The Physiological and Pathophysiological Role of IL-6/STAT3-Mediated Signal Transduction and STAT3 Binding Partners in Therapeutic Applications

Abstract

The interleukin 6 (IL-6) family of cytokines is defined by the usage of gp130, a common β-receptor signaling subunit, which promotes a variety of signals. They induce many biological functions on many cell types, including immune and inflammatory cells. They also exhibit hormone-like features, which are involved in homeostatic processes. Signal transducer and activator of transcription 3 (STAT3) is a significant signaling molecule fundamental in regulating IL-6/gp130 and is highly implicated in pathological conditions; therefore, STAT3 activation is tightly regulated through various mechanisms and at multiple levels. There is a large amount of information about STAT3-interacting proteins, which positively or negatively regulate STAT3 activity. This review is focused on IL-6-mediated signal transduction and the introduction of novel STAT3-binding partners. The review will help develop new strategies for clinically controlling the functions of IL-6/STAT3.

1. INTRODUCTION

In 1986, cDNA encoding B cell stimulatory factor-2 (BSF-2), which induces immunoglobulin production, was cloned.¹⁾ At the same time, interferon-β₂ and fibroblast-derived 26 kDa protein were independently cloned and found to be identical to BSF-2.²⁾ In addition, hybridoma/plasmacytoma growth factor and hepatocyte stimulating factor were also reported to have the same structure as BSF-2.^3,4) Although multiple names were given to these molecules based on their diverse biological functions, they all were unified as interleukin 6 (IL-6) based on structural identity.^5–7) Subsequently, more IL-6 functions were described, including its involvement in developing chronic inflammatory diseases, such as rheumatoid arthritis and Castleman’s disease^8,9) (Fig. 1A).

Fig. 1. Physiological and Pathophysiological Role of IL-6/STAT3-Mediated Signaling and Current Perspectives on Tocilizumab’s Applications

A. IL-6 participates in a broad spectrum of biological events, such as cell proliferation, differentiation, survival, and apoptosis, through STAT3. The IL-6-STAT3-mediated signaling plays roles in the immune, the endocrine, the nervous and the hematopoietic systems, and on bone metabolism. B. Current perspectives on Tocilizumab for the treatment of rheumatoid arthritis and other systemic autoimmune diseases. Tocilizumab, a humanized monoclonal antibody that inhibits IL-6 receptor (IL-6R) function, successfully improved clinical symptoms of patients with rheumatoid arthritis, Castleman disease, systemic juvenile idiopathic arthritis (JIA), adult-onset Still’s disease (AOSD), giant cell arteritis (GCA), Takayasu arteritis and cytokine release syndrome (CRS). IL-6: interleukin 6; CTL: Cytotoxic T cell; Th17: T helper type 17; Tfh: T follicular helper; Treg: T regulatory; TAF: T cell-activating factor; BSF-2: B-cell stimulatory factor 2; HPGF: Hybridoma/plasmacytoma growth factor; HSF: hepatocyte-stimulating factor; KGF: keratinocyte growth factor; CSF-309: colony-stimulating factor-309; MGI-2A: macrophage and granulocyte inducer, type 2A; SAA: serum amyloid A; FIB: fibrinogen; AAT: Alpha-1-antitrypsin; ALB: albumin; CRP: C-reactive protein; ACTH: adrenocorticotrophic hormone; GH: growth hormone; PRL: prolactin; LH: luteinising hormone; NGF: nerve growth factor; VEGF: vascular endothelial growth factor; RANKL: receptor activator of nuclear factor of κB (NF-κB) ligand; STAT3: signal transducer and activator of transcription 3; AIDS: acquired immunodeficiency syndrome.

Naïve CD4⁺ T cells differentiate into effector/helper T (Th) cells after activation by antigen-presenting cells.¹⁰⁾ Among Th subsets, the differentiation to Th17 cells, whose excessive and prolonged activation promotes autoimmune and inflammatory diseases, requires IL-6-stimulation together with transforming growth factor (TGF)-β.¹¹⁾ In contrast, IL-6 inhibits TGF-β-induced differentiation of regulatory T cells.¹²⁾ Additionally, IL-6 and IL-21 are essential for T follicular helper cell differentiation.¹³⁾ Therefore, blocking IL-6 activity has been proposed as a new therapeutic approach for several disease conditions^14–17) (Fig. 1B). For example, tocilizumab, a humanized monoclonal antibody that inhibits IL-6 receptor (IL-6R) function, successfully improved clinical symptoms of patients with rheumatoid arthritis, adult-onset Still’s disease, Takayasu arteritis, and Castleman’s disease.^18,19) Furthermore, the inhibition of IL-6 and IL-6R could successfully treat cytokine release syndrome induced by chimeric antigen receptor T cell therapy (CAR-T) and coronavirus disease 2019 (COVID-19) infection.^20–23)

Patients lacking functional IL-6 receptor components or its downstream signaling molecules (for example, signal transducer and activator of transcription 3, STAT3 mutation) exhibit insufficient humoral/cellular immunity, eosinophilia, decreased acute-phase reactions, high susceptibility to fungal infections, and epidermal afflictions.^24–29) Clinical usage of tocilizumab has uncovered unexpected biological functions of IL-6, such as controlling lipid, glucose, and iron metabolism as well as involvement in regulating mitochondrial activity, appetite, and fatigue.^17–19,30) Tocilizumab treatment downregulates hepcidin levels that result in recovery from anemia in patients with chronic inflammatory diseases. Moreover, tocilizumab-treatment influences serum cholesterol and triglyceride levels, along with increases in body weight. These IL-6-mediated metabolic processes are further validated by data from Il-6-deficient mice, which develop maturity-onset obesity, triglyceride elevation, and glucose intolerance.³¹⁾ Similarly, IL-6 affects tissue turnover, regeneration, and repair, as partly observed in cases with failure of anti-IL-6 therapy in inflammatory bowel diseases.^14,15)

The progression, severity, and duration of diseases on cellular and molecular levels are determined by the location, time, and mechanism of IL-6 activation. Here, I describe the IL-6/IL-6R system and its downstream signaling, particularly STAT3. This review will help develop new treatment options to improve the clinical symptoms of patients with various disease conditions, such as immune and/or inflammatory abnormalities as well as malignancies.

2. THE IL-6 RECEPTOR SYSTEM—THREE MECHANISTIC MODELS

The IL-6R system is composed of an 80 kDa α-receptor subunit (IL-6R; also known as CD126), which recognizes IL-6, and a 130 kDa signal-transducing β-receptor subunit (gp130; also known as CD130).^15,22,32) During IL-6-stimulation, the IL-6/IL-6R/gp130 complex is formed, followed by their clustering to make the functional dimer structure. Gp130 plays essential roles in signal transduction for development, hematopoiesis, cell survival, and growth; therefore, gp130-deficient mice are embryonic lethal. Gp130 also functions as a β-cytokine receptor for the IL-6 family of cytokines, including IL-11, IL-27, oncostatin-M, ciliary neurotrophic factor, cardiotrophin-1, leukemia inhibitory factor, and cardiotrophin-like cytokine 1 (Fig. 2A). Thus, gp130 is ubiquitously expressed in various cells beyond the immune system. In contrast, IL-6R expression is mostly limited to hepatocytes, leukocytes, and megakaryocytes. Thus, both IL-6R- and IL-6-deficient mice are viable and exhibit no severe phenotypes, although these mice show some phenotypic differences in wound healing, the severity of induced colitis, and insulin sensitivity. Notably, an ancestral IL-6-like cytokine system is prevalent even at the Drosophila melanogaster level³³⁾ (Fig. 2B). After bacterial infection, an IL-6-like unpaired-3 (upd-3) forms a signaling complex with a gp130-like Domeless as well as Hopscotch, a Drosophila homolog of Jak, and Marelle, a Drosophila homolog of STAT, to provide innate immunity. Upd-3 from Drosophila macrophages also regulates glucose and tissue homeostasis, similar to mammalian IL-6. Additionally, human herpesvirus 8 expresses a viral form of IL-6, with approximately 25% amino acid similarity with human IL-6,³⁴⁾ that can suppress the recruitment of neutrophils.³⁵⁾ The viral IL-6 can transduce signals through a single chain of the heterodimeric IL-6 receptor, promoting cellular proliferation and preventing apoptosis.³⁶⁾

Fig. 2. Receptor Usage of IL-6 Family Cytokines and Three Modes of IL-6-Mediated Signaling

A. IL-6 family cytokines use gp130 to transduce their signals through gp130 homodimers or gp130-containing heterodimers. IL-6, IL-11, IL-27, LIF, OSM, CNTF and CLCF1 require binding of their nonsignaling receptor to transduce signals. CNTFRα, a glycosylphosphatidylinositol-anchored protein that does not directly contribute to signaling beyond facilitation of ligand binding. B. In Drosophila, the IL-6-like unpaired 3 (Upd3) protein binds to a gp130-like cytokine receptor, Domeless (Dome), which shares functional and sequence similarity with the mammalian cytokine class I receptors. Moreover, Drosophila has a single Jak, Hopscotch (Hop) and one STAT, Stat92E. Drosophila SOCS proteins (Socs36E and Socs44A), dPIAS, and a protein tyrosine phosphatase, PTP61F mediate the negative feedback loop for Drosophila Jak-STAT signaling. C. IL-6R exerts its biological effects via three different signaling modes. In the ‘classical signaling’, the cytokine interacts with IL-6R in cells that also express gp130. In the ‘trans-signaling’, the IL-6– soluble IL-6R (sIL-6R) complex binds to gp130, forming a dimer that initiates intracellular signaling. ‘Cluster signaling’ is specific to dendritic cells, in which the IL-6-IL-6R complex binds to gp130 expressed on T cells to induce pathogenic Th17 cells. IL: interleukin; OSM: oncostatin M; LIF: leukaemia inhibitory factor; CNTF: ciliary neutrophic factor; OSM: oncostatin M; CLCF1: cardiotrophin-like cytokine factor 1; PIAS: Protein inhibitor of activated STAT; SOCS: suppressor of cytokine signaling.

Three mechanistic models of IL-6 signal transduction have been proposed; ‘classical signaling,’ ‘trans-signaling,’ and ‘cluster signaling^14–17,37)’ (Fig. 2C). ‘Classical signaling’ depends on the binding between circulating IL-6 and membrane-bound IL-6R and only influences cells expressing both IL-6R and gp130, such as hepatocytes, leukocytes, megakaryocytes, myocytes, and adipocytes. ‘Classical signaling’ regulates central homeostasis and immune responses, such as hematopoiesis, acute-phase reactions, neuroendocrine system, glucose metabolism, fatigue, appetite loss, and hyperthermia. ‘Trans-signaling’ occurs when IL-6R is processed to a soluble form of the receptor (sIL-6R), which has the capacity to bind to circulating IL-6. The IL-6/sIL-6R complex increases the half-life of circulating IL-6 and promotes its bioavailability. sIL-6R shares 60% identity with the IL-12p40 subunit and represents an ancestral link to other heterodimeric cytokines IL-12, IL-23, and IL-27.³⁸⁾ Therefore, ‘trans-signaling’ likely improves IL-6-responsiveness to many cell types expressing gp130, leading to the expansion of cells under the influence of IL-6. The original identification of gp130 as the β-subunit of the IL-6 receptor was facilitated by the fact that IL-6 binds to gp130 in the presence of recombinant sIL-6R. A biologically active form of sIL-6R can be purified from human urine and plasma, and the serum concentration of sIL-6R (normal range: 25–35 ng/mL) increases during inflammation.³⁰⁾ ‘Trans-signaling’ is usually related to the enhancement of proinflammatory effects of IL-6 because sIL-6R production is associated with enzymatic cleavage by a disintegrin and metalloprotease (ADAM) family of proteases or alternative splicing of IL6ST mRNA that occurs in activated immune cells.³⁹⁾ sIL-6R is released by monocytes and activated T cells. In addition, sIL-6R may be classified as an alarmin released by neutrophils and promotes potential danger responses to cause innate and adaptive immune-related diseases. Thus, ‘trans-signaling’ is involved in the recruitment and apoptosis of leukocytes, the maintenance of effector T cells, and the inflammation of stromal tissues in experimental models for infection, allergy, colitis, fibrosis, arthritis, neuroinflammation, cardiovascular diseases, and cancers. IL-6R expression in CD4⁺ T cells is limited to naive and central memory populations. However, CD4⁺ T cells recovered from disease sites largely lack IL-6R but remain responsive to ‘trans-signaling.’ This fact also supports the meaning of ‘trans-signaling’ in immune responses during disease progression. Indeed, mice carrying the rs2228145 mutation in IL6R exhibit elevated circulating levels of sIL-6R, leading to high levels of C-reactive protein as well as greater risks of obesity, insulin resistance, and cardiovascular diseases.^40,41) The last mode of IL-6 signaling is ‘cluster signaling.’ ‘Cluster signaling’ presents the IL-6/IL-6R complex of dendritic cells to gp130-expressing T cells.⁴²⁾ This type of signaling is primarily used among immune cells. A further layer of regulation may be supplied with a soluble form of gp130 (sgp130), which blocks the sIL-6R/IL-6 complex.⁴³⁾ Three, or possibly four, forms of sgp130 are produced through different patterns of splicing of IL6ST mRNA, although no unique function is assigned to any of these sgp130 isoforms.⁴⁴⁾

3. IL-6/STAT3-MEDIATED SIGNAL TRANSDUCTION

Various cytokines preferentially activate Janus kinases (Jaks), activating one or more STATs through their tyrosine phosphorylation^45–51) (Fig. 3A). The Jak family comprises four members in mammals: Jak1, Jak2, Jak3, and tyrosine-protein kinase 2 (Tyk2). The STAT family contains seven members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Jaks carry four functional domains: the F for 4.1 protein, E for ezrin, R for radixin, and M for moesin (FERM) domain and the Src Homology 2 (SH2)-like domain to interact with upstream receptor components; the pseudokinase domain to inhibit excess of kinase activity; and the kinase domain, which carries catalytic regions to induce tyrosine-phosphorylation of Jaks and STATs (Fig. 3B). STATs has seven functional domains: the N-terminal domain and the coiled-coil domain to promote protein–protein interactions for signal transduction; the DNA-binding domain, which directly interfaces with DNA and controls nuclear import-export signals; the linker domain, which is structurally essential to promote transcriptional activity; the SH2 domain, which is involved in dimerization and interaction with upstream receptor components; the transactivation domain, which carries the tyrosine-phosphorylation residues necessary for signal transduction (especially STAT3 at tyrosine 705: Y705); and the C-terminal domain, which carries the serine-phosphorylation sites to support its full activation (especially STAT3 at serine 727: S727) (Fig. 3B). Through the DNA binding domain, all STATs recognize a gamma-activated sequence, a palindromic TTCN_3-4GAA motif, within the promoter region of STAT-responsive target genes.

Fig. 3. Jak-STAT Signaling and the Classical IL-6 Signaling Pathway

A. The steric change of the cytokine receptor brings two Jaks constitutively bound to the cytokine receptor subunits into close proximity, thus facilitating trans-phosphorylation. The activated Jaks phosphorylate Signal transducer and activator of transcription (STAT) proteins as the major substrate. Once the conserved tyrosine toward the C-terminal transactivation domain of the STAT has been phosphorylated, it can act as dimerization interface in conjunction with the Src homology 2 (SH2) domains of another STAT. These activated STAT dimers are then translocated to the nucleus and bind to specific DNA motifs to activate target gene transcription. B. Schematic diagram of structural and functional domains of human Tyk2 protein and STAT3. C. A schematic representation of the signal transduction activated by IL-6. IL-6 binds to the membrane-bound IL-6R, thus inducing formation of a hexameric complex consisting of two molecules each of IL-6, IL-6R, and gp130. The Jak/STAT3 signaling pathway is then activated, leading to the transcription of STAT3 target genes. The IL-6/IL-6R/gp130 complex can also activate the PI3K/Akt/mTOR and Ras/Raf/MEK/MAPK pathways. Activation of these pathways consequently induce the expression of different genes with crucial roles in a variety of physiological events, such as cell proliferation, differentiation, survival, and apoptosis. The Yap-Notch pathway is triggered by Src/Yes and controls tissue growth and regeneration. IL-6: interleukin 6; FERM; the four-point-one protein, ezrin, radixin, moesin domain, JH; Jak homology, SH2; Src homology 2, and TAD; C-terminal transactivation domain; MEK: mitogen activated protein kinase kinase; MAPK; mitogen activated protein kinase; Jak: janus kinase; Tyk2; tyrosine kinase 2; mTOR: mammalian target of rapamycin; PI3K: phosphatidylinositol-3-kinase; STAT: signal transducer and activator of transcription; Yap: yes-associated protein; SHP2: SH2-domain containing protein tyrosine phosphatase-2; SOCS: suppressor of cytokine signaling.

The membrane-proximal Boxes 1 and 2 in gp130 can bind to Jaks. Jak1, Jak2, and Tyk2 activation trigger the intracellular signaling via gp130^22,51) (Fig. 3C). The activated Jaks promote the distinct patterns of tyrosine phosphorylation and subsequent STAT1 and STAT3 activation (to a lesser extent, STAT5). Four distal tyrosine residues (Tyr767, Tyr814, Tyr905, and Tyr915) of gp130 are involved in STAT3 phosphorylation, whereas Tyr905 and Tyr915 stimulate STAT1. The consensus YXPQ and YXXQ sequences provide docking sites to activate STAT1 and STAT3, respectively. Gp130 Tyr759 plays an important role in the activation of Src homology domain 2 domain-containing phosphatase-2 (SHP2), which promotes signals through the Ras-Raf-mitogen-activated protein kinase (MAPK) pathway and the Yes/Src-Yap-Notch pathway. The Ras-Raf-MAPK pathway regulates cellular proliferation and differentiation. The activation of the Ras-Raf cascade is involved in the RAC-alpha serine/threonine-protein kinase (Akt) and mechanistic target of rapamycin complex 1 (mTORC1) pathways to regulate cell survival, apoptosis, and metabolism. SHP2-induced MAPK activation occurs via recruiting growth factor-receptor-bound protein/son of sevenless (Grb/SOS) complex and/or Grb2-associated binder-1 (Gab1) to the cell membrane. The Yes/Src-Yap-Notch signaling cascade is important for tissue regeneration and is strongly activated upon mucosal injury to promote wound healing and maintain barrier function.⁵²⁾

STAT3 mediates the majority of IL-6 functions; however, abnormal expression of STAT3 is detected in some cancer cells as well as autoimmune diseases.^49,53–55) Thus, STAT3 activation via gp130 is tightly regulated by multiple molecular mechanisms^51,56,57) (Fig. 4). One of the significant modules is the regulation by several phosphatases, such as CD45, SHP1, SHP2, and protein-tyrosine phosphatase 1B (PTP-1B; also known as PTPN1), T cell protein tyrosine phosphatase (TC-PTP; also known as PTPN2) and Dual-specificity phosphatases (DUSPs).^58–60) Another regulatory module is the protein inhibitor of the activated STAT (PIAS) family.⁶¹⁾ PIAS3 inhibits STAT3-mediated transactivation by interfering with the DNA binding of STAT3 in the nucleus. A third regulatory module is the suppressor of the cytokine signaling (SOCS) family. SOCS3, induced by STAT3, contributes to negative feedback of STAT3 activities.⁶²⁾ SOCS3 binds to a short glycine (Gly)-glutamic acid (Glu)-methionine (Met) (GQM) motif on Jak2, leading to direct blockage of Jak2-docking to gp130. Gp130 Tyr759 is critical for SHP2 and SOCS3 to exert their inhibitory activities on IL-6 signal transduction. In the absence of SOCS3, the effects of IL-6 tend to be similar to those of IL-10, a potent inhibitor of macrophages and dendritic cells.^63–65) Mice expressing mutant gp130 Y759F, which failed to bind to SOCS3, show more sustained signals mediated by STAT1 and STAT3 and develop exacerbated inflammation and cancers. The SH2 domains of the SOCS proteins are also responsible for specific interaction with phosphorylated or activated signaling molecules, and play a role in the mechanism of signal suppression.⁶⁶⁾ Smad7, a negative regulator of TGF-β-mediated signaling, directly interacts with gp130 and disrupts the gp130-SHP2 or gp130-SOCS3 complex, thereby upregulating STAT3 activation.⁶⁷⁾ During LIF stimulation, Smad7 also facilitates STAT3-mediated self-renewal of mouse embryonic stem cells. Ubiquitination of gp130 is promoted during IL-6 stimulation.⁶⁸⁾ Ubiquitination of gp130 is triggered by phosphorylation at Y759, resulting in an association with SHP2 and then an E3 ligase casitas B-lineage lymphoma (Cbl). The ubiquitinated gp130 interacts with Hrs, an endosomal-sorting protein required for transport (ESCRT) protein, followed by Hrs-dependent endosomal sorting and lysosomal degradation, terminating IL-6 signaling.

Fig. 4. Positive and Negative Regulators of IL-6/STAT3-Mediated Signaling

IL-6/ STAT3-mediated signaling is positively regulated by several STAT3 binding partners, such as STAP-2, ZIPK, Y14, and NF-κB (see text for details). Moreover, this signaling negatively regulated by a number of mechanisms. Suppressor of cytokine signalling 3 (SOCS3) binds to and inhibit the kinase activity of Jaks. SOCS3 is also a STAT3 target gene. By following its transcription, SOCS3 then acts as a component of a negative-feedback loop by maintaining tight regulation of this pathway. The following phosphatases also have a role in the negative regulation of this pathway: tyrosine-protein phosphatase non-receptor type 6 (PTPN6; also known as SHP1); tyrosine-protein phosphatase non-receptor type 11 (SHP2); dual specificity protein phosphatase 2 (DUSP2); dual specificity protein phosphatase 11 (DUSP11); receptor-type tyrosine-protein phosphatase, CD45; tyrosine-protein phosphatase non-receptor type 1 (PTPN1); tyrosine-protein phosphatase non-receptor type 2 (PTPN2; also known as TC-PTP). TRAF5, Hrs, and Smad7 targets gp130. Protein inhibitor of activated STAT3 (PIAS3) as well as PDZ and LIM domain protein 2 (PDLIM2) act as additional endogenous STAT3 inhibitors. IL-6/STAT3-mediated signaling is also posttransciptionally regulated by several RNA binding proteins or microRNAs. In the unstimulated cellular condition, Regnase-1 inhibits IL-6 production through degradation of IL-6 mRNA by binding at the 3′ untranslated region (UTR). Arid5a counteracts the destabilizing effect of regnase-1, resulting in positive feedback on IL-6 production. The expression of IL-6 is controlled by Lin28 through Let-7 microRNA inhibition. miR-18a targets PIAS3, whereas miR-19a target SOCS3. TLR, toll-like receptor; TRAF5, tumor necrosis factor receptor-associated factor 5; Arid5A: AT-rich interactive domain-containing protein 5A; miR: microRNA; UTR: untranslated region.

Post-transcriptional modifications of STAT3 (except for tyrosine phosphorylation) play essential roles in its complete transcriptional activity. STAT3 is phosphorylated at serine 727 (Ser727) by various serine/threonine protein kinases, such as MAPK members.^69–72) A STAT3 mutant carrying a substitution of Ser727 to alanine (Ala) has only half transcriptional activity compared with the wild type.⁷³⁾ Embryonic fibroblasts from mice carrying the mutation display approximately 50% of transcriptional cellular responses compared to wild-type mice. Of note, serine phosphorylation upregulates STAT3 activity through the interaction with some cofactors, such as histone acetyltransferase p300.⁷⁴⁾ P300 acetylates STAT3 at Lys685, reversible by type I histone deacetylase (HDAC).^75,76) STAT3 Lys685 acetylation is required to form stable STAT3 dimers, which are essential for its DNA binding and transcriptional activation, leading to the transactivation of cell growth-related genes. As for small ubiquitin-like modifier (SUMO) modification of STAT3, STAT3 Lys451 SUMOylation inhibits the interaction with protein tyrosine-phosphatase TC-PTP, leading to abnormally sustained STAT3 phosphorylation and activity.⁷⁷⁾ As for STAT3 methylation, STAT3 dimethylation occurs at Lys49 by the histone methyl transferase, enhancer of zeste homolog 2 (EZH2).⁷⁸⁾ Dimethylation of K49 modulates IL-6–responsive transcription. In humans with rheumatoid arthritis, expression levels of JMJD1C, a JmjC domain histone demethylase member in B cells, are negatively related to plasma cell frequency and disease severity.⁷⁹⁾ Indeed, hypermethylation of STAT3 at Lys140 by JMJD1C-deficiency inhibits the interaction between STAT3 and protein tyrosine-phosphatase SHP1 and resulted in sustained phosphorylation and activation of STAT3. In addition, STAT3 has a reversible S-palmitoylation at Cys108. Zinc finger DHHC-type containing 7 (DHHC7) palmitoylates STAT3 resulted in the promotion of its membrane recruitment and phosphorylation.⁸⁰⁾ Acyl protein thioesterase 2 (APT2, also known as LYPLA2) depalmitoylates the phosphorylated STAT3, leading to its translocation into the nucleus.

MicroRNAs (miRNAs), small non-coding RNAs, are the other key regulatory components of the IL-6/STAT3-mediated signaling pathway.^17,55) Through post-transcriptional gene regulation, they control inflammation and cancer progression.⁸¹⁾ Various miRNAs interfere with IL-6 signaling components to fine-tune the expression and bioavailability of specific components in the IL-6 receptor system and the Jak-STAT pathway.⁸²⁾ For example, Let-7 miRNA directly suppresses IL6 expression. Nuclear factor-kappaB (NF-κB) directly activates Lin28, an RNA-binding protein, which selectively inhibits Let-7 maturation.⁸³⁾ IL-6-mediated STAT3 activation promotes cellular transformation, and further, IL-6 activates NF-κB, forming a positive feedback loop. miR-19a targets SOCS3 to enhance IL-6 signal transduction via STAT3.⁸⁴⁾ PIAS3 is repressed by miR-18a, resulting in the elevated expression of STAT3-mediated genes, such as BCL2L1 and MYC.⁸⁵⁾

Post-transcriptional regulation of mRNA degradation has been reported to regulate IL-6 signaling.^86–89) Various external stimuli, such as TLR ligands, control IL-6 mRNA stability. Indeed, IL-6 mRNA is unstable and degraded with a half-life of 30 min in transfected COS7 cells.⁹⁰⁾ Like IL-6, many inflammatory cytokines have unstable mRNAs, which contain specific sequence/structural characteristics in their 3′ untranslated regions (3′UTR), termed cis-elements.^87,89) Among these cis-elements, the best-characterized motifs are AU-rich element (ARE) and stem-loop structures. AREs typically contain a repetitive AUUUA sequence. Several ARE-binding proteins exist, such as tristetraprolin, ZFP36L1, ZFP36L2, AU-rich binding factor 1, human antigen R, and KH-type splicing regulatory proteins. The stem-loop structure forms a hairpin-like shape and carries constitutive decay elements, which Roquin-1 and Roquin-2 target.^88,89) Both ARE and stem-loop structures exist in partially overlapping sets of inflammatory mRNAs. mRNAs carrying these characteristic sequences are degraded by a set of RNA binding proteins (RBPs), Regnase-1, and Roquin-1 and -2.^88,89) In contrast, AT-rich interactive domain-containing protein 5A (Arid5A) can counteract mRNA degradation.⁹¹⁾ Members of the Regnase family have PIN-like ribonuclease (RNase) domains and CCCH-type zinc finger domains. Regnase-1 recognizes ARE and the stem-loop structures in the 3′ UTR of inflammatory mRNAs, such as IL-6, and post-transcriptionally degrades them via RNase activity. For example, Regnase-1-deficient mice spontaneously develop severe autoimmune inflammatory diseases and die within 12 weeks, showing elevated serum immunoglobulin levels and autoantibody production.⁹²⁾ In addition, Regnase-1-deficient macrophages secrete large amounts of IL-6 after TLR ligand stimulation.⁹³⁾

4. STAT3-BINDING PARTNERS

We have identified and analyzed novel STAT3-binding partners, including death domain-associated protein (Daxx),^94,95) zipper-interacting protein kinase (ZIPK),^96,97) Krüppel-associated box-associated protein 1 (KAP1),^98–100) Y14,¹⁰¹⁾ PDZ and LIM-containing protein 2 (PDLIM2),^102,103) and signal transducing adaptor protein-2 (STAP-2).^104,105) These molecules positively or negatively regulate essential steps of STAT3-mediated signals through a unique mechanism (Fig. 4). Studying the interactions of these STAT3-binding partners will provide novel regulatory mechanisms of STAT3 activities. Although many investigations have proposed the physiological and/or pathological significance of STAT3, the clinical significance of the interactions between STAT3 and its interacting partners requires further clarification.

4.1. Daxx

Daxx is primarily located in the nucleus and regulates transcriptional activities through association with various transcription factors, such as Ets1, paired box gene 5 (Pax5), glucocorticoid receptor, RelA, RelB, transcription factor 4 (Tcf4), Smad4, CCAAT/enhancer binding protein (C/EBP), and autoimmune regulator (Aire).^106–114) In addition, Daxx also regulates epigenetic modification by binding to histone deacetylases, DNA methyltransferases and their associated proteins, and the chromatin-modifying α-thalassemia syndrome protein.^115–119)

Daxx can regulate STAT3 transactivation. The pretreatment suppresses IL-6-induced STAT3 transactivation with interferon (IFN).¹²⁰⁾ In this situation, Daxx directly binds to STAT3 within the nucleus to suppress STAT3 transactivation. Indeed, Daxx-knockdown significantly upregulates IL-6/STAT3-mediated gene expression. In lymphocytes, STAT3 contributes to IL-6- and/or IL-27-dependent cell growth; STAT3 is also involved in pro-B cell survival and efficient production of B lymphocytes.¹²²⁾ Of note, Daxx suppresses the binding of STAT3 to the consensus DNA sequences of its target genes through its constitutive association with STAT3. An inverse correlation between Daxx and Bcl-2 expression levels is often observed during lymphocyte apoptosis. When progenitor B lymphocytes are treated with IFN-β, the expression and nuclear localization of Daxx are enhanced in parallel to Bcl-2 down-regulation. Thus, Daxx functions to negatively control STAT3 activity and Bcl-2 expression after cytokine stimulation.

4.2. ZIPK

ZIPK becomes enzymatically active by aggregating through its leucine zipper domain.⁹⁷⁾ Overexpression of wild-type ZIPK, but not the kinase-inactive mutant ZIPK K42A, promotes apoptosis in NIH 3T3 fibroblast cells, indicating that ZIPK-induced apoptosis requires its catalytic activity.⁹⁶⁾ The kinase domain of ZIPK has high sequence homology with death-associated protein kinase (DAPK), and these proteins consist of a family with DAPK2/DRP-1, DRAK1, and DRAK2, all of which are involved in apoptosis.^122–124) Collaborating with Daxx and Par-4, ZIPK induces apoptosis within nuclear PML oncogenic domains (PODs).¹²⁵⁾ Therefore, IL-6/STAT3-mediated signals are likely to promote apoptotic activity via inducing the translocation of ZIPK into PODs, together with PML and Daxx.

We identified ZIPK as a new STAT3-binding protein using a yeast two-hybrid screen using the C-terminal region of STAT3 as bait.^72,126) ZIPK specifically associates with STAT3 but not other types of STAT proteins. In addition, the kinase domain of ZIPK binds to both the DNA-binding and C-terminal domains of STAT3. ZIPK phosphorylates STAT3 at Ser727 in the nucleus, leading to functional enhancement of the transactivation of STAT3 after IL-6- and LIF-stimulation. ZIPK promotes the phosphorylation of STAT3 Ser727 and enhances STAT3-mediated transactivation. However, ZIPK K42A fails to induce STAT3 Ser727 phosphorylation in the early but not late phase of IL-6-stimulation, suggesting that other kinases may be involved in the late phase of STAT3 Ser727 phosphorylation. STAT3 Ser727 phosphorylation enhances STAT3-mediated transactivation through the associations with co-activators, such as p300.⁷⁵⁾ ZIPK also binds to p300 and forms a STAT3/ZIPK/p300 complex, leading to enhanced transcription.

4.3. KAP1

KAP1, also known as transcriptional intermediary factor 1β and Tripartite motif-containing 28, is a co-repressor of Krüppel-associated box-domain-containing zinc finger proteins.^98–100) KAP1 coordinates a variety of components involved in gene silencing by regulating the HDAC complex and histone methyltransferase.^127–130) Thus, KAP1 inhibits the transactivation of its target genes via orchestrating functions of the co-repressor complexes.

We identified KAP1 as a STAT3-interacting protein using a yeast two-hybrid screening of a mouse embryo cDNA library and confirmed their binding using co-immunoprecipitation experiments.¹³¹⁾ Endogenous KAP1 is present within the nucleus even in the absence of stimulation. After IL-6 stimulation, STAT3 predominantly translocates into the nucleus, which overlaps with KAP1. In hepatoma Hep3B cells, KAP1-knockdown by a specific small interfering RNA (siRNA) significantly upregulates not only STAT3 activities but also mRNA expression of SOCS3 and C/EBPδ after IL-6-stimulation. Thus, KAP1 negatively regulates IL-6/STAT3-mediated transcription and gene expression. Phosphorylation of STAT3 Ser727, but not STAT3 Tyr705, increases in parallel to the decrease of KAP1 expression. Coincident with these data, the decrease of KAP1 expression promotes enhanced nuclear accumulation of phosphorylated STAT3 at Ser727. Thus, KAP1 seems to recruit protein phosphatases to dephosphorylate STAT3 Ser727 in the nucleus. Alternatively, the direct interaction of KAP1 with HDACs may be another mechanism for KAP1-mediated transcriptional repression. Thus, KAP1 can inhibit the transcriptional activities of STAT3 in multiple ways.

4.4. Y14

Y14 is an RNA-binding protein to form an exon-junction complex (EJC) with Magoh.¹⁰¹⁾ This complex preferentially recognizes spliced forms of mRNAs immediately upstream of exon-exon junctions. In general, mRNAs produced by splicing are translated very efficiently.^132–134) The EJC, in part, contributes to this translational upregulation because both Y14 and Magoh recognize the spliced form of mRNAs until the translation is over; in addition, Y14 shuttles mRNAs to interact with Magoh.^135,136)

We found that endogenous Y14 directly associates with STAT3 and influences STAT3 transactivation activity at several steps of IL-6-mediated signaling, including tyrosine-phosphorylation, nuclear accumulation, and the DNA-binding of STAT3.¹³⁷⁾ In addition, Magoh suppresses complex formation between STAT3 and Y14, and Magoh-knockdown by a specific siRNA upregulates IL-6-induced gene expression.¹³⁸⁾ Thus, Y14 positively regulates IL-6-induced STAT3 activation, and Magoh interferes with this effect by displacing Y14 from STAT3.

4.5. PDLIM2

PDLIM2 (also known as SLIM or mystique) contains PDZ (postsynaptic density 65-discs large-zonula occludens 1) and LIM (abnormal cell lineage 11-isket 1-mechanosensory abnormal 3) domains.^103,104) PDLIM2 is a nuclear ubiquitin E3 ligase, which binds to and degrades STAT3 in a proteasome-dependent manner.¹³⁹⁾ Consistently, PDLIM2 deficiency causes insufficient STAT3 degradation, resulting in the nuclear accumulation of STAT3 and high expression of STAT3 target genes. In general, ubiquitination reactions are established by three types of enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). RING-type E3 ligases provide the polyubiquitin chain from E2 to their substrate by binding their RING-finger domain to E2 and the interaction of the other domain with substrate proteins.¹⁴⁰⁾ The LIM domain forms a zinc finger structure and plant homeodomain (PHD) finger domains that generally possess ubiquitin E3 ligase activity and polyubiquitinate their target proteins. Indeed, the PDLIM2 LIM domain can recognize and polyubiquitinate STAT3 proteins. PDLIM2 associates with phosphorylated and unphosphorylated STAT3; however, it preferentially associates with only phosphorylated STAT4.¹⁰²⁾ Thus, PDLIM2 may control STAT3 protein levels through a different mechanism for STAT4.

4.6. STAP-2

We originally isolated STAP-2 as a c-FMS-interacting protein. STAP-2 consists of a pleckstrin homology (PH) domain in its N-terminal region and an SH2 domain in its central region.^104,105,141) A proline-rich region and YXXQ, a STAT3-binding motif, are also present in its C-terminal region. STAP-2 is structurally an adaptor protein and is a murine homolog of BKS, a substrate of breast tumor kinase (BRK).^141,142) Upon stimulation with epidermal growth factor, STAP-2 is tyrosine-phosphorylated and moves to the plasma membrane in fibroblasts.¹⁴¹⁾ IL-6, as well as lipopolysaccharide, strongly promotes STAP-2 mRNA in hepatocytes. STAP-2 is a novel STAT3-binding partner through its YXXQ motif. In STAP-2-deficient hepatocytes, mRNA expression of acute-phase proteins and the tyrosine-phosphorylation of STAT3 are specifically diminished after IL 6 stimulation. Overexpression of STAP-2 significantly upregulates STAT3 activities. Of note, STAP-2 tyrosine-250 (Tyr250), a major tyrosine phosphorylation site, is essential to upregulate STAT3 activity.¹⁴³⁾ Indeed, a STAP-2 Y250F mutant, in which Tyr250 is substituted to phenylalanine, fails to influence STAT3 transcriptional activity. STAP-2 is a substrate of BRK, a non-receptor tyrosine kinase composed of an SH3 domain, an SH2 domain, and a tyrosine kinase catalytic domain but lacks a myristoylation site in its N-terminal region.^142,144) Several malignancies, such as metastatic melanomas and colon, prostate, and breast cancers, express BRK.^145–149) In the mammary gland, some types of breast cancer cells express BRK, but not normal mammary cells¹⁵⁰⁾; also, breast cancer cell growth partly depends on BRK expression.¹⁵¹⁾ STAP-2 functions to enhance BRK-mediated STAT3 activation.¹⁵²⁾ Indeed, in T47D breast cancer cells, STAP-2 associates with both BRK and STAT3, and STAP-2-knockdown by specific siRNA greatly diminished STAT3 activities induced by BRK. In addition, an artificial STAP-2-BRK fusion protein shows robust kinase activity and strongly promotes the activation and tyrosine phosphorylation of STAT3. Thus, STAP-2 can enhance BRK-mediated STAT3 activation by reinforcing the interactions between BRK and STAT3. STAP-2 also upregulates epidermal growth factor receptor (EGFR)/STAT3-mediated signals through the stabilization of EGFR protein, resulting in massive tumor formation of DU145 prostate cancer cells.¹⁵³⁾ STAP-2 directly associates with and stabilizes EGFR, leading to enhanced EGFR/STAT3-signaling. EGFR dimerizes upon its ligand ligation and subsequently associates with Grb2, followed by Ras activation. Activated Ras then induces the activation of MAPK.¹⁵⁴⁾ Cell surface expression of EGFR is essential to promote Ras and MAPK activation; however, EGFR is internalized and degraded after EGF stimulation. In prostate cancer cells, STAP-2 inhibits EGFR ubiquitination by Cbl, resulting in EGFR restoration. Indeed, STAP 2 deficiency promotes the quick disappearance of EGFR protein from the cell surface after EGF stimulation. STAP-2 fails to interact with a dimerization-deficient mutant EGFR K721A; therefore, STAP-2 is likely to interact with EGFR after its dimerization process. In addition to EGFR signaling, STAT3 is activated by IL-6R-mediated signals, and the blockade of IL-6R significantly inhibits tumor cell growth.¹⁵⁵⁾ STAP-2-knockdown successfully inhibits prostate cancer cell growth through synergistic inhibition of EGFR- and IL-6R-signals. Recently, we have found that 2D5 peptide, a STAP-2-derived peptide, inhibited STAP-2-EGFR interactions and suppressed EGFR-mediated cell proliferation in a variety of cancer cell lines, indicating that 2D5 peptide is a novel anti-cancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses cancer progression¹⁵⁶⁾

5. CROSSTALK BETWEEN STAT3 AND NF-κB

Both NF-κB and STAT3 are central signaling hubs in inflammation and oncogenesis.^157–161) NF-κB influences the expression of several genes of anti-apoptosis/proinflammatory cytokines and chemokines.^162–164) Like STAT3, constitutively activated NF-κB is related to many types of malignancies. Target genes controlled by positive or negative crosstalk between STAT3 and NF-κB are gradually increasing. In immune cells, activated STAT3 induces the serine-phosphorylation and subsequent proteasome-dependent degradation of IκBα, leading to the activation of NF-κB.¹⁶⁵⁾ In cardiomyocytes and non-small cell lung cancer cells, activated NF-κB upregulates STAT3 expression.^166,167) The DNA-binding domain of STAT3 can directly interact with the transactivation domain of NF-κB.^168,169) Besides nuclear translocation upon cytokine stimulation, STAT3 continuously shuttles between the cytoplasm and the nucleus, independently of its tyrosine phosphorylation condition. Unphosphorylated STAT3 interacts with NF-κB, binds to DNA, and distinctively drives gene expression from phosphorylated STAT3.¹⁷⁰⁾ Thus, STAT3 regulates gene expression in several forms of NF-κB-dependent genes via its direct interactions with NF-κB.

6. THERAPEUTIC STRATEGIES FOR IL-6/STAT3-MEDIATED SIGNALING

A recent genome-wide association study (GWAS) has proposed that most cytokine receptors are involved in the initiation and development of many types of immunological diseases (especially T cell-dependent diseases) through the downstream Jak-STAT pathway.^171–174) Thus, targeting signals from cytokine receptors, such as IL-6R, has a powerful influence^15,16,19) (Fig. 5). The therapeutic strategies based on inhibitory monoclonal antibodies are best described for IL-6. Indeed, multiple myeloma patients receiving the treatment with anti-IL-6 antibodies advocate its efficacy in inhibiting myeloma cell expansion because the levels of IL-6 and sIL-6R indicate tumor severity.^175,176) Siltuximab, sirukumab, clazakizumab, and olokizumab are anti-IL-6 blocking antibodies. Tocilizumab and sarilumab are anti-IL-6R blocking antibodies.^16,19) An engineered chimeric sgp130 protein, olamkicept recognizing the IL-6-IL-6R complex also inhibits IL-6 trans-signaling.^17,20,177) These are recommended for treating rheumatoid arthritis, juvenile idiopathic arthritis, adult-onset Still disease, and multicentric Castleman disease. Treatment with therapeutic monoclonal antibodies is very effective in extracellularly regulating cytokines and/or their receptors; however, intracellular signaling proteins, such as Jaks and STATs, can also be targeted in clinical settings. Several pharmacological inhibitors have been developed and approved for treating patients with various autoimmune and inflammatory diseases.¹⁷⁸⁾ For example, JAK inhibitors, such as tofacitinib, baricitinib, ruxolitinib, upadacitinib, oclacitinib, and peficitinib, mainly bind to the ATP binding pocket of the catalytically active kinase JH1 domain. Because the catalytic domain is well conserved, the inhibitors often influence multiple Jaks with low selectivity (Fig. 5). Their clinical benefits are confirmed in many types of diseases, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, myeloproliferative neoplasms, and graft-versus-host disease; therefore, they have been approved by the Food and Drug Administration.

Fig. 5. Inhibitors of the IL-6/STAT3-Mediated Signaling

Siltuximab, sirukumab, clazakizumab, and olokizumab are anti-IL-6 blocking antibodies. Tocilizumab and sarilumab are blocking antibodies that target IL-6R. These antibodies inhibit both the classical and trans-signaling pathways. The gp130-Fc fusion protein, olamkicept inhibits IL-6 trans-signalling but not the classical signaling pathway. Tofacitinib, baricitinib, ruxolitinib, upadacitinib, oclacitinib, and peficitinib are small-molecule protein tyrosine kinase inhibitors that target Jaks, preventing phosphorylation of STAT3. Both niclosamide and nitidine chloride inhibit STAT3 phosphorylation. Src homology domain 2 (SH2) domain inhibitors such as Stattic and C188-9, interfere with STAT3 dimerization. N-4-Hydroxyphenylretinamide also targets STAT3 dimerization. The STAT3 antisense oligonucleotide AZD9150 binds to and induces the destruction of STAT3 mRNA. MPT0B098 acts as a SOCS3 stabilizer. IL-6: interleukin 6; STAT3: signal transducer and activator of transcription 3; SOCS: suppressor of cytokine signaling.

Abundant evidence has proposed that STAT3 might be a suitable molecular target to treat patients with immune and/or inflammatory and malignant diseases^53,55,179) (Fig. 5). Some possible strategies to inhibit STAT3 functions include: (1) the blockage of upstream signaling mechanisms, (2) the inhibition of STAT3 activation through its binding to the SH2 domain, (3) the inhibition of STAT3 phosphorylation, (4) nuclear-targeted siRNAs to silence STAT3 gene, (5) the modification of the interactions with STAT3-binding partners. Many inhibitors have been designed to indirectly block STST3 activation by suppressing the IL-6/IL-6R system and JAK functions. Manipulating intrinsic STAT3 inhibitors, such as SOCS and PTPs, might be a potential strategy to repress the STAT3 signaling pathway. For example, MPT0B098 inhibits the JAK2/STAT3 signaling pathway by modulating SOCS3 stability.¹⁸⁰⁾ Direct STAT3 inhibitors have been developed based on the binding to the STAT3 functional domains, with a major focus on the SH2 domain. For example, N-4-hydroxyphenylretinamide can tightly bind to the dimerization site of STAT3.¹⁸¹⁾ Stattic, a STAT3 SH2-domain inhibitor discovered by high-throughput chemical library screening, significantly reduces tumor invasion and outgrowth by targeting STAT3.¹⁸²⁾ C188-9, a potent STAT3 inhibitor, which targets the SH2 domain, suppresses STAT3 phosphorylation promoted by granulocyte colony-stimulating factor and kills acute myeloid leukemia cells.¹⁸³⁾ However, the STAT3 SH2 domain has high homology with other family members; therefore, STAT3 SH2-domain inhibitors may exhibit severe off-target toxicity.^55,157,173) The other direct STAT3 inhibitors interfere with STAT3 phosphorylation. For example, niclosamide and nitidine chloride inhibit STAT3 phosphorylation at Tyr705 and transcription of its target genes.^184,185) Post-transcriptional inhibition of STAT3 synthesis using siRNAs and peptide inhibitors designed from amino acid residues of the STAT3 target-specific domains are also possible methods to inhibit the STAT3 pathway. AZD9150 is an antisense oligonucleotide carrying a restricted ethyl modification targeting STAT3.¹⁸⁶⁾ It decreases STAT3 expression and has antitumor activity in malignant lymphoma, neuroblastoma, and lung cancer. As mentioned above, some novel STAT3-binding partners regulate STAT3 activities, and their manipulation is likely to control STAT3 activities during disease progression. Especially, STAP-2 may be a suitable target because STAP-2-deficiency inhibits characteristic phenotypes after stimulation of cells or mice, but no obvious phenotype is observed under steady-state conditions.^55,104,105) New STAT3 regulatory strategies to impede disease progression will be developed shortly.

7. CONCLUSION

I reviewed IL-6/IL-6R/STAT3 signaling mechanisms and components and the possible role of their inhibition in the development of clinical therapeutic strategies. Both IL-6 and STAT3 are involved in physiological and pathophysiological events within the body. The IL-6/IL-6R systems play essential roles in immune homeostasis, defense against infection, and tissue repair after injury; therefore, their components are measured as diagnostic indicators and markers of disease activity. Analysis of the Jak-STAT pathway has improved our understanding of how cells and tissues are influenced during health and disease. Although therapeutic antibodies against IL-6 or IL-6R are approved for clinical application to treat immune/inflammatory diseases and IL-6-related malignancies, the development of small molecular compounds to regulate STAT3 activity is still underway.^55,173)

Further studies are required to gather precise information about drug delivery, avoiding adverse effects, and translation research guiding clinical applications. Special attention must be paid to studying the adverse effects of STAT3 inhibitors. STAT3 has high sequence and structural similarity with other STAT members, increasing the risks associated with off-target effects.^55,157,173) Because STAT3-deficient mice are embryonic lethal,¹⁸⁷⁾ risks of blocking STAT3 require attention. Therefore, the inhibition of new STAT3-binding partners may control STAT3 activity under malignancy or inflammation without severe adverse effects because their effect on STAT3 activity is mild. Hence, further experiments, including establishing low molecular compounds to inhibit their interaction with STAT3, could give us helpful information about their clinical utility and physiological and/or pathological significance. In conclusion, a comprehensive understanding of the molecular mechanisms of the IL-6/IL-6R/STAT3 signaling is essential to develop novel targeting strategies that can effectively suppress these signals with high selectivity and specificity.

Acknowledgments

The author thanks Dr. K. Oritani for his careful reading of this manuscript and his many insightful comments and suggestions. Further, the author would like to thank all members of my past and present laboratories at the Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University as well as many collaborators for conducting the above-mentioned research. The author also appreciates the financial support in part from the Grant-in-Aid for Scientific Research (KAKENHI) of Japan Society for the Promotion of Science and the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of Interest

The author declares no conflict of interest.

Notes

This review of the author’s work was written by the author upon receiving the 2022 Pharmaceutical Society of Japan Award.

REFERENCES

• 1) Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K, Koyama K, Iwamatsu A, Tsunasawa T, Sakiyama F, Matsui H, Takahara Y, Taniguchi T, Kishimoto T. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin. Nature, 324, 73–76 (1986).
• 2) Gauldie J, Richards C, Harnish D, Lansdorp P, Baumann H. Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells. Proc. Natl. Acad. Sci. U.S.A., 84, 7251–7255 (1987).
• 3) Van Damme J, Opdenakker G, Simpson RJ, Rubira MR, Cayphas S, Vink A, Billiau A, Van Snick J. Identification of the human 26-kD protein, interferon beta 2 (IFN-beta 2), as a B cell hybridoma/plasmacytoma growth factor induced by interleukin 1 and tumor necrosis factor. J. Exp. Med., 165, 914–919 (1987).
• 4) Andus T, Geiger T, Hirano T, Northoff H, Ganter U, Bauer J, Kishimoto T, Heinrich PC. Recombinant human B cell stimulatory factor 2 (BSF-2/IFNβ2) regulates β-fibrinogen and albumin mRNA levels in Fao-9 cells. FEBS Lett., 221, 18–22 (1987).
• 5) Matsuda T, Hirano T. Interleukin-6 (IL-6). Biotherapy, 2, 363–373 (1990).
• 6) Kang S, Narazaki M, Metwally H, Kishimoto T. Historical overview of the interleukin-6 family cytokine. J. Exp. Med., 217, e20190347 (2020).
• 7) Hirano T. Interleukin 6 and its receptor: ten years later. Int. Rev. Immunol., 16, 249–284 (1998).
• 8) Hirano T, Matsuda T, Turner M, Miyasaka N, Buchan G, Tang B, Sato K, Shimizu M, Maini R, Feldmann M, Kishimoto T. Excessive production of interleukin 6/B cell stimulatory factor-2 in rheumatoid arthritis. Eur. J. Immunol., 18, 1797–1802 (1988).
• 9) Yoshizaki K, Matsuda T, Nishimoto N, Kuritani T, Taeho L, Aozasa K, Nakahata T, Kawai H, Tagoh H, Komori T, Kishimoto S, Hirano T, Kishimoto T. Pathogenic significance of interleukin 6 (IL-6/BSF-2) in Castleman’s disease. Blood, 74, 1360–1367 (1989).
• 10) Mosmann TR, Coffman RL. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol., 7, 145–173 (1989).
• 11) Bettelli E, Korn T, Oukka M, Kuchroo VK. Induction and effector functions of T(H)17 cells. Nature, 453, 1051–1057 (2008).
• 12) Zhou L, Lopes JE, Chong MM, Ivanov II, Min R, Victora GD, Shen Y, Du J, Rubtsov YP, Rudensky AY, Ziegler SF, Littman DR. TGF-beta-induced Foxp3 inhibits T_H17 cell differentiation by antagonizing RORgammat function. Nature, 453, 236–240 (2008).
• 13) Eto D, Lao C, DiToro D, Barnett B, Escobar TC, Kageyama R, Yusuf I, Crotty S. IL-21 and IL-6 are critical for different aspects of B cell immunity and redundantly induce optimal follicular helper CD4 T cell (Tfh) differentiation. PLoS ONE, 6, e17739 (2011).
• 14) Hunter CA, Jones SA. IL-6 as a keystone cytokine in health and disease. Nat. Immunol., 16, 448–457 (2015).
• 15) Jones SA, Jenkins BJ. Recent insights into targeting the IL-6 cytokine family in inflammatory diseases and cancer. Nat. Rev. Immunol., 18, 773–789 (2018).
• 16) Garbers C, Heink S, Korn T, Rose-John S. Interleukin-6: designing specific therapeutics for a complex cytokine. Nat. Rev. Drug Discov., 17, 395–412 (2018).
• 17) Millrine D, Jenkins RH, Hughes STO, Jones SA. Making sense of IL-6 signalling cues in pathophysiology. FEBS Lett., 596, 567–588 (2022).
• 18) Kang S, Tanaka T, Narazaki M, Kishimoto T. Targeting Interleukin-6 signaling in clinic. Immunity, 50, 1007–1023 (2019).
• 19) Choy EH, De Benedetti F, Takeuchi T, Hashizume M, John MR, Kishimoto T. Translating IL-6 biology into effective treatments. Nat. Rev. Rheumatol., 16, 335–345 (2020).
• 20) Moore JB, June CH. Cytokine release syndrome in severe COVID-19. Science, 368, 473–474 (2020).
• 21) Kishimoto T. IL-6: from arthritis to CAR-T-cell therapy and COVID-19. Int. Immunol., 33, 515–519 (2021).
• 22) Hirano T. IL-6 in inflammation, autoimmunity and cancer. Int. Immunol., 33, 127–148 (2021).
• 23) Jones SA, Hunter CA. Is IL-6 a key cytokine target for therapy in COVID-19? Nat. Rev. Immunol., 21, 337–339 (2021).
• 24) Holland SM, DeLeo FR, Elloumi HZ, et al. STAT3 mutations in the hyper-IgE syndrome. N. Engl. J. Med., 357, 1608–1619 (2007).
• 25) Kreins AY, Ciancanelli MJ, Okada S, et al. Human TYK2 deficiency: mycobacterial and viral infections without hyper-IgE syndrome. J. Exp. Med., 212, 1641–1662 (2015).
• 26) Schwerd T, Twigg SRF, Aschenbrenner D, et al. A biallelic mutation in IL6ST encoding the GP130 co-receptor causes immunodeficiency and craniosynostosis. J. Exp. Med., 214, 2547–2562 (2017).
• 27) Shahin T, Aschenbrenner D, Cagdas D, Bal SK, Conde CD, Garncarz W, Medgyesi D, Schwerd T, Karaatmaca B, Cetinkaya PG, Esenboga S, Twigg SRF, Cant A, Wilkie AOM, Tezcan I, Uhlig HH, Boztug K. Selective loss of function variants in IL6ST cause Hyper-IgE syndrome with distinct impairments of T-cell phenotype and function. Haematologica, 104, 609–621 (2019).
• 28) Chen YH, Grigelioniene G, Newton PT, et al. Absence of GP130 cytokine receptor signaling causes extended Stuve-Wiedemann syndrome. J. Exp. Med., 217, e20191306 (2020).
• 29) Spencer S, Kostel Bal S, Egner W, et al. Loss of the interleukin-6 receptor causes immunodeficiency, atopy, and abnormal inflammatory responses. J. Exp. Med., 216, 1986–1998 (2019).
• 30) Jenkins RH, Hughes STO, Figueras AC, Jones SA. Unravelling the broader complexity of IL-6 involvement in health and disease. Cytokine, 148, 155684 (2021).
• 31) Wallenius V, Wallenius K, Ahrén B, Rudling M, Carlsten H, Dickson SL, Ohlsson C, Jansson JO. Interleukin-6-deficient mice develop mature-onset obesity. Nat. Med., 8, 75–79 (2002).
• 32) Hirano T, Matsuda T, Nakajima K. Signal transduction through gp130 that is shared among the receptors for the interleukin 6 related cytokine subfamily. Stem Cells, 12, 262–277 (1994).
• 33) Morin-Poulard I, Vincent A, Crozatier M. The Drosophila JAK-STAT pathway in blood cell formation and immunity. JAK-STAT, 2, e25700 (2013).
• 34) Moore PS, Boshoff C, Weiss RA, Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV. Science, 274, 1739–1744 (1996).
• 35) Fielding CA, McLoughlin RM, Colmont CS, Kovaleva M, Harris DA, Rose-John S, Topley N, Jones SA. Viral IL-6 blocks neutrophil infiltration during acute inflammation. J. Immunol., 175, 4024–4029 (2005).
• 36) Molden J, Chang Y, You Y, Moore PS, Goldsmith MA. A Kaposi’s sarcoma-associated herpesvirus-encoded cytokine homolog (vIL-6) activates signaling through the shared gp130 receptor subunit. J. Biol. Chem., 272, 19625–19631 (1997).
• 37) Jones SA, Scheller J, Rose-John S. Therapeutic strategies for the clinical blockade of IL-6/gp130 signaling. J. Clin. Invest., 121, 3375–3383 (2011).
• 38) Gearing DP, Cosman D. Homology of the p40 subunit of natural killer cell stimulatory factor (NKSF) with the extracellular domain of the interleukin-6 receptor. Cell, 66, 9–10 (1991).
• 39) Jones SA, Horiuchi S, Topley N, Yamamoto N, Fuller GM. The soluble interleukin 6 receptor: mechanisms of production and implications in disease. FASEB J., 15, 43–58 (2001).
• 40) IL6R Genetics Consortium Emerging Risk Factors Collaboration, Sarwar N, Butterworth AS, et al. Interleukin-6 receptor pathways in coronary heart disease: a collaborative meta-analysis of 82 studies. Lancet, 379, 1205–1213 (2012).
• 41) Ferreira RC, Freitag DF, Cutler AJ, Howson JM, Rainbow DB, Smyth DJ, Kaptoge S, Clarke P, Boreham C, Coulson RM, Pekalski ML, Chen WM, Onengut-Gumuscu S, Rich SS, Butterworth AS, Malarstig A, Danesh J, Todd JA. Functional IL6R 358Ala allele impairs classical IL-6 receptor signaling and influences risk of diverse inflammatory diseases. PLoS Genet., 9, e1003444 (2013).
• 42) Heink S, Yogev N, Garbers C, et al. Trans-presentation of IL-6 by dendritic cells is required for the priming of pathogenic T_H17 cells. Nat. Immunol., 18, 74–85 (2017).
• 43) Rabe B, Chalaris A, May U, Waetzig GH, Seegert D, Williams AS, Jones SA, Rose-John S, Scheller J. Transgenic blockade of interleukin 6 transsignaling abrogates inflammation. Blood, 111, 1021–1028 (2008).
• 44) Lamertz L, Rummel F, Polz R, Baran P, Hansen S, Waetzig GH, Moll JM, Floss DM, Scheller J. Soluble gp130 prevents interleukin-6 and interleukin-11 cluster signaling but not intracellular autocrine responses. Sci. Signal., 11, eaar7388 (2018).
• 45) Ihle JN. STATs: signal transducers and activators of transcription. Cell, 84, 331–334 (1996).
• 46) O’Shea JJ. Jaks, STATs, cytokine signal transduction, and immunoregulation: are we there yet? Immunity, 7, 1–11 (1997).
• 47) Darnell JE Jr. STATs and gene regulation. Science, 277, 1630–1635 (1997).
• 48) Leonard WJ, O’Shea JJ. Jaks and STATs: biological implications. Annu. Rev. Immunol., 16, 293–322 (1998).
• 49) Levy DE, Darnell JE Jr. STATs: transcriptional control and biological impact. Nat. Rev. Mol. Cell Biol., 3, 651–662 (2002).
• 50) Stark GR, Darnell JE Jr. The JAK-STAT pathway at twenty. Immunity, 36, 503–514 (2012).
• 51) Villarino AV, Kanno Y, O’Shea JJ. Mechanisms and consequences of Jak-STAT signaling in the immune system. Nat. Immunol., 18, 374–384 (2017).
• 52) Taniguchi K, Wu LW, Grivennikov SI, de Jong PR, Lian I, Yu FX, Wang K, Ho SB, Boland BS, Chang JT, Sandborn WJ, Hardiman G, Raz E, Maehara Y, Yoshimura A, Zucman-Rossi J, Guan KL, Karin M. A gp130-Src-YAP module links inflammation to epithelial regeneration. Nature, 519, 57–62 (2015).
• 53) Yu H, Lee H, Herrmann A, Buettner R, Jove R. Revisiting STAT3 signalling in cancer: new and unexpected biological functions. Nat. Rev. Cancer, 14, 736–746 (2014).
• 54) Matsuda T, Muromoto R, Sekine Y, Togi S, Kitai Y, Kon S, Oritani K. Signal transducer and activator of transcription 3 regulation by novel binding partners. World J. Biol. Chem., 6, 324–332 (2015).
• 55) Hashimoto S, Hashimoto A, Muromoto R, Kitai Y, Oritani K, Matsuda T. Central roles of STAT3-mediated signals in onset and development of cancers: tumorigenesis and immunosurveillance. Cells, 11, 2618 (2022).
• 56) O’Shea JJ, Gadina M, Schreiber RD. Cytokine signaling in 2002: new surprises in the Jak/STAT pathway. Cell, 109 (Suppl.), S121–S131 (2002).
• 57) Shuai K, Liu B. Regulation of JAK–STAT signalling in the immune system. Nat. Rev. Immunol., 3, 900–911 (2003).
• 58) Yamamoto T, Sekine Y, Kashima K, Kubota A, Sato N, Aoki N, Matsuda T. The nuclear isoform of protein-tyrosine phosphatase TC-PTP regulates interleukin-6-mediated signaling pathway through STAT3 dephosphorylation. Biochem. Biophys. Res. Commun., 297, 811–817 (2002).
• 59) Sekine Y, Tsuji S, Ikeda O, Sato N, Aoki N, Aoyama K, Sugiyama K, Matsuda T. Regulation of STAT3-mediated signaling by LMW-DSP2. Oncogene, 25, 5801–5806 (2006).
• 60) Böhmer FD, Friedrich K. Protein tyrosine phosphatases as wardens of STAT signaling. JAK-STAT, 3, e28087 (2014).
• 61) Shuai K, Liu B. Regulation of gene-activation pathways by PIAS proteins in the immune system. Nat. Rev. Immunol., 5, 593–605 (2005).
• 62) Babon JJ, Nicola NA. The biology and mechanism of action of suppressor of cytokine signaling 3. Growth Factors, 30, 207–219 (2012).
• 63) Croker BA, Krebs DL, Zhang JG, Wormald S, Willson TA, Stanley EG, Robb L, Greenhalgh CJ, Förster I, Clausen BE, Nicola NA, Metcalf D, Hilton DJ, Roberts AW, Alexander WS. SOCS3 negatively regulates IL-6 signaling in vivo. Nat. Immunol., 4, 540–545 (2003).
• 64) Lang R, Pauleau AL, Parganas E, Takahashi Y, Mages J, Ihle JN, Rutschman R, Murray PJ. SOCS3 regulates the plasticity of gp130 signaling. Nat. Immunol., 4, 546–550 (2003).
• 65) Yasukawa H, Ohishi M, Mori H, Murakami M, Chinen T, Aki D, Hanada T, Takeda K, Akira S, Hoshijima M, Hirano T, Chien KR, Yoshimura A. IL-6 induces an anti-inflammatory response in the absence of SOCS3 in macrophages. Nat. Immunol., 4, 551–556 (2003).
• 66) Linossi EM, Babon JJ, Hilton DJ, Nicholson SE. Suppression of cytokine signaling: the SOCS perspective. Cytokine Growth Factor Rev., 24, 241–248 (2013).
• 67) Yu Y, Gu S, Li W, Sun C, Chen F, Xiao M, Wang L, Xu D, Li Y, Ding C, Xia Z, Li Y, Ye S, Xu P, Zhao B, Qin J, Chen YG, Lin X, Feng XH. Smad7 enables STAT3 activation and promotes pluripotency independent of TGF-β signaling. Proc. Natl. Acad. Sci. U.S.A., 114, 10113–10118 (2017).
• 68) Tanaka Y, Tanaka N, Saeki Y, Tanaka K, Murakami M, Hirano T, Ishii N, Sugamura K. c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130. Mol. Cell. Biol., 28, 4805–4818 (2008).
• 69) Yokogami K, Wakisaka S, Avruch J, Reeves SA. Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR. Curr. Biol., 10, 47–50 (2000).
• 70) Chung J, Uchida E, Grammer TC, Blenis J. STAT3 serine phosphorylation by ERK-dependent and -independent pathways negatively modulates its tyrosine phosphorylation. Mol. Cell. Biol., 17, 6508–6516 (1997).
• 71) Jain N, Zhang T, Kee WH, Li W, Cao X. Protein kinase C delta associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. J. Biol. Chem., 274, 24392–24400 (1999).
• 72) Sato N, Kawai T, Sugiyama K, Muromoto R, Imoto S, Sekine Y, Ishida M, Akira S, Matsuda T. Physical and functional interactions between STAT3 and ZIP kinase. Int. Immunol., 17, 1543–1552 (2005).
• 73) Shen Y, Schlessinger K, Zhu X, Meffre E, Quimby F, Levy DE, Darnell JE Jr. Essential role of STAT3 in postnatal survival and growth revealed by mice lacking STAT3 serine 727 phosphorylation. Mol. Cell. Biol., 24, 407–419 (2004).
• 74) Schuringa JJ, Schepers H, Vellenga E, Kruijer W. Ser727-dependent transcriptional activation by association of p300 with STAT3 upon IL-6 stimulation. FEBS Lett., 495, 71–76 (2001).
• 75) Yuan ZL, Guan YJ, Chatterjee D, Chin YE. Stat3 dimerization regulated by reversible acetylation of a single lysine residue. Science, 307, 269–273 (2005).
• 76) Wang R, Cherukuri P, Luo J. Activation of Stat3 sequence-specific DNA binding and transcription by p300/CREB-binding protein-mediated acetylation. J. Biol. Chem., 280, 11528–11534 (2005).
• 77) Zhou Z, Wang M, Li J, Xiao M, Chin YE, Cheng J, Yeh ET, Yang J, Yi J. SUMOylation and SENP3 regulate STAT3 activation in head and neck cancer. Oncogene, 35, 5826–5838 (2016).
• 78) Dasgupta M, Dermawan JK, Willard B, Stark GR. STAT3-driven transcription depends upon the dimethylation of K49 by EZH2. Proc. Natl. Acad. Sci. U.S.A., 112, 3985–3990 (2015).
• 79) Yin Y, Yang X, Wu S, Ding X, Zhu H, Long X, Wang Y, Zhai S, Chen Y, Che N, Chen J, Wang X. Jmjd1c demethylates STAT3 to restrain plasma cell differentiation and rheumatoid arthritis. Nat. Immunol., 23, 1342–1354 (2022).
• 80) Zhang M, Zhou L, Xu Y, Yang M, Xu Y, Komaniecki GP, Kosciuk T, Chen X, Lu X, Zou X, Linder ME, Lin HA. STAT3 palmitoylation cycle promotes T_H17 differentiation and colitis. Nature, 586, 434–439 (2020).
• 81) Contreras J, Rao D. MicroRNAs in inflammation and immune responses. Leukemia, 26, 404–413 (2012).
• 82) Johnson DE, O’Keefe RA, Grandis JR. Targeting the IL-6/JAK/STAT3 signalling axis in cancer. Nat. Rev. Clin. Oncol., 15, 234–248 (2018).
• 83) Iliopoulos D, Hirsch HA, Struhl K. An epigenetic switch involving NF-kappaB, Lin28, Let-7 MicroRNA, and IL6 links inflammation to cell transformation. Cell, 139, 693–706 (2009).
• 84) Collins AS, McCoy CE, Lloyd AT, O’Farrelly C, Stevenson NJ. miR-19a: an effective regulator of SOCS3 and enhancer of JAK-STAT signalling. PLoS ONE, 8, e69090 (2013).
• 85) Wu W, Takanashi M, Borjigin N, Ohno SI, Fujita K, Hoshino S, Osaka Y, Tsuchida A, Kuroda M. MicroRNA-18a modulates STAT3 activity through negative regulation of PIAS3 during gastric adenocarcinogenesis. Br. J. Cancer, 108, 653–661 (2013).
• 86) Tanaka T, Narazaki M, Kishimoto T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb. Perspect. Biol., 6, a016295 (2014).
• 87) Anderson P. Post-transcriptional regulons coordinate the initiation and resolution of inflammation. Nat. Rev. Immunol., 10, 24–35 (2010).
• 88) Yoshinaga M, Takeuchi O. Post-transcriptional control of immune responses and its potential application. Clin. Transl. Immunology, 8, e1063 (2019).
• 89) Akira S, Maeda K. Control of RNA stability in immunity. Annu. Rev. Immunol., 39, 481–509 (2021).
• 90) Paschoud S, Dogar AM, Kuntz C, Grisoni-Neupert B, Richman L, Kühn LC. Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1. Mol. Cell. Biol., 26, 8228–8241 (2006).
• 91) Masuda K, Kishimoto T. A potential therapeutic target RNA-binding protein, Arid5a for the treatment of inflammatory disease associated with aberrant cytokine expression. Curr. Pharm. Des., 24, 1766–1771 (2018).
• 92) Matsushita K, Takeuchi O, Standley DM, Kumagai Y, Kawagoe T, Miyake T, Satoh T, Kato H, Tsujimura T, Nakamura H, Akira S. Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA decay. Nature, 458, 1185–1190 (2009).
• 93) Uehata T, Iwasaki H, Vandenbon A, Matsushita K, Hernandez-Cuellar E, Kuniyoshi K, Satoh T, Mino T, Suzuki Y, Standley DM, Tsujimura T, Rakugi H, Isaka Y, Takeuchi O, Akira S. Malt1-induced cleavage of regnase-1 in CD4⁺ helper T cells regulates immune activation. Cell, 153, 1036–1049 (2013).
• 94) Salomoni P, Khelifi AF. Daxx: death or survival protein? Trends Cell Biol., 16, 97–104 (2006).
• 95) Mahmud I, Liao D. DAXX in cancer: phenomena, processes, mechanisms and regulation. Nucleic Acids Res., 47, 7734–7752 (2019).
• 96) Kawai T, Matsumoto M, Takeda K, Sanjo H, Akira S. ZIP kinase, a novel serine/threonine kinase which mediates apoptosis. Mol. Cell. Biol., 18, 1642–1651 (1998).
• 97) Usui T, Okada M, Yamawaki H. Zipper interacting protein kinase (ZIPK): function and signaling. Apoptosis, 19, 387–391 (2014).
• 98) Agata Y, Matsuda E, Shimizu A. Two novel Kruppel-associated box-containing zinc-finger proteins, KRAZ1 and KRAZ2, repress transcription through functional interaction with the corepressor KAP-1 (TIF1b/KRIP-1). J. Biol. Chem., 274, 16412–16422 (1999).
• 99) Friedman JR, Fredericks WJ, Jensen DE, Speicher DW, Huang XP, Neilson EG, Rauscher FJ 3rd. KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev., 10, 2067–2078 (1996).
• 100) Kim SS, Chen YM, O’Leary E, Witzgall R, Vidal M, Bonventre JV. A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins. Proc. Natl. Acad. Sci. U.S.A., 93, 15299–15304 (1996).
• 101) Chuang TW, Lee KM, Tarn WY. Function and pathological implications of exon junction complex factor Y14. Biomolecules, 5, 343–355 (2015).
• 102) Tanaka T, Soriano MA, Grusby MJ. SLIM is a nuclear ubiquitin E3 ligase that negatively regulates STAT signaling. Immunity, 22, 729–736 (2005).
• 103) Guo ZS, Qu Z. PDLIM2: signaling pathways and functions in cancer suppression and host immunity. Biochim. Biophys. Acta Rev. Cancer, 1876, 188630 (2021).
• 104) Matsuda T, Oritani K. STAP-2 adaptor protein regulates multiple steps of immune and inflammatory responses. Biol. Pharm. Bull., 44, 895–901 (2021).
• 105) Matsuda T, Oritani K. Possible therapeutic applications of targeting STAP proteins in cancer. Biol. Pharm. Bull., 44, 1810–1818 (2021).
• 106) Li R, Pei H, Watson DK, Papas TS. EAP1/Daxx interacts with ETS1 and represses transcriptional activation of ETS1 target genes. Oncogene, 19, 745–753 (2000).
• 107) Emelyanov AV, Kovac CR, Sepulveda MA, Birshtein BK. The interaction of Pax5 (BSAP) with Daxx can result in transcriptional activation in B cells. J. Biol. Chem., 277, 11156–11164 (2002).
• 108) Lin DY, Lai MZ, Ann DK, Shih HM. Promyelocytic leukemia protein (PML) functions as a glucocorticoid receptor co-activator by sequestering Daxx to the PML oncogenic domains (PODs) to enhance its transactivation potential. J. Biol. Chem., 278, 15958–15965 (2003).
• 109) Park J, Lee JH, La M, Jang MJ, Chae GW, Kim SB, Tak H, Jung Y, Byun B, Ahn JK, Joe CO. Inhibition of NF-kappaB acetylation and its transcriptional activity by Daxx. J. Mol. Biol., 368, 388–397 (2007).
• 110) Croxton R, Puto LA, de Belle I, Thomas M, Torii S, Hanaii F, Cuddy M, Reed JC. Daxx represses expression of a subset of antiapoptotic genes regulated by nuclear factor-kappaB. Cancer Res., 66, 9026–9035 (2006).
• 111) Tzeng SL, Cheng YW, Li CH, Lin YS, Hsu HC, Kang JJ. Physiological and functional interactions between Tcf4 and Daxx in colon cancer cells. J. Biol. Chem., 281, 15405–15411 (2006).
• 112) Chang CC, Lin DY, Fang HI, Chen RH, Shih HM. Daxx mediates the small ubiquitin-like modifier-dependent transcriptional repression of Smad4. J. Biol. Chem., 280, 10164–10173 (2005).
• 113) Wethkamp N, Klempnauer KH. Daxx is a transcriptional repressor of CCAAT/enhancer-binding protein beta. J. Biol. Chem., 284, 28783–28794 (2009).
• 114) Meloni A, Fiorillo E, Corda D, Incani F, Serra ML, Contini A, Cao A, Rosatelli MC. DAXX is a new AIRE-interacting protein. J. Biol. Chem., 285, 13012–13021 (2010).
• 115) Hollenbach AD, McPherson CJ, Mientjes EJ, Iyengar R, Grosveld G. Daxx and histone deacetylase II associate with chromatin through an interaction with core histones and the chromatin-associated protein Dek. J. Cell Sci., 115, 3319–3330 (2002).
• 116) Puto LA, Reed JC. Daxx represses RelB target promoters via DNA methyltransferase recruitment and DNA hypermethylation. Genes Dev., 22, 998–1010 (2008).
• 117) Muromoto R, Sugiyama K, Takachi A, Imoto S, Sato N, Yamamoto T, Oritani K, Shimoda K, Matsuda T. Physical and functional interactions between Daxx and DNA methyltransferase 1-associated protein, DMAP1. J. Immunol., 172, 2985–2993 (2004).
• 118) Xue Y, Gibbons R, Yan Z, Yang D, McDowell TL, Sechi S, Qin J, Zhou S, Higgs D, Wang W. The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies. Proc. Natl. Acad. Sci. U.S.A., 100, 10635–10640 (2003).
• 119) Tang J, Wu S, Liu H, Stratt R, Barak OG, Shiekhattar R, Picketts DJ, Yang X. A novel transcription regulatory complex containing death domain-associated protein and the ATR-X syndrome protein. J. Biol. Chem., 279, 20369–20377 (2004).
• 120) Muromoto R, Nakao K, Watanabe T, Sato N, Sekine Y, Sugiyama K, Oritani K, Shimoda K, Matsuda T. Physical and functional interactions between Daxx and STAT3. Oncogene, 25, 2131–2136 (2006).
• 121) Muromoto R, Kuroda M, Togi S, Sekine Y, Nanbo A, Shimoda K, Oritani K, Matsuda T. Functional involvement of Daxx in gp130-mediated cell growth and survival in BaF3 cells. Eur. J. Immunol., 40, 3570–3580 (2010).
• 122) Kögel D, Prehn JH, Scheidtmann KH. The DAP kinase family of pro-apoptotic proteins: novel players in the apoptotic game. BioEssays, 23, 352–358 (2001).
• 123) Shohat G, Shani G, Eisenstein M, Kimchi A. The DAP-kinase family of proteins: study of a novel group of calcium-regulated death-promoting kinases. Biochim. Biophys. Acta, 1600, 45–50 (2002).
• 124) Inbal B, Cohen O, Polak-Charcon S, Kopolovic J, Vadai E, Eisenbach L, Kimchi A. DAP kinase links the control of apoptosis to metastasis. Nature, 390, 180–184 (1997).
• 125) Kawai T, Akira S, Reed JC. ZIP kinase triggers apoptosis from nuclear PML oncogenic domains. Mol. Cell. Biol., 23, 6174–6186 (2003).
• 126) Sato N, Kamada N, Muromoto R, Kawai T, Sugiyama K, Watanabe T, Imoto S, Sekine Y, Ohbayashi N, Ishida M, Akira S, Matsuda T. Phosphorylation of threonine-265 in Zipper-interacting protein kinase plays an important role in its activity and is induced by IL-6 family cytokines. Immunol. Lett., 103, 127–134 (2006).
• 127) Underhill C, Qutob MS, Yee SP, Torchia J. A novel nuclear receptor corepressor complex, N-CoR, contains components of the mammalian SWI/SNF complex and the corepressor KAP-1. J. Biol. Chem., 275, 40463–40470 (2000).
• 128) Schultz DC, Friedman JR, Rauscher FJ 3rd. Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2 α subunit of NuRD. Genes Dev., 15, 428–443 (2001).
• 129) Satou A, Taira T, Iguchi-Ariga SM, Ariga H. A novel transrepression pathway of c-Myc. Recruitment of a transcriptional corepressor complex to c-Myc by MM-1, a c-Myc-binding protein. J. Biol. Chem., 276, 46562–46567 (2001).
• 130) Schultz DC, Ayyanathan K, Negorev D, Maul GG, Rauscher FJ 3rd. SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev., 16, 919–932 (2002).
• 131) Tsuruma R, Ohbayashi N, Kamitani S, Ikeda O, Sato N, Muromoto R, Sekine Y, Oritani K, Matsuda T. Physical and functional interactions between STAT3 and KAP1. Oncogene, 27, 3054–3059 (2008).
• 132) Kataoka N, Yong J, Kim VN, Velazquez F, Perkinson RA, Wang F, Dreyfuss G. Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell, 6, 673–682 (2000).
• 133) Palacios IM. RNA processing: splicing and the cytoplasmic localisation of mRNA. Curr. Biol., 12, R50–R52 (2002).
• 134) Chang YF, Imam JS, Wilkinson MF. The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem., 76, 51–74 (2007).
• 135) Kim VN, Yong J, Kataoka N, Abel L, Diem MD, Dreyfuss G. The Y14 protein communicates to the cytoplasm the position of exon-exon junctions. EMBO J., 20, 2062–2068 (2001).
• 136) Kataoka N, Diem MD, Kim VN, Yong J, Dreyfuss G. Magoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex. EMBO J., 20, 6424–6433 (2001).
• 137) Ohbayashi N, Taira N, Kawakami S, Togi S, Sato N, Ikeda O, Kamitani S, Muromoto R, Sekine Y, Matsuda T. An RNA binding protein, Y14 interacts with and modulates STAT3 activation. Biochem. Biophys. Res. Commun., 372, 475–479 (2008).
• 138) Muromoto R, Taira N, Ikeda O, Shiga K, Kamitani S, Togi S, Kawakami S, Sekine Y, Nanbo A, Oritani K, Matsuda T. The exon-junction complex proteins, Y14 and MAGOH regulate STAT3 activation. Biochem. Biophys. Res. Commun., 382, 63–68 (2009).
• 139) Tanaka T, Yamamoto Y, Muromoto R, Ikeda O, Sekine Y, Grusby M, Kaisho T, Matsuda T. PDLIM2 inhibits T helper 17 cell development and granulomatous inflammation through degradation of STAT3. Sci. Signal., 4, ra85 (2011).
• 140) Liu YC. Ubiquitin ligases and the immune response. Annu. Rev. Immunol., 22, 81–127 (2004).
• 141) Minoguchi M, Minoguchi S, Aki D, Joo A, Yamamoto T, Yumioka T, Matsuda T, Yoshimura A. STAP-2/BKS, an adaptor/docking protein, modulates STAT3 activation in acute-phase response through its YXXQ motif. J. Biol. Chem., 278, 11182–11189 (2003).
• 142) Mitchell PJ, Sara EA, Crompton MR. A novel adaptor-like protein which is a substrate for the non-receptor tyrosine kinase, BRK. Oncogene, 19, 4273–4282 (2000).
• 143) Ikeda O, Miyasaka Y, Sekine Y, Mizushima A, Muromoto R, Nanbo A, Yoshimura A, Matsuda T. STAP-2 is phosphorylated at tyrosine-250 by Brk and modulates Brk-mediated STAT3 activation. Biochem. Biophys. Res. Commun., 384, 71–75 (2009).
• 144) Mitchell PJ, Barker KT, Martindale JE, Kamalati T, Lowe PN, Page MJ, Gusterson BA, Crompton MR. Cloning and characterisation of cDNAs encoding a novel non-receptor tyrosine kinase, brk, expressed in human breast tumours. Oncogene, 9, 2383–2390 (1994).
• 145) Lee ST, Strunk KM, Spritz RA. A survey of protein tyrosine kinase mRNAs expressed in normal human melanocytes. Oncogene, 8, 3403–3410 (1993).
• 146) Derry JJ, Prins GS, Ray V, Tyner AL. Altered localization and activity of the intracellular tyrosine kinase BRK/Sik in prostate tumor cells. Oncogene, 22, 4212–4220 (2003).
• 147) Easty DJ, Mitchell PJ, Patel K, Florenes VA, Spritz RA, Bennett DC. Loss of expression of receptor tyrosine kinase family genes PTK7 and SEK in metastatic melanoma. Int. J. Cancer, 71, 1061–1065 (1997).
• 148) Llor X, Serfas MS, Bie W, Vasioukhin V, Polonskaia M, Derry J, Abbott CM, Tyner AL. BRK/Sik expression in the gastrointestinal tract and in colon tumors. Clin. Cancer Res., 5, 1767–1777 (1999).
• 149) Schmandt RE, Bennett M, Clifford S, Thornton A, Jiang F, Broaddus RR, Sun CC, Lu KH, Sood AK, Gershenson DM. The BRK tyrosine kinase is expressed in high-grade serous carcinoma of the ovary. Cancer Biol. Ther., 5, 1136–1141 (2006).
• 150) Barker KT, Jackson LE, Crompton MR. BRK tyrosine kinase expression in a high proportion of human breast carcinomas. Oncogene, 15, 799–805 (1997).
• 151) Harvey AJ, Crompton MR. Use of RNA interference to validate Brk as a novel therapeutic target in breast cancer: Brk promotes breast carcinoma cell proliferation. Oncogene, 22, 5006–5010 (2003).
• 152) Ikeda O, Sekine Y, Mizushima A, Nakasuji M, Miyasaka Y, Yamamoto C, Muromoto R, Nanbo A, Oritani K, Yoshimura A, Matsuda T. Interactions of STAP-2 with Brk and STAT3 participate in cell growth of human breast cancer cells. J. Biol. Chem., 285, 38093–38103 (2010).
• 153) Kitai Y, Iwakami M, Saitoh K, Togi S, Isayama S, Sekine Y, Muromoto R, Kashiwakura JI, Yoshimura A, Oritani K, Matsuda T. STAP-2 protein promotes prostate cancer growth by enhancing epidermal growth factor receptor stabilization. J. Biol. Chem., 292, 19392–19399 (2017).
• 154) Avraham R, Yarden Y. Feedback regulation of EGFR signalling: decision making by early and delayed loops. Nat. Rev. Mol. Cell Biol., 12, 104–117 (2011).
• 155) Heo T-H, Wahler J, Suh N. Potential therapeutic implications of IL-6/IL-6R/gp130-targeting agents in breast cancer. Oncotarget, 7, 15460–15473 (2016).
• 156) Maemoto T, Kitai Y, Takahashi R, Shoji H, Yamada S, Takei S, Ito D, Muromoto R, Kashiwakura JI, Handa H, Hashimoto A, Hashimoto S, Ose T, Oritani K, Matsuda T. A peptide derived from adaptor protein STAP-2 inhibits tumor progression by downregulating epidermal growth factor receptor signaling. J. Biol. Chem., 299, 102724 (2022).
• 157) Yu H, Jove R. The STATs of cancer—new molecular targets come of age. Nat. Rev. Cancer, 4, 97–105 (2004).
• 158) Lee H, Herrmann A, Deng JH, Kujawski M, Niu G, Li Z, Forman S, Jove R, Pardoll DM, Yu H. Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors. Cancer Cell, 15, 283–293 (2009).
• 159) Yu H, Pardoll D, Jove R. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer, 9, 798–809 (2009).
• 160) He G, Karin M. NF-κB and STAT3—key players in liver inflammation and cancer. Cell Res., 21, 159–168 (2011).
• 161) Fan Y, Mao R, Yang J. NF-κB and STAT3 signaling pathways collaboratively link inflammation to cancer. Protein Cell, 4, 176–185 (2013).
• 162) Ghosh S, May MJ, Kopp EB. NF-kappaB and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol., 16, 225–260 (1998).
• 163) Darnell JE Jr. Transcription factors as targets for cancer therapy. Nat. Rev. Cancer, 2, 740–749 (2002).
• 164) Chen LF, Greene WC. Shaping the nuclear action of NF-kappaB. Nat. Rev. Mol. Cell Biol., 5, 392–401 (2004).
• 165) Welte T, Zhang SS, Wang T, Zhang Z, Hesslein DG, Yin Z, Kano A, Iwamoto Y, Li E, Craft JE, Bothwell AL, Fikrig E, Koni PA, Flavell RA, Fu XY. STAT3 deletion during hematopoiesis causes Crohn’s disease-like pathogenesis and lethality: a critical role of STAT3 in innate immunity. Proc. Natl. Acad. Sci. U.S.A., 100, 1879–1884 (2003).
• 166) Lu Y, Zhou J, Xu C, Lin H, Xiao J, Wang Z, Yang B. JAK/STAT and PI3K/AKT pathways form a mutual transactivation loop and afford resistance to oxidative stress-induced apoptosis in cardiomyocytes. Cell. Physiol. Biochem., 21, 305–314 (2008).
• 167) Dalwadi H, Krysan K, Heuze-Vourc’h N, Dohadwala M, Elashoff D, Sharma S, Cacalano N, Lichtenstein A, Dubinett S. Cyclooxygenase-2-dependent activation of signal transducer and activator of transcription 3 by interleukin-6 in non-small cell lung cancer. Clin. Cancer Res., 11, 7674–7682 (2005).
• 168) Yu Z, Kone BC. The STAT3 DNA-binding domain mediates interaction with NF-kappaB p65 and inducible nitric oxide synthase transrepression in mesangial cells. J. Am. Soc. Nephrol., 15, 585–591 (2004).
• 169) Kesanakurti D, Chetty C, Rajasekhar Maddirela D, Gujrati M, Rao JS. Essential role of cooperative NF-kappaB and Stat3 recruitment to ICAM-1 intronic consensus elements in the regulation of radiation-induced invasion and migration in glioma. Oncogene, 32, 5144–5155 (2013).
• 170) Yang J, Stark GR. Roles of unphosphorylated STATs in signaling. Cell Res., 18, 443–451 (2008).
• 171) O’Shea JJ, Schwartz DM, Villarino AV, Gadina M, McInnes IB, Laurence A. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annu. Rev. Med., 66, 311–328 (2015).
• 172) Villarino AV, Kanno Y, Ferdinand JR, O’Shea JJ. Mechanisms of Jak/STAT signaling in immunity and disease. J. Immunol., 194, 21–27 (2015).
• 173) Hu X, Li J, Fu M, Zhao X, Wang W. The JAK/STAT signaling pathway: from bench to clinic. Signal Transduct. Target. Ther., 6, 402 (2021).
• 174) Tanaka Y, Luo Y, O’Shea JJ, Nakayamada S. Janus kinase-targeting therapies in rheumatology: a mechanisms-based approach. Nat. Rev. Rheumatol., 18, 133–145 (2022).
• 175) Bataille R, Barlogie B, Lu ZY, Rossi JF, Lavabre-Bertrand T, Beck T, Wijdenes J, Brochier J, Klein B. Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma. Blood, 86, 685–691 (1995).
• 176) Lu ZY, Brailly H, Wijdenes J, Bataille R, Rossi JF, Klein B. Measurement of whole body interleukin-6 (IL-6) production: prediction of the efficacy of anti-IL-6 treatments. Blood, 86, 3123–3131 (1995).
• 177) Rose-John S, Winthrop K, Calabrese L. The role of IL-6 in host defence against infections: immunobiology and clinical implications. Nat. Rev. Rheumatol., 13, 399–409 (2017).
• 178) Schwartz D, Kanno Y, Villarino A, Ward M, Gadina M, O’Shea JJ. JAK inhibition as a therapeutic strategy for immune and inflammatory diseases. Nat. Rev. Drug Discov., 16, 843–862 (2017).
• 179) Huynh J, Chand A, Gough D, Ernst M. Therapeutically exploiting STAT3 activity in cancer—using tissue repair as a road map. Nat. Rev. Cancer, 19, 82–96 (2019).
• 180) Peng HY, Cheng YC, Hsu YM, Wu GH, Kuo CC, Liou JP, Chang JY, Jin SL, Shiah SG. MPT0B098, a microtubule inhibitor, suppresses JAK2/STAT3 signaling pathway through modulation of SOCS3 stability in oral squamous cell carcinoma. PLOS ONE, 11, e0158440 (2016).
• 181) Mallery SR, Wang D, Santiago B, Pei P, Schwendeman SP, Nieto K, Spinney R, Tong M, Koutras G, Han B, Holpuch A, Lang J. Benefits of multifaceted chemopreventives in the suppression of the oral squamous cell carcinoma (OSCC) tumorigenic phenotype. Cancer Prev. Res. (Phila), 10, 76–88 (2017).
• 182) Schust J, Sperl B, Hollis A, Mayer TU, Berg T. Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem. Biol., 13, 1235–1242 (2006).
• 183) Redell MS, Ruiz MJ, Alonzo TA, Gerbing RB, Tweardy DJ. Stat3 signaling in acute myeloid leukemia: ligand-dependent and -independent activation and induction of apoptosis by a novel small-molecule Stat3 inhibitor. Blood, 117, 5701–5709 (2011).
• 184) Ren X, Duan L, He Q, Zhang Z, Zhou Y, Wu D, Pan J, Pei D, Ding K. Identification of niclosamide as a new small-molecule inhibitor of the STAT3 signaling pathway. ACS Med. Chem. Lett., 1, 454–459 (2010).
• 185) Chen J, Wang J, Lin L, He L, Wu Y, Zhang L, Yi Z, Chen Y, Pang X, Liu M. Inhibition of STAT3 signaling pathway by nitidine chloride suppressed the angiogenesis and growth of human gastric cancer. Mol. Cancer Ther., 11, 277–287 (2012).
• 186) Hong D, Kurzrock R, Kim Y, et al. AZD9150, a next-generation antisense oligonucleotide inhibitor of STAT3 with early evidence of clinical activity in lymphoma and lung cancer. Sci. Transl. Med., 7, 314ra185 (2015).
• 187) Takeda K, Noguchi K, Shi W, Tanaka T, Matsumoto M, Yoshida N, Kishimoto T, Akira S. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc. Natl. Acad. Sci. U.S.A., 94, 3801–3804 (1997).