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A Role for NF-κB in the Induction of β-R1 by Interferon-β

2001; Elsevier BV; Volume: 276; Issue: 48 Linguagem: Inglês

10.1074/jbc.c100417200

1083-351X

M.R. Sandhya Rani, Ashok R. Asthagiri, Aakash Singh, Nywana Sizemore, Swati Sathe, Xiaoxia Li, Joseph DiDonato, George R. Stark, Richard M. Ransohoff,

Immune Response and Inflammation

Previous experiments have suggested that induction of the β-R1 gene by interferon (IFN)-β required transcription factor ISGF-3 (IFN-stimulated gene factor-3) and an additional component. We now provide evidence that nuclear factor-κB (NF-κB) can serve as this component. Site-directed mutagenesis of an NF-κB binding site in the β-R1 promoter or over-expression of an IκBα super-repressor abrogated IFN-β-mediated induction of a β-R1 promoter-reporter. IFN-β treatment did not augment abundance of NF-κB but did lead to phosphorylation of the p65 NF-κB subunit. It is proposed that IFN-β-mediated enhancement of the transactivation competence of NF-κB components is required for inducible transcription of the β-R1 promoter. These results provide a novel insight into the role of NF-κB in the transcriptional response to IFN-β. Previous experiments have suggested that induction of the β-R1 gene by interferon (IFN)-β required transcription factor ISGF-3 (IFN-stimulated gene factor-3) and an additional component. We now provide evidence that nuclear factor-κB (NF-κB) can serve as this component. Site-directed mutagenesis of an NF-κB binding site in the β-R1 promoter or over-expression of an IκBα super-repressor abrogated IFN-β-mediated induction of a β-R1 promoter-reporter. IFN-β treatment did not augment abundance of NF-κB but did lead to phosphorylation of the p65 NF-κB subunit. It is proposed that IFN-β-mediated enhancement of the transactivation competence of NF-κB components is required for inducible transcription of the β-R1 promoter. These results provide a novel insight into the role of NF-κB in the transcriptional response to IFN-β. interferon-β IFN-stimulated gene IFN-stimulated gene factor-3 IFN-stimulated response element mutant ISRE nuclear factor-κB electrophoretic mobility shift assay tumor necrosis factor polyacrylamide gel electrophoresis glutathione S-transferase wild type phosphatidylinositol 3-kinase signal transducers and activators of transcription-3 Interferons (IFNs)1elicit multiple biological responses mediated by the proteins encoded by interferon-stimulated genes (ISGs) (1Gresser I. J. Invest. Dermatol. 1990; 95 (. S-71S): 66Abstract Full Text PDF PubMed Scopus (93) Google Scholar). IFN-α and -β activate transcription of ISGs through the transcription factor interferon-stimulated gene factor-3 (ISGF-3), which interacts with the interferon-stimulated response element (ISRE) present in the promoters of ISGs (2Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5061) Google Scholar). We previously reported the selective induction of β-R1 by IFN-β but not IFN-α in human fibrosarcoma cells (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). ISGF-3 was essential but not sufficient for IFN-β-dependent induction of β-R1 (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In the current report we show that the transcription factor nuclear factor-κB (NF-κB) is also involved in the induction of this gene by IFN-β. NF-κB is a dimer of members of the Rel family of proteins, including p50, p52, c-Rel, RelA/p65, and RelB. NF-κB dimers bind to DNA segments collectively referred to as κB elements (5Chen F.E. Ghosh G. Oncogene. 1999; 18: 6845-6852Crossref PubMed Scopus (266) Google Scholar). In unstimulated cells, NF-κB is sequestered in the cytosol with oligomeric inhibitory components, termed IκB. Stimulation of cells with appropriate inducers results in the rapid degradation of IκB and translocation of NF-κB to the nucleus, where it binds target sequences to initiate transcription. Increasing evidence suggests that a second signaling pathway, independent of IκBα degradation (6Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar), culminates in phosphorylation of the p65/RelA subunit in its transactivation domain (11Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). A role for NF-κB in the induction of ISG in response to IFNs has not been reported. While studying IFN-β activation of β-R1 promoter, we found that modification of pre-existing NF-κB by phosphorylation was essential for IFN-β-induced transcription of β-R1. The human fibrosarcoma cell line UI.wt was maintained as described (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Purified recombinant IFN-β-1b (2 × 108 IU/mg) was from Berlex Biosciences (Richmond, CA). Purified IFN-γ (1.9 × 107 IU/mg) was from Genentech (South San Francisco, CA). TNF-α, from Collaborative Biomedical Products (Bedford, MA), was used at a final concentration of 50 ng/ml. IFN-β and -γ were used at 1000 and 500 units/ml, respectively, unless mentioned otherwise. Human embryonic kidney 293 cells transfected with interleukin-1R were maintained in Dulbecco's modified Eagle's medium with 10% calf serum (12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar). Z5, a mutant cell line derived from 293-TK/Zeo cells (12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar), 2S. S. Sathe, X. Li, and G. R. Stark, unpublished data. exhibits constitutive NF-κB activity as shown by EMSA assays and expression of an NF-κB-dependent promoter. Total RNA was prepared from IFN-treated cells (13Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNase protection experiments were performed as described (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). The probes used, β-R1 (protects 400 bases), 6-16 (protects 190 bases), and γ-actin (protects 130 bases), are as described (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Site-directed mutagenesis of the ISRE and κB sequence in pGL3-wt-β-R1 (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) was achieved by multiple rounds of polymerase chain reaction using pGL3-wt-β-R1 as template and appropriate primers (14Zhou Z.H. Chaturvedi P. Han Y.L. Aras S. Li Y.S. Kolattukudy P.E. Ping D. Boss J.M. Ransohoff R.M. J. Immunol. 1998; 160: 3908-3916PubMed Google Scholar). Three rounds of polymerase chain reaction were performed using two sets of primers to obtain mISRE-β-R1. The first pair of primers used to make the pGL3-mISRE mutant were 5′-GAAGAGAACAccACAtAAACTCTTGGAAGC-3′ (forward primer with ISRE sequence underlined and mutagenized nucleotides in lowercase) and BglII reverse primer 5′-TTGGAAGATCTAGTAGAAATG-3′. The second pair of primers wereSacI primer 5′-ATACGAGCTCTCCGCTGC-3′ and 5′-GCTTCCAAGAGTTTaTGTggTGTTCTCTTC-3′ (backward primer with ISRE sequence underlined and mutagenized nucleotides in lowercase). The first pair of primers used to make pGL3-mκB mutant were 5′-GCATGACTCAAAGAGtGAAATTaCTGTGCCAT-3′ (forward primer with NF-κB sequence underlined and mutagenized nucleotides in lowercase) and BglII reverse primer 5′-TTGGAAGATCTAGTAGAAATG-3′. The second pair of primers were SacI primer 5′-ATACGAGCTCTCCGCTGC-3′ and 5′- ATGGCACAGtAATTTCaCTCTTTGAGTCATGC-3′ (backward primer with κB binding sequence underlined and mutagenized nucleotides in lowercase). Plasmid DNA from two clones for each set of cloning was sequenced to verify the nucleotide sequence. Transient transfections were done as described (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), with 10 μg of β-R1 reporter plasmid, 10 μg of test plasmid DNA and 1 μg of pCH110 β-galactosidase plasmid DNA. The plasmid construct pCMV-IκBα (with Ser32 and Ser36 mutated to Ala) was as reported earlier (15DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar); pCMV-50 was kindly provided by Dr. M. Kieran (16Kieran M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israel A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (602) Google Scholar). The pCMV-65 plasmid was generously given by Dr. D. W. Ballard (17Brockman J.A. Scherer D.C. McKinsey T.A. Hall S.M. Qi X. Lee W.Y. Ballard D.W. Mol. Cell. Biol. 1995; 15: 2809-2818Crossref PubMed Google Scholar). The p561-luciferase construct containing the ISRE from p56 ISG and the 6xIP-10κB-luciferase construct has been reported earlier (4Rani M.R. Gauzzi C. Pellegrini S. Fish E.N. Wei T. Ransohoff R.M. J. Biol. Chem. 1999; 274: 1891-1897Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar). For electrophoretic mobility shift assays (EMSA) the NF-κB binding site (5′-GAGCAGAGGGAAATTCCGTAACTT-3′) from thehIP-10 gene was used as a probe. Nuclear extracts were prepared and gel-shift assays performed as described earlier (18Majumder S. Zhou L.Z. Chaturvedi P. Babcock G. Aras S. Ransohoff R.M. J. Immunol. 1998; 161: 4736-4744PubMed Google Scholar). U1.wt cells were labeled with [32P]orthophosphate (200 μCi/ml) as described (7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar). Cells were stimulated for 5 min with IFN-β (5000 units/ml) or TNF-α (50 ng/ml). Lysis and immunoprecipitations were performed using anti-p65 (Santa Cruz Biotechnology, Santa Cruz, CA) as described (7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar,11Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Phosphorylated proteins were separated by 10% SDS-PAGE and autoradiographed. The carboxyl terminus of p65 fused to GST obtained from Dr. H. Sakurai (8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar) was expressed in E. coli and cells lysed in 0.5% Nonidet P-40 lysis buffer as described (19Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) and bound to glutathione-Sepharose beads (Amersham Pharmacia Biotech, Uppsala, Sweden). In vitro phosphorylation was performed using ∼1 μg of GST-fused p65 protein in 23 μl of kinase buffer containing 3 μCi [γ-32P]ATP (19Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Nuclear extracts from IFN-β-, IFN-α-, or TNF-α-treated cells (3 μg of protein in a volume of 2 μl) were added to the reaction and incubated at 30 °C for 30 min (8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 19Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Following the kinase reaction, phosphorylated protein was separated by 10% SDS-PAGE and autoradiographed. Western analysis with anti-GST antibody (Amersham Pharmacia Biotech) was used to quantitate the total amount of p65 protein. We performed a functional analysis of the β-R1 ISRE-like element using wt-β-R1 and mutant mISRE-β-R1 (Fig.1 a). In transient transfection assays, IFN-β responsiveness of mISRE-β-R1 was reduced by more than 80% compared with the wt-β-R1 promoter (Fig. 1 a). This result confirmed the requirement of this ISRE-like element for the induction of β-R1 by IFN-β. The induction of mISRE-β-R1 activity by IFN-γ was also significantly reduced by about 70% compared with wt-β-R1 (Fig. 1 a). These results are consistent with our earlier observation in U2A cells (which lack the p48/IRF-9 ISRE recognition factor) of loss of induction of β-R1 by IFN-β or IFN-γ (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We proposed that a second cis-element might be involved in the induction of the β-R1 gene (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We had previously observed 3M. R. S. Rani and R. M. Ransohoff, unpublished data. synergistic induction of β-R1 by IFN-β and TNF-α, the latter being a well characterized activator of transcription factor NF-κB. Hence, a salient choice for analysis was the κB element on the promoter of β-R1. Using transient transfection assays, wt-β-R1 or mκB-β-R1 promoter-reporters were analyzed for IFN-β responsiveness. Mutation of the κB element on the promoter of β-R1 markedly reduced IFN-β induction of the promoter, by 70% (Fig. 1 b). Induction of the wild-type and mκB-β-R1 constructs by IFN-γ differed only slightly (Fig. 1 b). This result indicated that the κB element was specifically required for IFN-β but not IFN-γ-mediated induction. We used EMSAs to address whether the requirement for the κB binding site was associated with IFN-β induced nuclear translocation and DNA binding by NF-κB. Analysis of extracts of U1.wt cells revealed, as previously reported (20Plattner R. Gupta S. Khosravi-Far R. Sato K.Y. Perucho M. Der C.J. Stanbridge E.J. Oncogene. 1999; 18: 1807-1817Crossref PubMed Scopus (50) Google Scholar, 21Wiest J.S. Burnett V.L. Anderson M.W. Reynolds S.H. Oncogene. 1994; 9: 2449-2454PubMed Google Scholar), basal NF-κB DNA binding activity (Fig.2 a). Super-shift experiments showed complexes composed of p65 homodimers, and p65/p50 heterodimers (results not shown). No IFN-β-inducible increase in DNA binding activity was detected by densitometric analysis of the ratios of the specific NF-κB complexes to a nonspecific band (Fig.2 b). To address the possibility that basal nuclear NF-κB activity might be essential for induction of β-R1 by IFN-β, we analyzed two sibling cell lines that differed in basal NF-κB. Wild-type 293 cells did not contain basal nuclear NF-κB as detected by EMSA, whereas Z5 cells contained constitutive NF-κB (Fig. 2 c). The major κB binding complex in Z5 cells consisted of p65 and p50 subunits (Fig.2 d). IFN-β treatment did not produce a detectable change in NF-κB in either cell line. RNase protection analysis documented the induction of β-R1 by IFN-β in Z5 cells but not in 293 cells (Fig. 2 e), although another type I IFN-inducible gene (6Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 11Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 12Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, 13Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar, 14Zhou Z.H. Chaturvedi P. Han Y.L. Aras S. Li Y.S. Kolattukudy P.E. Ping D. Boss J.M. Ransohoff R.M. J. Immunol. 1998; 160: 3908-3916PubMed Google Scholar, 15DiDonato J.A. Mercurio F. Karin M. Mol. Cell. Biol. 1995; 15: 1302-1311Crossref PubMed Google Scholar, 16Kieran M. Blank V. Logeat F. Vandekerckhove J. Lottspeich F. Le Bail O. Urban M.B. Kourilsky P. Baeuerle P.A. Israel A. Cell. 1990; 62: 1007-1018Abstract Full Text PDF PubMed Scopus (602) Google Scholar) could be induced equally well in both cell lines. TNF-α, in combination with IFN-β, mediated synergistic induction of β-R1 in both Z5 and 293 cells, confirming the existence of an intact NF-κB pathway in both cell lines. We also over-expressed super-repressor mutant isoform of IκBα in U1.wt cells, resulting in diminished induction of the wt-β-R1 promoter-reporter by IFN-β (Fig. 2 f). To exclude nonspecific effects of IκBα over-expression, we analyzed IFN-β-mediated induction of a promoter-reporter construct derived from the p561 gene (a type I IFN-induced gene). Induction of the 561 promoter-reporter (which lacks a κB site) was unaffected by IκBα (Fig. 2 f). In transient transfection assays, a 6xκB promoter-reporter construct was not activated by IFN-β, but TNF-α gave a 6-fold induction (Fig. 2 g). We asked whether homodimers of p50 (which lack a transactivation domain and are transcriptionally inactive) were sufficient to promote β-R1 transcription by virtue of occupancy at the κB site. For these experiments, p50 was over-expressed in U1.wt cells along with the wt-β-R1 promoter-reporter, and reporter gene activation in response to IFN-β was examined. IFN-β responsiveness of the promoter was reduced by 60% to a level similar to that obtained by disruption of the NF-κB binding site (TableI). This result indicated that p50 homodimers were insufficient to support transcription of β-R1 and suggested the requirement of transcriptionally competent NF-κB complexes.Table IRequirement of p65 but not p50 for the induction of β-R1 by IFN-βPlasmidsNormalized luciferase activity1-aLuciferase activity was normalized for transfection efficiency as described under “Experimental Procedures” and expressed in arbitrary units. All co-transfections were done as reported under “Experimental Procedures.”Relative reporter expression1-bLuciferase activity on transfection of wt-β-R1 in the absence of treatment was set at 1. This value was used to normalize the effects of treatment with IFN-β or over-expression of NF-κB subunits.-Fold increase with IFN-β−IFN-β+IFN-β−IFN-β+IFN-β1. wt-β-R10.02 ± 0.0050.11 ± 0.01715.35.32. wt-β-R1 + pCMV-651-c0.25 μg of pCMV-65 was used along with 10 μg of wt-β-R1 promoter-reporter construct. Basal activity was higher for cells transfected with p65 in the absence of treatment.0.18 ± 0.0301.08 ± 0.0169.258.06.33. wt-β-R1 + pCMV-500.02 ± 0.0030.04 ± 0.00612.02.01-d-Fold induction in luciferase activity by IFN-β was significantly less upon co-transfection of wt-β-R1 and pCMV-50 compared to wt-β-R1 with empty vector (p < 0.001, paired t test).1-a Luciferase activity was normalized for transfection efficiency as described under “Experimental Procedures” and expressed in arbitrary units. All co-transfections were done as reported under “Experimental Procedures.”1-b Luciferase activity on transfection of wt-β-R1 in the absence of treatment was set at 1. This value was used to normalize the effects of treatment with IFN-β or over-expression of NF-κB subunits.1-c 0.25 μg of pCMV-65 was used along with 10 μg of wt-β-R1 promoter-reporter construct. Basal activity was higher for cells transfected with p65 in the absence of treatment.1-d -Fold induction in luciferase activity by IFN-β was significantly less upon co-transfection of wt-β-R1 and pCMV-50 compared to wt-β-R1 with empty vector (p < 0.001, paired t test). Open table in a new tab Co-expression of p65 with the wt-β-R1 promoter increased the basal activity of the promoter by 9-fold (Table I). IFN-β treatment further increased promoter activity by 6-fold. Taken together these results indicated that transcriptionally competent NF-κB complexes were required for maximal transcription of the β-R1 gene in response to IFN-β. Basal NF-κB activity was essential for IFN-β-mediated transcription of β-R1, although IFN-β treatment did not lead to increased NF-κB (by EMSA). Recent reports (6Wang D. Baldwin A.S. J. Biol. Chem. 1998; 273: 29411-29416Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar, 8Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 9Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 10Zhong H. SuYang H. Erdjument-Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar, 11Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 27Jefferies C.A. O'Neill L.A. J. Biol. Chem. 2000; 275: 3114-3120Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) have shown that phosphorylation of p65 augments NF-κB-dependent gene transcription. To address IFN-β-mediated phosphorylation of NF-κB proteins, cells were metabolically labeled with [32P]orthophosphate, and whole cell extracts were immunoprecipitated with anti-p65 antibodies, which co-precipitated p65, p50, and IκB (Fig. 3 a). The identity of the phosphorylated proteins was confirmed by Western analysis (results not shown). This analysis revealed increase in p65 phosphorylation in cells treated with 1FN-β or TNF-α (Fig.3 a). Phosphorylation of p65 in untreated U1.wt cells was anticipated, as these cells contain basal NF-κB nuclear activity. We also detected IFN-β-mediated increases in p50 and IκB phosphorylation. Strikingly, phosphorylated IκBα remained intact in IFN-β-treated cells but was rapidly degraded in TNF-α-treated cells. Between 5 min and 1 h, we observed rapid IκBα degradation in response to TNF-α but not to IFN-β (Fig. 3 c). Experiments using GST-fused p65 revealed that an inducible p65 phosphorylating activity was present in nuclear extracts of IFN-β- or TNF-α treated cells (Fig. 3 b) but not in IFN-α-treated cells (Fig. 3 d). Here, we address the role of NF-κB in the induction of β-R1 by IFN-β. We had previously hypothesized that ISGF-3 was essential but not sufficient for the induction of the gene and proposed that an additional component might be required (3Rani M.R. Foster G.R. Leung S. Leaman D. Stark G.R. Ransohoff R.M. J. Biol. Chem. 1996; 271: 22878-22884Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). We studied NF-κB because of the striking synergy between TNF-α and IFN-β for induction of β-R1, despite the fact that neither TNF-α nor interleukin-1β alone induced β-R1. Site-directed mutagenesis of the κB element in the β-R1 promoter or over-expression of a super-repressor mutant of IκBα blocked the induction of the β-R1 gene by IFN-β, indicating that NF-κB components were required. However, IFN-β did not induce increased NF-κB DNA binding activity by EMSA. Further IFN-β treatment did not activate transcription from a NF-κB reporter-construct. To address the question of whether the physical presence of κB binding activity was sufficient to enhance the transcriptional response of the β-R1 gene to IFN-β, we individually over-expressed transcriptionally inert p50 or transcriptionally active p65. Over-expression of p50 suppressed β-R1 transcription, whereas over-expression of p65 elevated basal β-R1 promoter activity but supported a further 6-fold increase in response to IFN-β. These results confirmed a requirement for active NF-κB complexes to drive maximal transcription of the β-R1 gene. In Daudi and other cell lines, type I IFN was reported to signal to NF-κB via a pathway involving PI3K and serine-threonine kinase Akt (22Yang H.C. Murti A. Pfeffer S.R. Kim J.G. Donner D.B. Pfeffer L.M. J. Biol. Chem. 2001; 276: 13756-13761Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The output of this pathway comprised markedly increased NF-κB. IFN-mediated endogenous NF-κB-dependent gene expression was not documented in these or prior reports (22Yang H.C. Murti A. Pfeffer S.R. Kim J.G. Donner D.B. Pfeffer L.M. J. Biol. Chem. 2001; 276: 13756-13761Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 23Yasumoto K. Okamoto S. Mukaida N. Murakami S. Mai M. Matsushima K. J. Biol. Chem. 1992; 267: 22506-22511Abstract Full Text PDF PubMed Google Scholar, 24Moitreyee C.K. Suraksha A. Swarup A.S. Mol. Cell Biochem. 1998; 178: 103-112Crossref PubMed Scopus (9) Google Scholar). The signaling pathway described in this report (22Yang H.C. Murti A. Pfeffer S.R. Kim J.G. Donner D.B. Pfeffer L.M. J. Biol. Chem. 2001; 276: 13756-13761Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar), implicated STAT3 as a docking site for PI3K components. Such signaling is not observed in HT1080 cells, where STAT3 activation is dispensable for generating PI3K lipid kinase activity and where physical interaction between STAT3 and PI3K does not occur (25Rani M.R. Leaman D.W. Han Y. Leung S. Croze E. Fish E.N. Wolfman A. Ransohoff R.M. J. Biol. Chem. 1999; 274: 32507-32511Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Given these dissimilar signaling characteristics of type I IFN receptor activation in HT1080 cells as compared with Daudi cells (26Yang C.H. Murti A. Pfeffer S.R. Basu L. Kim J.G. Pfeffer L.M. Proc. Natl. Acad. Sci. U. S. 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Because there is no cytokine-dependent increase in NF-κB DNA binding activity, IFN-β-treated cells represent an excellent system in which to investigate post-translational modification of p65.