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and IFN-
as Modulators of TNF- Signaling in Macrophages: Regulation and Functional Implications of the TNF Receptor 1:STAT-1 Complex1
Department of Cell Biology, University of Alabama, Birmingham, AL 35294
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Abstract |
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and IFN-
cooperate in the activation of macrophages. TNF--dependent activation of NF-
B is stronger in the presence of IFN-. STAT-1 associates with TNFR1 in TNF--treated cells, and this association attenuates TNF--mediated NF-B activation. We hypothesized that nuclear localization of STAT-1 due to IFN- signaling would preclude it from being recruited to the TNFR1 and therefore enhance TNF--induced NF-B activation. In the RAW264.7 macrophage cell line, TNF- treatment indeed recruits STAT-1 to the TNFR1, and this association is abrogated when cells are exposed to IFN-. TNF- treatment induces a more robust activation of NF-B in STAT-1-deficient cells, and restoration of STAT-1 inhibits TNF--dependent NF-B activation. Our results suggest that a receptor-proximal level of cross-talk exists between these two cytokine pathways: IFN- limits STAT-1 availability to the TNFR1 by depleting STAT-1 from the cytoplasm, thus allowing for optimal NF-B activation upon TNF- ligation. |
Introduction |
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is a pleiotropic cytokine that elicits a broad spectrum of cellular responses including macrophage activation, fever, acute phase response, as well as cell proliferation, differentiation, and apoptosis (for review, see Ref. 1). Of the two types of TNFRs characterized (TNFR1 and TNFR2), TNFR1 is responsible for most of the biological properties of TNF- (1). The cytoplasmic domain of TNFR1, when activated by ligand binding, recruits the adaptor protein TNFR1-associated death domain protein (TRADD)3; the TNFR1-TRADD complex leads to at least two opposing downstream signaling cascades. The TNFR1-TRADD complex forms the death-initiated signaling complex by recruiting Fas-associated death domain protein (FADD), leading to caspase activation and apoptosis (2). Alternatively, the TNFR1-TRADD complex can recruit receptor interacting protein and/or TNFR-associated factor 2, leading to mitogen-activated protein kinase (MAPK) and NF-B activation (3, 4). NF-B activation is critical for the expression of proinflammatory cytokines, chemokines, adhesion molecules, acute phase proteins, and costimulatory molecules, as well as regulators of apoptosis and cell proliferation (for review, see Ref. 5).
The central biological actions described for IFN- are macrophage activation, antiviral activity, antiproliferative activity on tumor cells, production of free radicals, and expression of adhesion molecules as well as class I and II MHC Ags (for review, see Ref. 6). Produced mainly by Th1 and NK cells (6), IFN-, when bound to the IFN- receptor, induces the transphosphorylation of Janus kinase 1(JAK1) and JAK2, which are associated with the IFN- receptor subunits. The subsequent phosphorylation of a tyrosine residue on the IFN- receptor by activated JAKs provides a docking site for latent STAT-1. Bound STAT-1 becomes tyrosine phosphorylated by activated JAKs, dissociates from the receptor complex, homodimerizes, then translocates to the nucleus to induce transcription via binding to the -activation site in IFN--inducible promoters (for review, see Ref. 7). One of the STAT-1-inducible genes is IFN-regulatory factor (IRF)-1 (8). IRF-1 is itself a transcription factor that recognizes the sequence termed IFN stimulation response element and plays, like STAT-1, a central role in IFN- inducible gene expression (9).
IFN- and TNF- synergize in the production of a large number of proinflammatory cytokines, chemokines, and costimulatory molecules (for review, see Ref. 10). Cooperativity between these pathways occurs at multiple levels. Most, but not all, of the promoters induced synergistically by IFN- and TNF- contain binding sites for STAT-1 or IRF-1 and NF-B (10). For these genes, synergism occurs by the binding of TNF--activated NF-B and IFN--activated STAT-1 or IRF-1 to their respective sites within promoters, thereby affecting gene expression at the transcriptional level (10). Another level of cross-talk exists at the level of NF-B activation. Though not known to activate NF-B directly, IFN- enhances and prolongs TNF--dependent NF-B activation (10); enhanced IB
degradation upon costimulation is a likely molecular reason for the synergy (11). dsRNA-activated protein kinase R is involved in the synergistic activation of NF-B by TNF- and IFN- (12, 13).
In the RAW264.7 macrophage cell line, IFN- and TNF- synergize in the activation of NF-B (14, 15). In these cells, IFN- synergizes with endogenously produced TNF- to give stronger and more prolonged NF-B activation than does exogenous TNF- treatment or IFN- treatment alone (achieved by the use of a neutralizing anti-TNF- Ab to inhibit signaling by endogenously produced TNF-) (14, 15). These results prompted us to search for mechanisms by which these two signaling pathways could communicate to enhance NF-B activation. Recently, in an Ab array screen for STAT-1 binding proteins, TNFR1 was shown to interact with STAT-1 in a TNF--dependent manner in Hela cells and A549 lung epithelial cells (16). When associated with the TNFR1, STAT-1 inhibited TNF--mediated NF-B activation (16). Because STAT-1 is a critical component of IFN- signaling, we hypothesized that IFN- may attenuate the ability of STAT-1 to interact with the TNFR1, thereby augmenting TNF--mediated NF-B activation. We demonstrate via coimmunoprecipitation experiments that TNF- treatment leads to an association between STAT-1 and the TNFR1, and that this complex is attenuated in the presence of IFN-. Immunofluorescence studies show that STAT-1 is located in the cytoplasm in both untreated and TNF--treated cells, where it is found to grossly colocalize with TNFR1. IFN- leads to a pronounced STAT-1 nuclear translocation, suggesting a potential mechanism for the ability of IFN- to inhibit the STAT-1:TNFR1 complex. Coupled with these findings, exposure of cells to IFN- allows cells to respond more vigorously to constitutively present endogenous TNF- by enhanced activation of NF-B and NF-B responsive genes. We also demonstrate that TNF- treatment leads to enhanced NF-B activation in cells deficient in STAT-1, and upon restoration of STAT-1, TNF--dependent NF-B activation and RANTES (an NF-B-dependent gene) expression is inhibited. These data suggest that STAT-1 interacts with the TNFR1 and acts as a repressor of TNF--dependent NF-B activation. Additionally, IFN--dependent control of STAT-1 availability to the TNFR1 may contribute to the regulation of NF-B activation through TNFR1. We propose that this represents a novel form of cross-talk that contributes to the synergistic proinflammatory response by these two cytokines.
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Materials and Methods |
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Recombinant murine IFN- was purchased from Genzyme (Boston, MA). Murine and human TNF- were purchased from Endogen (Woburn, MA) and Calbiochem (La Jolla, CA), respectively, and neutralizing anti-murine TNF- Ab was purchased from Endogen (Woburn, MA). Rabbit polyclonal TNFR1 Ab, mouse monoclonal STAT-1 Ab and consensus NF-B binding oligonucleotide were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-STAT-1 Ab was purchased from Upstate Biotechnology (Lake Placid, NY). Mouse monoclonal anti-FADD Ab was purchased from BD PharMingen (San Diego, CA). Biotinylated anti-mouse TNFR1 was purchased from Caltag Laboratories (Burlingame, CA). Streptavidin-conjugated HRP was purchased from Southern Biotechnology Associates (Birmingham, AL).
Cells
The murine macrophage cell line RAW264.7 was maintained in DMEM supplemented with 10% FBS as previously described (17). The U3A cells (a generous gift from Dr. G. Stark, The Cleveland Clinic, Cleveland, OH) were maintained with 10% FBS in DMEM/F12 as previously described (18).
Stable transfection of U3A cells
STAT-1 and pcDNA3 stable transfectants were created by transfecting U3A cells with either STAT-1 (a generous gift of Dr. J. Darnell, Rockefeller University, New York, NY) or the pcDNA3 backbone vector as previously described (18). STAT-1 expression was determined by immunoblotting for STAT-1.
Nuclear extracts and EMSA
EMSA was performed with 5–10 µg of nuclear extract as previously described (15). For supershift experiments, 1 µg of anti-p50, anti-p65 or isotype-matched control Abs (Santa Cruz Biotechnology, Santa Cruz, CA) were included in the reaction mixture. For competition experiments, 100x unlabeled NF-B consensus probe or 100x of a probe (Santa Cruz Biotechnology) containing a mutation in the NF-B binding site were added to the reaction mixture. Bound and free DNA were then resolved by electrophoresis through a 6% polyacrylamide gel in 0.5x Tris-borate-EDTA buffer at 250 V for 1 h.
Coimmunoprecipitation and Western blotting
RAW264.7 cells were incubated with medium, IFN- (10 ng/ml), TNF- (50 ng/ml), or a combination of IFN- and TNF- for various time periods, followed by lysis in cold radioimmunoprecipitation assay (RIPA) buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS). Lysates were then cleared of debris by centrifugation (12,000 x g, 10 min) and 900 µg of protein were precleared with 0.25 µg of rabbit IgG Ab and 30 µl of protein A/G bead slurry (Pierce, Rockford, IL) for 2.5 h. Cleared lysates were then incubated overnight with 1.5 µg of rabbit anti-TNFR1 Ab and an additional 3 h with 30 µl of protein A/G bead slurry. Beads were then washed five times with cold RIPA buffer, boiled in sample buffer, separated on 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a monoclonal anti-STAT-1 Ab for detection of endogenous TNFR1:STAT-1 association. For detection of immunoprecipitated TNFR1, membranes were stripped and reprobed with a biotinylated anti-TNFR1 Ab. Following which, membranes were incubated with a streptavidin-HRP conjugate. For detection of components of the TNFR1 signaling pathway, RIPA lysates were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with Abs to TNFR1, TRADD, FADD, and NF-B p65 (Santa Cruz Biotechnology) as well as an anti-actin Ab (Sigma-Aldrich, St. Louis, MO) to control for loading. The ECL method was used for protein detection.
Immunofluorescence
RAW264.7 cells were grown in two-chamber culture slides and were incubated in the absence or presence of IFN- (10 ng/ml), TNF- (50 ng/ml), or both for 30 min. Cells were permeabilized/fixed in a 1:1 mixture of methanol/acetone at -20°C for 10 min, air-dried, and blocked in 1.5% goat serum in PBS for 30 min. Cells were then stained with polyclonal TNFR1 for 1 h, rinsed three times in PBS, and then stained with goat anti-rabbit Alexa Fluor 488 (Molecular Probes, Eugene, OR) for 45 min. Cells were again rinsed with PBS followed by staining with mouse monoclonal anti-STAT-1 Ab. Cells were then rinsed three times in PBS and stained with goat anti-mouse Alexa Fluor 594 (Molecular Probes) for 45 min. Cells were again rinsed three times and counterstained with Hoechst 33258 (Sigma-Aldrich) followed by a brief rinsing. Images were obtained with an Olympus IX70 digital camera (Melville, NY) and analyzed with IPLab 3.2 software (Scanalytics, Fairfax, VA).
RNA isolation, riboprobes, and RNase protection assay (RPA)
Total cellular RNA was isolated from confluent monolayers of cells. The riboprobes for Bcl-xL, Bcl-xS, RANTES, L32, and GAPDH were purchased from BD PharMingen and prepared following the manufacturer’s guidelines. The IRF-1 riboprobe was prepared as previously described (15). Twenty micrograms of total RNA were hybridized with Bcl-xL, Bcl-xS, RANTES, IRF-1, L32, and GAPDH riboprobes as described previously (15). Quantification of the protected RNA fragments was performed by scanning with the PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Values for Bcl-xL, Bcl-xS, IRF-1, and RANTES mRNA expression were normalized to GAPDH or L32 mRNA levels for each experimental condition.
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Results |
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and IFN- effects on TNFR1:STAT-1 complex formation
RAW264.7 cells were treated with medium, IFN-, TNF-, or IFN- plus TNF- for 30 min and then assayed for TNFR1:STAT-1 interaction via coimmunoprecipitation by immunoprecipitating TNFR1 and probing for STAT-1. As shown in Fig. 1A, treatment with IFN- had no effect on complex formation, while treatment with exogenous TNF- lead to an association of TNFR1 and STAT-1. Of most interest was the observation that inclusion of IFN- inhibited TNF--mediated TNFR1:STAT-1 complex formation (Fig. 1A). Immunoprecipitation with an isotype-matched control Ab was without effect (Fig. 1A). To determine the tyrosine phosphorylation status of STAT-1 with these treatments, 10% of the input samples were subject to Western blot analysis and probed with an Ab specific for STAT-1 that was phosphorylated on the Y701 residue. As shown in Fig. 1B, IFN-, but not TNF-, leads to the tyrosine phosphorylation of STAT-1. These results indicate that TNF- induces the association of STAT-1 and TNFR1 in a tyrosine phosphorylation-independent manner, and that formation of the TNFR1:STAT-1 complex is inhibited upon inclusion of IFN-.
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complex formation, cells were treated with medium, TNF-, or a combination of TNF- and IFN- for 0.5–6 h (Fig. 1C), and then were assayed for TNFR1:STAT-1 interaction via coimmunoprecipitation as described above. The TNFR1:STAT-1 complex was induced by 0.5 h and was stable for up to 6 h. Shown as well is that inclusion of IFN- inhibits TNF--mediated TNFR1:STAT-1 complex formation at all time points tested (Fig. 1C). To determine the dose dependency of this effect, cells were left untreated, or were incubated with increasing doses of TNF- (1–100 ng/ml), as well as increasing doses of IFN- (0.1–100 ng/ml) in the presence of TNF- (100 ng/ml). Complex formation was observed using 1 ng/ml TNF-, and increasing the dose of TNF- did not result in a stronger signal (Fig. 1D). IFN- treatment at all concentrations tested inhibited complex formation (Fig. 1D). We have also seen inhibition of complex formation with IFN- at 0.01 ng/ml (data not shown). These results suggest that the TNF--induced TNFR1:STAT-1 complex is induced to form with a threshold amount of TNF-, and that IFN- is a potent inhibitor of this process.
IFN- depletes cytoplasmic availability of STAT-1 and attenuates TNFR1:STAT-1 colocalization
Immunofluorescence technology was used to confirm TNFR1 and STAT-1 localization under the influence of TNF- and IFN-. In these experiments, cells were left untreated or given IFN-, TNF-, or both for 30 min, fixed, and then stained for TNFR1 (green) followed by staining for STAT-1 (red). Cells were also counterstained with the Hoechst DNA binding dye (blue). As shown in Fig. 2, STAT-1 and TNFR1 appear to grossly colocalize in unstimulated cells, as manifested by the yellow coloration in the red/green overlays (Fig. 2A). Upon TNF- treatment, STAT-1 remains in the cytoplasm where it remains grossly colocalized with TNFR1 (Fig. 2C). IFN- treatment induces nuclear translocation of STAT-1, and colocalization of STAT-1 and TNFR1 is attenuated (Fig. 2, B and D). Because there is such scant cytoplasm in monocytic cells, discriminating between cytoplasmic STAT-1 and membrane-bound TNFR1 is not straightforward. These results qualitatively show a significant proportion of IFN- mediated STAT-1 nuclear translocation, and suggest that the ability of IFN- to inhibit the formation of the TNFR1:STAT-1 complex may be related to its ability to localize STAT-1 to the nucleus, thereby depleting availability to the cytoplasmic domains of the TNFR1.
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exposure allows for optimal TNF- dependent NF-B activation by constitutively produced endogenous TNF-
The possibility of STAT-1 as a component of the TNF- signaling pathway led to the speculation that STAT-1 may be a central mediator of receptor proximal cross-talk between these two signaling pathways. The effect of IFN- activation of STAT-1 on TNF--mediated NF-B activation was examined. We have previously shown that IFN- synergizes with endogenously secreted TNF- in RAW264.7 cells to mediate optimal NF-B activation (14, 15). Initially, we hypothesized that IFN- activated NF-B by up-regulating the expression of TNF-, which subsequently signals to the cells in an autocrine fashion to activate NF-B. However, IFN--mediated activation of NF-B, to some degree, precedes IFN- induction of TNF- as measured by ELISA (15). These results indicate that although IFN--induced TNF- secretion contributes to IFN--mediated activation of NF-B, there may be additional mechanisms responsible for the more rapid IFN--mediated NF-B activation.
To examine the effect of IFN- and TNF- on NF-B activation, EMSA was performed on nuclear extracts from RAW264.7 cells treated with TNF-, IFN-, TNF- plus IFN-, or IFN- in the presence of a neutralizing anti-TNF- mAb. Using a consensus NF-B binding sequence as a probe, a small level of constitutive NF-B activity was observed, which is due to the fact that these cells constitutively secrete TNF- (Fig. 3A, lane 1). Addition of exogenous TNF- slightly enhanced NF-B activation (Fig. 3A, lane 2), while IFN- treatment clearly enhanced NF-B activation (Fig. 3A, lane 3). The combined treatment with IFN- and TNF- was additive with respect to NF-B activation compared with either cytokine alone (Fig. 3A, lane 4). This result suggests that exposure of RAW264.7 cells to IFN- allows constitutively expressed, endogenous TNF- to more actively induce NF-B activation. The ability of IFN- to induce NF-B activation was completely abrogated when endogenously produced TNF- was blocked by addition of a neutralizing anti-TNF- Ab (Fig. 3A, lane 5) (an isotype-matched control Ab has no effect, Fig. 3A, lane 6). Anti-p50 and anti-p65 Abs were used to determine the composition of the NF-B complex. These Abs acted as competitors of nucleotide binding compared with an isotype-matched control Ab (Fig. 3A, lanes 7–10), and suggest that the complex studied consists of the p65 and p50 NF-B isoforms. Abs that act as competitors of binding instead of inducing a supershift have been observed previously (14, 15). Competition analysis using cold competitor DNA and mutant DNA was performed as well to demonstrate the specificity of this assay for the NF-B binding sequence (Fig. 3A, lanes 11 and 12).
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B activation was assessed by examining expression of the NF-B regulated Bcl-x gene via RPA of both Bcl-xL and Bcl-xS mRNA splice variants. As shown in Fig. 3B, mRNA for Bcl-xS was constitutively expressed in these cells (Fig. 3B, lane 1), slightly augmented by TNF- treatment (Fig. 3B, lane 2), and highly up-regulated by IFN- treatment (Fig. 3B, lane 3: 8.3x induction). A simultaneous treatment with TNF- plus IFN- (Fig. 3B, lane 4) did not increase expression (8.5x) over that seen with IFN- alone (Fig. 3B, lane 3). The enhancement of Bcl-xS by IFN- was due to the presence of endogenous TNF-, because the response was inhibited in the presence of a neutralizing anti-TNF- Ab (Fig. 3B, lane 5), while the isotype-matched control Ab was without effect (Fig. 3B, lane 6). Comparable results were observed for the expression of Bcl-xL mRNA (Fig. 3B). These results collectively indicate that the ability of IFN- treatment to activate Bcl-x gene expression, which is a NF-B-dependent response (19, 20), is actually IFN- working in concert with constitutively expressed TNF-. It should be noted that inclusion of neutralizing anti-TNF- Ab does not interfere with IFN--induced, STAT-1-dependent expression of IRF-1 mRNA (Fig. 3C).
TNF--mediated NF-B activation is inhibited by STAT-1
To directly test the influence of STAT-1 on NF-B activation, we used a cell line deficient in STAT-1 (U3A cells) and stably transfected them with either STAT-1 (U3A-STAT-1) or the pcDNA3 vector (U3A-pcDNA3). These cells were then used to determine whether TNF--induced NF-B activation was affected when STAT-1 was absent (U3A-pcDNA3) and then restored (U3A-STAT-1). Using EMSA, we observed a strong activation of NF-B by TNF- in the U3A-pcDNA3 cells (Fig. 4A, lanes 2–6), however, in the U3A-STAT-1 cells, TNF--mediated NF-B activation was partially attenuated (Fig. 4A, lanes 8–12). To validate this phenomenon, we investigated whether the presence of STAT-1 affected the ability of TNF- to regulate a NF-B responsive gene. Using an RPA, TNF- treatment led to a 5.0- to 6.1-fold induction of RANTES mRNA at 6–12 h, respectively, in the U3A-pcDNA3 cells (Fig. 4B, lanes 1–3), whereas only a 3.0-fold induction of TNF--induced RANTES mRNA levels was observed in the U3A-STAT-1 cells (Fig. 4B, lanes 5–8). Western blotting confirmed that protein levels of components of the TNFR1 signaling pathway (TNFR1, TRADD, FADD, and NF-B p65) remain constant in the absence or presence of STAT-1 (Fig. 4C). These results demonstrate that STAT-1 indeed inhibits TNF--mediated activation of NF-B.
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Discussion |
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and TNF- synergize in the activation of NF-B (for review, see Ref. 10). Activated NF-B is a key part of the promotion of autoimmune and chronic inflammatory disorders mediated by the otherwise helpful immune system (for review, see Ref. 5). The synergistic activity of IFN- and TNF- is well-documented and occurs on many different levels within the cell. Herein, we elucidate a novel mechanism of cross-talk between these two signaling pathways occurring at the receptor level that may contribute to the observed enhancement of TNF--dependent NF-B activation in the presence of IFN-.
Consistent with results from other cell types (16), we demonstrate that TNF- treatment leads to the physical association of STAT-1 and TNFR1 (Fig. 1). Immunofluorescence staining reveals that STAT-1 is localized to the cytoplasm and grossly colocalizes with the TNFR1 in resting cells, and that STAT-1 remains cytoplasmic upon treatment with exogenous TNF- where gross colocalization with TNFR1 remains intact (Fig. 2). Notably, lysates for the coimmunoprecipitation studies were prepared using SDS-containing RIPA buffer and the interaction of endogenous STAT-1 and TNFR1 was detectable under these stringent conditions (Fig. 1). A previous report has demonstrated a direct association of JAK1 and JAK2 with the TNFR1 upon TNF- stimulation (21). In contrast, we were unable to detect any TNFR1:JAK1 or TNFR1:JAK2 association via coimmunoprecipitation in the absence or presence of either TNF- or IFN- (data not shown). However, this does not rule out the possibility that JAKs may still be involved in this process. Sorting out the molecular complexes formed with TNF- and IFN- stimulation will no doubt shed light on the integration of these signaling pathways, and is the topic of ongoing research.
Interestingly, when cells are exposed to IFN-, whether or not TNF- is provided, gross colocalization of STAT-1 and the TNFR1 can no longer be demonstrated, either by coimmunoprecipitation (Fig. 1), or by immunofluorescence (Fig. 2, B and D). STAT-1 has been shown to interact with the TNFR1 complex and has been implicated as a negative regulator of TNF--mediated NF-B activation while in this configuration (16). Consistent with the hypothesis that IFN--dependent removal of STAT-1 from the TNFR1 complex may sensitize cells to TNF--mediated NF-B activation, exposure of cells to IFN- allows endogenous, constitutively present TNF- to activate NF-B to a greater degree than the addition of exogenous TNF- in RAW264.7 cells (Fig. 3A). To rule out the possibility that IFN- may be merely up-regulating TNF- expression that would in turn signal in an autocrine fashion to give rise to NF-B activation, ELISA experiments were done to show that TNF- protein levels were unchanged for at least 3 h after IFN- exposure in RAW264.7 cells (15). Additionally, IFN- enhancement of TNF--dependent NF-B activation was still observed in the presence of the protein synthesis inhibitor puromycin (data not shown), suggesting that protein synthesis was not required for the effect of IFN- on TNF--mediated NF-B activation.
To further investigate the role of STAT-1 as a negative regulator of TNF--mediated NF-B activation, we sought to measure TNF--mediated NF-B activation in the absence and presence of STAT-1 using a STAT-1-deficient cell line that was reconstituted with either STAT-1 or the pcDNA3 vector. TNF- was found to induce a stronger NF-B activation in cells lacking STAT-1 than in the STAT-1 -reconstituted cells as measured by EMSA (Fig. 4A). As well, TNF--induced mRNA levels of RANTES, an NF-B responsive gene, were also inhibited in the presence of STAT-1 (Fig. 4B).
It has been known for some time that synergism between the IFN- and TNF- signaling pathways occurs by the binding of TNF--activated NF-B and IFN--activated STAT-1 or IRF-1 to their respective sites within promoters, thereby affecting gene expression at the promoter level (10) (Fig. 5). The observation that STAT-1 binds to TNFR1 and inhibits TNF--mediated NF-B activation, together with the finding that IFN- inhibits the TNF--induced TNFR1:STAT-1 complex, prompted us to propose a model that is consistent with the observed enhancement of TNF--dependent NF-B activation seen in the presence of IFN-. In this model (Fig. 5), resting macrophages have cytoplasmic STAT-1 available to interact with TNFR1 to serve as a negative regulator of TNF--dependent NF-B activation. When TNF- is available in the absence of IFN-, STAT-1 associates with TNFR1 to aid in receptor-proximal negative regulation of NF-B activation. However, in the presence of IFN-, STAT-1 is tyrosine phosphorylated and subsequently localizes to the nucleus where it is no longer able to interact with TNFR1, thus allowing for stronger TNF--dependent NF-B activation. Consistent with the hypothesis that STAT-1 tyrosine phosphorylation and nuclear localization is important for IFN--mediated inhibition of the TNFR1:STAT-1 complex for up to 6 h (Fig. 1C), we have previously shown that IFN--mediated STAT-1 tyrosine phosphorylation was detectable for up to 6 h in these cells (15). Additionally, STAT-1 was still detected in the nucleus through this 6 h time point (data not shown).
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has on the TNF- signaling pathway. Our results may represent a contributing factor to the general phenomenon of IFN- sensitization of macrophages to TNF- effects. In this regard, work from another laboratory has provided evidence that the effect of STAT-1 at the TNFR1 complex is not limited to NF-B modulation. It was shown that when STAT-1 interacted with the TNFR1, TNF- was a more potent inducer of apoptosis (16) (see Fig. 5). In light of these results, we tested the effect of IFN- on TNF--dependent apoptosis in RAW264.7 cells. The interpretation of the results from these experiments was problematic as IFN- induces a degree of apoptosis in these cells independent of TNF- treatment (data not shown). Additional experiments demonstrated that, unlike NF-B activation, IFN- had no effect on TNF--mediated MAPK activation (data not shown).
An intriguing question that remains is how TNF- ligation causes STAT-1 to associate with TNFR1. A previous study demonstrated that in Hela cells, TNF- induces tyrosine phosphorylation of STAT-1 and this is necessary for TNF--induced formation of a complex of STAT-1 and the adapter protein TRADD (16). However, although STAT-1 tyrosine phosphorylation was implied to be necessary, the authors of this study did not directly test whether STAT-1 tyrosine phosphorylation was a requirement for the TNFR1:STAT-1 complex formation (16). We have shown that TNF- does not induce tyrosine phosphorylation in RAW264.7 cells (Fig. 1), but still induces TNFR1:STAT-1 complex formation/colocalization (Figs. 1 and 2). Moreover, the STAT-1 that is complexed to TNFR1 in the coimmunoprecipitation studies does not react with Abs to tyrosine phosphorylated nor serine phosphorylated forms of STAT-1 (data not shown). Thus, an important difference between our results and those reported previously (16), is regarding the role of STAT-1 tyrosine phosphorylation. The results presented herein clearly demonstrate that tyrosine phosphorylation is not required for TNF--mediated TNFR1:STAT-1 complex formation as TNF- does not induce tyrosine phosphorylation in RAW264.7 cells. At this point, the mechanism of TNF--dependent TNFR1:STAT-1 complex formation remains unknown, and is under study.
The studies represented herein define a novel level of cross-talk between the IFN- and TNF- signaling pathways, and advance the concept that control of STAT-1 availability to TNFR1, or perhaps other TNFR signaling components by IFN- may in part mediate the balance and/or control the threshold of TNF--mediated processes.
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Acknowledgments |
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expression construct. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Etty N. Benveniste, Department of Cell Biology, University of Alabama, 1530 3rd Avenue South, McCallum Basic Health Sciences Building 395, Birmingham, AL 35294-0005. E-mail address: tika{at}uab.edu
3 Abbreviations used in this paper: TRADD, TNFR1-associated death domain protein; FADD, Fas-associated death domain protein; MAPK, mitogen-activated protein kinase; JAK, Janus kinase; IRF, IFN-regulatory factor; RPA, RNase protection assay.
Received for publication March 24, 2003. Accepted for publication September 2, 2003.
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 S. Keslacy, O. Tliba, H. Baidouri, and Y. Amrani Inhibition of Tumor Necrosis Factor-{alpha}-Inducible Inflammatory Genes by Interferon-{gamma} Is Associated with Altered Nuclear Factor-{kappa}B Transactivation and Enhanced Histone Deacetylase Activity Mol. Pharmacol., February 1, 2007; 71(2): 609 - 618. [Abstract] [Full Text] [PDF]
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 S. K. Frankel, G. P. Cosgrove, S.-I. Cha, C. D. Cool, M. W. Wynes, B. L. Edelman, K. K. Brown, and D. W. H. Riches TNF-{alpha} Sensitizes Normal and Fibrotic Human Lung Fibroblasts to Fas-Induced Apoptosis Am. J. Respir. Cell Mol. Biol., March 1, 2006; 34(3): 293 - 304. [Abstract] [Full Text] [PDF]
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 J.-T. Chen, J.-B. Liang, C.-L. Chou, M.-W. Chien, R.-C. Shyu, P.-I Chou, and D.-W. Lu Glucosamine Sulfate Inhibits TNF-{alpha} and IFN-{gamma}-Induced Production of ICAM-1 in Human Retinal Pigment Epithelial Cells In Vitro Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 664 - 672. [Abstract] [Full Text] [PDF]
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 H. S. Kim and M.-S. Lee Essential Role of STAT1 in Caspase-Independent Cell Death of Activated Macrophages through the p38 Mitogen-Activated Protein Kinase/STAT1/Reactive Oxygen Species Pathway Mol. Cell. Biol., August 1, 2005; 25(15): 6821 - 6833. [Abstract] [Full Text] [PDF]
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