Interleukins (ILs) are a group of cytokines that play an important role in inflammation and immunity (Mizel 1989). They act as ligands by binding to the receptors and generating a response from the cell. The signal received from ILs by their receptors is carried forward by a family of proteins called signal transducers and activators of transcription (STATs), which alter the expression of genes in cells exposed to ILs (Levy and Darnell 2002a). In IL-stimulated cells, the STAT family members are phosphorylated and then translocate to the nucleus, where they act as transcription factors. STAT6, which is a member of the STAT family, was first discovered in 1988 by Boothby et al., (Boothby et al. 1988) as a DNA-binding factor in B cells in which its activity increased in the presence of IL4. Further research in the following years recognized tyrosine phosphorylation as the key feature in the protein’s fast activation and translocation to the nucleus, where it binds to a specific DNA sequence motif. These characteristics supported the idea that this factor belongs to the STAT family, which at the time was mostly associated with interferon signaling. Strengthening this suggestion is the fact that the protein has been shown to share similar structural properties, such as the Src homology domain 2 (SH2) and DNA-binding domain (DBD), with already established STATs (Hou et al. 1994).
Variants and Structure
The STAT family consists of seven members, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6. These share many similar properties in terms of structure and domains. These include a coiled-coil domain (CCD, a section that contains α-helices coiled together), a central DBD, a linker domain, an SH2 domain, and a C-terminal transactivation domain (TAD), with each domain serving a different function (Zhuang 2013). In addition, the N-terminus is composed of a 130 amino acid (aa) long region that is conserved in all of the STAT family. This conserved region is required for the tetramerization of dimerized STATs such as STATs 1, 4, and 5 (Levy and Darnell 2002b).
In STAT6, the SH2 domain is of high importance as it works in a dual manner: firstly, it allows the STAT6 binding to the receptor’s phosphorylated tyrosines, and, secondly, it allows the dimerization of STAT6, an important step in signal transduction and gene transcription (Hebenstreit et al. 2006). This domain spans from aa position 532 to 632. By using the DBD, most STATs recognize a palindromic sequence separated by a 3-base-pair (bp) spacer (TTCNNNNGAA) (N3 site), known as an IFNγ activating sequence (GAS), in the chromosomal DNA. However, the DBD of STAT6, which spans aa position from 267 to 448, can only bind to this sequence with low affinity and would preferentially bind to a site with a 4-bp spacer (TTCNNNGAA) (N4 site) with much higher affinity. This is an entirely unique characteristic of STAT6 and is related to the IL4 responsive promoters that mainly consist of N4 sites (Schindler et al. 1995). The C-terminal TAD of STAT6 also differs from that in most other STAT proteins; it is relatively bigger but proline rich like that in other transcription factors; a similar structure is also seen in STAT2 (Hoey and Schindler 1998). The STAT6 TAD can also interact with a number of cofactors and thereby increase the target specificity of STAT6. In addition, the CCD of STAT6 has also been shown to have a potential role in the activated (phosphorylated) STAT6 localization by having the nuclear localization signal (NLS). It is suggested that the aa 136–140 (RLQHR) in this domain are considered to be the NLS (Chen and Reich 2010). Consistent with this, the CCD mutants lacking either NLS or had aa changes in this entire region (aa135–140) show only cytoplasmic localization. However, the point mutations in this region do not affect entry of STAT6 to the nucleus, suggesting the potential of the NLS acting in a structural manner rather than through individual aa (Chen and Reich 2010).
The human STAT6 gene can be found on chromosome 12q13.3-q14.1, spanning 19Kb and containing 23 exons and 22 introns; STAT2 is also found on the same chromosome (Hebenstreit et al. 2006). Interestingly, murine STAT6 and STAT2 are also located on the same chromosome (chromosome 10). Another common characteristic between human and murine STAT6 is that both have the Nab2 gene in close proximity. The STAT6 protein consists of 847aa in human and 837aa in murine, and its calculated molecular mass is about 94 kDa (Hebenstreit et al. 2006).
STAT6 Activation and Regulation
Activation Through IL4 and IL13 Stimulation
The IL4 receptor (IL4R)α mediates STAT6 activation by the cytokine IL4 (Hebenstreit et al. 2006). Upon IL4 binding, IL4Rα gets activated through phosphorylation of the tyrosine residues and then heterodimerizes with the common γ chain (γc). Janus kinases (JAKs), specifically Jak1 and Jak3, bind to the receptor and, upon cytokine stimulation, phosphorylate the IL4Rα on three tyrosine residues (Y575, Y603, Y631). It’s important to note that IL4Rα can also dimerize with another cytokine receptor, the IL13 receptor (IL13Rα1)α1. This dimer can be formed when IL4 binds to IL4Rα or IL13 binds to IL13Rα1, indicating that STAT6 can also be activated by IL13 stimulation. When the IL4Rα-IL13Rα1 dimer is formed, IL13Rα1 is phosphorylated through the tyrosine kinase 2 (Tyk2) (Hebenstreit et al. 2006). However, the other receptor for IL13, IL13Rα2, does not exhibit similar signaling characteristics to IL13Rα1 and so does not have a role in STAT6 activation and signaling.
The phosphorylation of the receptors promotes STAT6 binding to them as the phosphotyrosines of the receptors provide docking sites. The receptor-bound STAT6 is then phosphorylated by Jak and Tyk2 kinases on tyrosine residue Y641, which is located at C-terminus of the SH2 domain. This allows the homodimerization of STAT6 thereby efficient binding to the DNA sequences through the DBD (Hebenstreit et al. 2006). A serine residue at position 756 (located in the TAD) is also phosphorylated with no functional role identified so far for this change (Wang et al. 2004). The STAT6 homodimer then translocates to the nucleus, where it exhibits its transcription factor properties. Small molecules can usually diffuse through the nuclear pore complex (NPC); however, STAT proteins require facilitated import due to their size. An in vitro analysis has shown that STAT6 nuclear translocation can be assisted by importin-α3 and appears to be mediated by the importin-α3:importin-β1 complex (Reich 2013). Once in the nucleus, it can demonstrate its transcription factor properties by binding to specific DNA regions in the genome; the binding to these regions can be both for promoting and repressing gene expression. An example for STAT6 targets is the expression of IL4Rα. This is a positive feedback loop, as transport of the receptor to the plasma membrane will elicit a higher IL4-dependent STAT6 response (Goenka and Kaplan 2011).
Association with Transcription Factors
Since gene expression requires a number of factors in order to occur, STAT6 is not the only factor responsible for the transcription of its target genes. An example for this can be found in B cells for regulation of IgE class switching. This requires not only STAT6 activation through IL4 stimulation but also NF-κB activation through CD40 ligation. The promoter of IgE gene contains one STAT6 binding site and two NF-κB binding sites (p50/p65 and p50/relB). STAT6 associates with the NF-κB dimers and then the complexes bind to their respective promoter regions. The receptor of IgE, Fcer2a, is also regulated by NF-κB and STAT6. Other targets that are dependent on these two factors are activation-induced cytidine deaminase (AID) and eotaxin (CCL11) (Iciek et al. 1997; Goenka and Kaplan 2011).
Other transcription factors that couple their activity with the STAT6 signaling pathway have also been found. One of these is the transcription factor PU.1, which has been seen to participate in regulation of the Iε promoter with binding sites for both PU.1 and STAT6. The essential components for this action are the DBD and TAD of both these factors. C/EBP (CCAAT-enhancer binding protein) beta has also been found to have a role for induction of the Iε promoter by stabilizing STAT6 to its promoter element (Pesu et al. 2003).
Regulation of the Activity
Regulation on the activity of STAT6 is of critical importance as continuous or decreased activity could present with problems leading to diseases. Posttranslational modifications are the first step in regulating its activity. An example of this is SH2 domain-containing tyrosine phosphatase-1 (SHP-1), which is a member of the protein tyrosine phosphatase (PTP) family (Hanson et al. 2003). SHP-1 has been shown to associate with the cytoplasmic tail of the IL4Rα subunit at the phosphotyrosine 713 (pY713) (Hanson et al. 2003). As a phosphatase, it works by dephosphorylating its targets such as STAT6. There are reports to suggest that this does not happen in all cases; for example, SHP-1 dephosphorylates STAT6 in CD8+ T cells but not in CD4+ T cells or bone marrow-derived mast cells. This suggests that SHP-1 could act in a cell-type-specific manner. Another group has suggested that the action of SHP-1 can be carried out only in the nucleus, as it contains a nuclear localization sequence (Hebenstreit et al. 2006). The dephosphorylation of STAT6 through SHP-1 negates the activity of STAT6; thus, it plays a regulatory role in the action of STAT6. Another possible posttranslational modification that can occur is cleavage of STAT6 by proteases.
A number of other factors that can influence STAT6 activity have also been discovered; some of them are coactivators which are recruited in order to form what is called an enhanceosome. The function of the enhanceosome is to recruit more coactivators while also bridging the enhancer to the basal transcriptional machinery. An example for this is the CREB-binding protein (CBP)/p300 complex (Mcdonald and Reich 1999). These are two different proteins that both contain a bromodomain; however, due to the fact that both can be seen in the same complexes, they are most often mentioned together. CBP/p300 complex acts as coactivator in the histone acetylation process occurring through histone acetyltransferases (HATs). This complex has been found to cooperate with STAT6 to regulate STAT6-dependent transcription (Mcdonald and Reich 1999). An example of the transcriptional control of the CBP/p300 complex can be seen in the 15-lipoxygenase-1 (15-LOX-1) gene expression (Shankaranarayanan et al. 2001). It has been shown that the complex activity is increased by IL4 stimulation, and its cofactor functions are essential for the acetylation of both histones and STAT6. These results illustrate another important regulation of STAT6 activity, recognizing acetylation as an important step. Therefore, CBP/p300 can have an important role in the STAT6 activity.
The Role of STAT6 in Health and Diseases
STAT6 Role in Health
As STAT6 has been shown to be a direct downstream factor after IL4 stimulation, it is critical for IL4-dependent cellular functions such as T and B cell proliferation. An additional action of STAT6 after IL4 stimulation is the prevention of apoptosis. This is through the stimulation of expression of anti-apoptotic genes such as the Bcl-xL gene. In addition, it has been shown to be critical for Th2 cell development and differentiation by regulating Gata3, which is considered to be the master regulator for Th2 differentiation (Ansel et al. 2006). Other than promoting immunoglobulin class switching as previously mentioned, STAT6 also promotes cell surface molecules, like major histocompatibility complex II (MHC II), CD80, CD86, and CD23, expression in B cells (Shimoda et al. 1996; Takeda et al. 1996b).
The activity of STAT6 has also been observed in macrophages and dendritic cells. In macrophages, the involvement of STAT6 is similar to that in T and B cells, promoting IL-4-induced differentiation for alternatively activated macrophages (AAM) and gene expression such as MHC II. However, the activity in AAM has been linked with decreased proliferation of T cells. The function of STAT6 in dendritic cells largely differs from that in other cell types, by decreasing the production of IL10 and increasing that of IL12 (Yao et al. 2005; Takeda et al. 1996a; Martinez et al. 2009). As STAT6 is involved in the production of cytokines, it has also been suggested to play a critical role in inflammatory and immune responses for the resident cells.
STAT6 in Allergy
The Jak/STAT pathway has been involved in a number of diseases and allergic reactions (Walford and Doherty 2013). One of these is asthma. Increased levels of phosphorylated STAT6 have been found in the bronchial epithelium of asthmatic people, in addition of having higher levels of IL4 and IL13 (Mullings et al. 2001). Further evidence for the involvement of STAT6 in the pathogenesis of asthma comes from mouse studies, where STAT6 knockout mice did not develop either airway hyperresponsiveness or any lung pathology that would be associated to asthma (Darcan-Nicolaisen et al. 2009). It has also been shown that the development of asthma can be preventable by blocking the recruitment of STAT6 to the IL4Rα and the subsequent signaling cascade that would lead to asthma-related gene transcription (Wang et al. 2011). In addition, IL9 has been characterized as a candidate gene for asthma; this further enhances the role of STAT6 in asthma development as IL9 is a target gene of STAT6 (Levitt et al. 1999).
Atopic dermatitis (AD) is another allergic disease where STAT6 has been implicated. In keratinocytes, STAT6 regulates the expression of genes loricrin and involucrin, which have been implicated in the development of the disease. It has also been shown that mice with constitutively active STAT6 exhibit signs that resemble AD (Sehra et al. 2010; Kim et al. 2008).
In summary, overexpression or increased action of STAT6 predisposes toward allergic disease, while a lack of STAT6 expression or its activity greatly reduces the severity of a number of diseases such as food allergies and eosinophilic esophagitis.
STAT6 in Other Diseases
STAT6 has been reported to be involved in a number of diseases, including cancer and tumor development. One example comes from Kaposi’s sarcoma-associated herpesvirus (KSHV), which has been related to Kaposi’s sarcoma and primary effusion lymphoma (PEL) among other diseases. A recent study has shown that STAT6 is constitutively activated in PEL cells due to high levels of IL13 and a reduced expression of SHP-1. The presence of KSHV alters the Jak/Stat pathway, leading to cell proliferation and survival (Wang et al. 2015).
Doyle et al. (2014) also investigated the involvement of STAT6 in dedifferentiated liposarcoma (Doyle et al. 2014). The presence and amplification of STAT6 was detected in a number of samples tested. Furthermore, a previous report from the same group suggested that an intrachromosomal rearrangement leads to a NAB2-STAT6 oncogene fusion, which can also be detected in some cases of dedifferentiated liposarcomas and in a number of solitary fibrous tumors.
A number of antibodies for STAT6 have been developed and are commercially available. A rabbit monoclonal antibody has been developed by Abcam (#ab32520) to target the C-terminus of STAT6 and is used to detect both the human and mouse origin protein by flow cytometry, immunocytochemistry, immunohistochemistry, immunoprecipitation, and Western blotting. Thermo Fisher has also developed a similar product (#701110), a rabbit monoclonal antibody which binds to amino acids 630–638.
The antibodies specific for phosphorylated STAT6 are also commercially available. These are used to identify whether STAT6 has been activated through phosphorylation. Thermo Fisher has developed a rabbit monoclonal anti-phosphoSTA6 antibody (#700247), which binds to aa 636–645 when STAT6 is phosphorylated and used for detecting the activated human or mouse STAT6 by various methods.
STAT6 is a member of the signal transducer and activator of transcription family, with the main function of it being to trigger a response in immune cells through IL4 and IL13 signaling. Stimulation with IL4 or IL13 causes the phosphorylation and dimerization of STAT6, which leads to transcriptional regulation through the Jak/STAT pathway. As with many signaling proteins, its activity is tightly regulated in a number of ways, such as through CBP/p300 complex, which acetylates STAT6 and thereby promotes its activity, or SHP-1, which dephosphorylates STAT6 and renders it inactive. In B and T cells, STAT6 promotes differentiation through an IL4-stimulated response. It has also been seen to be critical in Th2 differentiation, as it regulates Gata3. In the resident cells, STAT6 promotes chemokine and cytokine production in order to produce an inflammatory response in the specific location. The irregularities that can take place in this signal cascade can lead to the generation of allergies or other diseases.
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