STATs are targeted by MAP kinases

Recent studies have indicated that serine phosphorylation regulates the activities of STAT1 and STAT3. However, the kinase(s) responsible and the role of serine phosphorylation in STAT function remain unresolved. In the present studies, the growth factor-dependent serine phosphorylation of STAT1 and STAT3 were examined. The ERK family of mitogen-activated protein kinases (see Drosophila Rolled), but not JNK or p38, specifically phosphorylates STAT3 at serine 727 in response to growth factors. Evidence for additional mitogen-regulated serine phosphorylation is also provided. STAT1 is a relatively poor substrate for all MAP kinases tested both in vitro and in vivo. STAT3 serine phosphorylation, not its tyrosine phosphorylation, results in retarded mobility of the STAT3 protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Importantly, serine 727 phosphorylation negatively modulates STAT3 tyrosine phosphorylation, which is required for dimer formation, nuclear translocation, and the DNA binding activity of this transcriptional regulator. Interestingly, the cytokine interleukin-6 also stimulates STAT3 serine phosphorylation, but in contrast to growth factors, this occurs by an ERK-independent process (Chung, 1997).

Turnover of STATs

Cytokines induce the tyrosine phosphorylation and associated activation of signal transducers and activators of transcription (Stat). The mechanisms by which this response is terminated are largely unknown. Among a variety of inhibitors examined, the proteasome inhibitors MG132 and lactacystin affect Stat4, Stat5 and Stat6 turnover by significantly stabilizing the tyrosine-phosphorylated form. However, these proteasome inhibitors did not affect downregulation of the tyrosine-phosphorylated Stat1, Stat2 and Stat3. With Stat5 isoforms, tyrosine-phosphorylated carboxyl-truncated forms of Stat5 proteins are considerably more stable than phosphorylated wild-type forms of the protein. Also, the C-terminal region of Stat5 could confer proteasome-dependent downregulation to Stat1. With a series of C-terminal deletion mutants, a relatively small, potentially amphipathic alpha-helical region has been defined that is required for the rapid turnover of the phosphorylated Stat5 proteins. The region is also required for transcriptional activation, suggesting that the functions are linked. The results are consistent with a model in which the transcriptional activation domain of activated Stat5 is required for its transcriptional activity and downregulation through a proteasome-dependent pathway (Wang, 2000).

Nuclear import of STATs

In response to interferon-gamma (IFN-gamma), Stat1 is tyrosine phosphorylated and translocates to the nucleus where it activates transcription. In this study, factors are identified that mediate the nuclear import of Stat1. Tyrosine-phosphorylated Stat1 associates with the beta subunit (a 97 kDa component) of the nuclear pore-targeting complex via the NPI-1 family, but not the Rch1 family, of alpha subunit (a 58 kDa component) as a result of IFN-gamma stimulation. Antibodies against NPI-1 or beta subunit consistently inhibit the IFN-gamma-dependent nuclear import of Stat1 in living cells, although antibodies reactive to Rch1 have no effect. Solution binding assays with deletion mutants of NPI-1 show that the Stat1-binding domain of NPI-1 is located in the carboxy-terminal region, which is clearly distinct from the SV40 large T antigen nuclear localization signal (NLS)-binding region. These results indicate that the extracellular signal-dependent nuclear transport of Stat1 is mediated by NPI-1, but not Rch1, in conjunction with the beta subunit, and that these factors participate in the conditional (as well as the constitutive) nuclear import of proteins (Sekimoto, 1997).

Signal transducers and activators of transcription (STATs) reside in a latent state in the cytoplasm of the cell, but accumulate in the nucleus in response to cytokines or growth factors. Localization in the nucleus occurs following STAT tyrosine phosphorylation and dimerization. A direct interaction of importin-alpha5 with tyrosine-phosphorylated STAT1 dimers is reported, and evidence is provided that a nuclear localization signal (NLS) exists in an inactive state within a STAT1 monomer. A mutation in STAT1 leucine 407 (L407A) is characterized, which generates a protein that is accurately tyrosine phosphorylated in response to interferon, dimerizes and binds DNA, but does not localize to the nucleus. The import defect of STAT1(L407A) appears to be a consequence of the inability of this protein to be recognized by its import shuttling receptor. In addition, STAT1 binding to specific target DNA effectively blocks importin-alpha5 binding. This result may play a role in localizing STAT1 to its destination in the nucleus, and in releasing importin-alpha5 from STAT1 for recycling back to the cytoplasm (McBride, 2002).

Developmentally regulated demethylation of genes encoding components of the JAK-STAT pathway

DNA methylation is a major epigenetic factor that has been postulated to regulate cell lineage differentiation. Conditional gene deletion of the maintenance DNA methyltransferase I (Dnmt1) in neural progenitor cells (NPCs) results in DNA hypomethylation and precocious astroglial differentiation. The developmentally regulated demethylation of astrocyte marker genes as well as genes encoding the crucial components of the gliogenic JAK-STAT pathway is accelerated in Dnmt1-/- NPCs. Through a chromatin remodeling process, demethylation of genes in the JAK-STAT pathway leads to an enhanced activation of STATs, which in turn triggers astrocyte differentiation. This study suggests that during the neurogenic period, DNA methylation inhibits not only astroglial marker genes but also genes that are essential for JAK-STAT signaling. Thus, demethylation of these two groups of genes and subsequent elevation of STAT activity are key mechanisms that control the timing and magnitude of astroglial differentiation (Fan, 2005).

Transcription targets of STATs

Transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) have opposite effects on diverse cellular functions, but the basis for this antagonism is not known. TGF-beta signals through a receptor serine kinase that phosphorylates and activates the transcription factors Smads 2 and 3, whereas the IFN-gamma receptor and its associated protein tyrosine kinase Jak1 mediate phosphorylation and activation of the transcription factor Stat1. A basis for the integration of TGF-beta and IFN-gamma signals is presented. IFN-gamma inhibits the TGF beta-induced phosphorylation of Smad3 and its attendant events, namely, the association of Smad3 with Smad4, the accumulation of Smad3 in the nucleus, and the activation of TGFbeta-responsive genes. Acting through Jak1 and Stat1, IFN-gamma induces the expression of Smad7, an antagonistic SMAD, which prevents the interaction of Smad3 with the TGF-beta receptor. The results indicate a mechanism of transmodulation between the STAT and SMAD signal-transduction pathways (Ulloa, 1999).

STAT5 is a member of a family of transcription factors that participate in the signal transduction pathways of many hormones and cytokines. Although STAT5 is suggested to play a crucial role in the biological effects of cytokines, its downstream target(s) associated with cell growth control is largely unknown. In a human interleukin-3 (IL-3)-dependent cell line F-36P-mpl, the induced expression of dominant-negative (dn)-STAT5 and of dn-ras leads to inhibition of IL-3-dependent cell growth, accompanying the reduced expression of cyclin D1 mRNA. Also, both constitutively active forms of STAT5A (1*6-STAT5A) and ras (H-rasG12V) enable F-36P-mpl cells to proliferate without added growth factors. In NIH 3T3 cells, 1*6-STAT5A and H-rasG12V individually and cooperatively transactivate the cyclin D1 promoter in luciferase assays. Both dn-STAT5 and dn-ras suppress IL-3-induced cyclin D1 promoter activities in F-36P-mpl cells. Using a series of mutant cyclin D1 promoters, 1*6-STAT5A was found to transactivate the cyclin D1 promoter through the potential STAT-binding sequence at -481 bp. STAT5 binds to the element in response to IL-3. Furthermore, the inhibitory effect of dn-STAT5 on IL-3-dependent growth is restored by expression of cyclin D1. Thus STAT5, in addition to ras signaling, appears to mediate transcriptional regulation of cyclin D1, thereby contributing to cytokine-dependent growth of hematopoietic cells (Matsumura, 1999).

Decreased Fas expression during tumor progression often results in a loss of Fas-ligand (FasL)-mediated apoptosis. Human and mouse melanoma exhibit an inverse correlation between the degree of Fas cell surface expression, tumorigenicity, and metastatic capacity. The expression of dominant negative Stat3 or c-Jun in melanoma cells efficiently increases Fas expression and sensitizes cells to FasL-induced apoptosis. Stat3+/- as well as c-Jun-/- cells exhibit increased Fas cell surface expression and higher sensitivity to FasL-mediated apoptosis. Suppression of Fas expression by Stat3 and c-Jun is uncoupled from Stat3-mediated transcriptional activation. These findings indicate that Stat3 oncogenic activities could also be mediated through its cooperation with c-Jun, resulting in downregulation of Fas surface expression, which is implicated in the tumor's ability to resist therapy and metastasize (Ivanov, 2001).

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. A cytokine-responsive enhancer between -5.2 and -6.1 kb in the 5'-flanking hiNOS promoter DNA contains multiple NF-kappa B elements. The role of the IFN-Jak kinase-Stat 1 pathway for regulation of hiNOS gene transcription is described in this study. In A549 human lung epithelial cells, a combination of cytokines TNF-alpha, IL-1 beta, and IFN-gamma function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at -5.8 kb in the hiNOS promoter identifies a bifunctional NF-kappa B/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at -5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-kappa B activates hiNOS promoter-reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-kappa B function in these cells. These results indicate that both Stat 1 and NF-kappa B are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner (Ganster, 2001).

Murine ES cells can be maintained as a pluripotent, self-renewing population by IL6 family members such as cytokine leukemia inhibitory factor (LIF) ---> STAT3-dependent signaling. The downstream effectors of this pathway have not been defined. A key target of the LIF self-renewal pathway was identified by showing that STAT3 directly regulates the expression of the Myc transcription factor. Murine ES cells express elevated levels of Myc and following LIF withdrawal, Myc mRNA levels collapse and Myc protein becomes phosphorylated on threonine 58 (T58), triggering its GSK3§ dependent degradation. Maintained expression of stable Myc (T58A) renders self-renewal and maintenance of pluripotency independent of LIF. By contrast, expression of a dominant negative form of Myc antagonizes self-renewal and promotes differentiation. Transcriptional control by STAT3 and suppression of T58 phosphorylation are crucial for regulation of Myc activity in ES cells and therefore in promoting self-renewal. Together, these results establish a mechanism for how LIF and STAT3 regulate ES cell self-renewal and pluripotency (Cartwright, 2005).

Although LIF/STAT3 signaling is crucial for murine ES cell maintenance, this pathway does not appear to have a role in human ES cell self-renewal, indicating the existence of alternate self-renewal mechanisms. A role has been defined for Wnt-dependent signaling in self-renewal of human and murine ES cells that functions independently of LIF and STAT3. Moreover, suppression of GSK3beta, an antagonist of Wnt signaling, is sufficient to maintain self-renewal and pluripotency of human and murine ES cells in the absence of LIF and Wnt. These observations signify a common mechanism of self-renewal that may be further applicable to adult stem cell populations that require Wnt-dependent signaling (Cartwright, 2005 and references therein).

Although LIF and Wnt promote self-renewal by activation of separate signaling pathways, it was reasoned that they would converge on a common target(s). It was hypothesized that Myc could be a common effector on which these signals converge because the Myc gene is a transcriptional target of STAT3 in a number of biological contexts, and signals transduced by Wnt can activate the Myc transcription through a §-catenin/TCF-dependent mechanism. Myc belongs to a family of helix-loop-helix/leucine zipper transcription factors and together with its obligatory binding partner, Max, performs roles in control of cell proliferation, transformation, growth, differentiation and apoptosis. A potential role for Myc in ES cell maintenance is suggested by two reports. (1) Expression of an RLF/L-myc minigene that frequently arises from a chromosomal translocation event in human small lung carcinomas, delays ES cell differentiation and interferes with early embryonic development. (2) Elevated Myc activity is able to block the differentiation of multiple cell lineages. These lines of evidence prompted an investigation of whether Myc plays a role in ES cell self-renewal downstream of LIF and/or Wnt. This report shows that elevated Myc activity is required for ES cell maintenance and that Myc is a key effector of the LIF/STAT3 self-renewal pathway. The data indicate that signals transduced by LIF and possibly Wnt, converge on Myc to maintain ES cell identity (Cartwright, 2005).

The STAT3 transcription factor is an important initiator of mammary gland involution in the mouse. This work shows that the STAT3 target gene CCAAT/enhancer binding protein delta (C/EBPdelta) is a crucial mediator of pro-apoptotic gene expression events in mammary epithelial cells. In the absence of C/EBPdelta, involution is delayed and the pro-apoptotic genes encoding p53, BAK, IGFBP5 and SGP2/clusterin are not activated, while the anti-apoptotic genes coding for BFL1 and Cyclin D1 are not repressed. Consequently, p53 targets such as survivin, BRCA1, BRCA2 and BAX are not regulated appropriately and protease activation is delayed. Furthermore, expression of MMP3 and C/EBP during the second phase of involution is perturbed in the absence of C/EBP. In HC11 cells, C/EBP alone is sufficient to induce IGFBP5 and SGP2. It also suppresses Cyclin D1 expression and cooperates with p53 to elicit apoptosis. This study places C/EBP between STAT3 and several pro- and anti-apoptotic genes promoting the physiological cell death response in epithelial cells at the onset of mammary gland involution (Thangaraju, 2005).

Nuclear protein interactions of STATs

Protein tyrosine kinases activate the STAT (signal transducer and activator of transcription) signaling pathway, which can play essential roles in cell differentiation, cell cycle control, and development. However, the potential role of the STAT signaling pathway in the induction of apoptosis remains unexplored. Gamma interferon (IFN-gamma) activates STAT1 and induces apoptosis in both A431 and HeLa cells, whereas epidermal growth factor (EGF) activates STAT proteins and induces apoptosis in A431 but not in HeLa cells. EGF receptor autophosphorylation and mitogen-activated protein kinase activation in response to EGF are similar in both cell lines. The breast cancer cell line MDA-MB-468 exhibits a similar response to A431 cells, i.e., STAT activation and apoptosis correlatively results from EGF or IFN-gamma treatment. In addition, in a mutant A431 cell line in which STAT activation is abolished, no apoptosis is induced by either EGF or IFN-gamma. Both EGF and IFN-gamma induce caspase 1 (interleukin-1beta converting enzyme [ICE]) gene expression (See Drosophila Death caspase 1) in a STAT-dependent manner. IFN-gamma is unable to induce ICE gene expression and apoptosis in either JAK1-deficient HeLa cells (E2A4) or STAT1-deficient cells (U3A). However, ICE gene expression and apoptosis are induced by IFN-gamma in U3A cells into which STAT1 had been reintroduced. Moreover, both EGF-induced apoptosis and IFN-gamma-induced apoptosis are effectively blocked by Z-Val-Ala-Asp-fluoromethylketone (ZVAD) in all the cells tested; studies from ICE-deficient cells indicate that ICE gene expression is necessary for IFN-gamma-induced apoptosis. It is concluded that activation of the STAT signaling pathway can induce apoptosis through the induction of ICE gene expression (Chin, 1997).

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro, HTLV-I induces ligand-independent transformation of human CD4(+) T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo. In cell extracts of uncultured leukemic cells from 12 patients with ATLL, the status of both JAK/STAT phosphorylation and DNA-binding activity of STAT proteins was tested with DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of the 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins; the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. An association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression has been demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four of the patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth (Takemoto, 1997).

TFII-I is a transcription factor that was initially characterized as a factor that binds to the initiator sites of various promoters. It has been implicated in the initiation of transcription of TATA-less promoters and in cell-type-specific transcription as well. Deletions of TFII-I are closely associated with neurodevelopmental Williams-Beuren syndrome in humans. TFII-I can also bind to E-box elements and can interact with upstream regulatory factors, including USF1 and c-myc. In addition, TFII-I can associate with Bruton's tyrosine kinase, and TfII-I's phosphorylation on tyrosine is stimulated by BTK. The activity of TFII-I is regulated by phosphorylation, and one of the potential phosphorylation sites is a mitogen-activated protein (MAP) kinase phosphorylation site. These observations suggest that TFII-I may play a role in signal transduction as well as in transcriptional initiation. In addition, TFII-I associates with the serum response factor (SRF) and the Phox1 protein, which are both involved in the regulation of the c-fos promoter (Kim, 1998 and references).

Overexpression of TFII-I can enhance the response of the wild-type c-fos promoter to a variety of stimuli. This effect depends on the c-fos c-sis-platelet-derived growth factor-inducible factor binding element (SIE) and serum response element (SRE). There is no effect of cotransfected TFII-I on the TATA box containing the c-fos basal promoter. Three TFII-I binding sites can be found in the c-fos promoter. Two of these overlap the c-fos SIE and SRE, and another is located just upstream of the TATA box. Mutations that distinguish between serum response factor (SRF), STAT, and TFII-I binding to the c-fos SIE and SRE suggest that the binding of TFII-I to these elements is important for c-fos induction in conjunction with the SRF and STAT transcription factors. Moreover, TFII-I can form in vivo protein-protein complexes with the c-fos upstream activators SRF, STAT1, and STAT3. These results suggest that TFII-I may mediate the functional interdependence of the c-fos SIE and SRE elements. In addition, the ras pathway is required for TFII-I to exert its effects on the c-fos promoter; growth factor stimulation enhances tyrosine phosphorylation of TFII-I. These results indicate that TFII-I is involved in signal transduction as well as transcriptional activation of the c-fos promoter (Kim, 1998).

Using the coiled-coil region of Stat5b as the bait in a yeast two-hybrid screen, the association of Nmi, a protein of unknown function previously reported as an N-Myc interactor, was identifed. Nmi interacts with all STATs except Stat2. Two cytokine systems, IL-2 and IFNgamma, were evaluated and Nmi was demonstrated to augment STAT-mediated transcription in response to these cytokines. Interestingly, Nmi lacks an intrinsic transcriptional activation domain; instead, Nmi enhances the association of CBP/p300 coactivator proteins with Stat1 and Stat5, and together with CBP/p300 augments IL-2- and IFNgamma-dependent transcription. Therefore, these data not only reveal that Nmi can potentiate STAT-dependent transcription, but also suggest that it can augment coactivator protein recruitment to at least some members of a group of sequence-specific transcription factors (Zhu, 1999).

The cytokines LIF (leukemia inhibitory factor) and BMP2 (bone morphogenetic protein-2) signal through different receptors and transcription factors, namely STATs (signal transducers and activators of transcription) and Smads. LIF and BMP2 act in synergy on primary fetal neural progenitor cells to induce astrocytes. The transcriptional coactivator p300 interacts physically with STAT3 at its amino terminus in a cytokine stimulation-independent manner, and with Smad1 at its carboxyl terminus in a cytokine stimulation-dependent manner. The formation of a complex between STAT3 and Smad1, bridged by p300, is involved in the cooperative signaling of LIF and BMP2 and the subsequent induction of astrocytes from neural progenitors (Nakashima, 1999).

The signal transducers and activators of transcription (Stats) mediate many effects of cytokines and peptide growth factors. Stat1 is a major transcription factor in the IFN-ß and IFN-gamma (interferons) signal transduction pathways that lead to the activation of antiviral, antiproliferative and immunomodulatory functions. IFN-gamma stimulates gene expression through the phosphorylation of Stat1 by Janus kinases (JAKs) at the cell membrane, followed by homodimerization of Stat1, nuclear translocation and binding to IFN-gamma-activated sequence (GAS) elements. TTCNNNG/TAA is the GAS consensus. Homodimerization of Stat1 is mediated by the binding of the phosphorylated Tyr701 of one Stat1 monomer to the Src homology 2 (SH2) domain of another. Analysis of mRNA levels in cells that express or lack signal transducers and activators of transcription 1 (Stat1) reveals that Stat1 mediates the constitutive transcription of many genes. Expression of the low molecular mass polypeptide 2 (LMP2), which requires Stat1, has been studied in detail. The overlapping interferon consensus sequence 2/gamma-interferon-activated sequence (ICS-2/GAS) elements in the LMP2 promoter bind to interferon regulatory factor 1 (IRF1) and Stat1, and are occupied constitutively in vivo. The point mutant of Stat1, Y701F, which does not form dimers involving SH2-phosphotyrosine interactions, binds to the GAS element and supports LMP2 expression. Unphosphorylated Stat1 binds to IRF1 directly and it is concluded that this complex uses the ICS-2/GAS element to mediate constitutive LMP2 transcription in vivo. The promoter of the IRF1 gene, which also contains a GAS site but not an adjacent ICS-2 site, is not activated by Stat1 Y701F. The promoters of other genes whose constitutive expression requires Stat1 may also utilize complexes of unphosphorylated Stat1 with IRF1 or other transcription factors (Chatterjee-Kishore, 2000).

The mechanisms by which neural stem cells give rise to neurons, astrocytes, or oligodendrocytes are beginning to be elucidated. However, it is not known how the specification of one cell lineage results in the suppression of alternative fates. In addition to inducing neurogenesis, the bHLH transcription factor neurogenin (Ngn1: Drosophila homolog Target of Pox-n) inhibits the differentiation of neural stem cells into astrocytes. While Ngn1 promotes neurogenesis by functioning as a transcriptional activator, Ngn1 inhibits astrocyte differentiation by sequestering the CBP-Smad1 transcription complex away from astrocyte differentiation genes, and by inhibiting the activation of STAT transcription factors that are necessary for gliogenesis. Thus, two distinct mechanisms are involved in the activation and suppression of gene expression during cell-fate specification by neurogenin (Sun, 2001).

Neuronal differentiation is promoted by both platelet-derived growth factor (PDGF) and by neurotrophin-3 (NT3). The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) are potent inducers of astrocyte production, and thyroid hormone induces oligodendrocyte differentiation. LIF and CNTF exert their effects primarily via the JaK/STAT signaling pathway. LIF and CNTF bind to related receptors, which activate a receptor-associated tyrosine kinase, the Janus kinase (JaK1). Activated JaK1 phosphorylates two cytoplasmic proteins, the signal transducers and activators of transcription 1 and 3 (STAT1 and STAT3). This leads to STAT dimerization and translocation to the nucleus where the STATs activate cell type and stimulus-specific programs of gene expression (Sun, 2001 and references therein).

Other factors, such as bone morphogenetic protein (BMP), can enhance both neuronal and astrocyte differentiation, depending on the age of the stimulated cortical progenitors. BMP-induced astrocyte differentiation appears to be mediated by the downstream Smad signaling proteins. BMPs bind a multimeric receptor, which in turn results in the direct phosphorylation of Smad1. This permits Smad1 to dimerize with Smad4 and to translocate to the nucleus, where these factors cooperate with STATs to activate glial-specific programs of gene expression (Sun, 2001 and references therein).

The cooperation between Smads and STATs on glial promoters such as the glial fibrillary acidic protein (GFAP) promoter appears to be facilitated by a family of coactivator proteins termed p300/CBP. CBP (CREB binding protein) and p300 are ubiquitously expressed and are involved in the transcriptional coactivation of many different transcription factors. STATs and Smads bind to different domains of CBP/p300, and the STAT/p300/Smad complex, acting at the STAT binding element in the astrocyte-specific GFAP promoter, is particularly effective at inducing astrocyte differentiation in neural stem cells (Sun, 2001 and references therein).

Many transcription factors require CBP/p300 in order to activate transcription, and there is evidence that the levels of CBP/p300 are limiting, i.e., that there is competition among the various families of transcription factors for CBP/p300 binding. For example, nuclear steroid receptors indirectly inhibit AP-1-dependent transcription by sequestering CBP/p300 away from AP-1 and onto sites where the nuclear receptors are bound. Similarly, the anti-adenoviral actions of interferon are attributed to interferon's ability to activate STATs, which then sequester CBP/p300 away from the adenoviral transcription factor E1A. During early cortical development, endogenous Ngn1 associates with both CBP and Smad1, and the presence of neurogenin blocks STAT binding to CBP. Xenopus neurogenin has been shown to recruits CBP/p300 to the NeuroD promoter to activate transcription and induce neurogenesis. The characterization of the domains of CBP that interact with neurogenin reveal that both an N- and a C-terminal domain are involved. Interestingly, the neurogenin binding domains of CBP overlap with the STAT binding sites on CBP (but not with the Smad binding sites). This is consistent with the finding that neurogenin competes with STAT proteins for binding to CBP. By sequestering CBP, neurogenin may not only inhibit STAT-mediated transcription, but may also inhibit the function of other CBP-dependent transcription factors. Ngn1 also inhibits AP-1-dependent transcription. This may be relevant to Ngn's ability to inhibit astrocyte differentiation since the analysis of the GFAP promoter identifies multiple sites, including an AP-1 site, that contribute to neurogenin's inhibition of the GFAP promoter. Taken together, these findings suggest that CBP/p300 may orchestrate broad programs of gene expression that are relevant to cell fate determination. The effect of CBP/p300 on cell fate may then be determined by the relative binding affinity and abundance of different transcription factors that either compete or cooperate with one another for binding to CBP/p300 (Sun, 2001 and references therein).

In addition to sequestering the CBP-Smad1 complex, neurogenin also inhibits the activation of astrocyte-specific genes by blocking STAT activation. The mechanism by which Ngn1 reduces the level of phospho-STAT1 and -STAT3 is unknown. The Ngn1 deficient in binding DNS can also inhibit STAT phosphorylation, though not to the extent seen with wild-type Ngn1. This suggests that Ngn1 inhibits STAT phosphorylation only in part by a mechanism that is independent of Ngn1 binding to DNA (Sun, 2001).

STAT6 is a central mediator of IL-4-induced gene responses. STAT6-mediated transcription is dependent on the C-terminal transcription activation domain (TAD), but the mechanisms by which STAT6 activates transcription are poorly understood. The staphylococcal nuclease (SN)-like domain and tudor domain (TD) containing protein p100 has been identified as a STAT6 TAD interacting protein. The TD is found in proteins with putative functions in RNA-binding or protein-binding functions during RNA metabolism and transport. p100 was originally characterized as a transcriptional coactivator for Epstein-Barr virus nuclear antigen 2. STAT6 interacts with p100 in vitro and in vivo. The interaction is mediated by the TAD domain of STAT6 and the SN-like domain of p100. p100 does not affect the immediate activation events of STAT6, but enhances STAT6-mediated transcriptional activation and the IL-4-induced Ig gene transcription in human B-cell line. Finally, p100 associates with the large subunit of RNA polymerase II and mediates interaction between STAT6 and RNA polymerase II. These findings identify p100 as a novel coactivator for STAT6 and suggest that p100 functions as a bridging factor between STAT6 and the basal transcription machinery (Yang, 2002)

PAF, which is composed of Paf1 (see Drosophila Paf1), Cdc73, Ctr9, Leo1, and Rtf1, is a novel complex with multiple functions in transcription-related activities. The PAF complex interacts with histone-modifying enzymes and RNA polymerase II to regulate transcription. With general transcription regulatory potential in yeast, Hyrax/Cdc73 has been reported to associate with beta-catenin to control Wnt/Wg signal-specific transcription in Drosophila. This study presents evidence of IL-6 signal-specific transcriptional regulation by SH2BP1/CTR9 in mammals. Upon LPS injection of mice, transient induction of the mammalian PAF complex is observed in the liver. Inhibition of CTR9 specifically abrogated expression of IL-6-responsive genes, but had no effect on genes constitutively expressed or induced by interferon-beta, TNFalpha, or IL-1beta. The PAF complex was found in the promoter regions of IL-6-responsive HP and FGGgamma, but not in the promoter region of constitutively active GAPDH. Transcriptional activation by STAT3 was inhibited when CTR9 siRNA was introduced, whereas transcriptional activation was enhanced by mCtr9 overexpression. IL-6-activated Stat3 was found to co-localize and interact with CTR9. In CTR9-depleted cells, decreased STAT3 association with the promoter regions, as well as impaired K4-trimethylation of histone H3 in the coding regions, of target genes was observed. These data suggest that CTR9 participates in the transcription of IL-6-responsive genes through the regulation of DNA association of STAT3 and modification of histone methylation (Youn, 2007).

STATs interact with protein inhibitor of activated STAT (PIAS) family proteins

STAT proteins are latent cytoplasmic transcription factors that become activated by tyrosine phosphorylation in response to cytokine stimulation. Tyrosine phosphorylated STATs dimerize and translocate into the nucleus to activate specific genes. Different members of the STAT protein family have distinct functions in cytokine signaling. Biochemical and genetic analysis has demonstrated that Stat1 is essential for gene activation in response to interferon stimulation. Although progress has been made toward understanding STAT activation, little is known about how STAT signals are down-regulated. The isolation of a family of PIAS (protein inhibitor of activated STAT) proteins is reported. PIAS1, but not other PIAS proteins, block the DNA binding activity of Stat1 and inhibit Stat1-mediated gene activation in response to interferon. Coimmunoprecipitation analysis shows that PIAS1 is associated with Stat1 but not Stat2 or Stat3 after ligand stimulation. The in vivo PIAS1-Stat1 interaction requires phosphorylation of Stat1 on Tyr-701. These results identify PIAS1 as a specific inhibitor of Stat1-mediated gene activation and suggest that there may exist a specific PIAS inhibitor in every STAT signaling pathway (Liu, 1998).

Androgen signaling influences the development and growth of prostate carcinoma. The transcriptional activity of androgen receptor (AR) is regulated by positive or negative transcriptional cofactors. PIAS1, PIAS3, and PIASy of the protein inhibitor of activated STAT (PIAS) family, which are expressed in human prostate, display distinct effects on AR-mediated gene activation in prostate cancer cells. While PIAS1 and PIAS3 enhance the transcriptional activity of AR, PIASy acts as a potent inhibitor of AR in prostate cancer cells. The effects of PIAS proteins on AR are competitive. PIASy binds to AR but does not affect the DNA binding activity of AR. An NH2-terminal LXXLL signature motif of PIASy, although not required for PIASy-AR interaction, is essential for the transrepression activity of PIASy. These results identify PIASy as a transcriptional corepressor of AR and suggest that different PIAS proteins have distinct effects on AR signaling in prostate cancer cells (Gross, 2001).

STATs, cell cycle and oncogenesis

Stat3 activation has been associated with cytokine-induced proliferation, anti-apoptosis, and transformation. Constitutively activated Stat3 has been found in many human tumors as well as v-abl- and v-src-transformed cell lines. Because of these correlations, the relationship of activated Stat3 to cellular transformation was examined directly. Wild-type Stat3 enhances the transforming potential of v-src; in contrast to this, three dominant negative Stat3 mutants inhibit v-src transformation. Stat3 wild-type or mutant proteins do not affect v-ras transformation. It is concluded that Stat3 has a necessary role in v-src transformation (Bromberg, 1998).

The signal transducer and activator of transcription molecules (STATs) play key roles in cytokine-induced signal transduction. STAT3 plays a key role in the G1 to S phase cell-cycle transition induced by the cytokine receptor subunit gp130, through the upregulation of cyclins D2, D3 and A, and cdc25A, and the concomitant downregulation of p21 and p27. Furthermore, unexpectedly, gp130 can induce the expression of p21 when STAT3 activation is suppressed. Such contradictory signals regulating cell-cycle progression could be simultaneously delivered from distinct cytoplasmic regions of gp130. An 'orchestrating model' is proposed for cytokine and growth factor action in which contradictory signals are orchestrated to produce a specific effect in a target cell (Fukada, 1998).

Although signal transducers and activators of transcription (STATs) were originally discovered as intracellular effectors of normal signaling by cytokines, increasing evidence also points to a role for STAT transcription factors in oncogenesis. Previous studies have demonstrated that one STAT family member, Stat3, possesses constitutively elevated tyrosine phosphorylation and DNA-binding activity in fibroblasts stably transformed by the Src oncoprotein. To determine if this Stat3 activation by Src can induce Stat3-mediated gene expression, luciferase reporter constructs based on synthetic and authentic promoters were transfected into NIH 3T3 cells. Activation of endogenous cellular Stat3 by the Src oncoprotein induces gene expression through a Stat3-specific binding element (TTCCCGAA) present in the C-reactive protein gene promoter. A naturally occurring splice variant of human Stat3 protein, Stat3beta, with a deletion in the C-terminal transactivation domain abolishes this gene induction in a dominant negative manner. Expression of Stat3beta does not have any effect on a reporter construct based on the c-fos serum response element, which is not dependent on Stat3 signaling, indicating that Stat3beta does not nonspecifically inhibit other signaling pathways or Src function. Transfection of vectors expressing Stat3beta, together with Src, blocks cell transformation by Src, as measured in a quantitative focus formation assay using NIH 3T3 cells. In contrast, Stat3beta has a much less pronounced effect on focus formation induced by the Ras oncoprotein, which does not activate Stat3 signaling. Three independent clones of NIH 3T3 cells stably overexpressing Stat3beta were generated and characterized, demonstrating that Stat3beta overexpression does not have a toxic effect on cell viability. These Stat3beta-overexpressing clones are deficient in Stat3-mediated signaling and refractory to Src-induced cell transformation. It is concluded that Stat3 activation by the Src oncoprotein leads to specific gene regulation and that Stat3 is one of the critical signaling pathways involved in Src oncogenesis. These findings provide evidence that oncogenesis-associated activation of Stat3 signaling is part of the process of malignant transformation (Turkson, 1998).

The propagation of pluripotent mouse embryonic stem (ES) cells depends on signals transduced through the cytokine receptor subunit gp130. Signaling molecules activated downstream of gp130 in ES cells include STAT3, the protein tyrosine phosphatase SHP-2, and the mitogen-activated protein kinases ERK1 and ERK2. A chimaeric receptor in which tyrosine 118 in the gp130 cytoplasmic domain was mutated does not engage SHP-2 and fails to activate ERKs. However, this receptor does support ES cell self-renewal. In fact, stem cell colonies form at 100-fold lower concentrations of cytokine than the unmodified receptor. Moreover, altered ES cell morphology and growth are observed at high cytokine concentrations. These indications of deregulated signaling in the absence of tyrosine 118 are substantiated by sustained activation of STAT3. Confirmation that ERK activation is not required for self-renewal was obtained by propagation of pluripotent ES cells in the presence of the MEK inhibitor PD098059. In fact, the growth of undifferentiated ES cells is enhanced by culture in PD098059. Thus activation of ERKs appears actively to impair self-renewal, suggesting that inhibitors of the Ras/MAPK pathway should promote the propagation of undifferentiated ES cells. These data imply that the self-renewal signal from gp130 is a finely tuned balance of positive and negative effectors (Burdon, 1999).

During the past several years, reports have accumulated indicating that human tumor samples contain constitutively activated Stats (1, 3, and 5 most frequently). Likewise, a consistent activation of Stat proteins, particularly Stat3, has been described in cells transformed in culture by known oncogenes or in cell lines started from human tumors. The requirement of Stat3 activation for the maximal transformation of cells by v-src was demonstrated using dominant-negative Stat3 to suppress transformation frequency and soft agar colony formation. In addition, maximal growth rates of squamous carcinoma cell lines were suppressed by expression of Stat3 dominant-negative constructs. All of these observations suggest at least a supplementary role for constitutively active wild-type Stat3 in tumorgenesis. However, until the present studies, it remained unsettled whether constitutively active Stat3 could by itself act as a transforming agent (Bromberg, 1999).

Since wild-type Stat3 requires tyrosine phosphorylation for activation, persistent tyrosine phosphorylation by wild-type protein would be required for constitutive activation. Sustained activation of wild-type Stat3 would appear logically to require a mutant protein that resists dephosphorylation or a mutation that renders a receptor-kinase complex continually active. Lacking knowledge of how to effect either of these changes, the attempt was made to effect activation (dimerization and DNA binding) without tyrosine phosphorylation by the insertion of cysteines in the SH2 domain. It appeared that the amino acid loops downstream from the crucial R608 (the phosphotyrosine-binding site in the SH2 domain) afforded a region of close proximity of the two chains into which cysteines might be introduced with the desired effect. While sulhydryls do not form easily in the reducing intracellular milieu, it is not an unknown event. Since the intracellular status of STAT molecules was not known accurately, it was assumed that proximity of these molecules might somehow allow disulfide bridges to form. It has, for example, been reported that some Stat3 nonphosphorylated dimers exist in cells without ligand stimulation and this might allow for cysteine-cysteine cross-links. Substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors (Bromberg, 1999).

The events downstream from constitutively active Stat3 (Stat3-C) that promote tumorgenesis are unclear but could include enhancing conditions for cell cycle progression and/or providing protection against apoptosis. A large number of cell cycle regulators, both inhibitors or promoters of cycling, are now known and form one important group of genes that might be affected by an activated Stat3 molecule. In examining this class of molecules it was found that both cyclin D1 mRNA and c-myc mRNA were elevated 3- to 5-fold in Stat3-C transformed cells, as compared to untransformed 3Y1 cells (cyclin A mRNA was not elevated). In addition, transfection of the cyclin D1 promoter appended to a luciferase reporter gene was transcriptionally activated by Stat3-C in transfection experiments. In addition to positive effectors (or inhibitors of negative effectors) of cell cycle regulation, inducible genes that affect apoptosis are also crucial in the survival of transformed cells. Stat3 has been clearly demonstrated to have an antiapoptotic role in T cells and in monocytes during their differentiation. Induction of Stat3-/- precursor cells in these lineages leads to cell death of the Stat3-/- cells. Likewise, cultured human multiple myeloma cells that have incorporated a Stat3 dominant-negative gene undergo apoptosis. And it was found that Stat3-C transformed cells have elevated levels of Bcl-XL mRNA. Thus, transformation by constitutively active Stat3 may generally contribute to stimulating cell cycle progression and provide protection against apoptosis (Bromberg, 1999 and references).

A noncanonical Frizzled2 pathway regulates epithelial-mesenchymal transition and metastasis

Wnt signaling plays a critical role in embryonic development, and genetic aberrations in this network have been broadly implicated in colorectal cancer. This study found that the Wnt receptor Frizzled2 (Fzd2; see Drosophila Frizzled) and its ligands Wnt5a/b (see Drosophila Wingless) are elevated in metastatic liver, lung, colon, and breast cancer cell lines and in high-grade tumors and that their expression correlates with markers of epithelial-mesenchymal transition (EMT). Pharmacologic and genetic perturbations reveal that Fzd2 drives EMT and cell migration through a previously unrecognized, noncanonical pathway that includes Fyn and Stat3 (see Drosophila Src42A and Stat92E). A gene signature regulated by this pathway predicts metastasis and overall survival in patients. An antibody was developed to Fzd2 that reduces cell migration and invasion and inhibits tumor growth and metastasis in xenografts. It is proposed that targeting this pathway could provide benefit for patients with tumors expressing high levels of Fzd2 and Wnt5a/b (Gujral, 2014).

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