1 transcription factor nf- b: a nuclear factor in b cells and beyond. fano, centro di biotecnologie...

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TRANSCRIPTION FACTOR NF-B:A NUCLEAR FACTOR IN B CELLS

AND BEYOND.

Fano, Centro di Biotecnologie04 Aprile 2008

© Linda Palma, 2008

Università degli Studi di Urbino

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● NF-B IDENTITY DOCUMENT

● NF-B REGULATION

● NF-B TARGET GENES

● NF-B FUNCTIONS

● NF-B AND CANCER

● NF-B AS THERAPEUTIC TARGET



SUMMARY

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GENE REGULATORY ELEMENTS

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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 LIGHT CHAIN GENE ENHANCER IN B CELLS

DISCOVERING NF-B

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NF-B/Rel AND IB 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 IB proteins, thebinding of which to the RHD interferes with the function of the NLS.

All IBs contain 6–7 ankyrin repeats,which mediate their binding to RHDs.

Three family members, p65, cRel and RelB,contain transcriptional activation domains (TAD) at their C-terminus, and are therefore able to directly activate transcription.

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THE NF-B ACTIVATION PATHWAY (1)

Several stimuli are able to activate NF-B through

the engagement of various cell-surface receptors:

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Several adaptor proteins are recruited to the cytosolic

domains of cell receptors upon their engagement:

IRAKsRIP

RIP MALT

Bcl10

TAB2/3

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IRAKsRIP

RIP MALT

Bcl10

TAB2/3

Transduction of extracellular stimuli converge on either

of two intracellular signaling cascades, referred to as

CLASSICAL NF-B ACTIVATION PATHWAY and ALTERNATIVE NF-B ACTIVATION PATHWAY

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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:

IKK, catalytic

IKK, catalytic

IKK(NEMO), regulatory

Homo- and heterodimers containing the IKK subunit are responsible of activation

of the classical pathway, IKK homodimers are involved in the alternative pathway.

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ALTERNATIVE ACTIVATION PATHWAYCLASSICAL ACTIVATION PATHWAY

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NF-B TARGET GENES (1)

BAFF *CXCL 11 IL-1b IL-13 *LIX (mouse) ENA-78 (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-1alphaHOXA9 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 BtkCathepsin 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-9matrix 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 330 *Clone 156 *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 *GS3686 Hair K5 keratin *HCCS1 HMG14 IBABP IMP2 K3 Keratin K6b Keratin K15 Keratin *Lactoferrin Laminin B2 ChainLipocalin-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 channelsSkp2 *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-1 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

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● Degeneration

● Redundancy

● Sequence-dimer pairs

● Exchange of dimers


B MOTIF: 9-11 mer

GGGRNNYYCC(R = purine; N = any nucleotide; Y = pyrimidine)

B MOTIF: 9-11 mer

GGGRNNYYCC(R = purine; N = any nucleotide; Y = pyrimidine)

● 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

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● Growth factors, ligands and their modulators ● Transcription factors and Regulators ● Enzymes ● Cyclins

● Stress response genes ● Regulators of apoptosis

● Cytokines/Chemokines and their modulators ● Immunoreceptors ● Proteins involved in antigen presentation ● Cell adhesion molecules ● Acute phase proteins

● Viruses● Early response genes ● NF-B proteins● Cell-surface receptors

● IkBs● A20

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NF-B FUNCTIONS:APOPTOSIS (1)

Fas / DR4 / DR5

FADDPROCASPASE 8

PROCASPASE 10C-FLIP

TRADDTRAF2TRAF5RIP1

TNFR1 / DR3 /DR6 / EDAR

● Death Receptors:

APOPTOTIC RESPONSE

NF-B

FLIPc-IAP1c-IAP2XIAP

Bcl-2 family members

NF-B INDUCES GENES WHOSE FUNCTIONS INTERFERE WITH THOSE OF THE

DEATH-INDUCING SIGNALING COMPLEXES

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DEPOLARIZATION

CASPASE-DEPENDENTAND –INDEPENDENT

MECHANISMS

● Mitochondria:

APOPTOTIC RESPONSE

NF-B

NF-B INDUCES GENES WITHANTIOXIDANT FUNCTIONS

REACTIVEOXYGENSPECIES

(ROS)

glutathione-S-transferaseMn SOD

Ferritin heavy chainMetallothionein

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NF-B, 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 tovarious stimuli.

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NF-B FUNCTIONS: IMMUNITY (1)

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

NF-NF-B INDUCERSB INDUCERS

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Pathogen INNATE IMMUNITYINNATE IMMUNITY

INFLAMMATION AND COMPLEMENT INFLAMMATION AND COMPLEMENT CASCADECASCADE

Epithelial cells from lung, liver and Epithelial cells from lung, liver and intestinal mucosa and professional intestinal mucosa and professional immune cells (macrophages, immune cells (macrophages, neutrophils, dendritic cells, mast cells, neutrophils, dendritic cells, mast cells, eosinophils, basophils and natural eosinophils, basophils and natural killer cells).killer cells).

ADAPTIVE IMMUNITYADAPTIVE IMMUNITY

LYMPHOID ORGANS DEVELOPMENT,LYMPHOID ORGANS DEVELOPMENT,

ANTIGEN PRESENTATION ANDANTIGEN PRESENTATION AND

MAINTENANCE OF MEMORY CELLSMAINTENANCE OF MEMORY CELLS

APCs, T and B lymphocytes.APCs, T and B lymphocytes.

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NF-B

NF-B

Inflammatory genes

mRNA

Inflammatory genes

mRNA

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NF-B DYSFUNCTION (1)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

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NF-B DYSFUNCTION (2)Cancer type

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

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NF-B 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

INFLAMMATION IS INVOLVED IN

TUMOR INITIATION, PROMOTION

AND PROGRESSION

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-1 and IL-1RA and GASTRIC CANCER

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NF-B AND CANCER (2)

● INADEQUATE PATHOGEN

ERADICATION

● PROLONGED INFLAMMATORY

SIGNALING

● DEFECTIVE ANTI-INFLAMMATORY

MECHANISMS

ENHANCED PROLIFERATION

AND

PROTECTION OF PRE-

NEOPLASTIC /

MALIGNANT CELLS FROM

APOPTOSIS

AND NECROSIS

PERSISTENT NF-B

ACTIVATION

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NF-B 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

metastasisChronic infection

IL-8 / VEGF /MMPs

Angiogenesis and metastases

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NF-B AND CANCER (4)

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NF-B AS TARGET FORANTICANCER DRUGS (1)

MAJOR ISSUES:

● UPTAKE

● STABILITY

● EFFICACY IN PATIENTS

● SIDE EFFECTS

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NF-B AS TARGET FORANTICANCER DRUGS (2)

1 transcription factor nf- b: a nuclear factor in b cells and beyond. fano, centro di biotecnologie...

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