Inhibitors of Protein Kinase C (PKC) Prevent Activated Transcription

In pulmonary A549 cells, the protein kinase C (PKC) inhibitor, Ro 31-8220, and the phosphotidylcholine-specific phospholipase C inhibitor, D609, prevent NF-κB-dependent transcription, yet NF-κB DNA binding is unaffected (Bergmann, M., Hart, L., Lindsay, M., Barnes, P. J., and Newton, R. (1998) J. Biol. Chem. 273, 6607–6610). We now show that this effect also occurs in BEAS-2B bronchial epithelial cells as well as with other PKC inhibitors (Gö 6976, GF109203X, and calphostin C) in A549 cells. Similarly, phorbol ester, a diacylglycerol mimetic, activates NF-κB-dependent transcription and potentiates tumor necrosis factor α (TNFα)-induced NF-κB-dependent transcription, yet unlike TNFα, poorly activates IκB kinase (IKK) activity, IκBα degradation, or NF-κB DNA binding in both A549 and BEAS-2B cells. As phorbol ester-induced NF-κB-dependent transcription was relatively insensitive to the proteasome inhibitor, MG-132, PKC may affect NF-κB-dependent transcription via mechanisms other than the core IKK-IκB pathway. This is supported by Gal4 one hybrid analysis of p65/RelA transactivation, which was potentiated by TNFα and phorbol ester and was inhibited by Ro 31-8220 and D609. Additionally, a number of PKC isoforms, particularly the novel isoform PKCϵ, induced p65/RelA transactivation. Phosphorylation of p65/RelA and cAMP-responsive element-binding protein (CREB)-binding protein (CBP) was increased by TNFα treatment and, in the case of CBP, was prevented by Ro 31-8220 or D609. However, p65/RelA-CBP interactions were unaffected by either compound. As this effect was not limited to NF-κB, but was a more general feature of inducible gene transcription, we suggest PKC isoforms may provide a point of intervention in diseases such as inflammation, or cancer, where activated gene expression is prominent. In pulmonary A549 cells, the protein kinase C (PKC) inhibitor, Ro 31-8220, and the phosphotidylcholine-specific phospholipase C inhibitor, D609, prevent NF-κB-dependent transcription, yet NF-κB DNA binding is unaffected (Bergmann, M., Hart, L., Lindsay, M., Barnes, P. J., and Newton, R. (1998) J. Biol. Chem. 273, 6607–6610). We now show that this effect also occurs in BEAS-2B bronchial epithelial cells as well as with other PKC inhibitors (Gö 6976, GF109203X, and calphostin C) in A549 cells. Similarly, phorbol ester, a diacylglycerol mimetic, activates NF-κB-dependent transcription and potentiates tumor necrosis factor α (TNFα)-induced NF-κB-dependent transcription, yet unlike TNFα, poorly activates IκB kinase (IKK) activity, IκBα degradation, or NF-κB DNA binding in both A549 and BEAS-2B cells. As phorbol ester-induced NF-κB-dependent transcription was relatively insensitive to the proteasome inhibitor, MG-132, PKC may affect NF-κB-dependent transcription via mechanisms other than the core IKK-IκB pathway. This is supported by Gal4 one hybrid analysis of p65/RelA transactivation, which was potentiated by TNFα and phorbol ester and was inhibited by Ro 31-8220 and D609. Additionally, a number of PKC isoforms, particularly the novel isoform PKCϵ, induced p65/RelA transactivation. Phosphorylation of p65/RelA and cAMP-responsive element-binding protein (CREB)-binding protein (CBP) was increased by TNFα treatment and, in the case of CBP, was prevented by Ro 31-8220 or D609. However, p65/RelA-CBP interactions were unaffected by either compound. As this effect was not limited to NF-κB, but was a more general feature of inducible gene transcription, we suggest PKC isoforms may provide a point of intervention in diseases such as inflammation, or cancer, where activated gene expression is prominent. In inflammation, the binding of proinflammatory cytokines, such as TNFα 1The abbreviations used are: TNFα, tumor necrosis factor α; 4α-PMA, 4α-phorbol 12-myristate 13-acetate; IKK, IκB kinase; CREB, cAMP-responsive element-binding protein; CBP, CREB-binding protein; CRE, cAMP response element; COX, cyclooxygenase; DAG, diacylglycerol; EMSA, electrophoretic mobility shift assay; GRE, glucocorticoid response element; HAT, histone acetyltransferase; NF-κB, nuclear factor-κB; PC-PLC, phosphatidyl-specific phospholipase C; PDBu, phorbol 12,13-dibutrate; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; TRE, 12-O-tetradecanoylphorbol-13-acetate response element; TSA, tricostatin A; IL, interleukin. or IL-1β, to their respective receptors results in the rapid activation of the transcription factor nuclear factor κB (NF-κB). This process involves various signaling molecules and leads to the activation of the IκB kinase (IKK) complex, which consists of two closely related kinases, IKKα and IKKβ, and the structural protein IKKγ (1Li Q. Verma I.M. Nat. Rev. Immunol. 2002; 2: 725-734Google Scholar). Phosphorylation and activation of this complex, particularly IKKβ, leads to the phosphorylation, ubiquitination and subsequent degradation of the NF-κB inhibitor protein, IκBα. Loss of IκBα releases NF-κB, typically heterodimers of p50 (NFκB1) and p65 (RelA), which can then translocate to the nucleus and activate transcription. However, there is considerable data to suggest that this process is not sufficient for transcriptional action (2Schmitz M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Google Scholar). For example, we have previously reported that the protein kinase C inhibitor, Ro 31-8220, and the phosphotidycholine-specific phospholipase C (PC-PLC) inhibitor, D609, had no effect on the induction of NF-κB DNA binding by TNFα or IL-1β yet totally ablated NF-κB-dependent transcription (3Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Google Scholar). A number of similar observations have also been made using inhibitors of the p38 mitogen-activated protein kinase (4Beyaert R. Cuenda A. Vanden Berghe W. Plaisance S. Lee J.C. Haegeman G. Cohen P. Fiers W. EMBO J. 1996; 15: 1914-1923Google Scholar, 5Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Google Scholar, 6Wesselborg S. Bauer M.K. Vogt M. Schmitz M.L. Schulze-Osthoff K. J. Biol. Chem. 1997; 272: 12422-12429Google Scholar), phosphatidylinositol 3-kinase (7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Google Scholar), protein kinase A (8Zhong H. SuYang H. Erdjument Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Google Scholar), tyrosine kinases (9Nasuhara Y. Adcock I.M. Catley M. Barnes P.J. Newton R. J. Biol. Chem. 1999; 274: 19965-19972Google Scholar), and others (see Ref. 2Schmitz M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Google Scholar). Taken together these data suggest that a number of additional signal transduction pathways are required, which impact on events post-DNA binding, but are nevertheless necessary for NF-κB-dependent transcription. Candidate protein targets for these additional activation pathways include components of the transcriptional apparatus (5Carter A.B. Knudtson K.L. Monick M.M. Hunninghake G.W. J. Biol. Chem. 1999; 274: 30858-30863Google Scholar), co-activator molecules (8Zhong H. SuYang H. Erdjument Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Google Scholar), as well as NF-κB itself (2Schmitz M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Google Scholar). In this context, p65 is widely reported to exist as a phosphoprotein, and a number of studies have documented its signal-induced phosphorylation (10Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Google Scholar, 11Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Google Scholar, 12Diehl J.A. Tong W. Sun G. Hannink M. J. Biol. Chem. 1995; 270: 2703-2707Google Scholar, 13Bird T.A. Schooley K. Dower S.K. Hagen H. Virca G.D. J. Biol. Chem. 1997; 272: 32606-32612Google Scholar). Similarly, p50 may also be phosphorylated (11Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Google Scholar). While the exact role (or roles) of NF-κB phosphorylation is currently equivocal, an initial report that phorbol ester-induced phosphorylation of p65, within the C-terminal activation domain, correlated with increased transactivation potential has set the prevailing theme (10Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Google Scholar). Likewise, inhibitors of the p38 mitogen-activated protein kinase and the extracellular reglated kinase pathway were able to reduce p65-dependent transactivation (14Berghe W.V. Plaisance S. Boone E. De Bosscher K. Schmitz M.L. Fiers W. Haegman G. J. Biol. Chem. 1998; 273: 3285-3290Google Scholar), while the PI3K/Atk (protein kinase B) pathway was implicated in p65 phosphorylation and p65-dependent transactivation (7Sizemore N. Leung S. Stark G.R. Mol. Cell. Biol. 1999; 19: 4798-4805Google Scholar, 15Madrid L.V. Wang C.Y. Guttridge D.C. Schottelius A.J. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Google Scholar, 16Madrid L.V. Mayo M.W. Reuther J.Y. Baldwin Jr., A.S. J. Biol. Chem. 2001; 276: 18934-18940Google Scholar). Analysis and mapping of phosphorylated residues in p65 has variously revealed serines 529 and 536, within the transactivation domain as being phosphorylated following TNFα treatment (17Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Google Scholar, 18Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar). Phosphorylation of these residues, at least in some studies, is believed to occur via the IKKs and appears to enhance transcriptional activation (17Wang D. Baldwin Jr., A.S. J. Biol. Chem. 1998; 273: 29411-29416Google Scholar, 18Sakurai H. Chiba H. Miyoshi H. Sugita T. Toriumi W. J. Biol. Chem. 1999; 274: 30353-30356Google Scholar, 19Yang F. Tang E. Guan K. Wang C.Y. J. Immunol. 2003; 170: 5630-5635Google Scholar). This contrasts with reports that implicate serine 276 in phosphorylation and transcriptional activation by protein kinase A (PKA) via a mechanism that involves enhanced association with the transcriptional co-activator CREB-binding protein (CBP) (8Zhong H. SuYang H. Erdjument Bromage H. Tempst P. Ghosh S. Cell. 1997; 89: 413-424Google Scholar, 20Zhong H. Voll R.E. Ghosh S. Mol. Cell. 1998; 1: 661-671Google Scholar). In the present manuscript, we have extended our previous observations by further exploring the mechanism of inhibition of NF-κB-dependent transcription by the PKC inhibitor, Ro 31-8220, and the PC-PLC inhibitor, D609 (3Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Google Scholar). We provide evidence of a novel activation pathway, which impacts on transcriptional competency at a point downstream of transcription factor binding to DNA. Cell Culture and Drugs—A549 and BEAS-2B cells were obtained from European Collection of Cell Cultures and were cultured without antibiotics as previously described (21Newton R. Hart L.A. Stevens D.A. Bergmann M. Donnelly L.E. Adcock I.M. Barnes P.J. Eur. J. Biochem. 1998; 254: 81-89Google Scholar). TNFα and IL-1β were both from R & D Systems (Oxon, UK) and phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), and 4α-phorbol 12-myristate 13-acetate (4α-PMA) were from Sigma (Poole, UK). Ro 31-8220, Gö 6976, GF109203X, wortmannin, LY294002, and H-89 (all from Calbiochem, Nottingham, UK) were dissolved in dimethyl sulfoxide (Me2SO). D609 (Alexis, Nottingham, UK) and MG-132 (Calbiochem) were dissolved in Hanks' balanced salt solution (Sigma). Drugs were added 10 min prior to stimulation. Trichostatin A (TSA) (Sigma) was dissolved in ethanol and added 60 min prior to stimulation. In all cases final concentrations of Me2SO and ethanol were no more than 0.1% (v/v), and at this level there was no effect on activation of NF-κB or NF-κB-dependent transcription (data not shown). Cells were utilized at confluence and were incubated overnight in serum free media prior to treatments. EMSA, Western Blot Analysis, and IKK Kinase Assay—EMSA, Western blot analysis, and IKK kinase were as previously described (9Nasuhara Y. Adcock I.M. Catley M. Barnes P.J. Newton R. J. Biol. Chem. 1999; 274: 19965-19972Google Scholar, 21Newton R. Hart L.A. Stevens D.A. Bergmann M. Donnelly L.E. Adcock I.M. Barnes P.J. Eur. J. Biochem. 1998; 254: 81-89Google Scholar). NF-κB probe (consensus NF-κB site GGG GAC TTT CCC) was from Promega (Southhampton, UK). Western blot analysis was performed using conventional SDS-PAGE prior to transfer to Hybond-ECL membranes (Amersham Biosciences, Chalfont, UK) and detection of proteins was performed using ECL (Amersham Biosciences). Antibodies used for Western blot analysis: were p65 (Sc-109 or Sc-372, Santa Cruz Biotechnology), IκBα (Sc-371, Santa Cruz Biotechnology) and Ser-32 phospho-IκBα (Sc-9240, Cell Signaling, Hitchin, UK), IKKα (H744, Santa Cruz Biotechnology), IKKβ (H740, Santa Cruz Biotechnology), CBP (A22 and C-20, Santa Cruz Biotechnology) and acetylated histone H4 (AHP418, Serotec). All buffers for Western blot or EMSA analysis were supplemented with 0.5 mm phenylmethylsulfonyl fluoride, 25 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mm Na3VO4, and 50 mm NaF. Luciferase Reporter Constructs and Stable Transfectants—A549 cells harboring the NF-κB-dependent reporter, 6κBtkluc, which contains six copies of the consensus NF-κB binding site (GGG ACT TTC C) has previously been described (3Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Google Scholar). Likewise the CRE and TRE A549 cells were generated by transient transfection of 8 μg of CRE and TRE luciferase reporter plasmids into preconfluent A549 or BEAS-2B cells in a T-75 using Tfx50 (Promega) as previously described (3Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Google Scholar, 21Newton R. Hart L.A. Stevens D.A. Bergmann M. Donnelly L.E. Adcock I.M. Barnes P.J. Eur. J. Biochem. 1998; 254: 81-89Google Scholar). These plasmids have six CRE or TRE (AP-1) sites, respectively, positioned upstream of a minimal β-globin promoter driving a luciferase gene as well as a separate neomycin gene to confer resistance to G-418 (22Himmler A. Stratowa C. Czernilofsky A.P. J. Recept. Res. 1993; 13: 79-94Google Scholar, 23Stratowa C. Machat H. Burger E. Himmler A. Schafer R. Spevak W. Weyer U. Wiche-Castanon M. Czernilofsky A.P. J. Recept. Signal. Transduct. Res. 1995; 15: 617-630Google Scholar). Cells were cultured in the presence of 0.5 mg/ml (A549) G-418 until foci of transfected cells appeared. These were harvested to create a heterogenous population, randomized for integration site, and the lines expanded for generation of stocks and experimental procedures. The constitutively active SV40 A549 cells contain the plasmid pGL3control.neo, which was generated by inserting a ∼1.15-kb SalI/XhoI fragment containing the neomycin gene expression cassette from pMC1(Poly(A)) (Stratagene) into the SalI site downstream of, and in the same orientation as, the luciferase gene on pGL3control (Promega). A similar procedure was used to generate pGL3basic.neo from pGL3basic (Promega). The TATA-driven luciferase reporter, pGL3.neo.TATA, was generated by inserting a double-stranded oligonucleotide (sense strand 5′-AGC TTT CGA CCT TGG GTA TAA AAG GCA GAG CAC TGC AGC TGC TGC TTA CA), which corresponds to a modified (C to T substitution underlined) minimal β-globin promoter, into the HindIII site of pGL3basic.neo. The 2×GRE reporter, pGL3.neo.TATA.2GRE, was generated by digestion of pGL3.neo.TATA at the SmaI site, upstream of the minimal promoter, and insertion of a double-stranded oligonucleotide (sense strand 5′-GCT GTA CAG GAT GTT CTA GGC TGT ACA GGA TGT TCT AG-3′) containing two tandem copies of a consensus GRE site (underlined) (24Strahle U. Klock G. Schutz G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7871-7875Google Scholar). Generation of the SV40, TATA, and GRE A549 reporter lines was achieved by transient transfection of the plasmids pGL3Control.neo, pGL3.neo.TATA, and pGL3.neo.TATA.2GRE and G-418 selection of stable recombinants as above. The BEAS-2B 6κBtk reporter cell line was also generated by this approach using the plasmid 6κBtk.neo and a G-418 concentration of 0.075 mg/ml as described above (3Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Google Scholar). In all cases luciferase reporter cell lines were incubated overnight in serum-free G-418-free medium prior to stimulation. Reporter cells were harvested in reporter lysis buffer (Promega) 6 h after treatments and luciferase activity was determined using a commercial kit (Promega). One-hybrid p65 Transactivation Assay—The Gal4-dependent reporter, Gal4-luc, which contains two copies of a yeast Gal4 binding site cloned upstream of a minimal promoter driving a luciferase gene, and the expression vector containing the full-length human p65 fused downstream of a Gal4 DNA binding domain (Gal4-p65) have been previously described (10Schmitz M.L. dos Santos Silva M.A. Baeuerle P.A. J. Biol. Chem. 1995; 270: 15576-15584Google Scholar, 14Berghe W.V. Plaisance S. Boone E. De Bosscher K. Schmitz M.L. Fiers W. Haegman G. J. Biol. Chem. 1998; 273: 3285-3290Google Scholar). Preconfluent A549 cells were transfected in 6-well plates with 0.1 μg of Gal4-luc and 0.5 μg of Gal4-p65 fusion using Tfx50 according to the manufacturers instructions (Promega). After 24 h cells were treated with stimuli or drugs and after a further 6-h luciferase activity was measured as above. For analysis of constitutively active PKC isoforms, cells were transfected with 0.1 μg of Gal4-luc and 0.5 μg of Gal4-p65 plus variable amounts, up to a maximum of 2 μg, of previously described expression plasmids for constitutively active PKC isoforms α, β1, δ, ϵ, η, and ζ (25Schonwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Mol. Cell. Biol. 1998; 18: 790-798Scopus (684) Google Scholar). In all cases the total DNA content was maintained at 2.6 μg/well by the addition of empty vector. After 24 h cells were harvested and luciferase activity measured. Immunoprecipitation and Metabolic Labeling of p65 and CBP—Metabolic labeling and immunoprecipitation was essentially as described (9Nasuhara Y. Adcock I.M. Catley M. Barnes P.J. Newton R. J. Biol. Chem. 1999; 274: 19965-19972Google Scholar). Prior to stimulation, cells were incubated for 2 h in phosphate-free medium (Sigma) and for 4 h in phosphate-free medium supplemented with 0.25 mCi/ml [32P]orthophosphate (Amersham Biosciences). For p65, cells were lysed in RIPA buffer (1 × phosphate-buffered saline, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS). After preclearing for 1 h, 4 °C with 1 μg of normal rabbit IgG (Santa Cruz Biotechnology) and 20 μl of protein A-agarose (Santa Cruz Biotechnology), immunoprecipitation was performed with 5 μl of p65 agarose-conjugated antibody (SC-109AC, Santa Cruz Biotechnology) for 2 h at 4 °C supplemented where indicated with 50 μl of blocking peptide (SC-109P, Santa Cruz Biotechnology). For CBP, cells were lysed in mild lysis buffer (0.5% Nonidet P-40, 50 mm Tris, pH 8.0, 150 mm NaCl, 5 mm EDTA). After preclearing, immunoprecipitation was performed overnight at 4 °C using 2 μg of CBP antibody (C20, Santa Cruz Biotechnology). Precipitates were washed four times with the lysis buffer, and after boiling in Western sample buffer, each sample was divided into halves and subjected to SDS-PAGE and either autoradiography or Western blot analysis (p65-Sc-372, CBP-A22). In Vivo Histone Acetyltransferase (HAT) Assay—Cells at 50% confluence were incubated in serum free media for 24–48 h until synchronized and total histone acetylation assessed as described previously (26Ito K. Barnes P.J. Adcock I.M. Mol. Cell. Biol. 2000; 20: 6891-6903Google Scholar). After incubated with 0.005 mCi/ml [3H]acetic acid, cells were stimulated and harvested in 10 mm Tris-HCl, pH 6.5, 50 mm sodium bisulfite, 10 mm MgCl2, 8.6% sucrose, 2% Triton X-100, 100 ng/ml TSA supplemented with protease inhibitor mixture (Roche Applied Science). Pure nuclei were obtained by three further washes in lysis buffer and one wash in 10 mm Tris-Cl, pH 7.4, 13 mm EDTA, 100 ng/ml TSA. Histone proteins were acid extracted at 4 °C for 1 h in 0.2 m HCl, 0.4 m H2SO4, and incorporation of 3H was measured by liquid scintillation counting. Results were normalized to protein content. For Western blot analysis of acetylated histone H4, histone proteins were acetone concentrated overnight at -20 °C, and 10 μg was used. Immunoprecipitation-HAT Assay—CBP immunoprecipitation was performed as described above, and precipitates were washed with HAT buffer (50 mm Tris-HCl, pH 8.0, 10% glycerol, 0.1 mm EDTA) supplemented with 1 mm dithiothreitol. Precipitates were resuspended in 30 μl of HAT buffer supplemented with 0.2 mg/ml histone II-A (Sigma). HAT assay was started by adding 0.1 μCi of [3H]acetyl-CoA (PerkinElmer Life Sciences) and incubated for 45 min at 30 °C. The reaction was stopped by spotting samples onto Whatman P81 paper (Whatman). After rinsing twice with cold HAT buffer filters were subject to liquid scintillation counting. Northern Blot Analysis—RNA extraction and Northern blot analysis were carried out using standard procedures as previously described (27Newton R. Seybold J. Kuitert L.M.E. Bergmann M. Barnes P.J. J. Biol. Chem. 1998; 273: 32312-32321Google Scholar). Probes for COX-2 and glyceraldehyde-3-phosphate dehydrogenase were as previously described (27Newton R. Seybold J. Kuitert L.M.E. Bergmann M. Barnes P.J. J. Biol. Chem. 1998; 273: 32312-32321Google Scholar). In each case other probes were generated by reverse transcription polymerase chain reaction amplification using the indicated primers, cloning, and verification by sequencing. Excised inserts were 32P-radiolabeled using the random primed method (Ready-Prime kit) (Amersham Biosciences, Little Chalfont, Bucks, UK). Primer pairs (5′ > 3′) were: c-fos (K00650), TTC ATT CCC ACG GTC ACT GCC ATC (forward) and GTC TTC AGC TCC ATG CTG ATG CTC (reverse); c-jun (J04111), CTA TGA CGA TGC CCT CAA CGC CTC (forward) and GGA TTA TCA GGC CCA GCT (reverse); IκBα (M69043), GGA CTC CAT GAA AGA CGA GGA (forward) and AAG TCT CGG AGC TCA GGA TCA (reverse); IL-8 (M26383), CTA GGA CAA GAG CCA GGA AGA (forward) and AAC CCT CTG CAC CCA GTT TTC (reverse); p50/p105 (M58603), GCA AAG GTT ATT GTT CAG TT (forward) and GCT TGC AAA TAG GCA AGG TC (reverse). Inhibitors of Protein Kinase C Prevent NF-κB-dependent Transcription but Not p65 Translocation—In our previous studies (3Bergmann M. Hart L. Lindsay M. Barnes P.J. Newton R. J. Biol. Chem. 1998; 273: 6607-6610Google Scholar), we showed that the induction of NF-κB-dependent transcription by TNFα was completely blocked by D609, a reported PC-PLC inhibitor, and Ro 31-8220, a PKC inhibitor, yet the activation of NF-κB DNA binding or IκBα degradation was unaltered. To further explore this effect, we have also tested the known inhibitors of PKC, Gö 6976, GF109203X, as well as the structurally unrelated inhibitor, calphostin C. In each case, there was no effect on DNA binding as determined by EMSA, whereas robust inhibition of NF-κB-dependent transcription was observed (Fig. 1, A and B). This result was confirmed by Western blotting of nuclear extracts, which revealed no effect of either Ro 31-8220 or Gö 6976 on nuclear translocation of p65 (Supplemental Fig. S-1). Similarly, confocal microscopy revealed that neither the Gö 6976 nor D609 had any inhibitory effect on the nuclear translocation of p65 following TNFα stimulation of A549 cells (Supplemental Fig. S-2). Ro 31-8220 and D609 Prevent NF-κB-dependent Transcription in BEAS-2B Cells—To examine the possibility that these effect were due to a peculiarity of the A549 cells system, the bronchial epithelial cell line, BEAS-2B, was also treated with TNFα in the presence or absence of both Ro 31-8220 or D609. In neither case was there any effect on the induction of NF-κB DNA binding activity (Fig. 1C), yet activation of the NF-κB-dependent reporter, 6κBtk, was substantially repressed (Fig. 1D) suggesting that our data are more generally applicable. Phorbol Ester Activates NF-κB-dependent Transcription by a Mechanistically Distinct Process—To further substantiate the role of PKC, 6κBtk A549 cells were stimulated with the DAG mimetic, PMA, which is a potent activator of PKC. This markedly induced NF-κB-dependent transcription and was also prevented by inhibitors of PKC (Fig. 1B). Analysis of the concentration-response characteristics for PMA revealed that a maximal response was achieved at 10-7m (Fig. 2A), a value that is consistent with other PKC-mediated events. A combination of a maximally effect dose of TNFα (10 ng/ml) with a maximally effect dose of PMA (10-7m) resulted in a strongly enhanced luciferase response suggesting that these two treatments act via independent pathways to activate transcription (Fig. 2A). Similarly, PDBu (10-7m) also induced NF-κB-dependent transcription, whereas the inactive analog 4α-PMA (10-7m) was without effect suggesting that these effects are indeed specific (data not shown). Analysis of NF-κB DNA binding was consistent with our previous findings in showing a strong increase following TNFα or IL-1β treatment and only a very weak induction following PMA treatment (Fig. 2B) (28Newton R. Adcock I.M. Barnes P.J. Biochem. Biophys. Res. Commun. 1996; 218: 518-523Google Scholar, 29Catley M.C. Chivers J.E. Cambridge L.M. Holden N. Slater D.M. Staples K.J. Bergmann M.W. Loser P. Barnes P.J. Newton R. FEBS Lett. 2003; 547: 75-79Google Scholar). To our initial surprise the combination of TNFα plus PMA resulted in an apparent decrease in DNA binding activity with respect to TNFα alone. However, this result could be explained by the increased NF-κB-dependent transcription following combined TNFα + PMA treatment resulting in elevated expression of the NF-κB-dependent IκBα gene (30Ito C.Y. Kazantsev A.G. Baldwin Jr., A.S. Nucleic Acids Res. 1994; 22: 3787-3792Google Scholar). This in turn would accelerate feedback inhibition of NF-κB DNA binding. To examine the ability of TNFα and PMA to activate the primary IKK-IκBα pathway involved in NF-κB activation, the IKK complex was immunoprecipitated and kinase activity assessed. As is normally observed (31DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Google Scholar, 32Mercurio 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: 860-866Google Scholar), TNFα produced a profound activation of IKK activity that was maximal within 5 min of treatment (Fig. 2C). This activity was mirrored in the rapid phosphorylation (mobility shift) of IκBα observed at 5-min post-stimulation and the complete degradation within 15 min of treatment (Fig. 2D). In marked contrast, PMA failed to induce IKK kinase activity to any great extent and did not result in substantial loss of IκBα (Fig. 2, C and D). Analysis of the 6κBtk BEAS-2B cells also revealed maximally effective concentrations of 10-7m (EC50 = 1.43 10-8m) and 10 ng/ml (EC50 = 0.16 ng/ml) for PMA and TNFα, respectively (data not shown), and these when added together resulted in a slight (but nonsignificant) enhancement of reporter activity. Consistent with the A549 study, PMA produced a very minor increase in NF-κB DNA binding over basal levels and failed to induce both IκBα degradation or the appearance of S-32 phosphorylated IκBα (Fig. 2, F and G). Conversely, TNFα strongly activated both NF-κB DNA binding, phosphorylation of IκBα, and subsequent loss of IκBα (Fig. 2, F and G). Therefore the data from these two cell line models demonstrate that, whereas TNFα robustly activates the IKK-IκBα pathway to result in NF-κB nuclear translocation and transcriptional activation, this is not primarily the mechanism that accounts for transcriptional activation by phorbol esters. To test the requirement for proteasome activity in the response to PMA, A549 cells and 6κBtk A549 cells were treated with TNFα or PMA in the presence or absence of the proteasome inhibitor, MG-132 (33Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Google Scholar). This has previously shown to be effective in A549 cells against TNFα-induced activation of NF-κB and IL-8 expression (34Fiedler M.A. Wernke Dollries K. Stark J.M. Am. J. Respir. Cell Mol. Biol. 1998; 19: 259-268Google Scholar). Western blot analysis of IκBα degradation revealed a marked inhibition by MG-132 (Fig. 3A). No effect of MG-132 was observed on IκBα following PMA treatment due to the lack of obvious degradation by PMA alone. Likewise, MG-132 was an effective inhibitor of TNFα-induced NF-κB-dependent transcription at concentrations (30 μm) that also inhibited IκBα degradation (Fig. 3B). Little or no effect was observed on PMA-induced NF-κB-dependent transcription, suggesting that that proteasome degradation does not play a role in this pathway. Role of Protein Kinase A and Phosphatidylinositol 3-Kinase in NF-κB-dependent Transcription—As both the PKA and PI3K pathways have been implicated in the activation of NF-κB-dependent transcription (see Ref. 2Schmitz M.L. Bacher S. Kracht M. Trends Biochem. Sci. 2001; 26: 186-190Google Scholar and references therein), we tested the effect H-89, a potent inhibitor of PKA, and wortmannin and LY294002, both potent inhibitors of PI3K. Whereas, the PKC inhibitor, Ro 31-8220, was effective at inhibiting either TNFα- or PMA-mediated NF-κB-dependent transcription between 10-7 and 10-6m (EC50 values 1.53 10-7m and 4.63 10-7m, respectively), H-89 was at least 10–100-fold less effective with the respective EC50 values lying between 10-4 and 10-5m (Fig. 3C). As the Ki value for H-89 on PKA is around 48 nm, and this drug is known to inhibit PKC isofor