A Cell-penetrating Peptide Suppresses Inflammation by Inhibiting NF-κB Signaling

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1 originl rticle A Cell-penetrting Peptide Suppresses Inflmmtion y Inhiiting NF-κB Signling Yu Fu Wng 1,2, Xing Xu 1, Xi Fn 1, Chun Zhng 1, Qing Wei 1, Xi Wng 1, Wei Guo 1, Wei Xing 1, Jin Yu 3, Jing-Long Yn 2 nd Hu-Ping Ling 1 1 Stte Key Lortory of Trum, Burn nd Comined Injury, Reserch Institute of Surgery nd Dping Hospitl, Third Militry Medicl University, Chongqing, People s Repulic of Chin; 2 Deprtment of Orthopedics, First Affilited Hospitl of Hrin Medicl University, Hrin, People s Repulic of Chin; 3 Deprtment of Pthology, University of Pittsurgh, Pittsurgh, Pennsylvni, USA Nucler fctor-κb (NF-κB) is centrl regultor of immune response nd potentil trget for developing nti-inflmmtory gents. Mechnistic studies suggest tht compounds tht directly inhiit NF-κB DNA inding my lock inflmmtion nd the ssocited tissue dmge. Thus, we ttempted to discover peptides tht could interfere with NF-κB signling sed on highly conserved DNA-inding domin found in ll NF-κB memers. One such smll peptide, designted s ntiinflmmtory peptide-6 (), ws chrcterized in the current study. directly intercted with p65 nd displyed n intrinsic cell-penetrting property. This peptide demonstrted significnt nti-inflmmtory effects in vitro nd in vivo. In vitro, inhiited the DNA-inding nd trnscriptionl ctivities of the p65 NF-κB suunit s well s the production of inflmmtory meditors in mcrophges upon stimultion. Locl dministrtion of significntly inhiited inflmmtion induced y zymosn in mice. Collectively, our results suggest tht is promising led peptide for the development of specific NF-κB inhiitors s potentil nti-inflmmtory gents. Received 28 Octoer 21; ccepted 2 April 211; pulished online 1 My 211. doi:1.138/mt Introduction Nucler fctor-κb (NF-κB) represents group of five structurlly relted nd evolutionrily conserved mmmlin proteins, nmely RelA (p65), RelB, c-rel, p5 (nd its precursor p15), nd p52 (nd its precursor p). 1 Most memers of this fmily cn homodimerize s well s heterodimerize with one nother. The most prevlent form of NF-κB is heterodimer consisting of the p5 nd p65 suunits. As uiquitous trnscription fctor, NF-κB plys key role in regulting the trnscription of mny genes relted to inflmmtion, such s cytokines, chemokines, nd inflmmtory enzymes. 2 4 A numer of proteins regulted y NF-κB lso induce the ctivtion of NF-κB, therey forming positive regultory loop tht mplifies inflmmtory responses. Anorml ctivtion of NF-κB hs een implicted in the pthogenesis of mny inflmmtory diseses, such s rheumtoid rthritis, sthm, sepsis, virl infections, nd inflmmtory owel diseses, nd its role hs een confirmed in experimentl models involving oth cute nd chronic inflmmtion. 5 9 Suppression of NF-κB is likely to e effective for the tretment of inflmmtory diseses. NF-κB is one of the points involved in cross-tlk etween multiple signl trnsduction pthwys. Activted NF-κB leds to trnscriptionl upregultion of multiple genes. In unstimulted cells, NF-κB is locted in the cytoplsm in n inctive form nd is ound to inhiitor proteins, nmely IκBs, including IκBα, IκBβ, nd IκBε. A vriety of molecules, including proinflmmtory cytokines such s tumor necrosis fctor-α (TNF-α) nd interleukin-1β (IL-1β) nd cteril products such s lipopolyscchrides, nd virl infection induce phosphoryltion of IκB t specific NH 2 -terminl serine residues. Phosphorylted IκB is then uiquitinted nd degrded y the protesome; thus, the ound NF-κB is relesed. NF-κB then trnsloctes to the nucleus, inds to κb elements with consensus sequence of 5 -GGGRNYYYCC-3, nd induces the expression of trget genes. 2,1 The recognition nd inding of NF-κB to κb elements is key step in the process of NF-κB ctivtion. The p5 suunit of the p5-p65 heterodimer minly intercts with the κb elements. 11,12 The RXXRXRXXC motif in p5, conserved in ll Rel/NF-κB proteins, is essentil in DNA recognition nd inding. 13 Most NF-κB inhiitors developed to dte inhiit either NF-κB protein ctivtion or IκB phosphoryltion nd degrdtion, thus preventing the relese of free NF-κB nd its entry into the nucleus. Another pproch tht directly inhiits NF-κB DNA inding, y interfering with the DNA inding of NF-κB to the promoter of trgeted genes, lso ppers menle to designing specific inhiitors. However, limited numer of gents hve een developed sed on this pproch. 14 We resoned tht short peptides resemling the DNA-inding motif RXXRXRXXC in p5 might selectively ind with κb elements nd prevent the inding of ctive NF-κB complexes. With this in mind, we designed nd synthesized The first two uthors contriuted eqully to this work. Correspondence: Hu-Ping Ling, The Stte Key Lortory of Trum, Burn nd Comined Injury, Reserch Institute of Surgery nd Dping Hospitl, Third Militry Medicl University, 1 Brnch Rod, Chngjing Street, Chongqing 442, People s Repulic of Chin. E-mil: huping_ling@yhoo.com.cn or Jing-Long Yn, Deprtment of Orthopedics, First Affilited Hospitl of Hrin Medicl University, 23 Youzheng Street, Nngng District, Hrin, 151, PR Chin. E-mil: yjlg4@yhoo.com.cn Moleculr Therpy vol. 19 no. 1, oct

2 series of peptides. Intriguingly, one of these peptides, designted nti-inflmmtory peptide-6 (), inhiited NF-κB trnscriptionl ctivity ut did not ind with the κb motif. In the current study, we show tht the peptide intercts directly with p65 to inhiit the DNA-inding nd trnscriptionl ctivities of NF-κB nd the production of inflmmtory meditors in vitro. Interestingly, possesses n intrinsic cell-penetrting property. Locl dministrtion of inhiited inflmmtory responses induced y zymosn in the joints nd soft tissues in mice. Tken together, our findings suggest tht cn e used s led peptide to develop nti-inflmmtory gents for the tretment of diseses cused y norml ctivtion of NF-κB. Results inds to nd inhiits the DNA-inding ctivity of the NF-κB p65 suunit We discovered tht is potentil inhiitor of NF-κB signling y using ioinformtic pproch sed on the conserved RXXRXRXXC motif tht is required for DNA recognition nd inding of NF-κB fmily memers. 13 We first determined whether could lock the inding of NF-κB nd κb elements in vitro y using n ssy sed on the enzyme-linked immunosorent ssy (ELISA). or negtive control peptide () t vrious concentrtions ws preincuted with Jurkt nucler extrct [phorol, 12-myristte, 13 cette (TPA) + clcium ionophore-stimulted] for 3 minutes efore eing dded to microtiter pltes coted with κb site oligonucleotides. t 25 μmol/l ws found to inhiit the DNA-inding ctivity of NF-κB in dose-dependent mnner ut did not (Figure 1). Interestingly, when, even t 4 μmol/l, ws preincuted in microtiter pltes coted with κb oligonucleotides efore the ddition of Jurkt nucler extrct (TPA + clcium ionophore-stimulted), no inhiition of the DNAinding ctivity of NF-κB ws found (Supplementry Figure S1). This suggested tht inds to one or more NF-κB suunits rther thn to the κb site. We first used surfce plsmon resonnce spectroscopy to mesure the inding of with recominnt p65 or p5. ound to p65 (Figure 1) ut not to p5 (Supplementry Figure S2). Gel shift ssy showed tht interfered with the inding ctivity of NF-κB suunit p65 to the κb sites in dose-dependent mnner (Figure 1c) ut did not ffect tht of the p5 suunit (Supplementry Figure S2). Next, we performed supershift ssys to nlyze the effects of on DNA inding of p5/p65 heterodimers, which mke up the predominnt NF-κB complex. inhiited the interctions etween the p5/p65 heterodimers nd DNA (Figure 1d). As expected, n excess of cold proe completely locked this interction (Figure 1d). These results suggested tht does not ind to the κb element ut disrupts the inding etween NF-κB nd the κb element through direct interction with p65, not with p5. Inhiition (%) d Extrct 32 P leled proe Cold proe Anti-p65 ntiody 25 µmol/l 4 µmol/l Response (rc seconds) c Supershift Peptide concentrtion (µmol/l) Shift Extrct P leled proe Cold proe Anti-p5 ntiody Supershift Time (minutes) Shift p µmol/l 25 µmol/l 4 µmol/l NF-κB DNA complex Figure 1 Effects of on the DNA-inding ctivity of NF-κB p65. () The effect of on the DNA-inding ctivity of NF-κB ws mesured y ELISA. s were preincuted with 2.5 μg Jurkt nucler extrcts for 3 minutes. The mixture ws then dded to ech well to detect the DNAinding ctivity of NF-κB. The inhiition rtio (%) of vrious concentrtions of peptides ws clculted nd plotted y ELISA nlysis. () Interction nlysis of with the p65 NF-κB suunit y using surfce plsmon resonnce mesurements. Recominnt NF-κB p65 ws used in EMSA nd surfce plsmon resonnce mesurement. (c) The effect of on the DNA-inding ctivity of p65 mesured y EMSA. (d) Effect of on the DNA-inding ctivity of the NF-κB p5/p65 heterodimer ws nlyzed y supershift ssy y using Jurkt nucler extrcts with p65 or p5 ntiody. Results re expressed s men ± SEM (n = 3). P <.5 versus inhiition rtio of., nti-inflmmtory peptide-6; ELISA, enzyme-linked immunosorent ssy; EMSA, electrophoretic moility shift ssys;, negtive control peptide; NF-κB, nucler fctor-κb vol. 19 no. 1 oct. 211

3 effectively trnsduces cells in vitro nd in vivo To study the effects of on cells, we first used cell-permele peptide trnsctivtor of trnscription (TAT) derived from the HIV to synthesize TAT- fusion peptide. nd TAT- were leled with fluorescein isothiocynte (FITC) nd nlyzed for uptke in RAW mcrophges. Confocl microscopy detected strong fluorescence in the cytoplsm nd nucleus of lmost ll RAW cells minutes fter incution with FITC-leled peptides (Figure 2). The cellulr uptke ws similr etween the nd TAT- groups. To exclude the possiility tht trnsduction ws due to phgocytosis in mcrophges, similr experiments were conducted in LoVo cells derived from humn colorectl cncer line. Confocl microscopy showed tht TAT-,, nd TAT, ut not, effectively entered the LoVo cells (Figure 2). These results indicted tht hs n intrinsic cell-penetrting property similr to tht of TAT. We therefore continued to use in our study. To exmine the uptke of in vivo, we used mouse model with zymosn-induced locl inflmmtion. FITC-leled (6 μg/pw, in 5 µl) or phosphte-uffered sline (PBS) ws injected into the hind pws of mice tht hd een chllenged with zymosn efore 24 hours. Frozen tissue sections were evluted y confocl microscopy. FITC-leled efficiently entered the cells 1 hour or 4 hours fter injection. No fluorescence signl ws detected in smples otined from PBS-treted mice. Therefore, efficiently trnsduces cells in vitro nd in vivo (Figure 2c). inhiits NF-κB ctivtion nd production of proinflmmmtory meditors Knowing tht trnsduces cells nd interferes with the DNAinding ctivity of NF-κB, we determined the potentil ntiinflmmtory ctivity of in zymosn-ctivted mcrophges. The levels of two representtive proinflmmtory meditors, TNF-α nd prostglndin E 2 (PGE 2 ), in the medium of RAW cells were mesured y ELISA. Zymosn tretment significntly incresed the levels of TNF-α nd PGE 2. Pretretment with, ut not, decresed the production of TNF-α nd PGE 2 in dose-dependent mnner (Figure 3). Zymosn-induced production of cytokines is the result of signling cscde tht culmintes in the ctivtion, nucler trnsloction, nd trnscriptionl ctivtion of NF-κB. 15 Therefore, we next investigted whether interferes with the NF-κB ctivtion cscde. In untreted cells, p65 ws minly found in the cytoplsm. Zymosn (.1 mg/ml) tretment for 1 hour induced nucler trnsloction of NF-κB. Pretretment of cells with s did not significntly ffect zymosn-induced nucler import of NF-κB p65 in the cells (Figure 3). Nonetheless, inhiited the DNA-inding ctivity of NF-κB in stimulted RAW nucler extrct in dose-dependent mnner. hd no effect on the DNA-inding ility of NF-κB t ll concentrtions tested (Figure 3c). Experiments with trnsfected NF-κB luciferse reporter lso indicted tht zymosn-stimulted NF-κB trnscriptionl ctivity diminished upon tretment with (Figure 3d). t 15 μmol/l did not exert ny inhiitory effect on NF-κB trnscriptionl ctivtion. Furthermore, did not ffect ctivtor protein (AP)-1- or signl trnsducer nd ctivtor of trnscription-1/3-medited trnscription (Supplementry Figures S3,). lso effectively inhiited NF-κB trnscriptionl ctivtion induced y TPA, strong ctivtor of NF-κB, through the protein kinse C pthwy 16 (Supplementry Figure S3c). Pretretment with did not ffect zymosn-induced degrdtion of IκB proteins or phosphoryltion of IκBα in RAW FITC-TAT FITC-TAT- FITC- FITC TI Merge c FITC- Merge TI DAPI FITC FITC- FITC-TAT- FITC-TAT Merge DAPI FITC 1 hour 4 hours Control Figure 2 Cellulr uptke of with or without TAT sequence. () Cellulr uptke of TAT- nd in RAW mcrophges. Cells were treted with FITC-leled TAT, FITC-leled, or FITC-leled TAT- (green) for minutes with nuclei counterstined with DAPI (lue). Representtive confocl imges with FITC, DAPI, phse contrst, nd merged FITC/DAPI chnnels. Br = 2 μm. () Trnsduction of into colon cncer cells. The cells were treted nd nlyzed similrly s. Br = 2 μm. (c) Cellulr uptke of in mice. After 1-hour nd 4-hour injection intervls of or PBS (control), soft tissues were removed from the pws of zymosn-treted mice. Frozen sections were nlyzed y confocl microscopy with nuclei counterstined with DAPI. Br = 2 μm., nti-inflmmtory peptide-6; CPP, cell-permele peptide; DAPI, 4,6-dimidino- 2- phenylindole; FITC, fluorescein isothiocynte; PBS, phosphte-uffered sline; TAT, trnsctivtor of trnscription; TI, trnsmission imge. Moleculr Therpy vol. 19 no. 1 oct

4 TNF-α relese (ng/ml) Zymosn stimultion Untreted Merge DAPI p65 No peptide (1.5 µmol/l) (15 µmol/l) (15 µmol/l) (15 µmol/l) PGE 2 relese (ng/ml) Zymosn stimultion Untreted No peptide (1.5 µmol/l) (15 µmol/l) Zymosn stimultion Untreted No peptide (15 µmol/l) (15 µmol/l) (15 µmol/l) (15 µmol/l) e c NF-κB DNA complex d RLU Zymosn stimultion µmol/l # 2 p4-κb-luc Zymosn (1 µg/ml) (µmol/l) (µmol/l) Zymosn lκ Bα lκ Bβ lκ Bε β-ctin Zymosn (µmol/l) p-lκ Bα Totl lκ Bα (µmol/l) Figure 3 Effect of on trnscriptionl ctivity of NF-κB in zymosn-ctivted mcrophges. () Effects of s on zymosn-induced production of inflmmtory meditors. RAW cells were treted with t indicted concentrtion or (15 μmol/l) nd stimulted with zymosn (.1 mg/ml) for 24 hours. The production of TNF-α nd PGE 2 in culture superntnts ws mesured y ELISA. Results re expressed s men ± SEM (n = 3), P <.5 zymosn versus untreted; # P <.5 zymosn + versus zymosn. () Effects of on nucler trnsloction of p65. Representtive confocl imges of p65 (green) locliztion with nuclei counterstined with DAPI (lue) in control (untreted) RAW cells nd zymosn-treted RAW cells for 3 minutes with or without t indicted concentrtions. Br = 2 µm. (c) Effects of on the DNA-inding ctivity of p65 ws mesured y EMSA in RAW cells. Cells were incuted t vrious concentrtions of s or s for 2 hours, followed y zymosn tretment for 1 hour. Nucler extrcts were prepred to nlyze NF-κB ctivtion y EMSA. (d) The effect of on the expression of n NF-κB driven luciferse reporter. RAW cells trnsfected with p4-κb-luciferse reporter were pretreted with different doses of or (15 µmol/l) for 2 hours nd stimulted with zymosn for 16 hours. The luciferse ctivity nd NF-κB trnscriptionl ctivity were plotted s reltive luminescence units (RLU). P <.5 zymosn versus untreted; # P <.5 zymosn + versus zymosn. (e) Effects of on the expression nd phosphoryltion of IκB. RAW cells were treted with t indicted concentrtions for 1 hour nd stimulted with zymosn for 45 minutes (left) or 15 minutes (right). The expressions of vrious IκBs or p-iκbα were nlyzed y western lot nlysis., nti-inflmmtory peptide-6; DAPI, 4,6-dimidino-2-phenylindole; ELISA, enzyme-linked immunosorent ssy; EMSA, electrophoretic moility shift ssy; I-κB, inhiitory κb;, negtive control peptide; NF-κB, nucler fctor-κb; TNF, tumor necrosis fctor; PGE 2, prostglndin E 2 ; TNF-α, tumor necrosis fctor-α Control (medium lone) 1 4 (15 µmol/l) 1 4 (15 µmol/l) Cell viility (%) PI PI PI Peptide concentrtion (µmol/l) Annexin V-FITC Annexin V-FITC Annexin V-FITC Figure 4 Effects of nd negtive control peptide on cell prolifertion nd viility. RAW cells were incuted with zymosn (.1 mg/ ml) with (white) or (lck) t indicted concentrtions for 24 hours. () Cell prolifertion ws nlyzed y WST-1 ssy. Results re expressed s men ± SEM (n = 3) nd normlized to control (%) (cells not treted with peptide s %). () Apoptosis ws nlyzed y flow cytometry fter stining with nnexin V nd PI. Cells were treted with no peptide, (15 μmol/l), or (15 μmol/l)., nti-inflmmtory peptide-6; FITC, fluorescein isothiocynte;, negtive control peptide; PI: propidium iodide; WST-1, wter-solule tetrzolium vol. 19 no. 1 oct. 211

5 PBS (control) 6 µg/pw 3 µg/pw 1.5 µg/pw 6 µg/pw DEX 5 µg/pw 25 # c 5 Swelling volume (µl) 2 15 Joint cell infiltrtion score dy 1 dy 3 dys 5 dys 7 dys Norml PBS Dys fter zymosn injection Joint Norml Zymosn Zymosn + Zymosn + d NF-κB p65 ctivtion (OD 45 nm) # Pw Norml PBS (1.5 µg/pw) (3 µg/pw) (6 µg/pw) (6 µg/pw) e Norml PBS f Norml PBS TNF-α/GAPDH IL-1β/GAPDH 1 4 2, 1,5 1, 5 il-6/gapdh inos/gapdh COX-2/GAPDH TNF-α relese (ng/ml) IL-1β relese (ng/ml) IL-6 relese (ng/ml).5 NO relese (ng/ml) PGE 2 relese (ng/ml) 4 2 Figure 5 Effects of on zymosn-induced inflmmtion in vivo. () Effects of on joint nd pw edem. PBS,, or in 5 μl ws dministered to the hindpws (oth left nd right) of mice t different time points (8 hours, 1 dy, 3 dys, nd 5 dys) fter zymosn injection. Results re expressed s men ± SEM (n = 9). P <.5 /dexmethsone versus PBS t the sme point; # P <.5, versus dexmethsone. () Representtive HE-stined sections of nkle joints (upper) nd hindpws (lower) from mice with indicted tretments t dy 7: norml group, zymosn injected group, zymosn + (6 μg/pw)-treted group, nd zymosn + (6 μg/pw)-treted group. Br = 5 µm for joint pictures (upper) nd 1 µm for hindpw pictures (lower). (c) Effects of on the inflmmtion in joints. Inflmmtory cell infiltrtion ws scored t scle from (no cells) to 4 (severe cell influx in joint cvity nd synovium). Results re expressed s men ± SEM (n = 5).P <.5 versus PBS or. (d) Effects of on NF-κB ctivtion in vivo. Nucler extrcts were prepred from soft tissues from ech experiment group t dy 7, nd the DNA-inding ctivity ws mesured y ELISA y using the p65 nucler trnsfctor kit, s in Figure 1. Results re expressed s men ± SEM (n = 4). P <.5 versus PBS or ; # P <.5 zymosn + PBS versus untreted. (e,f) Effects of on the expression of inflmmtory meditors. Totl RNA or protein extrcts isolted from ech experiment group were collected 7 dys fter zymosn injection. or (6 μg/pw) ws injected t 8 hours, 1 dy, 3 dys, nd 5 dys fter zymosn tretment. The mrna levels of indicted genes were mesured y rel-time PCR. Protein levels were mesured y ELISA. Results re expressed s men ± SEM (n = 4). P <.5 versus PBS or., nti-inflmmtory peptide-6; COX-2, cyclooxygense-2; Dex, dexmethsone; ELISA, enzyme-linked immunosorent ssy; GAPDH, glycerldehyde-3-phosphte dehydrogense; HE, hemtoxylin nd eosin; IL, interleukin; inos, inducile nitric oxide synthse;, negtive control peptide; OD, Opticl density; PBS, phosphte-uffered sline; PGE 2, prostglndin E 2 ; TNF, tumor necrosis fctor cells (Figure 3e). nd t 15 μmol/l did not significntly ffect cell prolifertion or poptosis, s mesured y the wter-solule tetrzolium ssy (Figure 4) or y flow cytometry (Figure 4). Together, these findings indicted tht selectively inhiits NF-κB medited trnscription nd hs low nonspecific cytotoxicity t effective concentrtions. Moleculr Therpy vol. 19 no. 1 oct

6 suppresses zymosn-induced inflmmtion in mice Bsed on the ove nti-inflmmtory ctivities of in vitro, we decided to nlyze its efficcy in n experimentl inflmmtion model in mice. 12 Zymosn injection in the feet of mice led to time-dependent swelling of the joints nd pws, which peked t 24 hours. Swelling strted to decrese nd remined ove control levels until 7 dys (Figure 5). From dy 1 to dy 7, inhiition of swelling y (6 μg/pw) ws etter thn or comprle to tht induced y dexmethsone (5 μg/pw). injection significntly inhiited the formtion of swelling t different time points, including the swelling pek t 24 hours, in dose-dependent mnner. The optiml inhiitory effect of dexmethsone ws oserved 3 dys fter zymosn injection. did not show ny effect t ny time point. We lso histologiclly ssessed inflmmtion in the joints nd pws. Zymosn induced significnt mononucler cell infiltrtion, thickening of the synovil memrne, nd severe inflmmtion of soft tissues, ll of which ws suppressed y (6 μg/pw) (Figure 5). Lower levels of inflmmtory cell infiltrtion were found in the joints of mice treted with compred with other groups (Figure 5c). The reduced inflmmtory response in mice treted with ws ssocited with suppressed NF-κB p65 ctivtion. Using nucler extrcts prepred from mice in vrious tretment groups t dy 7, we mesured the DNA-inding ctivity of NF-κB p65 with n ELISA-sed ssy. Zymosn cused nerly fourfold increse in DNA inding, which ws suppressed y, ut not (Figure 5d). As expected, injection strongly inhiited zymosn-induced expression of NF-κB trget genes nd inflmmtory meditors, including TNF-α, IL-1β, IL-6, inducile nitric oxide synthse, cyclooxygense-2, PGE 2, nd nitric oxide (Figure 5e,f). Discussion NF-κB regultes the trnscription of mny genes involved in immune response nd is considered potentil therpeutic trget in inflmmtory diseses. In this study, we hve discovered nd chrcterized pentpeptide (RLRWR) tht prevents the inding of NF-κB with κb elements through specific interction with p65. suppressed zymosn- or TPA-induced NF-κB ctivtion nd the production of inflmmtory meditors in mcrophges. The inhiitory effects of on NF-κB ppered specific nd did not involve IκB degrdtion, IκBα phosphoryltion, or p65 nucler import. Using zymosn-induced inflmmtion model, we showed tht locl injection impired cute inflmmtion responses through inhiition of NF-κB ctivtion nd production of proinflmmtory meditors. Importntly, displys n intrinsic nd strong cell-penetrting property in vitro nd in vivo. A vriety of gents exhiit vrious degrees of effectiveness in suppressing NF-κB signling. However, few of these compounds re specific, nd some side effects hve een reported. For exmple, glucocorticoids, which re considered s the most powerful nonspecific inhiitors of NF-κB, resulted in reduced one formtion nd suppression of hypothlmic pituitry drenl xis function. 18,19 Therefore, there is cler need for nti-inflmmtory gents tht lck steroid ction nd hve reduced side effects. NF-κB ctivtion or function cn e inhiited y more specific mens. For exmple, the NF-κB essentil modifier (NEMO)- inding domin peptide inhiits p65 phosphoryltion, nd the SN5 peptide inhiits NF-κB nucler trnsport Among vrious strtegies imed t inhiiting NF-κB ctivtion, locking the inding of the p65 or p5 suunit with the κb site my e more specific 23,24 ecuse higher degree of specificity my e chieved y the inhiition of NF-κB medited trnscription. In this study, we designed peptide sed on sequence required for DNA inding, which is conserved in NF-κB fmily memers. Unexpectedly, this peptide selectively intercted with the p65 NF-κB suunit ut not the κb motif, nd it functioned s selective inhiitor of NF-κB medited trnscription. did not pper to ffect the phosphoryltion or degrdtion of IκB proteins or the nucler import of p65. The moleculr mechnism of -medited inhiition of the DNA-inding ctivity of p65 is not fully understood; my induce conformtionl chnges in the protein 25 or prevent the formtion of the trnscriptionlly competent p65/p5 complex. The results of our ELISA-sed trnsfctor ssys mke us speculte is competitive inhiitor ginst NF-κB trnscription ctivity tht will require reltively higher quntity to e effective. Thus, to keep nti-inflmmtory ction of, hs to e delivered frequently to mintin its in vivo concentrtion. The exct mechnism my e determined through dditionl moleculr nd structurl studies. Nevertheless, this property my help void undesirle effects y reducing interference with the sl ctivity of NF-κB, which my e essentil for the survivl of severl cell types. 2 Inhiitors of trnscription fctors cn only exert iologicl inhiitory effects fter their efficient uptke into cells. Trnsduction medited y cell-penetrting peptides (CPPs) cn deliver ioctive regents directly into living cells. Commonly used CPPs include the HIV-1 TAT protein, penetrtin, nd polyrginine. 26 Our dt showed tht itself possesses n intrinsic cell-penetrting property independent of phgocytosis. This unique property my e ttriuted to the presence of severl positively chrged rginine residues nd the low moleculr weight nd moderte hydrophoicity of. 27,28 Other preferred mechnisms for CPP-medited trnsduction hve een proposed, including energy-independent memrne trnsloction, endocytosis, nd specific receptors. 29 In ddition, the toxicity nd unsuspected side effects of severl CPPs hve een reported in recent yers A similr study reported tht 16-AA, CPP, ws found to suppress inflmmtory response in vitro nd in vivo y inhiiting the ctivtion of NF-κB. 34 The fct tht is cell penetrting nd contins only 5-AA residues with no ovious toxicity mkes it n ttrctive led structure for future drug development. It might lso e prcticl short CPP. Of course, we cnnot deny the potentil toxicity of tht we did not test in this study, nd we therefore need more work to ssess the sfety of efore it cn e developed to therpeutic gent. suppressed zymosn-induced inflmmtion tht is ssocited with reduced levels of inflmmtory meditors nd well-known NF-κB trgets, including TNF-α, IL-1 β, IL-6, inducile nitric oxide synthse, nd cyclooxygense-2. 4,35,36 Locl tretment with inhiited NF-κB ctivtion in this model, which suggested tht locks excessive NF-κB ctivtion nd vol. 19 no. 1 oct. 211

7 highlighted the importnce of the inhiition of NF-κB medited trnscription in overll nti-inflmmtory effects. Becuse did not ffect AP-1 or signl trnsducer nd ctivtor of trnscription-medited trnscription of reporters, it is ruled out s generl inhiitor of trnscription. However, our study could not rule out the possiility tht interferes with other pthwys. As discussed efore, few inhiitors of NF-κB re specific. For exmple, most IκB kinse-β inhiitors cn exert their function vi ATP competition, nd SN5 lso prevents nucler trnsloction of AP-1, nucler fctor of ctivted T-cells nd signl trnsducer nd ctivtor of trnscription-1. 23,37 Gene expression profiling coupled with chromtin immunoprecipittion ssy could led to etter understnding of the nti-inflmmtory mechnisms of. In summry, our study shows tht, cell-penetrting pentpeptide, inhiits the DNA-inding ctivity of NF-κB nd efficiently ttenutes inflmmtory responses in vitro nd in vivo. These results suggest tht is promising led structure for the development of specific NF-κB inhiitors s potentil ntiinflmmtory gents. Mterils nd Methods Chemicls nd mterils. All peptides, including FITC-leled TAT protein of HIV, FITC-leled, nd FITC-leled, used in this study were synthesized y HD Biosciences Compny, Shnghi, Chin. nd were purified to more thn 95% purity y using high pressure liquid chromtogrphy. The sequences of the peptides were s follows:, Arg- Leu-Arg-Trp-Arg (RLRWR);, Leu-Arg-Trp-Arg-Lys (LRWRK); nd TAT, Arg-Leu-Arg-Trp-Arg (YGRKKRRQRR-RLRWR). Other chemicls nd mterils re listed in Supplementry Mterils nd Methods. Cell lines. RAW mcrophges nd LoVo colon cncer cells were initilly purchsed from the Americn Type Culture Collection (Mnsss, VA). RAW mcrophges nd LoVo cells were cultured in Dulecco s modified Egle s medi supplemented with 1% het-inctivted fetl ovine serum, enzylpenicillin potssium (143 U/ml), nd streptomycin sulfte ( μg/ml) under 37 C nd 5% CO 2 tmosphere. ELISA-sed NF-κB p65 trnsfctor ssy. To determine whether hs the ility to ind with κb elements, we designed n experiment using n ELISA-sed Trnsfctor p65 kit (Active Motif, Crlsd, CA). Aout 3 µl of inding uffer (Active Motif) contining or t vrious concentrtions (25, 5,, 2, nd 4 μmol/l) ws dded to ech well of the corresponding microtiter plte coted with n oligonucleotide contining the κb site of the immunogloulin light chin gene promoter (GGGACTTTCC) nd incuted for 3 minutes t room temperture. After wshing three times with inding uffer, 3 μl inding uffer nd 2 μl complete lysis uffer (Active Motif) contining 2.5 μg Jurkt nucler extrct (TPA + clcium ionophore) were dded to the corresponding microtiter plte. The following steps were performed ccording to the mnufcturer s instructions. Briefly, the plte ws incuted for 1 hour t room temperture on rocking pltform. After wshing, NF-κB p65 ntiody (1:1,) ws dded nd incuted for 1 hour t room temperture. The wells were then wshed, nd horserdish peroxidse-conjugted ntiody (1:1,) ws dded nd incuted for 1 hour t room temperture. After wshing, sustrte solution ws dded to the wells nd incuted t room temperture for 1 minutes, protected from light. Stop solution (2NH 2 SO 4 ) ws dded, nd the opticl density ws mesured t 45 nm. Inhiition rtio (%) = [(opticl density vlue of positive control group opticl density vlue of peptide-treted group)/opticl density vlue of positive control group]. The wells to which only Jurkt nucler extrct ws dded were set s the positive control group. To detect the inhiitory effect of on the inding ctivity of NF-κB, nother experiment ws performed. To detect the inding ctivity of to p65, 3 μl inding uffer contining or t vrious concentrtions (25, 5,, 2, nd 4 μmol/l) ws mixed with 2 μl complete lysis uffer contining 2.5 μg Jurkt nucler extrct nd incuted for 3 minutes t room temperture. The mixtures were then dded to the corresponding microtiter plte, nd the plte ws incuted for 1 hour t room temperture on rocking pltform. The steps descried ove were then performed. The effect of on the nucler levels of NF-κB p65 in locl inflmmtory tissue ws lso determined using ELISA Trnsfctor p65 kits. Nucler extrcts were collected using the regents supplied in the Nucler Extrct Kit (Active Motif). Proteins were quntified using the icinchoninic cid ssy nd sujected to ELISA-sed NF-κB p65 trnsfctor ssy. All the procedures were performed ccording to the mnufcturer s instructions. Electrophoretic moility shift ssy. To identify the inhiitory effect of on the inding ctivity of the p65 or p5 suunit, NF-κB proe (5 -AGTTGAGGGGACTTTCCCAGGC-3 ) ws phosphorylted with T4 polynucleotide kinse in the presence of [γ- 32 P] ATP nd ws purified. Recominnt p65 protein (2 ng) nd recominnt p5 protein were used for electrophoretic moility shift ssy. Rection mixtures contining inding uffer (15 mmol/l Tris-HCl (ph 7.5), 75 mmol/l NCl, 1.5 mmol/l EDTA, 1.5 mmol/l dithiothreitol, 7.52% glycerol, nd.3% Nonidet P-4), 1 µg of poly (di-dc)-(di-dc), nd either recominnt p65 protein or recominnt p5 protein were kept on ice for 1 minutes. (25, 5,, nd 2 µmol/l) or (25, 5,, nd 2 µmol/l) ws dded to the mixtures nd incuted t room temperture. After 3 minutes, 1 µmol/l 32 P-leled NF-κB proe ws dded to the mixtures nd further incuted t room temperture for 3 minutes. For the supershift ssys, 2.5 µl ntip65 or nti-p5 ntiody ws dded to the rection mixture simultneously with the Jurkt nucler extrct, nd (25 nd 4 µmol/l) ws dded to the mixtures. The mixture ws incuted s descried ove. Susequently, 2 µl of the mixture ws loded onto 5% ntive polycrylmide gel prepred in.5 tris-orte-edta nd electrophoresed for 2.5 hours. The gel ws dried nd then sujected to utordiogrphy. In nother experiment, RAW cells were pretreted with s (9.375, 75, 15, nd 3 µmol/l) or s (15 nd 3 µmol/l) for 2 hours nd stimulted with zymosn (.1 mg/ml) for 1 hour. The nucler extrct ws prepred from these cells nd then sujected to electrophoretic moility shift ssy. Surfce plsmon resonnce spectroscopy. To identify the specific interction etween nd the p65 or p5 NF-κB suunit, surfce plsmon resonnce mesurements were performed using BIAcore 3 instrument (Bicore, Pisctwy, NJ). The running uffer ws protein inding uffer [5 mmol/l Tris-HCl (ph 7.2),.1 mol/l NCl, 1 mmol/l MgCl 2, 1 µmol/l ZnCL 2, 1 mmol/l dithiothreitol,.1% (v/v) NP4]. Dt cquisition ws then performed, nd seline dt ws gthered for severl minutes. The croxymethylted dextrn surfce of CM5 sensor chip (BIAcore AB) ws ctivted y injecting coupling solution of N-hydroxysuccinimide nd 1-ethyl-3-(dimethylminopropyl) crodiimide hydrochloride (BIAcore AB). Recominnt p65 or p5 proteins were diluted to 25 µg/ml in 1 mmol/l cette uffer (ph 5.) nd injected over the surfce of the sensor chip for 8 minutes t flow rte of 5 µl/minute. The unrected sites of the sensor chip surfce were then quenched y injection of 4 µl of 1 M Tris-HCl (ph 8.). Unimmoilized p65 nd p5 were wshed 3 times with 4 µl of 1 mmol/l HCl. nd were diluted to 1 µg/ml in protein inding uffer nd injected over the surfce of the sensor chip for 5 min t flow rte of 1 µl/ min. The unound nd were wshed with protein inding uffer. The resonnt ngle response ws recorded. The chip surfce ws then regenerted y dding 1 mmol/l HCl until the response signl returned to seline to proceed with nother inding cycle. Confocl fluorescence microscopy. All the smples were nlyzed under lser scnning confocl microscope (Zeiss LSM51, Oerkochen, Moleculr Therpy vol. 19 no. 1 oct

8 Germny). Cells were treted under stndrd condition of 5% CO 2 in humidified incutor t 37 C. To investigte the trnsduction of in vitro, RAW cells nd LoVo cells were cultured in the presence of FITC-leled TAT, FITC-leled TAT-, FITC-leled, nd FITC-leled t concentrtion of 15 µmol/l for minutes. After wshing three times, nuclei were stined with 4,6-dimidino-2-phenylindole. These cells were then wshed two times, nd the trnsduction of ws ssyed y confocl fluorescence nlysis. Confocl fluorescence nlysis ws lso performed to investigte the trnsduction of in vivo. Suderml injections of 5 µl of 15 µmol/l FITC-leled (6 µg/pw) were performed in the inflmed hindpws of mice (t 24 hours fter zymosn injection). Control nimls received suderml injections of 5 µl of PBS into inflmed hindpws. Mice were nesthetized with chlorl hydrte nd killed 1 hour or 4 hours fter the injection. Soft smples from hindpws were removed immeditely nd fixed in 4% prformldehyde. Smples were cut on cryostt t 2 µm, nd DNA dye (4,6-dimidino-2-phenylindole) ws dded. The sections were then nlyzed under confocl microscopy. Confocl lser scnning microscopy ws lso used to oserve whether ffected the nucler import of NF-κB p65. RAW cells were pretreted with 15 or 15 µmol/l for 2 hours nd stimulted with zymosn (.1 mg/ml) for 1 hour. These cells were then fixed in 4% prformldehyde, permeilized in.5% Triton X-, nd locked in 1% ovine serum lumin in PBS. For immunostining, the cells were incuted with nti-nf-κb p65 ntiody (1:3) for 2 hours nd then stined with FITC-leled secondry ntiody (1:8) for 1 hour. For stining the nuclei, the cells were incuted with 4,6-dimidino-2-phenylindole solution. Luciferse reporter ssys. RAW cells were plted t density of cells/well in 24-well pltes nd llowed to rech up to 7 8% confluence. The p4-κb-luciferse plsmid (1 μg) ws trnsfected using Lipofectmine Plus regent (Invitrogen, Crlsd, CA) ccording to the mnufcturer s instructions. Therefter, the cells were treted for 24 hours with (, 9.375, 18.75, 37.5, 75, nd 15 μmol/l) or (15 μmol/l) nd then stimulted with 1 μg/ml zymosn. After 16 hours of incution, cells were lysed nd luciferse ctivity ws mesured using luminometer (Promeg, Fitchurg, WI) ccording to the mnufcturer s instructions. AP-1-Luc nd m67-luc vectors were used to detect AP-1 or STAT-dependent trnscription ctivities. The effect of on NF-κB trnscription ctivities in TPA-stimulted mcrophges re descried in Supplementl Mterils nd Methods. Western lot nlysis. The cells were pretreted with 37.5 or 15 µmol/l for 2 hours, stimulted with zymosn (.1 mg/ml) for 15 minutes or 45 minutes, nd then lysed in lysis uffer. Whole cell lystes were sujected to sodium dodecyl sulfte polycrylmide gel electrophoresis nd then trnsferred to polyvinylidene difluoride memrne. The lots were usully incuted t 4 C overnight with primry ntiodies in 5% ovine serum lumin in 1 mmol/l tris-uffered sline Tween-2 [1 mmol/l Tris (ph 8.), 5 mmol/l NCl,.5% (v/v) Tween-2]. The primry ntiodies were nti-iκbα (dilution, 1:5), nti-iκbβ (1:5), nti-iκbε (1:5), nti-p-iκbα (1:2), nd nti-glycerldehyde-3-phosphte dehydrogense (1:2,). The lots were then incuted with secondry ntiody, nmely, horserdish peroxidse-leled nti-rit IgG ntiody (1:1,), t room temperture for 2 5 hours. Immune complexes on the lots were finlly visulized y rdiogrphy fter rection with n enhnced chemiluminescence regent (GE Helthcre, Little Chlfont, UK). Ech lot presented is representtive of findings in t lest three similr independent experiments. Cell prolifertion nd poptosis ssys. The cytotoxicity of nd ws ssessed y using wter-solule tetrzolium ssy. RAW mcrophges (5 1 4 /well) were incuted with vrious concentrtions of or for 24 hours. They were exposed to wter-solule tetrzolium of 2-(4-iodophenyl)-3- (4-nitrophenyl)-5-(2, 4- disulfophenyl)-2h-tetrzolium for 3 hours, nd then sornce vlues were mesured t 45 nm. Apoptosis ws ssessed y flow cytometry. The RAW cells fter eing incuted with 15 µmol/l of or for 24 hours were hrvested y trypsin-edta followed y two wshes with ice-cold PBS nd were susequently stined with ntiody to nnexin V-FITC nd DNA/ RNA dye propidium iodide. Smples were nlyzed y flow cytometry (FACScn; Becton Dickinson; Mountin View, CA) ccording to the mnufcturer s protocol. The red (61 nm, propidium iodide) nd green (525 nm, nnexin V) fluorescence emissions were seprted opticlly y seprte photomultipliers. Zymosn-induced inflmmtion in mice. Femle C57BL/6J mice (21 23 g) were provided y the Animl Breeding Center of Third Militry Medicl University (Chongqing, Chin). All the nimls were mintined in plstic cges t 2 ± 2 C with free ccess to food nd wter nd were kept on 12-hour light/drk cycle. Experiments with nimls were conducted in ccordnce with the guidelines of the Ntionl Institutes of Helth nd Third Militry Medicl University for lortory niml cre. For the inflmmtion model, 15 mg/ml suspension of zymosn ws mde in PBS, nd the suspension ws utoclved efore injection. Animls received suderml injections of 75 μg zymosn (5 μl) in the left nd right hindpws. The volume of edem (including hindpw nd nkle joint) ws mesured efore zymosn injection (time ) nd t different time points fter stimultion using volume meter/plethysmometry (plethysmometer 714; Ugo Bsile; Comerio, Itly). Mice were treted with suderml dministrtion of the test gents (5 μl) into the hindpws t 8 hours, 1 dy, 3 dys, nd 5 dys. The test gents used in this experiment were 15 μmol/l (6 μg/pw), 75 μmol/l (3 μg/pw), 37.5 μmol/l (1.5 μg/pw), 15 μmol/l (6 μg/pw), nd 15 μmol/l dexmethsone (5 μg/pw). For the negtive control group, mice were given n equl volume of PBS. The mice were killed y cervicl disloction immeditely fter the volume ws mesured (t 7 dys fter zymosn injection), nd the pws nd nkle joints were removed. Some soft tissues from pws were recovered y sclpel nd immeditely processed to otin tissue extrcts or to isolte totl RNA. Histologicl nlysis. Pws nd nkle joints were dissected nd fixed for 2 weeks in 1% uffered formlin. Fixed tissues were declcified for 2 weeks in 15% EDTA, dehydrted, nd emedded in prffin. Tissue sections (1 μm) were cut nd stined with hemtoxylin/eosin for the detection of inflmmtion. Joint inflmmtion ws scored y the influx of inflmmtory cells in the joint cvity nd synovium. Histologicl ssessment of joints ws scored y two oservers linded to the tretment, ccording to methods descried erlier. 38 A score of indicted no cell influx, nd 1 4 ws scored ccording to the degree of cell influx. Quntittive rel-time reverse trnscription PCR. Totl RNA ws isolted from pw soft tissues y using TRIzol, ccording to the mnufcturer s instructions. The extrcted RNA ws kept t 8 C efore use. Reltime PCR ws performed t finl volume of 2 μl in cpillry tues in LightCycler instrument (Roche Dignostics, Mnnheim, Germny). The primers used for PCR re given in Supplementry Tle S1. Rection mixtures contined 2 μl of LightCycler Fst-Strt DNA mstermix for SYBR Green I (Roche Dignostics),.5 μmol/l of ech primer, 4 mmol/l MgCl 2, nd 2 μl of templte DNA. All cpillries were seled, centrifuged t 5g for 5 seconds, nd then mplified in LightCycler instrument with ctivtion of polymerse (95 C for 1 minutes), followed y 45 cycles of 1 seconds t 95 C, 1 seconds t C, nd 1 seconds t 72 C. The temperture trnsition rte ws 2 C/second for ll steps. Doule-strnded PCR products were mesured during the extension step t 72 C y detection of fluorescence ssocited with the inding of SYBR Green I to the product. Fluorescence curves were nlyzed with LightCycler softwre v. 3.. For quntittive nlysis of TNF-α, IL-1β, IL-6, inducile nitric oxide synthse, nd cyclooxygense-2 mrna, LightCycler (Roche Dignostics) ws used. The expression levels of TNF-α, IL-1β, IL-6, inducile nitric oxide synthse, nd cyclooxygense-2 were clculted nd corrected for the vlues of the vol. 19 no. 1 oct. 211

9 endogenously expressed housekeeping gene control (glycerldehyde-3- phosphte dehydrogense). Melting curve nlysis ws performed immeditely fter the mplifiction protocol under the following conditions: second (hold time) t 95 C, 15 seconds t C, nd second (hold time) t 95 C. Temperture chnge rtes were 2 C/second, except in the finl step, in which it ws.1 C/second. The melting pek generted represented the specific mplified product. The crossing point ws defined s the mximum of the second derivtive from the fluorescence curve. Negtive controls were lso included nd contined ll the elements of the rection mixture, except templte DNA. All smples were processed in duplicte. ELISA. The mcrophges were pretreted with s or s for 2 hours nd then stimulted with zymosn (.1 mg/ml) for 24 hours. The levels of TNF-α nd PGE 2 were determined in the hrvested superntnts using corresponding ELISA kits, ccording to the mnufcturer protocols. Levels of TNF-α, IL-1β, IL-6, PGE 2, nd nitric oxide in the tissue extrcts were evluted y ELISA using corresponding nti-mouse ntiodies nd iotinylted secondry ntiodies, ccording to the mnufcturer s instructions. Sttisticl nlysis. Results re expressed s mens ± SE. The inhiitory effect of nd t vrious concentrtions ws compred y using two-tiled Student s t-test. Differences etween multiple groups were evluted y using one-wy nlysis of vrince, followed y Dunnett s post hoc or Tukey comprisons. Results were considered sttisticlly significnt t P <.5. SUPPLEMENTARY MATERIAL Figure S1. Effects of on the DNA-inding ctivity of NF-κB ws mesured y ELISA. Figure S2. Effects of on DNA inding ctivity of NF-κB p5. Figure S3. Effects of on TPA (phorol, 12-myristte, 13 cette)-induced AP-1 (), TPA-induced NF-κB (), nd interferon (IFN)-γ induced signl trnsducer nd ctivtor of trnscription (STAT) ctivtion (c) in RAW mcrophges. Tle S1. Primers nd prmeters used in rel-time PCR. Mterils nd Methods. ACKNOWLEDGMENTS We thnk Jv Vtsyyn, Deprtment of Cell Biology nd Physiology, University of Pittsurgh School of Medicine, Rngos Reserch Center t Children s Hospitl of Pittsurgh of UPMC, for helpful discussion nd criticl review. This study ws supported y n estlishment grnt from the Ntionl High Technology Reserch nd Development Progrm ( 863 Progrm, No. 28AA2Z44) of Chin, the Ntionl Nture Science Foundtion of Chin (NSFC, No ; No. 8132), Ntionl Institutes of Helth grnts R1CA nd U1DK8557, ACS grnt RGS CCE, nd FAMRI grnt 3229_YCSA. The uthors declred no conflict of interest. REFERENCES 1. Ghosh, S nd Krin, M (22). Missing pieces in the NF-kppB puzzle. Cell 19 Suppl: S81 S Bldwin, AS Jr (21). Series introduction: the trnscription fctor NF-kppB nd humn disese. J Clin Invest 17: Srkr, FH, Li, Y, Wng, Z nd Kong, D (28). NF-kppB signling pthwy nd its therpeutic implictions in humn diseses. Int Rev Immunol 27: Tin, B nd Brsier, AR (23). Identifiction of nucler fctor kpp B-dependent gene network. 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