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Inhibition of NF-B Activity by a Membrane-Transducing Mutant of IB¹

Panagiotis S. Kabouridis^2,*, Maemunah Hasan^*, Justine Newson, Derek W. Gilroy and Toby Lawrence

^* Bone and Joint Research Unit and Department of Experimental Pathology, Barts and London School of Medicine and Dentistry, London, United Kingdom

	Abstract

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The transcription factor NF-B is regulated by the IB family of proteins. The nonphosphorylatable, nondegradable superrepressor IB (srIB) mutant is a potent inhibitor of NF-B activity when expressed in cells. We generated a form of srIB in which its N terminus is fused to the protein transduction domain of HIV TAT (TAT-srIB). Purified TAT-srIB protein rapidly and efficiently entered HeLa or Jurkat T cells. TAT-srIB, when exogenously added to HeLa cells, inhibited in a dose-dependent manner TNF-- or IL-1-induced NF-B activation and binding of NF-B to its consensus DNA sequence. TAT-srIB was coimmunoprecipitated with the p65 subunit of NF-B, and this interaction was resistant to stimulation with IL-1. Therefore, TAT-srIB-mediated inhibition could result from its nonreversible binding and sequestration of endogenous NF-B. In contrast, exogenously added TAT-srIB did not inhibit IL-1-induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 mitogen-activated protein kinases or the phosphorylation and degradation of endogenous IB. These results identify a novel way for direct regulation of NF-B activity in diverse cell types that may be useful for therapeutic purposes.

	Introduction

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The NF-B/Rel family of transcription factors is a major mediator of inflammation (1). Five members of this family have been identified in mammals: p50 (NF-B-1), p52 (NF-B-2), p65 (RelA), c-Rel, and RelB. They are present in cells as homo- or heterodimers; however, the most common transcription-competent form is the p50/p65 dimer. All members share a Rel homology domain, which mediates dimerization, nuclear translocation, DNA binding, and interaction with the IB family of proteins (2, 3). IB proteins interact with NF-B via their ankyrin repeats to retain the transcription factor in the cytoplasm in nonstimulated cells. After cell stimulation, IB proteins are phosphorylated in an N-terminal recognition motif by the IB kinase (IKK)³ complex, which comprises two kinases, IKK (IKK-1) and IKK (IKK-2), and a third molecule, IKK or NF-B essential modulator (NEMO), which couples upstream activators to the IKK complex (4, 5, 6). Phosphorylated IBs become polyubiquitinated and are subsequently degraded by the 26S proteasome. The best-characterized member is IB, which is phosphorylated on serines 32 and 36 by the IKK complex. Degradation of IB exposes a nuclear localization signal on NF-B, which mediates its translocation to the nucleus to initiate gene transcription. One of the NF-B-responsive genes is IB itself, which upon synthesis binds to NF-B and terminates its transcriptional activity (4, 5). The S32,36A double mutant of IB is not phosphorylated or degraded and remains constitutively attached to NF-B. This mutant, when expressed in cells, strongly inhibits NF-B activity and for this reason is termed superrepressor IB (srIB) (7, 8, 9, 10).

Proinflammatory cytokines such as TNF- and IL-1 mediate their action by activating NF-B. These cytokines are prevalent in sites of inflammation, as is the case in rheumatoid arthritis (RA), where it has been shown that inhibition of TNF- action by neutralizing Abs or soluble TNF-R ameliorates the severity of the disease (11, 12). The expression of many proinflammatory genes is regulated by NF-B, such as the cytokines TNF-, IL-1, IL6, and IL8; adhesion molecules; matrix remodelling enzymes; cyclooxygenase-2; and inducible NO synthase. NF-B activity regulates expression of IL6 and IL8 in RA synovial fibroblasts (13) and links inflammation to hyperplasia in the arthritic joint (14). In addition, a peptide that inhibited nuclear localization of NF-B improved inflammatory disease in an animal model (15). Genetic studies in knockout mice have shown that loss of p50 ablates eosinophilic airway response in allergen-induced asthma and that lack of c-Rel decreased airway hyperresponsiveness, eosinophil infiltration, and serum IgE levels in the same model of asthma (16, 17). Therefore, methods that reduce NF-B activity will be beneficial in chronic inflammatory conditions.

Certain proteins have the capacity to cross the plasma membrane of mammalian cells via yet unknown mechanism (18, 19, 20). Three such proteins are the HIV-TAT (21), VP22 from herpes simplex virus (22), and the Antennapedia protein from Drosophila (23). The segment of the protein that exhibits such capability has been named protein transduction domain (PTD), and in the case of TAT is confined to amino acids 47–57 (19). It is rich in positively charged amino acids, which may interact with the negatively charged phospholipids of the plasma membrane (19). Polyarginine peptides are also capable of entering cells, although they are less potent than the TAT PTD (24) as well as those that correspond to the hydrophobic region of a signal peptide sequence (reviewed in Ref. 25). Recently, it was demonstrated that TAT PTD domain genetically fused to heterologous proteins can mediate their rapid and efficient entry into cells (26, 27). Importantly, under certain conditions the transduced proteins acquire their physiological function inside cells (27). TAT-mediated protein transduction has also been demonstrated in vivo, where a TAT--galactosidase (TAT--gal) fusion protein injected in mice transduced a wide collection of tissues (28). These experiments have opened a new avenue for regulating intracellular functions by introducing specific protein modulators into cells.

In this report, we describe the generation and properties of a TAT-srIB fusion protein. TAT-srIB efficiently enters cells, associates with p65, and inhibits NF-B-mediated transcription. This is a novel way to regulate NF-B activity and could have useful applications in pathological conditions such as inflammation.

	Materials and Methods

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Expression constructs

The superrepressor S32,36A double mutant of human IB was a generous gift from Prof. R. Hay (University of St. Andrews, Fife, U.K.) (10). The entire coding region of srIB cDNA was amplified with PCR using the forward primer 5'-GGAGGTACCTTCCAGGCGGCCGAGCGCC-3' and the reverse primer 5'-GGAGCATGCTCATAACGTCAGACGCTGG-3'. The PCR product was digested with KpnI/SphI restriction enzymes and subcloned in frame into the corresponding sites of the pRSET-TAT-HA vector. The pRSET-HA-TAT vector has been described previously and contains an N-terminal 6x His epitope for purification of the fusion proteins, the TAT PTD, and the HA tag (27). Both strands of the TAT-HA-srIB coding region were verified by sequencing. Empty pRSET-TAT-HA vector and vector containing green fluorescent protein (GFP) or -gal were kindly provided by Dr. S. Dowdy (Washington University, St. Louis, MO) (27).

Expression and purification of fusion proteins

Purification of fusion proteins was performed as previously described (27) with some modifications. Briefly, BL21(DE3)pLysS bacteria (Novagen, Madison, WI) transformed with the TAT-srIB-, TAT-GFP-, or TAT--gal-containing constructs were grown to OD₆₀₀ of 1 in Luria-Bertani/ampicillin medium. At that point cultures were induced with 1 mM iso-propyl--D-thiogalactopyranoside (Sigma-Aldrich, Dorset, U.K.) for 3–4 h, and the bacterial pellet was resuspended in buffer Z (8 M urea, 100 mM NaCl, and 20 mM Tris, pH 8) plus a mixture of protease inhibitors (5 µg each of pepstatin A, chymostatin, leupeptin, and 1 mM PMSF). Lysates were sonicated three times for 15 s and clarified by centrifugation at 12,000 x g for 10 min. Ni²⁺-agarose (Qiagen, Valencia, CA) was added and incubation was conducted for 1 h at 4°C. The beads were washed extensively with buffer Z containing 20 mM imidazole, and bound material was eluted with buffer Z containing 1 M imidazole. The eluted fusion proteins were dialyzed extensively against PBS, adjusted to 10% glycerol, and stored at -70°C. In all preparations, the concentration of purified protein was determined with a bicinchoninic acid kit (Pierce, Rockford, IL) and purity was assessed by SDS-PAGE.

Cells, Abs, and immunoprecipitations

The human leukemic T cell line Jurkat was maintained in RPMI 1640/5% FCS and 57A HeLa cells were maintained in DMEM/10% FCS medium. 57A HeLa cells were a kind gift from Prof. R. Hay and are stably transfected with the luciferase reporter gene under the control of NF-B regulatory elements (29). Abs to IB, c-Jun N-terminal kinase 1 (JNK1), and p65 were from Santa Cruz Biotechnology (Santa Cruz, CA), anti-GFP was from Clontech Laboratories (Palo Alto, CA), and the anti-HA mAb 12CA5 was from Babco (Lakeside, CA). Abs specific for the phosphorylated forms of IB, extracellular signal-regulated kinase 1/2 (ERK1/2), JNK1/2, and p38 were from Cell Signaling Technology (Beverly, MA). For immunoprecipitations and Western analyses cells were lysed in 50 mM Tris (pH 8), 150 mM NaCl, 1% Nonidet P-40 buffer containing protease inhibitors. Aliquots of cell lysates were either resolved in SDS-PAGE or used for immunoprecipitations with the indicated Abs or with Ni²⁺-agarose. Proteins were transferred onto polyvinylidene difluoride membranes and immunodetected with the indicated Abs. HRP-conjugated secondary Abs and ECL were used to develop the Western blots as previously described (30, 31).

Luciferase assay

57A HeLa cells were seeded in 96-well plates and cultured overnight. Cells were preincubated with various concentrations of TAT-fusion proteins for the times shown in figures. After preincubation, the medium in the wells was changed to serum-free medium and cells were left unstimulated or were stimulated with 10 ng/ml TNF- or 10 ng/ml IL-1 (PeproTech, London, U.K.) for 6 h. During stimulation, TAT-fusion proteins were present in the culture medium at the same concentrations as during preincubation. After stimulation, cells were washed and cell lysates were assayed for luciferase activity with a commercial kit (Promega, Madison, WI), following the manufacturer’s instructions, in a Dynex Technologies (Chantilly, VA) luminometer. For each experiment, all of the treatment conditions shown were done in duplicates.

EMSA

EMSAs were performed as previously described (32). Briefly, HeLa 57A cells were incubated in six-well plates with fusion proteins for the indicated times and then were stimulated with 10 ng/ml IL-1 for an additional hour at 37°C. Basal NF-B binding was assessed in nonstimulated cells. After stimulation cells were disrupted in lysis buffer (20 mM HEPES, pH 8, 350 mM KCl, 0.6% Nonidet P-40, 1 mM MgCl₂, 5 mM EDTA, 20% glycerol, and 5 mM DTT) containing protease inhibitors. NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') and OCT-1 (5'-TGTCGAATGCAAATCACTAGAA-3') consensus oligonucleotides purchased from Promega were labeled with [-³²P]ATP (ICN Biochemicals, Oxfordshire, U.K.), and binding reactions were performed for 20 min by mixing 0.04 pmol of labeled oligonucleotide with 40 µg of protein extract in binding buffer (5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl, 0.5 µg poly(dI · dC), 2.5% glycerol, and 2% Ficoll) to a final volume of 20 µl. Reaction mixtures were resolved by 5% nondenaturing PAGE, and protein/oligonucleotide complexes were visualized by autoradiography.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TAT-chimeric proteins cross the plasma membrane

For expression of chimeric proteins, we used the pRSET-TAT-HA vector described by Nagahara et al. (27), which contains the 6x His epitope for protein purification, the TAT PTD flanked by glycine residues for increased flexibility, and the HA tag. A schematic representation of the three TAT chimeras used in this study is shown on Fig. 1A. Bacterially expressed proteins were purified under denaturing conditions in 8 M urea-containing buffer (see Materials and Methods). It has been previously suggested that unfolded proteins upon entry into cells can readopt their native conformation, possibly with the assistance of endogenous chaperones, and hence can regain physiological function. In contrast, exogenously added proteins if in their native conformation may be preferentially targeted for degradation (27). On SDS-PAGE, purified TAT-srIB migrates at 50 kDa, whereas TAT-GFP and TAT--gal proteins migrate as 35- and 120-kDa bands, respectively (Fig. 1B). All three of the purified proteins reacted with the anti-HA mAb 12CA5 as expected (Fig. 1C). To assess whether TAT-fusion proteins cross the plasma membrane, Jurkat T cells were incubated with 300 nM TAT--gal, TAT-GFP, or -gal that lacks the TAT PTD. Subsequently, cells were washed and cell lysates were incubated with Ni²⁺-agarose. Ni²⁺-bound proteins were resolved on SDS-PAGE and probed with the anti-HA mAb. As shown in Fig. 2A, both TAT--gal and TAT-GFP entered Jurkat cells, whereas, as expected, -gal protein without the TAT PTD was excluded. Next, Jurkat cells were incubated with 300 nM, 150 nM, or 20 nM TAT-srIB or with 300 nM TAT-GFP for 2 h at 37°C. In cells incubated with TAT-srIB, a 50-kDa, Ni²⁺-bound protein was detected with the anti-HA mAb that was not present in cells incubated with PBS, whereas a 37-kDa protein was detected in cells treated with TAT-GFP (Fig. 2B). The intensity of the 50-kDa band was reduced in cells treated with 150 nM TAT-srIBa and was very low (seen only in longer exposures) in cells treated with 20 nM TAT-srIB (Fig. 2B). Therefore, intracellular TAT-srIB levels are directly proportional to its concentration in the culture medium. Similarly, TAT-srIB and TAT-GFP fusion proteins transduced 57A HeLa cells when added exogenously as shown in Fig. 2C. In this experiment, TAT-srIB and TAT-GFP were detected with anti-IB and anti-GFP Abs, respectively.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Purified proteins containing the TAT PTD. A, Schematic representation of the TAT-fusion proteins used in this study. B, Coomassie blue stain of bacterially purified proteins. C, Purified TAT-fusion proteins were transferred onto membrane and probed with the anti-HA mAb, 12CA5. Migration of molecular mass markers is shown on the right.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. TAT-fusion proteins can transduce cells. A, 10 x 106 Jurkat T cells were left untreated or were incubated for 2 h at 37°C with 300 nM TAT-GFP, TAT--gal, or -gal, which lacks the TAT PTD. Cells were washed extensively and lysed, and Ni2+-bound proteins were analyzed in a Western blot with the anti-HA mAb 12CA5. The positions where TAT--gal and TAT-GFP migrate on the gel are shown on the left, whereas molecular mass markers are on the right. The star between markers for 150 and 100 kDa indicates a product in the TAT-GFP preparation that reacts with the anti-HA mAb. B, Jurkat cells were incubated with TAT-srIB or TAT-GFP at the concentration shown, and Western analysis of Ni2+-bound proteins was done as in A. The star indicates a band with a slightly lower mobility than TAT-srIB that nonspecifically reacts with 12CA5. C, 57A HeLa cells either received no addition or were treated with 300 nM TAT-srIB or TAT-GFP for 2 h at 37°C. Cell lysates from each cell culture were blotted with anti-IB or anti-GFP Abs, respectively. The migration distances of exogenously added TAT-srIB and of endogenous IB are shown with arrows.

TAT-srIB inhibits NF-B-driven transcription

To determine whether TAT-srIB could inhibit the transcriptional activity of NF-B, we treated 57A HeLA cells, which are stably transfected with the luciferase reporter under NF-B responsive elements, for 3 h with various concentrations of TAT-fusion proteins followed by stimulation with TNF- for an additional 6 h. TAT-srIB exogenously added inhibited TNF--induced luciferase production in a dose-dependent manner (Fig. 3A). In the experiment shown in Fig. 3A, the presence of 800 nM TAT-srIB in the culture medium resulted in 70% inhibition. Addition of equivalent amounts of TAT-GFP (Fig. 3A) or TAT--gal (data not shown) to the culture medium had no effect on TNF--induced luciferase activity. In the absence of TNF-, addition of TAT proteins did not induce NF-B activity.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. TAT-srIB inhibits NF-B-mediated transcription. A, 57A HeLa cells were preincubated for 3 h with the indicated concentrations of purified TAT-srIB or TAT-GFP proteins, and cells were stimulated with 10 ng/ml TNF- for 6 h and luciferase activity in cell lysates was determined. Luciferase activity in nonstimulated cells was 49 ± 10 arbitrary luminometer units, and in cells treated with only TAT-GFP or TAT-srIB the counts were 99 ± 7 and 58 ± 6, respectively. B, Cells were treated with the indicated purified proteins as in A, and stimulation was performed with 10 ng/ml IL-1 for 6 h and luciferase activity was determined as in A. Activity in nonstimulated cells was 60 ± 26 luciferase units. C, 57A HeLa cells were preincubated with 800 nM TAT-GFP or TAT-srIB overnight (O.N.) or for 3 h, or the TAT-fusion proteins were added to the cells simultaneously with 10 ng/ml TNF-. After 6 h of stimulation, luciferase activity in cell lysates was measured. Each point shown in the graphs is the mean of duplicate cell cultures, and where error bars are not visible it is because they are smaller than the symbol.

TAT-srIBa inhibits binding of NF-B to its consensus sequence

To test whether TAT-srIB inhibits the binding of NF-B to its consensus DNA sequence, HeLa 57A cells were incubated overnight with 600 nM TAT-srIB or TAT-GFP as control, and then cells were stimulated with 10 ng/ml IL-1 for 1 h. DNA binding of NF-B or of the unrelated transcription factor OCT-1 to their corresponding consensus sequences was detected with EMSA (Fig. 4A). TAT-srIB but not TAT-GFP addition dramatically reduced the DNA binding activity of NF-B. In contrast, binding of OCT-1 remained unaltered, indicating that TAT-srIB-mediated inhibition is selective for NF-B (Fig. 4A). From the experiment in Fig. 3C, it was noted that the length of incubation time with TAT-srIB correlated with its ability to inhibit NF-B-mediated transcription. Therefore, EMSAs were performed on lysates from HeLa cells that were incubated with TAT proteins for 1 and 3 h before stimulation with IL-1. One hour of preincubation with TAT-srIB produced a small but detectable inhibition in the DNA binding activity of NF-B, whereas 3 h of preincubation had a profound effect (Fig. 4B). However, the presence of TAT-srIB overnight produced the most dramatic inhibition. The extent of inhibition of NF-B binding after IL-1 stimulation correlated with the concentration of TAT-srIB present in the cell culture. As shown in Fig. 4C, overnight incubation with as little as 75 nM TAT-srIB, although not completely abolishing the inducible DNA binding of NF-B as did the 600- and 300-nM doses, did produce a dramatic reduction in binding. The results from the EMSA experiments correlate with those obtained using the reporter gene assay shown in Fig. 3.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Inhibition of NF-B binding to DNA by TAT-srIB. A, HeLa 57A cells were treated overnight with 600 nM TAT-srIB or TAT-GFP, and then cells were left unstimulated to detect basal level of NF-B binding or they were stimulated with 10 ng/ml IL-1 for 1 h. NF-B or OCT-1 binding to their consensus oligonucleotides was detected with EMSA. B, Cells were incubated with TAT-fusion proteins for 1 or 3 h before stimulation with IL-1, and EMSA on cell lysates was performed as in A. C, Cells treated overnight with 600 nM TAT-GFP or the indicated concentrations of TAT-srIB were stimulated for 1 h with IL-1, and the DNA binding activity of NF-B was assessed by EMSA.

TAT-srIB associates with the p65 subunit of NF-B

To assess whether exogenously added TAT-srIB can interact with cellular NF-B, 57A HeLa cells were treated with TAT-srIB or TAT-GFP for 1 or 3 h, and the p65 subunit of NF-B was immunoprecipitated. TAT-srIB coimmunoprecipitated with p65, suggesting its association with endogenous NF-B (Fig. 5A, top panel), whereas no TAT-GFP was detected in p65 immunoprecipitates (Fig. 5A, middle panel). Comparable levels of p65 protein were precipitated from the various treatment conditions (Fig. 5A, bottom panel). From these experiments we conclude that TAT-srIB that enters cells is capable of associating with NF-B, and in this regard it resembles the function of endogenously produced IB. We next investigated the effect that stimulation might have on the interaction of TAT-srIB with NF-B. 57A HeLa cells were pretreated for 3 h with 600 nM TAT-srIB or TAT-GFP and then were stimulated with IL-1 for 0, 10, and 30 min, and p65 was immunoprecipitated. In TAT-GFP-treated cells, p65 associated with endogenous IB, and upon IL-1 stimulation this interaction was strongly diminished due to the degradation of IB. In TAT-srIB-treated cells, p65 could associate with both the endogenous IB and the exogenously added TAT-srIB, and after IL-1 stimulation, although the endogenous IB was degraded, the p65/TAT-srIB interaction remained unchanged (Fig. 5B). The lower panel in Fig. 5B shows that comparable levels of p65 protein were immunoprecipitated.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. TAT-srIB associates with NF-B. A, HeLa cells were left untreated or were incubated with 600 nM TAT-srIB or TAT-GFP for 1 or 3 h. Cells were washed and lysed, and the p65 subunit of NF-B was immunoprecipitated. Immune complexes were resolved in SDS-PAGE, transferred onto membrane, and sequentially probed with anti-IB, anti-GFP, and anti-p65 Abs. B, HeLa cells were incubated with 600 nM TAT-srIB for 3 h or received no exogenous protein and then were stimulated with 10 ng/ml IL-1 for the indicated times. p65 was immunoprecipitated from cell lysates and immune complexes were probed with anti-IB Abs. The positions of endogenous IB and of exogenously added TAT-srIB are shown.

TAT-srIBa does not inhibit stimulus-induced degradation of endogenous IBa or activation of the mitogen-activated protein (MAP) kinase pathways

IL-1 and TNF-, in addition to inducing phosphorylation and degradation of IB, also stimulate activation of the ERK, JNK, and p38 MAP kinase pathways. To investigate potential effects of TAT-srIB on these events, HeLa 57A cells were incubated for 3 h with TAT-srIB or TAT-GFP as control and then were stimulated with IL-1 for the times shown in Fig. 6. Cell lysates were probed with Abs specific to the phosphorylated forms of IB, ERK1/2, JNK1/2, and p38. From Fig. 6 it is clear that TAT-IB does not affect the IL-1-induced phosphorylation and degradation of the endogenous IB protein (this conclusion can also be deduced from Fig. 5B) or the activation of the three MAP kinase pathways. Interestingly, after IL-1 stimulation of HeLa 57A cells, ERK activation peaked at around 10 min, whereas for the JNK and p38 pathways their activity was still increasing by 30 min, the latest time point investigated.

View larger version (42K): [in this window] [in a new window]   FIGURE 6. The activation of MAP kinase pathways is not affected by TAT-srIB. HeLa 57A cells were incubated for 3 h with 600 nM of the indicated TAT-fusion protein and then were stimulated with 10 ng/ml IL-1 for the times shown. Cell lysates were Western blotted (WB) with the phosphospecific or other Abs as indicated. The anti-JNK1 immunoblot shows that an equivalent amount of lysate was loaded in each lane.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Here, we describe a novel way to introduce the srIB inhibitor into cells to inhibit aberrant NF-B activity. We make use of the ability of TAT PTD to transport proteins of any size (28), or even large particles (35), into cells when it is genetically fused or covalently attached to them. The engineered TAT-srIB protein efficiently transduced cells and inhibited NF-B activity in direct relation to its concentration in the culture medium. TAT-srIB most likely exerts its inhibitory function, by associating nonreversibly with NF-B components inside cells. Only a portion of TAT-srIB that enters cells was found to interact with p65, so it is possible that only a portion of the protein that enters cells refolds appropriately to regain physiological function. What determines successful refolding of the protein inside the cell is not known and currently this is the limiting step in our ability to fully exploit this methodology. Presently we are testing the effects of TAT-srIB in animal models of inflammation. Our results agree with data presented in a recent publication in which the properties of two separate TAT-IB mutants were investigated during osteoclastogenesis (36). In this report it was shown that IB mutants that lack tyrosine 42, an Src kinase target, when delivered as TAT-fusion proteins into osteoclast precursors, inhibited nuclear localization of NF-B and prevented their maturation into osteoclasts (36).

Other labs have used bioactive peptides, which interrupt critical processes during NF-B signaling, fused to a membrane-permeable peptide to inhibit NF-B activity in vitro and in vivo. May et al. (37) have identified the NEMO binding domain (NBD) on IKK, a six-amino-acid-long motif conserved among IKK and IKK. A synthetic peptide comprising the NBD and a cell-permeable sequence derived from the Antennapedia homeodomain was shown to disrupt the NEMO/IKK interaction and to inhibit NF-B activity. However, the concentration of NBD peptide needed for this inhibition was approximately an order of magnitude greater when compared with TAT-srIB used in this paper (37). Cell-permeable peptides that inhibit nuclear translocation of NF-B were also used as means to regulate its activity. One such peptide corresponding to the nuclear localization sequence (NLS) of p50 was fused to the hydrophobic region of a signal peptide sequence and, when delivered into endothelial or monocytic cells that were stimulated with LPS or TNF-, was effective in inhibiting nuclear localization of NF-B (38). Furthermore, a cyclic version of the same peptide was shown to be three times more potent than the linear form (39). However, the inhibitory action of the p50 NLS peptide may not be exclusive for NF-B but, as was shown in T lymphocytes, it can limit nuclear translocation of the transcription factors AP-1, NFAT, and STAT1 as well (40). A cell-permeable version of the NLS that corresponds to the SV40 large T Ag, synthesized in a D-amino acid form, was also used to inhibit NF-B activity in vitro and inflammation in two animal models in vivo (15).

One could identify potential advantages and disadvantages in regulating intracellular functions in vivo using protein transduction technology. A major advantage is that it allows for the application, in various pathological conditions, of our accumulated knowledge on intracellular functions and particularly signaling pathways. Second, the levels of protein inside cells are directly regulated so that maximum benefit can be achieved with minimal side effects. Third, it is reversible and treatment can be terminated or can resume after a resting period as deemed necessary. Among the disadvantages is lack of targeting specificity because PTD chimeras enter all cell types and, therefore, specificity must be built in the protein transduced, which implies that in certain cases complex bioengineering has to be applied. Immunogenicity could be another serious drawback because delivered proteins will be in an unfolded state and it is possible that newly exposed epitopes could elicit an immune response, which will prevent repeated administration of the protein. A third disadvantage is our incomplete knowledge of the molecular mechanisms via which proteins enter cells and how they regain function once inside. Nevertheless, protein transduction presents a promising way to directly manipulate intracellular functions and a novel way to manipulate NF-B activity that could be useful for therapeutic purposes.

	Acknowledgments

 
We thank Dr. Steven Dowdy for the pTAT-HA expression vector and the TAT--gal and TAT-GFP constructs and Prof. Ronald Hay for the srIkB cDNA and 57 HeLa cells. We also thank Prof. Yuti Chernajovsky and Dr. Vassilis Pachnis for critically reviewing the manuscript and for their suggestions.

	Footnotes

 
¹ This work was supported by a Wellcome Trust Career Development Award to P.S.K. (Reference No. 058408).

² Address correspondence and reprint requests to Dr. Panagiotis S. Kabouridis, Bone and Joint Research Unit, Barts and London School of Medicine and Dentistry, Queen Mary, Charterhouse Square, London EC1M 6BQ, U.K. E-mail address: p.s.kabouridis{at}qmul.ac.uk

³ Abbreviations used in this paper: IKK, IB kinase; NEMO, NF-B essential modulator; srIB, superrepressor IB; RA, rheumatoid arthritis; PTD, protein transduction domain; -gal, -galactosidase; GFP, green fluorescent protein; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; MAP, mitogen-activated protein; NBD, NEMO binding domain; NLS, nuclear localization sequence.

Received for publication January 18, 2002. Accepted for publication June 20, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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