Stat & psoriasis by yousry

1M.Y.Abdel-Mawla,MD zagazig

Signal Transducer Signal Transducer &Activator of &Activator of Transcription( STAT)-Transcription( STAT)-3 :Role in Psoriasis3 :Role in Psoriasis

Prof. M.YOUSRY ABDEL-Prof. M.YOUSRY ABDEL-MAWLA,MDMAWLA,MDZAGAZIG FACULTY OF MEDICINE,EGYPTZAGAZIG FACULTY OF MEDICINE,EGYPT

M.Y.Abdel-Mawla,MD zagazig

2

INTRODUCTIONINTRODUCTION


SignalSignal transducerstransducers andand activatorsactivators ofof transcriptiontranscription) )

Stat) FamilyStat) Family StatsStats areare latentlatent inin thethe cytoplasmcytoplasm untiluntil

theythey are are activatedactivated byby extracellularextracellular signalingsignaling ligandsligands, , includingincluding cytokinescytokines, , growthgrowth factorsfactors andand hormones.hormones.

Binding of these extracellular ligands to Binding of these extracellular ligands to the specific receptors leads to activation of the specific receptors leads to activation of various various tyrosine kinases (TKs). tyrosine kinases (TKs).

They include JAKs, receptor TKs, and non-They include JAKs, receptor TKs, and non-receptor TKs such as Src and ABL, which receptor TKs such as Src and ABL, which can directly phosphorylate Stat proteins in can directly phosphorylate Stat proteins in the absence of ligand-induced receptor the absence of ligand-induced receptor signaling signaling



• 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


STAT 1

STAT 2

STAT 3

STAT 4

STAT 5

STAT 6

SH2DNAHelix750

851

DNA SH2Helix770

SH2DNAHelix748

SH2DNAHelix734

SH2DNAHelix848

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


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.


• 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.


Ang IIAng IINH2

NUCLEUS

c-fos

YIPP COOH

STAT 1 /

Fyn +

SHP-2 + JAK2

SHP-2

JAK2P

STAT 1 /

STAT1 /

STAT1 /P

P

STAT1 /

STAT1 /P

P

FynP

P

SHP-2

MKP-1

STAT 1 /

P P

Fyn

PP

JAK2-STAT1 activation


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)



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

FUNCTIONSFUNCTIONS

Cytokines receptor signaling moleculesCytokines receptor signaling molecules biological functionbiological function

IL-2 IL-2R Jak1/3, Stat5/3IL-2 IL-2R Jak1/3, Stat5/3 T cell T cell growth and peripheral growth and peripheral tolerance; NK activity; B cell tolerance; NK activity; B cell differentiationdifferentiation

IL-4IL-4 IL-4R Jak1/3, Stat6 IL-4R Jak1/3, Stat6 Th2 Th2 differentiation; B cell differentiation; B cell differentiationdifferentiation

IL-7IL-7 IL-7R Jak1/3,sata5/3 IL-7R Jak1/3,sata5/3 T-cell T-cell homeostasis; T and B homeostasis; T and B development (mouse)development (mouse)

IL-9IL-9 IL-9R Jak1/3, Stat5/3 IL-9R Jak1/3, Stat5/3 Airway Airway mucus production; mucus production; mast cell proliferationmast cell proliferation

IL-15IL-15 IL-15R Jak1/3, Stat5/3 IL-15R Jak1/3, Stat5/3 CD8 CD8 homeostasis, NK cell homeostasis, NK cell developmentdevelopment

IL-21IL-21 IL-21R Jak1/3, Stat3,5,1 IL-21R Jak1/3, Stat3,5,1 B-cell B-cell differentiation; T and differentiation; T and NK activationNK activation



STAT3 ActivationSTAT3 Activation Signal transducer and activator of transcription 3 (Stat3) is a Signal transducer and activator of transcription 3 (Stat3) is a

latent latent cytoplasmic protein that conveys signals to the nucleus upon cytoplasmic protein that conveys signals to the nucleus upon stimulation with IL-6, EGF, and many other cytokines/growth stimulation with IL-6, EGF, and many other cytokines/growth factors, factors, leading to transcriptional activation of the downstream genes. leading to transcriptional activation of the downstream genes.

Stat3 is activated by cytokines of the IL-6 family such as IL-6, Stat3 is activated by cytokines of the IL-6 family such as IL-6, IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic IL-11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), oncostatin M and cardiotropin I .factor (CNTF), oncostatin M and cardiotropin I .

Stat3 is the major signal transducer downstream of Stat3 is the major signal transducer downstream of gp130-like receptorsgp130-like receptors . .

Other extracellular signaling ligands such as IL-10 family Other extracellular signaling ligands such as IL-10 family members, epidermal growth factor (EGF), platelet derived members, epidermal growth factor (EGF), platelet derived growth factor (PDGF), hepa- tocyte growth factor (HGF), growth factor (PDGF), hepa- tocyte growth factor (HGF), granulocyte colony- stimulating factor (G-CSF) and leptin granulocyte colony- stimulating factor (G-CSF) and leptin have also known to activate Stat3. have also known to activate Stat3.




Mechanism of Stat3 Mechanism of Stat3 signalingsignaling

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


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

migrationmigration..


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

These families not present in fungi or plants, hintingat an important evolutionary divergence leading to animals.


STATs - a signal responsive TF STATs - a signal responsive TF familyfamily

STATs: Signal Transducers and STATs: Signal Transducers and Activators of TranscriptionActivators of Transcriptiontwo functions given in the nametwo functions given in the name

1. Transducers for signals from many 1. Transducers for signals from many cytokinescytokinesBroad spectrum of biological effectsBroad spectrum of biological effects

2. Transcriptional activators2. Transcriptional activatorscharacteristic activation mechanismcharacteristic activation mechanismactivation at the cell membrane, activation at the cell membrane,

response in the nucleusresponse in the nucleus

Rapid signal responseRapid signal responseThe activation/deactivation cycle The activation/deactivation cycle of of

STAT molecules is quite short, STAT molecules is quite short, about 15 min for an individual about 15 min for an individual molecule. molecule.


Simple signalling pathwaySimple signalling pathway


REGULATION of Cell REGULATION of Cell SignalingSignaling


The JAK-STAT signalling The JAK-STAT signalling pathwaypathway

Function: regulation of gene expression in response to cytokinesFunction: regulation of gene expression in response to cytokines1. cytokines bind and aggregate the cytokine receptors in the 1. cytokines bind and aggregate the cytokine receptors in the

cell membranecell membrane2. associated JAK-type tyrosine kinases are activated by 2. associated JAK-type tyrosine kinases are activated by

aggregation and tyrosine-phosphorylates neighbouring-JAK aggregation and tyrosine-phosphorylates neighbouring-JAK (transphosphorylation) as well as the C-terminal tail of the (transphosphorylation) as well as the C-terminal tail of the receptor (multiple sites)receptor (multiple sites)

3. Tyr-phosphates recruit inactive STAT-factors in the 3. Tyr-phosphates recruit inactive STAT-factors in the cytoplasm which are bound through their SH2-domainscytoplasm which are bound through their SH2-domains

4. STATs become tyrosine-phosphorylated by JAK4. STATs become tyrosine-phosphorylated by JAK5. phosphorylated STATs dissociate, dimerize (homo-/hetero-) 5. phosphorylated STATs dissociate, dimerize (homo-/hetero-)

and migrate to the nucleusand migrate to the nucleus6. STAT-dimers bind DNA and activates target genes6. STAT-dimers bind DNA and activates target genes


Canonical JAK–STAT Canonical JAK–STAT pathwaypathway

Sequential tyrosine Sequential tyrosine phosphorylations phosphorylations Receptor dimerization allows Receptor dimerization allows

transphosphorylation and transphosphorylation and activation of Janus kinases activation of Janus kinases (JAKs). (JAKs).

This is followed by This is followed by phosphorylation of receptor tails phosphorylation of receptor tails and the recruitment of the STAT and the recruitment of the STAT proteins through their SH-2 proteins through their SH-2 domains. STAT tyrosine domains. STAT tyrosine phosphorylation then occurs. phosphorylation then occurs.

Dimerization of activated Dimerization of activated (tyrosine phosphorylated) STAT is (tyrosine phosphorylated) STAT is followed by nuclear entry.followed by nuclear entry.


IFN-response: two variantsIFN-response: two variants Signalling pathway first discovered in studies Signalling pathway first discovered in studies

of interferon-response (IFN)of interferon-response (IFN)

IFNIFN// IFNIFN// activation of Jak1+Tyk2 activation of Jak1+Tyk2 DNA-binding DNA-binding

complexes (trimer: STAT1+STAT2+p48, together complexes (trimer: STAT1+STAT2+p48, together designated ISGF3) designated ISGF3) activation of target genes activation of target genes with ISRE (IFN-stimulated response element)with ISRE (IFN-stimulated response element)

IFNIFN IFNIFN activation of Jak1+Jak2 activation of Jak1+Jak2 DNA-binding DNA-binding

complex (dimer: 2x STAT1) complex (dimer: 2x STAT1) activation of target activation of target genes having GAS elements (IFNgenes having GAS elements (IFN activated activated sequence)sequence)


IFN-response: IFN-response: two variantstwo variants


STAT-family membersSTAT-family members STAT1 STAT1 - - involved in IFNinvolved in IFN//- and IFN- and IFN-response-response STAT2STAT2 - - involved in IFNinvolved in IFN//-response-response

Mainly acting as partner for STAT1/p48Mainly acting as partner for STAT1/p48 STAT3STAT3 - - involved in response to several cytokines including involved in response to several cytokines including

IL6. It activates several genes involved in acute phase IL6. It activates several genes involved in acute phase responseresponse Important in growth regulation, embryonic development & Important in growth regulation, embryonic development &

organogenesisorganogenesis Activation of STAT3 correlated with cell growth, link to cancer, bind c-Activation of STAT3 correlated with cell growth, link to cancer, bind c-

JunJun STAT4STAT4 - - involved in IL12-responseinvolved in IL12-response STAT5a & 5bSTAT5a & 5b - - involved in response to several cytokinesinvolved in response to several cytokines

including prolactin, IL-2, and regulates expression of milk including prolactin, IL-2, and regulates expression of milk proteins in breast tissue in response to prolactinproteins in breast tissue in response to prolactin

STAT6STAT6 - - involved in IL4-responseinvolved in IL4-response non-mammalian family membersnon-mammalian family members (e.g. Drosophila (e.g. Drosophila))


STAT-membersSTAT-members

SH2 Y


STAT-STAT interaction occurs STAT-STAT interaction occurs through reciprocal phospho-through reciprocal phospho-

Tyr - SH2 interactionsTyr - SH2 interactions SH2-domainSH2-domain

SH2 = “Src-homology domain 2”SH2 = “Src-homology domain 2” function: phospho-tyrosine bindingfunction: phospho-tyrosine binding Three important functions in Three important functions in

STATs:STATs: important for recruitment of STAT important for recruitment of STAT

to receptorto receptor important for interaction with the important for interaction with the

JAK kinaseJAK kinase important for dimerization of important for dimerization of

STATs to an active DNA-binding STATs to an active DNA-binding formform

Tyr-701Tyr-701 conserved key Tyr residue located conserved key Tyr residue located

just C-terminal to SH2just C-terminal to SH2 essensiell for dimerdannelse to an essensiell for dimerdannelse to an

active DNA-binding formactive DNA-binding form function: Tyrfunction: TyrPP bindingssted for bindingssted for

SH2 in partnerSH2 in partner

YP

YP

Y

Y

+


Dimerization via SH2-TyrDimerization via SH2-TyrPP

TyrP from the left monomer SH2 from the right monomer


STAT-membersSTAT-members

SH2 Y


STATs - structure and STATs - structure and functionfunction

DimerizationDimerization Reciprocal SH2- TyrReciprocal SH2- TyrPP interaction interaction HomodimersHomodimers

(STAT1)(STAT1)22

HeterodimersHeterodimers STAT1-STAT2STAT1-STAT2 STAT1-STAT3STAT1-STAT3

DNA-binding domainDNA-binding domain DBD located in the middle of the proteinDBD located in the middle of the protein Unique motif - se next slideUnique motif - se next slide All DBDs bind similar motifs in DNAAll DBDs bind similar motifs in DNA

symmetric inverted half sitessymmetric inverted half sites Only difference to STATs: preference for central nucleotideOnly difference to STATs: preference for central nucleotide

GAS= TTN5-6AA

ISRE= AGTTTN3TTTCC


STAT-STRUCTURESTAT-STRUCTURE Known structuresKnown structures

[STAT1][STAT1]22-DNA and [STAT3-DNA and [STAT3]]22-DNA, as well as an N--DNA, as well as an N-terminal of STAT4terminal of STAT4

Characteristic feature :Characteristic feature : Symmetry-axis through DNA, each monomer Symmetry-axis through DNA, each monomer

contacts a separate half sitecontacts a separate half site structure resembles NFstructure resembles NFB and p53 B and p53

(immunoglobuline fold). The dimer forms a C-(immunoglobuline fold). The dimer forms a C-shaped ”clamp” around DNA.shaped ”clamp” around DNA.

The dimer is kept together by reciprocal SH2- TyrThe dimer is kept together by reciprocal SH2- TyrPP interactions between the SH2 domain in one interactions between the SH2 domain in one monomer and the phosphorylated Tyr in the other.monomer and the phosphorylated Tyr in the other.

The SH2 domain in each monomer is closely linked The SH2 domain in each monomer is closely linked to the core DBD and is itself close to DNA, and is to the core DBD and is itself close to DNA, and is assumed also to contribute to DNA-binding.assumed also to contribute to DNA-binding.

N-terminal coiled-coil region not close to DNA, N-terminal coiled-coil region not close to DNA, probably involved in prot-prot interaction with probably involved in prot-prot interaction with flexible positionflexible position


3D3D

STAT STAT domain domain structurstructure and e and protein protein binding binding sites.sites.


Promoter recognition and Promoter recognition and selectivityselectivity Mechanisms to achieve specific trx Mechanisms to achieve specific trx

responses.responses. Inverted repeat TTNInverted repeat TTN5–65–6AA motif AA motif

common. Binding specificity to common. Binding specificity to individual elements based on evolved individual elements based on evolved preferences for specific positions. preferences for specific positions.

In the ISGF3 heterotrimeric complex, In the ISGF3 heterotrimeric complex, STAT1–STAT2 heterodimers bind to a STAT1–STAT2 heterodimers bind to a third protein, p48/ISGF3third protein, p48/ISGF3, a TF that , a TF that recognizes the ISRE sequence. recognizes the ISRE sequence.

STAT N-domains mediate dimer–STAT N-domains mediate dimer–dimer interactions allowing high-dimer interactions allowing high-avidity binding to tandemly arranged avidity binding to tandemly arranged low-affinity GAS elements. low-affinity GAS elements.

Adjacent response elements bind to Adjacent response elements bind to other TFs. Cooperativity and synergy.other TFs. Cooperativity and synergy.

STAT directly recruit co-activators STAT directly recruit co-activators that alter chromatin dynamics. that alter chromatin dynamics.


Transactivation Transactivation Domain(TAD)Domain(TAD)

Transactivation domainTransactivation domain C-terminal part of the protein, less conservedC-terminal part of the protein, less conserved variants generated by alternative splicing + variants generated by alternative splicing +

proteolysisproteolysis STAT1STAT1 lacking the last 38aa has all functions retained lacking the last 38aa has all functions retained

except transactivationexcept transactivation Regulation through TAD-modificationRegulation through TAD-modification

Activity of TAD is regulated through Ser Activity of TAD is regulated through Ser phosphorylation (LPMSP-motif)phosphorylation (LPMSP-motif)

Ser727 in STAT1Ser727 in STAT1 Kinase not identified - candidates: p38, ERK, JNKKinase not identified - candidates: p38, ERK, JNK A role in recruitment of GTF/coactivatorA role in recruitment of GTF/coactivator Proteins identified that bind TAD in a Ser-dependent Proteins identified that bind TAD in a Ser-dependent

mannermanner MCM5MCM5 BRCA1BRCA1

TAD in STAT2 binds C/H-rich region of CBPTAD in STAT2 binds C/H-rich region of CBP STAT2 carries the principal TAD of the ISGF3-complexSTAT2 carries the principal TAD of the ISGF3-complex


Other functional domainsOther functional domains The N-domain is important for The N-domain is important for

stabilizing interactions between stabilizing interactions between STAT dimers, bound to tandemly STAT dimers, bound to tandemly arranged response elementsarranged response elements


PsoriasisPsoriasis


PsoriasisPsoriasis

Psoriasis is a chronic inflammatory skin Psoriasis is a chronic inflammatory skin disease characterized by excessive disease characterized by excessive proliferation, abnormal differentiation of proliferation, abnormal differentiation of epidermal keratinocytes, vascular epidermal keratinocytes, vascular proliferation, and leukocyte infiltration in proliferation, and leukocyte infiltration in the dermis and epidermis .the dermis and epidermis .

It has been considered that psoriasis It has been considered that psoriasis results from complex, aberrant results from complex, aberrant relationships between the skin and relationships between the skin and immune system immune system as well as genetic as well as genetic predisposition and environmental factorspredisposition and environmental factors


Psoriasis Clinical Psoriasis Clinical PresentationPresentation


Psoriasis as an immune Psoriasis as an immune disorderdisorder


T-Cell in PsoriasisT-Cell in Psoriasis


Immunomodulator(Alefacet) Immunomodulator(Alefacet) effect on Psorasis Area Score effect on Psorasis Area Score

Index(PASI)Index(PASI)


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




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


The in vivo role for Stat3 in the skin was The in vivo role for Stat3 in the skin was elucidated elucidated using keratinocyte-specific Stat3 gene knockout using keratinocyte-specific Stat3 gene knockout mice, referred to as Stat3-disrutped mice.mice, referred to as Stat3-disrutped mice.

It was shown that Stat3 activation contributed to It was shown that Stat3 activation contributed to skin wound healing, keratinocyte migration, hair skin wound healing, keratinocyte migration, hair follicle growth, and resistance to UV follicle growth, and resistance to UV irradiation-induced apoptosis. irradiation-induced apoptosis.

Stat3-disrupted mice did not develop any skin Stat3-disrupted mice did not develop any skin tumors. In contrast, transgenic mice with tumors. In contrast, transgenic mice with a constitutive active form of Stat3 (K5.Stat3C a constitutive active form of Stat3 (K5.Stat3C mice) developed squamous cell carcinoma (SCC) mice) developed squamous cell carcinoma (SCC) with a shorter latency and in much greater with a shorter latency and in much greater number compared to control mice. K5.Stat3C number compared to control mice. K5.Stat3C mice were found to be psoriasis-prone. mice were found to be psoriasis-prone.


A role for Stat3 protein has been A role for Stat3 protein has been suggested not suggested not only in early stages of skin carcinogenesis only in early stages of skin carcinogenesis but also in driving malignant progression but also in driving malignant progression in vivo. in vivo.

Moreover, Stat3 was consistently Moreover, Stat3 was consistently activated in epidermal keratinocytes in activated in epidermal keratinocytes in human psoriatic lesions, which has been human psoriatic lesions, which has been assumed to recapitulate a condition of assumed to recapitulate a condition of persistent wound healing reaction.persistent wound healing reaction.

Finally, it was demonstrated that an Finally, it was demonstrated that an inhibition of Stat3 activation inhibition of Stat3 activation ameliorated these pathological ameliorated these pathological conditions, i.e., skin carcinogenesis and conditions, i.e., skin carcinogenesis and psoriasis. psoriasis.



Stat3 in keratinocytes(KC)Stat3 in keratinocytes(KC)


Prepsoriasis StatePrepsoriasis State


Prepsoriasis StatePrepsoriasis State


Immunopathology of Immunopathology of PsoriasisPsoriasis


Stat3 links activated keratinocytes Stat3 links activated keratinocytes and immunocytes required for and immunocytes required for

development of psoriasisdevelopment of psoriasis Epidermal keratinocytes in psoriatic lesions are Epidermal keratinocytes in psoriatic lesions are

characterized by activated Stat3. characterized by activated Stat3. Transgenic mice with keratinocytes expressing a Transgenic mice with keratinocytes expressing a

constitutively active Stat3 (constitutively active Stat3 (K5.Stat3C miceK5.Stat3C mice) develop a ) develop a skin phenotype either spontaneously, or in response to skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis.wounding, that closely resembles psoriasis.

Keratinocytes from K5.Stat3C mice show upregulation Keratinocytes from K5.Stat3C mice show upregulation of several molecules linked to the pathogenesis of of several molecules linked to the pathogenesis of psoriasis.psoriasis.

In addition, the development of psoriatic lesions in In addition, the development of psoriatic lesions in K5.Stat3C mice requires cooperation between Stat3 K5.Stat3C mice requires cooperation between Stat3 activation in keratinocytes and activated T cells. activation in keratinocytes and activated T cells.

Finally, abrogation of Stat3 function by a decoy Finally, abrogation of Stat3 function by a decoy oligonucleotide inhibits the onset and reverses oligonucleotide inhibits the onset and reverses established psoriatic lesions in K5.Stat3C mice. established psoriatic lesions in K5.Stat3C mice. Thus, Thus, targeting Stat3targeting Stat3 may be potentially therapeutic in the may be potentially therapeutic in the treatment of psoriasis.treatment of psoriasis.


New InformationsNew Informations



Development of PsoriasisDevelopment of Psoriasis


Manipulation of PsorasisManipulation of Psorasis


Do Other Therapies Work Within This Do Other Therapies Work Within This Framework?Framework?

Anti–T-cell agents could affect Th17 Anti–T-cell agents could affect Th17 cells as they would other T cells, cells as they would other T cells, but this needs to be clarified but this needs to be clarified

Anti-TNF agents could decrease Anti-TNF agents could decrease activity of Th17 cells or work activity of Th17 cells or work directly on keratinocyte responsesdirectly on keratinocyte responses



Studies on Stat 3 in Studies on Stat 3 in PsoriasisPsoriasis


Stat 3 in PsoriasisStat 3 in Psoriasis




New Prospectives New Prospectives &Directions&Directions



68

Tyrosine kinasesTyrosine kinases


The JAK-family of tyrosine The JAK-family of tyrosine kinaseskinases

Family membersFamily members JAK1 (135 kDa)JAK1 (135 kDa) JAK2 (130 kDa)JAK2 (130 kDa) JAK3 (120 kDa)JAK3 (120 kDa) Tyk2 (140 kDa)Tyk2 (140 kDa)

Common featureCommon feature C-terminal kinase + pseudokinaseC-terminal kinase + pseudokinase ≠ ≠ RTK by lacking transmembrane domains and SH2, SH3, PTB, PHRTK by lacking transmembrane domains and SH2, SH3, PTB, PH several regions homologous between JAK-membersseveral regions homologous between JAK-members Associated with cytokine receptors (type in and II)Associated with cytokine receptors (type in and II)

FunctionFunction Associated with cytokine receptors in non-stimulated cells in an Associated with cytokine receptors in non-stimulated cells in an

inactive forminactive form


The role of the kinases in the The role of the kinases in the signalling pathwaysignalling pathway

INF-signalling INF-signalling


The cytokine-receptor The cytokine-receptor superfamilysuperfamily

A receptor-family that mediates response to A receptor-family that mediates response to more than 30 different cytokinesmore than 30 different cytokines

Common feature: conserved extracellular Common feature: conserved extracellular ligand-binding domainligand-binding domain

Are associated with tyrosine-kinases in the Are associated with tyrosine-kinases in the JAK-familyJAK-family

Ligand-binding Ligand-binding Receptor dimerization or Receptor dimerization or oligomerization leads to JAK apposition oligomerization leads to JAK apposition associated JAK Tyr kinases are activated associated JAK Tyr kinases are activated transphosphorylation of neighbour-JAKs transphosphorylation of neighbour-JAKs tyrosine-phosphorylation of C-terminal tail of tyrosine-phosphorylation of C-terminal tail of receptors on multiple sites receptors on multiple sites several cellular several cellular substrate-proteins associate (including STATs) substrate-proteins associate (including STATs) multiple signalling pathways are activated multiple signalling pathways are activated


The role of the kinases in the The role of the kinases in the signalling pathwaysignalling pathway

INF-signalling INF-signalling


Specificity in responseSpecificity in response Specific cytokines activate distinct STATs and lead to a specific Specific cytokines activate distinct STATs and lead to a specific

response - what mediate specificity?response - what mediate specificity? each cytokine activates a subgroup STATeach cytokine activates a subgroup STAT some cytokines activate only one specific STATsome cytokines activate only one specific STAT

One contribution: the SH2 - receptor interaction specific for One contribution: the SH2 - receptor interaction specific for certain combinationscertain combinations swaps-experiments of SH2 between STATs change specificityswaps-experiments of SH2 between STATs change specificity affinity of the SH2-receptor interaction is affected by the sequence affinity of the SH2-receptor interaction is affected by the sequence

context of the Tyrcontext of the Tyr

Another contribution: different STAT-dimers bind different Another contribution: different STAT-dimers bind different response elements in the genome and turn on different genesresponse elements in the genome and turn on different genes

STAT1 knock-out mice illustrate biological specificitySTAT1 knock-out mice illustrate biological specificity STAT1STAT1-/- -/- phenotype: total lack of IFN-response phenotype: total lack of IFN-response highly sensitive to highly sensitive to

virus-infectionvirus-infection


Several signalling pathways Several signalling pathways linkedlinked

STATs may also be Tyr-phosphorylated and hence activated by other receptor STATs may also be Tyr-phosphorylated and hence activated by other receptor familiesfamilies receptor tyrosine kinases (RTKs) such as EGF-receptor may phosphorylate receptor tyrosine kinases (RTKs) such as EGF-receptor may phosphorylate

STATsSTATs EGF stimulation EGF stimulation activation of STAT1, STAT3 activation of STAT1, STAT3

non-receptor tyrosine kinases such as Src and Abl may also phosphorylate non-receptor tyrosine kinases such as Src and Abl may also phosphorylate STATs (?)STATs (?)

G-protein coupled 7TMS receptors such as angiotensine receptor (?)G-protein coupled 7TMS receptors such as angiotensine receptor (?) STAT may also be modified by Ser-phosphorylationSTAT may also be modified by Ser-phosphorylation

DNA-binding reduced (STAT3)DNA-binding reduced (STAT3) Transactivationdomain Ser-phosphorylated (important for transactivation in Transactivationdomain Ser-phosphorylated (important for transactivation in

STAT1 and STAT3)STAT1 and STAT3) Responsible kinases not identified - MAPkinases candidates, probably also Responsible kinases not identified - MAPkinases candidates, probably also

othersothers JAKs may activate other signalling pathways than STATsJAKs may activate other signalling pathways than STATs

TyrTyrPP will recruit several protein-substrates and lead to phosphorylation and will recruit several protein-substrates and lead to phosphorylation and activation of other signalling pathwaysactivation of other signalling pathways

e.g. JAK activation e.g. JAK activation activation of MAP-kinases activation of MAP-kinases e.g. substrates: IRS-1, SHC, Grb2, HCP, Syp, Vave.g. substrates: IRS-1, SHC, Grb2, HCP, Syp, Vav


Cross TalksCross Talks

Y

PP

PP

P

P

P

P

JAK

SH2YY

YCytokinereceptor

P

P

P

Receptortyrosineekinase

MAPK

Alternative inputsAlternative inputs STATs may be STATs may be

Tyr-Tyr-phosphorylated phosphorylated by RTKsby RTKs

Alternative Alternative outputsoutputs JAK may JAK may

phosphorylate phosphorylate other targets and other targets and thus activate thus activate signal signal transduction transduction pathways other pathways other than through than through STATsSTATs


Variations in mechanisms of Variations in mechanisms of STAT activationSTAT activation


77

SMAD familySMAD family


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

familyfamily The Smad-factors mediate response to The Smad-factors mediate response to

TGFTGF-related growth- and -related growth- and differentiation factorsdifferentiation factors

STAT-related logicSTAT-related logic Membrane-bound receptors (such as the Membrane-bound receptors (such as the

TGFß-receptor) are activated by binding of TGFß-receptor) are activated by binding of ligand (TGFligand (TGF). The receptors here are ). The receptors here are transmembrane serine/threonine-kinasestransmembrane serine/threonine-kinases

Activated kinases phosphorylate specific Activated kinases phosphorylate specific Smad-factorsSmad-factors

phosphorylated Smad-factors associate with phosphorylated Smad-factors associate with a common Smad-factor (Smad4)a common Smad-factor (Smad4)

The generated heteromeric complexes The generated heteromeric complexes migrate to the nucleus as transcription migrate to the nucleus as transcription factorsfactors


TGFTGF effectors effectors

Latent cytoplasmic Latent cytoplasmic TFs activated by TFs activated by serine serine phosphorylation at phosphorylation at their cognate their cognate receptorsreceptors

This family This family transduces signals transduces signals from the transforming from the transforming growth factor-growth factor- (TGF- (TGF-) superfamily of ) superfamily of ligandsligands..


ClassificationClassification Smad-factors - design and Smad-factors - design and

classificationclassification Nine different Smad-factors Nine different Smad-factors

identified in vertebratesidentified in vertebrates common conserved domains: N-common conserved domains: N-

terminalt MH1-domain (DBD) + C-terminalt MH1-domain (DBD) + C-terminalt MH2-domainterminalt MH2-domain

Can be divided into three groupsCan be divided into three groups 1. Receptor-activated Smad-1. Receptor-activated Smad-

factors - become phosphorylated factors - become phosphorylated by activated receptors in their C-by activated receptors in their C-terminal (SSXS)terminal (SSXS)

2. common Smad-factors 2. common Smad-factors associated with activated Smad-associated with activated Smad-factors and participate in several factors and participate in several signalling pathwayssignalling pathways

3. Inhibitoriske Smad-factors 3. Inhibitoriske Smad-factors


SMAD-signalling pathwaySMAD-signalling pathway

Effector SMADs(R-SMADs)

Co-SMADs Repressor SMADs


Three groups of SMADsThree groups of SMADs First group: The effector SMADs (also called the R-SMADs) First group: The effector SMADs (also called the R-SMADs)

become serine-phosphorylated in the C-terminal domain by the become serine-phosphorylated in the C-terminal domain by the activated receptoractivated receptor..

Smad1, Smad5, Smad8, and Smad9 become phosphorylated in Smad1, Smad5, Smad8, and Smad9 become phosphorylated in response to bone morphogenetic morphogenetic protein (BMP) response to bone morphogenetic morphogenetic protein (BMP) and growth and differentiation factor (GDF), and Smad2 and and growth and differentiation factor (GDF), and Smad2 and Smad3 become phosphorylated in response to the activin/nodal Smad3 become phosphorylated in response to the activin/nodal branch of the TGF-branch of the TGF- pathway pathway..

Second group: regulatory or co-SMADs (common SMADsSecond group: regulatory or co-SMADs (common SMADs). ). There are two regulatory SMADs: Smad4 and Smad4There are two regulatory SMADs: Smad4 and Smad4 (also (also

called Smad10)called Smad10) . .Smad4 binds to, and is essential for, the function of Smad1 and Smad4 binds to, and is essential for, the function of Smad1 and

Smad2. The regulatory Smad4 binds to all effector SMADs in Smad2. The regulatory Smad4 binds to all effector SMADs in the formation of transcriptional complexes, butthe formation of transcriptional complexes, but it does not it does not appear to be required for nuclear translocation of the effector appear to be required for nuclear translocation of the effector moleculesmolecules . .

Third group: two inhibitory SMADs, Smad6 and Smad7Third group: two inhibitory SMADs, Smad6 and Smad7..provide negative regulation of the pathway by blocking Smad4 provide negative regulation of the pathway by blocking Smad4

bindingbinding..


SMAD-signalling pathwaySMAD-signalling pathway


Final steps - target gene Final steps - target gene activationactivation

Once an activated, serine-phosphorylated Once an activated, serine-phosphorylated effector SMAD binds Smad4 and escapes effector SMAD binds Smad4 and escapes the negative influences of Smad6 and Smad7, the negative influences of Smad6 and Smad7, nuclear accumulation and regu-lation of specific nuclear accumulation and regu-lation of specific target genes can occur. target genes can occur. In most cases, SMADs require partner In most cases, SMADs require partner

transcription factors with strong DNA binding transcription factors with strong DNA binding capacity that determine the gene to be capacity that determine the gene to be activated. The DNA binding is then activated. The DNA binding is then strengthened by association with SMADs that strengthened by association with SMADs that on their own bind weakly to adjacent DNA on their own bind weakly to adjacent DNA sites. The SMADs furnish transcriptional sites. The SMADs furnish transcriptional activation capacity. activation capacity.

The specificity of response among different The specificity of response among different ligands can be partially explained by the ligands can be partially explained by the choice of DNA binding partner proteins. For choice of DNA binding partner proteins. For example, activin activation of SMADs results in example, activin activation of SMADs results in combinations with FAST1 and a particular set combinations with FAST1 and a particular set of genes is activated. Signaling by BMP of genes is activated. Signaling by BMP ligands results in association of activated ligands results in association of activated SMADs with a DNA binding proteinSMADs with a DNA binding protein called OAZ. called OAZ.


The Smad-factors activate their The Smad-factors activate their target genes in combination target genes in combination

with other TFswith other TFs


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