Title: Lymphotoxin ॆ Receptor Induces Sequential Activation of Distinct NF-κB Factors via Separate Signaling Pathways
Abstract: Lymphotoxin ॆ receptor (LTॆR)-induced activation of NF-κB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IκB kinase (IKK)ॆ- and IKKγ-dependent degradation of IκBα and resulted in the rapid but transient activation of primarily RelA-containing NF-κB dimers. The alternative or second pathway proceeded via NF-κB-inducing kinase (NIK)-, IKKα-, and protein synthesis-dependent processing of the inhibitory NF-κB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-κB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKγ regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-κB-induced resynthesis of IκBα, resulted in a pronounced temporal change in the nature of the NF-κB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-κB activity in mouse embryo fibroblasts in response to LTॆR stimulation. Based on the phenotype of mice deficient in various components of the LTॆR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures. Lymphotoxin ॆ receptor (LTॆR)-induced activation of NF-κB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IκB kinase (IKK)ॆ- and IKKγ-dependent degradation of IκBα and resulted in the rapid but transient activation of primarily RelA-containing NF-κB dimers. The alternative or second pathway proceeded via NF-κB-inducing kinase (NIK)-, IKKα-, and protein synthesis-dependent processing of the inhibitory NF-κB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-κB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKγ regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-κB-induced resynthesis of IκBα, resulted in a pronounced temporal change in the nature of the NF-κB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-κB activity in mouse embryo fibroblasts in response to LTॆR stimulation. Based on the phenotype of mice deficient in various components of the LTॆR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures. nuclear factor-κB inhibitor of κB IκB kinase complex NF-κB-inducing kinase lymphotoxin lymphotoxin ॆ receptor alymphoplasia tumor necrosis factor mouse embryo fibroblast electrophoretic mobility shift assay B cell-activating factor belonging to the TNF family NF-κB1 transcription factors are critical mediators in the fight of the host against invading pathogens (reviewed in Refs. 1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 3Caamaño J. Hunter C.A. Clin. Microbiol. Rev. 2002; 15: 414-429Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). These factors are integral parts of the innate machinery that translates initial detection of foreign pathogens, for example by epithelial cells, into activation of these cells, including production of chemokines and cytokines to in turn attract and activate professional immune cells. The innate system further involves NF-κB factors to produce antipathogenic effectors as well as chemokines and cytokines to mediate evolving cell-cell communications needed to coordinate responses. Depending on the exact nature of the initial innate response, NF-κB factors then help to develop the appropriate adaptive responses by lymphocytes. In the final phase of the immune response, NF-κB factors have important roles during the expansion and differentiation of lymphocytes involved in the adaptive response. Beyond the innate and adaptive antipathogenic responses, NF-κB factors are also essential during development and maintenance of lymphoid organ structures (6Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Google Scholar, 7Caamaño J.H. Rizzo C.A. Durham S.K. Barton D.S. Raventos-Suarez C. Snapper C.M. Bravo R. J. Exp. Med. 1998; 187: 185-196Google Scholar), and they make important contributions during the development of hematopoietic cells, including B cells and osteoclasts (8Franzoso G. Carlson L. Xing L. Poljak L. Shores E.W. Brown K.D. Leonardi A. Tran T. Boyce B.F. Siebenlist U. Genes Dev. (1997). 1997; 11: 3482-3496Google Scholar, 9Gugasyan R. Grumont R. Grossmann M. Nakamura Y. Pohl T. Nesic D. Gerondakis S. Immunol. Rev. 2000; 176: 134-140Google Scholar). To carry out its diverse physiologic roles, NF-κB factors not only help to induce expression of various factors and effectors, but depending on the cellular context, they also transcriptionally induce proteins that function to protect cells from apoptosis and that help to stimulate proliferation (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar, 10Karin M. Lin A. Nat. Immunol. 2002; 3: 221-227Google Scholar). NF-κB is a collective term for a family of dimeric complexes comprised of combinations of five polypeptides, RelA, c-Rel, RelB, p50/NF-κB1, and p52/NF-κB2. p50 and p52 are the N-terminal parts of the longer p105/NF-κB1 and p100/NF-κB2 proteins, respectively, and they are generated by proteolytic processing (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar,4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). High levels of p50 are produced constitutively by a cotranslational mechanism. In contrast, usually only small amounts of p52 exist in cells, but higher amounts may be induced by select signals. To activate NF-κB, appropriate environmental signals must bring about the release of NF-κB dimers from their bound cytoplasmic inhibitors, in particular from the prototypical inhibitor IκBα and its close relatives, IκBॆ and IκBκ (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). NF-κB factors are in addition subject to various direct and indirect mechanisms that modulate their ability to stimulate transcription, dependent also on promoter context (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar), but the release from the inhibitors is a first and necessary step in the activation process. Most of the NF-κB activation signals, and in particular inflammatory cytokines, such as TNFα and IL-1, induce the phosphorylation of the IκBs followed by the rapid ubiquitin- and proteasome-mediated degradation of the inhibitors, thus freeing NF-κB dimers to migrate to the nucleus to initiate gene transcription (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). IκBs are phosphorylated on two conserved serines by the IκB kinase (IKK) complex. IKKs consist of the catalytic subunits, IKKα and IKKॆκ, and the regulatory subunit IKKγ (also known as Nemo). Most signals have been shown to activate NF-κB by the classical, IKK-dependent pathway and, in particular, to be dependent on the IKKॆ catalytic and IKKγ/Nemo regulatory subunit to bring about the degradation of small IκB inhibitors (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar, 11Smahi A. Courtois G. Rabia S.H. Doffinger R. Bodemer C. Munnich A. Casanova J.L. Israel A. Hum. Mol. Genet. 2002; 11: 2371-2375Google Scholar). In addition to the small IκBs, the long forms of the NF-κB1 and NF-κB2 proteins, p105 and p100, can also act as cytoplasmic inhibitors of bound Rel proteins due to the presence of IκB-like inhibitory ankyrin domains in their C-terminal halves (1Ghosh S. Karin M. Cell. 2002; 109: S81-S96Google Scholar, 2Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar, 4Karin M. Ben-Neriah Y. Annu. Rev. Immunol. 2000; 18: 621-663Google Scholar, 5Brown K. Claudio E. Siebenlist U. Targeted Therapies in Rheumatology. Martin Dunitz Ltd., London, UK2002: 381-401Google Scholar). p105 may be completely degraded in response to some signals in a manner similar to that of small IκBs, including IKKॆ/IKKγ-induced phosphorylation of two serines embedded in a small IκB-like phosphorylation motif (12Heissmeyer V. Krappmann D. Hatada E.N. Scheidereit C. Mol. Cell. Biol. 2001; 21: 1024-1035Google Scholar). Recently, a second or alternative signaling path has been reported to liberate NF-κB activity via induced processing of p100 inhibitor (13Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Google Scholar, 14Senftleben U. Cao Y. Xiao G. Greten F.R. Krähn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Google Scholar). Although physiologic signals for this pathway were not reported, processing was mediated by the NF-κB-inducing kinase (NIK) and IKKα. In the present report, we demonstrate that physiologic signaling via the lymphotoxin ॆ receptor (LTॆR) in stromal cells induced the degradation of IκBα via the classical pathway, and it induced processing of p100 via an alternative pathway. p100 processing was shown to be dependent on NIK and IKKα but independent of IKKॆ and IKKγ/Nemo. Therefore, the p100 processing pathway was entirely independent of the IKK complex, not just of the IKKॆ kinase subunit. We also demonstrate that transient activation of the classical pathway caused the transient activation of p50-RelA dimers, whereas the delayed and protein synthesis-dependent p100 processing led to the delayed and sustained liberation of p50-RelB and p52-RelB complexes. We also provide an explanation and supporting evidence for how p100 processing liberated p50-RelB complexes. IKKα−/− and IKKॆ−/− mouse embryonic fibroblasts (MEFs) were kindly provided by Drs. Q. Li and I. M. Verma, and IKKγ−/− MEFs were kindly provided by Drs. M. Pasparakis and K. Rajewsky. NF-κB1−/− and NF-κB2−/− MEFs were kindly provided by Dr. E. Claudio. To prepare embryonic fibroblasts from wild-type and aly/alymice, 12-day-old embryos were dissected, heads and inner organs were removed, and remaining parts were minced, filtered, and subjected to trypsin (0.257) digestion for 10 min at 37 °C. The resulting cells were filtered and washed in Dulbecco's modified Eagle's medium (Invitrogen). Fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 107 heat-inactivated fetal bovine serum and antibiotics. Fibroblasts were plated into 6-well plates at 105 cells/well 24 h prior To Whom It May Concern: stimulation. After treatment, cells were lysed in 100 mm Tris, pH 6.8, 47 SDS, 207 glycerol, sonicated, and subjected to SDS-PAGE. MEFs were transfected with LipofectAMINE 2000 (Invitrogen). Cells were analyzed 24 h after transfection. Expression vectors for IKKα, IKKॆ, and NIK were kindly provided by Drs. R. Geleziunas and W. C. Greene (15Geleziunas R. Ferrell S. Lin X. Mu Y. Cunningham Jr., E.T. Grant M. Connelly M.A. Hambor J.E. Marcu K.B. Greene W.C. Mol. Cell. Biol. 1998; 18: 5157-5165Google Scholar). Human p100 was excised from a previously described expression vector (16Bours V. Burd P.R. Brown K. Villalobos J. Park S. Ryseck R.P. Bravo R. Kelly K. Siebenlist U. Mol. Cell. Biol. 1992; 12: 685-695Google Scholar) and inserted into pcRSV (Invitrogen). For each nuclear preparation, 5 × 105 cells were plated 24 h prior to stimulation. Stimulation was done under serum-free conditions. Following stimulation, nuclear and cytoplasmic extracts were prepared essentially as described (17Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar). Briefly, fibroblasts were mechanically removed, washed twice in phosphate-buffered saline, and resuspended in 400 ॖl of low salt buffer (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mmEGTA, supplemented with protease and phosphatase inhibitors). After a 15-min incubation on ice, Triton-X-100 was added to a final concentration of 0.67, and the suspension was vigorously vortexed for 10 s. The nuclei were pelleted, and the supernatant served as cytoplasmic extract. The pelleted nuclei were resuspended in 50 ॖl of high salt buffer (20 mm Hepes, pH 7.9, 400 mmNaCl, supplemented with protease and phosphatase inhibitors) and incubated for an additional 15 min on ice. 2.5 ॖl of this preparation were used in DNA binding reactions. An NF−κB-binding site from the κ light chain enhancer was used as a probe: 5′-AGTTGAGGGGACTTTCCCAGGC-3′ (Promega, Madison, WI). Oct1 oligonucleotides were used in controls: 5′-TGTCGAATGCAAATCACTAGAA-3′. Complementary and annealed oligonucleotides were end-labeled with [γ– 32P]ATP. Approximately 20,000 cpm of probe were used per assay. The binding reaction was carried out at room temperature for 15 min in a total volume of 25 ॖl containing 10 mm Tris, pH 7.5, 50 mm NaCl, 1 mmMgCl2, 0.5 mm dithiothreitol, 50 ॖg/ml poly(dI-dC)·poly(dI-dC), 47 glycerol. For supershift analyses, nuclear extracts were preincubated with antibodies for 30 min on ice prior to adding the probe. For Western blot analyses, 20 ॖl of each preparation were subjected to SDS-PAGE. The efficiency of the nuclear-cytoplasmic fractionation was confirmed in several ways, including by the fact that entry of factors was dependent on stimulation and by the fact that RelA did not enter nuclei in NEMO-deficient cells, whereas RelB did (see Fig.6, A andC). For immunoprecipitation, cytoplasmic and nuclear preparations were generated from 1.5 × 106 cells/condition. These extracts were adjusted to 150 mm NaCl and mixed with anti-RelB antibodies (SC-848) or anti-NEMO antibodies (SC-8330) (Santa Cruz Biotechnology, Santa Cruz, CA) as a negative control that had been conjugated to agarose beads (Pierce). Following a 2-h incubation at 4 °C, the agarose beads were washed four times in 150 mmNaCl, 25 mm Hepes, pH 7.3, 107 glycerol, 17 Triton-X-100, and the immunoprecipitated preparations were subjected to SDS-PAGE. The agonistic monoclonal anti-murine LTॆR antibodies were kindly provided by Dr. J. Browning and used at 10 ॖg/ml. For EMSA supershift experiments, the following antibodies to detect murine proteins were used: anti-RelA (SA-171) (Biomol, Plymouth Meeting, PA); anti-RelB (SC-226X), anti-c-Rel (SC-71X), anti-NF-κB1 (SC-114X), anti-NF-κB2 (SC-848X) (Santa Cruz Biotechnology). For Western analyses, the following antibodies were used: anti-IκBॆ (SC-945), anti-IκBα (SC-371), anti-RelB (SC-226), anti-c-Rel (SC-71) (Santa Cruz Biotechnology). Polyclonal anti-murine RelA, anti-murine p105, and anti-human p100 antibodies (also detects murine p100) were raised against the 13 C-terminal amino acids (RelA), the 15 N-terminal amino acids (p105), and the 398 N-terminal amino acids (p100), respectively. TNFα was purchased from PeproTech (Rocky Hill, NJ); Light and platelet-derived growth factor (PDGF-BB) were purchased from R&D Systems (Minneapolis, MN). Light was used at 20 ng/ml. The IKKॆ-specific inhibitor PS1145 was kindly provided by Dr. J. Adams, Millennium Pharmaceuticals (Cambridge, MA). To inhibit protein synthesis, cells were pretreated with 50 ॖm cycloheximide (Sigma) for 30 min prior to stimulation. NF-κB2-deficient mice are impaired in their splenic microarchitecture, they lack Peyer's patches, and they are severely impaired in lymph node formation, primarily due to defects within the stromal compartment (6Franzoso G. Carlson L. Poljak L. Shores E.W. Epstein S. Leonardi A. Grinberg A. Tran T. Scharton-Kersten T. Anver M. Love P. Brown K. Siebenlist U. J. Exp. Med. 1998; 187: 147-159Google Scholar, 7Caamaño J.H. Rizzo C.A. Durham S.K. Barton D.S. Raventos-Suarez C. Snapper C.M. Bravo R. J. Exp. Med. 1998; 187: 185-196Google Scholar, 18Poljak L. Carlson L. Cunningham K. Kosco-Vilbois M.H. Siebenlist U. J. Immunol. 1999; 163: 6581-6588Google Scholar, 19Paxian S. Merkle H. Riemann M. Wilda M. Adler G. Hameister H. Liptay S. Pfeffer K. Schmid R.M. Gastroenterology. 2002; 122: 1853-1868Google Scholar). These deficiencies in secondary lymphoid organs, which also include loss of follicular dendritic cell networks, in turn contribute to defective immune responses in these mutant mice. Similar deficiencies have been noted inaly/aly mice (20Matsumoto M. Iwamasa K. Rennert P.D. Yamada T. Suzuki R. Matsushima A. Okabe M. Fujita S. Yokoyama M. J. Immunol. 1999; 163: 1584-1591Google Scholar, 21Yamada T. Mitani T. Yorita K. Uchida D. Matsushima A. Iwamasa K. Fujita S. Matsumoto M. J. Immunol. 2000; 165: 804-812Google Scholar) and in mice lacking lymphotoxin ॆ (LTॆ) receptor or its ligands (LTακॆ and Light) (20Matsumoto M. Iwamasa K. Rennert P.D. Yamada T. Suzuki R. Matsushima A. Okabe M. Fujita S. Yokoyama M. J. Immunol. 1999; 163: 1584-1591Google Scholar, 22Futterer A. Mink K. Luz A. Kosco-Vilbois M.H. Pfeffer K. Immunity. 1998; 9: 59-70Google Scholar, 23Fu Y.-X. Chaplin D.D. Annu. Rev. Immunol. 1999; 17: 399-433Google Scholar, 24Scheu S. Alferink J. Potzel T. Barchet W. Kalinke U. Pfeffer K. J. Exp. Med. 2002; 195: 1613-1624Google Scholar, 25Tamada K. Ni J. Zhu G. Fiscella M. Teng B. van Deursen J.M. Chen L. J. Immunol. 2002; 168: 4832-4835Google Scholar), members of the TNF receptor/ligand family.aly/aly mice are mutated in NIK (26Shinkura R. Kitada K. Matsuda F. Tashiro K. Ikuta K. Suzuki M. Kogishi K. Serikawa T. Honjo T. Nat. Genet. 1999; 22: 74-77Google Scholar). Overexpression of the wild-type form of NIK induces processing of the p100 protein of NF-κB2 to p52, but its mutant form (aly) does not (13Xiao G. Harhaj E.W. Sun S.-C. Mol. Cell. 2001; 7: 401-409Google Scholar). Furthermore, splenocytes fromaly/aly mice contain much less of the p52 protein of NF-κB2 than aly/+ mice while maintaining normal levels of p100 (21Yamada T. Mitani T. Yorita K. Uchida D. Matsushima A. Iwamasa K. Fujita S. Matsumoto M. J. Immunol. 2000; 165: 804-812Google Scholar). NIK-induced processing in B cells depends on IKKα (14Senftleben U. Cao Y. Xiao G. Greten F.R. Krähn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Google Scholar), and thus IKKα-deficient B cells contain much less p52 protein (14Senftleben U. Cao Y. Xiao G. Greten F.R. Krähn G. Bonizzi G. Chen Y. Hu Y. Fong A. Sun S.-C. Karin M. Science. 2001; 293: 1495-1499Google Scholar). Finally, although IKKα-deficient animals die perinatally, which limits their analysis, it could nevertheless be shown that these mutant mice are deficient in Peyer's patches organogenesis (27Matsushima A. Kaisho T. Rennert P.D. Nakano H. Kurosawa K. Uchida D. Takeda K. Akira S. Matsumoto M. J. Exp. Med. 2001; 193: 631-636Google Scholar). Based on these data, we hypothesized that critical functions of the LTॆ receptor on stromal cells depend on signaling via NIK, IKKα, and NF-κB2 and thus may involve processing of p100. To test for this possibility, we subjected MEFs to an agonistic antibody directed against the LTॆR. We investigated with Western analyses for the expression of the NF-κB2 proteins p100 and p52, as well as the NF-κB1 proteins p105 and p50, the inhibitor of NF-κB (IκB)α, IκBॆ, RelA, RelB, and c-Rel at six time points during an 8-h stimulation with anti-LTॆR antibodies (Fig.1). We also tested for expression of these proteins at 15 min and 8 h of stimulation with TNFα. The experiments revealed a marked decrease in p100 and a concomitant increase in p52, beginning just before 4 h and maximal by 8 h of stimulation via the LTॆR. No such changes in p100 and p52 levels were seen with TNFα stimulation. TNFα instead caused the nearly complete degradation of IκBα and the partial degradation of IκBॆ by 15 min of stimulation; IκBα levels were partly restored by 8 h of stimulation. LTॆR stimulation induced only a partial degradation of IκBα, which showed a delayed onset when compared with that induced by TNFα. The amounts of IκBα began to increase again after 2 h of stimulation via the LTॆR and were above starting levels by 8 h. Most likely this was due to increased synthesis in response to activated NF-κB, in the absence of continued degradation of this inhibitor (see below). We failed to observe consistent changes in the amounts of the other proteins analyzed in Fig. 1, with the exceptions of RelB, whose amounts were increased, and p105, whose amounts were modestly decreased after 8 h of stimulation with TNFα. In contrast to p52, the amounts of the p50 form of NF-κB1 did not increase after LTॆR stimulation. These data indicated that LTॆ receptor stimulation in MEFs caused processing of p100 to generate p52 and a more modest degradation of the IκBα inhibitory protein, implying engagement of two pathways to activate NF-κB. Light and the membrane-bound LTॆ2α are natural ligands of the LTॆ receptor. In addition to the agonistic antibody, Light could also be shown to induce processing of p100 to p52 in MEFs, whereas platelet-derived growth factor did not (Fig.2). Given the delayed onset of processing, we asked whether the underlying mechanisms might involve intermediate steps requiring protein synthesis. Light-induced processing of p100 was sensitive to the protein synthesis inhibitor cycloheximide (Fig. 2), and this result was confirmed when cells were stimulated with the agonistic antibody to the LTॆ receptor (data not shown). Thus, signal-induced processing of p100 required the new or continued synthesis of a protein, which could explain the slow onset of processing upon stimulation. Next, we investigated the mechanism underlying LTॆR-induced processing by taking advantage of mutant mice impaired or lacking in various signaling components. We generated MEFs fromaly/aly mice, which carry a mutation in NIK. Agonistic antibodies to the LTॆR failed to induce processing of p100 to p52 in aly/aly MEFs (Fig.3A). Therefore, NIK was required for LTॆR-mediated processing of p100, consistent with the ability of NIK to induce processing in transfected cells and the impaired LTॆR-induced NF-κB transcriptional activity in NIK-mutated and NIK-deficient MEFs (27Matsushima A. Kaisho T. Rennert P.D. Nakano H. Kurosawa K. Uchida D. Takeda K. Akira S. Matsumoto M. J. Exp. Med. 2001; 193: 631-636Google Scholar, 28Yin L. Wu L. Wesche H. Arthur C.D. White J.M. Goeddel D.V. Schreiber R.D. Science. 2001; 291: 2162-2165Google Scholar). NIK was shown previously to depend on IKKα to induce processing in B cells, and consistent with this, LTॆR signaling also failed to induce processing in MEFs from IKKα-deficient mice (Fig. 3A; the 8-h-stimulated lane for these mutant cells contained slightly more protein, but the ratio of p100 to p52 did not change). Interestingly, MEFs from mice lacking IKKॆ or those lacking IKKγ (also known as Nemo) were permissive for LTॆR-induced processing of p100, as were MEFs deficient in NF-κB1 (Fig. 3A). (IKKα/IKK1-, IKKॆ/IKK2-, and Nemo/IKKγ-deficient mice are described in Refs.29Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Google Scholar, 30Li Q. Van Antwerp D. Mercurio F. Lee K.F. Verma I.M. Science. 1999; 284: 321-325Google Scholar, 31Schmidt-Supprian M. Bloch W. Courtois G. Addicks K. Israel A. Rajewsky K. Pasparakis M. Mol. Cell. 2000; 5: 981-992Google Scholar.) The regulatory subunit IKKγ and the two catalytic subunits IKKα and IKKॆ together constitute the classical IKK core complex. Therefore, although LTॆR-induced processing did require the IKKα subunit, it was nevertheless independent of the classical, IKKγ (Nemo)-containing IKK complex that controls degradation of the small IκB inhibitors in response to many signals. By comparison with wild-type MEFs, the amounts of p100 appeared to be somewhat reduced in NF-κB1- and IKKॆ-deficient MEFs but were especially reduced in IKKγ-deficient MEFs. Nevertheless, processing still occurred in response to LTॆR stimulation, resulting in a more substantial depletion of p100 in the mutant versus wild-type cells (a longer exposure of the IKKγ-deficient MEFs is shown in Fig.3A, lower panel). Basal and LTॆR-induced activation of NF-κB via the classical IKK to IκB degradation path may be required for optimal p100 expression. MEFs deficient in IKKγ may have contained especially low levels of p100 because the classical activation pathway was completely blocked in these mutant cells, whereas residual activity may have persisted in the IKKॆ-deficient mutants due to the presence of IKKα. We also used an IKKॆ-specific inhibitor, PS1145, to provide further support for the suggested role of the classical activation route in maintaining p100 levels while having no role in processing. To control for the activity of this inhibitor, we confirmed a dose-dependent inhibition of TNFα-induced degradation of IκBα after 15 min of stimulation (Fig.3B). Increasing amounts of PS1145 also decreased the amounts of p100 after 8 h of stimulation via the LTॆ receptor, presumably due to reduced new synthesis of p100, whereas processing to p52 was essentially unaffected (Fig. 3B). Therefore, optimal expression of p100 depended on basal and induced activation of the classical, IKK-mediated pathway for NF-κB, but processing of p100 did not and instead only depended on the IKKα subunit. It remained theoretically possible that the inability to process p100 in IKKα-deficient and NIK-impaired MEFs in response to LTॆ stimulation was not directly related to loss of IKKα or NIK function. We therefore tested whether transfection of these mutant and wild-type MEFs with p100 together with IKKα, IKKॆ, or NIK could confirm the conclusions reached with Fig. 2. Overexpression of IKKα and especially of NIK in wild-type MEFs induced processing of p100 to generate p52, whereas IKKॆ did not (Fig.4A). IKKα and NIK were similarly able to induce processing in IKKγ (Nemo)-deficient (Fig.4B), IKKॆ-deficient (Fig. 4D), and NIK-impaired (aly/aly) (Fig. 4E) MEFs. This confirmed that the classical Nemo/IKKॆ pathway was irrelevant for processing and that the inability of aly/aly MEFs to allow processing could be overcome simply by supplying wild-type NIK or IKKα. This latter result also placed IKKα downstream of NIK, which was confirmed by the fact that overexpression of IKKα in IKKα-deficient MEFs resulted in processing, whereas overexpression of wild-type NIK did not (Fig. 4C). Therefore, we concluded that processing of p100 to p52 as induced by LTॆR stimulation depended on and proceeded via NIK and then IKKα but was independent of classical IKKγ (Nemo) and IKKॆ-dependent NF-κB activation; classical IKK activity did, however, help maintain p100 levels. Next, we investigated LTॆR-initiated NF-κB activation in EMSAs designed to determine the composition of activated NF-κB. Wild-type MEFs contained some basal κB DNA binding activity composed primarily of p50 homodimers and p50-RelA heterodimers (faster and slower migrating shifted bands, respectively), as assessed in EMSA supershift experiments with antibodies to the various NF-κB subunits (Fig. 5A). Approximately equal amounts of extracts were loaded, and this was confirmed in separate EMSAs in which DNA binding activity to the cognate site for the Octamer-1 transcription factor was assessed (data not shown). LTॆR stimulation for 2 h resulted in increased amounts of DNA binding activity composed primarily of p50-RelA and, to a lesser degree, p50-RelB (Fig. 5B; RelA supershifts marked). After 8 h of stimulation, the binding activity of p50-RelA dimers had decreased, whereas p50-RelB activity had increased further, and p52-RelB activity could be detected as well (Fig. 5C; RelB and p52 supershifts marked). In addition, p50 homodimer binding activity appeared to have increased somewhat. No significant c-Rel DNA binding activity was noted in these MEFs, although this antibody was able to detect c-Rel binding in lymphoid cel