Title: BIO 5051 Foundations in Immunology
1BIO 5051Foundations in Immunology
- Signaling in lymphocytes (transcription factors)
- November 04, 2005
- Robert H. Arch
- arch_at_wustl.edu
- phone 747-4681
2Inflammation
- 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
3Inflammation
- 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
4Inflammation
- 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
5Inflammation
- 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
6Inflammation
- 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
7Glucocorticoids activate transcriptionof
anti-inflammatory genes
HSP-90
HSP-90
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0536
8Acetylation of core histones regulates gene
repression and transcription
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0533
9Glucocorticoids inhibit transcriptionof
pro-inflammatory genes
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0535
10Transcription 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
11Transcription 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
12Nuclear 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
13Li 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
14Chen 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
15Li 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
16NF-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
17NF-kB activation by LTbR vs. TNFR-I
Yilmaz et al., (2003) EMBO J. Vol 22121-30
2003 267
18NF-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
19A central role for ubiquitinin multiple
signalling pathways
Chen (2005). Nature Cell Biol. 7758-65
2005 0532
20The 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
21ikka-/- 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
22IKKa-/-
Hu et al. (1999), Science 284316
2003 0350
23IKKa-/-
Hu et al. (1999), Science 284316
2003 0350
24IKKb-/-
Li et al. (1999), J. Exp. Med. 1891839
2003 0351
25Li and Verma (2002), Nature Reviews 2725
2002 006
26ikba-/-
- 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
27rela-/-
- 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
28rela-/-
- 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
29p50-/- (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
30Histone acetylation regulatesNF-kB-induced
transcription
Adcock et al. (2004). Proc. Am. Thorac. Soc.
1247-54
2005 0534
31Activating 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
32Signals leading toIL-2 expression in CD4 cells
Foletta et al (1998) J. Leukoc. Biol. 63139.
2004 0474
33Interactions between AP-1 proteins and other
transcription factors
Foletta et al (1998) J. Leukoc. Biol. 63139.
2004 0475
34Co-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
35Nuclear 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
36The NFAT family
renal atrophy and lack of tonicity-responsive
gene expression
modified from Macián et al. (2001) Oncogene
202476.
2004 0472
37Signal transduction byCa2, calcineurin and NF-AT
Crabtree (1999) Cell 96611.
2004 0473
38Signal transduction byCa2, calcineurin and NF-AT
Macian (2005) Nature Reviews Immunology 5, 472-84.
2004 0473
39Macián et al. (2001) Oncogene 202476.
40Analysis of NFAT1 Phosphorylation.Okamura et al.
(2000) Mol. Cell 6539.
ST1
ST4
ST5
ST2
ST8
41The SRR-1 Region Regulates the Active
Conformation of NFAT1.Okamura et al. (2000) Mol.
Cell 6539.
42Macián et al. (2001) Oncogene 202476.
43JAK/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
44STAT1 is activated by IFNg
McBride et al. (2000), EMBO J. 196196-206
2003 0376
45STAT domain structureand protein binding sites
Levy and Darnell. (2003),Nature Rev. Mol. Cell
Biol. 3651-62
2003 0368
46Leptomycin B inhibits nuclear exportof STAT1
McBride et al. (2000), EMBO J. 196196-206
2003 0372
47Intracellular localization ofSTAT1 DNA binding
mutant
McBride et al. (2000), EMBO J. 196196-206
2003 0373
48Identification of STAT1 nuclear export signal
McBride et al. (2000), EMBO J. 196196-206
2003 0373
49Effect of NES placement outside ofthe STAT1 DNA
biding domain
McBride et al. (2000), EMBO J. 196196-206
2003 0374
50Grogan and Locksley (2002), Curr. Opin. Immunol.
14366
51Transcription factors in the helper T cell
differentiation
Serfling et al. (2000) Biochim. Biophys. Acta.
14981.
2004 0476