Title: Persistent Tumor Necrosis Factor Signaling in Normal Human Fibroblasts Prevents the Complete Resynthesis of IκB-α
Abstract: Transcription factor NF-κB is normally sequestered in the cytoplasm, complexed with IκB inhibitory proteins. Tumor necrosis factor (TNF) and interleukin-1 induce IκB-α phosphorylation, leading to IκB-α degradation and translocation of NF-κB to the nucleus where it activates genes important in inflammatory and immune responses. TNF and interleukin-1 actions are typically terminated by desensitization, and IκB-α reappearance normally occurs within 30–60 min. We found that in normal human FS-4 fibroblasts maintained in the presence of TNF, IκB-α protein failed to return to base-line levels for up to 15 h. Removal of TNF at any time during the 15-h period resulted in complete IκB-α resynthesis, suggesting that IκB-α reappearance was prevented by continued TNF signaling. Long term exposure of FS-4 fibroblasts to TNF led to a persistent presence of IκB-α mRNA, sustained IκB kinase activation, continuous proteasome-mediated degradation of IκB-α, and sustained nuclear localization of NF-κB. Continuous exposure of FS-4 cells to TNF did not lead to a sustained activation of p38 or ERK mitogen-activated protein kinases, suggesting that not all TNF-induced signaling pathways are persistently activated. These findings challenge the notion that all cytokine-mediated signals are rapidly terminated by desensitization and illustrate the need to elucidate the process of deactivation of TNF-induced signaling. Transcription factor NF-κB is normally sequestered in the cytoplasm, complexed with IκB inhibitory proteins. Tumor necrosis factor (TNF) and interleukin-1 induce IκB-α phosphorylation, leading to IκB-α degradation and translocation of NF-κB to the nucleus where it activates genes important in inflammatory and immune responses. TNF and interleukin-1 actions are typically terminated by desensitization, and IκB-α reappearance normally occurs within 30–60 min. We found that in normal human FS-4 fibroblasts maintained in the presence of TNF, IκB-α protein failed to return to base-line levels for up to 15 h. Removal of TNF at any time during the 15-h period resulted in complete IκB-α resynthesis, suggesting that IκB-α reappearance was prevented by continued TNF signaling. Long term exposure of FS-4 fibroblasts to TNF led to a persistent presence of IκB-α mRNA, sustained IκB kinase activation, continuous proteasome-mediated degradation of IκB-α, and sustained nuclear localization of NF-κB. Continuous exposure of FS-4 cells to TNF did not lead to a sustained activation of p38 or ERK mitogen-activated protein kinases, suggesting that not all TNF-induced signaling pathways are persistently activated. These findings challenge the notion that all cytokine-mediated signals are rapidly terminated by desensitization and illustrate the need to elucidate the process of deactivation of TNF-induced signaling. nuclear factor κB tumor necrosis factor interleukin-1 inhibitor of NF-κB IκB kinase mitogen-activated protein kinase Dulbecco's modified Eagle's medium fetal bovine serum polyacrylamide gel electrophoresis extracellular signal-regulated kinase tumor necrosis factor receptor phosphate-buffered saline glutathioneS-transferase Tris-buffered saline TNF receptor-associated death domain TNF receptor-associated factor 2 receptor interacting protein TRAF-interacting protein silencer of death domain The transcription factor NF-κB1 is important in the regulation of genes involved in the immune and inflammatory responses, including genes encoding inflammatory cytokines (e.g. TNF, IL-1, IL-6, and IL-8), cell adhesion molecules (e.g. ICAM-1 and E-selectin), acute phase proteins, and many other proteins participating in host defenses, e.g. inducible nitric oxide synthase, major histocompatibility complex class I, and major histocompatibility complex class II (reviewed in Refs. 1Baldwin Jr., A.S. Annu. Rev. Immunol. 1996; 14: 649-683Crossref PubMed Scopus (5551) Google Scholar, 2Ghosh S. May M.J. Kopp E.B. Annu. Rev. Immunol. 1998; 16: 225-260Crossref PubMed Scopus (4584) Google Scholar, 3Rothwarf, D. M., and Karin, M. (1999) Science's STKE, www.stke.org/cgi/content/full/OC_sigtrans; 1995/5/rel.1–16.Google Scholar). The NF-κB family is comprised of several proteins, including p65/RelA, RelB, c-Rel, p50/p105, and p52/p100. These Rel family members share an ∼300 N-terminal amino acid sequence (the Rel homology domain), involved in subunit dimerization, DNA binding, and in the interaction of NF-κB proteins with members of the inhibitor κB (IκB) family of proteins. NF-κB is normally sequestered within the cytoplasm due to its interaction with IκB proteins, which bind to the NF-κB Rel homology domain and mask its nuclear localization sequence. Proteins comprising the IκB family include IκB-α, IκB-β, IκB-γ, IκB-ε, and Bcl-3. A variety of stimuli, including the inflammatory cytokines TNF and IL-1, and bacterial lipopolysaccharide cause the inducible phosphorylation of N-terminal serines in IκB (Ser-32 and Ser-36 in the IκB-α isoform) leading to the subsequent ubiquitination of neighboring lysines and the proteasome-mediated degradation of IκB-α (4Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1311) Google Scholar, 5Scherer D.C. Brockman J.A. Chen Z. Maniatis T. Ballard D.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11259-11263Crossref PubMed Scopus (501) Google Scholar, 6Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar). TNF initiates a signaling cascade that leads to IκB degradation by binding to cell surface TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Subsequent signaling (reviewed in Refs. 7Darnay B.G. Aggarwal B.B. J. Leukocyte Biol. 1997; 61: 559-566Crossref PubMed Scopus (168) Google Scholar and 8Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1123) Google Scholar)) occurs through the recruitment of cytosolic signaling proteins including TNF receptor-associated death domain protein (TRADD), TNF receptor-associated factor 2 (TRAF2), and receptor interacting protein (RIP), eventually leading to the activation of the IκB kinase complex (IKK). The 700–900-kDa IKK complex, comprising the IKKα, IKKβ, and IKKγ subunits, directly phosphorylates IκB (9Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 10Karin M. J. Biol. Chem. 1999; 274: 27339-27342Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 11Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (303) Google Scholar). Unmasking of the nuclear localization sequence following proteasome-mediated degradation of IκB permits NF-κB translocation to the nucleus, leading to transcriptional activation of a variety of genes. One of the genes transcriptionally activated by NF-κB is the gene encoding IκB-α (12Sun S.C. Ganchi P.A. Ballard D.W. Greene W.C. Science. 1993; 259: 1912-1915Crossref PubMed Scopus (952) Google Scholar), which upon its translation in the cytoplasm returns to the nucleus to dissociate NF-κB-DNA ternary complexes (13Turpin P. Hay R.T. Dargemont C. J. Biol. Chem. 1999; 274: 6804-6812Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), thereby turning off transcription of NF-κB-driven genes. In addition, newly synthesized IκB-α interacts with NF-κB dimers in the cytoplasm and prevents NF-κB translocation to the nucleus. This autoregulatory loop serves to terminate NF-κB activation so as to prevent the protracted expression of mediators of host defense and inflammation that are regulated by NF-κB. There exist other mechanisms to prevent the deleterious effects that would likely result from persistent cytokine signaling. In view of the well known acute toxicity and chronic inflammatory disorders resulting from TNF overexpression (14Tracey K.J. Cerami A. Annu. Rev. Med. 1994; 45: 491-503Crossref PubMed Scopus (948) Google Scholar, 15Feldmann M. Brennan F.M. Maini R. Int. Rev. Immunol. 1998; 17: 217-228Crossref PubMed Scopus (112) Google Scholar, 16Kollias G. Douni E. Kassiotis G. Kontoyiannis D. Immunol. Rev. 1999; 169: 175-194Crossref PubMed Scopus (235) Google Scholar), it is not surprising that upon their extended exposure to TNF cells often become desensitized to TNF action. The most common mechanism of desensitization involves the down-modulation of cell surface TNFR expression, which can occur by receptor-mediated endocytosis (17Tsujimoto M. Yip Y.K. Vilcek J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 7626-7630Crossref PubMed Scopus (354) Google Scholar, 18Imamura K. Spriggs D. Kufe D. J. Immunol. 1987; 139: 2989-2992PubMed Google Scholar, 19Higuchi M. Aggarwal B.B. J. Immunol. 1994; 152: 3550-3558PubMed Google Scholar), by TNFR shedding (20Brakebusch C. Varfolomeev E.E. Batkin M. Wallach D. J. Biol. Chem. 1994; 269: 32488-32496Abstract Full Text PDF PubMed Google Scholar, 21Porteu F. Nathan C. J. Exp. Med. 1990; 172: 599-607Crossref PubMed Scopus (377) Google Scholar, 22Madge L.A. Sierra-Honigmann M.R. Pober J.S. J. Biol. Chem. 1999; 274: 13643-13649Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), or by mechanisms that have not been fully characterized (23Decoster E. Vanhaesebroeck B. Boone E. Plaisance S. De Vos K. Haegeman G. Grooten J. Fiers W. J. Biol. Chem. 1998; 273: 3271-3277Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Alternatively, TNF signaling may be blocked at some point along the intracellular signaling cascade by factors that inhibit the association or function of signaling intermediates in the TNF pathway, such as TRAF-interacting protein (TRIP (24Lee S.Y. Lee S.Y. Choi Y. J. Exp. Med. 1997; 185: 1275-1285Crossref PubMed Scopus (170) Google Scholar)) and the recently identified silencer of death domain (SODD) protein (25Jiang Y. Woronicz J.D. Liu W. Goeddel D.V. Science. 1999; 283: 543-546Crossref PubMed Scopus (343) Google Scholar). In most cells, TNF induces IκB-α degradation within 15 min, which is followed by IκB-α resynthesis and complete reappearance of IκB-α protein within approximately 30 min to 2 h (12Sun S.C. Ganchi P.A. Ballard D.W. Greene W.C. Science. 1993; 259: 1912-1915Crossref PubMed Scopus (952) Google Scholar, 26de Martin R. Vanhove B. Çhen Q. Hofer E. Csizmadia V. Winkler H. Bach F.H. EMBO J. 1993; 12: 2773-2779Crossref PubMed Scopus (290) Google Scholar). Complete reappearance of IκB-α protein commonly occurs even if cells are maintained in the continuous presence of TNF (27Beg A.A. Finco T.S. Nantermet P.V. Baldwin Jr., A.S. Mol. Cell. Biol. 1993; 13: 3301-3310Crossref PubMed Google Scholar, 28Schwenger P. Alpert D. Skolnik E.Y. Vilcek J. Mol. Cell. Biol. 1998; 18: 78-84Crossref PubMed Google Scholar, 29Alpert D. Schwenger P. Han J. Vilcek J. J. Biol. Chem. 1999; 274: 22176-22183Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and this has been ascribed to the previously mentioned autoregulatory NF-κB loop and to cellular desensitization to TNF action. We show here that in normal human diploid FS-4 fibroblasts stimulated with TNF, IκB-α is rapidly degraded but IκB-α reappearance is incomplete because cells do not become desensitized to TNF signaling, and newly synthesized IκB-α continues to be phosphorylated and degraded. Continued signaling by TNF in these cells is evidenced by persistent activation of the IKK complex, ongoing proteasome-mediated IκB-α degradation, continued nuclear localization of p65/RelA, and the persistent presence of IκB-α mRNA. We also demonstrate that not all TNF signaling pathways are persistently activated in FS-4 cells, as TNF did not produce a sustained activation of the ERK and p38 MAP kinases. Our results challenge the paradigm that TNF signaling (and cytokine signaling in general) is always rapidly terminated by desensitization. Normal human FS-4 diploid fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% heat-inactivated fetal bovine serum (FBS). For use in experiments cells at passage 14 were split into 10-cm plates and serum-starved for 2–5 days in DMEM containing 0.25% FBS. COS-1 African Green Monkey kidney cells were maintained in DMEM containing 10% FBS and were serum-starved for 1–2 days in DMEM, 0.5% FBS prior to stimulation. DMEM was purchased from Life Technologies, Inc. Recombinant human TNF-α was a gift from Masafumi Tsujimoto of the Suntory Institute for Biomedical Research (Osaka, Japan). Recombinant human IL-1α was obtained from the NCI, National Institutes of Health, Bethesda, MD. The TNFR1-specific TNF mutein (Trp-32/Thr-86) was a gift of Dr. Werner Lesslauer (Hoffmann-La Roche). The TNFR2-specific TNF mutein was a gift from Dr. B. Aggarwal (The University of Texas, MD Anderson Cancer Center, Houston). The proteasome inhibitor MG132 (benzyloxycarbonyl-Leu-Leu-Leu-CHO) was obtained from Biomol Research Laboratories (Plymouth Meeting, PA). Rabbit polyclonal antibodies against IκB-α, p38, ERK, p65/RelA, and IKKα were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-p38 (a rabbit polyclonal antibody that detects the phosphorylated Thr-180 and Tyr-182 residues in p38) and anti-phospho-IκB-α (which detects the phosphorylated Ser-32 residue in IκB-α) were purchased from New England Biolabs (Beverly, MA). Anti-phospho-ERK was purchased from Promega (Madison, WI). Texas Red-conjugated goat anti-rabbit IgG for use in immunolocalization studies was purchased from Vector Laboratories (Burlingame, CA). Protein A-Sepharose was purchased fromZymed Laboratories Inc. (South San Francisco, CA), and protein A/G-agarose from Santa Cruz Biotechnology. A GST-IκB-α construct encoding the N-terminal 62 amino acids of the human IκB-α sequence was provided by Deborah Alpert (New York University School of Medicine). Paraformaldehyde, dimethyl sulfoxide, Hoechst nuclear dye, and Triton X-100 were obtained from Sigma. All radioactive isotopes, including [α-32P]dCTP, [γ-32P]dATP, and35S-Express Protein Labeling Mix, were purchased from NEN Life Science Products. Immunoblotting analysis was performed as described previously (30Schwenger P. Alpert D. Skolnik E.Y. Vilcek J. J. Cell. Physiol. 1999; 179: 109-114Crossref PubMed Scopus (45) Google Scholar). Briefly, cells were lysed in an Nonidet P-40-based lysis buffer (1% Nonidet P-40, 50 mm HEPES, pH 7.5, 100 mm NaCl, 2 mm EDTA, 1 mmpyrophosphate, 10 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, and 100 mm sodium fluoride). Lysates were boiled for 5 min in denaturing sample buffer, separated via SDS-polyacrylamide gel electrophoresis, transferred to Immobilon-P membranes (Millipore), blocked for 1 h at room temperature in Tris-buffered saline (TBS, 10 mm Tris, pH 7.4, 150 mm NaCl) with 5% milk, and incubated for 16 h with shaking with the appropriate antibody at 4 °C. All antibody dilutions were prepared in TBS containing 5% bovine serum albumin and 0.02% sodium azide. Anti-IκB-α was used at a dilution of 1:250, and anti-phospho-IκB-α was used at 1:1000. Antibodies against both p38 and phospho-p38 were used at a 1:1000 dilution, anti-ERK was used at 1:500, and anti-phospho-ERK was used at 1:20,000. Detection was accomplished with the use of horseradish peroxidase-conjugated goat anti-rabbit IgG or horseradish peroxidase-protein A secondary antibodies (Bio-Rad), both at 1:3000 in TBS, 5% milk for 1 h at room temperature. Bands were visualized using a chemiluminescence substrate kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD). Cells were lysed in denaturing solution (4 m guanidine isothiocyanate, 25 mmsodium citrate, 0.5% Sarkosyl, and 0.7% β-mercaptoethanol). RNA was extracted using phenol/chloroform as described previously (31Gerecitano J. Perle M.A. Vilcek J. J. Interferon Cytokine Res. 1999; 19: 393-405Crossref PubMed Scopus (8) Google Scholar). RNA pellets were dissolved in formamide, and 12–15 μg of RNA was electrophoretically separated and transferred via capillary action onto nylon membranes (Nytran, Schleicher & Schuell). RNA was cross-linked to the membranes, and membranes were prehybridized for 45 min at 68 °C with QuikHyb (Stratagene). Murine IκB-α and human glyceraldehyde-3-phosphate dehydrogenase cDNA probes (31Gerecitano J. Perle M.A. Vilcek J. J. Interferon Cytokine Res. 1999; 19: 393-405Crossref PubMed Scopus (8) Google Scholar) were labeled with [α-32P]dCTP using the RediPrime random primer labeling kit (Amersham Pharmacia Biotech) for 30 min at 37 °C. Labeled probes were boiled with salmon sperm DNA, added to the QuikHyb solution, and hybridized with the membranes for 1–3 h at 68 °C. Membranes were then washed in 2× SSC (0.3 mNaCl, 30 mm sodium citrate) with 0.1% SDS at room temperature for 30 min, followed by washes in 1× SSC, 0.1% SDS at room temperature, 1× SSC, 0.1% SDS at 65 °C, and finally 0.2× SSC, 0.1% SDS at 65 °C. Autoradiography was then performed. Whole cell lysates were prepared as described for immunoblotting. The IκB kinase (IKK) complex was immunoprecipitated with an antibody to IKKα (3 μg) for 1–2 h at 4 °C, and immunocomplexes were collected with protein A/G-agarose (1–2 h, 4 °C). The agarose beads were then washed three times with Nonidet P-40 lysis buffer and once with kinase buffer (20 mm HEPES, pH 7.6, 20 mmMgCl2, 20 mm β-glycerophosphate, 10 mm sodium fluoride, 0.2 mm sodium orthovanadate, and 0.2 mm dithiothreitol). Half of the immunoprecipitated fraction was separated via SDS-PAGE and immunoblotted with the IKKα antibody (1:500) to ascertain that equal amounts of the IKK complex were immunoprecipitated for all treatment conditions. The remainder of the immunoprecipitated IKK complexes was then incubated with a kinase buffer mixture containing 10 μm ATP, 10 μCi of [γ-32P]ATP/sample, and 5 μg of GST-IκB-α/sample for 30 min at 30 °C. The kinase reaction was terminated by boiling the samples in reducing sample buffer. Samples were separated on 12% SDS-PAGE; the gel was dried, and autoradiography was performed. To quantify their intensity, autoradiography bands were subjected to densitometric analysis (NIH Image 1.61). Serum-starved FS-4 fibroblasts were plated on glass coverslips precoated with 0.1% gelatin (150,000 cells/coverslip) in 24-well plates. After the appropriate treatments cells were washed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature, and permeabilized with PBS, 0.2% Triton X-100 for 20 min at room temperature. The permeabilized cells were blocked in TBS, 5% bovine serum albumin (1–2 h, room temperature), and then incubated with anti-p65/RelA antibody (1 μg/ml final concentration) for 1–2 h at room temperature. Cells were then washed with PBS, 0.1% Triton X-100, and incubated with a Texas Red-conjugated goat anti-rabbit IgG secondary antibody (12 μg/ml final concentration) for 1–2 h at room temperature while shielded from light. Cells were next washed with PBS, 0.1% Triton X-100 and with PBS, counterstained with Hoechst nuclear dye (1 μg/ml) for 10–15 min, and washed with PBS. Coverslips were then mounted on microscope slides and examined by fluorescence microscopy. Serum-starved FS-4 cells were either left untreated or stimulated with TNF as indicated. The cells were then washed and incubated for 1 or 2 h in methionine- and cysteine-free labeling DMEM (Cellgro) containing 0.5 mCi of 35S-Express Protein Labeling Mix. The labeling medium was then removed, and the cells were either immediately lysed in Nonidet P-40 lysis buffer (for control and t = 0 time points) or “chased” in complete DMEM, 0.25% FBS medium for the indicated times. Cells were then lysed, and supernatants were precleared with normal rabbit serum (Santa Cruz Biotechnology) and protein A-Sepharose. IκB-α was then immunoprecipitated from supernatants with a rabbit polyclonal antibody against IκB-α (4 μg/sample, 1–2 h, 4 °C) and collected with protein A-Sepharose (1–2 h, 4 °C). The beads were washed three times with RIPA buffer (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.4% SDS, 50 mm Tris, pH 7.5). The beads were then boiled in the presence of reducing sample buffer and separated on 10% SDS-PAGE, and the gel was dried and exposed to film. The autoradiography bands were subjected to densitometric analysis, and all values were normalized to and expressed as a fraction of thet = 0 value (designated as “mean normalized arbitrary densitometric units”) and plotted versus time. Logarithmic or exponential curves were fit to the data points and used to calculate estimates of the IκB-α half-life under different treatment conditions. A fraction of the immunoprecipitate was separated by SDS-PAGE and immunoblotted with the IκB-α antibody to ascertain that equal amounts of lysate were loaded for all treatment conditions (not shown). Normal human diploid FS-4 fibroblasts were treated with either TNF or IL-1 for periods ranging from 15 min to 15 h. The cells were then lysed, and the presence of IκB-α was determined by immunoblot analysis (Fig.1). A 15-min exposure to either TNF or IL-1 led to the complete disappearance of IκB-α. Upon further incubation of cells in the presence of TNF or IL-1, IκB-α gradually reappeared. However, in cells incubated with TNF, IκB-α protein remained below the level seen in unstimulated cells. In contrast, in cells treated continuously with IL-1, the reappearance of IκB-α protein over time was more complete, with IκB-α reaching a level similar to that seen in unstimulated cells by about 4 h. To explain the difference in the extent of IκB-α reappearance in cells treated with TNF and IL-1, we considered the possibility that IL-1 might be more rapidly depleted from the culture medium than TNF. We therefore treated cells with either TNF or IL-1 for 15 h and then restimulated cells with fresh TNF or IL-1 for 15 min (Fig.2). When fibroblasts were restimulated with the homologous ligand, there was no significant decrease in IκB-α, whereas subsequent treatment with the heterologous ligand (i.e. addition of IL-1 to TNF-treated cultures or addition of TNF to IL-1-treated cultures) led to the complete disappearance of IκB-α in 15 min. These findings indicate that following 15 h of continuous treatment with either TNF or IL-1, FS-4 cells are refractory to restimulation with the homologous ligand but are responsive to restimulation with the heterologous ligand. These results further suggested that the complete IκB-α reappearance in IL-1-treated cells was not due to depletion of IL-1 from the culture medium or the loss of its biological activity. As restimulation of cells with the heterologous ligand led to a rapid and complete IκB-α degradation, it is also reasonable to conclude that the apparatus necessary for the phosphorylation, ubiquitination, and degradation of IκB-α is intact in cells treated for 15 h with TNF or IL-1. Analysis of IκB-α levels in cells treated for various lengths of time with both TNF and IL-1 simultaneously showed that results at the early time points resembled those seen in TNF-treated cells, whereas the 15-h result was similar to that seen in cells treated with IL-1 alone (data not shown). We next sought to determine whether the failure of complete IκB-α reappearance in TNF-treated cells was reversible. We therefore treated FS-4 cells with TNF for periods ranging from 15 min to 15 h. In one group of cultures, TNF was removed after the initial 15 min treatment (i.e. after IκB-α disappearance), and the cells were then incubated in TNF-free medium for the remainder of the indicated times, whereas other groups were maintained in the continuous presence of TNF until the end of the incubation period (Fig.3 A). This experiment showed that IκB-α protein returned to levels seen in the control cultures by about 2 h following the removal of TNF. In contrast, in the continuous presence of TNF, IκB-α levels remained below control levels for up to 15 h. TNF is known to signal through two cell surface receptors, with TNFR1 being the main signaling receptor (7Darnay B.G. Aggarwal B.B. J. Leukocyte Biol. 1997; 61: 559-566Crossref PubMed Scopus (168) Google Scholar, 8Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1123) Google Scholar). To determine whether TNFR1 accounts for the failure of complete IκB-α reappearance in TNF-treated FS-4 cells, we repeated the preceding experiment with a TNF mutein protein that can bind to TNFR1 but not to TNFR2 (32Van Ostade X. Vandenabeele P. Everaerdt B. Loetscher H. Gentz R. Brockhaus M. Lesslauer W. Tavernier J. Brouckaert P. Fiers W. Nature. 1993; 361: 266-269Crossref PubMed Scopus (166) Google Scholar, 33Schwenger P. Skolnik E.Y. Vilcek J. J. Biol. Chem. 1996; 271: 8089-8094Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The results of this experiment (Fig. 3 B) were virtually identical to the findings obtained with wild-type TNF. A TNFR2-selective mutant TNF protein failed to induce IκB-α degradation in the FS-4 cells (not shown). Together, these data suggested that TNFR1 plays a major, if not an exclusive, role in the ability of TNF to modulate the reappearance of IκB-α following its TNF-driven degradation. Our next series of experiments was designed to investigate the mechanism whereby complete IκB-α reappearance was curtailed in the continuous presence of TNF. In one group of experiments we used Northern blot analysis to study steady-state IκB-α mRNA levels in FS-4 cells incubated for periods ranging from 30 min to 18 h, either in the continuous presence of TNF or with TNF removed from the cultures following an initial 15-min stimulation. Peak steady-state IκB-α mRNA levels were reached at 1 h under both continuous and “pulse treatment” conditions (Fig. 4). In the continuous presence of TNF, IκB-α mRNA was sustained near this peak level for up to 18 h. In contrast, IκB-α mRNA levels decreased rapidly upon TNF removal from the culture medium. This result showed that the failure of IκB-α protein to return to control levels in the continuous presence of TNF was not due to a decreased availability of IκB-α mRNA. In fact, our observation that IκB-α mRNA levels were sustained in the continuous presence of TNF, but not when TNF was removed after 15 min, is the opposite of what is seen at the protein level (Fig. 3 A). Increased IκB-α mRNA levels in cells treated continually with TNF probably reflect persistent TNF signaling, with ongoing TNF-mediated NF-κB activation likely to be driving the transcription of IκB-α under these conditions. We then considered the possibility that ongoing proteasome-mediated degradation of IκB-α in cells treated continually with TNF may explain the failure of complete IκB-α reappearance, despite the presence of higher levels of IκB-α mRNA in such cells. FS-4 cells were stimulated with TNF for periods ranging from 5 min to 15 h and treated with the proteasome inhibitor MG132 as indicated (Fig. 5). Cell lysates were subjected to immunoblot analysis with antibodies directed against IκB-α and N-terminally phosphorylated IκB-α. Pretreatment of cells with MG132 decreased TNF-induced IκB-α degradation (Fig. 5, lanes 4and 6, upper and middle panels). Importantly, in cells incubated with TNF for 1, 4, and 15 h, MG132 treatment resulted in the appearance of the phosphorylated IκB-α species as well as in greatly increased levels of IκB-α. These findings indicate that proteasome-mediated degradation of inducibly phosphorylated IκB-α is likely to be important in the failure of IκB-α to return to control levels in cells maintained in the presence of TNF. Seeking further evidence for persistent TNF signaling, we examined the activity of the IKK complex. The IKK complex includes the IKKα and IKKβ subunits and is responsible for the inducible phosphorylation of IκB-α (3Rothwarf, D. M., and Karin, M. (1999) Science's STKE, www.stke.org/cgi/content/full/OC_sigtrans; 1995/5/rel.1–16.Google Scholar, 9Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 10Karin M. J. Biol. Chem. 1999; 274: 27339-27342Abstract Full Text Full Text PDF PubMed Scopus (618) Google Scholar, 11Zandi E. Karin M. Mol. Cell. Biol. 1999; 19: 4547-4551Crossref PubMed Scopus (303) Google Scholar). FS-4 cells were incubated for periods ranging from 15 min to 15 h, either in the continuous presence of TNF or with TNF present only during the initial 15 min. The IKK complex was immunoprecipitated, incubated with GST-IκB-α in an in vitro kinase reaction, then resolved on SDS-PAGE, and subjected to autoradiography (Fig.6). Results from this experiment indicated that IKK was very strongly activated at 15 min after TNF stimulation. In cells maintained in the continuous presence of TNF, IKK activity decreased gradually at later times but was still detectable after 15 h. Densitometric analysis revealed that the strength of the phosphorylated band at 15 h was roughly 6% of the level observed after a 15-min stimulation. IKK-mediated IκB-α phosphorylation decreased faster when TNF was removed after a 15-min stimulation, with no kinase activity detectable by 15 h. To investigate further the effect of TNF stimulation on NF-κB activation, we examined the subcellular localization of p65/RelA in TNF-treated cells (Fig. 7). Cells were treated with TNF for the times shown, and where indicated TNF was removed after an initial 20-min stimulation. Fixed and permeabilized cells were incubated with an antibody against p65/RelA and a Texas Red-conjugated secondary antibody and were examined by fluorescence microscopy. Whereas p65/RelA was present in the cytoplasm of untreated cells, cells treated with TNF for 20 min, 1 h, or 15 h showed p65/RelA exclusively in the nucleus, consistent with a persist