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*
Department of Microbiology and Immunology and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, IN 46202; and
Department of Pathology, University of Tennessee College of Medicine, Memphis, TN 38163
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first step in TNF action is binding to specific receptors that are expressed on virtually all nucleated cells. Two distinct receptors for TNF have been identified, the 55-kDa type 1 receptor (TNFR1)3 and the 75-kDa type 2 receptor (TNFR2), and their cDNAs cloned (3, 4, 5). The extracellular domains of the receptors share homologies with one another and with a group of cell surface proteins that include the FAS Ag, the low affinity nerve growth factor receptor, the murine cDNA clone 4-1BB from induced helper and cytolytic T cells, the B cell surface Ag CD40, the OX40 Ag of activated CD4--positive rat lymphocytes, and the T2 Ag of the Shope fibrosarcoma virus (6). The intracellular domains of the TNF receptors are dissimilar (3, 4, 5), and the receptors induce a distinct but overlapping repertoire of responses: TNFR1 promotes cytotoxicity, fibroblast proliferation, and antiviral responses, and plays a preeminent role in the host defense against microorganisms and pathogens (7, 8, 9, 10, 11); TNFR2 also plays a role in cytotoxicity, modulates hemopoiesis, and is involved in the proliferation of monocytes and T cells (12, 13, 14, 15, 16).
The proximal steps in the pathways through which TNF induces such
responses are incompletely understood. Neither TNF receptor contains
protein tyrosine kinase activity or any motif suggesting a biochemical
activity (3, 4, 5). However, the induction of specific tyrosine
phosphorylations is associated with cellular responses to TNF. Such
phosphorylations have been related to alterations of cellular
sensitivity to TNF-mediated cytotoxicity (17, 18). In addition,
inhibitors of protein tyrosine kinases suppress TNF-stimulated
phosphorylation of a 23-kDa nuclear protein (19), DNA fragmentation
(20), activation of nuclear factor-
B, and expression of endothelial
cell adhesion molecules (21, 22). We recently demonstrated that in
3T3-L1 adipocytes, TNF rapidly induces the tyrosine phosphorylation of
a group of cytoplasmic proteins, including insulin receptor
substrate-1. These results show that early activation of tyrosine
kinase activity is a component of the cellular response to TNF (23).
The priming of neutrophils by TNF is accompanied by tyrosine
phosphorylation events believed to participate in the transduction of
signals that direct the cells to undergo a respiratory burst (24, 25).
In neutrophils, TNF, granulocyte CSF, and granulocyte-macrophage CSF
induce the tyrosine phosphorylation of a common group of proteins (26, 27), which may indicate that the cytokines act, in part, through common
signaling pathways.
The present study was initiated to identify protein tyrosine kinases involved in TNF signal transduction. In this regard, our attention was drawn to a family of cytoplasmic protein tyrosine kinases, Janus (Jak) kinases (28, 29, 30), which promote signaling by cytokine/hemopoietin receptors activated by ligands such as granulocyte-macrophage CSF and granulocyte CSF, and mediate the tyrosine phosphorylation of a number of intracellular targets including insulin receptor substrate-1 (31, 32, 33, 34). We have found that by acting through TNFR1, TNF promotes the phosphorylation and activation of Jak protein tyrosine kinases in 3T3-L1 adipocytes. These kinases interact directly with TNFR1 in a cell-free system and in vivo. Activation of Jak kinases by TNF was accompanied by the tyrosine phosphorylation of members of the STAT family of transcription factors and the induction of STAT3 DNA-binding activity. The Jak/STAT signaling pathway represents a mechanism through which TNF transmits signals from the cell surface to the nucleus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant murine TNF (mTNF) was from R&D Systems (Minneapolis, MN). Recombinant human TNF (hTNF) and agonist Abs against mTNFR1 were gifts from Genentech (South San Francisco, CA). Ab to hTNFR1 was from Genzyme Corp. (Cambridge, MA). Jak1 cDNA in pRc/CMV and Jak2 cDNA in Prk5 were gifts from Dr. James Ihle, St. Jude’s Children’s Research Hospital, Memphis, TN. Horseradish peroxidase-conjugated anti-phosphotyrosine mAb (RC20) was from Transduction Laboratories (Lexington, KY). Abs to Jak1, Jak2, Jak3, and Tyk2 were from Upstate Biotechnology (Lake Placid, NY). Abs to STAT1, STAT3, STAT5b, and STAT6 were from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture
3T3-L1 fibroblast cells from American Type Culture Collection (Rockville, MD) were grown in 100-mm dishes in DMEM supplemented with 10% heat-inactivated FBS, nonessential amino acids, L-glutamine, penicillin (100 U/ml), and streptomycin (100 U/ml) in a humidified incubator under 5% CO2 at 37°C. Differentiation to adipocytes was conducted as described (35). Maximal adipose conversion was achieved 10 to 12 days after initiation of differentiation.
Immunoprecipitation and immunoblotting tyrosine-phosphorylated Jaks and STAT proteins
After treatment with TNF, cells were washed twice with ice-cold PBS and lysed by incubation in 50 mM HEPES, pH 7, 150 mM NaCl, 10% glycerol, 1.2% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM sodium o-vanadate for 30 min at 4°C. The lysate was centrifuged (150,000 x gmax, 1 h) and the infranatant was removed from the fat and passed through a 22-µm filter. Extracts (1–1.5 mg of protein) were shaken with antiserum overnight at 4°C, 40 µl of a slurry of protein G-plus/protein A-agarose was added, and the incubation was continued for 2 h at 4°C. The incubate was centrifuged briefly at 10,000 x g, and the agarose was washed with 20 mM HEPES, pH 7, 10% glycerol, 0.1% Triton X-100, 150 mM NaCl, and 1 mM sodium o-vanadate and recentrifuged. This was repeated four times, after which Laemmli medium was added and the agarose was heated at 100°C for 5 min. Immunoprecipitated proteins were fractionated on 7.5% acrylamide gels and blotted onto Immobilon-P (Millipore, Marlborough, MA) during an overnight transfer. Immunoblots were blocked by incubation in TBST (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 0.1% Tween-20) containing 1% BSA and then incubated with the primary Ab for 1 to 2 h at room temperature, washed three times with TBST, and, in experiments not using a primary Ab conjugated to horseradish peroxidase, incubated with anti-mouse or anti-rabbit secondary Abs for 1 h. The enhanced chemiluminescent detection system (Amersham, Arlington Heights, IL) was used for protein detection. To probe immunoblots with a second antiserum, membranes were stripped by incubation in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-ME for 30 min at 50°C. The blots were then incubated with antiserum and processed as described above.
Nuclear extracts and gel shift assays
Nuclei were extracted from 3T3-L1 adipocytes with buffer containing 20 mM Tris-HCl, pH 7.85, 250 mM sucrose, 0.4 M KCl, 1.1 mM MgCl2, 5 mM 2-ME, and 0.4 mM PMSF, and extracts were frozen on dry ice and stored at -80°C. For gel shift analysis, the nuclear extracts were incubated with a 32P end-labeled promoter probe for either the high affinity c-sis-inducible element (SIE) from the c-fos gene (5'-AGCTTCATTTCCCGTAATCCCTAAAGCT-3') or the IFN-stimulated response element (ISRE) from ISG15 (5'-GATCCATGCCTCGGGAAGGGAAACCGAAACTGAAGCC-3') at 25°C for 30 min, and the free probe was separated from protein-DNA complexes on 5% polyacrylamide gels. For supershift assays, nuclear extracts were preincubated with a 1/50 dilution of normal rabbit serum, anti-STAT1, or anti-STAT3 at 25°C for 0.5 h. Gels were quantitated by phosphor image autoradiography.
In vitro binding assays
Three GST-TNFR1IC fusion constructs were prepared by amplifying and inserting desired portions of TNFR1IC into pGEX-2T (Pharmacia Biotech, Piscataway, NJ) (36): 1) the full-length cytoplasmic domain of TNFR1IC (amino acids 205–413, GST-TNFR1IC); 2) part of the N-terminal half of TNFR1IC (amino acids 243–315, GST-TNFR1NH2); and 3) the C-terminal half of TNFR1IC (amino acids 316–413, GST-TNFR1COOH). After induction, cells were grown at 30°C for 3 h, suspended in lysis buffer (20 mM Tris, pH 8, 200 mM NaCl, 10% glycerol, 0.5% Nonidet P-40, 1 mM PMSF, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.1% 2-ME), and sonicated. The lysate was centrifuged (12,000 rpm, 30 min), and one-tenth volume of 50% GST-agarose slurry (Sigma Chemical Co., St. Louis, MO) was incubated with the supernatant for 1 h at 4°C. The beads were washed three times with lysis buffer, and the purity of the GST fusion proteins was confirmed by SDS-PAGE.
The troponin T-coupled rabbit reticulocyte lysate system (Promega Corp., Madison, WI) was used for transcription/translation of Jak1 cDNA in PRc/CMV or Jak2 cDNA in Prk5 (under control of the T7 or SP6 promoter, respectively), according to the instructions of the manufacturer. Rabbit reticulocyte was mixed with 1 µg of Jak cDNA along with SP6 or T7 RNA polymerase, after which an amino acid mixture without methionine plus [35S]methionine was added. After 2 h at 30°C, the purity and level of expression of each Jak were analyzed by SDS/PAGE and phosphor image analysis (Bio-Rad, Hercules, CA). Approximately 5 µg of each agarose-bound GST-TNFR1IC construct was added into binding buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 1.0 mM sodium o-vanadate, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and 40 µl of reticulocyte lysate that contained [35S]methionine-labeled Jak. After 1 h at 4°C, the agarose beads were washed three times with binding buffer before addition of SDS sample buffer. The samples were subjected to electrophoresis and phosphor image analysis.
GST-TNFR1IC was also used to bind Jak kinases in lysates of control- or TNF-treated 3T3-L1 adipocytes. In this assay, 5 µg of agarose-bound GST-TNFR1IC was incubated with cell lysates for 1 h at 4°C in 50 mM HEPES, pH 7, 150 mM NaCl, 10% glycerol, 1.2% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM sodium o-vanadate. The beads were washed three times in lysis buffer before bound proteins were eluted into Laemmli medium, fractionated by SDS-PAGE, and transferred to Immobilon-P (Millipore), which was probed with Abs to Jak kinases.
In vivo association of TNFR1 and Jak2
HeLa cells in DMEM supplemented with 10% FBS were treated with vehicle or TNF (1 nM) for 15 min at 37°C, washed twice with ice-cold PBS, and lysed by incubation in 50 mM HEPES, pH 7, 150 mM NaCl, 10% glycerol, 1.2% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM sodium o-vanadate for 30 min at 4°C. Extracts were shaken with antiserum to human TNFR1 for 16 h at 4°C, after which 40 µl of a slurry of protein G-plus/protein A-agarose were added and incubation was continued for 2 h at 4°C. The incubate was centrifuged briefly at 10,000 x g, and the agarose was washed with lysis buffer and recentrifuged. This process was repeated four times, after which Laemmli medium was added and the agarose was heated at 100°C for 5 min. Immunoprecipitated proteins were fractionated on 7.5% acrylamide gels and blotted onto Immobilon-P during an overnight transfer. Immunoblots were blocked by incubation in PBS containing 5% fat-free milk and then incubated with the primary Ab to Jak2 for 1 to 2 h at room temperature. Blots were washed three times with TBST and then incubated with anti-mouse secondary Ab for 1 h. The enhanced chemiluminescent detection system was used for protein detection. To reprobe immunoblots, the membranes were stripped by incubation in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM 2-ME for 30 min at 50°C. The blots were then incubated with Ab to TNFR1 and processed as described above.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, mTNF promoted the phosphorylation of
Jak1 (top panel), Jak2 (middle
panel), and Tyk2 (bottom panel).
Reproducibly, Jak2 became more highly tyrosine phosphorylated than Jak1
or Tyk2 after cell stimulation with mTNF. Jak3 phosphorylation was not
detected in 3T3-L1 adipocytes, as this kinase is not expressed in this
cell type (data not shown).
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). To accomplish this, Jak1 or Jak2 was
immunoprecipitated from control or mTNF-treated 3T3-L1 adipocytes and
then reacted with ATP to permit autophosphorylation. Tyrosine
phosphorylation was determined by Western blot analysis using Abs to
phosphotyrosine, which showed that mTNF promotes substantial
autophosphorylation of the Jak1 and Jak2 kinases. In the case of Jak1,
some low level of Jak phosphorylation was detected in cells exposed to
TNF alone, probably due to Jak autophosphorylation with endogenous ATP.
Because of the higher level of Jak2 autophosphorylation induced by TNF
in the presence of ATP, kinase activation induced by TNF alone could
only be detected by grossly overexposing the autoradiograph (data not
shown).
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, top)
and STAT3 (Fig. 3, middle), but did not affect STAT2
phosphorylation (data not shown). In addition, TNF strongly induced
phosphorylation of STAT5 and weakly induced phosphorylation of STAT6
(Fig. 3, bottom), but was unable to promote
phosphorylation of STAT4 (data not shown). These observations show that
STAT protein phosphorylation induced by TNF is specific and
selective.
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shows that TNF rapidly induced (within
15 min of addition) DNA-binding activity in nuclear lysates prepared
from TNF-treated adipocytes incubated with the SIE probe. No DNA
binding to the SIE was detected in the presence of excess unlabeled SIE
oligonucleotide, and binding was not competed by excess ISRE
oligonucleotide. Taken together, these results indicate that the
binding to the probe was specific. In contrast, no DNA-binding activity
could be detected in nuclear lysates prepared from TNF-treated
adipocytes incubated with the ISRE probe (which detects STAT1/STAT2
heterodimers, ISGF3). To detect specific STAT proteins in the
TNF-inducible DNA-protein complexes, we performed gel supershift assays
with STAT-specific antisera. Anti-STAT3 supershifted the DNA-binding
complex formed with the SIE probe in TNF-treated adipocytes, while
neither control normal rabbit serum nor anti-STAT1 shifted any of
the TNF-induced SIE DNA-protein complexes. Antisera to STAT2 also
failed to shift any of the TNF-induced SIE DNA-protein complexes (data
not shown). Thus, TNF induced a DNA-binding activity in adipocytes
attributable to STAT3.
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, top),
which shows that TNFR1 promoted these events. Consistent with the
conclusion that TNFR1 mediates phosphorylation of Jak kinases are
experiments in which 3T3-L1 adipocytes were stimulated with agonist
antisera specific to mTNFR1 (anti-mTNFR1). Jak1 and Jak2 were then
immunoprecipitated from cell lysates and fractionated by SDS-PAGE, and
Western blots were probed with antisera to phosphotyrosine. As shown in
Figure 5 (middle), activation of TNFR1 with
anti-mTNFR1 resulted in the time-dependent tyrosine phosphorylation
of Jak1 and Jak2. Since Jak kinase phosphorylation promoted by TNF has
not been demonstrated previously, we compared the extent of such
activation with that promoted by human growth hormone, a hormone
previously demonstrated to elicit such a response (38). Figure 5
(bottom) illustrates that mTNF, which
activates both TNFR subtypes, and agonist antiserum to TNFR1 both
significantly induced phosphorylation of Jak2, although the magnitude
of this response was less than that induced by human growth
hormone.
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) shows that signaling
through this receptor induces STAT phosphorylation. However, our
results do not preclude the possibility that signaling through TNFR2
might also activate the Jak/STAT pathway.
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, top), suggesting
that TNFR1 and Jak kinases interact directly, without the intermediacy
of other proteins. GST fusion proteins encoding the N- and C-terminal
regions of TNFR1 also bound Jak1 and Jak2, although the
NH2-terminal fusion protein was more effective in this
regard than the COOH-terminal fusion protein. These results suggest the
presence of a primary Jak binding site in the N-terminal portion of
TNFR1, which contains a proline-rich motif reminiscent of the box 1
motif with which these kinases are known to interact, as well as a
secondary Jak kinase binding site in the C-terminal half of TNFR1IC.
GST-TNFR1IC was also used to bind Jak1 and Jak2 in lysates of control-
and mTNF-treated 3T3-L1 adipocytes (Fig. 7, bottom).
Results from this experiment suggest that TNFR1 and Jak kinases
interact in vivo.
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show that
this Jak kinase constitutively associates with TNFR1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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acts through Jak1 and Tyk2, whereas IFN-
acts through Jak1 and Jak2 (as summarized in 39 . Prolactin and
growth hormone receptors appear to function exclusively through
Jak2 (39).
Among the substrates for activated Jak kinases are the STAT family of
latent cytoplasmic transcription factors. Activation of cytokine
receptors and their associated Jak kinases is rapidly followed by the
tyrosine phosphorylation of STAT proteins, which subsequently dimerize
and translocate to the nucleus. Within the nucleus, STATs bind DNA and
activate the transcription of distinct groups of genes, many of which
are important to inflammatory responses (28, 29, 30). Among these are genes
for acute phase reactant proteins, IFN-regulatory factor-1,
intercellular adhesion molecule 1, the ß-chain of the IL-2R, Igs, and
FcRI (40, 41, 42, 43). The alteration in gene expression plays an important
role in the innate immunity of organisms (44, 45) and in the regulation
of cell growth and viability (46, 47, 48, 49).
The STAT protein family is presently composed of six distinct, but
homologous members designated STAT1 through STAT6 (28, 29, 30, 39). Most
STATs are widely expressed in a variety of cell types. Each STAT may be
activated in response to cell stimulation by multiple ligands; however,
the process is not promiscuous, in that particular ligands tend to act
on a restricted array of STAT proteins. For example, IFN-
preferentially activates STAT1, IL-6 preferentially activates STAT3,
and IL-4 preferentially activates STAT6. Jak-STAT signaling is also
induced in cells transformed by the human T-lymphotropic virus-1 and by
the v-Src or v-Abl oncoproteins, and by signaling through the G
protein-coupled receptor for angiotensin II (50, 51, 52, 53). These
observations show that signaling pathways other than those initiated by
ligation of cytokine/hemopoietin receptors can lead to Jak-STAT
activity.
TNF exists primarily as a trimer in solution. Consequently, its binding to the extracellular domain of TNFR1 brings the cytoplasmic domains of the receptor into proximity such that they interact and generate the signal necessary for transmission of the TNF message to downstream structures (54, 55, 56, 57). One region in the C terminus of the cytoplasmic domain of TNFR1 plays an obligate role in signaling cytotoxicity. This death domain (58, 59) associates with itself and with cytoplasmic proteins implicated in apoptosis (60, 61, 62). Thus, as with other cytokine/hemopoietin receptors, aggregation of TNFR1 is important to TNF action. The essential role of self-association in the function of TNFR1, the numerous reports implicating tyrosine kinases in cellular responses to TNF, and the absence of intrinsic tyrosine kinase activity in either TNF receptor subtype led us to test whether TNF might activate Jak/STAT signaling.
The present study shows that TNF induces the tyrosine phosphorylation and activation of Jak1, Jak2, and Tyk2 in 3T3-L1 adipocytes. The effect of TNF was most evident on Jak2 phosphorylation, with the effect on tyrosine phosphorylation of Jak1 and Tyk2 less pronounced. TNF also promoted the phosphorylation of Jak2 in 3T3-L1 fibroblasts and C3H10T1/2 fibroblasts (data not shown). The ability of mTNF to promote the tyrosine phosphorylation of Jak2 in 3T3-L1 adipocytes was recapitulated by an agonist antiserum that specifically activates TNFR1, showing that this TNFR subtype can activate this cellular response. Consistent with this conclusion are experiments demonstrating that hTNF, which specifically binds TNFR1 on murine cells (37), also promotes Jak kinase phosphorylation. Together these observations show that Jak kinase activation is a common cellular response to TNF and that this response is mediated, at least in part, through the type 1 TNFR.
A GST fusion protein encompassing the full-length cytoplasmic domain of TNFR1 precipitated Jak2 from a cell-free translation system. This result shows that TNFR1 binds Jak2 directly and without the intermediacy of another protein. GST-TNFR1 also precipitated Jak2 from adipocyte cell lysates, showing that these proteins can interact within the intracellular milieu. Finally, TNFR1 and Jak2 were coprecipitated from cell lysates, a result that adds the Jak kinases to the list of proteins presently known to bind this TNF receptor.
In a previous study (58), we demonstrated that the C-terminal death domain in TNFR1 self-associates and mediates receptor-receptor interactions. Larger peptides that contained sequences from the N-terminal half of the cytoplasmic domain of TNFR1 interacted less strongly. These observations suggested that the conformation of the longer peptides might mask the aggregation domain, thereby diminishing receptor-receptor interactions. In the present study, we found that a fusion protein containing the full-length cytoplasmic domain of TNFR1 appears to bind Jak2 less efficiently than a protein containing only the N-terminal portion of this receptor region. This result suggests that the primary binding site to which the Jak kinases bind in the cytoplasmic domain of TNFR1 resides in its N-terminal half. Furthermore, these observations suggest that different domains in TNFR1IC may positively and negatively regulate the ability of the intracellular domain to interact with cytoplasmic signaling proteins that promote cellular responses to TNF.
Our data indicate that TNF may utilize Jak kinases to mediate some of its cellular responses. Consistent with this possibility is the recent report that CD40, another member of the TNF/nerve growth factor receptor superfamily, associates with and utilizes Jak3 as a component of its signaling mechanism (63). However, further experimentation will be required to demonstrate the roles that individual Jak kinases play in the manifold actions of TNF. This is necessitated by results showing that although Tyk2, Jak1, and Jak2 are all phosphorylated in response to IL-6 (64, 65), experiments with Jak-deficient cells demonstrate that only Jak1 plays a significant role in the IL-6-dependent phosphorylation of the gp130 subunit, STAT activation, and transcriptional induction of the IFN-regulatory factor-1 gene (66).
In addition to promoting Jak kinase activity, TNF also induced the tyrosine phosphorylation of a select group of STAT proteins. In 3T3-L1 adipocytes, TNF strongly induced the tyrosine phosphorylation of STAT3 and STAT5; phosphorylation of STAT1 and STAT6 was demonstrable, but less pronounced. Tyrosine phosphorylation of STAT2 and STAT4 could not be detected. The effects of mTNF on STAT protein phosphorylation were reproduced using agonist antisera directed against TNFR1, demonstrating that it induces this cellular response.
Electromobility shift assays demonstrated that treatment of 3T3-L1 adipocytes with mTNF induced DNA-binding activity attributable to STAT3. This result, together with the ability of TNF to activate Jak kinases, shows that TNF activates the Jak/STAT signal transduction pathway. Surprisingly, we were not able to detect DNA-binding activity attributable to STAT1 in 3T3-L1 adipocytes, despite the ability of TNF to promote the tyrosine phosphorylation of this transcription factor. A recent study by Ivashkiv et al. (67) identified DNA-binding activity in activated T cells that resulted from activation of STAT1 and STAT3. Induction of cAMP signaling in these cells inhibited the DNA binding of STAT1, but not STAT3, showing that the function of these transcription factors can be regulated by cross-talk with different signaling pathways. Previous studies have also shown that serine phosphorylation may also be important for the full activation of DNA-binding activity or transcriptional activity of STAT proteins (68, 69, 70, 71). Our results show that detection of STAT phosphorylation is insufficient in itself to demonstrate that these proteins become functional, and suggest the possibility that Jak/STAT signaling induced by TNF may be regulated by other signaling events. However, these complexities aside, our observations definitively demonstrate for the first time that TNF activates Jak/STAT signaling in cells.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. David B. Donner, The Walther Oncology Center, 1044 West Walnut Street, Indiana University School of Medicine, Indianapolis, IN 46202.
3 Abbreviations used in this paper: TNFR, 55-kDa tumor necrosis factor receptor; GST, glutathione S-transferase; h, human; ISRE, interferon-stimulated response element; m, murine; SIE, c-sis-inducible element.
Received for publication December 10, 1996. Accepted for publication November 25, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation induced by tumor necrosis factor requires membrane-associated components: comparison with pathway induced by ceramide. J. Biol. Chem. 269:25369.2 integrin-dependent tyrosine phosphorylation of paxillin in human neutrophils treated with tumor necrosis factor. J. Cell Biol. 127:1477. and granulocyte colony stimulating factor. Arch. Biochem. Biophys. 298:703.[Medline]
engages insulin receptor substrate-1 to associate with phosphatidylinositol 3-kinase. J. Biol. Chem. 270:15938.-induced transcription of the high-affinity Fc receptor for IgG requires assembly of a complex that includes the 91-kDa subunit of transcription factor ISGF3. Proc. Natl. Acad. Sci. USA 90:4314.B activation. Cell 81:495.[Medline]
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 A. C. Bharti, S. Shishodia, J. M. Reuben, D. Weber, R. Alexanian, S. Raj-Vadhan, Z. Estrov, M. Talpaz, and B. B. Aggarwal Nuclear factor-{kappa}B and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis Blood, April 15, 2004; 103(8): 3175 - 3184. [Abstract] [Full Text] [PDF]
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O. Tliba, S. Tliba, C. Da Huang, R. K. Hoffman, P. DeLong, R. A. Panettieri Jr., and Y. Amrani Tumor Necrosis Factor {alpha} Modulates Airway Smooth Muscle Function via the Autocrine Action of Interferon {beta} J. Biol. Chem., December 12, 2003; 278(50): 50615 - 50623. [Abstract] [Full Text] [PDF]
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D. R. Wesemann and E. N. Benveniste STAT-1{alpha} and IFN-{gamma} as Modulators of TNF-{alpha} Signaling in Macrophages: Regulation and Functional Implications of the TNF Receptor 1:STAT-1{alpha} Complex J. Immunol., November 15, 2003; 171(10): 5313 - 5319. [Abstract] [Full Text] [PDF]
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A. C. Bharti, N. Donato, and B. B. Aggarwal Curcumin (Diferuloylmethane) Inhibits Constitutive and IL-6-Inducible STAT3 Phosphorylation in Human Multiple Myeloma Cells J. Immunol., October 1, 2003; 171(7): 3863 - 3871. [Abstract] [Full Text] [PDF]
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D. R. Wesemann, Y. Dong, G. M. O'Keefe, V. T. Nguyen, and E. N. Benveniste Suppressor of Cytokine Signaling 1 Inhibits Cytokine Induction of CD40 Expression in Macrophages J. Immunol., September 1, 2002; 169(5): 2354 - 2360. [Abstract] [Full Text] [PDF]
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 S. Cottet, P. Dupraz, F. Hamburger, W. Dolci, M. Jaquet, and B. Thorens cFLIP Protein Prevents Tumor Necrosis Factor-{alpha}-Mediated Induction of Caspase-8-Dependent Apoptosis in Insulin-Secreting {beta}Tc-Tet Cells Diabetes, June 1, 2002; 51(6): 1805 - 1814. [Abstract] [Full Text] [PDF]
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 F. Machinal-Quelin, M. N. Dieudonne, M. C. Leneveu, R. Pecquery, and Y. Giudicelli Proadipogenic effect of leptin on rat preadipocytes in vitro: activation of MAPK and STAT3 signaling pathways Am J Physiol Cell Physiol, April 1, 2002; 282(4): C853 - C863. [Abstract] [Full Text] [PDF]
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 D.-c. Kang, R. V. Gopalkrishnan, Q. Wu, E. Jankowsky, A. M. Pyle, and P. B. Fisher mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties PNAS, January 22, 2002; 99(2): 637 - 642. [Abstract] [Full Text] [PDF]
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 S. Miscia, M. Marchisio, A. Grilli, V. Di Valerio, L. Centurione, G. Sabatino, F. Garaci, G. Zauli, E. Bonvini, and A. Di Baldassarre Tumor Necrosis Factor {alpha} (TNF-{alpha}) Activates Jak1/Stat3-Stat5B Signaling through TNFR-1 in Human B Cells Cell Growth Differ., January 1, 2002; 13(1): 13 - 18. [Abstract] [Full Text] [PDF]
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B. Emanuelli, P. Peraldi, C. Filloux, C. Chavey, K. Freidinger, D. J. Hilton, G. S. Hotamisligil, and E. Van Obberghen SOCS-3 Inhibits Insulin Signaling and Is Up-regulated in Response to Tumor Necrosis Factor-alpha in the Adipose Tissue of Obese Mice J. Biol. Chem., December 14, 2001; 276(51): 47944 - 47949. [Abstract] [Full Text] [PDF]
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J. Zamorano, A. L. Mora, M. Boothby, and A. D. Keegan NF-{kappa}B activation plays an important role in the IL-4-induced protection from apoptosis Int. Immunol., December 1, 2001; 13(12): 1479 - 1487. [Abstract] [Full Text] [PDF]
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N. M. Rizk, D. Stammsen, G. Preibisch, and J. Eckel Leptin and Tumor Necrosis Factor-{{alpha}} Induce the Tyrosine Phosphorylation of Signal Transducer and Activator of Transcription Proteins in the Hypothalamus of Normal Rats In Vivo Endocrinology, July 1, 2001; 142(7): 3027 - 3032. [Abstract] [Full Text] [PDF]
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J. Hoeck and M. Woisetschlager STAT6 Mediates Eotaxin-1 Expression in IL-4 or TNF-{{alpha}}-Induced Fibroblasts J. Immunol., April 1, 2001; 166(7): 4507 - 4515. [Abstract] [Full Text] [PDF]
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 E. Hiraoka, S. Kawashima, T. Takahashi, Y. Rikitake, T. Kitamura, W. Ogawa, and M. Yokoyama TNF-{alpha} induces protein synthesis through PI3-kinase-Akt/PKB pathway in cardiac myocytes Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1861 - H1868. [Abstract] [Full Text] [PDF]
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 S. Bhattacharya, B. A. Stout, M. E. Bates, P. J. Bertics, and J. S. Malter Granulocyte Macrophage Colony-Stimulating Factor and Interleukin-5 Activate STAT5 and Induce CIS1 mRNA in Human Peripheral Blood Eosinophils Am. J. Respir. Cell Mol. Biol., March 1, 2001; 24(3): 312 - 316. [Abstract] [Full Text] [PDF]
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R. Allendoerfer and G. S. Deepe Jr. Regulation of Infection with Histoplasma capsulatum by TNFR1 and -2 J. Immunol., September 1, 2000; 165(5): 2657 - 2664. [Abstract] [Full Text] [PDF]
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 Y. Wang, T. R. Wu, S. Cai, T. Welte, and Y. E. Chin Stat1 as a Component of Tumor Necrosis Factor Alpha Receptor 1-TRADD Signaling Complex To Inhibit NF-kappa B Activation Mol. Cell. Biol., July 1, 2000; 20(13): 4505 - 4512. [Abstract] [Full Text]
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 R. Baetta, M. Soma, C. De-Fraja, C. Comparato, C. Teruzzi, L. Magrassi, and E. Cattaneo Upregulation and Activation of Stat6 Precede Vascular Smooth Muscle Cell Proliferation in Carotid Artery Injury Model Arterioscler. Thromb. Vasc. Biol., April 1, 2000; 20(4): 931 - 939. [Abstract] [Full Text] [PDF]
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S. Matsukura, C. Stellato, J. R. Plitt, C. Bickel, K. Miura, S. N. Georas, V. Casolaro, and R. P. Schleimer Activation of Eotaxin Gene Transcription by NF-{kappa}B and STAT6 in Human Airway Epithelial Cells J. Immunol., December 15, 1999; 163(12): 6876 - 6883. [Abstract] [Full Text] [PDF]
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K. Sato, H. Nagayama, K. Tadokoro, T. Juji, and T. A. Takahashi Extracellular Signal-Regulated Kinase, Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase, and p38mapk Are Involved in IL-10-Mediated Selective Repression of TNF-{alpha}-Induced Activation and Maturation of Human Peripheral Blood Monocyte-Derived Dendritic Cells J. Immunol., April 1, 1999; 162(7): 3865 - 3872. [Abstract] [Full Text] [PDF]
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G. Constantin, C. Laudanna, S. Brocke, and E. C. Butcher Inhibition of Experimental Autoimmune Encephalomyelitis by a Tyrosine Kinase Inhibitor J. Immunol., January 15, 1999; 162(2): 1144 - 1149. [Abstract] [Full Text] [PDF]
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J. E. Towne, C. M. Krane, C. J. Bachurski, and A. G. Menon Tumor Necrosis Factor-alpha Inhibits Aquaporin 5 Expression in Mouse Lung Epithelial Cells J. Biol. Chem., May 25, 2001; 276(22): 18657 - 18664. [Abstract] [Full Text] [PDF]
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R. Craig, A. Larkin, A. M. Mingo, D. J. Thuerauf, C. Andrews, P. M. McDonough, and C. C. Glembotski p38 MAPK and NF-kappa B Collaborate to Induce Interleukin-6 Gene Expression and Release. EVIDENCE FOR A CYTOPROTECTIVE AUTOCRINE SIGNALING PATHWAY IN A CARDIAC MYOCYTE MODEL SYSTEM J. Biol. Chem., July 28, 2000; 275(31): 23814 - 23824. [Abstract] [Full Text] [PDF]
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Y. Morita, T. Naka, Y. Kawazoe, M. Fujimoto, M. Narazaki, R. Nakagawa, H. Fukuyama, S. Nagata, and T. Kishimoto Signals transducers and activators of transcription (STAT)-induced STAT inhibitor-1 (SSI-1)/suppressor of cytokine signaling-1 (SOCS-1) suppresses tumor necrosis factor alpha -induced cell death in fibroblasts PNAS, May 9, 2000; 97(10): 5405 - 5410. [Abstract] [Full Text] [PDF]
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