BIO 5051 Foundations in Immunology

1. BIO 5051Foundations in Immunology Signaling in lymphocytes (transcription factors) November 04, 2005 Robert H. Arch arch@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 ofsoluble 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 ofsoluble 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 ofsoluble 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 ofsoluble 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. dexamethasone cortisol, hydrocortisone Glucocorticoids activate transcriptionof anti-inflammatory genes HSP-90 HSP-90 Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54 2005 0536

8. Acetylation of core histones regulates gene repression and transcription Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54 2005 0533

9. Glucocorticoids inhibit transcriptionof pro-inflammatory genes Adcock et al. (2004). Proc. Am. Thorac. Soc. 1:247-54 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. 2:725-34 • 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 IkB, and can be blocked by proteasome inhibitors 2003 0379

14. Chen et al. (1998), Nature 391:410-3 • 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 IkB, and can be blocked by proteasome inhibitors 2003 0379

15. Li and Verma (2002), Nature Rev. Immunol. 2:725-34 • 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 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 22:121-30 2003 267

18. 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 NF-kB-regulated genes 2003 0358

19. A20 CYLD A central role for ubiquitinin multiple signalling pathways Chen (2005). Nature Cell Biol. 7:758-65 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. 7:758-65 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 284:316 2003 0350

23. IKKa-/- Hu et al. (1999), Science 284:316 2003 0350

24. IKKb-/- Li et al. (1999), J. Exp. Med. 189:1839 2003 0351

25. Li and Verma (2002), Nature Reviews 2:725 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. Doi et al. (1997), J. Exp. Med. 185:953 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. 1:247-54 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. 63:139. 2004 0474

33. Interactions between AP-1 proteins and other transcription factors Foletta et al (1998) J. Leukoc. Biol. 63:139. 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 20:2476. 2004 0472

37. Signal transduction byCa2+, calcineurin and NF-AT Crabtree (1999) Cell 96:611. 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 20:2476.

40. Analysis of NFAT1 Phosphorylation.Okamura et al. (2000) Mol. Cell 6:539. ST1 ST4 ST5 ST2 ST8

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

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

43. O’Shea et al. (2004), Nature Rev. Drug Disc. 3:555-64 O’Shea et al. (2004), Nature Rev. Drug Disc. 3:555-64 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 101:2940-54 2003 0366

44. STAT1 is activated by IFNg McBride et al. (2000), EMBO J. 19:6196-206 2003 0376

45. STAT domain structureand protein binding sites Levy and Darnell. (2003),Nature Rev. Mol. Cell Biol. 3:651-62 2003 0368

46. Leptomycin B inhibits nuclear exportof STAT1 McBride et al. (2000), EMBO J. 19:6196-206 2003 0372

47. Intracellular localization ofSTAT1 DNA binding mutant McBride et al. (2000), EMBO J. 19:6196-206 2003 0373

48. Identification of STAT1 nuclear export signal McBride et al. (2000), EMBO J. 19:6196-206 2003 0373

49. Effect of NES placement outside ofthe STAT1 DNA biding domain McBride et al. (2000), EMBO J. 19:6196-206 2003 0374

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