BIO 5051Foundations in Immunology
Signaling in lymphocytes (transcription factors)
November 04, 2005
Robert H. Arch
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
ilm
az e
t al
., (2
003)
EM
BO
J. V
ol 2
2:12
1-30
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
en (
2005
). N
atur
e C
ell B
iol.
7:75
8-65
CYLD
A20
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
ekli
et
al. (
2003
), B
lood
101
:294
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
e et
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.
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