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BIO 5051 Foundations in Immunology

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Title: BIO 5051 Foundations in Immunology


1
BIO 5051Foundations in Immunology
  • Signaling in lymphocytes (transcription factors)
  • November 04, 2005
  • Robert H. Arch
  • arch_at_wustl.edu
  • phone 747-4681

2
Inflammation
  • 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

2003 0390
3
Inflammation
  • 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

2003 0390
4
Inflammation
  • 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

2003 0390
5
Inflammation
  • 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

2003 0390
6
Inflammation
  • 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

2003 0390
7
Glucocorticoids activate transcriptionof
anti-inflammatory genes
HSP-90
HSP-90
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0536
8
Acetylation of core histones regulates gene
repression and transcription
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0533
9
Glucocorticoids inhibit transcriptionof
pro-inflammatory genes
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0535
10
Transcription factorsof the immune system
  • GR glucocorticoid receptor
  • NF-kB nuclear factor kappa B
  • NFAT nuclear factor of activated T cells
  • AP-1 activating protein-1
  • STAT signal 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
11
Transcription factorsof the immune system
  • GR glucocorticoid receptor
  • NF-kB nuclear factor kappa B
  • NFAT nuclear factor of activated T cells
  • AP-1 activating protein-1
  • STAT signal 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
12
Nuclear factor kappa B (NF-kB)
  • first described as a nuclear factor in B cells
    that binds to a 10 bp region of the k intronic
    enhancer and is pivotal for Ig k 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 (IkB)
  • release from IkB results in nuclear translocation

2003 0357
13
Li and Verma (2002), Nature Rev. Immunol. 2725-34
  • RHD Rel-homology domainTD transactivation
    domainN nuclear localization signalLZ leucine
    zipperGRR glycine-rich regionANK ankyrin
    repeats
  • transcriptionally activep65/p50, p65/p65,
    p50/c-Rel
  • transcriptionally inactivep50/p50, p52/p52
  • p100/p52 and l05/p50 are precursors
  • processing (signal-dependent and -independent
    pathways?) is ATP-dependent, requires
    poly-ubiquitination of IkB, and can be blocked by
    proteasome inhibitors

2003 0379
14
Chen et al. (1998), Nature 391410-3
  • RHD Rel-homology domainTD transactivation
    domainN nuclear localization signalLZ leucine
    zipperGRR glycine-rich regionANK ankyrin
    repeats
  • transcriptionally activep65/p50, p65/p65,
    p50/c-Rel
  • transcriptionally inactivep50/p50, p52/p52
  • p100/p52 and l05/p50 are precursors
  • processing (signal-dependent and -independent
    pathways?) is ATP-dependent, requires
    poly-ubiquitination of IkB, and can be blocked by
    proteasome inhibitors

2003 0379
15
Li and Verma (2002), Nature Rev. Immunol. 2725-34
  • RHD Rel-homology domainTD transactivation
    domainN nuclear localization signalLZ leucine
    zipperGRR glycine-rich regionANK ankyrin
    repeats
  • transcriptionally activep65/p50, p65/p65,
    p50/c-Rel
  • transcriptionally inactivep50/p50, p52/p52
  • p100/p52 and l05/p50 are precursors
  • processing (signal-dependent and -independent
    pathways?) is ATP-dependent, requires
    poly-ubiquitination of IkB, and can be blocked by
    proteasome inhibitors

2003 0379
16
NF-kB inhibitors (IkB)
  • IkBa, IkBb, IkBg, IkBd, IkBe, Bcl-3
  • central ankyrin repeat mediate interaction with
    rel-homology domains of NF-kB proteins
  • N-terminal domain is phosphorylated in response
    to NF-kB activating signals
  • phosphorylation of two conserved Ser residues is
    required for ubiquitylation and degradation
  • C-terminal PEST domain involved in basal turnover

2003 0384
17
NF-kB activation by LTbR vs. TNFR-I
Yilmaz et al., (2003) EMBO J. Vol 22121-30
2003 267
18
NF-kB-regulated genes
  • adhesion molecules
  • intercellular adhesion molecule-1 (ICAM-1)
  • vascular cell adhesion molecule-1 (VCAM-1)
  • E-selectin
  • cytokines
  • tumor necrosis factor a (TNF-a)
  • interleukin-1b
  • interleukin-6
  • interleukin-11
  • granulocyte-macrophage colony stimulating factor
    (GMCSF)
  • chemokines
  • interleukin-8
  • CCL3 (macrophage inflammatory protein (MIP)-1a )
  • CCL7 (monocyte chemotactic protein (MCP)-3)
  • CCL5 (RANTES)
  • CCL11 (eotaxin)
  • enzymes
  • inducible nitric oxide synthase (iNOS)
  • cyclooxygenase-2 (COX-2)
  • cytosolic phospholipase A2
  • 5-lipidoxygenase (5-LOX)
  • anti-apoptotic proteins
  • TNFR-associated factors (TRAF) 1 and TRAF2
  • cellular inhibitor of apoptosis (c-IAP) 1 and
    c-IAP2
  • bcl-2 homologues AI/Bfl-1 and bcl-xL
  • NF-kB and IkB family members
  • IkBa
  • NF-kB1 (p105/p50)
  • NF-kB2 (p100/p52)
  • RelB

2003 0358
19
A central role for ubiquitinin multiple
signalling pathways
Chen (2005). Nature Cell Biol. 7758-65
2005 0532
20
The NF-kB signalling pathways
IL-1R, TLR
BAFF-R, LTbR, CD40
TNFRs
canonical
non-canonical
modified from Chen (2005). Nature Cell Biol.
7758-65
2005 0531
21
ikka-/- and ikkb-/-
  • 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-a-/- animals

22
IKKa-/-
Hu et al. (1999), Science 284316
2003 0350
23
IKKa-/-
Hu et al. (1999), Science 284316
2003 0350
24
IKKb-/-
Li et al. (1999), J. Exp. Med. 1891839
2003 0351
25
Li and Verma (2002), Nature Reviews 2725
2002 006
26
ikba-/-
  • 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-a, G-CSF and VCAM
  • not all genes induced by NF-kB are upregulated

27
rela-/-
  • embryonic lethality between E15 and E16 due to
    fetal hepatocyte apoptosis induced by TNF-a
  • embryonic lethality can be rescued by crossing
    with TNFR-I-/- and TNF-a-/- animals
  • reconstitution of SCID mice with fetal
    hepatocytes revealed defects in mitogen-induced
    proliferation and isotype switching but normal
    lymphopoiesis

28
rela-/-
  • embryonic lethality between E15 and E16 due to
    fetal hepatocyte apoptosis induced by TNF-a
  • embryonic lethality can be rescued by crossing
    with TNFR-I-/- and TNF-a-/- animals
  • reconstitution of SCID mice with fetal
    hepatocytes revealed defects in mitogen-induced
    proliferation and isotype switching but normal
    lymphopoiesis

29
p50-/- (NFKB1-/-)
  • 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

30
Histone acetylation regulatesNF-kB-induced
transcription
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0534
31
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
32
Signals leading toIL-2 expression in CD4 cells
Foletta et al (1998) J. Leukoc. Biol. 63139.
2004 0474
33
Interactions between AP-1 proteins and other
transcription factors
Foletta et al (1998) J. Leukoc. Biol. 63139.
2004 0475
34
Co-operative DNA bindingof NFAT and AP-1 proteins
Monomeric NFAT and heterotrimeric AP-1
transcription factors havelow affinity for their
respectivebinding sites. Interactions
betweenNFAT and AP1 stabilize theNFAT-AP1-DNA
complex.
Fig 11.24 Lodish et al. Molecular Cell Biology
2004 0471
35
Nuclear factor of activated T cells (NFAT)
  • first identified in T cells as rapidly inducible
    nuclear factor binding to the IL-2 promoter
  • family of transcription factors related to NF-kB
  • 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
36
The NFAT family
renal atrophy and lack of tonicity-responsive
gene expression
modified from Macián et al. (2001) Oncogene
202476.
2004 0472
37
Signal transduction byCa2, calcineurin and NF-AT
Crabtree (1999) Cell 96611.
2004 0473
38
Signal transduction byCa2, calcineurin and NF-AT
Macian (2005) Nature Reviews Immunology 5, 472-84.
2004 0473
39
Macián et al. (2001) Oncogene 202476.
40
Analysis of NFAT1 Phosphorylation.Okamura et al.
(2000) Mol. Cell 6539.
ST1
ST4
ST5
ST2
ST8
41
The SRR-1 Region Regulates the Active
Conformation of NFAT1.Okamura et al. (2000) Mol.
Cell 6539.
42
Macián et al. (2001) Oncogene 202476.
43
JAK/STAT signal transduction
  • Janus kinases
  • Jak1
  • Jak2
  • Jak3
  • Tyk2
  • Signal transducer andactivator of transcription
  • Stat1
  • Stat2
  • Stat3
  • Stat4
  • Stat5a
  • Stat5b
  • Stat6

Benekli et al. (2003), Blood 1012940-54
2003 0366
44
STAT1 is activated by IFNg
McBride et al. (2000), EMBO J. 196196-206
2003 0376
45
STAT domain structureand protein binding sites
Levy and Darnell. (2003),Nature Rev. Mol. Cell
Biol. 3651-62
2003 0368
46
Leptomycin B inhibits nuclear exportof STAT1
McBride et al. (2000), EMBO J. 196196-206
2003 0372
47
Intracellular localization ofSTAT1 DNA binding
mutant
McBride et al. (2000), EMBO J. 196196-206
2003 0373
48
Identification of STAT1 nuclear export signal
McBride et al. (2000), EMBO J. 196196-206
2003 0373
49
Effect of NES placement outside ofthe STAT1 DNA
biding domain
McBride et al. (2000), EMBO J. 196196-206
2003 0374
50
Grogan and Locksley (2002), Curr. Opin. Immunol.
14366
51
Transcription factors in the helper T cell
differentiation
Serfling et al. (2000) Biochim. Biophys. Acta.
14981.
2004 0476
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