Identification of Nucleophosmin as an NF-κB Co-activator for the Induction of the Human SOD2 Gene
2004; Elsevier BV; Volume: 279; Issue: 27 Linguagem: Inglês
10.1074/jbc.m403553200
ISSN1083-351X
AutoresSanjit K. Dhar, Bert C. Lynn, Chotiros Daosukho, Daret K. St. Clair,
Tópico(s)NF-κB Signaling Pathways
ResumoManganese superoxide dismutase (MnSOD) is an antioxidant enzyme essential for the survival of life. We have reported that NF-κB is essential but not sufficient for the synergistic induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. To further identify transcription factors and co-activators that participate in the induction of MnSOD, we used NF-κB affinity chromatography to isolate potential NF-κB interacting proteins. Proteins eluted from the NF-κB affinity column were subjected to proteomic analysis and verified by Western analysis. Nucleophosmin (NPM), a nucleolar phosphoprotein, is the most abundant single protein identified. Co-immunoprecipitation studies suggest a physical interaction between NPM and NF-κB proteins. To verify the role of NPM on MnSOD gene transcription, cells were transfected with constructs expressing NPM in sense or antisense orientation as well as interference RNA. The results indicate that an increase NPM expression leads to increased MnSOD gene transcription in a dose-dependent manner. Consistent with this, expression of small interfering RNA for NPM leads to inhibition of MnSOD gene transcription but does not have any effect on the expression of interleukin-8, suggesting that the effect of NPM is selective. These results identify NPM as a partner of the NF-κB transcription complex in the induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme essential for the survival of life. We have reported that NF-κB is essential but not sufficient for the synergistic induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. To further identify transcription factors and co-activators that participate in the induction of MnSOD, we used NF-κB affinity chromatography to isolate potential NF-κB interacting proteins. Proteins eluted from the NF-κB affinity column were subjected to proteomic analysis and verified by Western analysis. Nucleophosmin (NPM), a nucleolar phosphoprotein, is the most abundant single protein identified. Co-immunoprecipitation studies suggest a physical interaction between NPM and NF-κB proteins. To verify the role of NPM on MnSOD gene transcription, cells were transfected with constructs expressing NPM in sense or antisense orientation as well as interference RNA. The results indicate that an increase NPM expression leads to increased MnSOD gene transcription in a dose-dependent manner. Consistent with this, expression of small interfering RNA for NPM leads to inhibition of MnSOD gene transcription but does not have any effect on the expression of interleukin-8, suggesting that the effect of NPM is selective. These results identify NPM as a partner of the NF-κB transcription complex in the induction of MnSOD by phorbol 12-myristate 13-acetate and cytokines. Superoxide dismutases (SODs) 1The abbreviations used are: SOD, superoxide dismutase; MnSOD, manganese superoxide dismutase; NPM, nucleophosmin; NF-κB, nuclear factor κB; AP, activating protein; Sp1, specificity protein 1; PMA, phorbol 12-myristate 13-acetate; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; DTT, dithiothretol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; C/EBP, CAAT-enhancer binding protein. 1The abbreviations used are: SOD, superoxide dismutase; MnSOD, manganese superoxide dismutase; NPM, nucleophosmin; NF-κB, nuclear factor κB; AP, activating protein; Sp1, specificity protein 1; PMA, phorbol 12-myristate 13-acetate; TNF-α, tumor necrosis factor α; IL-1β, interleukin-1β; DTT, dithiothretol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline; EMSA, electrophoretic mobility shift assay; RT, reverse transcriptase; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-α; C/EBP, CAAT-enhancer binding protein. are the first line of cellular defense against the damaging effects of superoxide anion radicals (1Halliwel B. Gutteridge J.M. Methods Enzymol. 1990; 186: 1-85Crossref Scopus (4392) Google Scholar). MnSOD is a highly regulated SOD exclusively localized in mitochondria but coded by the SOD2 gene located on human chromosome 6q25.3 (2Dougall W.C. Nick H.S. Endocrinology. 1991; 129: 2379-2384Crossref Scopus (114) Google Scholar, 3Suzuki K. Tatsumi H. Satoh S. Sendra T. Naskata T. Fuji J. Takiguchi N. Am. J. Physiol. 1993; 265: H1173-H1178PubMed Google Scholar). Accumulating studies show that increased cellular levels of MnSOD are cytoprotective against oxidative stress (4Del Maestro R. McDonald W. Mech. Aging Dev. 1989; 48: 15-31Crossref PubMed Scopus (42) Google Scholar), inflammatory responses, tumor necrosis factor α (TNF-α) (5Wong G.H. Elwell J. Overly L. Goeddel D.V. Cell. 1989; 58: 923-931Abstract Full Text PDF PubMed Scopus (759) Google Scholar, 6Wong G.H. Goeddel D.V. Science. 1988; 242: 941-944Crossref PubMed Scopus (832) Google Scholar), interleukin-1 β (IL-1 β) (7Visner G.A. Dougall W.C. Wilson J.M. Burr I.M. Nick H.S. J. Biol. Chem. 1990; 256: 2856-2864Abstract Full Text PDF Google Scholar), 12-O-tetradecanoylphorbol-13-acetate (8Fuji J. Taniguchi N. J. Biol. Chem. 1991; 266: 23142-23146Abstract Full Text PDF PubMed Google Scholar), ionizing radiation (9Akashi M. Hachiya M. Paquette R.L. Osawa Y. Shimizu S. Suzuki G. J. Biol. Chem. 1995; 270: 15864-15869Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 10Eastgate J. Moreb J. Nick H.S. Suzuki K. Taniguchi N. Zucali J.R. Blood. 1993; 81: 639-646Crossref PubMed Google Scholar), and neurotoxins (11Manganaro F. Chopra V.S. Mydlarski M.B. Bernatchez G. Schipper H.M. Free Radic. Biol. Med. 1995; 19: 823-835Crossref PubMed Scopus (42) Google Scholar, 12Baker K. Marcus C.B. Huffman K. Kruk H. Malfroy B. Doctrow S.R. J. Pharmacol. Exp. Ther. 1998; 284: 215-221PubMed Google Scholar). The critical role of MnSOD as a cytoprotective enzyme is illustrated in both MnSOD knockout and transgenic animal models. For instance, MnSOD knockout mice develop cardiomyopathy and die within 10 days after birth (13Li Y. Huang T.T. Carison E.J. Melov S. Ursell P.C. Olson J.L. Noble L.J. Yoshimura M.P. Berger C. Chan P.H. Nat. Genet. 1995; 11: 376-381Crossref PubMed Scopus (1435) Google Scholar). MnSOD knockout mice treated with a SOD mimetic were protected from systemic toxicity and from neonatal lethality (14Melov S. Schneider J.A. Day B.J. Hinerfeld D. Coskun P. Mirra S.S. Crapo J.D. Wallace D.C. Nat. Genet. 1998; 18: 159-163Crossref PubMed Scopus (437) Google Scholar). Conversely, transgenic mice overexpressing human MnSOD experienced less toxic injury resulting from inflammation (15Wispe J.R. Warner B.B. Clark J.C. Dey C.R. Neuman J. Glasser S.W. Crapo J.D. Chang L. Whitselt J.A. J. Biol. Chem. 1992; 267: 23937-23941Abstract Full Text PDF PubMed Google Scholar), acute adriamycin-induced cardiac injury (16Yen H.-C. Oberley T.D. Vichitbandha S. Ho Y.-S. St. Clair D.K. J. Clin. Investig. 1996; 98: 1253-1260Crossref PubMed Scopus (394) Google Scholar), and ischemia-induced brain injury (17Keller J.N. Kindy M.S. Holtsberg F.W. St. Clair D.K. Yen H.-C. Germeyer A.M. Steiner S.M. Bruce-Keller A.J. Hutchins J.B. Mattson M.P. J. Neurosci. 1998; 18: 687-697Crossref PubMed Google Scholar). Additionally, several contemporary studies suggest that MnSOD may act as a tumor-suppressor gene (18Church S.L. Grant J.M. Ridnour L.A. Oberley L.W. Swanson P.E. Meltzer P.S. Trent J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3113-3117Crossref PubMed Scopus (452) Google Scholar, 19Safford S.E. Oberley T.D. Urano M. St. Clair D.K. Cancer Res. 1994; 54: 4261-4265PubMed Google Scholar, 20Urano M. Kuroda M. Reynolds R. Oberley T.D. St. Clair D.K. Cancer Res. 1995; 55: 2490-2493PubMed Google Scholar, 21Zhong W. Oberley L.W. Oberley T.D. St. Clair D.K. Oncogene. 1997; 14: 481-490Crossref PubMed Scopus (217) Google Scholar) by modulating redox-sensitive transcription factors (22Kiningham K.K. St. Clair D.K. Cancer Res. 1997; 57: 5265-5271PubMed Google Scholar). The human MnSOD (SOD2) gene is a single-copy gene consisting of five exons interrupted by four introns with a typical splice junction (23Wan X.S. Devalaraja M.N. St. Clair D.K. DNA Cell Biol. 1994; 13: 1127-1136Crossref PubMed Scopus (191) Google Scholar). The SOD2 gene from human, bovine, rat, and mouse share more than 90% homology in the coding sequence; the 5′-flanking regions are less homologous between human and other species (24Meyrick B. Magnuson M.A. Am. J. Respir. Cell Mol. Biol. 1994; 10: 113-121Crossref PubMed Scopus (42) Google Scholar). Sequence analysis of the 5′- and 3′-flanking regions reveal multiple potential regulatory motifs for NF-κB, AP-1, AP-2, and Sp1 bindings. The 5′ proximal promoter in the SOD2 gene is characterized by a lack of TATA or CAAT box, but it is rich in GC boxes. The basal promoter of the SOD2 gene has multiple transcription factor binding motifs containing Sp1 and AP-2 binding sites. Functional studies in different cell lines with different levels of Sp1 protein suggest that cellular levels of this protein might be differentially regulated via GC binding motifs in the human MnSOD promoter. Site-directed mutagenesis of Sp1 and AP-2 binding sites shows that Sp1 is essential for transcription of the SOD2 gene, whereas AP-2 plays a negative role on transcription. AP-2 down-regulates the transcription of MnSOD through interaction with Sp1 in the promoter region (25Zhu C.-H. Huang Y. Oberley L.W. Domann F.E. J. Biol. Chem. 2001; 276: 14407-14413Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 26Zhu C. Huang Y. Weydert C.J. Oberley L.W. Domann F.E. Antioxid. Redox Signaling. 2001; 3: 387-395Crossref PubMed Scopus (19) Google Scholar, 27Xu Y. Porntadavity S. St. Clair D.K. Biochem. J. 2002; 362: 401-412Crossref PubMed Scopus (110) Google Scholar). The binding of Sp1 and AP-2 proteins to their recognition sites generates a specific DNA looping structure required for cooperative activation of other regulatory proteins and recruitment of co-activators of the MnSOD gene (28Mastrangelo I.A. Courey A.J. Wall J.S. Jackson S.P. Hough P.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5670-5674Crossref PubMed Scopus (202) Google Scholar). In addition to Sp1 and AP-2, the basal promoter also contains binding sites for transcription modulators including the early growth protein, Egr-1. Interestingly, Egr-1 appears to play a negative role on basal promoter activity without affecting the 12-O-tetradecanoylphorbol-13-acetate-mediated activation of the human MnSOD promoter (29Porntadavity S. Xu Y. Kiningham K.K. Rangnekar V.M. Prachayasitikul V. St. Clair D.K. DNA Cell Biol. 2001; 20: 473-481Crossref PubMed Scopus (36) Google Scholar). The SOD2 gene of mice and humans contains enhancer elements in the second intron of the gene (30Jones P.L. Ping D. Boss J.M. Mol. Cell. Biol. 1997; 17: 6970-6981Crossref PubMed Scopus (210) Google Scholar, 31Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.-C. Majima H. Kasarskis E.J. St. Clair D.K. DNA Cell Biol. 1999; 18: 709-722Crossref PubMed Scopus (202) Google Scholar). Functional analyses of enhancer elements demonstrate that the intronic element consists of binding motifs for NF-κB, C/EBP, and NF-1, which are responsive to TNF-α and IL-1β. The activation of NF-κB is essential but not sufficient for the induction of MnSOD by TNF-α and IL-1β (32St. Clair D.K. Porntadavity S. Xu Y. Kiningham K.K. Methods Enzymol. 2002; 349: 306-312Crossref PubMed Scopus (49) Google Scholar). NF-κB forms various homo- and heterodimer units among the mammalian subunits of p50, p52, p65 (Rel A), c-Rel, and Rel B. These dimer units in turn bind to a group of NF-κB DNA binding sites with different affinities within the target gene (33Grilli M. Chiu J.J. Lenardo M.J. Int. Rev. Cytol. 1993; 143: 1-62Crossref PubMed Scopus (880) Google Scholar, 34Zabel U. Schreck R. Baeuerle P.A. J. Biol. Chem. 1991; 266: 252-260Abstract Full Text PDF PubMed Google Scholar). The role of Sp1 on NF-κB-mediated induction of the SOD2 gene is unclear. Whereas it has been shown that Sp1 interferes with the DNA binding sites of NF-κB (35Hirano F. Tanaka H. Hirano Y. Hiramoto M. Hanada H. Makino I. Scheidereit C. Mol. Cell. Biol. 1998; 18: 1266-1274Crossref PubMed Scopus (144) Google Scholar), other studies demonstrate that Sp1 bound to distal enhancer regions can interact with Sp1 bound at sites proximal to the promoter and synergistically activates transcription (36Pascal E. Tjian R. Genes Dev. 1991; 5: 1646-1656Crossref PubMed Scopus (354) Google Scholar). Mastrangelo et al. (28Mastrangelo I.A. Courey A.J. Wall J.S. Jackson S.P. Hough P.V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5670-5674Crossref PubMed Scopus (202) Google Scholar) have shown that this synergism is likely a direct consequence of interactions between the distal and proximal Sp1 protein through a loop formation. Loop formation may lead to increased concentration of the activator protein. Such double stem-loop structures favor activation of some specific proteins (such as RNA binding proteins) to bind with the loop structure. An important RNA-binding protein, nucleophosmin (NPM) is ubiquitously expressed and continuously shuttled between nucleus and cytoplasm. It was originally identified as functioning in the assembly and transport of ribosome. Recently, expression of NPM has been implicated in modulating active cellular processes such as transcription, DNA replication, and apoptosis (37Carrier F. Gatignol A. Hollander M.C. Jeang K.T. Fornace Jr., A.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1544-1558Crossref PubMed Scopus (25) Google Scholar). Nucleophosmin can relieve the transcription response of several mammalian genes by the transcription factor YY1 (38Inouye C.J. Seto E. J. Biol. Chem. 1994; 269: 6506-6510Abstract Full Text PDF PubMed Google Scholar). The relief of YY1-induced transcriptional repression is achieved by its ability to interact with the YY1 protein. It has been shown that NPM regulates the stability of the transcriptional activity of p53 (39Colombo E. Marine J.C. Danovi D. Fulini B. Pelicci P.G. Nat. Cell Biol. 2002; 4: 529-533Crossref PubMed Scopus (431) Google Scholar). Furthermore, NPM rapidly up-regulates and has been considered to be an immediate early response gene induced by damaged DNA. Interestingly, expression of nucleophosmin is increased in human myometrium during labor (40Chan E.C. Fraser S. Yin S. Yeo G. Kwek K. Fairclough R.J. Smith R. J. Clin. Endocrinol. Metab. 2002; 87: 2435-2441Crossref PubMed Scopus (69) Google Scholar). The increase in NPM levels is associated with an increase in MnSOD levels during labor, suggesting a link between NPM and MnSOD expression. However, it is not known whether and how the expression of nucleophosmin affects the expression of MnSOD. In this study, we use proteomic, immunoprecipitation, site-directed mutagenesis, gene transfection, and gene knock-down approaches to demonstrate that NPM enhances MnSOD transcription by interacting with NF-κB. The results identify NPM as an NF-κB co-activator that regulates the expression of MnSOD by interacting with NF-κB in response to PMA and cytokine treatment. It is possible that NPM may be a key factor that enables the induction of the housekeeping gene. Cell Culture—The human hepatocarcinoma cell line HepG2 was purchased from American Type Culture Collection (Manassas, VA). Cells were grown in a 5% CO2 incubator at 37 °C in media consisting of Dulbecco's modified Eagle's/Ham's F-12 medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (Hyclone Inc., Logan, UT), 1% (w/v) l-glutamine (Invitrogen), 1% PSN antibiotic (Invitrogen), and 1 mg/ml insulin (Invitrogen) as a growth factor. Reagents—Unless otherwise stated, all antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin monoclonal antibody and anti-NPM-V5-epitope antibodies were purchased from Sigma and Invitrogen, respectively. Rabbit polyclonal MnSOD antibody and GAPDH antibodies were purchased from Upstate Biotechnologies (Lake Placid, NY). All chemicals were purchased from Sigma, unless otherwise indicated. Construction of Plasmid—A BamHI fragment (B7) containing a 3.4-kb 5′-flanking region was used to generate the –555 to +24 basal MnSOD promoter by polymerase chain reaction. To create this construct, PCR primers with recognition sequences KpnI (upstream) and BglII (downstream) restriction sites were added for subcloning at the upstream of the luciferase reporter gene. To generate the intronic fragment (I2E) (1742 to 2083) constructs, a BamHI-digested 39b λ phase DNA, containing the entire human MnSOD gene (8074 bp), was used as the template for PCR amplification with a primer having a BglII artificial restriction site. The PCR product was then ligated to the BglII site of the –555 to +24 basal promoter containing pGL3 vector, which yielded the natural orientation of the gene. The following primer sets were used to generate the aforementioned constructs (the underlined sequences are the artificial KpnI and BglII recognition sites in the forward and reverse strand primers, respectively, or the BglII recognition site in both forward and reverse strands): forward primer (–555), 5′-CGGGGTACCCGCTGGCTCTACCCTCAGCTCATA-3′; reverse primer (+24), 5′-GGAAGATCTGCCGAAGCCACCACAGCCACGAGT-3′; forward primer (+1742), 5′-GGAAGATCTCGGGGTTATGAAATTTGTTGAGTA-3′; reverse primer (+2083), 5′-GGAAGATCTCCACAAGTAAAGGACTGAAATTAA-3′. After amplification of the human MnSOD basal promoter (–555 to +24), the system was subcloned into the pGL3 basic vector (Promega, Madison, WI) containing the luciferase gene. PCR amplified I2E fragments were directly subcloned downstream of the human MnSOD basal promoter. Site-specific mutations on the NF-κB binding region in the I2E construct were prepared by the Chameleon™ double-stranded site-directed mutagenesis kit (Stratagene, La Jolla, CA) as previously reported (31Xu Y. Kiningham K.K. Devalaraja M.N. Yeh C.-C. Majima H. Kasarskis E.J. St. Clair D.K. DNA Cell Biol. 1999; 18: 709-722Crossref PubMed Scopus (202) Google Scholar). pcDNA3.1/NPM(B23), a cDNA clone, is a ras-gene™-constructed NPM expression vector obtained from Invitrogen. The presence of a cDNA insert within the vector was confirmed by restriction digestion. For construction of antisense NPM, pcDNA3.1/NPM plasmid DNA was used as the PCR template and primer with HindIII and BglII artificial restriction sites (underlined sequences) in the forward and reverse strand primers, respectively, and were used as follows: forward primer, 5′-TCAAAGCTTTCACGGTTGTGAACTAAAGGCCGA-3′; reverse primer, 5′-ATCGGAAGATCTTTACCTTCGAATGGGTGACCT-3′. After amplification, the PCR product was subcloned into the pCMV vector at the multicloning site in the antisense orientation. The nucleotide sequence and orientations of all constructs were confirmed by automated DNA sequencing. Transient Transfection and Luciferase Assay—HepG2 cells were grown in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, l-glutamine, insulin, and PSN antibiotics. 70–80% confluent cells were achieved within 24 h by plating the cells at a density of 2 × 106 in a 100 × 20-mm cell culture dish. The cells were then transfected with plasmids following a modified calcium phosphate method as described previously (41Kiningham K.K. Xu Y. Daosukho C. Popova B. St. Clair D.K. Biochem. J. 2001; 353: 147-156Crossref PubMed Scopus (100) Google Scholar). Cells were co-transfected with 6 μm plasmid DNA constructs containing the intronic enhancer fragment (I2E) of the human MnSOD gene in the pGL3 reporter gene and pRL-TK (Promega) containing Renilla cDNA at 1/10th the molar concentration of the I2E construct. Eight hours after transfection, the cells were washed twice with PBS and incubated in fresh medium. Twenty-four hours later, transfected cells were trypsinized and replated in a 24-well plate at a density of 105 cells per well. After an additional 24 h, cells were treated either independently or in combination with 200 IU/ml recombinant human TNF-α (R & D Systems, Minneapolis, MN), 100 nm PMA (Sigma), and 2 ng/ml IL-1β (Endogen, Woburn, MA). Twelve hours post-treatment, the cells were washed with PBS and lysed in passive lysis buffer (Promega). Similarly, NPM expression vector (pcDNA3.1/NPM) and antisense NPM vector (pCMV/ASNPM) were individually co-transfected with I2E containing pGL3 reporter vectors in subconfluent HepG2 cells. After 24 h, cells were washed with PBS, trypsinized, and replated in a 12-well plate at a density of 1.5 × 105 cells per well. After another 24 h, cells were treated with a PMA and cytokine combination for 12 h in the same manner as described above. Then the cells were lysed in passive lysis buffer (Promega); cell lysates were collected, and the samples were analyzed with the dual luciferase reporter assay system (Promega), in accordance with the manufacturer's instructions, in a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). Nuclear Extract Preparation—Nuclei were isolated from HepG2 cells as described by Dignam and co-workers (42Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). Subconfluent monolayers of cells were collected and centrifuged at 100 × g for 2 min at 4 °C. Cell pellets were resuspended in buffer A containing 10 mm HEPES (pH 7.9), 1.5 mm MgCl2, 10mm KCl, 0.5 mm dithiothretol (DTT), and 0.2 mm phenylmethylsulfonyl fluoride with the inclusion of protease inhibitors (pepstatin, aprotinin, and leupeptin) at a concentration of 1 μg/ml. Additionally, the phosphatase inhibitors NaF (5 mm) and Na3VO4 (1 mm) were included. The cell suspension was incubated on ice for 15 min. 12.5 μl of 10% Nonidet P-40 was added, and the mixture was vigorously vortexed for 15 s. The cytoplasmic and nuclear fractions were separated by centrifugation at 17,000 × g for 30 s at 4 °C. The nuclear pellet was subsequently resuspended in buffer B containing 20 mm HEPES (pH 7.9), 1.5 mm MgCl2, 420 mm NaCl, 0.2 mm EDTA, 35% glycerol, 0.5 mm DTT, 0.2 mm phenylmethylsulfonyl fluoride, and protease inhibitor (pepstatin, aprotinin, and leupeptin) at a concentration of 1 μg/ml, and incubated on ice for 20 min. Nuclear proteins in the supernatant fraction were collected by centrifugation at 14,000 × g for 2 min at 4 °C and stored at –80 °C until used. Nuclear extracts older than 2 weeks were not used in any of the experiments. Protein concentration was determined by a colorimetric assay using bovine serum albumin as the standard (Bio-Rad). Electrophoretic Mobility Shift Assay (EMSA)—The consensus double-stranded oligonucleotides of the NF-κB sequence (5′-GAGACTGGGGAATACCCCAGT-3′) were purchased from Promega. The oligonucleotides were radioactively labeled with [γ-32P]ATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The probes were purified on 20% native polyacrylamide gels. Double-stranded DNA probes were eluted overnight at 37 °C in 600 μl of TE buffer (pH 7.4) containing 10 mm Tris-HCl and 1 mm EDTA. The activity of a radiolabeled probe was counted and stored at –80 °C. Probes were used within 2 weeks after preparation. In each reaction, 5 μg of nuclear protein or 0.25 μg of affinity purified proteins and 6 μl of 5× binding buffer containing 20% (v/v) glycerol, 5 mm MgCl2, 2.5 mm EDTA, 5 mm DTT, 50 mm Tris-HCl (pH 7.5), 0.25 mg/ml poly(dI-dC), and 50,000 counts/min of radiolabeled probe were used. Samples were incubated at room temperature for 20 min. Supershift experiments to determine the components of the complex bound to the NF-κB consensus element following treatment were performed by adding 2 μg of the primary antibody (p50, p65, and c-Rel) to the binding reaction and extending the incubation to 1 h at room temperature. The reaction was stopped by the addition of 3 μlof10× DNA loading buffer (25 mm Tris-HCl, pH 7.5, 0.02% bromphenol blue, and 4% (v/v) glycerol). DNA-protein complexes were separated from unbound probes on 6% polyacrylamide native gel. Gels were dried and exposed to Kodak film at –80 °C for 6–12 h. Protein Purification by Affinity Chromatography—The consensus NF-κB oligonucleotide was synthesized and labeled with biotin at the 5′ end of the oligonucleotide (Invitrogen). The NF-κB binding sequence is 5′-GAGACTGGGGAATACCCCAGT-3′, in which biotin is cross-linked at the 5′ end of the primer. The complementary sequence, 5′-ACTGGGGTATTCCCCAGTCTC-3′, was synthesized and annealed with the biotin-labeled NF-κB oligomer in 10 mm HEPES (pH 7.8), 10 mm MgCl2, and 0.1 mm EDTA by heating at 65 °C for 5 min followed by cooling to room temperature. Double-stranded DNA formation was verified by 1.5% agarose gel electrophoresis, and only 95–100% annealed DNA was used to pull down NF-κB binding proteins. Extracts of nuclei treated with the PMA and cytokine (TNF-α and IL-1β) combination were suspended in binding buffer (12% glycerol, 60 mm KCl, 12 mm HEPES, pH 7.8, 0.12 mm EDTA, 5 mm MgCl2, 5 mm DTT, and 0.1% Triton X-100), and proteins were purified as described by Hagenbuchle et al. (43Hagenbuchle O. Wellauer P.K. Nucleic Acids Res. 1992; 20: 3553-3559Crossref Scopus (41) Google Scholar). Briefly, biotin-labeled double-stranded NF-κB oligonucleotide was added directly to the nuclear suspension in the presence of poly(dI-dC) to a final concentration of 0.25 mg/ml. Binding reactions were performed at 4 °C overnight with continuous gentle rotation in the presence of protease inhibitor (pepstatin, leupeptin, and aprotinin) at 1 μg/ml concentration. The reaction mixture was centrifuged at 3,000 rpm for 5 min to remove debris that may have clogged the affinity column. The affinity column was prepared using high-performance streptavidin-agarose beads (Amersham Biosciences). Briefly, a small column was packed with 500 μl of streptavidin-agarose-conjugated beads and washed five times with 1× binding buffer at 4 °C. The mixture of DNA-protein complex was passed through the column five times at a flow rate of 20 ml/h. The column was washed with binding buffer to remove nonspecific attachment of proteins. Additionally, the column was washed 5 times with 2-column volumes each of 1× binding buffer containing 60 mm KCl. Before elution, the column was further washed with binding buffer containing 150 mm KCl. NF-κB sequence-specific binding proteins or NF-κB interacting proteins were eluted with 450 mm KCl in 1× binding buffer. Purified proteins were then subjected to 10% SDS-polyacrylamide gel electrophoresis and visualized by silver staining. Silver Staining—Purified proteins were separated by SDS-polyacrylamide gel electrophoresis. Gels were preincubated in the fixing solution (50% methanol, 5% acetic acid) for 30 min with gentle shaking and then washed2hin distilled water with gentle shaking. Gels were sensitized with 0.02% Na2S2O3 solution for 1 min and washed 2 times with distilled water. Gels were incubated in 0.1% AgNO3 for 20 min, washed 2 times with distilled water, and then proteins were visualized by incubating the gel in 2% Na2CO3 and 0.04% formaldehyde solution. In-gel Digestion—Protein bands were excised from the silver-stained gel and washed with 100 mm NH4HCO3 dehydrated with acetonitrile and rehydrated with 100 mm NH4HCO3. This cycle was repeated three times. The gel slices were reduced with 10 mm DTT, 100 mm NH4HCO3 at 56 °C for 60 min. Samples were S-alkylated with 20 mm iodoacetamide at 25 °C in the dark for 30 min and vacuum dried. In-gel digestion of excised bands with trypsin (sequencing grade, Promega) and extraction of peptides were done according to Shevchenko et al. (44Shevchenko A. Wilm M. Vorm O. Jensen O.N. Podtelejnikov A.V. Neubauer G. Mortenson P. Mann M. Biochem. Soc. Trans. 1996; 24: 893-896Crossref PubMed Scopus (193) Google Scholar). Extracted peptide solutions were dried, rehydrated in 10 μl of 0.1% aqueous formic acid, and stored at –20 °C until analyzed. Mass Spectrometry—All mass spectra reported in this study were acquired by the University of Kentucky Mass Spectrometry Facility. LC/MS/MS spectra were acquired on a Finnigan LCQ “Classic” quadrupole ion trap mass spectrometer (Finnigan Co., San Jose, CA). Separations were performed with HP 1100 high performance liquid chromatography modified with a custom splitter to deliver 4 μl/min to a custom C18 capillary column (300 μm inner diameter × 15 cm), packed inhouse with Macrophere 300 5-μm C18 (Alltech Associates, Deerfield, IL). Gradient separations consisted of a 2-min isocratic step at 95% water and 5% acetonitrile (both phases contain 0.1% formic acid). The organic phase was increased to 20% acetonitrile over 8 min and then increased to 90% acetonitrile over 25 min; held at 90% acetonitrile for 8 min and then increased to 95% in 2 min; finally they were returned to the initial conditions in 10 min (total acquisition time 45 min with a 10 min recycle time). Tandem mass spectra were acquired in a data-dependent manner. Three microscans were averaged to generate the data-dependent full-scan spectrum. The most intense ion was subjected to tandem mass spectrometry, and three microscans were averaged to produce the MS/MS spectrum. Masses subjected to the MS/MS scan were placed on an exclusion list for 2 min. Data Base Analysis of Peptides—Tandem spectra used for protein identification from tryptic fragments were searched against the NCBI nonredundant protein data base using an in-house copy of the MASCOT search engine. For MS/MS spectra, the peptides were also assumed to be monoisotopic, oxidized at methionine residues, and carbamidomethylated at cysteine residues with a 0.8 Da mass tolerance. Probability-based MOWSE scores were estimated by comparing search results
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