Interleukin 1 Activates STAT3/Nuclear Factor-κB Cross-talk via a Unique TRAF6- and p65-dependent Mechanism
2004; Elsevier BV; Volume: 279; Issue: 3 Linguagem: Inglês
10.1074/jbc.m311498200
ISSN1083-351X
AutoresYasuhiro Yoshida, Arvind Kumar, Yoshinobu Koyama, Haibing Peng, Ahmet Arman, Jason A. Boch, Philip E. Auron,
Tópico(s)interferon and immune responses
ResumoInterleukins (IL) 1 and 6 are important cytokines that function via the activation, respectively, of the transcription factors NF-κB and STAT3. We have observed that a specific type of κB DNA sequence motif supports both NF-κB p65 homodimer binding and cooperativity with non-tyrosine-phosphorylated STAT3. This activity, in contrast to that mediated by κB DNA motifs that do not efficiently bind p65 homodimers, is shown to be uniquely dependent upon signal transduction through the carboxyl terminus of TRAF6. Furthermore, STAT3 and p65 are shown to physically interact, in vivo, and this interaction appears to inhibit the function of "classical" STAT3 GAS-like binding sites. The distinct p50 form of NF-κB is also shown to interact with STAT3. However, in contrast to p65, p50 cooperates with STAT3 bound to GAS sites. These data argue for a novel transcription factor cross-talk mechanism that may help resolve inconsistencies previously reported regarding the mechanism of IL-1 inhibition of IL-6 activity during the acute-phase response. Interleukins (IL) 1 and 6 are important cytokines that function via the activation, respectively, of the transcription factors NF-κB and STAT3. We have observed that a specific type of κB DNA sequence motif supports both NF-κB p65 homodimer binding and cooperativity with non-tyrosine-phosphorylated STAT3. This activity, in contrast to that mediated by κB DNA motifs that do not efficiently bind p65 homodimers, is shown to be uniquely dependent upon signal transduction through the carboxyl terminus of TRAF6. Furthermore, STAT3 and p65 are shown to physically interact, in vivo, and this interaction appears to inhibit the function of "classical" STAT3 GAS-like binding sites. The distinct p50 form of NF-κB is also shown to interact with STAT3. However, in contrast to p65, p50 cooperates with STAT3 bound to GAS sites. These data argue for a novel transcription factor cross-talk mechanism that may help resolve inconsistencies previously reported regarding the mechanism of IL-1 inhibition of IL-6 activity during the acute-phase response. Interleukins (ILs) 1The abbreviations used are: IL(s), interleukin(s); APR, acute phase response; C/EBP, CAAT enhancer-binding protein; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; GAS, γ-interferon activation sequence; HEK, human embryonic kidney; hSIE, high affinity sis-inducible element; ICAM, intercellular adhesion molecule; IκB, inhibitor of κB; IL1RI, IL-1 type I receptor chain; iNOS, inducible nitric-oxide synthase; LPS, lipopolysaccharide; luc, luciferase; MATH, meprin and TRAF homology; MHC, major histocompatibility complex; NF-κB, nuclear factor κB; SOCS, suppressor of cytokine synthesis; STAT, signal transducers and activators of transcription; STAT3aPTyr, aphosphotyrosine STAT3 monomer; TAD, transcription activation domain; TNF, tumor necrosis factor; TRAF, tumor necrosis factor receptor-associated factor. 1 and 6 play critical roles in inflammation and stress. One of the important homeostatic processes is the acute phase response (APR) in which various stress stimuli, such as microbiological products, injury, and neoplasia, result in the activation of endothelial cells, smooth muscle cells, and antigen-presenting cells such as macrophages, which in turn express cytokines such as IL-1, TNF, and IL-6. These cytokines can induce targets, such as endothelial, fibroblast, chondrocyte, osteoclast, and pituitary cells, to express factors important for both cellular and humoral immunity. These factors include IL-4, -5, -12, and -18 as well as secondary expression of additional IL-1, -6, and TNF. This organism-wide phenomenon has recently been referred to as the "danger response" (1.Matzinger P. Science. 2002; 296: 301-305Crossref PubMed Scopus (3427) Google Scholar). The liver is an important component of the APR and has been exhaustively studied (2.Baumann H. Gauldie J. Immunol. Today. 1994; 15: 74-80Abstract Full Text PDF PubMed Scopus (495) Google Scholar). Central to the activation of the liver is the induction of distinct gene collectives that are primarily under the control of three transcription factors, namely STAT3, NF-κB, and C/EBPβ. STAT3 is activated from a cytoplasmic non-tyrosine-phosphorylated monomer to a nuclear localized tyrosine-phosphorylated dimer by an IL-6-dependent signal transduction mechanism. Both C/EBPβ (also called NF-IL6) and NF-κB are activated either by IL-1 or TNF. C/EBPβ is activated by direct mitogen-activated protein kinase serine phosphorylation, which results in a conformational change of the constitutively inactive transcription factor (3.Kowenz-Leutz E. Twamley G. Ansieau S. Leutz A. Genes Dev. 1994; 8: 2781-2791Crossref PubMed Scopus (211) Google Scholar, 4.Williams S.C. Dillner B.M. Johnson P.F. EMBO J. 1995; 14: 3170-3183Crossref PubMed Scopus (200) Google Scholar). NF-κB is activated via either serine or tyrosine phosphorylation of the various forms of the IκB inhibitor that is subsequently released and usually degraded by the proteasome (5.Fan C. Li Q. Ross D. Engelhardt J. J. Biol. Chem. 2003; 278: 2072-2080Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Although IL-6 can also activate C/EBPβ, it cannot activate NF-κB. The IL-6 responses mediated by STAT3 are known to be somewhat independent of those associated with C/EBPβ and especially NF-κB. In fact, evidence has accumulated arguing that many of the IL-6 responses are inhibited by agents that activate NF-κB (6.Conti P. Bartle L. Barbacane R. Reale M. Sipe J. Mol. Cell. Biochem. 1995; 142: 171-178Crossref PubMed Scopus (17) Google Scholar, 7.Zhang Z. Fuller G. Biochem. Biophys. Res. Commun. 1997; 237: 90-94Crossref PubMed Scopus (52) Google Scholar, 8.Zhang Z. Fuller G. Blood. 2000; 96: 3466-3472Crossref PubMed Google Scholar, 9.Bode J. Fischer R. Haussinger D. Graeve L. Heinrich P. Schaper F. J. Immunol. 2001; 167: 1469-1481Crossref PubMed Scopus (50) Google Scholar). Yet, under some conditions the two responses are also cooperative. Although several models have been proposed to explain these apparently inconsistent observations, none is either entirely satisfactory or universally accepted (7.Zhang Z. Fuller G. Biochem. Biophys. Res. Commun. 1997; 237: 90-94Crossref PubMed Scopus (52) Google Scholar, 9.Bode J. Fischer R. Haussinger D. Graeve L. Heinrich P. Schaper F. J. Immunol. 2001; 167: 1469-1481Crossref PubMed Scopus (50) Google Scholar, 10.Ahmed S. Ivashkiv L. J. Immunol. 2000; 165: 5227-5237Crossref PubMed Scopus (121) Google Scholar, 11.Deon D. Ahmed S. Tai K. Scaletta N. Herrero C. Lee I. Krause A. Ivashkiv L. J. Immunol. 2001; 167: 5395-5403Crossref PubMed Scopus (87) Google Scholar). In the process of investigating such mechanisms, we discovered that an asymmetric functional relationship exists between STAT3 and NF-κB. This relationship is mechanistically dependent upon a protein-protein interaction between one form of NF-κB and monomeric aphosphotyrosine STAT3 (STAT3aPTyr). We have observed that NF-κB p65 homodimers can cooperate with STAT3aPTyr, when bound to a specific type of κB motif. Reciprocally, this interaction appears to inhibit function of "classical" STAT3 GAS-like binding sites. In contrast, NF-κB p50 can cooperate with STAT3 bound to STAT3 GAS sites. The specificity of the response is not only associated with the specific DNA binding site, but also the signal transduction pathway, which appears to rely upon the carboxyl-terminal meprin and TRAF homology (MATH) domain of TRAF6 for the selective activation of p65 homodimers. Together these data argue for a novel transcription factor cross-talk mechanism that may explain many of the controversial observations associated with APR and the activation of distinct forms of NF-κB. Cell Lines—Hep3B cells were obtained from the American Type Culture Collection (ATCC). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 4 mm l-glutamine, and 0.5% penicillin-streptomycin in humidified 10% CO2 and 90% air at 37 °C. HEK293R cells were derived from HEK293 cells (ATCC) and then stably transfected with pcDNA3.1 expression vector (Invitrogen) containing the cDNA sequence for the human IL-1 type I receptor chain (IL1RI) introduced by recombinant PCR subcloning. These cells generated a qualitatively similar response to the parental HEK293. However, the presence of IL1R1 amplified the response, generating a higher sensitivity. Selection for stable transfection was accomplished in the presence of 400 μg/ml G418. The resulting cells expressed high levels of IL-1 type I receptor and are referred to in the text as HEK293/IL1RI cells. Oligonucleotides and Antibodies—For all of the oligonucleotide sequences shown, the consensus factor binding sites are underlined. High affinity sis-inducible element (hSIE), which binds both STAT3 and STAT1 (12.Wagner B. Hayes T. Hoban C. Cochran B. EMBO J. 1990; 9: 4477-4484Crossref PubMed Scopus (554) Google Scholar), 5′-AGCTTGTGCATTTCCCGTAAATCTTGTCGTCGA-3′; STAT3-specific 4spC-like sequence (13.Seidel H. Milocco L. Lamb P. Darnell J. Stein R. Rosen J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3041-3045Crossref PubMed Scopus (385) Google Scholar), 5′-ACCTCTTATTCCCGAAAGTACAT-3′; human IL-8 promoter (14.Schulte R. Grassl G. Preger S. Fessele S. Jacobi C. Schaller M. Nelson P. Autenrieth I. FASEB J. 2000; 14: 1471-1484Crossref PubMed Google Scholar) (–88 to–65), 5′-CAAATCGTGGAATTTCCTCTGACA-3′; human ICAM-I promoter (15.Ledebur H. Parks T. J. Biol. Chem. 1995; 270: 933-943Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar) (–194 to–171), 5′-TTTAGCTTGGAAATTCCGGAGCTG-3′; human Bcl-x gene promoter (16.Chen C. Edelstein L. Gelinas C. Mol. Cell. Biol. 2000; 20: 2687-2695Crossref PubMed Scopus (700) Google Scholar) (–238 to–215), 5′-GCGGGGGGGACTGCCCAGGGAGTG-3′; human iNOS gene promoter (17.Taylor B. de Vera M. Ganster R. Wang Q. Shapiro R. Morris S. Billiar T. Geller D. J. Biol. Chem. 1998; 273: 15148-15156Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar) (–5790 to–5813), 5′-CTTGGTTCTGGGAAAGCCCTCTAG-3′; murine KBF1/H2TF1 (MHCκB) (10–11-bp NF-κB), 5′-GAGGCTGGGGATTCCCCATCTC-3′; and the wild-type and mutant GAS/κB sequences, as described in Fig. 4, were synthesized by Qiagen Operon (Valencia, CA). The GAS/κB (9-bp NF-κB) 5′-GATCCTTCTGGGAATTCCTAGATC-3′ used in Fig. 1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies obtained from Santa Cruz Biotechnology were as follows: anti-NH2-terminal STAT3α, anti-internal STAT3α, anti-COOH-terminal STAT3α, anti-p50, anti-p65, anti-STAT1, and anti-STAT3 phosphotyrosine 705. Electrophoretic Mobility Shift Assays (EMSAs)—EMSAs were performed essentially as described previously (18.Koyama Y. Tanaka Y. Saito K. Abe M. Nakatsuka K. Morimoto I. Auron P.E. Eto S. J. Immunol. 1996; 157: 5097-5103PubMed Google Scholar, 19.Tsukada J. Waterman W.R. Koyama Y. Webb A.C. Auron P.E. Mol. Cell. Biol. 1996; 16: 2183-2194Crossref PubMed Scopus (72) Google Scholar). Briefly, 10 μg of whole cell extracts or nuclear extracts was preincubated for 20 min at room temperature in 15 μl of buffer (10 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm β-mercaptoethanol, 4% glycerol, 40 mm NaCl) containing 1 μg of poly(dI-dC), and each oligonucleotide was labeled with T4 polynucleotide kinase (New England Biolabs, Beverly, MA) and [γ-32P]ATP (6,000 Ci/mmol; PerkinElmer Life Sciences). Protein-DNA complexes were resolved on 4% TBE polyacrylamide gels and analyzed with or without specific antibody or nonlabeled hSIE oligonucleotide. DNA Constructs—Luciferase reporter plasmids were constructed containing the sequences used as EMSA probes (see above) inserted upstream (between unique KpnI and NheI sites) of the minimal (–56 to +109) murine c-fos promoter (20.Gilman M.Z. Wilson R.N. Weinberg R.A. Mol. Cell. Biol. 1986; 6: 4305-4316Crossref PubMed Scopus (387) Google Scholar), which had previously been inserted into the pGL3 Basic promoter (Promega, Madison, WI). The GAS/κB reporter used for most of the studies harbored four copies of the 24-bp oligonucleotide containing the 9-bp κB sequence described, above, under "Oligonucleotides and Antibodies." These four sequences are separated by spacers of 12, 32, and 12 bp in order from the upstream-toward the downstream-most element. An additional 59 bp separates the last element from the–56 position of the c-fos promoter. The hSIE GAS reporter contained two copies of the oligonucleotide described, above, spaced 13 bp apart and separated from the c-fos promoter by 47 bp. The 4spC-like GAS reporter contained a single copy of the above described oligonucleotide located 94 bp upstream of the c-fos promoter. The wild-type and mutated GAS/κB reporters described in Fig. 4 each contained two copies of the same sequence located 47 bp upstream of the c-fos promoter. The MHCκB reporter (MHCκB-luc) was from Dr. T. Born (21.Born T.L. Thomassen E. Bird T.A. Sims J.E. J. Biol. Chem. 1998; 273: 29445-29450Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar) and contained four tandem copies of the described oligonucleotide located 3 bp upstream of the c-fos promoter. IL-8 reporter (IL-8-luc) was provided by Dr. T. A. Libermann, which contains the IL-8 promoter region from –1481 to +40 as reported by Zhao et al. (22.Zhao D. Keates A.C. Kuhnt-Moore S. Moyer M.P. Kelly C.P. Pothoulakis C. J. Biol. Chem. 2001; 276: 44464-44471Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). TRAF6 vectors were constructed by inserting PCR-amplified cDNA fragments into the pcDNA3.1 expression vector (Invitrogen). The creation of the COOH-terminal FLAG-tagged, v-Src myristoylation signal-TRAF6 fusion expression vectors used pFLAG-CMV-5a (Sigma) as a parent vector to which was added the v-Src myristoylation sequence (23.Dadgostar H. Cheng G. J. Biol. Chem. 2000; 275: 2539-2544Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) along with HindIII, XhoI, and NotI restriction sites (5′-GAAAGCTTCTCGAGAATACCATGGGTAGCAACAAGAGCAAGCCCAAGGATGCCAGCCAGCGGGGGGCGGCCGCTAAA-3′). This double stranded oligonucleotide was introduced into the HindIII/NotI site of the parent vector. Each TRAF mutant was constructed using PCR and appropriate primers and introduced into the NotI/BglII site of the vector. The cDNA for STAT3 was purchased (Invitrogen) and was inserted into pcDNA3.1. The mutant STAT3 at tyrosine 705 (mSTAT3) was made by PCR-based point mutation methods. The dominant negative IκBα was purchased (Upstate, Waltham, MA). The pCI p50 and p65 plasmids were provided by Dr. P. Oettgen. All plasmids used for transfections were prepared by means of the EndoFree Plasmid Maxi Kit (Qiagen, Valencia, CA). Transfections and Luciferase Assays—Cells were transfected with the transfection reagent FuGENE6 (Roche Applied Science) at 3 μl of reagent/μg of DNA according to the manufacturer's instructions. A total of 0.15–0.5 μg of DNA, including the luciferase reporter construct, expression vectors, and 0.01 μg of pRL-TK-RLuc (Promega), was added to about 90% confluent cells in 24-well tissue culture plates. The total amount of transfected DNA was adjusted to be constant in each experiment. At 24 h after transfection, cells were stimulated with or without 2 ng/ml of IL-1β (Cistron Biotechnology, Pine Brook, NJ) or 50 ng/ml IL-6 (Upstate, Waltham, MA). After an additional 3-h incubation, cells were lysed with Passive Lysis Buffer (Promega). The cytosolic fractions were used for a dual luciferase reporter assay system (Promega). Transfection efficiency was monitored by use of the internal control. Immunoblots—For preparation of whole cell lysis, cells were lysed in radioimmune precipitation assay buffer with 10 mg/ml phenylmethylsulfonyl fluoride, aprotinin, proteinase inhibitors (Complete™, EDTA-free; Roche Applied Science). Nuclear extracts were prepared using a kit (Sigma) according to the manufacturer's instructions. The protein amount for each sample was measured using a Bradford assay (Bio-Rad) and adjusted to be equal in all samples. Immunoprecipitation and Western blot analysis with enhanced chemiluminescent detection (SuperSignal WestFemto System, Pierce) were carried out by standard methods (24.Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, New York1994Google Scholar). SDS-PAGE was run with 40% acrylamide using a 29:1 ratio of acrylamide to bisacrylamide. Molecular weight standards (Bio-Rad) were used for determining molecular mass. Chromatin Immunoprecipitation (ChIP)—Hep3B cells were grown in 100 × 20-mm plates to 75% confluence and were treated with 10 ng/ml IL-1β and 50 ng/ml IL-6. After 15 min, ChIP was performed using a kit from Upstate USA. In brief, proteins of both treated and untreated cells were cross-linked to DNA by adding 37% formaldehyde to a final concentration of 1% for 15 min. Cells were washed with ice-cold phosphate-buffered saline containing protease inhibitor mixture (Sigma), scraped, and washed three times. Cells were lysed with 300 μl of SDS-lysis buffer containing protease inhibitor, and DNA was sonicated (10 W on a Fisher model 100 Sonic Dismembrator for 18 × 5 s to generate 200–1,000-bp lengths), and samples were centrifuged at 12,000 rpm × 8 min. Sonicated cell supernatant was diluted 10-fold with ChIP dilution buffer (Upstate USA). Immunoclearing was accomplished by incubating with sheared salmon sperm DNA and protein A/G-Sepharose. Samples were rotated at 4 °C for 30 min and incubated with specific antibodies overnight. Sepharose beads were harvested by centrifugation (3,000 rpm × 3 min) and washed with ChIP wash buffer (Upstate). Beads were incubated with proteinase K for 45 min. DNA was eluted from the beads with elution buffer (1% SDS, 0.1 m NaHCO3), and then DNA cross-links were reversed by incubating the sample 4 h to overnight at 65 °C in 0.2 m NaCl. Final purification of DNA used a PCR minielute kit (Qiagen) followed by PCR amplification using specific primers and Fast Start Taq polymerase (Roche Applied Science). The PCR amplification conditions were 4 min at 94 °C followed by 27 cycles of 30 s at 94 °C, 30 s at 54 °C, and 45 s at 72 °C. Synthesis was terminated by incubation for 15 min at 72 °C. The human IL-8 gene primers were TGCCATTAAAAGAAAATCATCCA (–359/–337 sense strand) and CCTTATGGAGTGCTCCGGT (+10/–9 antisense strand). IL-1 Induces a STAT3·p65 Complex to Bind DNA—It is known that IL-1β induces the transcription of many genes via members of the REL(NF-κB) family of transcription factors. The REL factors, such as p50, p52, p65, c-Rel, RelA, and RelB, function as dimers and have been shown to be both differentially activated (25.Thompson J. Phillips R. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1995; 80: 573-582Abstract Full Text PDF PubMed Scopus (701) Google Scholar, 26.Han Z. Ip Y. J. Biol. Chem. 1999; 274: 21355-21361Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 27.Yamazaki S. Muta T. Takeshige K. J. Biol. Chem. 2001; 276: 27657-27662Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar) and to possess distinct target DNA binding site specificities (28.Chen Y. Ghosh S. Ghosh G. Nat. Struct. Biol. 1998; 5: 67-73Crossref PubMed Scopus (205) Google Scholar, 29.Kunsch C. Rosen C. Mol. Cell. Biol. 1993; 13: 137-146Crossref Google Scholar) that depend upon dimer composition. The p50 and p52 proteins do not possess a transactivation domain (TAD) and can either inhibit as homodimers or potentiate the activity of other REL factors upon heterodimerization. Fig. 1 demonstrates that Hep3B cells treated for 15 min with IL-1β contain inducible DNA-binding complexes, the nature of which depends upon the sequence of the DNA probe (Fig. 1B and 1C, lanes 1 and 2). One probe contains a well characterized 10–11-bp-long KBF1/H2TF1 κB site (MHCκB) from the murine major histocompatibility complex class I locus enhancer that is known to be a good binding site for p50-containing REL family dimers (30.Scheinman R. Beg A. Baldwin A. Mol. Cell. Biol. 1993; 13: 6089-6101Crossref PubMed Google Scholar). The other has a GAS-like STAT factor binding site that overlaps a κB motif and is closely related to a sequence derived from the rat α2-macroglobulin gene (Fig. 1A). The 9-bp-long κB sequence in this probe is similar to that which was shown to be capable of binding NF-κB p65 homodimers strongly (28.Chen Y. Ghosh S. Ghosh G. Nat. Struct. Biol. 1998; 5: 67-73Crossref PubMed Scopus (205) Google Scholar). A fast migrating complex (complex I) was associated with both probes and reacted completely with either anti-p50 or p65 antibody and is likely a p50/p65 heterodimer (Fig. 1B, lanes 7 and 8, and 1C, lanes 3 and 4). The GAS/κB probe, but not the MHCκB probe, also generated a more slowly migrating complex (complex II) that was, surprisingly, recognized by antibodies specific for p65 and two STAT3 epitopes (the COOH-terminal transcription activation domain and the internal SH2 domain), but not by antibodies specific for p50, STAT1, and the NH2-terminal protein-protein interaction (31.Zhang X. Darnell J. J. Biol. Chem. 2001; 276: 33576-33581Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) domain of STAT3 (Fig. 1B, lanes 3–8). Unexpectedly, Complex II did not bind to an hSIE GAS oligonucleotide known to be a consensus sequence for STAT3 homo- and heterodimers (Fig. 1B, lane 9). As expected, EMSA analysis using the hSIE GAS probe revealed that, in contrast to IL-6, IL-1 could not induce STAT3 binding (data not shown). Interestingly, complex II could only be detected to bind MHCκB probe after very long exposure to film (not shown), arguing that the NF-κB site in the MHCκB probe binds only p50/p65 heterodimers strongly. It is unclear why there is an absence of an effect for N-STAT3 antibody (Fig. 1B, lane 5). All the antibodies were tested independently on EMSA using extracts from IL-6-treated Hep3B cells and a labeled hSIE probe (not shown). The EMSA revealed the expected three complexes corresponding to STAT3 and STAT1 homo- and heterodimers. All the antibodies reacted with the appropriate complexes, except for N-STAT3, which reacted with the STAT3 homodimer complex, but not with that corresponding to the STAT1/STAT3 heterodimer. It is not known whether this is because the antibody recognizes the likely complex that occurs between STAT amino termini or whether there is a distinct conformational change for the heterodimer. Because complex II appeared to be completely dependent upon both p65 and STAT3, we looked for a protein-protein interaction between these two proteins. Following IL-1 stimulation, cell lysates were prepared and immunoprecipitated with either anti-p65 or anti-STAT3 antibodies. Fig. 1D shows that STAT3 is coimmunoprecipitated with p65, suggesting that p65 interacts with STAT3 in vivo. The above results show that p65 can make a complex with STAT3 both in vitro, in the presence, and in vivo, in the absence of DNA. This complex binds to an overlapping GAS/κB motif, but not to the MHCκB sequence, which does not reveal any obvious GAS-like sites. STAT3 and p65 Cooperate Functionally on a Specific DNA Binding Site—To characterize functionally the in vitro DNA binding, we prepared luciferase reporter constructs containing multiple copies of various binding sites positioned immediately upstream of the murine c-fos core promoter. These included reporters containing the GAS/κB and MHCκB sequences, as well as consensus GAS sequences such as the hSIE and 4spC, which are devoid of any obvious κB-like motifs. These constructs were transfected into Hep3B cells, and luciferase activity was assayed in response to stimulation with either IL-1 or IL-6 (Fig. 2A). Treatment of Hep3B cells with IL-1 resulted in strong activity for the GAS/κB and MHCκB-luc reporters, but not for the two GAS-luc reporters. This activity appeared to be dependent upon NF-κB, as judged by its sensitivity to inhibition by overexpression of a mutated form of IκBα, which serves as a strong NF-κB dominant negative (dn-IκB in Fig. 2A). In contrast, IL-6 strongly activated the two GAS-luc reporters, but only weakly activated GAS/κB-luc. As expected, the MHCκB-luc reporter did not respond to IL-6. Fig. 1B shows that STAT3 was induced by IL-1 to bind to the GAS/κB sequence as an apparent complex with NF-κB p65. Therefore, we investigated whether the expression of STAT3 could induce the GAS/κB-luc reporter. Fig. 2B shows that STAT3 alone, as expected, could not induce GAS/κB-luc. This is in contrast to what was observed for cotransfection with an expression vector coding for NF-κB p65. This is probably the result of an expression level that exceeds that of the endogenous IκB inhibitors. Coexpression of both STAT3 and p65 resulted in a cooperative response. Interestingly, this cooperativity appeared to be independent of IL-6 stimulation as well as the ability of STAT3 to be tyrosine-phosphorylated, as judged by the activity of a nonphosphorylatable Y705F mutation in STAT3 (mSTAT3). To confirm that the product of the Y705F mutated STAT3 expression vector was not tyrosine-phosphorylated, the vector was cotransfected along with the GAS-luc reporter. Fig. 2C shows that the Y705F mutation resulted in dominant negative activity for the GAS-luc reporter, as reported previously (32.Wen Z. Zhong Z. Darnell Jr, J.E. Cell. 1995; 82: 241-250Abstract Full Text PDF PubMed Scopus (1770) Google Scholar, 33.Kapstein A. Paillard V. Saunders M. J. Biol. Chem. 1996; 271: 5961-5964Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). It is well known that the IL-6-induced tyrosine phosphorylation of STAT3 is necessary for dimerization and DNA binding. The observation that STAT3aPTyr can cooperate with p65 suggests that tyrosine phosphorylation is not necessary for formation of the STAT3·p65 complex. The cooperative behavior between STAT3 and p65 was only observed for GAS/κB-luc, not MHCκB-luc. The results shown in Fig. 2D argue that IL-1 could neither efficiently tyrosine phosphorylate STAT3, nor interfere with tyrosine phosphorylation by IL-6. This argues that IL-1 inhibition of GAS site activation during APR is not likely via induction of SOCS3 (34.Bode J. Nimmesgern A. Schmitz J. Schaper F. Schmitt M. Frisch W. Haussinger D. Heinrich P. Graeve L. FEBS Lett. 1999; 463: 365-370Crossref PubMed Scopus (177) Google Scholar), a Janus kinase inhibitor that mediates LPS- and TNF-dependent inhibition of STAT3. The observation that pretreatment of Hep3B cells with IL-1 inhibits IL-6 induction of a GAS-luc reporter, while not having an effect on STAT3 tyrosine phosphorylation, argues that SOCS3 is probably not involved in the observed activity inhibition. p65 Inhibits, Whereas p50 Activates, a GAS-dependent Promoter—The observation that IL-1 induces the formation of a functionally cooperative interaction between p65 and STAT3aPTyr on a composite GAS/κB-luc reporter, while inhibiting the activation of a GAS-luc reporter, prompted investigation of how different forms of NF-κB might affect the induction of a GAS-dependent reporter. Fig. 3 shows that cotransfection of expression vectors for either the p65 or p50 form of NF-κB resulted in distinct responses for a GAS-luc reporter. As expected, only IL-6 (and not IL-1, p50, and p65) induced transcription from the GAS-luc reporter. Expression of p65 inhibited the IL-6-dependent activity, whereas p50 cooperated with IL-6. As a positive control, expression of STAT3 in the presence of IL-6 revealed strong activity. These data argue that the activation of p65 by IL-1 may be responsible for both the inhibition of GAS-dependent genes as well as the observed cooperativity for the GAS/κB-luc reporter. Furthermore, it appears that TAD-deficient p50, in contrast to p65, can cooperate with IL-6 to activate a STAT3-dependent reporter containing APR class 2 GAS sites. Cooperativity between STAT3 and p65 Depends upon the Binding of p65 to DNA—The observation that the composite GAS/κB sequence, in contrast to that of Ig-κB, both binds and functionally cooperates with STAT3, taken together with the observation that STAT3 and p65 can interact in the absence of DNA (Fig. 1D), suggests that either the presence of the GAS motif or differences between the nature of the two different κB sequences may be responsible for the observed specificities. In an attempt to evaluate the importance of the intact GAS site in the overlapping GAS/κB sequence, mutations were introduced into the nonoverlapping portion of each half-site (Fig. 4A). Fig. 4B shows that mutation of the κB (mNFκB), but not the GAS (mGAS) half-site eliminates formation of both DNA-binding complexes. In contrast, mutation of the GAS half-site has no effect on DNA binding. To confirm this, the same sequences used as EMSA probe were incorporated into luciferase reporters and assayed for activity. Fig. 4C shows that IL-1 activates both the wild-type and mGAS-luc but not mNFκB-luc, which is consistent with the EMSA data. Furthermore, as in Fig. 2B, p65 and STAT3 Y705F cooperate on both the composite GAS/κB and mGAS-luc reporters (Fig. 4D). Taken together, the STAT3·p65 complex likely binds to DNA primarily through the 9-bp p65 homodimer κB motif and not the GAS site. This suggests that the presence of the GAS site may not be critical for the cooperativity between the two factors. Other Proinflammatory Genes Contain Similar p65 Sites That Recruit STAT3—It is known that numerous proinflammatory genes have 10–11-bp functional κB binding sites, of the form GGGN4–5CCC, in their promoters. Therefore, we examined a small collection of genes reported to be dependent upon NF-κB activation (Fig. 5A). Two genes (BCL-x and iNOS) fit the 10–11-bp consensus, whereas two others (IL-8 and ICAM-1) have the shorter 9-bp κB site, similar to th
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