**5. Stat-responsive cytokines genes**

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

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

Defining transcription factor binding sites was critical for revealing the structure of cis-regu‐ latory motifs that regulated transcriptional activity [53]. However, microarray analysis using different cell types determined that although several hundred genes were potential STAT3 target gene sites, only a small fraction of those STAT3-target gene sites turned out to be true

As previously indicated, p-STAT proteins do not act independently of one another and U- and p-STAT-protein interactions take various forms which enable them to bind efficiently to DNA [55]. These activated STAT-protein interaction types include, 1) the direct binding of activated STAT homodimers to DNA; 2) the interaction of activated STATs with non-STAT proteins to form activated STAT/non-STAT protein complexes which bind to DNA; and 3) activated STAT proteins interacting with other non-STAT transcription factors or co-activator proteins which bind to DNA [16, 53]. In addition, several novel mechanisms were described for the binding of U-STAT3 and U-STAT1 to DNA [54, 55]. In that regard, Cheon et al. [56] showed that U-STAT3 can drive expression of proteins not induced by p-STAT3, whereas U-STAT1 was shown to ex‐ tend and up-regulate the expression of a subset of genes initially responsive to p-STAT1 (e.g.,

proteins, the majority of T-cells in RA synovium were 'SOCS negative.'

whilst also ameliorating RA clinical signs and symptoms.

**4. Stat-DNA promoter-binding motifs**

direct STAT3-target genes [54].

376 Drug Discovery

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


Charles J. Malemud: Suppression of Pro-Inflammatory Cytokines via Targeting of

**6. Th1/Th2 Cells, Treg Cells, IL-2R, and IL-15**

protein interactions [71].

suppressed Foxp3 expression [76].

Th1 or Th17 cells [80].

that regulates the genesis and maintenance of immune tolerance [16].

Up-regulation of the Th1 and Th17 T-cell subsets and reduced levels of human T-regulatory (Treg) cells are known to occur in autoimmune diseases [16, 68]. In addition, Treg cells are a critical contributor to T-cell development in the thymus as well as being the T-cell subset

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

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

379

The IL-2Rα/IL-2Rβ subunits in complex with the common IL-2γ subunit make up the highaffinity IL-2 receptor, whereas homodimeric IL-2Rα results in a low-affinity receptor [69]. The functional significance of blocking the high-affinity IL-2R with the small molecule inhib‐ itor (SMI), SP4206 (Kd ~70nM) in response to IL-2 (Kd~10nM) was that JAK/STAT activation was inhibited [70]. This result could provide the impetus for development of the next gener‐ ation SMI designed to efficiently inhibit the IL-2/IL-2R pathway and this task should be fa‐ cilitated by employing recently developed technologies based on the principles of protein-

As indicated previously, the interaction of IL-2 with the high-affinity IL-2R causes activation of JAK/STAT with STAT5A and STAT5B, the principally activated STAT proteins. However, the eventual change in STAT5-gene responsiveness following IL-2 activation of STAT5 was shown to be dependent on the complexity of the promoter regions of those STAT5-target genes [72]. Interestingly, Tsuji-Takayama et al. [73] showed that IL-2-mediated JAK/STAT activation up-regulated the production of IL-10 by Treg cells. The production of IL-10 arose from the interaction of STAT5 with a STAT5-responsive element within intron 4, designated I-SRE-4 of the IL-10 locus which was considered to be an interspecies conserved enhancer sequence (Table 1). Of note, the clustered CpG regions around I-SRE-4 were under-methy‐ lated in IL-10-producing Treg cells, but not in other T-cell subsets. This result confirmed pre‐ vious results which showed that expression of Foxp3, a member of the forkhead/wingedhelix family of transcription factors and a biomarker for the development and function of Treg cells [47, 74] was also IL-2/STAT5-dependent [75]. Thus, development of Treg cells was regulated by the methylation status of CpG residues because methylation of CpG residues

Chen et al. [77] identified a novel set of IL-4/STAT6-target genes in mice that regulated the proliferation of activated T-cells. In addition, these genes were shown to regulate the pro‐ duction of the Th2 lineage as evinced by the finding that the cells isolated from wild-type mice produced Th2 whereas cells from STAT6-/- mice did not. Later, Lund et al. [78] showed that the IL-4/STAT6 pathway was also critical for the commitment of naïve T-cells to become either the Th1 or Th2 subset. In that regard, the ratio of Th1 to Th2 produced from naïve Tcells was found to be dependent on a set of STAT6-responsive genes which included the transcription factors STATB1, Bcl-6, and TCF7 [78, 79]. Moreover, the IL-4/STAT6-mediated pathway was also shown to be a strong modulator of human Treg cell production from either

Wurster et al. [81] were among the first to demonstrate that IL-4-mediated activation of STAT6 could also up-regulate IL-2Rα gene expression (Table 1). Because IL-2 is the major

STAT-Responsive Genes

1Cytokines that activate this STAT protein

2Activated STAT that becomes a transcription factor for the STAT-responsive cytokine/protein

3Function(s) of STAT-responsive cytokine/protein

4IL-6 Signal Transducer

5Leukemia Inhibitory Factor Receptor

6Oncostatin M Receptor

7p38 kinase

8C-Jun-N-terminal kinase

**Table 1.** STAT-Responsive Pro-Inflammatory Cytokine Gene Expression
