BIO 5051Foundations in Immunology

Signaling in lymphocytes (transcription factors)

November 04, 2005

Robert H. Arch

arch@wustl.edu

phone 747-4681

InflammationInflammation

2003 0390

• response to?microbial infections and tissue injury• local features include? upregulation of adhesion

molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation

• systemic features include? fever and acute phase response

• mediated and resolved by? a large array of soluble factors, cell surface molecules, and enzymes

InflammationInflammation

2003 0390

• response to infections, allergens and tissue injury• local features include? upregulation of adhesion

molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation

• systemic features include? acute phase response and fever

• mediated and resolved by? a large array of soluble factors, cell surface molecules, and enzymes

InflammationInflammation

2003 0390

• response to infections, allergens and tissue injury• local features include upregulation of adhesion

molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation

• systemic features include? acute phase response and fever

• mediated and resolved by? a large array of soluble factors, cell surface molecules, and enzymes

InflammationInflammation

2003 0390

• response to infections, allergens and tissue injury• local features include upregulation of adhesion

molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation

• systemic features include acute phase response and fever

• mediated and resolved by? a large array of soluble factors, cell surface molecules, and enzymes

InflammationInflammation

2003 0390

• response to infections, allergens and tissue injury

• local features include upregulation of adhesion molecules and enzymes, increased blood flow, and phagocytosis of debris and dead cellsas well as tissue repair by cell proliferation

• systemic features include acute phase response and fever

• mediated and resolved by a large array of soluble factors, cell surface molecules and enzymes

dexamethasone

2005 0536

Glucocorticoids activate transcriptionGlucocorticoids activate transcriptionof anti-inflammatory genesof anti-inflammatory genes

Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54

HSP-90

HSP-90

cortisol, hydrocortisone

2005 0533

Acetylation of core histones regulates Acetylation of core histones regulates gene repression and transcription gene repression and transcription

Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54

2005 0535

Glucocorticoids inhibit transcriptionGlucocorticoids inhibit transcriptionof pro-inflammatory genesof pro-inflammatory genes

Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54

2003 0349

Transcription factorsTranscription factorsof the immune systemof the immune system

• GR glucocorticoid receptor• NF-B nuclear factor kappa B• NFAT nuclear factor of activated T cells• AP-1 activating protein-1• STATsignal transducer and activator

of transcription• GATA-3 (A/T)GATA(A/G) consensus binding motif• T-bet T box expressed in T cells• p53 tumor suppressor p53• Smad Sma/Mad (C. elegans/Drosophila)

2003 0349

Transcription factorsTranscription factorsof the immune systemof the immune system

• GR glucocorticoid receptor• NF-B nuclear factor kappa B• NFAT nuclear factor of activated T cells• AP-1 activating protein-1• STATsignal transducer and activator

of transcription• GATA-3 (A/T)GATA(A/G) consensus binding motif• T-bet T box expressed in T cells• p53 tumor suppressor p53• Smad Sma/Mad (C. elegans/Drosophila)

Nuclear factor kappa B (NF-Nuclear factor kappa B (NF-B)B)

• first described as a nuclear factor in B cells that binds to a 10 bp region of the intronic enhancer and is pivotal for Ig light chain transcription

• can be found in the cytoplasm of most cell types

• family of dimeric transcription factors

• monomers have 300 aa Rel homology region required for dimerization, DNA binding, and interaction with inhibitor proteins (IB)

• release from IB results in nuclear translocation2003 0357

Li and Verma (2002), Nature Rev. Immunol. 2:725-34 2003 0379

• RHD: Rel-homology domainTD: transactivation domainN: nuclear localization signalLZ: leucine zipperGRR: glycine-rich regionANK: ankyrin repeats

• transcriptionally active:p65/p50, p65/p65, p50/c-Rel

• transcriptionally inactive:p50/p50, p52/p52

• p100/p52 and l05/p50 are precursors

• processing (signal-dependent and -independent pathways?) is ATP-dependent, requires poly-ubiquitination of IB, and can be blocked by proteasome inhibitors

Chen et al. (1998), Nature 391:410-3 2003 0379

• RHD: Rel-homology domainTD: transactivation domainN: nuclear localization signalLZ: leucine zipperGRR: glycine-rich regionANK: ankyrin repeats

• transcriptionally active:p65/p50, p65/p65, p50/c-Rel

• transcriptionally inactive:p50/p50, p52/p52

• p100/p52 and l05/p50 are precursors

• processing (signal-dependent and -independent pathways?) is ATP-dependent, requires poly-ubiquitination of IB, and can be blocked by proteasome inhibitors

Li and Verma (2002), Nature Rev. Immunol. 2:725-34 2003 0379

• RHD: Rel-homology domainTD: transactivation domainN: nuclear localization signalLZ: leucine zipperGRR: glycine-rich regionANK: ankyrin repeats

• transcriptionally active:p65/p50, p65/p65, p50/c-Rel

• transcriptionally inactive:p50/p50, p52/p52

• p100/p52 and l05/p50 are precursors

• processing (signal-dependent and -independent pathways?) is ATP-dependent, requires poly-ubiquitination of IB, and can be blocked by proteasome inhibitors

NF-NF-B inhibitors (IB inhibitors (IB)B)

• IB, IB, IB, IB, IB, Bcl-3 • central ankyrin repeat mediate interaction with

rel-homology domains of NF-B proteins• N-terminal domain is phosphorylated in

response to NF-B activating signals• phosphorylation of two conserved Ser residues

is required for ubiquitylation and degradation• C-terminal PEST domain involved in basal

turnover

2003 0384

2003 267Y

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NF-NF-B activation by LTB activation by LTR vs. TNFR-IR vs. TNFR-I

2003 0358

NF-NF-B-regulated genesB-regulated genesadhesion moleculesintercellular adhesion molecule-1 (ICAM-1)vascular cell adhesion molecule-1 (VCAM-1)E-selectin

cytokinestumor necrosis factor (TNF-)interleukin-1interleukin-6interleukin-11granulocyte-macrophage colony stimulating factor (GMCSF)

chemokinesinterleukin-8CCL3 (macrophage inflammatory protein (MIP)-1 )CCL7 (monocyte chemotactic protein (MCP)-3)CCL5 (RANTES)CCL11 (eotaxin)

enzymes

inducible nitric oxide synthase (iNOS)cyclooxygenase-2 (COX-2)cytosolic phospholipase A25-lipidoxygenase (5-LOX)

anti-apoptotic proteins

TNFR-associated factors (TRAF) 1 and TRAF2cellular inhibitor of apoptosis (c-IAP) 1 and c-IAP2bcl-2 homologues AI/Bfl-1 and bcl-xL

NF-B and IB family members

IBNF-B1 (p105/p50) NF-B2 (p100/p52)RelB

2005 0532

A central role for ubiquitinA central role for ubiquitinin multiple signalling pathways in multiple signalling pathways

Ch

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2005

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CYLD

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BAFF-R, LTR, CD40TNFRs

IL-1R, TLR

2005 0531

The NF-The NF-B signalling pathways B signalling pathways

modified from Chen (2005).Nature Cell Biol. 7:758-65

canonical non-canonical

• ikka-/- mice die post-natally due to multiple morphological defects; shiny taut skin prevents emergence of fore- and hind-limbs, absence of ears, truncation of head, skeletal abnormalities

• ikkb-/- mice die between E12.5 and E14.5 as a result of fetal hepatocyte apoptosis; embryonic lethality is rescued by crossing with TNFR-I-/-

and TNF--/- animals

ikkaikka-/- -/- and ikkband ikkb-/--/-

2003 0350

Hu et al. (1999), Science 284:316

IKKIKK-/--/-

2003 0350

Hu et al. (1999), Science 284:316

IKKIKK-/--/-

2003 0351

Li et al. (1999), J. Exp. Med. 189:1839

IKKIKK-/--/-

Li and Verma (2002), Nature Reviews 2:725 2002 006

• normal embryonic development, but mice die7-10 days post-natally due to severe widespread dermatitis and granulocytosis (delayed in DKO)

• increased expression of distinct pro-inflammatory cytokines and factors associated with granulocyte recruitment, such as TNF-, G-CSF and VCAM

• not all genes induced by NF-B are upregulated

ikbaikba-/--/-

• embryonic lethality between E15 and E16 due to fetal hepatocyte apoptosis induced by TNF-

• embryonic lethality can be rescued by crossing with TNFR-I-/- and TNF--/- animals

• reconstitution of SCID mice with fetal hepatocytes revealed defects in mitogen-induced proliferation and isotype switching but normal lymphopoiesis

relarela-/--/-

Doi et al. (1997), J. Exp. Med. 185:953

• embryonic lethality between E15 and E16 due to fetal hepatocyte apoptosis induced by TNF-

• embryonic lethality can be rescued by crossing with TNFR-I-/- and TNF--/- animals

• reconstitution of SCID mice with fetal hepatocytes revealed defects in mitogen-induced proliferation and isotype switching but normal lymphopoiesis

relarela-/--/-

• despite nearly ubiquitous expression and its role as major partner of p65 (Rel A), which is essential for embryogenesis, surprisingly normal development

• although not essential for hematopoiesis, multiple defects in functions of immune system

p50p50-/--/- (NFKB1 (NFKB1-/--/-))

2005 0534

Histone acetylation regulatesHistone acetylation regulatesNF-NF-B-induced transcription B-induced transcription

Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54

Activating protein 1 (AP-1)Activating protein 1 (AP-1)• family of dimeric transcription factors• expressed at low levels• usually constitutively bound to their DNA sites• rapid changes of complex composition upon

stimulation of cells due to de novo synthesis• phosphorylation by MAPK, e.g., c-Jun N-

terminal kinase (JNK), strongly enhances transactivating capacity

• play crucial roles in cell proliferation, apoptosis and oncogenesis

2003 0355

2004 0474

Signals leading toSignals leading toIL-2 expression in CD4IL-2 expression in CD4++ cells cells

Foletta et al (1998) J. Leukoc. Biol. 63:139.

2004 0475

Interactions between AP-1 proteins Interactions between AP-1 proteins and other transcription factorsand other transcription factors

Foletta et al (1998) J. Leukoc. Biol. 63:139.

Co-operative DNA bindingCo-operative DNA bindingof NFAT and AP-1 proteinsof NFAT and AP-1 proteins

2004 0471

Fig 11.24 Lodish et al. Molecular Cell Biology

Monomeric NFAT and heterotrimericAP-1 transcription factors havelow affinity for their respectivebinding sites. Interactions betweenNFAT and AP1 stabilize theNFAT-AP1-DNA complex.

Nuclear factor of activated T cells Nuclear factor of activated T cells (NFAT)(NFAT)

• first identified in T cells as rapidly inducible nuclear factor binding to the IL-2 promoter

• family of transcription factors related to NF-B

• expressed in most cells of the immune system, including lymphocytes, mast cells, basophils,NK cells and endothelial cells

• target genes include cytokines, cell surface receptors, signaling proteins and transcription factors

2003 0381

The NFAT familyThe NFAT family

2004 0472

modified from Macián et al. (2001) Oncogene 20:2476.

renal atrophy and lack of tonicity-responsive gene expression

2004 0473

Crabtree (1999) Cell 96:611.

Signal transduction bySignal transduction byCaCa2+2+, calcineurin and NF-AT, calcineurin and NF-AT

2004 0473

Signal transduction bySignal transduction byCaCa2+2+, calcineurin and NF-AT, calcineurin and NF-AT

Macian (2005) Nature Reviews Immunology 5, 472-84.

Macián et al. (2001) Oncogene 20:2476.

ST1

ST2

ST8

ST4

ST5

Analysis of NFAT1 Phosphorylation.Okamura et al. (2000) Mol. Cell 6:539.

The SRR-1 Region Regulates the Active Conformation of NFAT1.Okamura et al. (2000) Mol. Cell 6:539.

Macián et al. (2001) Oncogene 20:2476.

2003 0366

Ben

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2003

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0-54

JAK/STAT signal transductionJAK/STAT signal transduction

Janus kinaseso Jak1o Jak2o Jak3o Tyk2

Signal transducer andactivator of transcription

o Stat1o Stat2o Stat3o Stat4o Stat5ao Stat5bo Stat6

O’Shea et al. (2004), Nature Rev. Drug Disc. 3:555-64

O’Shea et al. (2004), Nature Rev. Drug Disc. 3:555-64

STAT1 is activated by IFNSTAT1 is activated by IFN

2003 0376

McBride et al. (2000), EMBO J. 19:6196-206

2003 0368

Levy and Darnell. (2003),Nature Rev. Mol. Cell Biol. 3:651-62

STAT domain structureSTAT domain structureand protein binding sitesand protein binding sites

Leptomycin B inhibits nuclear exportLeptomycin B inhibits nuclear exportof STAT1of STAT1

McBride et al. (2000), EMBO J. 19:6196-206

2003 0372

Intracellular localization ofIntracellular localization ofSTAT1 DNA binding mutantSTAT1 DNA binding mutant

2003 0373

McBride et al. (2000), EMBO J. 19:6196-206

Identification of Identification of STAT1 nuclear export signalSTAT1 nuclear export signal

McBride et al. (2000), EMBO J. 19:6196-206

2003 0373

Effect of NES placement outside ofEffect of NES placement outside ofthe STAT1 DNA biding domainthe STAT1 DNA biding domain

2003 0374

McB

rid

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al.

(200

0), E

MB

O J

. 19:

6196

-206

Grogan and Locksley (2002), Curr. Opin. Immunol. 14:366

2004 0476

Transcription factors in the helper T cell differentiation

Serfling et al. (2000) Biochim. Biophys. Acta. 1498:1.