STAT3 Protein in Psoriasis by M. Yousry Abdel-Mawla

Signal Transducer &Activator of Transcription( STAT)-3 :Role in Psoriasis : 

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

INTRODUCTION : 

M.Y.Abdel-Mawla,MD zagazig 2 INTRODUCTION

Signal transducers and activators of transcription) Stat) Family : 

3 M.Y.Abdel-Mawla,MD zagazig 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

Slide 4: 

4 M.Y.Abdel-Mawla,MD zagazig

Slide 5: 

5 M.Y.Abdel-Mawla,MD zagazig 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

Slide 6: 

6 M.Y.Abdel-Mawla,MD zagazig

Slide 7: 

7 M.Y.Abdel-Mawla,MD zagazig 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.

Slide 8: 

8 M.Y.Abdel-Mawla,MD zagazig 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.

Slide 9: 

9 M.Y.Abdel-Mawla,MD zagazig

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

10 M.Y.Abdel-Mawla,MD zagazig NK-22 cell-secreted cytokines stimulate epithelial cells toproliferate, release IL-10 and activate STAT1 and STAT3. M Cella et al. Nature 000, 1-4 (2008)

Slide 11: 

11 M.Y.Abdel-Mawla,MD zagazig

CYTOKINES, CYTOKINE RECEPTORS, ACTIVATED SIGNALING MOLECULES AND MAJOR BIOLOGICAL FUNCTIONS : 

12 M.Y.Abdel-Mawla,MD zagazig 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

Slide 13: 

13 M.Y.Abdel-Mawla,MD zagazig

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

14 M.Y.Abdel-Mawla,MD zagazig Contribution of keratinocyte migration to both wound healing and anagen progression. (B) Stat3- dependent and -independent keratinocyte migration.

Class IIB(3)(b)latent cytoplasmic factors : 

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

STATs - a signal responsive TF family : 

16 M.Y.Abdel-Mawla,MD zagazig 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.

Simple signalling pathway : 

17 M.Y.Abdel-Mawla,MD zagazig Simple signalling pathway

REGULATION of Cell Signaling : 

18 M.Y.Abdel-Mawla,MD zagazig REGULATION of Cell Signaling

The JAK-STAT signalling pathway : 

19 M.Y.Abdel-Mawla,MD zagazig 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

Canonical JAK–STAT pathway : 

20 M.Y.Abdel-Mawla,MD zagazig 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.

IFN-response: two variants : 

21 M.Y.Abdel-Mawla,MD zagazig 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)

IFN-response: two variants : 

22 M.Y.Abdel-Mawla,MD zagazig IFN-response: two variants

STAT-family members : 

23 M.Y.Abdel-Mawla,MD zagazig 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)

STAT-members : 

24 M.Y.Abdel-Mawla,MD zagazig STAT-members SH2 Y

STAT-STAT interaction occurs through reciprocal phospho-Tyr - SH2 interactions : 

25 M.Y.Abdel-Mawla,MD zagazig 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: TyrP bindingssted for SH2 in partner Y P Y P Y +

Dimerization via SH2-TyrP : 

26 M.Y.Abdel-Mawla,MD zagazig Dimerization via SH2-TyrP TyrP from the left monomer SH2 from the right monomer

STAT-members : 

27 M.Y.Abdel-Mawla,MD zagazig STAT-members SH2 Y

STATs - structure and function : 

28 M.Y.Abdel-Mawla,MD zagazig STATs - structure and function Dimerization Reciprocal SH2- TyrP 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= TTN5-6AA ISRE= AGTTTN3TTTCC

STAT-STRUCTURE : 

29 M.Y.Abdel-Mawla,MD zagazig STAT-STRUCTURE Known structures [STAT1]2-DNA and [STAT3b]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 NFkB and p53 (immunoglobuline fold). The dimer forms a C-shaped ”clamp” around DNA. The dimer is kept together by reciprocal SH2- TyrP 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

3D : 

30 M.Y.Abdel-Mawla,MD zagazig 3D STAT domain structure and protein binding sites.

Promoter recognition and selectivity : 

31 M.Y.Abdel-Mawla,MD zagazig Promoter recognition and selectivity Mechanisms to achieve specific trx responses. Inverted repeat TTN5–6AA 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/ISGF3g, 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.

Transactivation Domain(TAD) : 

32 M.Y.Abdel-Mawla,MD zagazig 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

Other functional domains : 

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

SMAD-family - a logic resembling the STAT-family : 

34 M.Y.Abdel-Mawla,MD zagazig SMAD-family - a logic resembling the STAT-family

SMAD family : 

M.Y.Abdel-Mawla,MD zagazig 35 SMAD family

SMAD-family - a logic resembling the STAT-family : 

36 M.Y.Abdel-Mawla,MD zagazig SMAD-family - a logic resembling the STAT-family The Smad-factors mediate response to TGFb-related growth- and differentiation factors STAT-related logic Membrane-bound receptors (such as the TGFß-receptor) are activated by binding of ligand (TGFb). 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

TGFb effectors : 

37 M.Y.Abdel-Mawla,MD zagazig TGFb effectors Latent cytoplasmic TFs activated by serine phosphorylation at their cognate receptors This family transduces signals from the transforming growth factor-b (TGF-b) superfamily of ligands.

Classification : 

38 M.Y.Abdel-Mawla,MD zagazig 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

SMAD-signalling pathway : 

39 M.Y.Abdel-Mawla,MD zagazig SMAD-signalling pathway

Three groups of SMADs : 

40 M.Y.Abdel-Mawla,MD zagazig 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-b pathway. Second group: regulatory or co-SMADs (common SMADs). There are two regulatory SMADs: Smad4 and Smad4b (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.

SMAD-signalling pathway : 

41 M.Y.Abdel-Mawla,MD zagazig SMAD-signalling pathway

Final steps - target gene activation : 

42 M.Y.Abdel-Mawla,MD zagazig 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.

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

43 M.Y.Abdel-Mawla,MD zagazig The Smad-factors activate their target genes in combination with other TFs

Several signalling pathways linked : 

44 M.Y.Abdel-Mawla,MD zagazig 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 TyrP 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

Cross Talks : 

45 M.Y.Abdel-Mawla,MD zagazig Cross Talks P P P P P P P P JAK SH2 Cytokine receptor 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

Variations in mechanisms of STAT activation : 

46 M.Y.Abdel-Mawla,MD zagazig Variations in mechanisms of STAT activation

STAT3 Activation : 

47 M.Y.Abdel-Mawla,MD zagazig 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.

Mechanism of Stat3 signaling : 

48 M.Y.Abdel-Mawla,MD zagazig 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

Psoriasis : 

49 M.Y.Abdel-Mawla,MD zagazig Psoriasis

Psoriasis : 

50 M.Y.Abdel-Mawla,MD zagazig 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

Psoriasis Clinical Presentation : 

51 M.Y.Abdel-Mawla,MD zagazig Psoriasis Clinical Presentation

Psoriasis as an immune disorder : 

52 M.Y.Abdel-Mawla,MD zagazig Psoriasis as an immune disorder

T-Cell in Psoriasis : 

53 M.Y.Abdel-Mawla,MD zagazig T-Cell in Psoriasis

Immunomodulator(Alefacet) effect on Psorasis Area Score Index(PASI) : 

54 M.Y.Abdel-Mawla,MD zagazig Immunomodulator(Alefacet) effect on Psorasis Area Score Index(PASI)

ANTI –Tumor Necrosis Factor (TNF) in Psoriasis : 

55 M.Y.Abdel-Mawla,MD zagazig ANTI –Tumor Necrosis Factor (TNF) in Psoriasis

Slide 56: 

56 M.Y.Abdel-Mawla,MD zagazig

Slide 57: 

57 M.Y.Abdel-Mawla,MD zagazig

Slide 58: 

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

Slide 59: 

59 M.Y.Abdel-Mawla,MD zagazig

Stat3 in keratinocytes(KC) : 

60 M.Y.Abdel-Mawla,MD zagazig Stat3 in keratinocytes(KC)

Prepsoriasis State : 

61 M.Y.Abdel-Mawla,MD zagazig Prepsoriasis State

Prepsoriasis State : 

62 M.Y.Abdel-Mawla,MD zagazig Prepsoriasis State

Immunopathology of Psoriasis : 

63 M.Y.Abdel-Mawla,MD zagazig Immunopathology of Psoriasis

Stat3 links activated keratinocytes and immunocytes required for development of psoriasis : 

64 M.Y.Abdel-Mawla,MD zagazig 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.

Slide 65: 

65 M.Y.Abdel-Mawla,MD zagazig Blocking the function of STAT3 using antisense oligo-nucleotides inhibited the onset of, and reversed, established psoriatic lesions. Further analysis revealed a dual requirement of both activated STAT3 in keratinocytes as well as in T cells, indicating that the pathogenesis of psoriasis is rooted in a co-operative process involving STAT3-regulated genes in both skin cells and the immune system . Phosphatyrosyl peptides block STAT3-mediated DNA binding activity, gene regulation and cell transformation. (Varadwaj et al 2010 Egyptian Dermatology Online Journal 6 (1) )

New Informations : 

66 M.Y.Abdel-Mawla,MD zagazig New Informations

Slide 67: 

67 M.Y.Abdel-Mawla,MD zagazig

Development of Psoriasis : 

68 M.Y.Abdel-Mawla,MD zagazig Development of Psoriasis

Manipulation of Psorasis : 

69 M.Y.Abdel-Mawla,MD zagazig Manipulation of Psorasis

Do Other Therapies Work Within This Framework? : 

70 M.Y.Abdel-Mawla,MD zagazig 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

Slide 71: 

71 M.Y.Abdel-Mawla,MD zagazig

Studies on Stat 3 in Psoriasis : 

72 M.Y.Abdel-Mawla,MD zagazig Studies on Stat 3 in Psoriasis

Stat 3 in Psoriasis : 

73 M.Y.Abdel-Mawla,MD zagazig Stat 3 in Psoriasis

Slide 74: 

74 M.Y.Abdel-Mawla,MD zagazig

Slide 75: 

75 M.Y.Abdel-Mawla,MD zagazig

New Prospectives &Directions : 

76 M.Y.Abdel-Mawla,MD zagazig New Prospectives &Directions

Slide 77: 

77 M.Y.Abdel-Mawla,MD zagazig

Message Home : 

78 M.Y.Abdel-Mawla,MD zagazig Message Home

THANK YOU : 

79 M.Y.Abdel-Mawla,MD zagazig THANK YOU