Mammary gland involution is characterized by extensive apoptosis of the epithelial cells. At the onset of involution, Stat3 is specifically activated. To address the function of this signaling molecule in mammary epithelial apoptosis, a conditional knockout of Stat3 was generated using the Cre-lox recombination system. Following weaning, a decrease in apoptosis and a dramatic delay of involution occurred in Stat3 null mammary tissue. Involution is normally associated with a significant increase in IGFBP-5 levels. This was observed in control glands, but not in the absence of Stat3. IGFBP-5 has been suggested to induce apoptosis by sequestering IGF-1 to casein micelles, thereby inhibiting its survival function. These findings suggest that IGFBP-5 is either a direct or indirect target for Stat3, and that IGFBP-5 upregulation is essential to normal involution. No marked differences are seen in the regulation of Stat5, Bcl-x(L), or Bax in the absence of Stat3. Precocious activation of Stat1 and increases in levels of p53 and p21 occur and may act as compensatory mechanisms for the eventual initiation of involution observed in Stat3 null mammary glands. Little is known about the regulation of IGFBP-5 transcription, and it remains unclear whether IGFBP-5 expression is directly dependent on Stat3 binding to the IGFBP-5 promoter. However, the human IGFBP-5 promoter does contain a consensus Stat-binding element in addition to consensus sequences for AP-1 (which increases dramatically during mammary involution) and AP-2. It will be interesting to investigate the transcriptional regulation of IGFBP-5 and whether AP-1 is central to the mechanism by which Stat3 regulates IGFBP-5. This is the first demonstration of the importance of a Stat factor in signaling the initiation of physiological apoptosis in vivo (Chapman, 1999).

Unregulated FGF receptor signaling results in bone malformations that affect both endochondral and intramembranous ossification, and is the basis for several genetic forms of human dwarfism. FGF signaling inhibits chondrocyte proliferation and the transcription factor STAT1 mediates the growth inhibitory effect of FGF in vitro. Genetic evidence suggests that STAT1 is a modulator of the negative regulation of bone growth by FGF in vivo. Stat1-/- mice were crossed with a transgenic mouse line overexpressing human FGF2 (TgFGF). TgFGF mice exhibit phenotypes characterized by chondrodysplasia and macrocephaly, both of which affect endochondral and intramembranous ossification. The chondrodysplasic phenotype of these mice results both from reduced proliferation and increased apoptosis of growth plate chondrocytes. Loss of STAT1 function in TgFGF mice leads to a significant correction of the chondrodysplasic phenotype, but does not affect the skull malformations. The reduced proliferation of TgFGF growth plate chondrocytes, as well as their excessive apoptosis, is restored to near-normal levels in the absence of STAT1 function. Unregulated FGF signaling in TgFGF mice also induces apoptosis in calvarial osteoblasts that is not, however, corrected by the absence of STAT1. Detailed analysis of Stat1-/- growth plates has uncovered a transient phenotype, characterized by an expansion of the proliferative zone and by acceleration of longitudinal bone growth, which attenuates as the animals grow older. These results document an essential role for STAT1 in FGF-mediated regulation of cell growth that is specific to the epiphyseal growth plate (Sahni, 2001).

To elucidate the biological role of Stat3 in the skin, conditional gene targeting using the Cre-loxP system was performed as germline Stat3 ablation leads to embryonic lethality. K5-Cre;Stat3(flox/-) transgenic mice, whose epidermal and follicular keratinocytes lack functional Stat3, are viable and the development of epidermis and hair follicles appears normal. However, hair cycle and wound healing processes are severely compromised. Furthermore, mutant mice express sparse hair and develop spontaneously occurring ulcers with age. Growth factor-dependent in vitro migration of Stat3-disrupted keratinocytes is impaired despite normal proliferative responses. It is therefore concluded that Stat3 plays a crucial role in transducing a signal required for migration but not for proliferation of keratinocytes, and that Stat3 is essential for skin remodeling, including hair cycle and wound healing (Sano, 1999).

STAT deficient mice show no overt developmental abnormalities, but display a complete lack of responsiveness to either IFN alpha or IFN gamma, and are highly sensitive to infection by microbes and viruses. In contrast, these mice respond normally to several other cytokines that activate STAT1 in vitro, revealing an unexpected level of physiological specificity for STAT1 action (Meraz, 1996).

The Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway is an important signaling pathway for interferons and cytokines. The activation of STAT proteins induced by interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or erythropoietin (EPO) were analyzed using the human leukemia cell line, UT-7, which requires these cytokines for growth. IL-3, GM-CSF, and EPO induce DNA-binding activity to the oligonucleotides corresponding to the sis-inducible elements (SIE) of c-fos, in addition to the beta-casein promoter (beta-CAP). SIE- and beta-CAP-binding proteins were identical to Stat1alpha and Stat3 complex and to Stat5 protein, respectively. This indicates that IL-3, GM-CSF, and EPO commonly activated Stat1alpha, Stat3, and Stat5 proteins in UT-7. However, EPO hardly activate Stat1alpha and Stat3 in UT-7/GM, which is a subline of UT-7 that grows slightly in response to EPO. Transfection studies reveal that UT-7/GM cells constitutively expressing Stat1alpha, but not Stat3, can grow as well in response to EPO as GM-CSF, suggesting that Stat1alpha is involved in the EPO-induced proliferation of UT-7. Thus, although Stat1alpha, Stat3, and Stat5 proteins are activated by GM-CSF, IL-3, and EPO, the data suggest that each STAT protein has a distinctive role in the actions of cytokines (Kirito, 1997).

Interleukin 2 (IL-2) induces tyrosine phosphorylation of STATs 3 and 5 (signal transducer and activator of transcription). IL-2 regulation of STAT3 proteins in T cells is a complex response involving activation of two forms of STAT3: 90-kDa STAT3alpha and an 83-kDa carboxyl-terminal truncated STAT3beta. The phosphorylation of STAT proteins on serine residues is also required for competent STAT transcription. A critical serine phosphorylation site in STAT3alpha is located at position 727. An antisera specific for STAT3alpha proteins phosphorylated on serine 727 was produced and used to monitor the phosphorylation of this residue during T lymphocyte activation. Phosphorylation of STAT3alpha on serine 727 is not constitutive in quiescent T cells but can be induced by the cytokine IL-2. Interestingly, triggering of the T cell antigen receptor complex or activation of protein kinase C with phorbol esters also induces phosphorylation of serine 727 but without simultaneously inducing STAT3 tyrosine phosphorylation or DNA binding. Hence, STAT3 serine phosphorylation can be regulated independently of the tyrosine phosphorylation of this molecule. IL-2 and T cell antigen receptor complex induction of STAT3alpha serine 727 phosphorylation is dependent on the activity of the MEK/ERK pathway. Previous studies have identified H-7-sensitive kinase pathways that regulate STAT3 DNA binding. H-7-sensitive pathways regulate STAT3 DNA binding in T cells. Nevertheless, t H-7-sensitive kinases do not regulate STAT3 tyrosine phosphorylation or phosphorylation of serine 727. These results thus show that STAT3 proteins are targets for multiple kinase pathways in T cells and can integrate signals from both cytokine receptors and antigen receptors (Ng, 1997).

The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines. These factors act through receptor complexes containing the signal transducer gp130. The downstream mechanisms that lead to ES cell self-renewal have not been delineated, however. In this study, chimeric receptors were introduced into ES cells. Biochemical and functional studies of transfected cells demonstrate a requirement for engagement and activation of the latent trancription factor STAT3. Detailed mutational analyses unexpectedly reveal that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was then investigated using the dominant interfering mutant, STAT3F. ES cells that express this molecule constitutively could not be isolated. An episomal supertransfection strategy was therefore used to allow an examination of the consequences of STAT3F expression. In addition, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogates self-renewal and promotes differentiation. These complementary approaches establish that STAT3 plays a central role in the maintenance of the pluripotential stem cell phenotype. This contrasts with the involvement of STAT3 in the induction of differentiation in somatic cell types. Cell type-specific interpretation of STAT3 activation thus appears to be pivotal to the diverse developmental effects of the LIF family of cytokines. Identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a first step in the molecular characterization of pluripotency (Niwa, 1998).

Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of the progenitor cells of neutrophilic granulocytes. The binding of G-CSF to its receptor specifically activates JAK1 and JAK2 kinases, as well as STAT3, a signal transducer and activator of transcription (STAT). To examine the role of STAT3 in G-CSF receptor-mediated signal transduction, two different forms of the dominant negative STAT3 were introduced into a mouse myeloid cell line that exogenously expresses the mouse G-CSF receptor. In response to G-CSF, the parental myeloid cells grow for about 4 days, and then they stop dividing and differentiate into cells with lobulated nuclei. During this period, the expression of the myeloperoxidase (MPO) gene is induced, while c-myc gene expression is down-regulated. In contrast, in the cells expressing the dominant negative STAT3, G-CSF can induce neither growth arrest nor morphological change. However, the induction of the MPO gene by G-CSF is not affected by the dominant negative STAT3. These results indicate that STAT3 activation is responsible for part of the G-CSF-induced differentiation of neutrophils but another pathway, involving the expression of the MPO gene that does not utilize the activated STAT3, is required in addition, to fully differentiate the cells (Shimozaki, 1997).

Gamma interferon (IFN-gamma) induces both the tyrosine and serine phosphorylation of Stat1. Stat1 serine phosphorylation is required for maximal transcriptional activity of Stat1. Stat1 tyrosine phosphorylation is not a prerequisite for Stat1 serine phosphorylation, although an active Jak2 kinase is required for both phosphorylation events. Stat1 serine phosphorylation occurs with a more delayed time course than tyrosine phosphorylation. The occurrence of serine phosphorylation without tyrosine phosphorylation suggests that serine phosphorylation takes place in the cytoplasm. Experiments performed with cells expressing either dominant-negative or constitutively active Ras protein indicate that the Ras-mitogen-activated protein kinase pathway is probably not involved in IFN-gamma-induced Stat1 serine phosphorylation. A kinase capable of correct Stat1 serine phosphorylation is detected in partially purified cytoplasmic extracts from both IFN-gamma-treated and untreated cells (Zhu, 1997).

In vascular smooth muscle cells, the induction of early growth response genes involves the Janus kinase (JAK)/signal transducer and activators of transcription (STAT) and the Ras/Raf-1/mitogen-activated protein kinase cascades. Electroporation of antibodies against MEK1 or ERK1 abolishes vascular smooth muscle cell proliferation in response to either platelet-derived growth factor or angiotensin II. However, anti-STAT1 or -STAT3 antibody electroporation abolishes proliferative responses only to angiotensin II and not to platelet-derived growth factor. AG-490, a specific inhibitor of the JAK2 tyrosine kinase, prevents proliferation of vascular smooth muscle cells, complex formation between JAK2 and Raf-1, the tyrosine phosphorylation of Raf-1, and the activation of ERK1 in response to either angiotensin II or platelet-derived growth factor. However, AG-490 has no effect on angiotensin II- or platelet-derived growth factor-induced Ras/Raf-1 complex formation. These results indicate that (1) STAT proteins play an essential role in angiotensin II-induced vascular smooth muscle cell proliferation, (2) JAK2 plays an essential role in the tyrosine phosphorylation of Raf-1, and (3) convergent mitogenic signaling cascades involving the cytosolic kinases JAK2, MEK1, and ERK1 mediate vascular smooth muscle cell proliferation in response to both growth factor and G protein-coupled receptors (Marrero, 1997).

IL-6 induces differentiation of PC12 cells pretreated with nerve growth factor (NGF). The signals required for neurite outgrowth of PC12 cells were explored by using a series of mutants of a chimeric receptor consisting of the extracellular domain of the granulocyte-colony stimulating factor (G-CSF) receptor and the cytoplasmic domain of gp130, a signal-transducing subunit of the IL-6 receptor. These mutants, incapable of activating the MAP kinase cascade, also fail to induce neurite outgrowth. Consistently, a MEK inhibitor, PD98059, inhibits neurite outgrowth, showing that activation of the MAP kinase cascade is essential for the differentiation of PC12 cells. In contrast, a mutation that abolishes the ability to activate STAT3 does not inhibit neurite outgrowth; rather, neurite outgrowth is stimulated. This mutant does not require NGF pretreatment for neurite outgrowth. Dominant-negative STAT3s mimics NGF pretreatment; NGF suppresses the IL-6-induced activation of STAT3, supporting the idea that STAT3 might negatively regulate the differentiation of PC12 cells. These results suggest that neurite outgrowth of PC12 cells is regulated by the balance of MAP kinase and STAT3 signal transduction pathways, and that STAT3 activity can be regulated negatively by NGF (Ihara, 1997).

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50cdc37 that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogate the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells (Ramana, 2000).

Leptin is involved in the hypothalamic control of food intake and body weight. Fos immunohistochemistry has been used to functionally map leptin target neurons involved in these regulatory processes. However, only a subset of hypothalamic neurons expressing the long form of the leptin receptor (Ob-Rb) also coexpress the neuronal activation marker Fos after leptin stimulation. To functionally map all leptin target neurons, regardless of whether leptin-mediated neuronal activation or inhibition occurs, the leptin-induced nuclear translocation of the signal transducer and activator of transcription molecule STAT3, which represents a crucial step in the regulation of leptin-dependent gene expression, was immunohistochemically investigated. Intracerebroventricular leptin treatment induces a time-dependent nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei, with strong nuclear STAT3 signals detectable in the arcuate nucleus, the lateral hypothalamus, and the ventromedial and dorsomedial hypothalamic nuclei. This leptin-induced STAT3 translocation pattern proved to be distinct from that induced by interleukin-6, another cytokine using STAT3 in its signaling pathway. Combined immunohistochemical STAT3 and Fos detection after leptin treatment reveal a higher number of STAT3-positive than Fos-positive cell nuclei in hypothalamic structures and show that Fos immunoreactivity colocalizes only in a subset of all leptin-responsive STAT3 nuclei. These results suggest that the detection of nuclear STAT3 immunoreactivity represents a new neuroanatomical tool to functionally map central leptin actions. They further support the importance of ventrally located caudal hypothalamic structures representing the main leptin targets involved in body weight regulation (Hubschle, 2001).

Activation of signal transducer and activator of transcription 3 (Stat3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal and also facilitates reprogramming to ground state pluripotency. Exactly how LIF/Stat3 signaling exerts these effects, however, remains elusive. This study identified gastrulation brain homeobox 2 (Gbx2) as a LIF/Stat3 downstream target that, when overexpressed, allows long-term expansion of undifferentiated mESCs in the absence of LIF/Stat3 signaling. Elevated Gbx2 expression also enhanced reprogramming of mouse embryonic fibroblasts to induced pluripotent stem cells. Moreover, overexpression of Gbx2 was sufficient to reprogram epiblast stem cells to ground state ESCs. Thee results reveal a novel function of Gbx2 in mESC reprogramming and LIF/Stat3-mediated self-renewal (Tai, 2013).

Vertebrate axis formation requires both the correct specification of cell fates and the coordination of gastrulation movements. The zebrafish Stat3 is activated on the dorsal side by the maternal Wnt/ß-catenin pathway. Zebrafish embryos lacking Stat3 activity display abnormal cell movements during gastrulation, resulting in a mispositioned head and a shortened anterior-posterior axis, but show no defects in early cell fate specification. Time course analysis, cell tracing, and transplantation experiments have revealed that Stat3 activity is required cell autonomously for the anterior migration of dorsal mesendodermal cells and non-cell autonomously for the convergence of neighboring paraxial cells. These results reveal a role for Stat3 in controlling cell movements during gastrulation (Yamashita, 2002).

Zebrafish Silberblick/Wnt11 has been shown to control convergent extension movements during gastrulation via a noncanonical Wnt pathway. These and other findings have suggested that a vertebrate equivalent of the Wingless/Frizzled/Dishevelled signaling cascade that controls Drosophila planar cell polarity is also required for the convergence and extension movements during gastrulation. This study shows that Stat3 also acts in the convergence and extension of paraxial mesoderm. wnt11 expression is normal in Stat3-depleted embryos, and Stat3 is activated normally in slb/wnt11 mutants. Moreover, Stat3 is required in axial mesoderm cells, whereas Slb/Wnt11 is required in more lateral cells. These results suggest that Slb/Wnt11 and Stat3 act in parallel in the control of convergence and extension. In Drosophila eye morphogenesis, ommatidial polarity is determined nonautonomously by JAK/STAT signaling via the regulation of a second signaling molecule. The ommatidia appear to sense the local gradient of the second signal and rotate accordingly. These results in zebrafish raise the possibility that not only conserved Wnt/fz/dsh interactions but also JAK/STAT signaling regulate both ommatidial polarity in Drosophila and convergence and extension in vertebrates (Yamashita, 2002).

Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine renders this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restores its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3 (Hart, 2001).

STAT factors act as signal transducers of cytokine receptors and transcriptionally activate specific target genes. The protein PIAS3 binds directly to STAT3 and blocks transcriptional activation. Experimental evidence is presented implementing the zinc finger protein Gfi-1 as a new regulatory factor in STAT3-mediated signal transduction. Gfi-1 (potential Drosophila homolog: CG11243) is a member of a protein family that includes Gfi-1b as well as the murine proteins Snail and Slug. All proteins of the Gfi-1 family share the six C-terminal C2-H2 zinc finger domains and the characteristic ‘SNAG’ domain that comprises the first 20 amino acid residues. The interaction between the two proteins first became evident in a yeast two-hybrid screen but is also seen in coprecipitation experiments from eukaryotic cells. Both Gfi-1 and PIAS3 colocalize in a characteristic nuclear dot structure. While PIAS3 exerts a profound inhibitory effect on STAT3-mediated transcription of target promoters, Gfi-1 can overcome the PIAS3 block and significantly enhances STAT3-mediated transcriptional activation. In primary T cells, Gfi-1 is able to amplify IL-6-dependent T-cell activation. As Gfi-1 is a known, dominant proto-oncogene, these findings bear particular importance for the recently described ability of STAT3 to transform cells malignantly and offers an explanation of the oncogenic potential of Gfi-1 in T lymphocytes (Rodel, 2000).

Transcriptional induction by interferons requires the tyrosine and serine phosphorylation of STAT transcription factors. The N-terminal region is highly homologous among the STAT proteins and surrounds a completely conserved arginine residue. Arginine methylation of STAT1 by the protein arginine methyl-transferase PRMT1 is a novel requirement for IFNalpha/beta-induced transcription. Methyl-thioadenosine, a methyl-transferase inhibitor that accumulates in many transformed cells, inhibits STAT1-mediated IFN responses. This inhibition arises from impaired STAT1-DNA binding due to an increased association of the STAT inhibitor PIAS1 with phosphorylated STAT1 dimers in the absence of arginine methylation. Thus, arginine methylation of STAT1 is an additional posttranslational modification regulating transcription factor function, and alteration of arginine methylation might be responsible for the lack of interferon responsiveness observed in many malignancies (Mowen, 2001).

STATs are activated by tyrosine phosphorylation on cytokine stimulation. A tyrosine-phosphorylated STAT forms a functional dimer through reciprocal Src homology 2 domain (SH2)-phosphotyrosyl peptide interactions. IFN treatment induces the association of PIAS1 and Stat1, which results in the inhibition of Stat1-mediated gene activation. The molecular basis of the cytokine-dependent PIAS1-Stat1 interaction has not been understood. A region near the COOH terminus of PIAS1 (amino acids 392-541) directly interacts with the NH(2)-terminal domain of Stat1 (amino acids 1-191). A mutant PIAS1 lacking the Stat1-interacting domain fails to inhibit Stat1-mediated gene activation. By using a modified yeast two-hybrid assay, it has been demonstrated that PIAS1 specifically interacts with the Stat1 dimer, but not tyrosine-phosphorylated or -unphosphorylated Stat1 monomer. In addition, whereas the NH(2)-terminal region of PIAS1 does not interact with Stat1, it serves as a modulatory domain by preventing the interaction of the COOH-terminal domain of PIAS1 with the Stat1 monomer. Thus, the cytokine-induced PIAS1-Stat1 interaction is mediated through the specific recognition of the dimeric form of Stat1 by PIAS1 (Liao, 2000).

STAT1 functions as both a constitutive transcriptional regulator and, in response to cytokine stimulation of cells, as an inducible tyrosine-phosphorylated transcription factor. A non-transferable nuclear targeting sequence has been identified and characterized in the STAT1 DNA-binding domain. This conserved signal is critical for the interferon-gamma (IFN-gamma)-induced nuclear import of phosphorylated STAT1 dimers and requires adjacent positively charged and hydrophobic residues for functioning. Additionally, the constitutive nucleocytoplasmic shuttling of STAT1 in the absence of IFN-gamma stimulation is revealed. Nuclear import and export of unphosphorylated STAT1 are demonstrated to be sensitive toward wheat germ agglutinin and to occur independently of the import receptor p97. Loss-of-function mutations of the dimer-specific import signal block nuclear entry of tyrosine-phosphorylated STAT1, which in turn also prevents induction of cytokine-inducible target genes. Nevertheless, nuclear import of unphosphorylated STAT1 continues and the STAT1-dependent constitutive expression of caspases and the tumor necrosis factor-gamma-mediated induction of apoptosis proceed unaltered. Thus, tyrosine-phosphorylated and unphosphorylated STAT1 molecules shuttle via independent pathways to distinct sets of target genes (Meyer, 2002).

A novel cDNA EZI isolated as an oncostatin M- inducible gene encodes a protein containing 12 C2H2-type zinc fingers. EZI transactivates the promoters that are also responsive to STAT3 and activate the acute phase response element (APRE) synergistically with STAT3. Co-immunoprecipitation has demonstrated the association of EZI with STAT3, which is mediated by the N-terminal region (1-183) of EZI. The EZI mutant lacking this region shows reduced transcriptional activity, indicating that EZI and STAT3 function cooperatively through physical interaction. While EZI predominantly localizes in the nucleus and enhances the nuclear localization of STAT3, the EZI mutant lacking 11 zinc finger motifs fails to translocate into the nucleus and also inhibits nuclear localization of STAT3 as well as STAT3-mediated transactivation. These results indicate that EZI is a novel nuclear zinc finger protein that augments STAT3 activity by keeping it in the nucleus (Nakayama, 2002).

A detailed time course of the assembly and disassembly of a STAT3-dependent, glucocorticoid-supplemented enhanceosome is described for the alpha2-macroglobulin (alpha2-M) gene and this is compared with a detailed time course of transcription of the gene by run-on analysis. The glucocorticoid receptor (GR) can associate with the enhanceosome without STAT3. Furthermore, the enhanceosome contains c-Jun/c-Fos and OCT-1 constitutively. All of these factors (GR, c-Jun, OCT-1) have transcription activation domains, but STAT3 is required for the observed transcriptional increase. The time course of enhanceosome occupation by GR and tyrosine-phosphorylated STAT3 shows that these transcription factors precede by ~5-10 min the arrival of RNA polymerase II (Pol II). The enhanceosome remains assembled for ~90 min in the continued presence of both inducers. When IL-6 and Dex are removed (after 30 min of treatment), the disappearance within an additional 30 min of the established enhanceosome indicates that renewal of STAT3 and GR binding must occur in the continued presence of IL-6+Dex. Compared with the total nuclear tyrosine-phosphorylated STAT3 capable of binding DNA, the chromatin-associated STAT3 resists dephosphorylation and appears to recycle to maintain the enhanceosome. Run-on transcription shows a lag after full enhanceosome occupation that can be largely but not completely explained by the ~30 min transit time of Pol II across the alpha2-Mlocus (Learner, 2003).

Bone remodeling is central to maintaining the integrity of the skeletal system, wherein the developed bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption. In the present study, a novel function of the Stat1 transcription factor is demonstrated in the regulation of bone remodeling. In the bone of the Stat1-deficient mice, excessive osteoclastogenesis is observed, presumably caused by a loss of negative regulation of osteoclast differentiation by interferon (IFN)-ß. However, the bone mass is unexpectedly increased in these mice. This increase is caused by excessive osteoblast differentiation, wherein Stat1 function is independent of IFN signaling. Actually, Stat1 interacts with Runx2 in its latent form in the cytoplasm, thereby inhibiting the nuclear localization of Runx2, an essential transcription factor for osteoblast differentiation. The new function of Stat1 does not require the Tyr 701 that is phosphorylated when Stat1 becomes a transcriptional activator. This study provides a unique example in which a latent transcription factor attenuates the activity of another transcription factor in the cytoplasm, and reveals a new regulatory mechanism in bone remodeling (Kim, 2003).

gp130-linked cytokines such as interleukin-6 (IL-6) stimulate the formation of tyrosine-phosphorylated STAT3 ( P-STAT3), which activates many genes, including the STAT3 gene itself. The resulting increase in the concentration of unphosphorylated STAT3 (U-STAT3) drives a second wave of expression of genes such as RANTES, IL6, IL8, MET, and MRAS that do not respond directly to P-STAT3. Thus, U-STAT3 sustains cytokine-dependent signaling at late times through a mechanism completely distinct from that used by P-STAT3. Many U-STAT3-responsive genes have kappaB elements that are activated by a novel transcription factor complex formed when U-STAT3 binds to unphosphorylated NFkappaB (U-NFkappaB), in competition with IkappaB. The U-STAT3/U-NFkappaB complex accumulates in the nucleus with help from the nuclear localization signal of STAT3, activating a subset of kappaB-dependent genes. Additional genes respond to U-STAT3 through an NFkappaB-independent mechanism. The role of signal-dependent increases in U-STAT3 expression in regulating gene expression is likely to be important in physiological responses to gp130-linked cytokines and growth factors that activate STAT3, and in cancers that have constitutively active P-STAT3 (Yang, 2007).

Experiments are reported that suggest a radical reorientation of tyrosine-phosphorylated parallel STAT1 dimers to an antiparallel form. Such a change in structure allows easy access to a phosphatase. With differentially epitope-tagged molecules, it is shown that the two monomers of a dimer remain together during dephosphorylation although they most likely undergo spatial reorientation. Extensive single amino acid mutagenesis within crystallographically established domains, manipulation of amino acids in an unstructured tether that connects the N-terminal domain (ND) to the core of the protein, and the demonstration that overexpressed ND can facilitate dephosphorylation of a core molecule lacking an ND all support this model: When the tyrosine-phosphorylated STAT1 disengages from DNA, the ND dimerizes and somehow assists in freeing the reciprocal pY-SH2 binding between the monomers of the dimer while ND - ND dimerization persists. The core of the monomers rotate allowing reciprocal association of the coiled:coil and DNA-binding domains to present pY at the two ends of an antiparallel dimer for ready dephosphorylation (Mertens, 2007).

Cytokines such as interferons (IFNs) activate signal transducers and activators of transcription (STATs) via phosphorylation. Histone deacetylases (HDACs) and the histone acetyltransferase (HAT) CBP dynamically regulate STAT1 acetylation. This study shows that acetylation of STAT1 counteracts IFN-induced STAT1 phosphorylation, nuclear translocation, DNA binding, and target gene expression. Biochemical and genetic experiments altering the HAT/HDAC activity ratio and STAT1 mutants reveal that a phospho-acetyl switch regulates STAT1 signaling via CBP, HDAC3, and the T-cell protein tyrosine phosphatase (TCP45). Strikingly, inhibition of STAT1 signaling via CBP-mediated acetylation is distinct from the functions of this HAT in transcriptional activation. STAT1 acetylation induces binding of TCP45, which catalyzes dephosphorylation and latency of STAT1. These results provide a deeper understanding of the modulation of STAT1 activity. These findings reveal a new layer of physiologically relevant STAT1 regulation and suggest that a previously unidentified balance between phosphorylation and acetylation affects cytokine signaling (Krämer, 2009).

Cytokine-dependent gene transcription greatly depends on the tyrosine phosphorylation ('activation') of Stat proteins at the cell membrane. This rapidly leads to their accumulation in the nucleus by an unknown mechanism. Microinjections of recombinant Stat1 protein were performed to show that nuclear accumulation of phosphorylated Stat1 can occur without cytokine stimulation of cells. Microinjection of Stat1 antibody and treatment of cells with kinase or phosphatase inhibitors reveals that nuclear accumulation is a highly dynamic process sustained by Stat1 nucleocytoplasmic cycling and continuous kinase activity. By characterizing nuclear accumulation mutants, it is demonstrated that nuclear import and nuclear retention are two separate steps leading up to nuclear accumulation, with nonspecific DNA binding of activated Stat1 being sufficient for nuclear retention. Critical for nuclear buildup of Stat1 and the subsequent nuclear export is the point of time of tyrosine dephosphorylation, because the data indicate that activated Stat1 is incapable of leaving the nucleus and requires dephosphorylation to do so. It is demonstrated that the inactivation of Stat1 is controlled by its exchange reaction with DNA, whereby DNA binding protects Stat1 from dephosphorylation in a sequence-specific manner. Thus, during nuclear accumulation, a surprisingly simple mechanism integrates central aspects of cytokine-dependent gene regulation, for example, receptor monitoring, promoter occupancy, and transcription factor inactivation (Meyer, 2003).

STAT proteins are cytoplasmic transcription factors that translocate to the nucleus and regulate gene expression upon activation of cytokine or growth factor receptors. While this translocation event is essential for gene regulation by STATs, their mechanism of transport through the cytoplasm to the nucleus has remained elusive. Cytoplasmic transport of Stat3 is an active process that requires receptor-mediated endocytosis. Stat3 co-localizes with endocytic vesicles in transit from the cell membrane to the perinuclear region in response to growth factor stimulation. Consistent with a role for receptor endocytosis in growth factor signaling, disruption of endocytosis with specific inhibitors blocks Stat3 nuclear translocation and Stat3-dependent gene regulation. These results indicate that receptor-mediated endocytosis may be a general mechanism of transport through the cytoplasm for a subset of cytoplasmic signaling proteins destined for the nucleus (Bild, 2002).

Receptor-mediated endocytosis allows the specific removal of cell surface receptors and their associated proteins from the plasma membrane and accumulates them in endosomes, where they are sorted for downregulation or recycling. This process is initiated upon ligand binding by recruitment of the receptor complex into a clathrin-coated pit at the plasma membrane, a structure formed by assembly of clathrin and clathrin adaptor protein 2 (AP-2) into a protein lattice on the membrane's cytosolic face. Clathrin and AP-2 bind to multiple components of the endocytic complex such as amphiphysin and epsin, which in turn bind to additional proteins that modulate formation and function of clathrin-coated pits. Expression of a fragment of amphiphysin 1 (Amph A1) leads to mislocalization of AP-2 and clathrin, with a resulting block in clathrin-mediated endocytosis. Overexpression of full-length Epsin 2a, another protein important for receptor-mediated endocytosis, mislocalizes endocytic complexes and also inhibits clathrin-mediated endocytosis (Bild, 2002 and references therein).

This study shows that Stat3 co-localizes with growth factor receptors in vesicles derived from endosomes at three locations in a time-dependent manner while in transit: the cell membrane, the cytosol and the perinuclear region. Inhibition of receptor-mediated endocytosis by Amph A1, Epsin 2a or a pharmacological inhibitor of endocytosis, phenylarsine oxide (PAO) has the following effects: abrogation of Stat3 transport to the perinuclear region, exclusion of functional Stat3 DNA-binding activity from the nucleus and suppression of Stat3-mediated transcriptional events. These results together demonstrate the important role of growth factor receptor-mediated endocytosis in Stat3 signaling. In addition, the accumulation of tyrosine phosphorylated Stat3, combined with the greatly reduced Stat3 DNA-binding activity when receptor endocytosis is blocked, strongly suggests that tyrosine phosphorylated Stat3 does not exist as randomly-diffusing active dimers in the cytoplasm but rather as monomers that remain bound to receptors following inhibition of endocytosis (Bild, 2002).

Intriguingly, a recent report confirms the presence of EGFR in the nucleus and suggests that nuclear EGFR regulates expression of genes, including cyclin D1. One possibility is that EGFR directly shuttles Stat3 into the nucleus, where Stat3 (perhaps still in a complex with EGFR) regulates gene expression. Consistent with this possibility is the finding that Stat3 regulates cyclin D1 gene expression. If this model is correct, then the primary role of EGFR translocation to the nucleus would be to serve as a scaffold for transport of signaling proteins like Stat3 directly to their nuclear targets involved in mediating growth factor responses. This raises the question of how receptors like EGFR and their associated proteins in the endocytic vesicles might translocate across the nuclear membrane. Certain components of the endocytic machinery, including Eps15, Eps15R and AP-180 possess FG repeats. FG-containing repeat regions have been shown to interact with several members of the importin family. Furthermore, Epsin 1 undergoes nucleo-cytoplasmic shuttling and its ENTH domain is structurally similar to Armadillo and HEAT repeats of the nuclear shuttling proteins ß-catenin and karyopherin-ß, respectively. Therefore, endocytic proteins may interact directly with the nuclear pore complex, thereby connecting the cell machineries that govern vesicle transport to those that mediate nucleo-cytoplasmic shuttling (Bild, 2002 and references therein).

Signal transducer and activator of transcription 3 (STAT3) is a latent transcription factor mainly activated by the interleukin-6 cytokine family. Previous studies have shown that activated STAT3 recruits p300, a coactivator whose intrinsic histone acetyltransferase activity is essential for transcription. This study investigated the function of the STAT3 NH(2)-terminal domain and how its interaction with p300 regulates STAT3 signal transduction. In STAT3(-/-) mouse embryonic fibroblasts, a stably expressed NH(2) terminus-deficient STAT3 mutant (STAT3-DeltaN) was unable to efficiently induce either STAT3-mediated reporter activity or endogenous mRNA expression. Chromatin immunoprecipitation assays were performed to determine whether the NH(2)-terminal domain regulates p300 recruitment or stabilizes enhanceosome assembly. Despite equivalent levels of STAT3 binding, cells expressing the STAT3-DeltaN mutant were unable to recruit p300 and RNA polymerase II to the native socs3 promoter as efficiently as those expressing STAT3-full length. It has been previously reported that the STAT3 NH(2)-terminal domain is acetylated by p300 at Lys-49 and Lys-87. By introducing K49R/K87R mutations, this study found that the acetylation status of the STAT3 NH(2)-terminal domain regulates its interaction with p300. In addition, the STAT3 NH(2)-terminal binding site maps to the p300 bromodomain, a region spanning from amino acids 995 to 1255. Finally a p300 mutant lacking the bromodomain (p300-DeltaB) exhibited a weaker binding to STAT3, and the enhanceosome formation on the socs3 promoter was inhibited when p300-DeltaB was overexpressed. Taken together, these data suggest that the STAT3 NH(2)-terminal domain plays an important role in the interleukin-6 signaling pathway by interacting with the p300 bromodomain, thereby stabilizing enhanceosome assembly (Hou, 2008).

STATs are cytoplasmic transcription factors that can be activated by the IL-6 cytokine family, a group of homologous peptides that include IL-6, oncostatin M (OSM), IL-11, and leukemia-inhibitory factor. The classic signaling pathway initiated by the IL-6 cytokine family is via ligand binding to cognate high affinity α chain receptors, e.g. IL-6Rα or OSMRα, that then complex with the ubiquitously expressed transmembrane protein gp130 β-subunit. The ligand-Rα-gp130 complex activates the cytoplasmic Janus and Tyk tyrosine kinases that phosphorylate the cytoplasmic domain of gp130, thereby providing docking sites for STAT1 and STAT3 isoforms. The recruited STATs are then phosphorylated by the same Janus/Tyk kinases at a specific tyrosine on the COOH-terminal transactivation domain (TAD), inducing their subsequent dimerization, nuclear translocation, and specific binding to IL-6 response elements. After the nuclear translocation of hetero- or homodimeric STATs, the magnitude of IL-6 signaling is further regulated by the recruitment of coactivators with histone acetyltransferase activity (Hou, 2008).

The crucial role of histone acetyltransferases in inducing chromatin remodeling and transcription activation has long been recognized. Several proteins with intrinsic histone acetyltransferase activity have been identified, including GCN5, p300/CREB-binding protein (CBP) homologs, p300/CBP-associated factor, and TAFII250. Histone acetyltransferases activate transcription by one or more of the following ways. (1) They are able to relax core nucleosome structure by acetylating the NH₂-terminal histone tails. (2) They can directly acetylate transcription factors and alter their transcription activities (3) They function as scaffold proteins to recruit other coactivators to the transcriptional apparatus. (4) They serve as bridging factors to physically connect sequence-specific transcription factors with multiple components in the basal transcription machinery. p300 and its homolog CBP are potent transcriptional coactivators that are actively involved in all of the four processes mentioned above. They have been shown to interact with several transcription factors, such as MyoD, p53, and E2F1, and regulate their activity by reversible acetylation (Hou, 2008).

There is strong evidence demonstrating that the histone acetyltransferase activity of p300 is required for STAT3 target gene activation. Previous studies have shown that overexpression of the p300 inhibitor adenovirus 12S E1A significantly inhibits the IL-6-induced activation of human angiotensinogen (hAGT), a vasoactive peptide and acute phase protein controlled by STAT3. The ectopic expression of p300 enhances the induction of hAGT reporter gene stimulated by IL-6. Conversely expression of a p300 mutant deficient in histone acetyltransferase activity functions as a dominant-negative inhibitor and strongly inhibits STAT3-dependent transcription. Moreover IL-6-inducible p300-STAT3 association causes an increase in histone H4 acetylation on the hAGT promoter, indicating that p300 recruitment augments STAT3 transactivation by acetylating histone tails, thereby relaxing chromatin structure (Hou, 2008).

p300 interacts with STAT3 within both its COOH-terminal TAD and NH₂-terminal domain, and this phenomenon has also been confirmed for STAT1 and STAT2. The STAT family shares a highly conserved modular structure that includes an NH₂-terminal domain, a coiled coil domain, a DNA-binding domain, a linker domain, an SH2 domain, and a COOH-terminal TAD. The coiled coil domain is actively involved in protein-protein interaction, and the SH2 domain mediates the STAT3 dimerization via intermolecular Tyr(P)-SH2 interactions. The COOH-terminal TAD contains a conserved single tyrosine residue that is phosphorylated in STAT activation and facilitates transcriptional activation. The function of the NH₂-terminal domain in STAT3, however, is poorly understood. In this study the STAT3 NH₂-terminal function was investigated by stably expressing an NH₂ terminus-deleted mutant (STAT3-ΔN) in STAT3^-/- MEFs. Both OSM-inducible γ-FBG reporter gene and endogenous mRNA expression including socs3, c-fos, and p21 were significantly reduced in response to STAT3-ΔN expression. Because the STAT3 NH₂-terminal domain is involved in p300 binding, the defective activity observed in STAT3-ΔN is probably caused by the reduced cooperation between STAT3 and p300. This hypothesis was then tested in native chromatin by chromatin immunoprecipitation (ChIP) assays that revealed a reduction in OSM-inducible p300 recruitment to the socs3 promoter in MEFs stably expressing STAT3-ΔN. At the same time, there was a decrease in RNA pol II binding to the socs3 promoter, indicating the STAT3 NH₂-terminal domain not only stabilizes coactivator association but also facilitates the assembly of transcription preinitiation complex (Hou, 2008).

Recent studies identified STAT3 not only as a binding partner of p300 but also as a substrate for its acetylation where p300 modifies STAT3 at multiple sites. A single acetylation on Lys residue Lys-685 localized in the COOH-terminal TAD is required for STAT3 dimerization and the subsequent DNA binding activity. This laboratory independently identified two other Lys residues, Lys-49 and Lys-87, in the STAT3 NH₂-terminal domain that are also inducibly acetylated by p300 in response to IL-6 and OSM. Although these NH₂-terminal acetylations have no effect on STAT3 DNA binding activity, they are essential for STAT3-dependent transcription because K49R/K87R substitutions significantly inhibit STAT3 target gene expression. It was also noticed that the K49R/K87R mutations decrease the association between p300 and STAT3, indicating that the inducible NH₂-terminal acetylations may augment STAT3-p300 interaction. This study further investigated the interaction between the STAT3 NH₂-terminal domain and p300 and found that the acetyl-Lys mimic substitutions (K49Q/K87Q) increase the STAT3 NH₂-terminal binding to p300, confirming the hypothesis that the NH₂-terminal acetylations stabilize the STAT3-p300 interaction. It was also discovered that the STAT3 NH₂-terminal binding site maps to the p300 bromodomain. The deletion of the bromodomain in p300 molecule decreased its ability to cooperate with STAT3. In addition, the bromodomain-deficient p300 mutant (p300-ΔB) exhibited weaker chromatin binding. Taken together, a model is proposed in which the IL-6- or OSM-inducible acetylations of STAT3 on Lys residues 49 and 87 trigger the recognition of the NH₂-terminal domain by the p300 bromodomain, resulting in a strengthened recruitment of p300 to the promoter of the STAT3 target gene, thereby facilitating subsequent enhanceosome assembly (Hou, 2008).