1. TRANSCRIPTION FACTOR NF-kB: A NUCLEAR FACTOR IN B CELLS
AND BEYOND.
Fano, Centro di Biotecnologie
04 Aprile 2008
Università degli Studi di Urbino
© Linda Palma, 2008

2. SUMMARY ● NF-kB IDENTITY DOCUMENT ● NF-kB REGULATION
● NF-kB TARGET GENES
● NF-kB FUNCTIONS
● NF-kB AND CANCER
● NF-kB AS THERAPEUTIC TARGET
Università degli Studi di Urbino
© Linda Palma, 2008

3. GENE REGULATORY ELEMENTS
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© Linda Palma, 2008

4. DISCOVERING NF-kB
In early 80’s, research studies on the mechanisms regulating
gene transcription led these authors to the discovery of an
apparently B cell-specific protein, able to bind to the kappa
light polypeptide gene enhancer.
They called it NUCLEAR FACTOR OF k LIGHT CHAIN GENE ENHANCER IN B CELLS
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© Linda Palma, 2008

5. Università degli Studi di Urbino
© Linda Palma, 2008

6. NF-kB/Rel AND IkB FAMILIES OF PROTEINS
The REL homology domain (RHD) mediates
their dimerization and binding to DNA.
The RHD also contains, at its carboxyl
terminus, a nuclear localization signal (NLS)
and is recognized by the IkB proteins, the
binding of which to the RHD interferes
with the function of the NLS.
Three family members, p65, cRel and RelB,
contain transcriptional activation domains (TAD)
at their C-terminus, and are therefore able to
directly activate transcription.
All IkBs contain 6–7 ankyrin repeats,
which mediate their binding to RHDs.
Università degli Studi di Urbino
© Linda Palma, 2008

7. THE NF-kB ACTIVATION PATHWAY (1)
Several stimuli are able to activate NF-kB through
the engagement of various cell-surface receptors:
Università degli Studi di Urbino
© Linda Palma, 2008

8. THE NF-kB ACTIVATION PATHWAY (2)
Several adaptor proteins are recruited to the cytosolic
domains of cell receptors upon their engagement:
IRAKs
RIP
MALT
Bcl10
TAB2/3
Università degli Studi di Urbino
© Linda Palma, 2008

9. THE NF-kB ACTIVATION PATHWAY (3)
Transduction of extracellular stimuli converge on either
of two intracellular signaling cascades, referred to as
CLASSICAL NF-kB ACTIVATION PATHWAY and ALTERNATIVE NF-kB ACTIVATION PATHWAY
IRAKs
RIP
MALT
Bcl10
TAB2/3
Università degli Studi di Urbino
© Linda Palma, 2008

10. THE NF-kB ACTIVATION PATHWAY (4)
The key regulator of both pathways is the Inhibitor of Kappa B Kinase (IKK),
a high molecular weight complex (900 kDa) composed of three kinds of subunits:
IKKa, catalytic
IKKb, catalytic
IKKg (NEMO), regulatory g b a g b a
Homo- and heterodimers containing the IKKb subunit are responsible of activation
of the classical pathway, IKKa homodimers are involved in the alternative pathway.
Università degli Studi di Urbino
© Linda Palma, 2008

11. THE NF-kB ACTIVATION PATHWAY (5)
CLASSICAL ACTIVATION PATHWAY
ALTERNATIVE ACTIVATION PATHWAY
Università degli Studi di Urbino
© Linda Palma, 2008

12. NF-kB TARGET GENES (1) Università degli Studi di Urbino
BAFF *CXCL 11 IL-1b IL *LIX (mouse) ENA (CXCL5) and GCP-2 (CXCL6) *mob-1 BRL-1 CD40 *ICOS MHC Class I HLA-B7 TREM-1 Fibronectin Complement factor B CCL5 Eotaxin IL-1
*IL-15 Lymphotoxin a Neutrophil activating peptide-78 CCR5 CD48 IL-2 receptor a-chain b2 Microglobulin Complement B ICAM-1 Complement factor C4 CCL15/Leukotactin/ SCYA15 Fractalkine
IL-2 IL-17 Lymphotoxin b RANTES CCR7 *IL-15 Lymphotoxin a Neutrophil activating peptide-78 CCR5 CD48 IL-2 receptor a-chain b2 Microglobulin Complement B ICAM-1 Complement factor C4
CCL15/Leukotactin/ SCYA15 Fractalkine IL-2 IL-17 Lymphotoxin b CCR7 CD83 Immunoglobulin Cgamma1 Nod2 Complement component 3 MadCAM-1 C-reactive protein CCL17 Gro alpha IL-6
IL-23A (p19) MCP-1/JE TCA3 T-cell activation gene 3 CXCR CD86 Immunoglobulin gamma1 Polymeric Ig receptor Complement Receptor 2 NCAM Hepcidin CCL19 Gro beta IL-8 IL-27 (p28) MIG
TNFalpha CXCR2 CD98 Immunoglobulin gamma4 PGRP-S Proteasome Subunit LMP2 P-selectin Lipopolysaccharide binding protein CCL20 Gro gamma IL-9 EBI3/IL-27BMIP-1a,b TNFbeta CD137 CD134
Immunoglobulin e heavy chain T-cell receptor b chain Peptide Transporter TAP1 Tenascin-C Pentraxin PTX3 CCL22 Gro-1 IL-10 beta-Interferon   TRAIL (aka Apo2 ligand) CD154 *F11-receptor
Immunoglobulin k light chain T-cell receptor/CD3gamma Tapasin VCAM-1 Serum amyloid A proteins (SAA1, SAA2, SAA3) CCL23/SYA23 *ICOS IL-11 IP-10 aka (LAG-1) TFF3 (Treefoil factor) CD3gamma
FcRn Invariant Chain II TLR-2 DC-SIGN Angiotensinogen (Angiotensin II) Serum amyloid A proteins (SAA1, SAA2, SAA3) CCL28 IFN-g IL-12B (p40) KC MIP-2 VEGI CD21 Fc epsilon receptor II (CD23)
Kinin B1 Receptor TLR9 ELAM-1 (CD62E, E-selectin) beta-defensin-2 Serum amyloid A proteins (SAA1, SAA2, SAA3) CINC-1 IL-1a IL-12A (p35) IiGp1 MIP-3alpha/CCL20 B7.1 (CD80) CD38
HLA-G MHC class I (H-2Kb) *TNF-Receptor p75/80 (CD120B) Endoglin C4b binding protein Tissue factor-1 *CYP2E1 CYP2C11 CYP7b COX-2 Ferritin H chain HSP90-alpha *5-Lipoxygenase (guinea pig)
12-Lipoxygenase Inducible NO-Synthase *MAP4K1   Cu/Zn SOD SOD1 SOD2 *Mx1 NAD(P)H quinone oxidoreductase (DT-diaphorase) Phospholipase A2 SEPS1 ABCA1 ABCC6 A1 adenosine receptor
A2A *ADAM19 Amiloride-sensitive sodium channel *alpha2B-adrenergic receptor Bradykinin B1-Receptor *CD23 CD69 DOR Epidermal Growth Factor Receptor ErbB2 Gal1 Receptor Lox-1 Ly49
Mdr1 Mu-opioid receptor mGlu2 Neuropeptide Y-Y1 receptor *NMDA receptor subunit 2A (rat) *NMDA receptor subunit NR-1 (GRIN1 gene) Oxytocin receptor PAF receptor 1 P-gp RAGE- receptor ASC
Bax Bfl1/A1 Bcl-xL Bcl-2 B7-H1 BNIP3 Caspase-11 Nr13 c-FLIP CD95 (Fas) *Fas-associated phosphatase-1 Fas-Ligand IAPs IEX-1L TRAF-1 TRAF-2 *TRAF-2 binding protein (Carp) XIAP Activin A
Angiopoietin BCAP BDNF BLyS BLNK BMP-2 BMP-4 CGRP FGF8 FLRG G-CSF GM-CSF *HGF/SF EPO *IGFBP-1 IGFBP-2 M-CSF (CSF-1) Midkine (neurite growth promoting factor-2) NGF NK-1R
NK4 Nrg1 OPN PDGF B chain PlGF Proenkephalin Prolactin Stem Cell Factor *Thrombospondin-1 (TSP-1) *Thrombospondin-2 (THBS2) VEGF C *WNT10B *B94 *Egr-1 p22/PRG1 *p62 *TIEG
A20 ABIN-3 Androgen receptor Bcl-3 BMI-1 CDX1 *c-fos (fish gene) c-myb c-myc c-rel C/EBPdelta *DC-SCRIPT Dmp1 E2F3a Elf3 *ELYS *ETR101 Gata-3 *Glucocorticoid receptor HIF-1alpha
HOXA9 IRF-1 IRF-2 IRF-4 IRF-7 IkB-a IkB-e junB jmjD3 Lef1 LZIP Mail nfkb2 nfkb1 NLRP2 NURR1 Osterix p53 Progesterone receptor relb Snail Sox9 Stat5a Tfec Twist WT1 YY1 ·
*ABC Transporters   *N-acetylglucosaminyltransferase I (rat gene)   ADH   AID   AMACR   ARF-related protein-1   Argininosuccinate synthetase   Aromatase (promoter II)   ART2.1   alpha 1ACT   BACE-1 Btk
Cathepsin B *Cathepsin L *Ceramide glycosyltransferase Chitinase 3-like protein *cis-retinoid/androgen dehydrogenase type 1 (CRAD1) *cis-retinoid/androgen dehydrogenase type 2 (CRAD2) Collagenase 1
*Dihydrodiol dehydrogenase *DYPD DNASIL2 EL *ENO2 *GAD67 GD3-synthase gp91 phox Gelatinase B GSTP1-1 Glutamate-cysteine ligase  GCLC *Glutamate-cysteine ligase modifier
*gamma glutamylcysteine synthetase *Glucose l-6-phosphate dehydrogenase Glucose-6-phosphatase GnRH II Granzyme B *Soluble Guanylyl cyclase alpha (1)   *Heparanase  HO-1 Hyaluronan synthase
11bHSD2 *17bHSD H+-K+ATPase alpha2 Iodothyronine deiodinase (type 2) Lipocalin-type prostaglandin D synthase (L-PGDS) Lysozyme Mthfr *MKP-1 MMP-3 matrix metalloproteinaase-3 MMP-9
matrix metalloproteinaase-9 MLCK iNOS n-NOS *PDE7A1 PIM-1 Plk3 PIK3CA *PP5 PKCdelta PLCdelta 1 *PTGIS prostaglandin synthase *PGES prostaglandin E synthase PTP1B PTHrP RACK1
*REV3 Slfn-2 Serpin 2A SIAT1 SNARK SSAT *SUV3 TERT Transglutaminase TTG Type II-secreted phospholipase A2 Uridine phosphorylase *Xanthine Dehydrogenase *ABCG5 (bovine) ABCG8 (bovine)
AbetaH-J-J alpha-1 acid glycoprotein alpha-fetoprotein AMH *beta-amyloid APOBEC2 Apolipoprotein C III Apolipoprotein D Apolipoprotein E AQP4 *Biglycan BRCA2 Calsarcin-1 *Caveolin-1
*Clone *Clone *Clone 68 *p21-CIP1 *Claudin-2 a2(I) collagen *Connexin32 Cyclin D1 Cyclin D2 *Cyclin D3 DIF2 DMT1 Elafin Endothelin 1 Ephrin-A1 Factor VIII Ferritin Heavy Chain
Gadd45beta Galpha i2 *GIF Galectin 3 GBP-1 epsilon-Globin zeta-Globin *GS Hair K5 keratin *HCCS1 HMG14 IBABP IMP2 K3 Keratin K6b Keratin K15 Keratin *Lactoferrin Laminin B2 Chain
Lipocalin-2 Mts1 Mir125b Mir146a, b Mir155 MNE1 Mucin (MUC-2) Myelin basic protein MCT1 Naf1 Neutrophil gelatinase-associated lipocalin NLF1 *p11 PA28 alpha PA28 beta PAI-1 *Pax8
*PCBD Perforin *PGK1 POMC Pregnancy-specific glycoprotein rnCGM3 Prodynorphin Prostate-specific antigen PTEN RAG-1 RAG-2 RbAp48 *RICK SerpinE2 S100A6 (calcyclin) SH3BGRL SK2 channels
Skp2 *Spergen-1 SWS1 Syncytin-1 Syndecan-4 TAUT TASK-2 *Tissue factor pathway inhibitor-2 (TFPI-2) *Transferrin (mosquito) TRIF TRPC1 *UBE2M *UCP-2 Uroplakin Ib  
25-hydroxyvtamin D3 1alpha hydroxylase Vimentin a1-antitrypsin Gro Adenovirus (E3 region) Avian Leukosis Virus Bovine Leukemia Virus CMV EBV (Wp promoter) HBV (pregenomic promoter)
HIV-1 HSV (ICP90, ICPO) JC Virus HPV type 16 SIV SV-40
Università degli Studi di Urbino
© Linda Palma, 2008

13. (R = purine; N = any nucleotide; Y = pyrimidine)
NF-kB TARGET GENES (2)
kB MOTIF: mer
GGGRNNYYCC
(R = purine; N = any nucleotide; Y = pyrimidine)
● Degeneration
● Redundancy
● Sequence-dimer pairs
● Exchange of dimers
● CONSTITUTIVELY AND IMMEDIATELY
ACCESSIBLE GENES (CIA)
● REGULATED LATE ACCESSIBILITY
GENES (RLA)
FINE TUNING OF TARGET GENE EXPRESSION RATES
IN A CELL- AND STIMULUS- SPECIFIC MANNER
Università degli Studi di Urbino
© Linda Palma, 2008

14. NF-kB TARGET GENES (3) ● Growth factors, ligands and their modulators
● Transcription factors
and Regulators
● Enzymes
● Cyclins
● IkBs
● A20
● Cytokines/Chemokines
and their modulators
● Immunoreceptors
● Proteins involved
in antigen presentation
● Cell adhesion molecules
● Acute phase proteins
● Stress response genes
● Regulators of apoptosis
● Viruses
● Early response genes
● NF-kB proteins
● Cell-surface receptors
Università degli Studi di Urbino
© Linda Palma, 2008

15. NF-kB FUNCTIONS: APOPTOSIS (1) NF-kB
● Death Receptors:
NF-kB
TNFR1 / DR3 /
DR6 / EDAR
Fas / DR4 /
DR5
TRADD
TRAF2
TRAF5
RIP1
FADD
PROCASPASE 8
PROCASPASE 10
C-FLIP
FLIP
c-IAP1
c-IAP2
XIAP
Bcl-2 family members
APOPTOTIC RESPONSE
NF-kB INDUCES GENES WHOSE FUNCTIONS
INTERFERE WITH THOSE OF THE
DEATH-INDUCING SIGNALING COMPLEXES
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© Linda Palma, 2008

16. NF-kB INDUCES GENES WITH ANTIOXIDANT FUNCTIONS
NF-kB FUNCTIONS:
APOPTOSIS (2)
● Mitochondria:
CASPASE-DEPENDENT
AND –INDEPENDENT
MECHANISMS
NF-kB
DEPOLARIZATION
glutathione-S-transferase
Mn SOD
Ferritin heavy chain
Metallothionein
REACTIVE
OXYGEN
SPECIES
(ROS)
NF-kB INDUCES GENES WITH
ANTIOXIDANT FUNCTIONS
APOPTOTIC RESPONSE
Università degli Studi di Urbino
© Linda Palma, 2008

17. NF-kB FUNCTIONS: APOPTOSIS (3) NF-kB, some members of its activation
pathway and some of its target gene products
are themselves substrates of caspases.
The ratio between pro-apoptotic and
anti-apoptotic activities can target a given
cell type to life or death in response to
various stimuli.
Università degli Studi di Urbino
© Linda Palma, 2008

18. NF-kB FUNCTIONS: IMMUNITY (1) NF-kB INDUCERS  Bacteria and Fungi
 Bacterial or Fungal Products
 Viruses
 Viral Products
 Eukaryotic parasite
 (Inflammatory) Cytokines
 Physiological (Stress) Conditions
 Physical Stress
 Oxidative Stress
 Environmental Hazards
 Therapeutically used drugs
 Modified Proteins
 Overexpressed Proteins
 Receptor Ligands
 Apoptotic Mediators
 Mitogens, Growth Factors and Hormones
 Physiological Mediators
 Chemical Agents
Università degli Studi di Urbino
© Linda Palma, 2008

19. NF-kB FUNCTIONS: IMMUNITY (2) Pathogen INNATE IMMUNITY
INFLAMMATION AND COMPLEMENT CASCADE
Epithelial cells from lung, liver and intestinal mucosa and professional immune cells (macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils and natural killer cells).
ADAPTIVE IMMUNITY
LYMPHOID ORGANS DEVELOPMENT,
ANTIGEN PRESENTATION AND
MAINTENANCE OF MEMORY CELLS
APCs, T and B lymphocytes.
Università degli Studi di Urbino
© Linda Palma, 2008

20. NF-kB FUNCTIONS: IMMUNITY (3) NF-kB Università degli Studi di Urbino
Inflammatory genes
mRNA
Università degli Studi di Urbino
© Linda Palma, 2008

21. NF-kB DYSFUNCTION (1) Università degli Studi di Urbino
Ageing
Headaches                                             
Pain                                                      
Complex Regional Pain Syndrome        
Cardiac Hypertrophy      
Muscular Dystrophy (type 2A)             
Muscle wasting                                     
Catabolic disorders
Diabetes, Type 1
Diabetes, Type 2                 
Obesity                                                      
Fetal Growth Retardation                     
Hypercholesterolemia                           
Atherosclerosis
Heart Disease
Chronic Heart Failure                            
Ischemia/reperfusion
Stroke                                                   
Cerebral aneurysm                                
Angina Pectoris
Pulmonary Disease
Cystic Fibrosis          
Acid-induced Lung Inury                       
Pulmonary hypertension                        
Chronic Obstructive Pulmonary
Disease (COPD)
Hyaline Membrane Disease                   
Kidney Disease
Glomerular Disease                               
Alcoholic Liver Disease                       
Leptospiriosis renal disease
Gut Diseases
Peritoneal endometriosis                
Skin Diseaes
Nasal sinusitis                                      
Mesothelioma                                   
Anhidrotic Ecodermal Dysplasia-ID    
Behcet's Disease                                  
Incontinentia pigmenti
Tuberculosis                                         
Asthma
Arthritis
Crohn's Disease
Colitis (rat)                                           
Ocular Allergy                                      
Glaucoma                                              
Appendicitis                                          
Paget's Disease                                      
Pancreatitis
Periodonitis
Endometriosis                                        
Inflammatory Bowel Disease
Inflammatory Lung isease                    
Sepsis
Silica-induced
Sleep apnoea                                         
AIDS (HIV-1)
Autoimmunity
Antiphospholipid Syndrome                  
Lupus
Lupus nephritis                                      
Waldenstrom macroglobulinemia         
Chronic Disease Syndrome                  
Familial Mediterranean Fever              
Hereditary Periodic Fever syndrome        
Psychosocial stress diseases                 
Neuropathological Diseases
Familial amyloidotic polyneuropathy, inflamm neuropathy
Traumatic brain injury                          
Spinal cord injury                                
Parkinson Disease                        
Multiple Sclerosis                           
Rheumatic Disease          
Alzheimers Disease
Amyotropic lateral sclerosis
Huntington's Disease                             
Retinal Disease                                     
Cataracts                                                
Hearing loss                                  
Cancer
Università degli Studi di Urbino
© Linda Palma, 2008

22. NF-kB DYSFUNCTION (2) Cancer type Università degli Studi di Urbino
Solid tumors (generally)
Breast
Cervix
Ovary
Vulva
Prostate
Kidney
Bladder                   
Lung    
Mesothelioma                                       
Non small-cell lung     
Liver
Pancreas
Esophygeal/gastric         
Laryngeal                                           
Stomach
Colon
Thyroid
Parathyroid
Melanoma
Endometrial (Uteris)
Squamous cell carcinoma                  
Head and neck  
Hilar Cholangiocarcinoma                
Oral carcinoma
Cylindromatosis
Brooke-Spiegler, Trichoepithelioma        
Glioblastoma
Hodgkin’s lymphoma
Astrocytoma/glioblastoma
Neuroblastoma  
Acute T-cell leukemia (+/-HTLV-1)
Acute Non-lymphocytic leukemia       
Acute lymphoblastic leukemia
Acute myelogenous leukemia
Mantle cell lymphoma                       
Myelodysplastic syndrome                
Chronic lymphocytic leukemia
Burkitts Lymphoma (EBV)
MALT lymphoma
Multiple myeloma
Diffuse large B-cell lymphoma
Università degli Studi di Urbino
© Linda Palma, 2008

23. INFLAMMATION IS INVOLVED IN TUMOR INITIATION, PROMOTION
NF-kB AND CANCER (1)
Epidemiological data have led to hypothesize an
association between INFLAMMATION and CANCER.
Examples:
silica / asbestos / cigarette smoke and LUNG CANCER
H. pylori infection and GASTRIC CANCER
IBDs and COLON CANCER
HPV and CERVICAL CANCER
EBV and BURKITT LYMPHOMA
viral hepatitis and LIVER CANCER
Genetic data exist, linking polymorphisms
of PRRs genes and other inflammatory genes
to increased cancer risk.
Examples:
TLR6 and TLR10 and PROSTATE CANCER
NOD2 locus and COLORECTAL CANCER
IL-1b and IL-1RA and GASTRIC CANCER
INFLAMMATION IS INVOLVED IN
TUMOR INITIATION, PROMOTION
AND PROGRESSION
Università degli Studi di Urbino
© Linda Palma, 2008

24. NF-kB AND CANCER (2) ● INADEQUATE PATHOGEN ERADICATION
● PROLONGED INFLAMMATORY SIGNALING
● DEFECTIVE ANTI-INFLAMMATORY MECHANISMS
PERSISTENT NF-kB ACTIVATION
ENHANCED PROLIFERATION AND
PROTECTION OF PRE-NEOPLASTIC /
MALIGNANT CELLS FROM APOPTOSIS
AND NECROSIS
Università degli Studi di Urbino
© Linda Palma, 2008

25. NF-kB AND CANCER (3) Tumorigenesis requires six essential
alterations to normal cell physiology:
● self-sufficiency in growth signals ● insensitivity to growth inhibition
● evasion of apoptosis ● immortalization
● sustained angiogenesis ● tissue invasion and metastasis
Chronic
infection
IL-8 / VEGF /
MMPs
Angiogenesis and
metastases
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© Linda Palma, 2008

26. NF-kB AND CANCER (4) Università degli Studi di Urbino
© Linda Palma, 2008

27. NF-kB AS TARGET FOR ANTICANCER DRUGS (1) MAJOR ISSUES: ● UPTAKE
● STABILITY
● EFFICACY IN PATIENTS
● SIDE EFFECTS
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© Linda Palma, 2008

28. NF-kB AS TARGET FOR ANTICANCER DRUGS (2)
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© Linda Palma, 2008