Abstract

P

W

, M

S

−J

, M

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The Role of Jak/STAT Signaling

in Rheumatoid Arthritis

Układ transmisji sygnału Jak/STAT

– rola w reumatoidalnym zapaleniu stawów

1_{Department of Balneology Silesian Piasts University of Medicine in Wrocław, Poland}

2 _{Department of Rheumatology and Internal Diseases Silesian Piasts University of Medicine in Wrocław, Poland}

Adv Clin Exp Med 2008, 17, 4, 447–452 ISSN 1230−025X

REVIEWS

© Copyright by Silesian Piasts University of Medicine in Wrocław

Abstract

The biological functions of cytokines, which play an important role in the pathogenesis of rheumatoid arthritis, are achieved by activating signal−transduction cascades that influence cellular activation, proliferation, differentiation, and survival. One of the most often utilized signal−transduction pathways is the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway. Jak is a novel family of cytoplasmic tyrosine kinases pre−associat− ed with cytokine receptors and catalyzing STAT activation by phosphorylation. STATs are latent monomeric pro− teins that upon activation serve as transcription factors and are present in the cytoplasm of numerous cell types. The mammalian STAT family comprises seven homologous members: STAT−1, −2, −3, −4, −5A, −5B, and −6, encod− ed by genes located in three chromosomal regions. The role of the STATs in the pathogenesis of rheumatoid arthri− tis is still being elucidated. In recent years the identification of signal−transduction pathways has suggested new therapeutic targets in rheumatoid arthritis. Modulation of STAT expression represents an alternative therapeutic target to cytokine antagonists, and their inhibition will probably prove more powerful than current treatment strate− gies (Adv Clin Exp Med 2008, 17, 4, 447–452).

Key words:rheumatoid arthritis, Jak/STAT, cytokines.

Streszczenie

Biologiczne funkcje cytokin, będących istotnym czynnikiem w patogenezie reumatoidalnego zapalenia stawów, są realizowane przez aktywację układów transmisji sygnału, które wpływają na aktywację, proliferację, różnicowa− nie i przeżycie komórek. Jednym z najczęściej aktywowanych układów transmisji sygnału jest układ kinazy Janus/ transmitera sygnału i czynnika transkrypcyjnego (Jak/STAT). Białka Jak są rodziną cytoplazmatycznych kinaz ty− rozynowych, przyłączonych do cytoplazmatycznej części receptora cytokinowego, które katalizują aktywację bia− łek STAT przez ich fosforylację. STAT są monomerycznymi białkami cytoplazmatycznymi, które po aktywacji słu− żą jako czynniki transkrypcyjne w wielu typach komórek. U ssaków rodzina białek STAT składa się z siedmiu ho− mologicznych białek: STAT−1, −2, −3, −4, −5A, −5B i −6, kodowanych przez geny umiejscowione w trzech regionach chromosomalnych. Znaczenie białek STAT w patogenezie nie zostało w pełni wyjaśnione. W ostatnich latach iden− tyfikacja układów transmisji sygnału stworzyła nowe możliwości badań w dziedzinie terapii reumatoidalnego za− palenia stawów. Modulowanie ekspresji białek STAT może okazać się alternatywą dla antagonistów cytokinowych, a hamowanie układów transmisji sygnału będzie prawdopodobnie dawało większe od obecnych możliwości tera− peutyczne (Adv Clin Exp Med 2008, 17, 4, 447–452).

Słowa kluczowe:reumatoidalne zapalenie stawów, Jak/STAT, cytokiny.

Cytokines have critical functions in regulating immune responses and play an important role in the pathogeneses of autoimmune and inflammato− ry diseases, including rheumatoid arthritis. The protein structure of cytokine molecules prevents

proliferation, differentiation, and survival. One of the most often utilized signal−transduction path− ways is the Janus kinase/signal transducer and activator of transcription (Jak/STAT) pathway.

General Characteristics

of the Jak/STAT Pathway

The Jak/STAT pathway was identified for the first time during studies on factors involved in interferon−dependent gene expression [1]. As a signal−transducing pathway, the Jak/STAT com− plex enables cytokines and growth factors to achieve their cellular effects. The binding of a par− ticular cytokine to its membrane receptor results in Jak activation. Active Jaks activate STATs by their phosphorylation, which is followed by dimeriza− tion of the STAT molecules and their translocation from the cytoplasm to the nucleus, where the inter− ference with DNA takes place.

Janus kinase is the general name for the mem− bers of a novel family of cytoplasmic tyrosine kinases that are pre−associated with membrane− proximal regions (termed box 1 and box 2) of cytokine receptors. The mammalian Jak family consists of only four different Jaks: Jak−1, Jak−2, Jak−3, and TYK−2. The structure of each Jak mole− cule contains three key regions: a catalytically active domain, a regulatory domain, and a receptor− binding domain, which are responsible for proper action during signal transduction [2]. It is now rec− ognized that all type I and II cytokine receptors activate various members of the Jak family [3, 4]. STATs (Signal Transducers and Activators of Transcription) are latent monomeric proteins that

upon activation serve as transcription factors and are present in the cytoplasm of numerous cell types. The mammalian STAT family comprises seven homologous members: STAT−1, −2, −3, −4, −5A, −5B, and −6, encoded by genes located in three chromo− somal regions (2, 12, 17) [5]. The STAT molecule structure is complex and contains a DNA−binding domain, a binding domain for other transcription factors and co−activators, a linker domain, a domain enabling dimer−dimer interactions, an SH2 domain, and a transcription activation domain [6].

The structures of most types of cytokine receptors are similar and consist of three different functional domains [7]. The extracellular domain enables the binding of the cytokine ligand. The transmembrane domain, characterized by hydrophobic properties, is responsible for keeping the whole receptor molecule in its proper position in relation to the membrane surface. The intracel− lular membrane activates the appropriate signal− transducing pathway and is thus responsible for the cellular effects of the particular cytokine.

The Mechanism of Jak/STAT

Signal Transduction

The binding of a cytokine ligand to the cog− nate receptor results in the receptor subunits’ dimerization. Jak molecules, physically associated with the receptor subunits, are therefore brought into close proximity and undergo activation as a result of tyrosine phosphorylation. Normally, the binding of a particular cytokine results in the acti− vation of a distinct pair of Jaks [8]. The signal− transduction cascade is thus initiated (Fig. 1).

Fig 1. Activation of the Jak/STAT pathway by a cytokine molecule. Cytokine ligation results in dimeriza− tion of plasma membrane receptors, activation of JAK kinases, and phos− phorylation of receptor cytoplasmic domain tyrosine residues. Latent cyto− plasmic STAT proteins are recruited to tyrosine residues and phosphorylat− ed, which causes their dimerization, translocation to the nucleus, and gene transcription activation

When Jaks are activated they catalyze the phos− phorylation of receptor subunits, which results in the creation of docking sites for various signaling molecules, including STATs [2, 6]. Monomers of STAT molecules bind to the phosphorylated sub− units via their SH2 domains; distinct STATs bind to particular cytokine receptors. While bound, STATs undergo tyrosine phosphorylation cat− alyzed by Jaks, then separate from the receptor subunits and dimerize. This change in STAT mol− ecule conformation is responsible for the release of a nuclear localizing signal (NLS), which is rec− ognized and bound to the importing protein [9]. The STAT dimer is transferred to the nucleus, where it dissociates from the complex with the importing protein and binds to the selected DNA fragment. STATs bind to two types of DNA motifs: IFN−stimulated response elements (ISREs, con− sensus AGTTTNCNTTTCC) and a γ−activated sequence (GAS elements, consensus TTC− NNNGAA) [6]. In general, different STATs recog− nize closely related DNA sequences [8]. After DNA binding, the STAT molecule undergoes dephosphorylation, what causes dissolution of the STAT−DNA complex. An active nuclear exporting signal (NES) then enables the binding of STAT molecules by exporting a protein called CRM1 and transfer of the complex to the cytoplasm [9].

The Role of Stats

in the Pathogenesis

of Rheumatoid Arthritis

The role of the STATs in the pathogenesis of the rheumatoid arthritis is still being elucidated. The suggested roles of various STATs in the pathogenesis of RA are presented below.

STAT−1

Raised expression of STAT−1 mRNA and STAT−1 protein was found in the synovial tissue of RA patients. Van der Pouw Kraan et al. revealed a higher number of STAT−1−positive cells in syn− ovial biopsy material from RA patients than in the tissues of osteoarthritis (OA) patients [10]. Positive cells were predominantly found in lym− phoid aggregates and intimal lining. Using the PCR technique they observed differences in STAT−1 mRNA expression among high, intermediate, and low gene expression profiles in RA patients. No significant differences in gene expression between the low RA and OA tissues were revealed [10]. An interesting observation was also made for erythro− cyte sedimentation rate (ESR); a statistically sig−

nificant difference was observed between the high RA group and the low and intermediate RA groups (p= 0.008) [10]. Kasperkovitz et al. evaluated the expression of STAT−1 in different synovial cells [11]. Strong positive staining for STAT−1 was mainly found in focal inflammatory infiltrates of B cells and T lymphocytic aggregates in the syn− ovial sublining and in the fibroblast−like synovio− cytes in the intimal layer. Only a limited number of macrophages was found to express STAT−1.

STAT−1 seems to have a dual nature. Its main role is to mediate IFN signaling. Experimentally induced deficiency of STAT−1 results in disability to handle infections, especially those of viral ori− gin. The antiviral and inflammatory effect of STAT−1 is realized by the induction of the major histocompatibility complex (MHC), costimulatory molecules, chemokines, complement, inducible nitric oxide synthase, and others [2]. The non− immune activity of STAT−1 focuses on the regula− tion of bone formation through its influence on osteoclasts and osteoblasts.

On the other hand, STAT−1’s proapoptotic activity is required for growth restraint by IFN−α and IFN−γ and the promotion of apoptosis [11]. Knowledge based on different models of arthritis (including human tissue) indicates its predominant anti−inflammatory activity. It is primarily activated by IFN−γ, but IL−6, IL−10, and IFN−α/βalso con− tribute to its activation [12]. It is known that STAT−1 and STAT−3 have antagonistic function and are both expressed in synovial fibroblasts. One hypoth− esis says that there is no proapoptotic signal from STAT−1 enough strong to inhibit the survival and expansion signal delivered from STAT−3 in syn− ovial fibroblasts. The mentioned dysregulation may contribute to maintaining the inflammatory process in RA.

STAT−3

part, maintaining myc expression and opposing a proapoptotic signal delivered by EGF and other factors [13].

Interleukin 6 (IL−6) is a cytokine of proin− flammatory profile in RA. Significantly elevated concentrations of IL−6 were detected in RA syn− ovial fluid [14]. IL−6 binds to its receptor IL−6Rα subunit (α chain), making a complex which induces the homodimerization of another subunit of the IL−6 receptor, the glycoprotein gp130 (βchain). Activation of gp130 leads to Jak activa− tion (Jak1, Jak2, Tyk2) and receptor phosphoryla− tion and results in the activation of the transcrip− tion factor STAT−3 in synovial fibroblasts. The IL−6−gp130−Jak−STAT−3 signaling cascade upreg− ulates WNT5Agene transcription. The product of the gene, glycoprotein WNT5A, activates canoni− cal WNT signaling through the Frizzled (FZD) receptor for synovial fibroblasts hypertrophy in RA [15]. Sen et al. described the downregulation of WNT5A expression in synovial fibroblasts by using a WNT5A anti−sense construct and the inhi− bition of FZD5 signaling using specific antibody inhibited rheumatoid synovial fibroblast acti− vation [16].

The important role of STAT−3 signaling in RA pathogenesis is supported by data from Shouda et al. showing that the overexpression of SOCS3 (an inhibitor of the Jak/STAT−3 pathway) suppressed experimental arthritis [17].

STAT−4

STAT−4 is activated by IL−12, IL−23, and IFN−α [2] and is down−regulated by IL−4 and IL−10 [18]. It is involved in regulating the differentiation of Th0 into Th1 cells, cell−mediated immune responses, and IFN−γ production. Most normal human peripheral blood leukocytes hardly express STAT−4 in their basal state, but the expression is markedly induced in T cells, macrophages, and dendritic cells following stimulation. In synovial macrophages from patients with rheumatoid arthritis, STAT−4 was also abundantly expressed [18]. STAT−4 was also strongly expressed in den− dritic cells from rheumatoid synovium [12, 19].

STAT−6

STAT−6 is activated by IL−4, IL−13, and, in B cells, by IFN−α[2]. It enables the differentiation of Th0 into Th2 cells, IgE production, and prolif− erative responses to IL−4. The expression and acti− vation of STAT−6 was shown in early and late stages of rheumatoid arthritis in synovial frozen sections using in situ hybridization and immuno− histochemistry [20]. In this study, the most inten−

sive expression of STAT−6 mRNA was seen in fol− licular inflammatory infiltrates, but synoviocytes, fibroblasts, and macrophages also expressed STAT−6 mRNA. However, in another study com− paring rheumatoid with osteo arthritic and normal synovium, there was no difference in STAT−6 pro− tein expression among the tissues [12]. According to this study, the high baseline level of STAT−6 might suggest its important homeostatic role with− in the joint.

Another hypothesis explaining both intense expressions of STAT−4 and STAT−6 in synovial tissue immune cells is based on ineffective anti−inflamma− tory processes. In the later stages of the immune reaction, anti−inflammatory cytokines (such as IL−4 and IL−13) are released, but the reaction lasts. In rheumatoid arthritis, dendritic cells may probably be inappropriately active in the late immune response as a result of inefficient signaling through IL−13. If signaling by IL−13 is inefficient, there will be a relative increase in Jak−3 and STAT−6 as the immune response attempts to downregulate its proinflammatory effects. The absence of successful inhibition will lead to continuous production of IL−12 and increased expression of STAT−4 [19].

Negative Regulation

of the Jak/STAT Pathway

The negative regulation of the Jak/STAT path− way is realized by three main groups of proteins: phosphatases, SOCSs (suppressors of cytokine sig− naling), and PIASs (protein inhibitors of activated STATs). Two members of the mammalian phos− phatases (SH2−containing phosphatase, SHP) were identified: SHP−1 and SHP−2. They both bind with their SH2 (Src homology 2) domains to phospho− tyrosine residues of the number of the cytokine receptors (during signal transduction, phosphoryla− tion of amino−acid residues is the common process). After binding to a phosphotyrosine motif, SHP becomes activated and able to dephosphory− late Jaks and receptors. SHP−1 influences cytokine signaling by dephosphorylating components such as the interleukin−4 receptor, the erythropoietin receptor, and Jak−2. It also acts in an SH2−indepen− dent manner (e.g. with the insulin receptor) [21]. SHP−2 seems to have a dual function as a positive regulator of signaling and a suppressor of cytokine signaling through the gp130 receptor [22].

different, depending on the SOCS protein. SOCS1 binds with its SH2 domain to phosphoty− rosine residues in Jaks. SOCS2, SOCS3, and CIS bind (also through the SH2 domain) directly to the cytokine receptors, blocking STAT docking and activation [23]. The structure of the protein includes a central SH2 domain, an N−terminal domain, and a C−terminal domain, the SOCS box.

Experiments with knockout mice revealed that different SOCSs have distinct functions. Mice lacking the genes encoding specific SOCS1 (SOSC1−/−) exhibited increased sensitivity to the inflammatory cytokine IFN−γ and prolonged STAT−1 activation and they died before three weeks of age from a complex disease; mice with SOCS3−/− died embryonically due to erythrocyto− sis or placental dysfunction [22].

The last group of proteins involved in the neg− ative regulation of Jak/STAT signaling is the PIAS

family. It consists of four members: PIAS1, PIAS3, PIASx, and PIASy. They all inhibit STAT protein, but it differs between family members [24]. PIAS1 and PIAS3 inhibit signaling by bind− ing STAT−1 and STAT−3, which prevents associa− tion with DNA. PIASx and PIASy inhibit STAT−4 and STAT−1 in different way (not affecting binding with DNA). All PIAS family members have also ligase activity known, as SUMO (E3 small ubiqui− tin−like modifier). The result of this activity (sumoylation) is similar to ubiquitylation but it is not connected with the degradation of the target.

In recent years the identification of signal transduction pathways has suggested new thera− peutic targets in rheumatoid arthritis. Modulation of STAT expression represents an alternative ther− apeutic target to cytokine antagonists as TNF−α and their inhibition will probably prove more powerful than current treatment strategies.

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Address for correspondence:

Marta Madej

Department of Rheumatology and Internal Diseases Silesian Piasts University of Medicine

Borowska 213 50−556 Wrocław Poland

E−mail: [email protected]

Conflict of interest: None declared