NF-B Signaling : Pros and Cons of Altering NF-B as a Therapeutic Approach

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  • NF-B Signaling

    Pros and Cons of Altering NF-B as aTherapeutic Approach

    LAURENCE J. EGANa AND MURAT TORUNERb

    aDepartment of Pharmacology and Therapeuticity, Clinical Science Institute,University College Hospital, Galway, IrelandbDepartment of Gastroenterology, Ankara University Medical School,Sihhiye - 06100 Anakara, Turkey

    ABSTRACT: The transcription factor, nuclear factor-B (NF-B), is adominant regulator of the expression of hundreds of genes, many of whichplay important roles in the regulation of inflammation and programmedcell death (apoptosis). Since the discovery of NF-B in the mid 1980s,this transcription factor has been the subject of intense investigation. Ex-cess or inappropriate activation of NF-B has been observed in humaninflammatory bowel disease and in a host of other inflammatory dis-eases and type of cancer. Functional studies in animals have shed lighton the role of NF-B in broader pathophysiological contexts. From suchstudies, it has become quite clear that NF-B plays unique and distinctfunctions in different cell types. Because of the importance of NF-B insignaling inflammation, and in inhibiting programmed cell death, manypharmaceutical companies are developing small-molecule inhibitors ofthis pathway. In this article, we evaluate the relative pros and cons ofblocking NF-B as a therapeutic approach for inflammatory bowel dis-ease. On the basis of the results of studies in animals that have primarilyused genetic approaches to inhibit NF-B activity, we suggest that thereare certain niche indications for blocking NF-B in inflammatory boweldisease that offer particular promise.

    KEYWORDS: nuclear factor-kappa B; inflammatory bowel disease

    INTRODUCTION

    Nuclear factor-B (NF-B) was first described in 1986 as a protein bindingto the enhancer in B lymphocytes.1 However, subsequent studies revealed

    Address for correspondence: Laurence J. Egan, M.D., Clinical Science Institute, University CollegeHospital, Galway, Ireland. Voice: +353-91-495370; fax: +353-91-495572.

    e-mail: laurence.egan@nuigalway.ie

    Ann. N.Y. Acad. Sci. 1072: 114122 (2006). C 2006 New York Academy of Sciences.doi: 10.1196/annals.1326.009

    114

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    that NF-B is in fact primarily a cytoplasmic factor that is expressed by al-most all cell types.2 Moreover, the function of NF-B as a transcription factorcan be induced by a wide variety of cellular stimuli that induce the nucleartranslocation and sequence-specific DNA binding of NF-B proteins. Thesestimuli most commonly include inflammatory cytokines, such as interleukin-1or tumor necrosis factor- (TNF-),3 infection with invasive microbes, such asSalmonella4 or viruses, bacterial cell wall products, such as lipopolysaccharide,peptidoglycan, and by other forms of cellular stress including oxidative stressand DNA double-strand breaks.5 The expression of over 150 genes has shownto be upregulated by NF-B. The encoded products of those genes are proteinsthat play important roles in immunity, apoptosis, and cellular proliferation.6

    NF-B IN INFLAMMATORY BOWEL DISEASE

    Pathways to NF-B Activation

    Two principal signaling pathways have been identified that control the activ-ity of NF-B.6 The best studied of these pathways positions the kinase IKK(inhibitor of NF-B kinase beta, also known as IKK2) as the focus point ofsignals emanating from a variety of sources including certain cell surface cy-tokine receptors (e.g., TNF- receptor type I) and bacterial pattern-recognitionreceptors (e.g., toll-like receptor 4), intracellular redox sensing mechanisms,and nuclear DNA damage signaling proteins (e.g., ATM). IKK is one compo-nent of the multisubunit IKK complex and, when activated, catalyzes the serinephosphorylation of IB on residues 32 and 36.7 IB retains NF-B proteins,principally the p50 and p65 subunits, in the cytoplasm. Upon phosphorylationby IKK, IB is targeted for degradation by a ubiquitinproteasomal path-way. This event represents the key cellular control point for the activation ofthe important NF-B p50/p65 heterodimer. For this reason, IKK has emergedas an attractive therapeutic target for the inhibition of NF-B.8

    The more recently described alternative pathway to NF-B activation ap-pears to be most important in lymphocytes. This pathway is stimulated byspecific lymphoid cytokines including BAFF, CD40 ligand, and lymphotoxin- and leads to the IKK-dependent proteolytic processing of NF-B p100 intothe active p52 form.9 p52 usually dimerizes with another NF-B protein RelBand translocates to the nucleus to induce expression of a set of genes importantin lymphoid structure and function.

    NF-B Is Inappropriately Activated in InflammatoryBowel Disease Mucosa

    When it was recognized that NF-B is important for controlling the expres-sion of many genes involved in signaling inflammation, investigators searched

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    for evidence of NF-B activation in numerous inflammatory diseases, includ-ing inflammatory bowel disease. Several papers have documented excess orinappropriate NF-B activation both in ulcerative colitis and Crohns diseaseand in animal models of inflammatory bowel disease.1012 NF-B activityhas been observed in lamina propria mononuclear cells and in epithelial cellsof the inflamed gut. The importance of NF-B activity in the pathogenesisof inflammatory bowel disease has also been hinted at by the fact that cer-tain anti-inflammatory drugs commonly used for inflammatory bowel diseaseappear to inhibit NF-B. Thus, corticosteroids have been shown to block nu-clear translocation of NF-B polypeptides and also to increase expressionof IB, the chief endogenous inhibitor of NF-B within cells.13 Moreover,sulfasalazine and 5-aminosalicylic acid drugs have also been shown to blockNF-B activation through inhibiting the activity of the IKK complex14and alsoby blocking functionally important posttranslational modifications of the p65NF-B subunit.15

    CELL TYPESPECIFIC FUNCTIONS OF NF-B

    Functional Studies of NF-B in Mice

    The overall importance of NF-B as an inhibitor of apoptosis was revealedby gene knockout studies. Mice that were engineered to lack expression of thep65 NF-B subunit or the essential subunits of the IKK complex, IKK16,17

    or IKK- 18 (also known as NF-B essential modulator, NEMO) all exhibit avery similar phenotype of embryonic lethality at day 13.5. This is due to mas-sive hepatocyte apoptosis caused by TNF- signaling. To further probe the invivo functions of NF-B, a number of laboratories have generated conditionalknockout mice that lack the ability to activate NF-B in specific cell types us-ing bone marrow chimeras or cre/loxP technology. Below we highlight someof the important results from studies of mice engineered to lack IKK expres-sion and hence NF-B activity, in cell types that are important in inflammatorybowel disease: lymphocytes, monocyte/macrophages, and intestinal epithelialcells.

    Lymphocyte-Specific IKK- Knockout Mice

    Mice have been engineered to lack IKK expression in both T lymphocytesand B lymphocytes. Deletion of IKK severely impairs NF-B activation in Tlymphocytes.19 This is accompanied by slightly lower overall levels of T lym-phocytes in both spleen and lymph nodes. However, more strikingly, there aredefects in the maturation of those cells in the absence of IKK, as evidenced bygreatly lower numbers of regulatory T cells and memory T cells.20 At the func-tional level, IKK was shown to be important for the inhibition of T lymphocyte

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    apoptosis induced by stimulation with TNF-.19 B lymphocytes that lack IKKsurvive poorly, as reflected by overall lower numbers of immature B cells, fol-licular B cells, and marginal zone B cells compared to IKK-sufficient cells.21

    This is functionally important because IKK-deficient B lymphocytes do notmount robust T celldependent or T cellindependent antibody responses uponantigenic stimulation.22 Thus, these observations indicate important roles forthe IKK- NF-B system in the survival, maturation, and activation of lympho-cytes, in their resistance to apoptosis, and in the generation of robust antibodyresponses. Recent data suggest that T lymphocytes from certain patients withinflammatory bowel disease might have acquired resistance to apoptotis.23,24 Itis unknown whether NF-B is involved in mediating this apoptotis resistance.

    Monocyte and Macrophages

    Monocytes and macrophages are important cells that link innate immune re-sponses to the adaptive immune system, primarily by the secretion of cytokinesthat act upon lymphocytes. NF-B can be robustly activated within mono-cytes and macrophages by many stimuli that are important in the pathogenesisof inflammatory bowel disease, such as bacteria-derived lipopolysaccharide,peptidoglycan, and immunostimulatory DNA. The importance of NF-B inmonocyte activation by those factors was revealed by the dramatically lowerlevels of interleukin-6 and interleukin-12 secretion by IKK-deficient mono-cytes after stimulation by those factors.25 Moreover, the chemokines MIP-2and KC are also strongly under the control of IKK in monocytes.26 Thefunctional importance of monocyte NF-B activation was revealed in experi-ments of colitis-associated colon cancer in mice. Those studies revealed a verystrong role for monocyte NF-B in their promotion of tumor growth.26 Thus, inmonocytes and macrophages, the IKKNF-B system is essential for produc-tion of proinflammatory cytokines, prostaglandins, and in tumor promotion inthe colon.

    Epithelial Cells

    Intestinal epithelial cells constitute a single cell barrier that separates thehost from the contents of the intestinal lumen and whose integrity is vital forhealth. The intestinal epithelial cells, while playing important protective andabsorptive functions, can also participate actively in mucosal immunity by theelaboration of cytokines and chemokines. Numerous studies in cell lines and ingenetically engineered mice have revealed a very important role for IKK andNF-B in the expression of inflammatory cytokines, such as TNF-, enzymessuch as cyclo-oxygenase-2 and chemokines, such as interleukin-8 by thesecells.2729 Moreover, NF-B activation in intestinal epithelial cells can be trig-gered by numerous forms of cellular stress typical of human disease, such as

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    ischemia/reperfusion, radiation injury, and chronic inflammation. Functionalstudies in mice have clearly revealed a very important role for IKK in block-ing apoptosis of intestinal epithelial cells under all of those conditions.26,30,31

    This is reflected in worse mucosal injury in animal models of colitis and is-chemia/reperfusion in the absence of NF-B activation.26,32 Although a failureto activate NF-B in intestinal epithelium is associated with worse morpho-logic injury in these disease models, the development of colon cancer wasstrongly inhibited. Thus, the IKKNF-B system in intestinal epithelial cellsis important for the elaboration of inflammatory cytokines, the inhibition ofapoptosis, promotion of ulcer healing, and carcinogenesis.

    PROS AND CONS OF NF-B INHIBITIONIN INFLAMMATORY BOWEL DISEASE

    Considering the results of the aforementioned studies, NF-B has emergedas an exciting potential therapeutic target. The predicted effects of blockingNF-B in different cell types within the context of inflammatory bowel diseaseare summarized in TABLE 1. NF-B inhibition would be predicted to decreasethe overactive state of the mucosal immune system in inflammatory boweldisease, by inactivating or killing lymphocytes and macrophages. In view ofthe relative resistance of lymphocytes to apoptosis in inflammatory boweldisease, blockade of NF-B should restore apoptotis sensitivity in those cells,an effect that is especially appealing. This beneficial effect may be partiallyoffset by immune suppression and the risks of opportunistic infections. The neteffect of blocking NF-B in intestinal epithelial cells is difficult to predict andmay depend on clinical factors, such as the presence of ulceration. It is likelythat NF-B inhibitors would decrease cytokine release from the epithelium, butwould also predispose cells to apoptosis, which might harm effective mucosalbarrier functions and delay ulcer healing. However, induction of intestinalepithelial cell death by NF-B blockade probably underlies the anticancereffects of this strategy, which would be a distinct benefit.

    TABLE 1. Predicted cell typespecific effects, beneficial and harmful of inhibition ofNF-B, in patients with inflammatory bowel disease

    Cell type Benefit Harm

    T lymphocyte activation Immune suppression cytokine expression apoptosis susceptibility

    B lymphocyte antibody production Immune suppressionMonocyte/macrophage cytokine expression Immune suppressionIntestinal epithelial cell cytokine expression ulcer healing

    apoptosis susceptibilityKeratinocyte risk of malignant transformationOsteoclast activation

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    While substantial evidence indicates a procarcinogenic role for NF-B inmany epithelia, including those throughout the gastrointestinal tract, this is nottrue in the skin. In fact, NF-B activity in keratinocytes appears to prevent thedevelopment of squamous cell carcinomas.33 Although the molecular mecha-nisms of this effect have not been fully elucidated, these findings suggest thatNF-B inhibitors might increase susceptibility to squamous cell skin cancer.

    THERAPEUTIC NICHES FOR NF-B INHIBITIONIN INFLAMMATORY BOWEL DISEASE

    1. Prevention of colonic neoplasia: 5-Aminosalicylic acid drugs are corner-stones of inflammatory bowel disease therapy. The use of these agentshas been shown in some epidemiological studies to be associated withlower rates of colonic neoplasia development in patients with inflamma-tory bowel disease.34 This fact, combined with powerful results fromgenetic studies indicating an essential role for NF-B activation inthe development of colitis-associated neoplasia26 suggests strongly thatNF-Binhibiting drugs could be used to prevent or treat early-stagecolitis-associated neoplasia. Thus, inflammatory bowel disease patientsat especially high risk for colonic neoplasia, including those with verylong-standing disease, a prior history of indefinite dysplasia, or with co-existing primary sclerosing cholangitis, might be good candidates forNF-Binhibiting therapy.

    2. Maintenance of remission: The intestines of mice lacking the ability toactivate NF-B in lymphocytes, monocytes/macrophages, or intestinalepithelial cells are normal. This indicates that NF-B in these cell typesdoes not have an important homeostatic role for intestinal morphogenesisor overall health. Early stages of inflammatory bowel disease activityare characterized by the expression in excess quantities of numerouscytokines, whose expression is controlled by NF-B. Therefore, the useof NF-B inhibition to prevent relapses of inflammatory bowel diseasewould be a rational objective that would not be hampered by the potentialfor NF-B inhibition to slow ulcer healing, a potentially detrimentaleffect.

    3. Treatment of inflammatory bowel disease: associated inflammatoryarthritisIn addition to the aforementioned functional roles, several stud-ies have pointed toward the importance of NF-B in osteoclastoge-nesis.35,36 In the absence of functional NF-B signaling, stimulatorsof bone resorption, such as the RANK ligand, are unable to initiatethe activation of osteoclasts, which are the cells that are important forbone resorption. Thus, patients with inflammatory bowel disease that isassociated with destructive arthritis, such as ankylosing spondylitis orinflammatory oligoarthritis might especially benefit from administrationof a systemic NF-B inhibitor.

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    CONCLUSIONS

    At present, our prediction of the effects of systemic NF-B inhibition onnormal physiological processes and within the context of disease pathophysi-ology requires us to piece together information derived from cell typespecificknockout mice and studies in cell lines. However, with the advent of potentand specific IKK- inhibitors, which are under development by many pharma-ceutical companies, we should be able to more accurately obtain informationregarding the overall functional effects of NF-B inhibition in disease models,and eventually in patients. There is much to be learned. In particular, significanteffort will have to be focused on studying the benefits and adverse effects ofIKK inhibitors in animal models and linking the inhibition of IKK to molec-ular effects, such as gene expression, and to cell biological events, such as cellmigration, proliferation, and death. Furthermore, it is likely that as the molec-ular subclassification of inflammatory bowel disease progresses, we will beable to identify subgroups of inflammatory bowel disease patients who mightbenefit especially from therapeutic strategies directed at selectively inhibitingNF-B.

    REFERENCES

    1. SEN, R. & D. BALTIMORE. 1986. Multiple nuclear factors interact with the im-munoglobulin enhancer sequences. Cell 46: 705716.

    2. SEN, R. & D. BALTIMORE. 1986. Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism. Cell 47: 921928.

    3. OSBORN, L., S. KUNKEL & G.J. NABEL. 1989. Tumor necrosis factor alpha and in-terleukin 1 stimulate the human immunodeficiency virus enhancer by activationof the nuclear factor kappa B. Proc. Natl. Acad. Sci. USA 86: 23362340.

    4. ELEWAUT, D. et al. 1999. NF-kappa B is a central regulator of the intestinal ep-ithelial cell innate immune response induced by infection with enteroinvasivebacteria. J. Immunol. 163: 14571466.

    5. LI, N. & M. KARIN. 1998. Ionizing radiation and short wavelength UV activateNF-kappaB through two distinct mechanisms. Proc. Natl. Acad. Sci. USA 95:1301213017.

    6. HAYDEN, M.S. & S. GHOSH. 2004. Signaling to NF-kappaB. Genes Dev. 18: 21952224.

    7. ZANDI, E. et al. 1997. The IkappaB kinase complex (IKK) contains two kinasesubunits, IKKalpha and IKKbeta, necessary for IkappaB phosphorylation andNF-kappaB activation. Cell 91: 243252.

    8. KARIN, M., Y. YAMAMOTO & Q.M. WANG. 2004. The IKK NF-kappa B system: atreasure trove for drug development. Nat. Rev. Drug Discov. 3: 1726.

    9. SENFTLEBEN, U. et al. 2001. Activation by IKKalpha of a second, evolutionaryconserved, NF-kappa B signaling pathway. Science 293: 14951499.

    10. ROGLER, G. et al. 1998. Nuclear factor kappaB is activated in macrophages andepithelial cells of inflamed intestinal mucosa. Gastroenterology 115: 357369.

  • EGAN & TORUNER: NF-B SIGNALING 121

    11. SCHREIBER, S., S. NIKOLAUS & J. HAMPE. 1998. Activation of nuclear factor kappaB inflammatory bowel disease. Gut 42: 477484.

    12. NEURATH, M.F. et al. 1996. Local administration of antisense phosphorothioateoligonucleotides to the p65 subunit of NF-kappa B abrogates established exper-imental colitis in mice. Nat. Med. 2: 9981004.

    13. AUPHAN, N. et al. 1995. Immunosuppression by glucocorticoids: inhibition ofNF-kappa B activity through induction of I kappa B synthesis. Science 270:286290.

    14. WAHL, C. et al. 1998. Sulfasalazine: a potent and specific inhibitor of nuclearfactor kappa B. J. Clin. Invest. 101: 11631174.

    15. EGAN, L.J. et al. 1999. Inhibition of interleukin-1-stimulated NF-kappaB RelA/p65phosphorylation by mesalamine is accompanied by decreased transcriptionalactivity. J. Biol. Chem. 274: 2644826453.

    16. LI, Z.W. et al. 1999. The IKKbeta subunit of IkappaB kinase (IKK) is essential fornuclear factor kappaB activation and prevention of apoptosis. J. Exp. Med. 189:18391845.

    17. LI, Q. et al. 1999. Severe liver degeneration in mice lacking the IkappaB kinase 2gene. Science 284: 321325.

    18. MAKRIS, C. et al. 2000. Female mice heterozygous for IKK gamma/NEMO defi-ciencies develop a dermatopathy similar to the human X-linked disorder incon-tinentia pigmenti. Mol. Cell. 5: 969979.

    19. SENFTLEBEN, U. et al. 2001. IKKbeta is essential for protecting T cells fromTNFalpha-induced apoptosis. Immunity 14: 217230.

    20. SCHMIDT-SUPPRIAN, M. et al. 2003. Mature T cells depend on signaling throughthe IKK complex. Immunity 19: 377389.

    21. PASPARAKIS, M., M. SCHMIDT-SUPPRIAN & K. RAJEWSKY. 2002. IkappaB kinasesignaling is essential for maintenance of mature B cells. J. Exp. Med. 196: 743752.

    22. LI, Z.W. et al. 2003. IKK beta is required for peripheral B cell survival and prolif-eration. J. Immunol. 170: 46304637.

    23. ITOH, J. et al. 2001. Decreased Bax expression by mucosal T cells favours resistanceto apoptosis in Crohns disease. Gut 49: 3541.

    24. INA, K. et al. 1999. Resistance of Crohns disease T cells to multiple apoptoticsignals is associated with a Bcl-2/Bax mucosal imbalance. J. Immunol. 163:10811090.

    25. CHU, W. et al. 2000. DNA-PKcs is required for activation of innate immunity byimmunostimulatory DNA. Cell 103: 909918.

    26. GRETEN, F.R. et al. 2004. IKKbeta links inflammation and tumorigenesis in amouse model of colitis-associated cancer. Cell 118: 285296.

    27. BERIN, M.C. et al. 2002. Role of EHEC O157:H7 virulence factors in the activa-tion of intestinal epithelial cell NF-kappaB and MAP kinase pathways and theupregulated expression of interleukin 8. Cell. Microbiol. 4: 635648.

    28. BERIN, M.C. et al. 2001. Regulated production of the T helper 2-type T-cellchemoattractant TARC by human bronchial epithelial cells in vitro and in humanlung xenografts. Am. J. Respir. Cell. Mol. Biol. 24: 382389.

    29. ECKMANN, L. et al. 2000. Analysis by high density cDNA arrays of alteredgene expression in human intestinal epithelial cells in response to infectionwith the invasive enteric bacteria Salmonella. J. Biol. Chem. 275: 1408414094.

  • 122 ANNALS NEW YORK ACADEMY OF SCIENCES

    30. EGAN, L.J. et al. 2004. IkappaB-kinasebeta-dependent NF-kappaB activation pro-vides radioprotection to the intestinal epithelium. Proc. Natl. Acad. Sci. USA101: 24522457.

    31. CHEN, L.W. et al. 2003. The two faces of IKK and NF-kappaB inhibition: pre-vention of systemic inflammation but increased local injury following intestinalischemia-reperfusion. Nat. Med. 9: 575581.

    32. EGAN, L.J. et al. 2003. Nuclear factor-kappa B activation promotes restitution ofwounded intestinal epithelial monolayers. Am. J. Physiol. Cell. Physiol. 285:C1028C1035.

    33. DAJEE, M. et al. 2003. NF-kappaB blockade and oncogenic Ras trigger invasivehuman epidermal neoplasia. Nature 421: 639643.

    34. BERNSTEIN, C.N. et al. 2002. Cancer prevention in inflammatory bowel diseaseand the chemoprophylactic potential of 5-aminosalicylic acid. Inflamm. BowelDis. 8: 356361.

    35. JIMI, E. et al. 2004. Selective inhibition of NF-kappa B blocks osteoclastogenesisand prevents inflammatory bone destruction in vivo. Nat. Med. 10: 617624.

    36. RUOCCO, M.G. et al. 2005. I{kappa}B kinase (IKK){beta}, but not IKK{alpha},is a critical mediator of osteoclast survival and is required for inflammation-induced bone loss. J. Exp. Med. 201: 16771687.