Inhibitors of Protein Kinase C (PKC) Prevent Activated Transcription ROLE OF EVENTS DOWNSTREAM OF NF- (cid:1) B DNA BINDING* □ S

In pulmonary A549 cells, the protein kinase C (PKC) inhibitor, Ro 31-8220, and the phosphotidylcholine-spe-cific phospholipase C inhibitor, D609, prevent NF- (cid:1) B-de-pendent transcription, yet NF- (cid:1) B DNA binding is unaffected (Bergmann, P. J., 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 (Go¨ 6976, GF109203X, and calphostin C) in A549 cells. Similarly, phorbol ester, a diacylglycerol mimetic, activates NF- (cid:1) B-dependent transcription and potenti-ates tumor necrosis


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 TNF␣ 1 or IL-1␤, to their respective receptors results in the rapid activation of the transcription factor nuclear factor B (NF-B). This process involves various signaling molecules and leads to the activation of the IB kinase (IKK) complex, which consists of two closely related kinases, IKK␣ and IKK␤, and the structural protein IKK␥ (1). Phosphorylation and activation of this complex, particularly IKK␤, leads to the phosphorylation, ubiquitination and subsequent degradation of the NF-B inhibitor protein, IB␣. Loss of IB␣ releases NF-B, typically heterodimers of p50 (NFB1) 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 (2). 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-B DNA binding by TNF␣ or IL-1␤ yet totally ablated NF-B-dependent transcription (3). A number of similar observations have also been made using inhibitors of the p38 mitogen-activated protein kinase (4 -6), phosphatidylinositol 3-kinase (7), protein kinase A (8), tyrosine kinases (9), and others (see Ref. 2). 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-B-dependent transcription.
Candidate protein targets for these additional activation pathways include components of the transcriptional apparatus (5), co-activator molecules (8), as well as NF-B itself (2). In this context, p65 is widely reported to exist as a phosphopro-tein, and a number of studies have documented its signalinduced phosphorylation (10 -13). Similarly, p50 may also be phosphorylated (11). While the exact role (or roles) of NF-B phosphorylation is currently equivocal, an initial report that phorbol ester-induced phosphorylation of p65, within the Cterminal activation domain, correlated with increased transactivation potential has set the prevailing theme (10). Likewise, inhibitors of the p38 mitogen-activated protein kinase and the extracellular reglated kinase pathway were able to reduce p65dependent transactivation (14), while the PI3K/Atk (protein kinase B) pathway was implicated in p65 phosphorylation and p65-dependent transactivation (7,15,16). Analysis and mapping of phosphorylated residues in p65 has variously revealed serines 529 and 536, within the transactivation domain as being phosphorylated following TNF␣ treatment (17,18). Phosphorylation of these residues, at least in some studies, is believed to occur via the IKKs and appears to enhance transcriptional activation (17)(18)(19). 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) (8,20).
In the present manuscript, we have extended our previous observations by further exploring the mechanism of inhibition of NF-B-dependent transcription by the PKC inhibitor, Ro 31-8220, and the PC-PLC inhibitor, D609 (3). We provide evidence of a novel activation pathway, which impacts on transcriptional competency at a point downstream of transcription factor binding to DNA.

EXPERIMENTAL PROCEDURES
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 (21). TNF␣ and IL-1␤ were both from R & D Systems (Oxon, UK) and phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), and 4␣-phorbol 12-myristate 13-acetate (4␣-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 (Me 2 SO). 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 Me 2 SO and ethanol were no more than 0.1% (v/v), and at this level there was no effect on activation of NF-B or NF-B-dependent transcription (data not shown). Cells were utilized at confluence and were incubated overnight in serum free media prior to treatments.
Luciferase Reporter Constructs and Stable Transfectants-A549 cells harboring the NF-B-dependent reporter, 6Btkluc, which contains six copies of the consensus NF-B binding site (GGG ACT TTC C) has previously been described (3). Likewise the CRE and TRE A549 cells were generated by transient transfection of 8 g of CRE and TRE luciferase reporter plasmids into preconfluent A549 or BEAS-2B cells in a T-75 using Tfx50 (Promega) as previously described (3,21). These plasmids have six CRE or TRE (AP-1) sites, respectively, positioned upstream of a minimal ␤-globin promoter driving a luciferase gene as well as a separate neomycin gene to confer resistance to G-418 (22,23). 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 ϳ1.15-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  5Ј-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 ␤-globin promoter, into the HindIII site of pGL3basic.neo. The 2ϫGRE 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 5Ј-GCT GTA CAG GAT GTT CTA GGC TGT ACA GGA TGT TCT AG-3Ј) containing two tandem copies of a consensus GRE site (underlined) (24). 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 6Btk reporter cell line was also generated by this approach using the plasmid 6Btk.neo and a G-418 concentration of 0.075 mg/ml as described above (3).
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 (10,14). Preconfluent A549 cells were transfected in 6-well plates with 0.1 g of Gal4-luc and 0.5 g 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 g of Gal4-luc and 0.5 g of Gal4-p65 plus variable amounts, up to a maximum of 2 g, of previously described expression plasmids for constitutively active PKC isoforms ␣, ␤1, ␦, ⑀, , and (25). In all cases the total DNA content was maintained at 2.6 g/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 (9). 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 [ 32 P]orthophosphate (Amersham Biosciences). For p65, cells were lysed in RIPA buffer (1 ϫ phosphate-buffered saline, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS). After preclearing for 1 h, 4°C with 1 g of normal rabbit IgG (Santa Cruz Biotechnology) and 20 l of protein A-agarose (Santa Cruz Biotechnology), immunoprecipitation was performed with 5 l of p65 agarose-conjugated antibody (SC-109AC, Santa Cruz Biotechnology) for 2 h at 4°C supplemented where indicated with 50 l 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 g 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 (26). After incubated with 0.005 mCi/ml [ 3 H]acetic acid, cells were stimulated and harvested in 10 mM Tris-HCl, pH 6.5, 50 mM sodium bisulfite, 10 mM MgCl 2 , 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 H 2 SO 4 , and incorporation of 3 H 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 Ϫ20°C, and 10 g 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 l of HAT buffer supplemented with 0.2 mg/ml histone II-A (Sigma). HAT assay was started by adding 0.1 Ci of [ 3 H]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 (27). Probes for COX-2 and glyceraldehyde-3-phosphate dehydrogenase were as previously described (27). 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 32 P-radiolabeled using the random primed method

Inhibitors of Protein Kinase C Prevent NF-B-dependent
Transcription but Not p65 Translocation-In our previous studies (3), we showed that the induction of NF-B-dependent transcription by TNF␣ was completely blocked by D609, a reported PC-PLC inhibitor, and Ro 31-8220, a PKC inhibitor, yet the activation of NF-B DNA binding or IB␣ 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-B-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 TNF␣ stimulation of A549 cells (Supplemental Fig. S-2).
Ro 31-8220 and D609 Prevent NF-B-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 TNF␣ in the presence or absence of both Ro 31-8220 or D609. In neither case was there any effect on the induction of NF-B DNA binding activity (Fig. 1C), yet activation of the NF-B-dependent reporter, 6Btk, was substantially repressed (Fig. 1D) suggesting that our data are more generally applicable.
Phorbol Ester Activates NF-B-dependent Transcription by a Mechanistically Distinct Process-To further substantiate the role of PKC, 6Btk A549 cells were stimulated with the DAG mimetic, PMA, which is a potent activator of PKC. This markedly induced NF-B-dependent transcription and was also pre- vented by inhibitors of PKC (Fig. 1B). Analysis of the concentration-response characteristics for PMA revealed that a maximal response was achieved at 10 Ϫ7 M ( Fig. 2A), a value that is consistent with other PKC-mediated events. A combination of a maximally effect dose of TNF␣ (10 ng/ml) with a maximally effect dose of PMA (10 Ϫ7 M) resulted in a strongly enhanced luciferase response suggesting that these two treatments act via independent pathways to activate transcription ( Fig. 2A). Similarly, PDBu (10 Ϫ7 M) also induced NF-B-dependent transcription, whereas the inactive analog 4␣-PMA (10 Ϫ7 M) was without effect suggesting that these effects are indeed specific (data not shown). Analysis of NF-B DNA binding was consistent with our previous findings in showing a strong increase following TNF␣ or IL-1␤ treatment and only a very weak induction following PMA treatment (Fig. 2B) (28,29). To our initial surprise the combination of TNF␣ plus PMA resulted in an apparent decrease in DNA binding activity with respect to TNF␣ alone. However, this result could be explained by the increased NF-B-dependent transcription following combined TNF␣ ϩ PMA treatment resulting in elevated expression of the NF-B-dependent IB␣ gene (30). This in turn would accelerate feedback inhibition of NF-B DNA binding.
To examine the ability of TNF␣ and PMA to activate the primary IKK-IB␣ pathway involved in NF-B activation, the IKK complex was immunoprecipitated and kinase activity as-sessed. As is normally observed (31,32), TNF␣ 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 IB␣ 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 IB␣ (Fig. 2, C and D).
Analysis of the 6Btk BEAS-2B cells also revealed maximally effective concentrations of 10 Ϫ7 M (EC 50 ϭ 1.43 10 Ϫ8 M) and 10 ng/ml (EC 50 ϭ 0.16 ng/ml) for PMA and TNF␣, 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-B DNA binding over basal levels and failed to induce both IB␣ degradation or the appearance of S-32 phosphorylated IB␣ (Fig. 2, F and G). Conversely, TNF␣ strongly activated both NF-B DNA binding, phosphorylation of IB␣, and subsequent loss of IB␣ (Fig. 2, F and G). Therefore the data from these two cell line models demonstrate that, whereas TNF␣ robustly activates the IKK-IB␣ pathway to result in NF-B 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 re- sponse to PMA, A549 cells and 6Btk A549 cells were treated with TNF␣ or PMA in the presence or absence of the proteasome inhibitor, MG-132 (33). This has previously shown to be effective in A549 cells against TNF␣-induced activation of NF-B and IL-8 expression (34). Western blot analysis of IB␣ degradation revealed a marked inhibition by MG-132 (Fig. 3A). No effect of MG-132 was observed on IB␣ following PMA treatment due to the lack of obvious degradation by PMA alone. Likewise, MG-132 was an effective inhibitor of TNF␣-induced NF-B-dependent transcription at concentrations (30 M) that also inhibited IB␣ degradation (Fig. 3B). Little or no effect was observed on PMA-induced NF-B-dependent transcription, suggesting that that proteasome degradation does not play a role in this pathway.  (Fig. 3C). As the K i value for H-89 on PKA is around 48 nM, and this drug is known to inhibit PKC isoforms at 31.7 M (Calbiochem data sheet), it is likely that these data point again to the involvement of PKC rather than PKA. This conclusion is supported by the additional finding that forskolin, an activator of PKA, or inhibitors of phosphodiesterase 4, which raise intracellular cAMP and would be expected to activate PKA, either show little effect or down-regulate activated NF-B-dependent transcription (data not shown). Likewise adenoviral overexpression of protein kinase inhibitor, a potent endogenous inhibitor of PKA (35), also showed no effect on NF-B-dependent transcription in A549 cells (data not shown).

Role of Protein Kinase
Similarly, the PI3K inhibitor, wortmannin, which shows an IC 50 value in the low nanomolar (5 nM) and is physiologically effective at 100 nM (Calbiochem data sheet), had little effect at up to 10 Ϫ5 M (Fig. 3C). Likewise, LY294008 (IC 50 1.4 M for PI3K, Calbiochem data sheet) also showed a similar lack of effect (data not shown) leading us to conclude that the PI3K pathway does not play a key role in the activation of NF-Bdependent transcription in this experimental system. p65 Transactivation Is Inhibited by D609 and Ro 31-8220 and Potentiated by PKC-The above data indicate that D609 and Ro 31-8220 act at a point downstream of NF-B translocation and DNA binding. To examine the effect on transactivation by p65, we established a Gal4 one-hybrid assay for p65 transactivation in A549 cells (10,36). In this system the transactivation potential of p65 was increased by both TNF␣ and PMA, and in each case both D609 and Ro 31-8220 substantially inhibited transactivation (Fig. 4, A and B). To determine whether PKC isoforms were actually able to increase p65-dependent transactivation, constitutively active versions of PKC␣, -␤1, -␦, -⑀, -, and -were overexpressed (25). In all cases, apart from PKC, some degree of potentiation was observed (Fig. 4C). This effect was most apparent for the novel isoforms with PKC⑀ giving rise to the greatest overall levels of activation. These data therefore support the idea that one or more PKC isoforms can act on or downstream of p65 to potentiate NF-B transcriptional potential.
Analysis of Potential Downstream Targets of Phosphorylation-To examine whether p65 itself was a downstream kinase target, cells were stimulated with TNF␣ in the presence of 32 P-labeled inorganic phosphate, and p65 was immunoprecipitated (Fig. 5A). This experiment clearly shows that the phosphorylation status of p65 is increased rapidly (around 3-fold) following TNF␣ stimulation. This effect was maximal within 15 min, and by 1 h levels of p65 phosphorylation were falling ( Fig.  5A and data not shown). However, as neither D609 nor Ro 31-8220 affected this increase (Fig. 5B), we conclude that the effect of these compounds must lie downstream of not only p65 translocation and DNA binding but also of p65 phosphorylation.
A potential candidate molecule that is known to interact with p65 and could be targeted by phosphorylation is CBP (37,38). This co-activator protein is believed to act a both as a physical link with the basal transcriptional machinery and to facilitate transcriptional activation by virtue of its associated HAT activity (38). Following 32 P metabolic labeling of proteins, CBP was immunoprecipitated and subject to autoradiography. This revealed a striking increase in radioactivity associated with CBP indicating increased phosphorylation (Fig. 6A). Unlike the rapid increase observed for p65, CBP phosphorylation increased gradually over 1 h until reaching a maximum 1-2 h post-TNF␣ stimulation. In each case, prior treatment with D609 or Ro 31-8220 markedly repressed this increase indicating that the event was downstream of processes that were targeted by these compounds (Fig. 6B).
Effect of D609 and Ro 31-8220 on CBP-associated HAT and Interaction with p65-To examine the possible mechanistic significance of CBP phosphorylation, the HAT activity associ-  1 M). After 20 min cells were harvested and total proteins prepared for Western blot analysis of IB␣. B, 6Btk.A549 cells were pretreated with various doses of MG-132 before stimulation with TNF␣ (10 ng/ml) or PMA (0.1 M). After 6 h cells were harvested for luciferase activity determination. Data (n ϭ 6 -10) were expressed as a percent of stimulated and plotted as means Ϯ S.E. C, 6Btk.A549 cells were pretreated with various concentrations of Ro 31-8220, H-89, wortmannin, or LY294008 before stimulation with either TNF␣ (10 ng/ml) or PMA (0.1 M) and luciferase determination as in B. In each case data (n ϭ 8) were expressed as a percent of stimulated and plotted as means Ϯ S.E. ated with CBP was assayed. As was previously found for IL-1␤ (26), CBP-associated HAT activity was increased by TNF␣ (Fig.  6C). This was essentially unaffected by prior treatment with Ro 31-8220 but was reduced to near basal levels by D609.
As phosphorylation has been documented as regulating the interaction of CBP with transcription factors (20), p65 was immunoprecipitated and then the precipitates probed for the presence of CBP (Fig. 6D). In this experiment, we could visualize a band corresponding to CBP from cells that had been treated with TNF␣, or with TNF␣ plus Ro 31-8220 or D609, but not from unstimulated cells. An equivalent result was also obtained in the converse experiment in which CBP was immunoprecipitated and the presence of p65 was assayed (Fig. 6E). These data support the existence of a signal-induced interaction between p65 and CBP and further suggest that this interaction is not disrupted by the prior treatment with these inhibitors.
Role of Global Histone Acetylation-Analysis of [ 3 H]acetate incorporation into total histone revealed an increase following TNF␣ treatment (Fig. 7A). As with CBP-associated HAT activity, this was reduced to near basal levels by prior treatment with D609 but not Ro 31-8220. Western blot analysis of acetylated histone H4 also confirmed this result (Fig. 7B).
To examine the link between histone acetylation and NF-Bdependent transcription, cells were treated with the histone deacetylase inhibitor, TSA. As expected this resulted in increased levels of acetylated histone H4 (Fig. 7B). Analysis of NF-B-dependent transcription revealed no effect of TSA on unstimulated transcription but resulted in a concentration-dependent enhancement of TNF␣-stimulated transcription (Fig.  7C). This confirms that increased overall HAT activity does promote NF-B transcriptional responses. By contrast the induction of NF-B-dependent transcription by PMA was only weakly potentiated by TSA, possibly because this pathway is already activated by PMA.
Effect on Various Transcriptional Responses-To examine the possibility that these compounds were merely causing nonspecific effects on transcription, the effects of D609 and Ro 31-8220 were tested on both low level constitutive (TATA) and high level constitutive (SV40) transcription (Fig. 8A). In each case these reporters revealed no responsiveness to a range of treatments including TNF␣ (10 ng/ml), IL-1␤ (1 ng/ml), PMA (10 Ϫ7 M), or dexamethasone (10 Ϫ6 M), and this situation was unchanged by the presence or absence of D609 or Ro 31-8220. By contrast A549 cells containing CRE-, TRE-(AP-1), or GREresponsive reporters revealed responsiveness to stimulation and in each case this was almost totally prevented by the prior addition of D609 or Ro 31-8220. As A549 cells respond rather poorly to cAMP elevating agents and the CRE reporter appears to act more like an AP-1/ATF-driven reporter (Fig. 8A and data not shown), the effect of these drugs was also analyzed on the CRE reporter in BEAS-2B cells. In these cells the CRE reporter responded robustly to a variety of cAMP-elevating agents, including salbutamol, prostaglandin E 2 , and the phosphodiesterase 4 inhibitor, rolipram, as well as to the cAMP analog 8-bromo-cAMP and the adenylyl cyclase activator, forskolin (data not shown). In present study, CRE-dependent transcription was dramatically enhanced by a combination of forskolin ϩ rolipram and was substantially reduced by both D609 and Ro 31-8220 (Fig. 8A).
To test whether these observations are also observed on bona fides genes, A549 cells were treated with IL-1␤, TNF␣, or PMA in the presence or absence of D609 or Ro 31-8220. Northern blot analysis revealed increased mRNA expression of c-fos, c-jun, and IB␣ at 1 h, and in each case expression was almost totally prevented by Ro 31-8220 (Fig. 8B). However, D609 resulted in potentiation of c-fos expression by IL-1␤ and TNF␣, little effect on c-jun expression, and repression of IB␣ expression. Similarly analysis of cyclooxygenase-2, IL-8 and p50/p105, revealed an up-regulation by each stimulus and a profound repression by Ro 31-8220 (Fig. 8B). D609 resulted in a further increase in COX-2 and IL-8 expression and showed little effect on p50/p105 expression.

DISCUSSION
In a previous study (3), the PKC inhibitor, Ro 31-8220 and the PC-PLC inhibitor, D609, were shown to profoundly inhibit NF-B-dependent transcription, yet no effect on loss of IB␣ or on induction of NF-B DNA binding was observed. This led to a hypothesis whereby activation pathways in addition to the core IKK-IB pathways are necessary for transcriptional activation of NF-B (3). In the current manuscript, we now confirm these findings in bronchial epithelial BEAS-2B cells and suggest that this effect is more generally applicable to such cells. Indeed numerous other studies have also proposed various "additional" events that impact on transcriptional activation by NF-B (3) (see Ref. 2). However, many of these reported events are readily distinguishable from the pathway presented here. Thus, contrary to models suggested by other experimental systems (7,8,15), the analysis of a number of kinase inhibitors in the present study excludes a role for PI3K and PKA, while a role for PKC is reinforced. The lack of a role for PKA is further strengthened by our finding that adenoviral overexpression of protein kinase inhibitor, an endogenous and potent inhibitor of PKA (39), or the PKA activator, forskolin, were without effect on NF-B-dependent transcription (data not shown). This is consistent with more recent reports documenting repression of NF-B transactivation by PKA (40). Similarly, the finding that the phorbol esters, PMA and PDBu, but not the inactive analog 4␣-PMA, are potent activators of NF-B-dependent transcription suggests the existence of a DAG-dependent effect and again raises the possibility of PKC involvement. In each case the induction of NF-B-dependent transcription was prevented by a number of inhibitors of PKC further supporting the involvement of this kinase. However, many of the reports implicating PKC in signaling to NF-B suggest a role upstream of NF-B DNA binding (2,41). For example the novel isoform, PKC ␦, can mediate IB␣ degradation and NF-B DNA binding (42), while the atypical isoforms, PKC, which may be activated by ceramide, and PKC, have been shown to phosphorylate and activate IKK␤ and NF-B DNA binding (43)(44)(45)(46)(47). Clearly this process is mechanistically distinct from the one observed in the present study as the inhibitors of PKC or the PC-PLC inhibitor, D609, have no effect on induction of NF-B nuclear transloca- tion or DNA binding. In addition, we have previously shown that ceramide is incapable of activating or potentiating the transcriptional activity of NF-B in A549 cells (48). Furthermore, despite proving to be effective at transcriptional induction, we found that PMA, which will only activate the DAGsensitive classical and novel PKC isoforms, is in fact a very poor of inducer of NF-B DNA binding and activation occurs gradually over a number of hours (28). Consistent with this, PMA failed to induce IKK activity and did not result in loss of IB␣ in either A549 or BEAS-2B cells, whereas TNF␣ proved to be highly effective at each of these, suggesting that the induction of NF-B transcriptional activity must occur via distinct cellular processes. This idea is strengthened by the observation that a maximally effect concentration of TNF␣ was further potentiated by PMA and the finding that this PMA-dependent pathway is relatively insensitive to proteasome inhibition. However, notwithstanding the above data, a low level of activation of NF-B DNA binding is observed in both cell lines following PMA treatment and this raises the possibility that one or more PMA-activated PKC isoforms, possibly PKC␣, may also way feed into the core IKK pathway (44).
An alternative explanation for PKC-dependent activation of NF-B has previously been found at the level of p65/RelA phosphorylation (2), an event that may regulate the ability of p65/RelA to associate, when unphosphorylated, with the transcriptional repressor HDAC1 or when phosphorylated with CBP (49). Thus phorbol ester-dependent phosphorylation of p65/RelA was previously shown to correlate with increased transactivation potential (10). More recently, PKC was implicated in p65/RelA phosphorylation, and increases in transactivation have now been correlated with phosphorylation of Ser-311 of p65 and a subsequent increase in the association with CBP (50, 51). However, neither of these mechanisms can ex-plain the effects in the present study as Ro 31-8220 failed to reverse TNF␣-induced phosphorylation of p65/RelA and overexpression of a constitutively active PKC was without effect.
The fact that a number of non-PKC DAG-activated proteins have been identified led us to directly test whether PKC isoforms were actually able to activate NF-B (52). However, the possibility that PKC isoforms may differentially input both onto NF-B DNA binding and onto transcriptional potential makes the analysis of these separate events difficult (2). To functionally separate these entities, a Gal4 one-hybrid transactivation assay for p65 was established in the A549 cells (10). In this system the transactivation potential that is assayed derives only from DNA binding via the Gal4 DNA binding domain, which is constitutively nuclear, and not from signalinduced release of p65. Thus events that affect the release of p65 are not important, allowing us to show that both TNF␣ and PMA can enhance p65 transactivation, and importantly, both Ro 31-8220 and D609 are potent inhibitors of p65-dependent transactivation. Consistent with the suggested role for PKC in the transactivation of p65, the overexpression of a number of constitutively active PKC isoforms produced a robust activation of p65 transactivation. Interestingly, the atypically isoform, PKC, failed to elicit a response and this is consistent with the fact that phorbol esters, being diacylglycerol mimetics, will only activate classical and novel isoforms of PKC. The ability to elevate p65 transactivation was most apparent with the novel isoforms, particularly PKC⑀, suggesting that these kinases may be responsible for regulating transactivation. Similarly, PKC⑀ has previously been shown to activate NF-B-dependent transcription in RAW 264.7 cells providing support for a role of this kinase (53).
To explore the mechanistic basis of this effect, the co-activator protein, CBP, which is known to be instrumental in NF-B transcriptional activation (37,54), was immunoprecipitated and shown to be inducibly phosphorylated following TNF␣ stimulation as has been previously shown for IL-1␤ stimulation (26). That this phosphorylation was abolished by Ro 31-8220 and D609 presents a rational explanation for the observed transcriptional effects. As previous studies have shown that the phosphorylation of p65/RelA is critical in transcriptional activation and complex formation with CBP (20,49) and that phosphorylation of CBP is also critical in transcriptional activation (55,56), we speculated that reciprocal phosphorylation on CBP could also be important. In this context, p300 is implicated as a downstream target of PKC, although in that case the effect was to repress transcriptional output (57). In the present study, we show that following TNF␣ treatment the p65-CBP interaction is unaffected by either Ro 31-8220 or D609 in A549 cells, suggesting that the inhibition of CBP phosphorylation must play an alternative role. In this context, CBP-associated HAT activity was examined and found to be induced by TNF␣ but largely unaffected by the PKC inhibitor Ro 31-8220. By contrast, D609 caused a marked repression in CBP-associated HAT activity, and this was reflected in global changes in histone acetylation. To functionally link changes in histone acetylation with NF-B-dependent transcription, cells were treated with the histone deacetylase inhibitor, trichostatin A. This increased both histone acetylation and potentiated NF-B-dependent transcription demonstrating a positive link between these events. In terms of the data here, while these effects may explain the ability of D609 to inhibit NF-B-dependent transcription, they do not appear to account for the repressive effect of PKC inhibitors.
Since CBP function is implicated in a variety of transcriptional responses in addition to NF-B (38), we speculated that our finding may not be restricted to just NF-B-dependent transcription and indeed analysis of AP-1, CRE, or GRE-dependent transcriptional responses confirms this and suggests a more general role in transcription. However, as low level, basal transcription, from just a TATA box or high level constitutive (SV40) transcription was unaffected by either compound, our data indicate that this repressive effect is restricted to activated transcription. This is consistent with the known roles of CBP and further supports the involvement of a signaling pathway that impacts on CBP function (38). In this context, we have encountered a number of reports detailing transcriptional activation of AP-1 and serum response element by novel PKC isoforms, which may also be accounted for by the mechanisms proposed above and raise the possibility of a wide application for this effect (58,59).
As our studies have primarily focused on artificial reporter constructs, we have also examined the effect of Ro 31-8220 and D609 on the expression of acute phase genes following proinflammatory or mitogenic stimulation. Consistent with the above reporter analysis, known NF-B-dependent genes, including IB␣ (30), COX-2 (29), IL-8 (60), and p50/p105 (61), were substantially repressed by the PKC inhibitor, Ro 31-8220. This effect also extended to c-fos and c-jun, which are predominantly regulated by Sis-inducible element, serum response element, and TRE (AP-1) sites (30,62), but not the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase, suggesting the validity of our reporter analyses. By contrast, D609 produced a highly variable response depending on the stimulus and the gene in question. Thus in many cases steady state mRNA levels were either unaffected or were potentiated. A similar effect was reported in respect of GM-CSF, expression of which was markedly increased by D609 via a mechanism that was at least partly due to a profound mRNA stabilization (63). This phenomenon, coupled with the global changes in histone acetylation observed following D609 treatment, have led us to treat these data with caution.
In conclusion, we present a body of data that support a role for additional regulatory signaling pathways in the transcriptional activation of NF-B. On the basis of inhibitor data, known activators and overexpression of various PKC isoforms, we predict a role for one or more isoforms of this kinase, and we further indicate CBP as a downstream target of this pathway. Analysis of additional luciferase reporters and real genes indicated that this effect is not limited to NF-B but is in fact more generally applicable to activated transcriptional responses. The further exploration of this novel pathway will undoubtedly reveal additional insights into the regulation of activated transcriptional responses. Finally, as basal transcription was unaffected; this pathway could provide possible targets that may be exploited therapeutically to damp down the high levels of activated gene expression observed in many diseases including inflammation or cancer.