Summary
Nuclear factor kappa B (NF-κB) is an inducible transcription factor that regulates the expression of many genes involved in normal immune and inflammatory responses. NF-κB activation is normally a rapid and transient response to pro-inflammatory stimuli however dysregulated constitutively active NF-κB signaling leads to chronic inflammation and provides a cell survival signal in many types of cancer. NF-κB signaling is therefore an important target for the development of novel anti-inflammatory or anti-cancer drugs. We previously identified and characterized a cell-permeable peptide that blocks NF-κB signaling by disrupting the critical upstream IκB kinase (IKK) complex. We describe in this chapter three separate methods to determine the effects of this NEMO-binding domain (NBD) peptide on pro-inflammatory NF-κB signaling in response to tumor necrosis factor (TNF).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Gilmore, T. D. (2006) Introduction to NF-kappaB: players, pathways, perspectivesOnco-gene25, 6680–6684.
Hayden, M. S. and Ghosh, S. (2004) Signaling to NF-kappaBGenes Dev18, 2195–2224.
Karin, M. and Greten, F. R. (2005) NF-kap-paB: linking inflammation and immunity to cancer development and progressionNat Rev Immunol5, 749–759.
Greten, F. R. and Karin, M. (2004) The IKK/ NF-kappaB activation pathway – a target for prevention and treatment of cancerCancer Lett206, 193–199.
Karin, M., Cao, Y., Greten, F. R., and Li, Z. W. (2002) NF-kappaB in cancer: from innocent bystander to major culpritNat Rev Cancer2, 301–310.
Gilmore, T. D. and Herscovitch, M. (2006) Inhibitors of NF-kappaB signaling: 785 and countingOncogene25, 6887–6899.
Karin, M., Yamamoto, Y., and Wang, Q. M. (2004) The IKK NF-kappa B system: a treasure trove for drug developmentNat Rev Drug Discov3, 17–26.
Hacker, H. and Karin, M. (2006) Regulation and function of IKK and IKK-related kinasesSci STKE2006, re13.
ScheIDereit, C. (2006) IkappaB kinase complexes: gateways to NF-kappaB activation and transcriptionOncogene25, 6685–6705.
May, M. J., D'Acquisto, F., Madge, L. A., Glockner, J., Pober, J. S., and Ghosh, S. (2000) Selective inhibition of NF-kappaB activation by a peptIDe that blocks the interaction of NEMO with the IkappaB kinase complexScience289, 1550–1554.
May, M. J., Marienfeld, R. B., and Ghosh, S. (2002) Characterization of the Ikappa B-kinase NEMO binding domainJ Biol Chem277, 45992–46000.
Strnad, J., McDonnell, P. A., Riexinger, D. J., Mapelli, C., Cheng, L., Gray, H., Ryseck, R. P. , and Burke, J. R. (2006) NEMO binding domain of IKK-2 encompasses amino acIDs 735–745J Mol Recognit19, 227–233.
Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranesJ Biol Chem269, 10444–10450.
KabourIDis, P. S. (2003) Biological applications of protein transduction technologyTrends Biotechnol21, 498–503.
Tilstra, J., Rehman, K. K., Hennon, T., Plevy, S. E., Clemens, P., and Robbins, P. D. (2007) Protein transduction: IDentification, charac-terization and optimizationBiochem Soc Trans35, 811–815.
Zorko, M. and Langel, U. (2005) Cell-penetrating peptIDes: mechanism and kinetics of cargo deliveryAdv Drug Deliv Rev57, 529–545.
Choi, M., Rolle, S., Wellner, M., Cardoso, M. C., ScheIDereit, C., Luft, F. C., and Ket-tritz, R. (2003) Inhibition of NF-kappaB by a TAT-NEMO-binding domain peptIDe accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosisBlood102, 2259–2267.
Corn, R. A., Aronica, M. A., Zhang, F., Tong, Y., Stanley, S. A., Kim, S. R., Stephenson, L., Ener-son, B., McCarthy, S., Mora, A., and Boothby, M. (2003) T cell-intrinsic requirement for NF-kappa B induction in postdifferentiation IFN-gamma production and clonal expansion in a Th1 responseJ Immunol171, 1816–1824.
Dai, S., Hirayama, T., Abbas, S., and Abu-Amer, Y. (2004) The I{kappa}B kinase (IKK) inhibitor, NEMO-binding domain peptIDe, blocks osteoclastogenesis and bone erosion in inflammatory arthritisJ Biol Chem279, 37219–37222.
De Plaen, I. G., Han, X. B., Liu, X., Hsueh, W., Ghosh, S., and May, M. J. (2006) Lipopol-ysaccharIDe induces CXCL2/macrophage inflammatory protein-2 gene expression in enterocytes via NF-kappaB activation: independence from endogenous TNF-alpha and platelet-activating factorImmunology118, 153–163.
Jimi, E., Aoki, K., Saito, H., D'Acquisto, F., May, M. J., Nakamura, I., Sudo, T., Kojima, T., Okamoto, F., Fukushima, H., Okabe, K., Ohya, K., and Ghosh, S. (2004) Selective inhibition of NF-kappa B blocks osteoclas-togenesis and prevents inflammatory bone destruction in vivoNat Med10, 617–624.
Oakley, F., Meso, M., Iredale, J. P. , Green, K., Marek, C. J., Zhou, X., May, M. J., Millward-Sadler, H., Wright, M. C., and Mann, D. A. (2005) Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apop-tosis and accelerated recovery from rat liver fibrosisGastroenterology128, 108–120.
Solt, L. A., Madge, L. A., Orange, J. S., and May, M. J. (2007) Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbetaJ Biol Chem282, 8724–8733.
Zaph, C., Troy, A. E., Taylor, B. C., Berman-Booty, L. D., Guild, K. J., Du, Y., Yost, E. A., Gruber, A. D., May, M. J., Greten, F. R., Eckmann, L., Karin, M., and Artis, D. (2007) Epithelial-cell-intrinsic IKK-beta expression regulates intestinal immune homeostasisNature446, 552–556.
Bharti, A. C., Donato, N., Singh, S., and Aggar-wal, B. B. (2003) Curcumin (diferuloylmeth-ane) down-regulates the constitutive activation of nuclear factor-kappa B and IkappaBalpha kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosisBlood101, 1053–1062.
Biswas, D. K., Shi, Q., Baily, S., Strickland, I., Ghosh, S., Pardee, A. B., and Iglehart, J. D. (2004) NF-kappaB activation in human breast cancer specimens and its role in cell proliferation and apoptosisProc Natl Acad Sci USA101, 10137–10142.
Liptay, S., Weber, C. K., Ludwig, L., Wagner, M., Adler, G., and SchmID, R. M. (2003) Mitogenic and antiapoptotic role of constitutive NF-kappaB/Rel activity in pancreatic cancerInt J Cancer105, 735–746.
Singh, S., Shi, Q., Bailey, S. T., Palczewski, M. J., Pardee, A. B., Iglehart, J. D., and Biswas, D. K. (2007) Nuclear factor-kappaB activation: a molecular therapeutic target for estrogen receptor-negative and epIDermal growth factor receptor family receptor-positive human breast cancerMol Cancer Ther6, 1973–1982.
Thomas, R. P. , Farrow, B. J., Kim, S., May, M. J., Hellmich, M. R., and Evers, B. M. (2002) Selective targeting of the nuclear factor-kappaB pathway enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated pancreatic cancer cell deathSurgery132, 127–134.
Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nucleiNucleic AcIDs Res11, 1475–1489.
Saksela, K. and Baltimore, D. (1993) Negative regulation of immunoglobulin kappa light-chain gene transcription by a short sequence homologous to the murine B1 repetitive element.Mol Cell Biol13, 3698–3705.
Acknowledgments
Work in the authors' laboratory was supported by RO1 HL080612
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Springer Science + Business Media, LLC
About this protocol
Cite this protocol
Madge, L.A., May, M.J. (2009). Inhibiting Proinflammatory NF-κB Signaling Using Cell-Penetrating NEMO Binding Domain Peptides. In: Kozlov, S.V. (eds) Inflammation and Cancer. Methods in Molecular Biology™, vol 512. Humana Press. https://doi.org/10.1007/978-1-60327-530-9_11
Download citation
DOI: https://doi.org/10.1007/978-1-60327-530-9_11
Publisher Name: Humana Press
Print ISBN: 978-1-60327-529-3
Online ISBN: 978-1-60327-530-9
eBook Packages: Springer Protocols