The physiological role of each individual STAT protein is now being examined through the study of ‘knockout’ (KO) mice, harboring a null allele for the particular gene. In contrast to other STATs deficient mice that are born alive, STAT3-deficient mice die during early embryogenesis. However, the role of STAT3 in adult tissues can be assessed by utilizing the Cre-loxP recombination system to ablate the gene in later life. Analyses of tissue-specific STAT3-deficient mice indicate that STAT3 plays a crucial role in a variety of biological functions including cell growth, suppression and induction of apoptosis, and cell motility.
The Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is activated in response to a larger number of cytokines, hormones and growth factors (Darnell, 1997). The JAK family of protein tyrosine kinases, which phosphorylate and activate STAT proteins, consists of Jak1, Jak3 and Tyk2. These JAK kinases are characterized by the possession of a kinase-like domain and a bona fide kinase domain in its C-terminal (Ihle, 1996). STAT proteins are a family of latent cytoplasmic transcription factors that contain the DNA binding domain in the middle, as well as an SH2 domain, a tyrosine phosphorylation site and a transcriptional activation domain in the C-terminal portion.
STAT3 was initially identified as APRF (acute phase response factor), an inducible DNA binding protein that binds to the IL-6 responsive element within the promoters of hepatic acute phase protein genes (Wegenka et al., 1993). APRF protein was purified from a pool of mouse liver nuclear extracts using DNA affinity chromatography, cDNA encoding mouse APRF was cloned based on the partial amino acid sequences (Akira et al., 1994). Mouse APRF cDNA encodes an open reading frame of 770 amino acids predicting a protein of 88 kD. A DNA data base search revealed that mouse APRF has a high degree of homology to STAT1 (52.5% homology). STAT3 was also independently cloned by Zhong et al. (1994).
Signal transductions that transmit for gp130
The receptors for the interleukin-6 (IL-6)-type cytokines, consisting of IL-6, IL-11, LIF, OSM, CNTF and cardiotrophin-1, utilize combinations of a common signal-transducing subunit, gp130, and various ligand-binding subunits (Kishimoto et al., 1995). Propagation of these cytokine signals requires gp130, which activates STAT3 and the Ras/MAP kinase pathway (Akira, 1997) (Figure 1). The binding of ligands to the ligand-binding subunit induces the homodimerization of gp130 and subsequently activates gp130-associated JAKs by transphosphorylation. JAKs then phosphorylate tyrosine residues in the cytoplasmic portion of gp130 as well as JAKs themselves. Phosphotyrosines on the receptor recruit src homology (SH2) domain-containing signaling molecules. Human gp130 has six tyrosine residues in the cytoplasmic domain. Among them, the membrane proximal second tyrosine residue (Y759) is responsible for activation of the MAP kinase cascade through SHP2 (Fukada et al., 1996), and each of other four tyrosine residues [the third(Y767), fourth(Y814), fifth(Y905) and sixth(Y915)] containing the YxxQ motif is required for STAT3 activation (Stahl et al., 1995).
Ras activation leads to activation of serine/threonine kinase Raf-1, followed by sequential phosphorylation of MEK and MAP kinases. Activated MAP kinases translocate to the nucleus, and phosphorylate transcription factors, which results in modulation of gene expression. Ras activation via cytokine receptors require several adaptor proteins. The adaptor protein Grb2 associates with the Ras GTP-GDP exchange factor Sos, and induces the conversion of inactive GDP-bound Ras to the active GTP-bound Ras. SHP2 (also called Syp, SHPTP-2, SHPTP-3, PTP2C, and PTP1D) is a protein tyrosine phosphatase containing two SH2 domains, and functions as an adaptor protein in recruitment of the Grb2/Sos complex to gp130. SHP2 is shown to be inducibly associated with gp130 (Fuhrer et al., 1995).
STAT3 proteins are initially present in inactive forms in the cytoplasm. Upon ligand binding, they become associated with gp130 via recognition of the receptor phosphotyrosines by the STAT SH2 domains. The activated JAK kinases then phosphorylate STAT proteins at their tyrosine residues. Thereafter, the phosphorylated STAT proteins detach from the receptor, become homodimerized or heterodimerized, and translocate to the nucleus to activate transcription by interaction with specific DNA sequences. In addition to the tyrosine phosphorylation required for both dimerization and translocation to the nucleus, STAT proteins also require serine phosphorylation for transcriptional activation (Wen et al., 1995; Zhang et al., 1995). The carboxy-terminal regions of STAT1, STAT3, STAT4, and STAT5 contain the MAPK consensus sequence, although there is no evidence showing that MAPKs are actually involved in the serine phosphorylation of STATs in vivo.
STAT3 is also activated in response to G-CSF and leptin, the receptors for which are both homologous to gp130. Furthermore, STAT3 is activated in response to stimulation of several receptor tyrosine kinases (epidermal growth factor, CSF-1, and PDGF), and by members of the interferon (IL-10, IFNγ, and IFNα) and IL-2 (IL-2, IL-7, and IL-15) families. These receptor molecules harbor a common STAT3 docking motif (YxxQ) in their cytoplasmic domain (Stahl et al., 1995).
Roles of STAT3 revealed in cell culture systems
The role of STAT3 has been investigated using the dominant-negative STAT3 mutant (STAT3DN) or a conditionally active form of STAT3. In the mouse myeloid leukemic M1 cells, overexpression of STAT3DN abolished the differentiation response to IL-6 or LIF, indicating that STAT3 activation is essential for IL-6 or LIF-mediated growth arrest and differentiation of M1 cells (Minami et al., 1996; Nakajima et al., 1996). In the mouse pro-B cell line BAF-B03, overexpression of STAT3DN did not proliferate and underwent cell death accompanied by DNA fragmentation, indicating that STAT3 transmits an anti-apoptotic signal (Fukada et al., 1996).
ES cells are nontransformed stem cells that can be continuously propagated in vitro in the presence of LIF. ES clones constitutively expressing STAT3DN showed an increased tendency to differentiate (Boeuf et al., 1997). Expression of STAT3DN using an inducible promoter in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation (Niwa et al., 1998). Recently, STAT3 activation is demonstrated to be sufficient for the self-renewal of ES cells by using a conditionally active form of STAT3, that is, a fusion protein between STAT3 and ER ligand binding domain in which STAT3 is activated in response to the synthetic ligand 4-hydroxytamoxifen (4HT) (Matsuda et al., 1999). ES cells expressing STAT5aER or STAT6ER did not maintain the undifferentiated state of ES cells in response to 4HT. Although STAT1 can be activated in response to LIF in ES cells, STAT1−/− ES cells retained responsiveness to LIF and remained LIF dependent for undifferentiated growth. These data show that self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3, and that STAT3 has a specific and nonredundant function in ES cells. It is also shown that SHP2 and MAP kinase activation through gp130 is dispensable for the self-renewal of ES cells.
STAT3 activation has further been shown to mediate IL-6- or LIF-induced astrocytic differentiation of primary cortical neuroepithelial cells (Bonni et al., 1998). It has also been shown that STAT3 is activated by hepatocyte growth factor and mediates epithelial tubulogenesis (Boccaccio et al., 1998).
Early embryonic lethality of STAT3-deficient mice
STAT3 activity is detected during early postimplantation development in the mouse, suggesting that STAT3 plays a role during early embryogenesis (Duncan et al., 1997). In fact, STAT3-deficient mice die early in embryogenesis, prior to gastrulation (Takeda et al., 1997). By 7.5 days post-coitum STAT3 mRNA is expressed in the extra embryonic visceral endoderm, which is the principal site of nutrient exchange between the maternal and embryonic environments. The timing of the degeneration of STAT3−/− embryos coincides with the onset of STAT3 expression in visceral endoderm in wild-type mice, suggesting that STAT3−/− lethality may be due to a defect in visceral endoderm function, such as nutritional insufficiency. The ligand that activates STAT3 in visceral endoderm remains unknown.
Tissue-specific targeting of STAT3
In an attempt to assess the role of STAT3 in adult tissues, we utilized the Cre-loxP recombination system, in which a specific region of DNA flanked by loxP sites can be deleted by expression of the Cre protein (Figure 2). We first generated floxed-STAT3 mice, in which two loxP sites were introduced 5′ and 3′ of the exon encoding the tyrosine residue critical for STAT activation. Floxed-STAT3 mice were mated with transgenic mice expressing Cre protein in specific tissues. For T cell-specific deletion, we used transgenic mice expressing Cre protein specifically in T cells under the control of the Lck promoter. STAT3-deficient T cells displayed a severely impaired proliferative response to IL-6 due to a defect in IL-6 mediated suppression of apoptosis, demonstrating the anti-apoptotic function of STAT3 (Takeda et al., 1998) (Figure 3). A similar result is shown in the mouse pro-B cell line BAF-B03. Different from the result with the pro-B cells in which STAT3 is involved in the expression of bcl-2, an anti-apoptotic gene, STAT3-mediated anti-apoptosis in T cells is not mediated by bcl-2 induction. The anti-apoptotic gene regulated by STAT3 in T cells remain to be unknown. The mechanisms of STAT3-mediated anti-apoptosis may be distinct, depending on the cell type.
STAT3-deficient T cells also show a partial defect in IL-2-induced proliferation. IL-2 receptors are composed of the combination of three distinct subunits, IL-2Rα, β and γ. IL-2Rα is required to convert intermediate-affinity receptors (containing IL-2Rβ and γ) into high-affinity receptors (containing all three chains), which confers a 100-fold increase in binding affinity for IL-2 as well as efficient cellular responsiveness to the low concentrations of IL-2. The partial defect in IL-2-induced proliferation of STAT3-deficient T cells are found to be due to a defect in IL-2-induced expression of IL-2Rα (Akaishi et al., 1998). Interestingly, high concentrations of IL-2 relieved the defect of proliferation. Thus, STAT3 is indirectly involved in T cell proliferation by upregulating the expression of IL-2Rα and forming high affinity receptors. The similar result is also demonstrated in STAT5a-deficient T cells (Nakajima et al., 1997). Therefore, both STAT3 and STAT5a are independently responsible for the IL-2Rα expression since the absence of one is not compensated by the other (Figure 3).
We have also generated mice in which STAT3 is deficient specifically in macrophages and neutrophils (Takeda et al., 1999). These mutant mice were highly susceptible to endotoxin shock and demonstrated increased production of inflammatory cytokines such as TNFα, IL-1, and IFNγ. Production of inflammatory cytokines from STAT3-deficient macrophages were dramatically augmented in response to LPS. The mice also showed a polarized immune response of the Th1 type as shown by increased secretion of IFNγ from splenic T cells. Aging mutant mice developed chronic enterocolitis. These phenotypes are quite similar to those seen in mice lacking IL-10, a cytokine with pleitorophic bioactivities, and is relatively unique in its ability to potentially inhibit production of proinflammatory cytokines (Kuhn et al., 1993). Indeed, the response to IL-10 was completely abolished in macrophages and neutrophils. These results indicate that STAT3 functions in vivo in macrophages and neutrophils to signal anti-inflammatory responses mediated by IL-10, and that IL-10-mediated anti-inflammatory response by macrophages and neutrophils plays a critical role in prevention of excessive Th1 response and chronic inflammation (Figure 4). IL-10 receptor system is composed of two subunits, IL-10Rα and IL-10Rβ. Binding of IL-10 to the extracellular domain of IL-10Rα activates STAT3. Murine IL-10Rα harbors two redundant STAT3 recruitment sites (427YQKQ430 and 477YLKQ480) (Weber-Nordt et al., 1996). Structure-function analysis of the intracellular domain of the IL-10Rα chain shows that two redundant STAT3 recruitment sites are required for all IL-10 dependent effects, whereas IL-10-dependent anti-inflammatory function requires the presence of a carboxy-terminal sequence on the intracellular domain of the IL-10Rα (Riley et al., 1999). This result indicates the IL-10-induced inhibition of TNFα production requires two distinct regions of the IL-10Rα intracellular domain and thereby establish a distinctive molecular basis for the proliferative versus the anti-inflammatory action IL-10.
The functional role of STAT3 in skin was assessed by crossing the floxed-STAT3 mice with mice expressing the Cre-transgene from the keratin 5 promoter (Sano et al., 1999). The mutant mice were viable and displayed no developmental alterations in the epidermis and hair follicles by postnatal day 11 (PD11), showing that STAT3 in keratinocytes is not involved in the morphogenesis of skin and hair follicles. However, the second hair cycle was impaired in STAT3-deficient mice. Follicular morphogenesis starts at 14.5 days post coitus through morphogenic mesenchymal-epithelial interactions. Hair follicular rudiments grow downwards and differentiate to develop the complex hair structure until around PD17. Then, follicles undergo cellular quiescent process (catagen) and finally, complete rest phase (telogen). Around PD21, the second anagen is initiated in response to remodeling mesenchymal signal. In STAT3-deficient mice, the second anagen was not observed and remained in a telogen stage. These results suggest that STAT3 is essential to the second and subsequent hair cycles (skin remodeling) although it is dispensable for the first hair cycle (morphogenesis). Skin wound healing was also severely impaired in STAT3-deficient mice when the mice were wounded with a biopsy punch and the process of healing was monitored. The fact that no difference was shown between STAT3-deficient mice and control mice in the dermal responses to wound such as granulation, inflammation and neovascularization indicates that the retarded wound healing is due not to an impairment in secondary development of dermal components but rather to a fault in epidermal regeneration. Since cell migration and proliferation are critical events in re-epithelialization of wounds, the motility and growth of keratinocyte was examined. Growth factor-dependent in vitro migration of STAT3-deficient epidermal cells was severely impaired, although proliferation was unaffected. This suggests that the defect in wound healing is due to the poor motility of epidermal cells. Furthermore, the mutant mice had sparse hair and developed ulcers spontaneously with age. Additionally the mutant mice expressed aberrant hair follicles, and marked hyperplasia of the epidermis (acanthosis) with hyperkeratosis and sclae-crusts. There was pronounced inflammatory infiltration and fibrosis throughout the dermis. The phenotype in aged STAT3-deficient mice appears to be consequence to the impaired wound healing and disorganized hair cycling.
Mammary gland involution is characterized by extensive apoptosis of the epithelial cells. STAT5 is activated during pregnancy and lactation but is rapidly down regulated during involution. By gene targeting, STAT5 has been shown to be essential for normal mammopoiesis and lactogenesis (Liu et al., 1997; Teglund et al., 1998). Conversely, STAT3 is specifically activated at the start of involution. The reciprocal activation of STAT3 and 5 at the onset of apoptosis suggests opposing roles for these STATs in the regulation of apoptosis in the mammary gland. The role of STAT3 in the mammary gland was addressed by crossing the floxed-STAT3 mice with mice expressing the Cre-transgene form the milk protein gene β-lactoglobulin (BLG) promoter (Chapman et al., 1999). Following weaning, a decrease in apoptosis and a dramatic delay of involution were observed in STAT3-deficient mammary tissue. No marked differences were seen in the regulation of Bcl-xL or Bax between the normal and STAT3-deficient mammary glands. Involution is normally associated with a significantly increased level of insulin-like growth factor-binding protein-5 (IGFBP-5), which has been suggested to induce apoptosis by sequestering insulin-like growth factor-1 (IGF-1) to casein micelles, thereby inhibiting its survival function. The increase in IGFBP-5 levels was strongly suppressed in STAT3-deficient mice, showing that IGFBP-5 is one target for STAT3. However, it remains unclear whether IGFBP-5 is directly dependent on STAT3 binding to the IGFBP-5 promoter, although the human IGFBP-5 promoter contains a consensus STAT-binding element.
Taken together, these analyses of tissue-specific STAT3-deficient mice demonstrate that STAT3 plays a crucial role in a variety of biological functions including cell growth, anti-apoptosis, apoptosis and cell motility depending on the cell type and stimulus (Table 1).
Targeted disruption has disclosed the specific function of each STAT protein. It is noteworthy that STAT3 and 5 are expressed in many cell types, activated by a variety of cytokines and growth factors, and play a role in various aspects of biological responses whereas other STAT proteins (STAT1, 2, 4, and 6) plays specific roles in host defenses. This suggests that the development of host defense mechanisms in mammals may have required STAT proteins to evolve specific roles in the immune response. In fact, the chromosomal localization of the mouse STAT genes suggests that the STAT1 and 4 genes cosegregating on chromosome 1, the STAT2 and 6 on chromosome 6 genes have arisen via a tandem duplication of an ancestral locus (probably chromosome 11 harboring the STAT3 and 5 genes).
Classical and tissue-specific targeting disruption of the STAT3 gene clearly showed the critical role of STAT3 in many aspects of biological functions. In the future the identification of target genes regulated by STAT3 will reveal the molecular mechanisms underlying the STAT3-mediated biological responses in various tissues.
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I thank Dr K Takeda for preparing the Figures, and T Aoki for excellent secretarial assistance. This work was in part supported by grants from the Ministry of Education of Japan.
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