The MAPK Kinase Kinase TAK1 Plays a Central Role in Coupling the Interleukin-1 Receptor to Both Transcriptional and RNA-targeted Mechanisms of Gene Regulation*

Helmut HoltmannDagger , Jost EnningaDagger , Solveig KälbleDagger , Axel ThiefesDagger , Anneke DörrieDagger , Meike BroemerDagger , Reinhard WinzenDagger , Arno WilhelmDagger , Jun Ninomiya-Tsuji§, Kunihiro Matsumoto§, Klaus ReschDagger , and Michael KrachtDagger

From the Dagger  Institute of Pharmacology, Medical School Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany and the § Department of Molecular Biology, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan

Received for publication, May 22, 2000, and in revised form, October 23, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Mechanisms of fulminant gene induction during an inflammatory response were investigated using expression of the chemoattractant cytokine interleukin-8 (IL-8) as a model. Recently we found that coordinate activation of NF-kappa B and c-Jun N-terminal protein kinase (JNK) is required for strong IL-8 transcription, whereas the p38 MAP kinase (MAPK) pathway stabilizes the IL-8 mRNA. It is unclear how these pathways are coupled to the receptor for IL-1, an important physiological inducer of IL-8. Expression of the MAP kinase kinase kinase (MAPKKK) TAK1 together with its coactivator TAB1 in HeLa cells activated all three pathways and was sufficient to induce IL-8 formation, NF-kappa B + JNK2-mediated transcription from a minimal IL-8 promoter, and p38 MAPK-mediated stabilization of a reporter mRNA containing IL-8-derived regulatory mRNA sequences. Expression of a kinase-inactive mutant of TAK1 largely blocked IL-1-induced transcription and mRNA stabilization, as well as formation of endogenous IL-8. Truncated TAB1, lacking the TAK1 binding domain, or a TAK1-derived peptide containing a TAK1 autoinhibitory domain were also efficient in inhibition. These data indicate that the previously described three-pathway model of IL-8 induction is operative in response to a physiological stimulus, IL-1, and that the MAPKKK TAK1 couples the IL-1 receptor to both transcriptional and RNA-targeted mechanisms mediated by the three pathways.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The proinflammatory cytokine IL-11 mediates its effects primarily by reprogramming gene expression in inflamed tissues. It is therefore likely that clarifying the molecular mechanisms of IL-1-induced gene expression will yield novel molecular targets for anti-inflammatory therapy (1). Among the many genes induced by IL-1 are those of chemokines, such as IL-8 (2). Blockade of IL-1 receptors by application of the IL-1 receptor antagonist consistently reduces tissue-neutrophilia in a variety of disease models presumably by preventing IL-1-induced synthesis of IL-8 and related chemokines (reviewed in Ref. 1), thus illustrating the pathophysiological importance of IL-1-induced expression of these genes.

IL-1 receptors have been molecularly cloned and are ubiquitously expressed. Binding of IL-1 to the cell surface results in formation of an IL-1 receptor complex consisting of the type I IL-1 receptor, the IL-1 receptor accessory protein, MyD88, TRAF6, and the protein kinase IRAK. TRAF6 couples the receptor complex to the I-kappa B kinase pathway, resulting in the release of I-kappa B from NF-kappa B and nuclear translocation of the latter (3, 4). Furthermore, IL-1 activates the c-Jun N-terminal kinase (JNK) and p38 MAP kinase (MAPK) pathways (5, 6). These MAP kinases are activated by dual-specificity MAP kinase kinases (MAPKKs). JNKs are activated by MKK7, whereas p38 MAPKs are activated by MKK3 and -6. Additionally, MKK4 activates both JNK and p38 MAPK (7, 8).

MAPKKs require phosphorylation in the conserved kinase domain VIII for activation. This is achieved by MAP kinase kinase kinases (MAPKKK). To date, a remarkably large group of distinct enzymes has been shown to fulfill this function, comprising Raf-1, A-Raf, B-Raf, Mos, NIK, MEKK1-4, MLK2, MLK3, DLK/MUK, ASK1, Tpl-2/Cot, and TAK1 (8). The JNK and p38 MAPK signaling pathways can be activated by ectopically expressed MEKK1-4, MLK2, MLK3, DLK/MUK, ASK1, Tpl-2/Cot, and TAK1, albeit to varying degrees (7, 8). Moreover, it is becoming increasingly evident that most of these MAPKKKs also directly activate the I-kappa B kinase/NF-kappa B signaling pathway in parallel, as demonstrated for MEKK1-3 (9-12), TAK1 (13-15), and Tpl-2/Cot (16). On the other hand, MAPKKKs exist that, like NIK, only activate NF-kappa B, but not JNK and p38 MAPK (17-19).

It is unclear which MAPKKKs are utilized by the activated IL-1 receptor complex. It is also unclear if different stimuli use the same MAPKKK for a particular biological response. IL-1 shares many biological responses with the other major proinflammatory cytokine TNF (1). Inactive mutants of NIK, MEKK1, ASK, and TAK1 suppressed TNF-dependent NF-kappa B, JNK, and p38 MAPK activation, suggesting a role for these MAPKKKs in biological responses to TNF (9, 14, 19-24). However, for IL-1, similar information is limited to NIK and TAK1 mutants (18, 13). An important question, therefore, that is yet to be answered is which MAPKKKs are physiologically involved in IL-1-induced gene expression. Changes in enzymatic activity of endogenous MAPKKKs in immunoprecipitation experiments have proven difficult to detect (25). Recently, however, endogenous TAK1 was shown to be activated by IL-1 as well as by TNF (13, 26). In that context it is interesting to note that TAK1 was originally found to function in signaling of transforming growth factor-beta (15, 27-30), a cytokine which has been described to have effects distinct from and in part opposing those of IL-1 and TNF (31, 32).

TAK1 binds to novel TAK binding proteins (TAB) 1 and 2. Its interaction with TAB1 is crucial for activation (33). Recently, it was shown that TAK1 and TAB1 associate with the adapter molecule TRAF6 of the IL-1 receptor complex in an IL-1-dependent fashion (13).

IL-8 induction represents an important (patho)physiological response to IL-1. IL-8 gene regulation is under complex transcriptional and post-transcriptional control, involving AP-1, CAAT/enhancer-binding protein, and NF-kappa B binding sites and AU-rich mRNA regions as cis-elements, respectively (17, 34). We have recently shown by expressing active forms of NIK, MKK7, and MKK6 that IL-8 transcription is controlled by the coordinated action of NF-kappa B and JNK pathways, whereas IL-8 mRNA degradation is regulated by the p38 MAPK pathway (17, 34).

Based on the above evidence for a role of TAK1 in IL-1 signaling we set up experiments to investigate the role of TAK1 in IL-1-induced IL-8 gene expression.


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Cells and Materials-- HeLa cells stably expressing the tet transactivator protein (35, kindly provided by H. Bujard) were cultured in Dulbecco's modified Eagle's medium complemented with 10% fetal calf serum. [gamma -32P]ATP was purchased from Hartmann Analytics. Rabbit antiserum SAK9 to the C terminus of JNK2 (36) was a kind gift of Jeremy Saklatvala. Polyclonal rabbit antibodies against TAK1 have been described (13) or were obtained from Santa Cruz (SC-M-579). Antibodies 12CA5 against hemagglutinin (HA) and 9E10 against C-MYC epitopes were from Roche Molecular Biochemicals. Horseradish peroxidase-coupled secondary antibodies against mouse, rabbit, and rat IgG were from Sigma. Protein A-, protein G-, and glutathione-Sepharose were from Amersham Pharmacia Biotech. Human recombinant IL-1-alpha was produced as described previously (36). Recombinant bacterially expressed HSP27 was a kind gift of M. Gaestel.

Plasmids-- The expression plasmid for GST-JUN (amino acids 1-135) was kindly provided by J. R. Woodgett. GST fusion proteins were expressed and purified from Escherichia coli by standard methods. pCDNA3MYC-MK2 was a kind gift of M. Gaestel. pCMV-TAB11-418 encoding C-terminally truncated TAB1, pEF-TAB1, pcDLRalpha HA-TAK1, and pcDLRalpha HA-TAK1K63W have been described elsewhere (13, 26, 33). pcDLRalpha HA-TAK11-77 was constructed by excising a SacI/SacI fragment encoding the C-terminal 520 amino acids of TAK1 from the pcDLRalpha HA-TAK1 plasmid. To generate expression vectors for GFP-TAK1 fusion proteins, the cDNAs (nucleotides 159-1896) of TAK1 or TAK1K63W were amplified by polymerase chain reaction using the oligonucleotides 5'-cgcggaattcgtcgacagcctccgccg-3' (sense) and 5'-cgcggaattctcatgaagtgccttgtcgtttc-3' (antisense) and subcloned into the EcoRI site of peGFPC1 (CLONTECH). peVHA-JNK2, peVHA-JNK2K55R, and pCS3FLAG-p38AGF have been described previously (17, 34). The NF-kappa B reporter plasmid pNF-kappa B-luc was obtained from Stratagene. pSV-beta -gal coding for SV40 promoter driven beta -galactosidase was from Promega. The IL-8 promoter-luciferase reporter plasmid pUHC13-3-IL-8pr (nucleotides 1348-1527 of the IL-8 gene) and the corresponding mutants in the NF-kappa B and AP-1 cis elements have been described in detail (17). The beta -globin-IL-8 hybrid mRNA, containing a fragment of the 3'-untranslated region of the IL-8 mRNA (nucleotides 972-1310), which includes the AU-rich regulatory region, was expressed under the control of the tetracycline-regulated promoter, using the ptetBBB-IL-8972-1310 plasmid (34).

Transfections and Reporter Assays for Transcription and mRNA Stability-- Transient transfections by the calcium phosphate method and determination of luciferase reporter gene activity were performed as described (17). Equal amounts of plasmid DNA within each experiment were obtained by adding empty vector. For determination of promoter activity cells (seeded at 5 × 105 per well of 6-well plates) were transfected with 0.25 µg of the IL-8-promoter or the 5xNF-kappa B luciferase reporter plasmid and 0.5 µg of pSV-beta -gal (Promega). beta -Galactosidase activity was determined (using reagents from CLONTECH) to allow normalization of luciferase activity in different transfections. RNA degradation kinetics were measured using the tet-off system as described (34). Briefly, cells (5 × 106 seeded per 9-cm-diameter dish) were transfected with the ptetBBB-IL-8972-1310 plasmid (3 µg). For each kinetics, cells from one transfection were distributed into parallel cultures. The next day transcription of the beta -globin-IL-8 DNA was stopped by addition of the tetracycline analog doxycycline (3 µg/ml). At the indicated times thereafter, total RNA was isolated, and Northern blot analysis was performed using a beta -globin antisense RNA probe labeled with digoxygenin (Roche Molecular Biochemicals). The RNA half-life was quantified as in a previous study (34) using a video-imaging system and the Molecular Analyst program (Bio-Rad).

For measurements of IL-8 secretion 2 × 105 cells per well were seeded in 6-well plates and transfected with 4 µl of LipofectAMINE (Life Technologies) and 2 µg of DNA according to the manufacturer's instructions. 24 h later the medium was changed and cells were stimulated with IL-1alpha (10 ng/ml) or left untreated. After 17 h, culture media were collected for IL-8 ELISA. Cell pellets were lysed, and transfected proteins were analyzed by Western blotting.

Preparation of Cell Extracts-- Cells were harvested and washed in ice-cold phosphate-buffered saline. For preparation of whole-cell extracts, cells were lysed in 10 mM Tris, pH 7.05, 30 mM NaPPi, 50 mM NaCl, 1% Triton X-100, 2 mM Na3VO4, 50 mM NaF, 20 mM beta -glycerophosphate, and freshly added 0.5 mM PMSF, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 mM para-nitrophenyl phosphate, 400 nM okadaic acid, 2 mM DTT (whole cell lysis buffer) at 4 °C. After 10 min on ice, lysates were cleared by centrifugation at 10,000 × g for 15 min at 4 °C. Nuclear and cytosolic extracts were prepared as described previously (17). Briefly, cells were suspended and pelleted in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.3 mM Na3VO4, 20 mM beta -glycerophosphate and freshly added 2.5 µg/ml leupeptin, 10 µM E-64, 300 µM PMSF, 1.0 µg/ml pepstatin, 5 mM DTT, 400 nM okadaic acid). The pellet was resuspended in buffer A + 0.1% Nonidet P-40, left on ice for 10 min, and then vortexed. After centrifugation at 10,000 × g for 5 min at 4 °C, supernatants were taken as cytosolic extracts. Pellets were resuspended in buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.3 mM Na3VO4, 20 mM beta -glycerophosphate, 2.5 µg/ml leupeptin, 10 µM E-64, 300 µM PMSF, 1.0 µg/ml pepstatin, 5 mM DTT, 400 nM okadaic acid). After 1 h on ice, samples were vortexed and cleared at 10,000 × g for 5 min at 4 °C. Supernatants were collected as nuclear extracts. Protein concentration of extracts was determined by the method of Bradford and samples stored at -80 °C.

Immune Complex Protein Kinase Assays-- Whole-cell extract (100-250 µg of protein) was diluted in 500 µl of ice-cold immunoprecipitation (IP) buffer A (20 mM Tris, pH 7.4, 154 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 1% Triton X-100). Samples were incubated for 3 h with 2 µl of antiserum SAK9 to immunoprecipitate endogenous JNK, 1 µg of 9E10 (anti-MYC) or 1 µg of 12CA5 (anti-HA) antibodies to immunoprecipitate epitope-tagged MYC-MK2 or HA-JNK2, respectively, followed by the addition of 20 µl of protein A-Sepharose beads (SAK9) or protein G-Sepharose beads (9E10, 12CA5) for 1-2 h at 4 °C. Beads were spun down, washed 3× in 1 ml of IP buffer A, and resuspended in 10 µl of the same buffer. Then 1 µg of recombinant protein substrates (GST-JUN or HSP27, respectively) in 10 µl of H2O and 10 µl of kinase buffer (150 mM Tris, pH 7.4, 30 mM MgCl2, 60 µM ATP, 4 µCi of [gamma -32P]ATP) were added. After 30 min at room temperature, SDS-PAGE sample buffer was added and proteins were eluted from the beads by boiling for 5 min. After centrifugation at 10,000 × g for 5 min, supernatants were separated on 10% SDS-PAGE. Phosphorylated proteins were visualized by autoradiography.

Western Blotting-- Cell extract proteins were separated on 10% SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon, Millipore). After blocking with 5% dried milk in Tris-buffered saline overnight, membranes were incubated for 4-24 h with primary antibodies, washed in Tris-buffered saline, and incubated for 2-4 h with the peroxidase-coupled secondary antibody. Proteins were detected by using the Amersham Pharmacia Biotech enhanced chemiluminescence system.

Electrophoretic Mobility Shift Assay-- Double-stranded oligonucleotides corresponding to the IL-8 promoter sequence 5'-ccatgggtggaatttcctctgacatg-3' (NF-kappa B site underlined) were end-labeled using [gamma -32P]ATP and T4 polynucleotide kinase and purified by gel filtration on S-200 spin columns (Amersham Pharmacia Biotech). Protein-DNA binding reactions were performed with 5-20 µg of nuclear extract protein, labeled oligonucleotide (1 × 105 cpm, 0.2-0.5 pmol), 1 µg of poly(dI-dC) in 10 mM Tris, pH 7.5, 10 mM EDTA, 0.05% (w/v) dried low-fat milk, 50 mM NaCl, 10 mM DTT, and 10% glycerol in a total volume of 10 µl. After incubation at room temperature for 30 min, protein-DNA complexes were resolved on 5% PAGE and visualized by autoradiography.

ELISA-- IL-8 protein concentrations in the cell culture medium were determined using the human IL-8 duo set kit (R&D Systems) exactly following the manufacturer's instructions.


    RESULTS
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ABSTRACT
INTRODUCTION
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DISCUSSION
REFERENCES

The NF-kappa B, JNK, and p38 MAPK Pathways Are Activated by Overexpressed TAK1 + TAB1-- IL-1 is a strong inducer of IL-8 synthesis. We previously suggested that the NF-kappa B and JNK pathways cooperate to activate transcription, whereas the p38 MAPK pathway stabilizes IL-8 mRNA (17, 34). How these downstream effects are coupled to the IL-1 receptor complex remained unclear. IL-1 has been shown to activate the MAPKKK TAK1 (13). Because it has been reported previously that wild-type TAK1 in a TAB1-dependent manner activates NF-kappa B, JNK, and also p38 MAPK in a variety of cell systems (13-15, 26, 30, 37-39), TAK1 is a candidate for a common upstream activator of signaling leading to transcriptional as well as post-transcriptional mechanisms involved in IL-8 induction.

Initially we confirmed that TAK1 activates all three signal transduction pathways within the same cell (Fig. 1). Simultaneous overexpression of TAK1 and its coactivator TAB1 in HeLa cells activated endogenous NF-kappa B, determined in EMSA with a radiolabeled probe corresponding to the NF-kappa B binding site of the IL-8 promoter sequence (17), JNK as determined by in vitro phosphorylation of its substrate GST-JUN, and p38 MAPK as determined by in vitro phosphorylation of HSP27 as substrate for the p38 MAPK-activated kinase MK2 (Fig. 1). Overexpression of a kinase-inactive mutant of TAK1, TAK1K63W (13), with TAB1 (Fig. 1) as well as overexpression of TAK1 without TAB1 (not shown) had no effect.



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Fig. 1.   Coexpression of TAK1 and its activator TAB1 activates endogenous NF-kappa B, JNK, and p38 MAPK signaling pathways. A and B, cells were transfected with empty vector or with pEF-TAB1 together with pcDLRalpha HA-TAK1 or pcDLRalpha HA-TAK1K63W (2.5 µg each). 24 h later cytosolic, and nuclear extracts were prepared. A, TAK1-dependent nuclear translocation and DNA binding of NF-kappa B to the IL-8 promoter was measured by EMSA using 20 µg of nuclear extracts. B, endogenous JNK was immunoprecipitated from cytosolic extracts with rabbit polyclonal anti-JNK antibodies, and its activity was measured in vitro using GST-JUN and [gamma -32P]ATP as substrates. C, cells were transfected as in A and 2.5 µg of pCDNA3MYC-MK2 in addition as indicated. Total amount of DNA was kept constant in all transfections by adding empty vector. 24 h later, cells were lysed in whole cell lysis buffer, and TAK1-dependent activation of the p38 MAPK pathway was measured by immune complex protein kinase assay of transfected epitope-tagged MK2 using HSP27 and [gamma -32P]ATP as substrates.

Overexpression of TAK1 + TAB1 Is Sufficient to Activate Transcription from a Minimal IL-8 Promoter in a JNK-dependent Manner-- The effect of TAK1 activation on IL-8 gene transcription was determined using a reporter construct in which a minimal IL-8 promoter encompassing the AP-1, NF-IL-6, and NF-kappa B binding sites was placed upstream of the luciferase cDNA (17). Expression of TAK1 was controlled by Western blot using anti-TAK1 antibodies (Fig. 2A). Cotransfection of cDNAs for TAK1 and TAB1 resulted in strong activation of luciferase activity (Fig. 2B). Consistent with earlier reports on the mode of action of TAK1, transfection of TAK1 or TAB1 alone had no significant effect. Transcriptional activation of the IL-8 promoter has been suggested by us to involve signaling through both NF-kappa B and JNK (17). To evaluate the contribution of the JNK pathway in TAB1-TAK1-induced transcription, a kinase-inactive mutant of JNK2 in which the ATP-binding lysine was changed to arginine, JNK2K55R, was cotransfected. This resulted in a strong and dose-dependent impairment of TAB1-TAK1-induced transcription (Fig. 2, C and D). On the other hand, a nonactivatable mutant of p38 MAPK in which the phosphorylatable threonine and tyrosine residues were changed to alanine and phenylalanine, p38AGF, had no significant effect (Fig. 2D). Thus activation of the JNK pathway appears crucial for TAB1-TAK1-induced transcriptional activation of the minimal IL-8 promoter.



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Fig. 2.   TAK1 + TAB1-induced IL-8 transcription requires the JNK but not the p38 MAPK pathway. HeLa cells were cotransfected with a minimal IL-8 promoter-luciferase cDNA construct (0.25 µg) (17), with 0.5 µg of pSV-beta -gal and with empty vector or (A, B) expression vectors for TAB1 and wild type or mutant TAK1 as described in the legend of Fig. 1, or (C, D) expression vectors for TAK1 + TAB1 and dominant negative JNK2 (JNK2K55R) or dominant negative p38 MAPK (p38AGF) (2.5 µg or indicated amounts of DNA). A, TAK1 overexpression was confirmed by Western blotting using polyclonal anti-TAK1 antibodies. B-D, luciferase activity was determined in cell lysates. Results normalized for transfection efficiency with beta -gal (for details see "Experimental Procedures") are expressed in relative light units (RLU), mean ± S.E. for triplicate determinations of one representative of two experiments for C, mean ± S.E. from three independent experiments performed in triplicates for B and D.

To compare the requirements for transcriptional activation by TAK1 + TAB1 and IL-1, the promoter was mutated at its NF-kappa B site or its AP-1 site or at both sites. Either mutation strongly impaired induction by TAB1-TAK1 as well as by IL-1, and mutation of both sites abolished inducibility by both stimuli (Fig. 3, A and B). These results show a similarity in the effect of TAB1-TAK1 and IL-1 on transcription and thus are in line with (but do not prove) involvement of the former in IL-1-induced transcription.



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Fig. 3.   Activation of the minimal IL-8 promoter by TAK1 + TAB1 or by IL-1 involves the AP-1 and NF-kappa B binding sites. HeLa cells were transfected with reporter plasmids containing the minimal IL-8 promoter (wt) (see legend to Fig. 2) or derivatives thereof with mutations in the AP-1 site (AP-1), NF-kappa B site (NFkappa B), or both sites (AP-1/NFkappa B) and cultured for 2 days. A, cells were cotransfected with empty vector (open bars) or TAK1 and TAB1 expression vectors (filled bars). B, cells were kept without (open bars) or with IL-1alpha (10 ng/ml, filled bars) for the final 5 h of the cultivation period. Luciferase activity was determined as for Fig. 2 (mean RLU ± S.E. from three independent experiments performed in triplicates).

Overexpression of TAK1 + TAB1 Is Sufficient to Induce Stabilization of a beta -Globin-IL-8 Reporter mRNA via the p38 MAPK Pathway-- Triggering of the p38 MAPK pathway can induce stabilization of IL-8 mRNA as measured with a tetracycline-controlled expression system, an effect that requires regulatory sequences in the 3'-untranslated region of the IL-8 transcript and occurs via MK2 (34). The degradation of a beta -globin mRNA, which carries the regulatory region of the IL-8 mRNA in its 3'-untranslated region, was monitored by sequential Northern blot analyses following stop of its transcription by adding the tetracycline analog doxycycline. The mRNA shows rapid basal degradation and IL-1-induced, p38 MAPK-dependent stabilization (34). As shown in Fig. 4 coexpression of TAK1 + TAB1 also induced marked stabilization of the mRNA. That stabilization was largely reversed by a dominant negative mutant of p38 MAPK, whereas the kinase-negative JNKK55R mutant, which strongly suppressed transcription (see Fig. 3), did not interfere with mRNA stabilization (Fig. 4). This indicates that activation of TAK1, just as exposure to IL-1 (17, 34), can induce mRNA stabilization via the p38 MAPK pathway.



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Fig. 4.   Ectopic expression of TAK1 + TAB1 induces stabilization of a beta -globin-IL-8 hybrid mRNA through the p38 MAPK pathway. HeLa cells constitutively expressing the tet transactivator protein were transfected with ptetBBB-IL-8972-1310 encoding a beta -globin mRNA with the regulatory region of the IL-8 mRNA inserted (BBB-IL-8) and cotransfected with empty vector and expression vectors for TAK1 + TAB1, inactive JNK2 (JNK2K55R) and inactive p38 MAPK (p38AGF) as indicated. The following day doxycycline (3 µg/ml) was added, and total RNA was isolated at the indicated times thereafter. Northern blot analysis was performed with a beta -globin antisense RNA probe. Ethidium bromide staining of 28 S rRNA (EtBR) is shown to allow comparison of RNA amounts loaded. mRNA half-life was determined as described under "Experimental Procedures."

Mutants of TAK1 and TAB1 Interfere with IL-1-induced Transcription and mRNA Stabilization-- Taken together, the data so far indicate that TAB1-TAK1 activates mechanisms of transcription and mRNA stabilization in a way similar to IL-1. The involvement of TAK1 in IL-1-induced activation of both modes of gene regulation was directly addressed using the kinase-inactive mutant TAK1K63W. Its coexpression largely abrogated the activation of NF-kappa B, JNK, and p38 MAPK in response to IL-1 (Fig. 5) (increased MK2 expression with increasing doses of TAK1 expression plasmid was not reproduced in other experiments). Furthermore, expression of TAK1K63W dose-dependently inhibited IL-1-induced activation of the minimal IL-8 promoter, with complete inhibition at 5 µg of transfected DNA (Fig. 6A). Likewise, IL-1-induced stabilization of the beta -globin-IL8 hybrid mRNA was impaired by the kinase-inactive mutant of TAK1 (Fig. 6B). These results provide strong evidence for a crucial role of TAB1-TAK1 in both IL-1-induced transcription and mRNA stabilization.



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Fig. 5.   A kinase-inactive mutant of TAK1, TAK1K63W, inhibits IL-1-induced activation of NF-kappa B, JNK, and p38 MAPK. A, cells were transfected with an NF-kappa B-luciferase reporter plasmid (0.25 µg), pSV-beta -gal (0.5 µg), and pcDLRalpha HA-TAK1K63W or empty vector (5 µg each). 43 h after transfection, cells were stimulated for 5 h with 10 ng/ml IL-1alpha or left untreated. Then cells were lysed and analyzed for luciferase activity. Shown are the mean RLU ± S.E. from two independent experiments performed in triplicates. B and C, cells were transfected with empty vector, 0.3-6 µg of pcDLRalpha HA-TAK1K63W and 2.5 µg of peVHA-JNK2 (B) or pCDNA3MYC-MK2 (C). 24 h later, cells were stimulated with 10 ng/ml IL-1alpha for 30 min or left untreated. Then cells were lysed in whole cell lysis buffer and ectopically expressed HA-JNK2 or MYC-MK2 immunoprecipitated by anti-epitope tag antibodies. Kinase activity of JNK (B) and MK2 (C) was measured in vitro with [gamma -32P]ATP and GST-JUN or HSP27, respectively, as substrates. 100 µg of extract proteins were separated on 10% SDS-PAGE and analyzed for expression of TAK1K63W, HA-JNK2, or MYC-MK2 with polyclonal rabbit anti-TAK1, anti-HA, or anti-MYC antibodies, respectively.



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Fig. 6.   IL-1-induced transcription and mRNA stabilization is blocked by the inactive TAK1K63W mutant. A, cells transfected with the minimal IL-8 promoter-luciferase plasmid, 0.5 µg of pSV-beta -gal, and the indicated amounts of pcDLRalpha HA-TAK1K63W were incubated for 48 h without (open bars) or with (filled bars) 10 ng/ml IL-1alpha added for the last 5 h. Then cells were lysed and luciferase activity was determined (mean RLU ± S.E. from three independent experiments performed in triplicates). B, cells transfected with ptetBBB-IL-8972-1310 and pcDLRalpha HA-TAK1K63W where indicated received doxycycline (3 µg/ml) alone or together with IL-1alpha (10 ng/ml). At the indicated times thereafter beta -globin-IL-8 hybrid mRNA (BBB-IL-8) was detected by Northern blotting as for Fig. 4 (EtBr, ethidium bromide staining of 28 S rRNA). mRNA half-life was determined as described under "Experimental Procedures."

In theory, TAK1K63W could exert its inhibitory function by binding to and masking downstream targets, thereby preventing their phosphorylation and activation by other more relevant MAPKKKs. Therefore, it was necessary to construct mutants that would act more selectively. Two such mutants were applied based on the following: first, TAK1 requires the interaction with TAB1 to function. TAB11-418, a C-terminally truncated form of TAB1, which lacks the TAK1 binding domain, acts as a dominant negative inhibitor in transforming growth factor-beta signaling (33). Second, the TAK1 N-terminal 22 amino acids have an inhibitory function, TAK1 lacking this region is constitutively active without requiring TAB1 (27, 29, 33). TAK11-77, an N-terminal peptide containing this inhibitory region should block TAK1 activity by masking a putative TAK1 activation domain, which interacts with the TAB1 C terminus (29, 33).

We initially confirmed that the TAK11-77 peptide and the TAB11-418 deletion mutant alone were functionally inactive in HeLa cells. Replacement of TAK1 by TAK11-77 or replacement of TAB1 by TAB11-418 resulted in loss of promoter activation by TAB1-TAK1 (Fig. 7A). Importantly, as shown in Fig. 7B, expressing either of the two mutants strongly and dose-dependently impaired promoter activation in response to IL-1. The extent of inhibition was comparable with that achieved with the full-length TAK1K63W mutant in parallel cell cultures (Fig. 7B). In contrast, wild type TAK1 or wild type TAB1 did not inhibit IL-1-induced IL-8 transcription, indicating a specific dominant negative effect of TAK11-77 and TAB11-418 (Fig. 7C). Additional experiments showed that TAK11-77 did not affect activation of the IL-8 promoter by constitutively active MEK1, a specific activator of the ERK pathway, thus ruling out a general suppressive effect of the TAK1 N terminus (data not shown).



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Fig. 7.   Expression of TAB1 lacking the TAK1-binding domain or of the putative autoinhibitory domain of TAK1 disrupts signaling from the IL-1 receptor to the IL-8 promoter. Cells were transfected with the IL-8 promoter-luciferase construct (0.25 µg), pSV-beta -gal (0.5 µg), and empty vector. pcDLRalpha HA-TAK1 (TAK1wt), pcDLRalpha HA-TAK1K63W (TAK1K63W), pEF-TAB1 (TAB1wt), pCMV-TAB11-418 (TAB11-418), or pcDLRalpha HA-TAK11-77 (TAK11-77) were cotransfected at 2.5 µg in the indicated combinations in A, 0.5, 2.5 and 5 µg in B and 5 µg in C. Cells were cultured for 48 h without further additions (A) or with IL-1alpha (10 ng/ml) for the final 5 h as indicated (B, C). Thereafter, cells were lysed and luciferase activity was measured (mean RLU ± S.E. from three (A, C), and five (B) independent experiments performed in triplicates).

Finally we assessed the relevance of TAK1 for inducible formation of IL-8 protein. Coexpression of TAK1 and TAB1, but not of each of them alone, resulted in a strong increase in IL-8 secretion (Fig. 8A), showing that TAB1-activated TAK1 is sufficient to activate the endogenous IL-8 gene. To analyze the effect of kinase-inactive TAK1 on IL-1-induced IL-8 formation, a liposome-based transfection procedure was employed that yielded higher rates of transfected cells. For these experiments the TAK1 cDNAs were fused to that of enhanced green fluorescent protein (GFP). The GFP-tagged wild type TAK1 and TAK1K63W behaved functionally similar to the respective HA-tagged forms in reporter gene assays (data not shown). Reproducibly more than 50% of cells expressed the GFP-TAK1 fusion proteins as estimated by fluorescence-activated cell sorting analysis (data not shown). Expression of GFP-TAK1 and GFP-TAK1K63W was also monitored by Western blot (Fig. 8C). In these cell cultures IL-1 induced a 10-fold increase in IL-8 secretion, which was hardly affected by expressing GFP-TAK1 (Fig. 8B). In contrast, expression of the kinase-inactive GFP-TAK1K63W resulted in marked inhibition of IL-1-induced IL-8 secretion (by 52-67% in three individual experiments). Taking into account the observed transfection efficiency, this result suggests that in cells expressing GFP-TAKK63W, IL-1-induced IL-8 secretion is almost completely inhibited.



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Fig. 8.   Formation of IL-8 protein is affected by TAK1. A, cells were transfected using LipofectAMINE with pcDLRalpha HA-TAK1 and pEF-TAB1 alone or in combination (1 µg each) and pCS3MT (vector), which was also used to adjust total amount of DNA to 2 µg per transfection. B, cells were transfected using LipofectAMINE with peGFPC1, peGFPC1-TAK1, or peGFPC1-TAK1K63W (2 µg each) as indicated. 24 h after transfection, medium was changed and cells were cultivated further in the presence or absence of 10 ng/ml IL-1alpha as indicated. Thereafter, IL-8 secreted into the cell culture supernatant was quantified by specific ELISA (see "Experimental Procedures" section for details). Results represent means ± S.E. from two (A) or three (B) independent experiments. C, equal expression of transfected GFP-TAK1 fusion proteins in the experiments summarized in B was verified by Western blotting of lysates using anti-TAK1 (M-579) antibodies. One Western blot representative for three is shown (end., endogenous).

The data presented in Figs. 5-8 show that IL-1-induced IL-8 gene expression is sensitive to disruption of IL-1 signaling by dominant negative mutants of both TAK1 and TAB1. In conclusion, our data therefore suggest that the MAPKKK TAK1, together with its coactivator TAB1, links the major mechanisms contributing to IL-8 gene expression to the IL-1 receptor.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Studying induction of IL-8 as an example for highly inducible gene expression, we have recently developed a model in which formation of large amounts of IL-8 requires the coordinated activation of at least three signaling pathways. First, activation of NF-kappa B alone was able to induce IL-8 transcription and secretion, but only to very low levels. Second, selective activation of the JNK pathway by expression of active mutants of MKK7 also induced low levels of IL-8 secretion and transcription (17). Activation of the JNK pathway is necessary for triggering IL-8 synthesis by its physiological inducer IL-1, because inhibition of that pathway by dominant negative JNK or its antisense mRNA strongly suppressed IL-1-induced IL-8 mRNA and protein expression (40). The NF-kappa B and JNK pathways markedly synergized on the transcriptional level and required intact AP-1 and NF-kappa B binding sites in the IL-8 promoter (17). Third, selective triggering of the p38 MAPK pathway by MKK6 stabilized IL-8 mRNA, and IL-1 induced mRNA stabilization in a p38 MAPK-dependent manner (17, 34). We now confirm and extend this model by demonstrating that IL-1 induces both transcription and mRNA stabilization through the MAPKKK TAK1 (Fig. 9).



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Fig. 9.   Scheme of signaling pathways cooperating in IL-1-induced IL-8 formation. For details see text.

TAK1, together with its coactivator TAB1, activated in parallel the NF-kappa B, JNK, and p38 MAPK pathways in HeLa cells and induced transcription from a minimal IL-8 promoter. The latter effect was inhibited by dominant negative JNK2 and required intact NF-kappa B and AP-1 sites. Thus TAB1-TAK1 activates transcription in a way suggested by us previously, namely through the coordinate function of both the NF-kappa B and JNK pathways. TAB1-TAK1 overexpression also induced mRNA stabilization. Although this effect was unaffected by dominant negative JNK2, it was largely abrogated by a dominant negative mutant of p38 MAPK.

Although the p38 MAPK pathway is capable of activating transcription by phosphorylation of several transcription factors (8), the dominant negative p38 MAPK mutant was devoid of any effect on TAK1-induced IL-8 transcription. The striking selectivity of effects of TAK1-activated JNK on IL-8 transcription and TAK1-activated p38 MAPK on IL-8 mRNA stabilization may be achieved by binding of the two MAPKs to specific scaffold proteins. Recently, three different scaffold protein families involved in JNK activation have been identified in mammalian cells, i.e. JIP1-3, JSAP1, and JNKBP1 (41-45). In yeast, scaffold proteins have been described also for the p38 MAPK homolog HOG1 (44). It appears that the FUS3 and HOG1 MAPK modules in yeast are activated by the same MAPKKK, Ste11, but are routed to distinct functions by different adapter proteins (8, 44). Our observations may therefore be explained by the formation of specific signaling complexes, which direct the cellular function of JNK and p38 MAPK in the IL-1-TAK1 signaling pathway toward transcriptional activation and mRNA stabilization, respectively.

IL-1 is an important physiological inducer of IL-8 formation. It simultaneously activates the NF-kappa B, JNK, and p38 MAPK signaling pathways. A number of different MAPKKKs have emerged as candidate molecules that serve as upstream activators of the JNK, p38, and NF-kappa B pathways. Very little information exists, however, that makes it possible to distinguish between the following hypothetical situations: 1) in response to a given stimulus several different MAPKKKs are activated and function in a promiscuous manner; 2) different MAPKKKs in a given setting activate distinct pathways selectively, implying a signal bifurcation point upstream of them; or 3) one MAPKKK species is activated and in turn activates the different downstream pathways, thus itself representing the bifurcation point.

Evidence supporting the latter model was obtained by investigating the involvement of TAK1 in IL-1-induced expression of IL-8. First we found that IL-1-induced activation of the NF-kappa B and JNK pathways was blocked by a kinase-inactive mutant of TAK1, thus confirming previous results (13). Furthermore, the mutant also blocked IL-1-induced activation of p38 MAPK. So far, no function has been demonstrated for TAK1-activated NF-kappa B, JNK, or p38 MAPK signaling pathways in IL-1-dependent cellular responses. As shown here, IL-1-induced transcriptional activation was blocked by the kinase-inactive TAK1 mutant, strongly suggesting a sequence of events involving the IL-1 receptor complex, TAK1 and JNK + NF-kappa B activation. The kinase-inactive form of TAK1 also inhibited IL-1-induced, p38 MAPK-dependent mRNA stabilization. Thus for both kinds of effects on gene expression, IL-1 appears to utilize the TAK1 MAPKKK.

Several other MAPKKKs that activate NF-kappa B, JNK, and p38 MAPK may be involved in IL-1 or TNF signaling (9, 18-24). Because the inactive TAK1 mutant could possibly interfere with signaling in a nonspecific way, i.e. by competing with those MAPKKKs through its interaction with common downstream targets, we sought to apply more specific inhibitors. TAK1 activation requires a protein-protein interaction with the TAB1 molecule (33). Available knowledge indicates that, within the group of MAPKKK enzymes, this mode of activation is unique for TAK1. In support of this, TAB1 has not been reported to bind to and activate other MAPKKKs. Mapping of the regions in TAK1 and TAB1 that are required for this interaction showed that a region within the first 303 amino acids of TAK1 binds to the C terminus of TAB1, which contains a region homologous to the serine-rich N terminus of TAK1 (33). Because deletion of the N-terminal 22 amino acids yields an activated form of TAK1 (29), it can be speculated that the N terminus exerts an autoinhibitory interaction with the same region in the TAK1 molecule that also binds TAB1. Binding of the N terminus would render the wild type TAK1 kinase inactive. TAB1 would activate TAK1 by dislodging the N terminus (and possibly exerting a stimulatory effect). The two mutants expected to act in a dominant negative fashion accordingly, the N-terminal TAK1 peptide and the C-terminally truncated TAB1, both effectively inhibited IL-1-induced IL-8 transcription. Therefore the results are indicative of a specific recruitment of TAK1 by the IL-1 receptor complex and suggest that IL-1-induced downstream effects are dependent on TAB1-mediated TAK1 activation.

Taken together the evidence presented in this study assigns a central role for IL-1-induced IL-8 formation in HeLa cells to the TAB1 and TAK1 molecules. Further proof will have to await the results of chemokine expression in mice deficient of TAB1 and TAK1. It will also be important to find out if this model holds true for other human cell types, stimuli, and induced genes. That information will shed light on the suitability of TAK1 and its coactivator TAB1 as targets for interference with gene induction by inflammatory stimuli.


    ACKNOWLEDGEMENTS

We thank Hermann Bujard for providing us with plasmids pUHD10-3 and pUHC13-3 and HeLa cells expressing the tet transactivator protein, James R. Woodgett for his gift of expression plasmid for GST-JUN, Jeremy Saklatvala for providing us with antiserum SAK9, Matthias Gaestel for MK2 plasmids and recombinant HSP27, and Eisuke Nishida for TAK1 and TAB1 expression plasmids.


    FOOTNOTES

* This work was supported by grants (Kr1143/2-3, SFB 244/B15, SFB 244/B18, Ho1116/2-1, III GK-GRK 99/2-98 (P4)) from the Deutsche Forschungsgemeinschaft (to H. H. and M. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-511-532-2800; Fax: 49-511-532-4081; E-mail: Kracht.Michael@MH-Hannover.de.

Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M004376200


    ABBREVIATIONS

The abbreviations used are: IL-1, interleukin-1; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; IP, immunoprecipitation; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; RLU, relative light units; TNF, tumor necrosis factor; TAB, TAK binding protein; GST, glutathione S-transferase; ELISA, enzyme-linked immunosorbent assay; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein.


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