**3. Stat proteins**

Gene analysis has revealed the existence of at least 6 STAT protein isoforms, namely, JAK1, JAK2, JAK3, JAK5A, JAK5B and JAK6 [20]. In normal homeostasis, phosphorylation of these STAT proteins is achieved via phosphorylation (i.e. activation) of specific JAK isoforms fol‐ lowing the interaction of various cytokines and growth factors with their specific receptors [16, 21]. In this manner, cytokine receptor-mediated JAK activation results in the conversion of latent cytoplasmic un-phosphorylated STAT (U-STAT) proteins into phosphorylated STAT (p-STAT) proteins which can form homo- or heterodimers and are then translocated to the nucleus where these activated STAT protein dimers act as potent transcription factors [17-20]. Although phosphorylation of specific STAT-tyrosine residues remains the primary requisite mechanism for p-STAT protein dimer formation, a second phosphorylation site was also recognized at a serine in the C-termini domain of the STAT protein [20, 22].

An amplification loop with potential major clinical significance in RA involves the transcrip‐ tional activity of p-STAT proteins which further regulate the expression of pro-inflammato‐ ry and anti-inflammatory cytokine genes as well as other genes of significance in cancer and autoimmune diseases [23-28]. In addition, p-STAT proteins can regulate other signaling pathways necessary for lymphocyte development, as well as the aberrant survival of activat‐ ed dendritic cells, monocytes, lymphocytes and synoviocytes in disorders of the immune system [29-33].

main. Leonard and O'Shea [18] identified a proline-rich conserved region in the cytokine re‐ ceptors, called Box1, that associated with JH7 whereas the catalytic phosphotyrosine kinase

**Figure 1.** JH domains and phosphorylation sites of JAK3: Structural analysis combined with functional studies of JAK3 showed that the JH4-JH7 region contained band 4.1 also known as the Four-point-one, Ezrin, Radixin, Moesin (FERM)

Additional structural analysis predicted that the JH2 domain was more than likely to be a pseudosubstrate domain [19]. In view of this latter finding the structural requirements for JAK activation was further clarified. Thus, the JH3-JH4 domain which shows a Src-homolo‐ gy-2-like structure had a shared homology with JH2. This finding indicated that the JH4-JH7 domains were, indeed, the critical regions required for regulating the interactions between the various JAK isoforms and other protein kinases. JH4-JH7 were also found to be essential for receptor binding, catalytic function, JAK autophosphorylation and even in some cases,

Gene analysis has revealed the existence of at least 6 STAT protein isoforms, namely, JAK1, JAK2, JAK3, JAK5A, JAK5B and JAK6 [20]. In normal homeostasis, phosphorylation of these STAT proteins is achieved via phosphorylation (i.e. activation) of specific JAK isoforms fol‐ lowing the interaction of various cytokines and growth factors with their specific receptors [16, 21]. In this manner, cytokine receptor-mediated JAK activation results in the conversion of latent cytoplasmic un-phosphorylated STAT (U-STAT) proteins into phosphorylated STAT (p-STAT) proteins which can form homo- or heterodimers and are then translocated to the nucleus where these activated STAT protein dimers act as potent transcription factors [17-20]. Although phosphorylation of specific STAT-tyrosine residues remains the primary requisite mechanism for p-STAT protein dimer formation, a second phosphorylation site

was also recognized at a serine in the C-termini domain of the STAT protein [20, 22].

An amplification loop with potential major clinical significance in RA involves the transcrip‐ tional activity of p-STAT proteins which further regulate the expression of pro-inflammato‐

domain. Reprinted by permission from [16].

inhibition of JAK activity.

**3. Stat proteins**

374 Drug Discovery

site, called YY was determined to correspond to the other JH domains (Figure 1).

It is noteworthy that during normal homeostasis, activation of STAT proteins induced the expression of Suppressor of Cytokine Signaling (SOCS) and Cytokine-Inducible SH-2 (CIS) proteins and it has been concluded that this is the negative feedback loop that underlies one of the mechanisms responsible for inhibiting JAK-mediated signaling by cytokines [34-38]. Thus, results of recently published experiments with human endothelial cells are germane to this point since the data in this paper provided a direct connection between the silencing of STAT3 with STAT3-specific silencing RNA and the suppression of SOCS3 [39].

The extent to which negative regulation of JAK-mediated signaling by SOCS/CIS may be in‐ activated in autoimmune diseases is a focus of current studies. In that regard, recent advan‐ ces in unraveling the details of mechanism(s) governing negative regulation of cytokine signaling by SOCS/CIS proteins have shed additional light on the extent to which SOCS/CISmediated down-regulation of pro- and/or anti-inflammatory cytokine JAK/STAT signaling may be compromised in inflammatory arthritis [40]. However, the results of some recent studies with osteoarthritic human cartilage have not clarified this issue. For example, one study showed that the level of SOCS2 and CIS-1, but not SOCS1 and SOCS3, were reduced in femoral head cartilages from subjects with osteoarthritis [41], whilst the results of another study [42] indicated that SOCS3, but not SOCS1 expression, was elevated in chondrocytes obtained from osteoarthritic cartilage compared to chondrocytes from cartilage obtained from patients who had femoral neck fracture.

The status of the activity of certain other negative regulators such as protein tyrosine phos‐ phatases, including SHP-1,-2 [43] and CD45 [44] and the 'Protein Inhibitor of Activated STAT' (PIAS) proteins [16, 45, 46] are also not precisely known in autoimmune diseases. These proteins could very likely suppress the activity of phosphorylated JAKs and p-STAT proteins by dephosphorylation or by interacting with p-STAT proteins in normal cells. However, these pathways may be compromised or markedly suppressed in arthritis.

It is also critical for gaining a further understanding of what alterations may occur in cyto‐ kine signaling in RA to recognize the fact that activation of JAK/STAT by any of the relevant cytokines can also activate other intracellular signaling pathways via the "cross-talk" mech‐ anism. Thus, "cross-talk" between JAK/STAT and other signaling pathways [16] can cause activation of the Stress-Activated Protein Kinase/Mitogen-Activated Protein Kinase (SAPK/ MAPK) pathway, the Phosphatidylinositol-3-Kinase/Akt/mammalian Target of Rapamycin (PI3K/Akt/mTOR) pathway [47], activation of signaling via Toll-like receptors [47, 48] and immunoreceptor tyrosine-based activation motifs (ITAMs) [49] as well as the NF-κB path‐ way [50]. These alternative signaling pathways which are all connected to inflammation have also been shown to significantly modulate many of the survival and/or apoptosis-sig‐ nals required to perpetuate abnormal proliferation and/or to cause the death of activated dendritic cells, lymphocytes, macrophages, synoviocytes and chondrocytes.

Evidence from a genome-wide analysis study (GWAS) of STAT-target genes showed that many of these genes regulated cellular proliferation, angiogenesis and metastasis in cancer cells [51]. These results when coupled with the data from another recent study [52] which highlighted the nature of the several forms of STAT-interacting proteins that bind to DNA suggested that GWAS could be employed to identify pro-inflammatory and/or anti-inflammatory cytokine STAT-tar‐ get gene structures and potentially additional STAT-interacting proteins present in RA joint tis‐ sues. Thus GWAS may be considered the next step in the development of future therapies for RA based on targeting STAT-responsive genes. This could be especially useful depending on the status of the activity of the SOCS/CIS protein family acting on cytokine-receptor-mediated sig‐ naling. For example, if SOCS/CIS activity is dampened or deregulated in RA then it would be un‐ likely that this negative regulator pathway for controlling cytokine signaling would be able to inhibit the amplification of pro-inflammatory cytokine-induced JAK/STAT signaling. To illus‐ trate this point, Isomäki et al. [40] showed that although SOCS-1 and SOCS-2 were up-regulated in T-cells recovered from peripheral blood, that SOCS-3 was found in peripheral blood mono‐ cytes and a significant number of synovial tissue macrophages expressed SOCS-1 and SOCS-3 proteins, the majority of T-cells in RA synovium were 'SOCS negative.'

interferon, IL-6), that result in more prolonged antiviral and/or immune responses. Thus, the results of these studies provided novel information regarding the functional significance for U-STAT1 and U-STAT3 acting as transcriptional activators and organizers of chromatin. These events have been shown to be important cellular mechanisms for regulating gene transcription

Suppression of Pro-Inflammatory Cytokines via Targeting of STAT-Responsive Genes

http://dx.doi.org/10.5772/52506

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The results of DNA sequencing studies originally demonstrated specific DNA-binding sites for STAT1 and STAT3 [57]. Boucheron et al. [58] then demonstrated that specific DNA binding sites existed for STAT5A and STAT5B homodimers despite the fact that STAT5A and STAT5B are evolutionarily conserved and encoded by 2 genes with a 91% homology in amino acid structure [59]. Moreover, targeted gene deletion of STAT5A and STAT5B in mice resulted in distinctive phenotypes [60]. This finding suggested a structural dissimilarity in the DNA-bind‐ ing motifs for these two STAT proteins. The results of studies reported in [60] were later con‐ firmed using the IL-3-dependent early pre-B cell line, Ba/F3 [61]. Here it was shown that both STAT isoforms bound to all of the promoters tested, but STAT5A and STAT5B bound with dif‐ fering kinetics [62]. This result suggested that DNA binding activity was likely at the root of

Ehret et al. [63] compared the specificity of STAT-DNA binding sites in specific STAT gene knockout mice showing distinct phenotypes with the STAT-DNA binding sites in a variety of cultured cells. From the *in vitro* analysis, Ehret et al. [63] also demonstrated that DNA binding site motifs for STAT1, STAT5A, STAT5B and STAT6 were essentially the same with only minor differences in DNA binding site specificity. However, STAT5A DNA-binding specificity was much more similar to STAT6 than was the preferential DNA-binding site for STAT1. Thus, the preferential DNA binding site for STAT6 contained a 4 base pair spacer (i.e. TTCNNNNGAA) (N4) which was defined as the weak DNA binding site. However, ad‐ ditional analyses showed that STAT6 bound to TTCNNNG-AA (N3) sites (i.e. the strong binding site) as well [63]. The binding of STAT1 and STAT5 to the N3 site was distinct from STAT5A which preferred N4. Of note, most of the STAT6 binding sites were found in IL-4 responsive promoters in the N4 sites [64-67]. These results reported by Ehret et al. [63] were extended by the findings of Moucadel and Constantinescu [64] who showed that STAT5B

This overview covering the specificity of STAT-DNA binding becomes especially important for improving our understanding of which cytokine gene expressional events are altered by p-STAT and U-STAT proteins. This section analyzes our current interpretation of several cy‐ tokines relevant to RA and other autoimmune diseases, namely, IL-2, IL-3, IL-4, IL-6, IL-15, IL-17, IL-19 and INF-γ, all of which have been shown to activate the JAK/STAT pathway [16]. Moreover, activation of JAK/STAT signaling by these cytokines was shown to result in altered patterns of transcriptional activity which lead to changes in the expression of the fol‐ lowing cytokine or cytokine-related genes, IL-2R, IL-3, IL-4, IL-6, IL-6ST (gp130), IL-10,

any differences in the biological activity of these two STAT protein isoforms.

in the nuclei of cells of the immune system and cancer cells.

bound to chromatin at both the N3 and the N4 site.

IL-18R1, INF-γ, oncostatin M (OSM) and TNF-α (Table 1).

**5. Stat-responsive cytokines genes**

For further discussion, this chapter will focus on the recent progress that has been made in furthering our understanding of how cytokine gene expression is regulated by both U-STAT and p-STAT proteins. The long-term prospect arising from the results of these studies would be to exploit this new knowledge to reduce the level of pro-inflammatory cytokines or to raise the level of anti-inflammatory cytokines in RA. By doing so this could potentially re‐ store the balance between these cytokine families and retard ongoing synovial joint damage whilst also ameliorating RA clinical signs and symptoms.
