The document summarizes the Signal Transducer and Activator of Transcription (STAT) protein family, with a focus on STAT3 and its role in psoriasis. It describes how STAT proteins are activated downstream of cytokine and growth factor receptors via phosphorylation by Janus kinases (JAKs). STAT3 in particular is activated by cytokines like IL-6 and plays roles in processes like acute phase response, cell growth, and embryonic development. Aberrant STAT3 activation has been implicated in conditions like psoriasis and cancer.

1. Signal Transducer &Activator of Transcription( STAT)-3 :Role in Psoriasis DR M.YOUSRY ABDEL-MAWLA,MD ZAGAZIG FACULTY OF MEDICINE,EGYPT

2. INTRODUCTION

3. Signal transducers and activators of transcription ) Stat) Family Stats are latent in the cytoplasm until they are activated by extracellular signaling ligands , including cytokines , growth factors and hormones. Binding of these extracellular ligands to the specific receptors leads to activation of various tyrosine kinases (TKs). They include JAKs, receptor TKs, and non-receptor TKs such as Src and ABL, which can directly phosphorylate Stat proteins in the absence of ligand-induced receptor signaling

5. There are four members of the JAK family in mammals, Jak1, Jak2, Jak3 and Tyk2. They are over 1000 amino acids in length, ranging in molecular weight from 120 to 130 kDa. Jak1, Jak2 and Tyk2 are expressed ubiquitously, whereas the expression of Jak3 is restricted to cells of the myeloid and lymphoid lineages

6. STAT 1 STAT 2 STAT 3 STAT 4 STAT 5 STAT 6 SH2 DNA Helix 750 851 DNA SH2 Helix 770 SH2 DNA Helix 748 SH2 DNA Helix 734 SH2 DNA Helix 848 Helix SH2 KGTGYIKTEL 701 ERRKYLKHRL 690 SAAPYLKTKF 705 GDKGYVPSVF AVDGYVKPQI KDGRGYVPAT 693 694 641 Activators IFN-  ,  , IFN  , EGF, PDGF, Insulin, Ang II, VEGF IFN-  ,  Ang II IL-6, LIF, CNTF, ET-1, serotonin, GM-CSF, IL-10, Leptin, EGF, PDGF, Ang II, FGF, VEGF IL-4 and IL- 12 Prolactin, GH, IL-3, Epo, IL- 5, Ang II, Insulin, GM-CSF IL- 4, IL-13, Ang II, PDGF, FGF

7. JAK/STAT Activation Upon ligand stimulation, receptors undergo conformational changes. These changes attract JAKs which subsequently are activated by trans-phosphorylation. Phosphorylated JAKs once activated phosphorylate downstream signaling molecules such as STATs.

8. Several studies have suggested that JAK’s associate with growth factor (e.g. Insulin, EGF and PDGF) and with G-protein coupled (e.g. Angiotensin II) receptors. These associations and JAK’s activation enables these receptors to activate the STAT’s.

9. Ang II NH 2 NUCLEUS c-fos YIPP COOH STAT 1  /  Fyn + SHP-2 + JAK2 SHP-2 JAK2 P STAT 1  /  STAT1  /  STAT1  /  P P STAT1  /  STAT1  /  P P Fyn P P SHP-2 MKP-1 STAT 1  /  P P Fyn P P JAK2-STAT1 activation

10. NK-22 cell-secreted cytokines stimulate epithelial cells to proliferate, release IL-10 and activate STAT1 and STAT3. M Cella et al. Nature 000, 1-4 (2008)

12. CYTOKINES, CYTOKINE RECEPTORS, ACTIVATED SIGNALING MOLECULES AND MAJOR BIOLOGICAL FUNCTIONS Cytokines receptor signaling molecules biological function IL-2 IL-2R Jak1/3, Stat5/3 T cell growth and peripheral tolerance; NK activity; B cell differentiation IL-4 IL-4R Jak1/3, Stat6 Th2 differentiation; B cell differentiation IL-7 IL-7R Jak1/3,sata5/3 T-cell homeostasis; T and B development (mouse) IL-9 IL-9R Jak1/3, Stat5/3 Airway mucus production; mast cell proliferation IL-15 IL-15R Jak1/3, Stat5/3 CD8 homeostasis, NK cell development IL-21 IL-21R Jak1/3, Stat3,5,1 B-cell differentiation; T and NK activation

14. Contribution of keratinocyte migration to both wound healing and anagen progression. (B) Stat3- dependent and -independent keratinocyte migration .

15. Class IIB(3)(b) latent cytoplasmic factors These families not present in fungi or plants, hinting at an important evolutionary divergence leading to animals.

16. STATs - a signal responsive TF family STATs: Signal Transducers and Activators of Transcription two functions given in the name 1. Transducers for signals from many cytokines Broad spectrum of biological effects 2. Transcriptional activators characteristic activation mechanism activation at the cell membrane, response in the nucleus Rapid signal response The activation/deactivation cycle of STAT molecules is quite short, about 15 min for an individual molecule.

17. Simple signalling pathway

18. REGULATION of Cell Signaling

19. The JAK-STAT signalling pathway Function: regulation of gene expression in response to cytokines 1. cytokines bind and aggregate the cytokine receptors in the cell membrane 2. associated JAK-type tyrosine kinases are activated by aggregation and tyrosine-phosphorylates neighbouring-JAK (transphosphorylation) as well as the C-terminal tail of the receptor (multiple sites) 3. Tyr-phosphates recruit inactive STAT-factors in the cytoplasm which are bound through their SH2-domains 4. STATs become tyrosine-phosphorylated by JAK 5. phosphorylated STATs dissociate, dimerize (homo-/hetero-) and migrate to the nucleus 6. STAT-dimers bind DNA and activates target genes

20. Canonical JAK–STAT pathway Sequential tyrosine phosphorylations Receptor dimerization allows transphosphorylation and activation of Janus kinases (JAKs). This is followed by phosphorylation of receptor tails and the recruitment of the STAT proteins through their SH-2 domains. STAT tyrosine phosphorylation then occurs. Dimerization of activated (tyrosine phosphorylated) STAT is followed by nuclear entry.

21. IFN-response: two variants Signalling pathway first discovered in studies of interferon-response (IFN) IFN  /  IFN  /   activation of Jak1+Tyk2  DNA-binding complexes (trimer: STAT1+STAT2+p48, together designated ISGF3)  activation of target genes with ISRE (IFN-stimulated response element) IFN  IFN   activation of Jak1+Jak2  DNA-binding complex (dimer: 2x STAT1)  activation of target genes having GAS elements (IFN  activated sequence)

22. IFN-response: two variants

23. STAT-family members STAT1 - involved in IFN  /  - and IFN  -response STAT2 - involved in IFN  /  -response Mainly acting as partner for STAT1/p48 STAT3 - involved in response to several cytokines including IL6. It activates several genes involved in acute phase response Important in growth regulation, embryonic development & organogenesis Activation of STAT3 correlated with cell growth, link to cancer, bind c-Jun STAT4 - involved in IL12-response STAT5a & 5b - involved in response to several cytokines including prolactin, IL-2, and regulates expression of milk proteins in breast tissue in response to prolactin STAT6 - involved in IL4-response non-mammalian family members (e.g. Drosophila )

24. STAT-members SH2 Y

25. STAT-STAT interaction occurs through reciprocal phospho-Tyr - SH2 interactions SH2-domain SH2 = “Src-homology domain 2” function: phospho-tyrosine binding Three important functions in STATs: important for recruitment of STAT to receptor important for interaction with the JAK kinase important for dimerization of STATs to an active DNA-binding form Tyr-701 conserved key Tyr residue located just C-terminal to SH2 essensiell for dimerdannelse to an active DNA-binding form function: Tyr P bindingssted for SH2 in partner Y P Y P Y + Y

26. Dimerization via SH2-Tyr P Tyr P from the left monomer SH2 from the right monomer

27. STAT-members SH2 Y

28. STATs - structure and function Dimerization Reciprocal SH2- Tyr P interaction Homodimers (STAT1) 2 Heterodimers STAT1-STAT2 STAT1-STAT3 DNA-binding domain DBD located in the middle of the protein Unique motif - se next slide All DBDs bind similar motifs in DNA symmetric inverted half sites Only difference to STATs: preference for central nucleotide GAS= TTN 5-6 AA ISRE= AGTTTN 3 TTTCC

29. STAT-STRUCTURE Known structures [STAT1] 2 -DNA and [STAT3  ] 2 -DNA, as well as an N-terminal of STAT4 Characteristic feature : Symmetry-axis through DNA, each monomer contacts a separate half site structure resembles NF  B and p53 (immunoglobuline fold). The dimer forms a C-shaped ”clamp” around DNA. The dimer is kept together by reciprocal SH2- Tyr P interactions between the SH2 domain in one monomer and the phosphorylated Tyr in the other. The SH2 domain in each monomer is closely linked to the core DBD and is itself close to DNA, and is assumed also to contribute to DNA-binding. N-terminal coiled-coil region not close to DNA, probably involved in prot-prot interaction with flexible position

30. 3D STAT domain structure and protein binding sites.

31. Promoter recognition and selectivity Mechanisms to achieve specific trx responses. Inverted repeat TTN 5–6 AA motif common. Binding specificity to individual elements based on evolved preferences for specific positions. In the ISGF3 heterotrimeric complex, STAT1–STAT2 heterodimers bind to a third protein, p48/ISGF3  , a TF that recognizes the ISRE sequence. STAT N-domains mediate dimer–dimer interactions allowing high-avidity binding to tandemly arranged low-affinity GAS elements. Adjacent response elements bind to other TFs. Cooperativity and synergy. STAT directly recruit co-activators that alter chromatin dynamics.

32. Transactivation Domain(TAD) Transactivation domain C-terminal part of the protein, less conserved variants generated by alternative splicing + proteolysis STAT1  lacking the last 38aa has all functions retained except transactivation Regulation through TAD-modification Activity of TAD is regulated through Ser phosphorylation (LPMSP-motif) Ser727 in STAT1 Kinase not identified - candidates: p38, ERK, JNK A role in recruitment of GTF/coactivator Proteins identified that bind TAD in a Ser-dependent manner MCM5 BRCA1 TAD in STAT2 binds C/H-rich region of CBP STAT2 carries the principal TAD of the ISGF3-complex

33. Other functional domains The N-domain is important for stabilizing interactions between STAT dimers, bound to tandemly arranged response elements

34. SMAD-family - a logic resembling the STAT-family

35. SMAD family

36. SMAD-family - a logic resembling the STAT-family The Smad-factors mediate response to TGF  -related growth- and differentiation factors STAT-related logic Membrane-bound receptors (such as the TGFß-receptor) are activated by binding of ligand (TGF  ). The receptors here are transmembrane serine/threonine-kinases Activated kinases phosphorylate specific Smad-factors phosphorylated Smad-factors associate with a common Smad-factor (Smad4) The generated heteromeric complexes migrate to the nucleus as transcription factors

37. TGF  effectors Latent cytoplasmic TFs activated by serine phosphorylation at their cognate receptors This family transduces signals from the transforming growth factor-  (TGF-  ) superfamily of ligands .

38. Classification Smad-factors - design and classification Nine different Smad-factors identified in vertebrates common conserved domains: N-terminalt MH1-domain (DBD) + C-terminalt MH2-domain Can be divided into three groups 1. Receptor-activated Smad-factors - become phosphorylated by activated receptors in their C-terminal (SSXS) 2. common Smad-factors associated with activated Smad-factors and participate in several signalling pathways 3. Inhibitoriske Smad-factors

39. SMAD-signalling pathway Effector SMADs (R-SMADs) C o-SMADs Repressor SMADs

40. Three groups of SMADs First group: The effector SMADs (also called the R-SMADs) become serine-phosphorylated in the C-terminal domain by the activated receptor . Smad1, Smad5, Smad8, and Smad9 become phosphorylated in response to bone morphogenetic morphogenetic protein (BMP) and growth and differentiation factor (GDF), and Smad2 and Smad3 become phosphorylated in response to the activin/nodal branch of the TGF-  pathway. Second group: regulatory or co-SMADs (common SMADs ). There are two regulatory SMADs: Smad4 and Smad4  (also called Smad10). Smad4 binds to, and is essential for, the function of Smad1 and Smad2. The regulatory Smad4 binds to all effector SMADs in the formation of transcriptional complexes, but it does not appear to be required for nuclear translocation of the effector molecules. Third group: two inhibitory SMADs, Smad6 and Smad7 . provide negative regulation of the pathway by blocking Smad4 binding.

41. SMAD-signalling pathway

42. Final steps - target gene activation Once an activated, serine-phosphorylated effector SMAD binds Smad4 and escapes the negative influences of Smad6 and Smad7, nuclear accumulation and regu-lation of specific target genes can occur. In most cases, SMADs require partner transcription factors with strong DNA binding capacity that determine the gene to be activated. The DNA binding is then strengthened by association with SMADs that on their own bind weakly to adjacent DNA sites. The SMADs furnish transcriptional activation capacity. The specificity of response among different ligands can be partially explained by the choice of DNA binding partner proteins. For example, activin activation of SMADs results in combinations with FAST1 and a particular set of genes is activated. Signaling by BMP ligands results in association of activated SMADs with a DNA binding protein called OAZ.

43. The Smad-factors activate their target genes in combination with other TFs

44. Several signalling pathways linked STATs may also be Tyr-phosphorylated and hence activated by other receptor families receptor tyrosine kinases (RTKs) such as EGF-receptor may phosphorylate STATs EGF stimulation  activation of STAT1, STAT3 non-receptor tyrosine kinases such as Src and Abl may also phosphorylate STATs (?) G-protein coupled 7TMS receptors such as angiotensine receptor (?) STAT may also be modified by Ser-phosphorylation DNA-binding reduced (STAT3) Transactivationdomain Ser-phosphorylated (important for transactivation in STAT1 and STAT3) Responsible kinases not identified - MAPkinases candidates, probably also others JAKs may activate other signalling pathways than STATs Tyr P will recruit several protein-substrates and lead to phosphorylation and activation of other signalling pathways e.g. JAK activation  activation of MAP-kinases e.g. substrates: IRS-1, SHC, Grb2, HCP, Syp, Vav

45. Cross Talks Alternative inputs STATs may be Tyr-phosphorylated by RTKs Alternative outputs JAK may phosphorylate other targets and thus activate signal transduction pathways other than through STATs P P P P P P P P JAK SH2 Cytokine receptor Y Y Y Y P P P Receptor tyrosinee kinase MAPK

46. Variations in mechanisms of STAT activation

47. STAT3 Activation Stat3 is activated by cytokines of the IL-6 family such as IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M and cardiotropin I . Stat3 is the major signal transducer downstream of gp130-like receptors . Other extracellular signaling ligands such as IL-10 family members, epidermal growth factor (EGF), platelet derived growth factor (PDGF), hepa- tocyte growth factor (HGF), granulocyte colony- stimulating factor (G-CSF) and leptin have also known to activate Stat3.

48. Mechanism of Stat3 signaling Different tyrosine kinases (TKs) can induce Stat3 activation. Growth factors such as EGF bind to receptor tyrosine kinases (RTKs), followed by phosphorylation of Stat3 through activation of intermediary kineases of the SRC and JAK families. Cytokines such as IL-6 family members bind to gp130, a common receptor subunit, thereby JAK families and subsequent Stat3 are activated. Non-receptor TKs such as SRC and ABL can directly phophorylate Stat3 in the absence of ligand-dependent receptor signaling. In any pathway, two tyrosine phosphorylated Stat3 proteins form dimers, enter the nucleus and bind DNA to activate transcription of the target genes

49. Psoriasis

50. Psoriasis Psoriasis is a chronic inflammatory skin disease characterized by excessive proliferation, abnormal differentiation of epidermal keratino- cytes, vascular proliferation, and leukocyte infiltration in the dermis and epidermis . It has been considered that psoriasis results from complex, aberrant relationships between the skin and immune system as well as genetic predisposition and environmental factors

51. Psoriasis Clinical Presentation

52. Psoriasis as an immune disorder

53. T-Cell in Psoriasis

54. Immunomodulator(Alefacet) effect on Psorasis Area Score Index(PASI)

55. ANTI –Tumor Necrosis Factor (TNF) in Psoriasis

58. STAT3 links activated keratinocytes and immunocytes required for development of psoriasis( Sano S. et al, 2005; Nature Medicine 11:43-49 (.

60. Stat3 in keratinocytes(KC)

61. Prepsoriasis State

62. Prepsoriasis State

63. Immunopathology of Psoriasis

64. Stat3 links activated keratinocytes and immunocytes required for development of psoriasis Epidermal keratinocytes in psoriatic lesions are characterized by activated Stat3. Transgenic mice with keratinocytes expressing a constitutively active Stat3 ( K5.Stat3C mice ) develop a skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis. Keratinocytes from K5.Stat3C mice show upregulation of several molecules linked to the pathogenesis of psoriasis. In addition, the development of psoriatic lesions in K5.Stat3C mice requires cooperation between Stat3 activation in keratinocytes and activated T cells. Finally, abrogation of Stat3 function by a decoy oligonucleotide inhibits the onset and reverses established psoriatic lesions in K5.Stat3C mice. Thus, targeting Stat3 may be potentially therapeutic in the treatment of psoriasis.

65. New Informations

67. Development of Psoriasis

68. Manipulation of Psorasis

69. Do Other Therapies Work Within This Framework? Anti–T-cell agents could affect Th17 cells as they would other T cells, but this needs to be clarified Anti-TNF agents could decrease activity of Th17 cells or work directly on keratinocyte responses

71. Studies on Stat 3 in Psoriasis

72. Stat 3 in Psoriasis

75. New Prospectives &Directions

77. Message Home

78. THANK YOU