance. We thank Luke O’Neill (University of Dublin) for providing expression plasmids and Douglas Hilton (The Walter and Eliza Hall Institute of Medical Research, Melbourne) for providing SOCS-3 antibodies. The MKK6, p38{alpha} MAPK and the TAKI-TABI fusion protein were expressed and purified by the protein production team of the division of signal transduction therapy, coordinated by Hilary McLaughlan and James Hastie.


   FOOTNOTES
 
The work was supported in part by grants from the Juvenile Diabetes Foundation International (Grant Nos. 1-2001-706 and 1-2004-736) (to N.B.). The UK Medical Research Council, The Royal Society, and Diabetes UK (to P.C.). H.F. was supported by a scholarship from the Medicon Valley Ph.D. program, and S.G.R. was supported by a Ph.D. fellowship from the University of Copenhagen.

First Published Online March 16, 2006

Abbreviations: CIS, Cytokine-inducible SH2 protein; FCS, fetal calf serum; HA, hemagglutinin; HEK, human embryonic kidney; LPS, lipopolysaccharide; IKK, I{kappa}B kinase; iNOS, inducible nitric oxide synthase; IRAK, IL-1R-activated kinase; JAK, Janus kinase; LPS, lipopolysaccharide; MEKK-1, MAPK/ERK kinase kinase-1; MKK, MAPK kinase; NF{kappa}B, nuclear factor {kappa} B; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TAB1 and 2, TAK1 binding protein 1 and 2; TAK, TGFß-activated kinase; TLR, Toll-like receptor; TRAF, TNF receptor-associated factor.

Received for publication July 21, 2005. Accepted for publication March 7, 2006.


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 INTRODUCTION
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 DISCUSSION
 MATERIALS AND METHODS
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