IκBα Degradation and Nuclear Factor-κB DNA Binding Are Insufficient for Interleukin-1β and Tumor Necrosis Factor-α-induced κB-dependent Transcription

Two closely related IκBα kinases as well as the upstream kinase, NIK, which integrates interleukin-1β (IL-1β)- and tumor necrosis factor (TNF)-α-dependent activation of the transcription factor NF-κB have recently been described. However, in this emerging pathway the role of previously identified components of cytokine-induced NF-κB activation, namely phosphatidylcholine-specific phospholipase C and protein kinase C, remains unclear. We now show that, in A549 human alveolar epithelial cells, the activation of a stably transfected NF-κB-dependent reporter gene by TNF-α and IL-1β is completely blocked by the phosphatidylcholine-specific phospholipase C inhibitor D609 and the protein kinase C inhibitor RO31-8220. However, IL-1β-induced IκBα degradation as well as NF-κB nuclear translocation and DNA binding, as determined by Western blot and electro-mobility shift assay, respectively, are not affected by these inhibitors. A similar effect, although less pronounced, is observed with the p38 mitogen-activated protein kinase inhibitor SB 203580. On the basis of these data we propose the existence of a second signaling pathway induced by IL-1β and TNF-α that is activated in parallel to the cascade leading to IκBα degradation and is specifically required for NF-κB-dependent transcriptional competency. Two closely related IκBα kinases as well as the upstream kinase, NIK, which integrates interleukin-1β (IL-1β)- and tumor necrosis factor (TNF)-α-dependent activation of the transcription factor NF-κB have recently been described. However, in this emerging pathway the role of previously identified components of cytokine-induced NF-κB activation, namely phosphatidylcholine-specific phospholipase C and protein kinase C, remains unclear. We now show that, in A549 human alveolar epithelial cells, the activation of a stably transfected NF-κB-dependent reporter gene by TNF-α and IL-1β is completely blocked by the phosphatidylcholine-specific phospholipase C inhibitor D609 and the protein kinase C inhibitor RO31-8220. However, IL-1β-induced IκBα degradation as well as NF-κB nuclear translocation and DNA binding, as determined by Western blot and electro-mobility shift assay, respectively, are not affected by these inhibitors. A similar effect, although less pronounced, is observed with the p38 mitogen-activated protein kinase inhibitor SB 203580. On the basis of these data we propose the existence of a second signaling pathway induced by IL-1β and TNF-α that is activated in parallel to the cascade leading to IκBα degradation and is specifically required for NF-κB-dependent transcriptional competency. The transcription factor nuclear factor-κB (NF-κB) 1The abbreviations used are: NF-κB, nuclear factor-κB; EMSA, electromobility shift assay; ERK, extracellular regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IKK, IκB kinase; IL, interleukin; MAPK, mitogen-activated protein kinase; PC-PLC, phosphatidylcholine-specific phospholipase C; PKC, protein kinase C; PTK, protein-tyrosine kinase; RT, reverse transcriptase; PCR, polymerase chain reaction; TNF, tumor necrosis factor; PMSF, phenylmethylsulfonyl fluoride. 1The abbreviations used are: NF-κB, nuclear factor-κB; EMSA, electromobility shift assay; ERK, extracellular regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IKK, IκB kinase; IL, interleukin; MAPK, mitogen-activated protein kinase; PC-PLC, phosphatidylcholine-specific phospholipase C; PKC, protein kinase C; PTK, protein-tyrosine kinase; RT, reverse transcriptase; PCR, polymerase chain reaction; TNF, tumor necrosis factor; PMSF, phenylmethylsulfonyl fluoride. plays a key role in the transcriptional regulation of adhesion molecules, enzymes, and cytokines involved in chronic inflammatory diseases (reviewed in Ref.1Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4256) Google Scholar). In epithelial cells, which play a major role in inflammation, pro-inflammatory cytokines, such as interleukin (IL)-1β, rapidly induce NF-κB DNA binding and cause up-regulation of NF-κB-dependent genes, including cyclooxygenase-2 (2Newton R. Kuitert L.M. Bergmann M. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1997; 237: 28-32Crossref PubMed Scopus (358) Google Scholar) and inducible nitric-oxide synthase (3Nunokawa Y. Oikawa S. Tanaka S. Biochem. Biophys. Res. Commun. 1996; 223: 347-352Crossref PubMed Scopus (80) Google Scholar). Because the potent anti-inflammatory effects of glucocorticoids have been linked to a functional antagonism between the NF-κB subunit p65 and the activated glucocorticoid receptor (4Scheinman R.I. Gualberto A. Jewell C.M. Cidlowski J.A. Baldwin A.S. Mol. Cell. Biol. 1996; 15: 943-953Crossref Google Scholar, 5Mukaida N. Morita M. Ishikawa Y. Rice N. Okamoto S. Kasahara T. Matsushima K. J. Biol. Chem. 1994; 269: 13289-13295Abstract Full Text PDF PubMed Google Scholar, 6Ray A. Prefontaine K.E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 752-756Crossref PubMed Scopus (917) Google Scholar), NF-κB activation pathways have attracted much attention as potential targets for new anti-inflammatory strategies. In resting cells, the inhibitory subunit IκBα is bound to the p50/p65 heterodimer of NF-κB in the cytoplasm. Treatment of cells with IL-1β or tumor necrosis factor (TNF)-α results in the specific phosphorylation of two serine residues on IκBα (7Scherer 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) followed by the ubiquitination (8Roff M. Thompson J. Rodriguez M.S. Jacque J.M. Baleux F. Arenzana Seisdedos F. Hay R.T. J. Biol. Chem. 1996; 271: 7844-7850Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) and degradation of this subunit (9Thanos D. Maniatis T. Cell. 1995; 80: 529-532Abstract Full Text PDF PubMed Scopus (1217) Google Scholar). This releases active NF-κB, which then translocates to the nucleus and activates transcription. Recently, two closely related kinases that directly phosphorylate IκBα have been described (10DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar, 11Maniatis T. Science. 1997; 278: 818-819Crossref PubMed Scopus (233) Google Scholar, 12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1841) Google Scholar). In addition, the upstream kinase, where the IL-1β and TNF-α signaling pathways converge prior to IκBα phosphorylation, has been identified as a mitogen-activated protein kinase kinase kinase and named NF-κB-inducing kinase (13Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1160) Google Scholar). At present, it remains unclear where other previously identified pathways activated by IL-1β and TNF-α feed into this emerging signal transduction cascade. For example, the phosphatidylcholine-specific phospholipase C (PC-PLC) as part of the sphingomyelin pathway upstream of the second messenger ceramide has been implicated in TNF activation of NF-κB (14Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). However, the signal transduction pathway leading to nuclear translocation of NF-κB after TNF stimulation was found to be intact in acidic sphingomyelinase-deficient mice (15Zumbansen M. Stoffel W. J. Biol. Chem. 1997; 272: 10904-10909Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Protein kinase C (PKC) isoforms have also been implicated in NF-κB activation. Transfection of a dominant negative mutant of the atypical isoform PKC-ζ severely impaired the activation of a NF-κB-dependent reporter gene plasmid by sphingomyelin (16Lozano J. Berra E. Municio M.M. Diaz Meco M.T. Dominguez I. Sanz L. Moscat J. J. Biol. Chem. 1994; 269: 19200-19202Abstract Full Text PDF PubMed Google Scholar), implicating a role of PKC-ζ downstream of the sphingomyelin pathway. In addition, PKC-ζ was shown to phosphorylate IκBα in vitro (17Diaz Meco M.T. Dominguez I. Sanz L. Dent P. Lozano J. Municio M.M. Berra E. Hay R.T. Sturgill T.W. Moscat J. EMBO J. 1994; 13: 2842-2848Crossref PubMed Scopus (219) Google Scholar). In contrast, the expression of highly purified PKC isoenzymes α, β, γ, δ, ε, and ζ in vivo failed to induce IκBα phosphorylation (18Janosch P. Schellerer M. Seitz T. Reim P. Eulitz M. Brielmeier M. Kolch W. Sedivy J.M. Mischak H. J. Biol. Chem. 1996; 271: 13868-13874Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, in vivo studies with constitutively active isoforms demonstrated novel PKC-ε to be a potent inducer of a NF-κB-dependent reporter gene (19Genot E.M. Parker P.J. Cantrell D.A. J. Biol. Chem. 1995; 270: 9833-9839Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). In addition to PC-PLC and PKC, the mitogen-activated protein kinase (MAPK), p38, has been implicated in NF-κB activation, because the selective inhibitor, SB203580, was able to inhibit the activation of a NF-κB-dependent reporter gene by TNF-α. However, NF-κB nuclear translocation and DNA binding was unaffected (20Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar). A similar effect has been observed with the protein-tyrosine kinase (PTK) inhibitor genistein, which was able to inhibit lipopolysaccharide-induced activation of NF-κB-dependent transcription (21Yoza B.K. Hu J.Y.Q. McCall C.E. J. Biol. Chem. 1996; 271: 18306-18309Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We have used human type II A549 pneumocyte cells to provide evidence of a second signaling pathway that is distinct from IκBα degradation and NF-κB nuclear translocation but is required for NF-κB transcriptional activation by IL-1β and TNF-α. A549 cells obtained from ECACC (code 86012804) were cultured as previously described (22Newton R. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1996; 218: 518-523Crossref PubMed Scopus (80) Google Scholar). Prior to transfection, cells were grown in T-75 culture flasks to 50–60% confluency. The NF-κB-dependent reporter, 6NF-κBtkluc, contains three tandem repeats of the sequence 5′-AGC TTA CAA GGG ACT TTC CGC TGG GGA CTT TCCAGG GA-3′, which harbors two copies of the NF-κB binding site (underlined) upstream of a minimal thymidine kinase promoter (−105 to +51) driving a luciferase gene as described before (23Israel N. Gougerot-Pocidalo M.A. Aillet F. Virelizier J.L. J. Immunol. 1992; 149: 3386-3393PubMed Google Scholar). Neomycin resistance was conferred by ligating a HincII (blunted)/PvuI fragment from pMC1neoPoly(A) (Stratagene, Cambridge, UK) into the PvuI site of 6NF-κBtkluc downstream of the luciferase gene. The resulting plasmid was named 6NF-κBtkluc.neo. Cells were washed with serum-free medium and incubated with medium containing 8 μg of plasmid and Tfx50 (Promega, UK) for 2 h. Subsequently, cells were cultured in fresh medium for 16 h before adding 0.5 mg/ml G-418 (Life Technologies, Inc.). Foci of stable transfected cells developed after approximately 14 days of culture in the presence of G-418. To create a heterogeneous population with regard to integration site, multiple clones were then harvested and used for experiments for another eight passages while maintained in medium containing 0.5 mg/ml G-418. Cells were stimulated with IL-1β and TNF-α (R & D Systems, Oxon, UK) at 1 and 10 ng/ml, respectively. Where used, RO31-8220 (Alexis, Nottingham, UK), SB203580, herbimycin A, and D609 (Calbiochem, Nottingham, UK) were added 5 min prior to stimulation. Cells were harvested 6 or 24 h later and assayed for luciferase activity using a commercially available luciferase reporter gene assay (Promega). RNA isolation, reverse transcription, PCR primers, conditions, and cycling parameters for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as described previously (24Newton R. Kuitert L.M. Slater D.M. Adcock I.M. Barnes P.J. Life Sci. 1997; 60: 67-78Crossref PubMed Scopus (135) Google Scholar). Luciferase primers were: 5′-GAG AGC AAC TGC ATA AGG CTA-3′ (forward) and 5′-TAC ATC GAC TGA AAT CCC TGG-3′ (reverse) (accession number M15077). Cycling parameters were: 94 °C, 20 s; 60 °C, 30 s; 72 °C, 30 s. The number of amplification cycles used was the number necessary to achieve exponential amplification where product formation is proportional to starting cDNA, and in each case this was determined as described (24Newton R. Kuitert L.M. Slater D.M. Adcock I.M. Barnes P.J. Life Sci. 1997; 60: 67-78Crossref PubMed Scopus (135) Google Scholar). Following amplification, products (10 μl) were run on 2.0% agarose gels stained with ethidium bromide. After densitometry, data were expressed as the ratio of luciferase/GAPDH as a percentage of IL-1β treated as means ± S.E. Because the transfected luciferase gene has no introns, identical control amplifications were performed from similar reverse transcriptions in which the reverse transcriptase had been omitted. In these cases no product was visible, indicating that any genomic contamination was below detectable levels (data not shown). A549 cells were grown to confluency in 6-well plates and incubated in serum-free medium for 24 h prior to treatment. Nuclear protein was isolated 1 h after stimulation with 1 ng/ml IL-1β or 10 ng/ml TNF-α as described previously (22Newton R. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1996; 218: 518-523Crossref PubMed Scopus (80) Google Scholar). Where used, inhibitors were added 5 min prior to stimulation. The consensus NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG-3′) and Oct-1 (TGT CGA ATG CAA ATC ACT AGA) probe were obtained from Promega. Specificity was determined by prior addition of 100-fold excess unlabeled consensus oligonucleotide. Reactions were separated on 7% native acrylamide gels before vacuum drying and autoradiography. Confluent A549 cells grown in 6-well plates were stimulated for the indicated times and harvested in 200 μl of lysing buffer (1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mm Tris-base, 75 mm NaCl, 10 mm EDTA, pH 7.4, supplemented with 0.5 mm PMSF, 2 mm sodium orthovanadate, 10 μg/ml leupeptin, 25 μg/ml aprotinin, 1.25 mm NaF, 1 mm sodium pyrophosphate). Prior to loading onto 10% SDS polyacrylamide gels, samples were denatured by boiling for 5 min. Gels were run at 200 mA for 40 min at 25 °C. Proteins were transferred onto Hybond-ECL nitro-cellulose paper (Amersham, Buckinghamshire, UK) in blotting buffer (20 mm Tris-base, 192 mm glycine, 20% methanol) at 400 mA for 1 h at 25 °C. Membranes were blocked for 1 h with a 5% (w/v) nonfat dry milk solution in TBS/T (10 mmTris-base, 150 mm NaCl, 0.1% Tween-20) before incubating the filter for 1 h with rabbit polyclonal anti-human IκBα (clone C21, Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1000. Membranes were washed five times with TBS/T and incubated for a further hour with goat anti-rabbit horseradish peroxidase-linked IgG (Dako, Bucks, UK) diluted 1:4000. After another five washes, antibody-labeled proteins were detected by ECL as described by the manufacturer (Amersham, Buckinghamshire, UK). Induction of two p50/p65 NF-κB DNA binding complexes by IL-1β and TNF-α has previously been shown in these cells (2Newton R. Kuitert L.M. Bergmann M. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1997; 237: 28-32Crossref PubMed Scopus (358) Google Scholar, 22Newton R. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1996; 218: 518-523Crossref PubMed Scopus (80) Google Scholar). To investigate the effects on κB-dependent transcription, a NF-κB-dependent reporter, 6NF-κBtkluc.neo, was stably transfected into A549 cells. As shown in Fig.1, the PTK inhibitor herbimycin A (25Kuo M.L. Chau Y.P. Wang J.H. Lin P.J. Mol. Pharmacol. 1997; 52: 535-541Crossref PubMed Scopus (17) Google Scholar), the PC-PLC inhibitor D609 (14Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar), the PKC inhibitor RO31-8220 (26Wilkinson S.E. Parker P.J. Nixon J.S. Biochem. J. 1993; 294: 335-337Crossref PubMed Scopus (495) Google Scholar), and the p38 MAPK inhibitor SB203580 (27Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1973) Google Scholar) inhibited both the IL-1β and TNF-α stimulation of NF-κB-dependent luciferase activity at concentrations previously shown to be selectively effective. The MAPK/extracellular regulated kinase kinase-1/2 inhibitor, PD 098059, at 10 μm, which potently inhibits downstream activation of the extracellular regulated kinase (ERK)1 and ERK2 (28Dudley D.T. Pang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2586) Google Scholar), and the PTK inhibitor, genistein (21Yoza B.K. Hu J.Y.Q. McCall C.E. J. Biol. Chem. 1996; 271: 18306-18309Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), at 100 μm had no effect on luciferase activity induced by IL-1β or TNF-α (data not shown). TableI depicts the EC50 for the various inhibitors; results are in the range of previously reported selectively active concentrations (see references cited above). Vehicle, 1 μl/ml Me2SO, had no effect on luciferase activity after IL-1β or TNF-α stimulation. Inhibitors alone had no effect on luciferase activity (data not shown).Table IEC50 of different inhibitors on IL-1β and TNF-α induced NF-κB-dependent transcriptionStimulusRO31–8220D609SB 203580Herbimycin AμmIL-1β (1 ng/ml)1.03112 (25.5 μg/ml)1.31.01TNF-α (10 ng/ml)0.8676 (17.4 μg/ml)1.833.93 Open table in a new tab To examine the effect of these inhibitors on NF-κB nuclear translocation and DNA binding electro mobility shift assays (EMSA) were performed. IL-1β-induced NF-κB DNA binding was only slightly altered by the nonselective PTK inhibitor herbimycin A, whereas the potent inhibitors of NF-κB-dependent transcription, RO31-8220, SB203580, and D609, had no effect (Fig.2 A). Specificity of the complex was shown by competing out the signal with a 100-fold excess of cold competitor (data not shown). Neither IL-1β nor any of the inhibitors under investigation had an effect on DNA binding activity of the noninducible transcription factor Oct-1 (data not shown). Likewise induction of NF-κB DNA binding by TNF-α was also unaffected by RO31-8220, D609, and SB203580 (Fig. 2 B). Because the changes in luciferase activity were observed at 24 h and the lack of change in NF-κB DNA binding was after a 1-h treatment, there remained the possibility that these drugs exert their effects by changing the longer term levels of active NF-κB. However, because the reporter assay produced identical data after a 6-h treatment, this possibility seemed remote (data not shown). Semi-quantitative RT-PCR was used to further examine this question. Consistent with the luciferase activity data, both RO31-8220 and D609 showed total repression of IL-1β-induced luciferase mRNA following a 1-h incubation (Fig.2 C). These data indicate that the changes in luciferase expression were the result of immediate changes in κB-dependent transcription and not due to effects on p50 or p65 expression or luciferase translation. Because NF-κB DNA binding was unaffected, these data suggest that phosphorylation and subsequent degradation of IκBα would also be unaffected by these compounds. The time course of IL-1β-induced IκBα degradation and resynthesis is shown in Fig.3. The IκBα signal is lost by 15 min post-stimulation except for a retarded band indicating phosphorylated but as yet undegraded IκBα. RO31-8220 and SB203580 appeared to have little effect on loss of IκBα, whereas D609 seemed to result in a marginally reduced loss of IκBα. These data are consistent with the EMSA data indicating no substantial effect of these compounds on NF-κB activation. By contrast both D609 and RO31-8220 delayed, by 30 and 60 min, respectively, the reappearance of IL-1β-induced IκBα, whereas SB203580 had no obvious effect. A significant step toward understanding the mechanism of NF-κB activation was the recent identification of two related IκBα kinases (IKKα and IKKβ) and the upstream kinase, NF-κB-inducing kinase, integrating IL-1β- and TNF-α-induced NF-κB activation (reviewed in Refs. 11Maniatis T. Science. 1997; 278: 818-819Crossref PubMed Scopus (233) Google Scholar, 29Stancovski I. Baltimore D. Cell. 1997; 91: 299-302Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, and 30Wallach D. Boldin M. Varfolomeev E. Beyaert R. Vandenabeele P. Fiers W. FEBS Lett. 1997; 410: 96-106Crossref PubMed Scopus (211) Google Scholar). However, because the pathways leading to IκBα phosphorylation and subsequent degradation are characterized in some detail now, the role of other previously identified components of cytokine-mediated NF-κB activation is becoming less clear. By employing a NF-κB-dependent luciferase reporter gene stably transfected into the human alveolar epithelial cell line A549, the compounds D609, RO31-8220, and SB203580 selective for the sphingomyelin, PKC, and p38 MAPK pathways, respectively, were shown to be potent inhibitors of NF-κB-dependent transcription after IL-1β or TNF-α stimulation. However, neither of these inhibitors had an effect on IκBα degradation or NF-κB nuclear translocation and DNA binding. We therefore propose the existence of a second pathway triggered by TNF-α and IL-1β in parallel to IκBα degradation, which confers NF-κB transcriptional competency. In the same assay system the PTK inhibitor herbimycin A was able to partially inhibit both the luciferase activity and DNA binding as determined by EMSA. The PTK inhibitor genistein, previously reported to inhibit NF-κB transactivation induced by lipopolysaccharide in a pro-monocytic cell line (21Yoza B.K. Hu J.Y.Q. McCall C.E. J. Biol. Chem. 1996; 271: 18306-18309Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), had no effect on NF-κB-dependent transcription in our model. The PTK p59fyn is a possible target for these inhibitors, because it was shown to be involved in NF-κB-mediated activation of the HIV long terminal repeat promoter (31Hohashi N. Hayashi T. Fusaki N. Takeuchi M. Higurashi M. Okamoto T. Semba K. Yamamoto T. Int. Immunol. 1995; 7: 1851-1859Crossref PubMed Scopus (14) Google Scholar). Discrepant findings with different PTK inhibitors as shown here for genistein and herbimycin A were recently described for the regulation of inducible nitric-oxide synthase mRNA in primary rat hepatocytes involving the PTK pp60c-src (25Kuo M.L. Chau Y.P. Wang J.H. Lin P.J. Mol. Pharmacol. 1997; 52: 535-541Crossref PubMed Scopus (17) Google Scholar), suggesting different target proteins for each inhibitor. In contrast to the partial effect exercised by herbimycin A and SB203580, the PC-PLC inhibitor D609 completely abolished NF-κB-dependent reporter gene activation by IL-1β and TNF α at doses of 50 μg/ml. The sphingomyelin pathway with its second mediator ceramide has previously been implicated in TNF-α-induced NF-κB activation (14Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). At doses of 100 μg/ml, D609 completely inhibited PC-PLC; however, doses of 250 μg/ml only partially affected NF-κB DNA binding (14Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). In contrast, the EC50 for the D609 effect on NF-κB-dependent transcription in this study is similar to the one calculated from the dose response curve for TNF-α-activated PC-PLC (14Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). Taking the data concerning the time course of IκBα degradation presented here into account, we conclude that the inhibitory effect of D609 on NF-κB activation is only marginally due to the inhibition of IκBα degradation and nuclear translocation. More importantly, D609, at doses that abolish PC-PLC activity completely, inhibits NF-κB-dependent transcription induced by IL-1β and TNF-α. The delayed resynthesis of IκBα observed here supports this view, because the IκBα promoter contains multiple NF-κB sites responsible for conferring TNF-α inducibility (32Le Bail O. Schmidt-Ullrich R. Israel A. EMBO J. 1993; 12: 5043-5049Crossref PubMed Scopus (290) Google Scholar, 33Ito C.Y. Kazantsev A.G. Baldwin Jr., A.S. Nucleic Acids Res. 1994; 22: 3787-3792Crossref PubMed Scopus (209) Google Scholar). Importantly, this hypothesis would also explain recent findings in sphingomyelinase-deficient mouse embryonic fibroblasts, where TNF-α-induced IκBα degradation and NF-κB nuclear translocation was unaffected (15Zumbansen M. Stoffel W. J. Biol. Chem. 1997; 272: 10904-10909Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Based on these findings, Zumbansen and Stoffel (15Zumbansen M. Stoffel W. J. Biol. Chem. 1997; 272: 10904-10909Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) questioned any role for acidic sphingomyelinase, which is downstream of PC-PLC (14Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar). However, NF-κB transactivation competency was not investigated and may be the crucial step mediated by PC-PLC and sphingomyelinase in response to TNF-α. The specificity of D609 for PC-PLC has recently been challenged by data showing the inhibition of platelet-derived growth factor-activated phospholipase D as well as PC-PLC (34van Dijk M.C. Muriana F.J. de Widt J. Hilkmann H. van Blitterswijk W.J. J. Biol. Chem. 1997; 272: 11011-11016Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, TNF-α-induced phopholipase D activity was not affected by D609 (14Schutze S. Potthoff K. Machleidt T. Berkovic D. Wiegmann K. Kronke M. Cell. 1992; 71: 765-776Abstract Full Text PDF PubMed Scopus (968) Google Scholar), raising the possibility of a platelet-derived growth factor-specific effect. Data concerning the role of PKC isoforms in TNF-α- or IL-1β-induced NF-κB activation have been conflicting. Here we find that the PKC-inhibitor RO31-8220 was able to completely block NF-κB-dependent transcription yet failed to block NF-κB nuclear translocation and DNA binding or IκBα degradation. The marked delay in IL-1β-dependent IκBα resynthesis further supports the hypothesis of a selective effect on NF-κB transcriptional competency. However, evidence for the involvement of PKC isoforms is only indirect, because RO31-8220 is not selective for PKC isoenzymes (36Alessi D.R. FEBS Lett. 1997; 402: 121-123Crossref PubMed Scopus (197) Google Scholar, 37Yeo E.J. Provost J.J. Exton J.H. Biochim. Biophys. Acta. 1997; 1356: 308-320Crossref PubMed Scopus (19) Google Scholar). Both MAPK-activated protein kinase 1β and p70 S6 kinase, which are activated in response to growth factors and phorbol esters, are also inhibited (36Alessi D.R. FEBS Lett. 1997; 402: 121-123Crossref PubMed Scopus (197) Google Scholar). Whether other kinases activated by cytokines are inhibited remains unclear. The cross-reactivity toward MAPK-activated protein kinase 1β was examined by testing the MAPK/extracellular regulated kinase kinase-1/2 specific inhibitor PD 098059, which blocks the upstream activation of ERK1 and ERK2. PD 098059 had no effect on NF-κB-dependent transcription. In summary, PC-PLC and PKC isoforms appear to be involved in a TNF-α- and IL-1β-induced pathway of NF-κB transcriptional activation that is distinct from the signaling pathway leading to IκBα degradation, NF-κB nuclear translocation, and DNA binding. Because cytokine-mediated phosphorylation of NF-κB subunits is shown to occur for p65 after TNF-α stimulation of HeLa cells (38Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar) and a serine kinase, which specifically phosphorylates NF-κB subunits and not IκBα, has been described (39Hayashi T. Sekine T. Okamoto T. J. Biol. Chem. 1993; 268: 26790-26795Abstract Full Text PDF PubMed Google Scholar), we speculate that both D609 and RO31-8220 may prevent p65 phosphorylation and lead to reduced transcriptional activity. Furthermore, Mercurio et al. (12Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1841) Google Scholar) identified a RelA kinase activity that was associated with the IKK signalsome and supports the hypothesis that cytokine-dependent phosphorylation of p65 (RelA) may be required for transcriptional activity. However, regulated phosphorylation of NF-κB subunits has been tested for the p38 inhibitor SB203580 (20Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Crossref PubMed Scopus (599) Google Scholar), and no change in NF-κB subunit phosphorylation was detected. Because the inhibition of NF-κB-dependent transcription by SB 203580 was the least pronounced effect observed in this study, future work needs to specifically address the effect of D609 and RO31-8220 on NF-κB subunit phosphorylation.