Identification of a p65 Peptide That Selectively Inhibits NF- (cid:1) B Activation Induced by Various Inflammatory Stimuli and Its Role in Down-regulation of NF- (cid:1) B-mediated Gene Expression and Up-regulation of Apoptosis*

Because of the critical role of the nuclear transcription factor NF- (cid:1) B in inflammation, viral replication, carcino-genesis, antiapoptosis, invasion, and metastasis, specific inhibitors of this nuclear factor are being sought and tested as treatments. NF- (cid:1) B activation is known to re-quire p65 phosphorylation at serine residues 276, 529, and 536 before it undergoes nuclear translocation. Small protein domains, termed protein transduction domains (PTDs), which are able to penetrate cell membranes can be used to transport other proteins across the cell mem-brane. We have identified two peptides from the p65 subunit of NF- (cid:1) B (P1 and P6 were from amino acid residues 271–282 and 525–537, respectively) that, when linked with a PTD derived from the third helix sequence of antennapedia, inhibited tumor necrosis factor (TNF)-induced NF- (cid:1) B activation in vivo . Linkage to the PTD was not, however, required to suppress the binding of the p50-p65-heterodimer to the DNA in vitro . PTD-p65-P1 had no effect on TNF-induced AP-1 activation. PTD-p65-P1 suppressed NF- (cid:1) B activation induced by lipopolysaccharide, inter-leukin-1,

NF-B represents a group of five proteins, namely c-Rel, RelA (p65), RelB, NF-B1 (p50 and p105), and NF-B2 (p52) (1). Ways to modulate NF-B expression therapeutically have focused on its active-inactive state transition mechanisms. NF-B is regulated by a family of inhibitors, called IB 1 (2). In an inactive state, NF-B is present in the cytoplasm as a heterotrimer consisting of p50, p65, and IB␣ subunits. In response to an activation signal, the IB␣ subunit is phosphorylated at serine residues 32 and 36, ubiquitinated at lysine residues 21 and 22, and degraded through the proteosomal pathway, thus exposing the nuclear localization signals on the p50-p65 heterodimer. The p65 is then phosphorylated, leading to the nuclear translocation and binding to a specific sequence in DNA, which in turn results in transcriptions of various genes including cyclin D1, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9.
NF-B has been shown to regulate the expression of a number of genes whose products are involved in inflammation, viral replication, carcinogenesis, antiapoptosis, invasion, and metastasis (3,4). Specific adhesion molecule, chemokine, inflammatory cytokine, and cell cycle regulatory gene are affected. Thus, agents that can suppress NF-B activation have the potential to be treatments for inflammatory diseases and cancer (4 -7).
Most proteins enter the cell through their specific cell surface receptors. Recent studies, however, indicate that certain short protein sequences can enter the cells without any receptors, and these have been named protein transduction domain (PTD) peptides (10,11). Importantly, proteins, peptides, and antisense oligonucleotides conjugated to these PTD have been shown to deliver these cargoes effectively, as demonstrated in several cell and animal models (10,11). Peptides derived from the third helix of the antennapedia homeodomain, herpes virus structural protein, and human immunodeficiency virus Tat protein have been used to deliver both small and large peptides of interest to the cells through an energy-and receptor-independent mechanism (12)(13)(14). Most of the PTD are arginine-rich peptides (15).
We designed specific inhibitors of NF-B activation based on the phosphorylation sites present in the p65. To permit the inhibitor to cross the cell membrane, the synthetic p65 peptide was linked with a protein transduction peptide, a short protein sequence that can enter cells without any receptors (10,11). Specifically, we linked the peptide to an antennapedia-derived PTD and investigated its ability to inhibit NF-B activation in vitro and in vivo and explored its mechanism of action. We report the identification of a novel peptide that suppressed NF-B activation induced by a variety of inflammatory stimuli and NF-B-mediated gene transcription and enhanced apoptosis induced by TNF and other apoptotic stimuli.
Cell Lines-We used the leukemic cell line KBM-5, which is phenotypically myeloid with monocytic differentiation and A 293 embryonic kidney cells. KBM-5 cells were maintained in Iscove's modified Dulbecco's medium supplemented with 15% fetal bovine serum, and A 293 cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin.
Peptide Synthesis-All peptides (see Fig. 1) were synthesized using an automated peptide synthesizer (Symphony Multiplex, Rainin Instruments, MA). The peptides were purified to more than 90% purity using high pressure liquid chromatography.
Electrophoretic Mobility Shift Assays (EMSA)-To measure NF-B activation, we performed EMSA as described previously (44,45). Briefly, nuclear extracts prepared from TNF-treated cells (1 ϫ 10 6 /ml) were incubated with 32 P-end-labeled 45-mer double-stranded NF-B oligonucleotides (10 g of protein with 16 fmol of DNA) from the human immunodeficiency virus long terminal repeat, 5Ј-TTGTTACAAGGGA-CTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3Ј (boldface type indicates NF-B binding sites) for 30 min at 37°C, and the DNA-protein complex formed was separated from free oligonucleotides on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5Ј-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCG-TGG-3Ј, was used to examine the specificity of binding of NF-B to the DNA. The specificity of binding was also examined by competition with the unlabeled oligonucleotides. To investigate the binding of recombi-nant p65 to DNA, poly(dI-dC) was excluded from the binding reaction buffer. For supershift assays, nuclear extracts prepared from TNFtreated cells were incubated with antibodies against either the p50 or p65 subunits of NF-B for 30 min at 37°C, and then the complex was analyzed by EMSA. Antibodies against cyclin D1 and preimmune serum were included as negative controls. The dried gels were visualized, and radioactive bands were quantitated by a PhosphorImager (Amersham Biosciences) using ImageQuant software.
Western Blot Analysis-To determine the levels of protein expression in the cytoplasm or the nucleus, we prepared extracts (46) from TNF-treated cells and fractionated them by SDS-PAGE. After electrophoresis, the proteins were electrotransferred to nitrocellulose membranes, blotted with each antibody, and detected by ECL regent (Amersham Biosciences).
IKK Assay-The IKK assay was performed by a method described previously (47). Briefly, IKK complex from whole-cell extract was precipitated with antibody against IKK-␣, followed by treatment with protein A/G-Sepharose beads (Pierce). After a 2-h incubation, the beads were washed with lysis buffer and then assayed in kinase assay mixture containing 50 mM HEPES (pH 7.4), 20 mM MgCl 2 , 2 mM dithiothreitol, 20 Ci of [␥-32 P]ATP, 10 M unlabeled ATP, and 2 g of substrate GST-IB␣ (residues 1-54). After incubation at 30°C for 30 min, the reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized by a PhosphorImager.
To determine the total amounts of IKK-␣ and IKK-␤ in each sample, 30 g of the whole-cell protein was resolved on 7.5% SDS-PAGE, electrotransferred to a nitrocellulose membrane, and then blotted with either anti-IKK-␣ or anti-IKK-␤ antibodies.
Cell-free phosphorylation of peptide by IKK was also determined using 10 g of peptides as a substrate in the kinase reaction mixture as described above and then fractionated on 20% SDS-PAGE in 2ϫ SDS electrophoresis buffer.
NF-B-dependent Reporter Secretory Alkaline Phosphatase (SEAP) Expression Assay-The effect of inhibitory peptides on TNF-, TNFR-, TRADD-, TRAF2-, NF-B-inducing kinase-, IKK-, and p65-induced NF-B-dependent reporter gene transcription was analyzed by SEAP assay as previously described (48). Briefly, A 293 cells (5 ϫ 10 5 cells/well) were plated in 6-well plates and transiently transfected by the calcium phosphate method with pNF-B-SEAP (0.5 g). To examine TNF-induced reporter gene expression, we transfected the cells with 0.5 g of the SEAP expression plasmid and 2 g of the control plasmid pCMVFLAG1 DNA for 24 h. Thereafter, the cells were treated for 24 h with 150 M peptides and then stimulated with 1 nM TNF for 24 h. The cell culture medium was then harvested and analyzed for alkaline phosphatase (SEAP) activity according to the protocol essentially as described by the manufacturer (Clontech, Palo Alto, CA) using a 96-well fluorescence plate reader (Fluoroscan II, Labsystems, Chicago, IL) with excitation set at 360 nm and emission at 460 nm.
Cytotoxicity Assay (MTT Assay)-The cytotoxic effects of TNF were determined by the MTT uptake method as described (47). Briefly, 5000 cells were incubated with synthetic peptides for 1 h in triplicate in 96-well plates and then treated with various concentrations of TNF for 72 h at 37°C. Thereafter, MTT solution was added to each well. After a 2-h incubation at 37°C, extraction buffer (20% SDS, 50% dimethylformamide) was added, the cells were incubated overnight at 37°C, and then the optical density was measured at 570 nm using a 96-well multiscanner (MRX Revelation; Dynex Technologies, Chantilly, VA).
Cytotoxicity Assay (Live/Dead® Assay)-The cytotoxic effects of TNF were also determined by the Live/Dead® assay (Molecular Probes, Inc., Eugene, OR). Briefly, 1 ϫ 10 5 cells were incubated with 100 M PTD-p65-P1 for 1 h and then treated with 1 nM TNF for 16 h at 37°C. Cells were stained with Live/Dead® reagent (5 M ethidium homodimer, 5 M calcein-AM) and then incubated at 37°C for 30 min. Cells were analyzed under a fluorescence microscope (Labophot-2, Nikon, Tokyo, Japan).
TUNEL Assay-The TNF-induced apoptosis was determined by TUNEL assay using In Situ Cell Death Detection reagent (Roche Applied Science). Briefly, 1 ϫ 10 5 cells were incubated with PTD-p65-P1 for 1 h and then treated with 1 nM TNF for 16 h at 37°C. Thereafter, cells were plated on a poly-L-lysine-coated glass slide by centrifugation using a Cytospin 4 centrifuge (Thermoshendon, Pittsburgh, PA), airdried, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate. After washing, cells were incubated with reaction mixture for 60 min in 37°C. Stained cells were mounted with mounting medium purchased from Sigma and analyzed under a fluorescence microscope (Labophot-2).

RESULTS
To design a peptide that can suppress NF-B activation, we targeted the phosphorylation sites of the p65 subunit of NF-B. The p65 consists of a DNA-binding and dimerization domain (RHD in Fig. 1; residues 191-290), nuclear localization domain (NLS; residues 291-325), and transactivation domain (TAD). The phosphorylation residue Ser 276 present in the DNA-binding and dimerization domain and Ser 529 and Ser 536 present in the transactivation domain were targeted (see Fig. 1A). The p65 peptides listed in Fig. 1 were linked with the PTD derived from the third helix of the antennapedia homeodomain (Fig. 1B). These peptides were tested for their ability to suppress NF-B activation induced by various proinflammatory stimuli.
Specific Amino Acid Sequence Is Required for Suppression of NF-B Activation in Vitro-We used the in vitro assay to determine the amino acid sequence needed for NF-B activation ( Fig. 2A). The p65-derived peptides in which Ser 276 was mutated (p65-P2) did not inhibit NF-B binding to the DNA. Peptides in which 5 amino acid residues from the C terminus (QLRRP, SD; p65-P4) or QLR-deleted (RP, SDREL, SE; p65-P5), were also inactive. The minimum peptide required for suppression of NF-B activation was QLRRPSDRELSE (p65-P1).
Whether p65-P6 can suppress the p50-p65 binding to the DNA was examined. Peptides in which Ser 529 was mutated (p65-P7) or Ser 536 was mutated (p65-P8) did not inhibit NF-B binding to the DNA (Fig. 2A). These results suggest that the inhibition of TNF-induced NF-B activation by p65-P6 requires the presence of both of the phosphorylation sites, Ser 529 and Ser 536 . In contrast, p65-P1 contains a single phosphorylation site, and it is needed to inhibit NF-B activity. All subsequent studies were performed with PTD-p65-P1.
Whether the p65-derived peptides also inhibit the binding of recombinant p65 protein to the DNA was investigated. To determine this, the recombinant purified p65 protein was incubated with various peptides and then examined for DNA binding. The p65-P1 and p65-P6 peptides, whether linked with or without PTD, inhibited the binding of recombinant p65 to the DNA in the same manner as natural p65/p50-NF-B complex (Fig. 2B).
The dose-dependent effects of PTD-p65-P1 and p65-P1 on the p50-p65 binding to the DNA were investigated. Nuclear extracts from TNF-treated cells were incubated with different concentrations of the peptide and then examined for DNA binding. The p65-P1 inhibited NF-B binding in a dose-dependent manner, and maximum inhibition occurred at 50 M (Fig.  2C). PTD-p65-P1 also inhibited NF-B binding at the same concentration. PTD alone had no effect.
PTD-p65-P1 Inhibits TNF-induced NF-B Activation in Vivo-To determine the effect of the peptide containing Ser 276 (PTD-p65-P1), we preincubated KBM-5 cells with the peptides for 1 h and then treated them with 0.1 nM TNF for the indicated times. Nuclear extracts were prepared, and we then analyzed NF-B activation by EMSA (Fig. 3A). TNF induced NF-B activation in a time-dependent manner, and pretreatment with PTD-p65-P1 completely abolished the TNF-induced NF-B activation. Neither PTD nor p65-P1 alone had any effect on TNF-induced NF-B activation, indicating that p65-P1 must be attached to a PTD for it to enter the membrane. We also investigated the minimum dose of PTD-p65-P1 required to suppress NF-B activation. PTD-p65-P1 suppressed TNF-induced NF-B activation by 25% at 100 M and completely at 150 M (Fig. 3B).
PTD-p65-P6 Also Inhibits TNF-induced NF-B Activation in Vivo-To determine the effect of the peptide containing Ser 529 and Ser 536 (PTD-p65-P6), we preincubated KBM-5 cells with various concentrations of peptides for 1 h and then treated them with 0.1 nM TNF for 30 min. PTD-p65-P6 inhibited TNF-induced NF-B activation in a dose-dependent manner. Neither PTD nor p65-P6 alone had any effect on TNF-induced NF-B activation (Fig. 3C). These results indicated that both Ser 539 and Ser 536 were important for inhibiting NF-B activation.
Specificity of PTD-p65-P1 Inhibiting Activity-Various combinations of Rel/NF-B protein can constitute an active NF-B heterodimer that binds to a specific sequence in the DNA (1). To show that the retarded band visualized by EMSA in TNF-treated cells was indeed NF-B, we incubated nuclear extracts from TNF-stimulated cells with antibodies to either the p50 (NF-B1) or the p65 (RelA) subunit of NF-B. Both shifted the band to a higher molecular mass (Fig. 3D), thus suggesting that the TNFactivated complex consisted of p50 and p65 subunits. Furthermore, 50 M p65-P1 inhibited the formation of the higher molecular mass band. Neither preimmune serum nor the irrelevant antibody anti-cyclin D1 had any effect. Excess unlabeled NF-B (100-fold; competitor) caused complete disappearance of the band, but mutant oligonucleotides did not.
TNF-induced AP-1 Activation Is Not Inhibited by PTD-p65-P1-Like NF-B, TNF is a potent activator of AP-1 (49). Whether PTD-p65-P1 affects TNF-induced AP-1 activation was also investigated. Cells were treated with 0.1 nM TNF for the indicated times, and nuclear extracts were prepared and assayed for AP-1 activation by EMSA (Fig. 4A). TNF activated AP-1, but PTD-p65-P1 had no effect on the activation of AP-1.
PTD-p65-P1 and PTD-p65-P6 Have No Effect on IB␣ Phosphorylation or Degradation-The translocation of NF-B to the nucleus is proceeded by the phosphorylation, ubiquitination, and proteolytic degradation of IB␣ (1). To determine whether PTD-p65-P1 inhibits TNF-induced NF-B activation by inhibiting IB␣ degradation and phosphorylation, we pretreated cells with the peptide for 1 h and then exposed them to 0.1 nM TNF for the indicated times. We then examined the cells for IB␣ status in the cytoplasm by Western blot analysis. TNF induced phosphorylation and degradation of IB␣, and pretreatment of cells with PTD-p65-P1 had no effect on either phosphorylation or degradation of IB␣ (Fig. 5A, upper panel). Similarly, PTD-p65-P6 also did not affect either phosphoryla-tion or degradation of IB␣ (Fig. 5B, upper panel).
PTD-p65-P1 and PTD-p65-P6 Inhibit Cytoplasmic p65 Phosphorylation and Nuclear Translocation-Western blot analysis showed that TNF induced nuclear translocation of p65 in a time-dependent manner. As early as 5 min after TNF stimulation, p65 was translocated to the nucleus, and the amount remained constant for 30 min (Fig. 5, A and B, middle panel), whereas cells treated with either PTD-p65-P1 or PTD-p65-P6 did not show p65 nuclear translocation. Our results also show that TNF induced the phosphorylation of cytoplasmic p65 in a time-dependent manner, whereas PTD-p65-P1 or PTD-p65-P6 suppressed it almost completely (bottom of Fig. 5, A and B). These results suggest that PTD-p65-P1 and PTD-p65-P6 suppressed TNF-induced NF-B activation by inhibiting phosphorylation and nuclear translocation of p65.
PTD-p65-P1 Has No Effect on the TNF-induced IKK Activation-Since IKK is required for TNF-induced NF-B activation, we next determined the effect of PTD-p65-P1 on TNF-induced IKK activation. Immune complex kinase assays showed that TNF activated IKK as early as 5 min after TNF treatment, and PTD-p65-P1 had no effect on this activation (Fig. 5C).
IKK Phosphorylates the p65 Peptides in a Cell-free System-p65-P1 or p65-P6 has one or two serine residues, respectively. Whether these residues could be phosphorylated by IKK was investigated. Whole-cell extracts from TNF-treated cells were immunoprecipitated with antibody against IKK and then sub- jected to the immunocomplex kinase assay using p65 peptides as a substrate. After reaction, samples were fractionated on 20% SDS-PAGE with 2-fold electrophoresis buffer. Fig. 5D shows that precipitated IKK complex phosphorylated p65-P1 and p65-P6, suggesting that synthetic peptides from the p65 subunit of NF-B can be phosphorylated by IKK complex. Our results also showed that p65 peptides in which Ser 276 , Ser 529 , or Ser 536 residues were mutated into alanine did not undergo phosphorylation by the IKK complex. These results suggested that Ser 276 , Ser 529 , and Ser 536 are necessary for p65 to be phosphorylated by IKK.
PTD-p65-P1 Inhibits TNF-induced NF-B-dependent Reporter Gene Expression-Although we had shown by EMSA that PTD-p65-P1 blocked NF-B activation, DNA binding does not always correlate with NF-B-dependent gene transcription, suggesting that there are additional regulatory steps (50). To determine the effect of PTD-p65-P1 on TNF-induced NF-B-dependent reporter gene expression, we transiently transfected the cells with the NF-B-regulated SEAP reporter construct, incubated them with the peptide, and then stimulated the cells with TNF. An almost 4-fold increase in SEAP activity over the vector control occurred upon stimulation with TNF, and peptide PTD-p65-P1 completely suppressed the TNF-induced increase. PTD or p65-P1 alone failed to suppress it (Fig. 6A). These results demonstrate that PTD-p65-P1 also repressed NF-B-dependent reporter gene expression induced by TNF.
TNF-induced NF-B activation is mediated through sequential interaction of the TNF receptor with TRADD, TRAF2, NF-B-inducing kinase, and IKK, resulting in phosphorylation of IB␣ (50,51). To delineate the site of action of PTD-p65-P1 in the TNF-signaling pathway leading to NF-B activation, we transfected cells with TNFR1-, TRADD-, TRAF2-, NF-B-inducing kinase-, IKK-, and p65-expressing plasmids and then monitored NF-B-dependent SEAP expression. As shown in Fig. 6B, NF-B-SEAP gene expression was induced in all of the plasmidtransfected cells, and PTD-p65-P1 suppressed NF-B reporter gene expression in every case. These results suggest that PTD-p65-P1 affected NF-B activation at the terminal step.
PTD-p65-P1 Potentiates Chemotherapy-induced Cytotoxicity-Chemotherapeutic agents are also known to activate NF-B and mediate chemoresistance (60). Whether suppression of NF-B by PTD-p65-P1 affects chemotherapy-induced cytotoxicity was investigated by the MTT assay. As shown in Fig. 7D, doxorubicin-induced (top panel) and cisplatin-induced (bottom panel) cytotoxicity was potentiated by PTD-p65-P1. These results suggest that PTD-p65-P1 increases the therapeutic effects of chemotherapeutic agents. DISCUSSION The nuclear transcription factor NF-B mediates inflammation, viral replication, carcinogenesis, antiapoptosis, invasion, and metastasis, so specific inhibitors of this factor should have therapeutic potential. In the present report, we identified a peptide from the p65 subunit of NF-B that contains phosphorylation sites (amino acids 271-282 or 524 -537) and that suppresses TNF-induced NF-B activation in vivo. We found that a membrane-penetrating peptide, specifically a PTD, was required to suppress NF-B activation in vivo but not in vitro. PTD-p65-P1 had no effect on TNF-induced AP-1 activation. The p65 peptide in which either of the serines was mutated or deleted did not inhibit DNA binding of NF-B. PTD-p65-P1 had no effect on TNF-induced IB␣ phosphorylation, IB␣ degradation, or IKK activation, but it blocked cytoplasmic p65 phosphorylation and nuclear translocation. In vitro PTD-p65-P1 underwent phosphorylation when incubated with IKK. PTD-p65-P1 was not specific in suppressing TNF-induced NF-B activation; it also suppressed activation induced by all of the seven inflammatory stimuli tested. NF-B-regulated reporter gene expression induced by TNF, TNFR1, TRADD, TRAF2, NF-B-inducing kinase, IKK, and p65 was also suppressed by PTD-p65-P1. Suppression of NF-B by PTD-p65-P1 enhanced the apoptosis induced by TNF and chemotherapeutic agents.
Our results indicate that the p65-P1 (QLRRPSDRELSE) amino acids sequence when linked with the cell delivery PTD can suppress NF-B activation. Deletion of residues either from the C terminus or N terminus abolished the NF-B sup- pressive activity of this peptide. Substitution of serine with an alanine residue also abolished the activity of the peptide. Previously, a sequence representing the nuclear localization of sequence of p50 has been reported to suppress NF-B activated by lipopolysaccharide and TNF (20 -23). A NEMO-binding domain peptide based on the carboxyl-terminal segment of IKK-␣ and IKK-␤ also has been reported to block NF-B activation (19). We showed that 150 M peptide is required to suppress NF-B activation. May et al. (19) showed complete suppression of NF-B at a 200 M concentration of their peptide. Interestingly, we found that the p65 peptide without the delivery peptide was quite effective in inhibiting binding of NF-B to the DNA in vitro and at a concentration lower than that required to suppress in vivo.
The p65-P1 (QLRRPSDRELSE) had no effect on AP-1 and FIG. 5. A, PTD-p65-P1 has no effect on the TNF-induced IB␣ phosphorylation or degradation but inhibits cyoplasmic p65 phosphorylation and nuclear translocation. Cells were incubated with 150 M PTD-p65-P1 for 1 h and treated with 0.1 nM TNF for the indicated times. Nuclear and cytoplasmic extracts were prepared and then fractionated on 10% SDS-PAGE. Western blot analysis was performed using with phospho-specific anti-IB␣, anti-IB␣, phospho-specific anti-p65, anti-p65, and ␤-actin. B, PTD-p65-P6 has no effect on the TNF-induced IB␣ phosphorylation or degradation but inhibits p65 phosphorylation and nuclear translocation. Cells were incubated with 150 M PTD-p65-P6 for 1 h and treated with 0.1 nM TNF for the indicated times. Nuclear and cytoplasmic extracts were prepared and then fractionated on 10% SDS-PAGE. Western blot analysis was performed using phospho-specific anti-IB␣, anti-IB␣, phospho-specific anti-p65, anti-p65, and ␤-actin. C, PTD-p65-P1 peptide has no effect on the TNF-induced IKK activation. Cells were incubated with 150 M PTD-p65-P1 for 1 h and treated with 0.1 nM TNF for the indicated times. Whole-cell extracts were prepared, incubated with anti-IKK-␣ antibody, and then immunoprecipitated using protein A/G-Sepharose beads. Immunocomplex kinase reaction was performed as described under "Materials and Methods." Whole-cell extracts were fractionated on 7.5% SDS-PAGE and immunoblotted using anti-IKK-␣ and anti-IKK-␤ antibodies. D, . Cells were treated with 1 nM TNF, and supernatants were collected and assayed for SEAP. B, A293 cells were transiently transfected with a NF-B-containing plasmid along with the indicated plasmids and then incubated with 150 M PTD-p65-P1. Cells were exposed to TNF, and supernatants of the culture medium were assayed for SEAP. Results are expressed as -fold activity of the vector control. C, KBM-5 cells were incubated with 150 M PTD-p65-P1 for 1 h and treated with 0.1 nM TNF for the indicated times. Nuclear and cytoplasmic extracts were prepared and then fractionated on 10% SDS-PAGE.
IKK phosphorylates p65 peptides in a cell-free system. Whole-cell extracts were prepared from TNF-treated cells and immunoprecipitated with antibody against IKK-␣. Thereafter, an immunocomplex kinase assay was performed in the presence of the peptides as a substrate. did not affect IKK or IB␣ phosphorylation or degradation. It did suppress cytoplasmic p65 phosphorylation and nuclear translocation. Whether p65 phosphorylation is needed for nuclear translocation is not fully understood. We also found that the peptide itself undergoes phosphorylation upon treatment with IKK. These results are consistent with previous reports that IKK can induce the phosphorylation of p65 (36 -38). Sakurai et al. (36) have shown that IKK phosphorylates p65 at serine residue 536.
We also found that peptide from 525-536 amino acids of p65 (p65-P6) suppressed TNF-induced NF-B activity, and substitution of serine with alanine residue at 529 or 536 also abolished the activity of the peptide, the same as p65-P1. However, both serine 529 and 536 amino acids were necessary to inhibit NF-B activity in p65-P6. These results showed different regulation mechanisms between p65-P1 and p65-P6 on NF-B activity. Why mutation of the serine phosphorylation sites to alanines should abolish the inhibitory activity of the peptides is not clear. It is possible that mutated peptide has low affinity for binding to p65 and thus is a poor decoy. These results are in agreement with a previous report showing that a point mutation within the NEMO-binding peptide from aspartic acid 738, tryptophan 739, or tryptophan 741 to alanine prevented the association with NEMO and thus lost the ability to inhibit NF-B activation (19). The presence of the dimerization domain of p65 between residues 191 and 290 also raises the possibility that mutation of Ser 276 altered the ability of the native peptide to modulate p65 dimerization. Zhong et al. (31) have shown that p65 phosphorylation enhances its binding to the DNA. Thus, if p65 phosphorylation is critical for binding to the DNA, a peptide with a mutated phosphorylation site may not inhibit the p65 phosphorylation. However, the latter possibility is less likely, because our experiments show that the binding of the recombinant p65 protein to the DNA is also inhibited by the native peptide and not by the mutated analogue (Fig. 2B).
Numerous reports suggest that NF-B mediates suppression of apoptosis. We showed that p65-P1 (QLRRPSDRELSE) could sensitize the cells to apoptosis induced by TNF, doxorubicin, and cisplatin. We showed that p65-P1 (QLRRPSDRELSE) suppresses TNF-induced NF-B-mediated reporter gene expression. Several genes that are involved in suppression of apoptosis are regulated by NF-B. These include cIAP, TRAF1, TRAF2, cFLIP, survivin, Bcl-x L , and XIAP. It is possible that p65-P1 (QLRRPS-DRELSE) potentiates apoptosis by suppressing the expression of one or more of these genes. Genes involved in tumorigenesis, metastasis, angiogenesis, and inflammation are all regulated by NF-B. These genes may also be target for p65-P1 (QLRRPS-DRELSE). Indeed we found that TNF-induced cyclin D1, COX-2, and MMP-9 expression is down-regulated by this peptide. Thus, the peptide p65-P1 (QLRRPSDRELSE) reported here has a wide ranging therapeutic potential.