STATs: An Old Story, Yet Mesmerizing
Signal transducers and activators of transcription (STATs) are cytoplasmic transcription factors that have a key role in cell fate. STATs, a protein family comprised of seven members, are proteins which are latent cytoplasmic transcription factors that convey signals from the cell surface to the nucleus through activation by cytokines and growth factors. The signaling pathways have diverse biological functions that include roles in cell differentiation, proliferation, development, apoptosis, and inflammation which place them at the center of a very active area of research. In this review we explain Janus kinase (JAK)/STAT signaling and focus on STAT3, which is transient from cytoplasm to nucleus after phosphorylation. This procedure controls fundamental biological processes by regulating nuclear genes controlling cell proliferation, survival, and development. In some hematopoietic disorders and cancers, overexpression and activation of STAT3 result in high proliferation, suppression of cell differentiation and inhibition of cell maturation. This article focuses on STAT3 and its role in malignancy, in addition to the role of microRNAs (miRNAs) on STAT3 activation in certain cancers.
Signal transducers and activators of transcriptions (STATs), originally discovered as DNA-binding proteins, mediate interferon-dependent gene expression (1-3). STATs are latent transcription factors activated by extracellular signaling ligands such as cytokines, growth factors and hormones (4, 5). These transducers become activated in the cytoplasm by Janus kinase (JAK), a family of tyrosine kinases (TKs). These signaling pathways have diverse biological functions which include roles in cell differentiation, proliferation, development, apoptosis, and inflammation that make them a very active area of research (6). In contrast to the restricted role of STATs 1, 2, 4 and 6 (Table 1,), STAT3 and STAT5 have broader functions in disease resistance to treatments. In the JAK/ STAT pathway, STAT3 has a broad role in cell function; its aberration contributes to excessive cell growth and proliferation. Interestingly, elevated levels of STAT3 have been observed in many human cancers and cancer cell lines (7). This review article presents an overview of the JAK/STAT pathway followed by an investigation of the role of STAT3 under normal and malignant conditions. Finally, we discuss the regulatory role of microRNAs (miRNAs) on STAT3 expression, as a new hot topic in therapeutics.
|Cytokines||Interferons||gp130 family||βc family||γc family||Homodimeric receptors||GPCRs|
|Type I IFNα/β||Type II IFN γ||Type III IL-10||IL-6, 11 LIF, G-CSF OSM||IL-12||Leptin||IL-3, IL-5 GM-CSF||IL-2, 7 IL-9, 15||IL-4||IL-13||GH||EPO Prl||TPO||Angi.||Serot.|
IFN; Interferon, IL; Interleukin, βc family; Common beta receptor subunit, γc family; Common gamma receptor subunit, G-CSF; Granulo- cyte colony stimulating factor, GH; Growth hormone, GM-CSF; Granulocyte macrophage colony-stimulating factor, EPO; Erythropoietin, TPO; Thrombopoietin, Prl; Prolactin, Angi.; Angiotensin, Serot.; Serotonin ,*; Activation by cytokine, LIF; Leukemia inhibitory factor, GP- CRs; G-protein-coupled receptors and TYK2; Tyrosine kinase 2.
Overview of JAK family structure and function
In contrast to other TK families, the JAK family is small. There are only four known mammalian JAKs-JAK1, JAK2, JAK3, and TYK2 that have been identified in the early 1990s by techniques that capitalized on homology of their kinase domains to other TKs (7, 8).
JAK1 is a member of a new class of protein-TKs (PTKs) characterized by the presence of a second phosphotransferase related domain immediately N-terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. JAK1 is a large, widely expressed membrane-associated phosphoprotein involved in the interferon-alpha/beta and -gamma signal transduction pathways. The reciprocal interdependence between JAK1 and TYK2 activities in the interferon-alpha pathway as well as between JAK1 and JAK2 in the interferon-gamma pathway may reflect a need for these kinases in the correct assembly of interferon receptor complexes. Binding of cytokines, growth factors and hormones to specific receptors leads to activation of various TKs. These kinases include JAKs, receptor TKs, and non-receptor TKs such as Src and ABL, which can directly phosphorylate STAT proteins without ligand-induced receptor signaling (9-11). They phosphorylate a tyrosine residue of STATs, followed by their dimerization through the reciprocal Src homology 2 (SH2)-phosphotyrosine interactions which lead to nuclear translocation and transcriptional activation of the target genes (12-15). The JAK protein are relatively large kinases with more than 1100 amino acids and apparent molecular weights of 120-130 kDa (Table 2,). JAK has seven defined regions of homology called the Janus homology domain (JH) 1-7 (Fig .1,). JH1 is a kinase domain important for JAK enzymatic activity where phosphorylation of its tyrosines leads to conformational changes in the JAK protein to facilitate substrate binding. JH2 is a pseudokinase domain, a domain structurally similar to a TK essential for normal kinase activity yet lacks enzymatic activity. The JH3-JH4 domains of JAKs share homology with SH2 domains. The amino terminal (NH2) end (JH4-JH7) of JAKs is called a FERM domain (short for band 4.1 ezrin, radixin and moesin); this domain is also found in the focal adhesion kinase (FAK) family and is involved in association of JAKs with cytokine receptors and/ or other kinases (16).
In summary it appears that specific JAK kinases, either alone or in combination with other JAKs, may be preferentially activated depending on the receptor that is being activated. Subsequentially different STATs will undergo activation.
|Member||Chromosomal location||Isoform||Gene size (bp)||mRNA size (bp)||Amino acid||MW (KDa)|
*; Canonical active member,**; STAT6, has transcript variant in addition of its isoforms and MW; Molecular weight.
STATs structure and activation
The seven mammalian STATs bear a conserved tyrosine residue (Y) near the C-terminus that is phosphorylated by JAKs. This phosphotyrosine allows for dimerization of a STAT (STATa) by a second STAT (STATb) through interaction with a conserved SH2 domain of the second STAT. Phosphotyrosine of the second STAT also interacts with the SH2 domain of STATa (Fig .2,). Phosphorylated and dimerization of STATs will occur. The STAT dimer enters the nucleus where it binds specific regulatory sequences to activate or repress transcription of target genes by direct DNA binding (Fig .3,) or by associating with other transcription factors (17, 18). The activity of STATs can be abolished by mutation of this critical tyrosine (19, 20). Each active homodimer STAT can induce the expressions of several target genes which are dependent upon both cell and STAT types. According to the Transcriptional Regulatory Element Database, some genes have more than one type of STAT transcription factor (Table 3,). The target genes of heterodimer STAT are unclear however they may depend on random binding of STATa or STATb to DNA which induces expression of target genes. In addition to gene expression by STAT, alterations can occur through association with other transcription factors and cofactors regulated by other signaling pathways. Thus integrating input from many signaling pathways must be considered.
|- Schematic structure of Janus kinases (JAK). JH; JAK homology domain. Kinase domain is located in JH1. JH2 has pseudokinase activity.|
Activation of the JAK/STAT pathway occurs
by binding of ligands to their receptors. These
ligands can activate different JAKs and STATs
(Table 1,). In addition to JAKs other non-receptor
TKs can be phosphorylated and activated by
interaction between ligands and their receptors
in the JAK/STAT pathway (Table 4,). The JAK
family (for mammals: JAK1, JAK2, JAK3 and
TYK2) activates when two JAKs are brought
into close proximity and trans-phosphorylation
is allowed. Once activated, JAKs can phosphorylate
additional targets which include both the
receptors and their major substrates, the STATs
(Fig .3,). Subsequently, phosphorylated STATs
are transported into the nucleus and modulate
expressions of several genes. In normal cells, after
modulating gene expression,
JAK/STAT pathway inhibitors
There are three major classes of negative regulators which inhibit JAK/STAT pathway. Signaling is also inhibited via two additional pathways.
|STAT1 target genes|
|Ligand||Receptor||JAK||Other TKs||STAT family members|
|IL-6||IL-6Ra+gp130||JAK1, 2, TYK2||Hck||STAT1, STAT3|
|IL-11||IL-11R+gp130||JAK1, 2, TYK2||Src, Yes||STAT3|
|CNTF, CT-1, LIF, OSM||CNTFR+gp130, CT-1R+gp130, LIFR+gp130, OSMR+gp130||JAK1, 2, TYK2||Src family||Predominant: STAT3 Secondary: STAT1, 5|
|IL-12 (p40+p35)||IL-12Rβ1+IL-12Rβ2||JAK2, TYK2||Lck||STAT4|
|Leptin||LeptinR||JAK2||NR||STAT3, 5, 6|
|IL-3||IL-3Rα+βc||JAK2||Fyn, Hck, Lyn||STAT3, 5, 6|
|IL-5||IL-5R+βc||JAK2||Btk||STAT3, 5, 6|
|GM-CSF||GM-CSFR+βc||JAK2||Hck, Lyn||STAT3, 5|
|Angiotensin||GPCR||JAK2, TYK2||NR||STAT1, 2, 3|
|IL-2||IL-2Rα+IL-2Rb+γc||JAK1, 2, 3||Fyn, Hck, Lck, Syk, Tec||STAT3, 5|
|IL-4||IL-4Rα+γcR or IL-4Rα+IL-13Rα1||JAK1, 3||Lck, Tec||STAT6|
|IL-7||IL-7R+γc||JAK1, 3||Lyn||STAT3, 5|
|IL-9||IL-9R+γc||JAK1, 3||NR||STAT1, 3 ,5|
|IL-13||IL-13Rα1+IL-4Rα||JAK1, 2, TYK2||Ctk||STAT6|
|IL-15||IL-15Rα+IL-2Rβ+γc||JAK1, 3||Lck||STAT3, 5|
|IL-20||IL-20Rα+IL-20Rβ, IL-22R+IL-20Rβ||JAK1, ?||NR||STAT3|
|IL-21||IL-21R+γc||JAK1, 3||NR||STAT1, 3, 5|
|IL-22||IL-22R+IL-10Rβ||JAK1, TYK2||NR||STAT1, 3, 5|
|IL-24||Same as IL-20||JAK1, ?||NR||STAT3|
|IL-27 (EBI3+p28)||gp130+WSX1||JAK1, 2, TYK2||NR||STAT1, 2, 3, 4, 5|
|IL-28A, IL-28B, IL-29||IL-28R+IL-10Rβ||JAK1, TYK2||NR||STAT1, 2, 3, 4, 5|
|IL-31||IL-31Rα+OSMR||JAK1, 2, TYK2||NR||STAT1, 3, 5|
|IL-35 (p35+EBI3)||gp130+WSX1||JAK1, 2, TYK2||NR||STAT1, 3, 5|
|GH||GHR||JAK2||Src family||STAT3, 5(mainly STAT5a)|
|Tpo||TpoR (c-Mpl)||JAK2, TYK2||Lyn||STAT1, 3, 5|
|Epo, Pro||EpoR, ProlactinR||JAK2||Src family||STAT5 (mainly STAT5a)|
|Interferon (IFNα/β)||IFNAR1+IFNAR2||JAK1, TYK2||Lck||Predominant: STAT1, 2 Secondary: STAT3, 4, 5|
|IFN-γ||IFN-gR1+IFN-γR2||JAK1, JAK2||Hck, Lyn||STAT1|
|IL-10||IL-10Rα+IL-10Rβ||JAK1, TYK2||NR||STAT1, 3, 5|
|TLSP||TLSPR and IL-7R||JAK1, possibly JAK2||NR||STAT3, 5|
|EGF||EGFR||JAK1||EGFR, Src||STAT1, 3, 5|
|PDGF||PDGFR||JAK1, 2||PDGFR, Src||STAT1, 3, 5|
NR; Not reported, bc; Common beta receptor subunit, gc; Common gamma receptor subunit, Epo; Erythropoietin receptor and Tpo; Thrombopoietin receptor.
|Upregulator||SOCS member||Inhibit signal induced by|
|IL-6, IFNγ||SOCS1||IL-2, 3 ,4, 6, IFNα, IFNγ, GH, Epo|
|IL-2, 6, IFNα, IFNγ, GH||SOCS2||IL-6, GH, Epo|
|IL-6, IFNγ||SOCS3||IL-2, 3, 4, 6, IFNα, IFNγ, GH, Epo|
IL; Interleukin, IFN; Interferon, NR; Not reported, GH; Growth hormone and Epo; Erythropoietin.
Protein inhibitors of activated STAT (PIAS) include PIAS1, PIAS2, PIAS3, PIAS4, PIASx and PIASy. These proteins have a Zn-binding ring-finger domain in the central portion. The PIAS proteins bind to activated STAT dimers and prevent them from binding DNA. PIAS1 and PIAS3 bind to STAT1 and STAT3, respectively. They inhibit transcriptional activity of the STATs, but do not affect phosphorylation. Just how specific they are in terms of regulating cytokine signaling remains to be determined; no knockouts have yet been reported (23).
Tyrosine phosphatases are the simplest way to reverse JAKs activity. The best characterized of these is the SH2 domain that contains protein tyrosine phosphatase-1 (SHP-1). It contains two SH2 domains and can bind to either phosphorylated JAKs or phosphorylated receptors to facilitate dephosphorylation of these activated signaling molecules.
SOCS proteins are a family of at least eight members that contain an SH2 domain and a SOCS box at the C-terminus. In addition, a small kinase inhibitory region located N-terminal to the SH2 domain has been identified for SOCS1 and SOCS3. The SOCS are responsible for a negative feedback loop in the JAK/STAT circuitry: activated STATs stimulate transcription of the SOCS genes. The resultant SOCS proteins bind phosphorylated JAKs and their receptors to turn off the pathway. SOCS can affect their negative regulation by three means: binding phosphortyrosines on the receptors (SOCS physically block the recruitment of signal transducers to the receptor), binding directly to JAKs, or to the receptors to specifically inhibit JAK kinase activity (Table 3,) (24).
In addition to SOCS, PIAS and SHIP-1 that have negative regulatory roles in active STATs, sumoylation (small ubiquitin-like modifier) is another system that controls STAT activity, however its exact mechanism is not known. Thus, it will be important to characterize the physiologic function of this family of molecules (23).
Activation of STATs and JAKs can mediate the recruitment of other molecules involved in signal transduction such as the Src-family kinases, protein tyrosine phosphatases, Mitogen-activated protein kinase (MAP) kinases, and Phosphoinositide 3-kinase (PI3K) kinase. These molecules process downstream signals via the Ras-Raf-MAP kinase and PI3 kinase pathway which results in the activation of additional transcription factors. The combined action of STATs and other transcription factors activated by these pathways dictate the phenotype produced by a given cytokine, interferon stimulation (25, 26). STATs have also been shown to play roles in the inflammatory signaling cascades triggered by lipopolysaccharide (LPS), interferon gamma (INFγ) and other cytokines (27-30). STAT1 and STAT3 have been implicated as key transcription factors in both immunity and inflammatory pathways (31, 32). In addition, it has been shown that LPS-induced interleukin-1β (IL-1β) production in macrophages is in part regulated through JAK2. The STAT3 pathway is activated in response to several cytokines such as IL-1β, IL-4 and IL-10 (33, 34). Additionally, STAT3 has a dual role in IL-6 mediated signaling; its activation may result in increased IL-6, but also IL-6 itself may lead to phosphorylation of STAT3 which results in diverse biological responses (6, 35). The DNA binding region of STATs resides within the central 171 amino acids, but relatively few direct contacts exist. Rather, the clamp-like structure is imparted by phosphotyrosine-SH2 interactions. STATs bind two types of DNA motifs: IFN-stimulated response elements (consensus: AGTTTNCNTTTCC) and IFNγ-activated sequence elements (consensus: TTCNNNGAA). STAT1, STAT2, and p48 bind to IFN-stimulated response elements whereas STAT1, STAT3, STAT4, STAT5a, and STAT5b bind to IFNγ-activated sequence element sites. STAT6 binds a similar but distinct site: TTCNNNNGAA (36). STAT1, STAT2, and STAT5 contain carboxy-terminal transcriptional activation domains. It has been shown that STAT1, STAT3, STAT4, and STAT5 are phosphorylated on serine residues in response to cytokine stimulation. For these proteins, a conserved site of serine phosphorylation that remains in a consensus sequence for MAPK-mediated phosphorylation has been mapped within the carboxy-terminal transcriptional activation domain. However the functional significance of STAT serine phosphorylation and the identity of the kinase(s) responsible for this event are controversial. Recently, a large number of reports have been published that STAT serine phosphorylation to the activation of various MAPKs. Notably they provide significantly divergent results, perhaps due to the differences in the STAT proteins investigated and in the systems utilized (37-40).
According to a PubMed search, until today more than 17700 STATs papers have been published. Most have discussed the direct and indirect functions of STATs which show the important role of STATs in molecular cell biology. The numbers of publications are as follows: STAT3 (40.5%), STAT1 (25%), STAT5 (18%), STAT6 (8.6%), STAT4 (4.5%), and STAT2 (3.4%). The large number of STAT3 publications possibly show contribution of STAT3 in the JAK/STAT pathway compared to other STATs. Here we focus on the biology of STAT3 and briefly describe the roles of this STAT on hemostasis and malignancies, including hematopoietic disorders.
The protein encoded by this gene is a member of the STAT protein family. STAT3 is activated through phosphorylation in response to various cytokines and growth factors that include IFNs, EGF, IL5, IL6, HGF, LIF, IL-11, Ciliary neurotrophic factor (CNTF), Macrophage colony-stimulating factor 1 (CSF-1), Platelet-derived growth factor (PDGF), Oncostatin-M (OSM) and Bone morphogenetic protein 2 (BMP2) (Tables1,, 4,). This protein mediates the expression of a variety of genes in response to cell stimuli and thus plays a key role in many cellular processes such as cell growth and apoptosis. The small GTPase Rac1 has been shown to bind and regulate the activity of this protein. PIAS3 protein is a specific inhibitor of STAT3. Three alternatively spliced transcript variants that encode distinct isoforms have been described (Table 2,). A number of factors regulate the JAK-STAT pathway including STAT dephosphorylation by phosphatases, altered nuclear import- export dynamics of STAT and STAT gene activation antagonists such as SOCS and PIAS (41, 42). STAT3 forms a homodimer or heterodimer with a related family member (at least STAT1). This molecule interacts with IL-31 receptor subunit alpha (IL31RA), Nuclear receptor coactivator 1 (NCOA1), Proline-, glutamic acidand leucinerich protein 1 (PELP1), Suppressor of cytokine signaling 7 (SOCS7), Hepatitis C (HCV) core protein and IL23R in presence of IL23. STAT3, via the SH2 domain, interacts with Serine/threonine-protein kinase NLK2 (NLK), Importin subunit alpha-3 (KPNA4), Importin subunit alpha-6 (KPNA5), Importin subunit alpha-3 (KPNA4), and Caveolin-2 (CAV2). It phosphorylates on serine region after DNA damage, probably by Serineprotein kinase ATM or Serine/threonine-protein kinase ATR. Serine phosphorylation is important for the formation of stable DNA-binding STAT3 homodimers and maximal transcriptional activity.
STAT3 in development and differentiation
Among the mammalian STAT proteins, STAT3 is the most diverse in cell biology. Embryonic stem 405 (ES) cells can be maintained in an undifferentiated state by the addition of leukemia inhibitory factor (LIF) but expression of a dominant negative form of STAT3 leads to the differentiation of ES cells, even when LIF is present (43).
Numerous cytokines induce expression of members of the anti-apoptotic regulator Bcl-2 family of proteins and STAT3 represses apoptosis in human myeloma cells by stimulating expression of Bcl-XL (44).
T helper 17 (Th17) development from naive precursors is dependent upon signal transduction through STAT3. In mice, RORC is a STAT3 target gene and Th17 differentiation is induced by STAT3 signaling cytokines, notably IL-6, IL-21 and IL-23, which can be abrogated effectively by a deficiency in STAT3 (45). In humans, STAT3 deficiency from dominant negative mutations in the
Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation, survival, and differentiation of myeloid progenitor cells towards neutrophilic granulocytes (51). The biological effects of G-CSF are mediated through a cell surface receptor (G-CSF-R) of the hematopoietin or class I cytokine receptor superfamily (52). G-CSF activates STAT1, STAT3, and STAT5 (53). Whereas the membrane-proximal cytoplasmic region of the G-CSF-R is sufficient for activation of STAT1 and STAT5, activation of STAT3 requires the membrane-distal C-terminal part of the receptor (54). The G-CSF-R C-terminus contains four conserved tyrosine residues (Y704, Y729, Y744, and Y764) and comprises a region that has specifically been implicated in the control of neutrophilic differentiation (55). These tyrosines are also important for differentiation and survival signals from the G-CSF-R (56). According to another study, IL-6 and OSM-induced growth inhibition of A375 melanoma cells is dependent on STAT3 activation and correlates with increased transcript levels of the cdk inhibitor p27Kip1 (57). Finally Silver et al. (58) have reported that STAT3 is involved in G-CSF-mediated differentiation, survival and regulation of p27 Kip1 expression. In addition, it has shown perturbations in the proliferation/differentiation balance of myeloid progenitor cells of p27-deficient mice in response to G-CSF. Based on these data, STAT3-mediated expression of p27 is proposed to represent one of the mechanisms by which G-CSF controls differentiation and survival of myeloid progenitor cells (58).
Inhibition of STAT3 activity in tumor-derived cell lines both
STAT3 inducer and inhibitor agents
Numerous JAK/STAT inhibitory pathways are inactivated in cancer cells which results in constitutively activated STATs. In addition to the canonical role of STATs in regulating transcription, STAT3 has other non-transcription based roles. Tyrosine phosphorylated STAT3 may be located at the leading edge of migrating cells, specifically at focal adhesions, where it promotes migration (65). Both JAKs and STATs can be associated with microtubules (66), and the interaction between STAT3 and microtubules promotes migration by competing with binding the microtubule associated protein stathmin (67). STAT3 is activated in 70% of breast tumors and often associated with both aggressive and invasive tumors (68). Inhibition of STAT3 leads to a reversion of the malignant phenotype of these cells, which indicates that it is a key mediator of breast cancer pathogenesis. Elucidating the role of STAT3 in breast cancer and identifying methods to inhibit STAT3 can be of benefit for developing cancer treatments. Microtubule-targeting agents are among the most active drugs used as breast cancer treatment. Two types are utilized: microtubule stabilizers such as paclitaxel (Taxol) and microtubule destabilizers such as vinorelbine (Navelbine). Since STAT3 is activated in most breast cancers and associates with microtubules, Taub (69) have shown that microtubule-targeted therapy modulates STAT3 signaling and function in breast cancer cells. ObR is a single transmembrane protein that belongs to the class I cytokine receptor superfamily (9). Leptin binding induces activation of JAK2 and
STAT3 signaling in malignancy
As one of the STAT family members, STAT3 is correlated with positive regulation of cell growth and highly activated in cancer cells (9,71). In cancers of epithelial origin, STAT3 is constitutively activated in head and neck squamous cell carcinoma (HNSCC) (72,73), breast cancer cell lines (74,75), ovarian cancer cell lines (76), lung cancer cell lines (77) and myeloma cell lines (14). In particular, STAT3 plays a critical role in the development of skin cancer (78). Activation of STAT3 signaling regulates the expression of numerous genes involved in growth control and survival. Studies have shown that numerous genes which encode for
STAT3 is a vital transcription factor activated by some ligands and IL-6 (103). It has important roles in mutagenesis and anti-apoptosis. STAT3 is involved in the transcriptional upregulation of many genes, not only acting by direct DNA binding, but in some cases as a coactivator of transcription factors such as activator protein-1 and hepatocyte nuclear factor-1 (104). STAT3 knockout results in early embryonic lethality, but conditional knockouts provide useful tools to examine the actions of STAT3 in specific tissues. In a study by Haga et al. (67), two animal models have been used to examine the effects of STAT3 modulation in Fas-mediated liver injury: mice injected with adenoviruses that expressed constitutively active STAT3 and other proteins, and mice with hepatocyte specific
In the past several years, compelling evidence has accumulated that highlights the role of STAT proteins in leukemogenesis. Constitutive activation of STATs has now been clearly demonstrated in acute and chronic leukemias. Elevated STAT3 activity has been observed in many spontaneous and experimentally established mammalian cancers, which demonstrates its critical role in tumorigenesis. Assessment of miRNAs expression patterns after the use of anticancer drugs can more precisely identify the molecular mechanisms of cancer cells.