Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. Expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.
TRK oncoproteins are chimeric versions of the NTRK1/NGF receptor and display constitutive tyrosine kinase activity leading to transformation of NIH3T3 cells and neuronal differentiation of PC12 cells. Signal Transducer and Activator of Transcription (STAT) 3 is activated in response to cytokines and growth factors and it has been recently identified as a novel signal transducer for TrkA, mediating the functions of NGF in nervous system. In this paper we have investigated STAT3 involvement in signalling induced by TRK oncogenes. We showed that TRK oncogenes trigger STAT3 phosphorylation both on Y705 and S727 residues and STAT3 transcriptional activity. MAPK pathway was involved in the induction of STAT3 phosphorylation. Interestingly, we have shown reduced STAT3 protein level in NIH3T3 transformed foci expressing TRK oncogenes. Overall, we have unveiled a dual role for STAT3 in TRK oncogenes-induced NIH3T3 transformation: i) decreased STAT3 protein levels, driven by TRK oncoproteins activity, are associated to morphological transformation; ii) residual STAT3 transcriptional activity is required for cell growth.
Signal transducer and activator of transcription-3 (STAT-3) is one of six members of a family of transcription factors. It was discovered almost 15 years ago as an acute-phase response factor. This factor has now been associated with inflammation, cellular transformation, survival, proliferation, invasion, angiogenesis, and metastasis of cancer. Various types of carcinogens, radiation, viruses, growth factors, oncogenes, and inflammatory cytokines have been found to activate STAT-3. STAT-3 is constitutively active in most tumor cells but not in normal cells. Phosphorylation of STAT-3 at tyrosine 705 leads to its dimerization, nuclear translocation, DNA binding, and gene transcription. The phosphorylation of STAT-3 at serine 727 may regulate its activity negatively or positively. STAT-3 regulates the expression of genes that mediate survival (survivin, bcl-xl, mcl-1, cellular FLICE-like inhibitory protein), proliferation (c-fos, c-myc, cyclin D1), invasion (matrix metalloproteinase-2), and angiogenesis (vascular endothelial growth factor). STAT-3 activation has also been associated with both chemoresistance and radioresistance. STAT-3 mediates these effects through its collaboration with various other transcription factors, including nuclear factor-κB, hypoxia-inducible factor-1, and peroxisome proliferator activated receptor-γ. Because of its critical role in tumorigenesis, inhibitors of this factor’s activation are being sought for both prevention and therapy of cancer. This has led to identification of small peptides, oligonucleotides, and small molecules as potential STAT-3 inhibitors. Several of these small molecules are chemo-preventive agents derived from plants. This review discusses the intimate relationship between STAT-3, inflammation, and cancer in more detail.
STAT-3; inflammation; cancer; chemoresistance
Signal transducer and activator of transcription 3 (STAT3) plays critical roles in neural development and is increasingly recognized as a major mediator of injury response in the nervous system. Cytokines and growth factors are known to phosphorylate STAT3 at tyrosine705 with or without the concomitant phosphorylation at serine727, resulting in the nuclear localization of STAT3 and subsequent transcriptional activation of genes. Recent evidence suggests that STAT3 may control cell function via alternative mechanisms independent of its transcriptional activity. Currently, the involvement of STAT3 mono-phosphorylated at residue serine727 (P-Ser-STAT3) in neurite outgrowth and the underlying mechanism is largely unknown.
In this study, we investigated the role of nerve growth factor (NGF) induced P-Ser-STAT3 in mediating neurite outgrowth. NGF induced the phosphorylation of residue serine727 but not tyrosine705 of STAT3 in PC12 and primary cortical neuronal cells. In PC12 cells, serine but not tyrosine dominant negative mutant of STAT3 was found to impair NGF induced neurite outgrowth. Unexpectedly, NGF induced P-Ser-STAT3 was localized to the mitochondria but not in the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NGF induced neurite outgrowth and the production of reactive oxygen species (ROS).
Taken together, the findings herein demonstrated a hitherto unrecognized novel transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 is involved in NGF induced neurite outgrowth.
The transcription factor, signal transducer and activator of transcription 3 (STAT3), is associated with proliferation, survival, and metastasis of cancer cells. We investigated whether gambogic acid (GA), a xanthone derived from the resin of traditional Chinese medicine, Gamboge hanburyi (mangosteen), can regulate the STAT3 pathway, leading to suppression of growth and sensitization of cancer cells. We found that GA induced apoptosis in human multiple myeloma cells that correlated with the inhibition of both constitutive and inducible STAT3 activation. STAT3 phosphorylation at both tyrosine residue 705 and serine residue 727 was inhibited by GA. STAT3 suppression was mediated through the inhibition of activation of the protein tyrosine kinases Janus-activated kinase (JAK) 1, and JAK2. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate reversed the GA-induced down-regulation of STAT3, suggesting the involvement of a PTP. We also found that GA induced the expression of the PTP SHP-1. Deletion of the SHP-1 gene by small interfering RNA suppressed the ability of GA to inhibit STAT3 activation and to induce apoptosis, suggesting the critical role of SHP-1 in its action. Moreover, GA down-regulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) proteins, and this correlated with suppression of proliferation and induction of apoptosis. Overall, these results suggest that GA blocks STAT3 activation, leading to suppression of tumor cell proliferation and induction of apoptosis.
Gambogic acid; STAT3; Apoptosis; Proliferation; Cancer
While 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 could 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 induced gene expression through a Stat3-specific binding element (TTCCCGAA) of the C-reactive protein gene promoter. A naturally occurring splice variant of human Stat3 protein, Stat3β, with a deletion in the C-terminal transactivation domain abolished this gene induction in a dominant negative manner. Expression of Stat3β did 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 Stat3β does not nonspecifically inhibit other signaling pathways or Src function. Transfection of vectors expressing Stat3β together with Src blocked cell transformation by Src as measured in a quantitative focus formation assay using NIH 3T3 cells. By contrast, Stat3β had a much less pronounced effect on focus formation induced by the Ras oncoprotein, which does not activate Stat3 signaling. In addition, three independent clones of NIH 3T3 cells stably overexpressing Stat3β were generated and characterized, demonstrating that Stat3β overexpression does not have a toxic effect on cell viability. These Stat3β-overexpressing clones were shown to be deficient in Stat3-mediated signaling and refractory to Src-induced cell transformation. We conclude 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. Our findings provide evidence that oncogenesis-associated activation of Stat3 signaling is part of the process of malignant transformation.
Stats (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that on a specific stimulus migrate to the nucleus and exert their transcriptional activity. Here we report a novel signaling pathway whereby RhoA can efficiently modulate Stat3 transcriptional activity by inducing its simultaneous tyrosine and serine phosphorylation. Tyrosine phosphorylation is exerted via a member of the Src family of kinases (SrcFK) and JAK2, whereas the JNK pathway mediates serine phosphorylation. Furthermore, cooperation of both tyrosine as well as serine phosphorylation is necessary for full activation of Stat3. Induction of Stat3 activity depends on the effector domain of RhoA and correlates with induction of both Src Kinase-related and JNK activities. Activation of Stat3 has biological implications. Coexpression of an oncogenic version of RhoA along with the wild-type, nontransforming Stat3 gene, significantly enhances its oncogenic activity on human HEK cells, suggesting that Stat3 is an essential component of RhoA-mediated transformation. In keeping with this, dominant negative Stat3 mutants or inhibition of its tyrosine or serine phosphorylation completely abrogate RhoA oncogenic potential. Taken together, these results indicate that Stat3 is an important player in RhoA-mediated oncogenic transformation, which requires simultaneous phosphorylation at both tyrosine and serine residues by specific signaling events triggered by RhoA effectors.
Signal transducer and transcription (STAT) factors are activated by tyrosine phosphorylation in response to a variety of cytokines, growth factors, and hormones. Tyrosine phosphorylation triggers dimerization and nuclear translocation of these transcription factors. In this study, the functional role of carboxy-terminal portions of the STAT family member acute-phase response factor/Stat3 in activation, dimerization, and transactivating potential was analyzed. We demonstrate that truncation of 55 carboxy-terminal amino acids causes constitutive activation of Stat3 in COS-7 cells, as is known for the Stat3 isoform Stat3beta. By the use of deletion and point mutants, it is shown that both carboxy- and amino-terminal portions of Stat3 are involved in this phenomenon. Dimerization of Stat3 was blocked by point mutations affecting residues both in the vicinity of the tyrosine phosphorylation site (Y705) and more distant from this site, suggesting that multiple interactions are involved in dimer formation. Furthermore, by reporter gene assays we demonstrate that carboxy-terminally truncated Stat3 proteins are incapable of transactivating an interleukin-6-responsive promoter in COS-7 cells. In HepG2 hepatoma cells, however, these truncated Stat3 forms transmit signals from the interleukin-6 signal transducer gp130 equally well as does full-length Stat3. We conclude that, dependent on the cell type, different mechanisms allow Stat3 to regulate target gene transcription either with or without involvement of its putative carboxy-terminal transactivation domain.
To explore the pathogenesis of chronic lymphocytic leukemia (CLL), we examined whether phosphorylation of one or more signal transducer and activator of transcription (STAT) factors was abnormal in cells from CLL patients. No constitutive tyrosine phosphorylation was detected on any STAT in CLL cells. To assess the phosphorylation of serine residues of STAT1 and STAT3 in CLL cells, we raised antibodies that specifically recognize the form of STAT1 phosphorylated on ser-727 and the form of STAT3 phosphorylated on ser-727. We found that in 100% of patients with CLL (n = 32), STAT1 and STAT3 were constitutively phosphorylated on serine. This was in contrast to normal peripheral blood B lymphocytes or CD5+) B cells isolated from tonsils, in which this phosphorylation was absent. Serine phosphorylation of STAT1 and STAT3 was seen occasionally in other leukemias, but it was a universal finding only in CLL. The serine phosphorylation of these STATs was a continuous process, as incubation of CLL cells with the kinase inhibitor H7 led to the dephosphorylation of these serine residues. The STAT serine kinase in CLL cells has not been identified, and appears to be neither mitogen-activated protein kinase nor pp70(s6k). In summary, the constitutive serine phosphorylation of STAT1 and STAT3 is present in all CLL samples tested to date, although the physiologic significance of this modification remains to be determined.
Both of the latent transcription factors activated by alpha interferon or gamma interferon contain the Stat1 protein. The role of Stat1 in virus interference mediated by interferons was directly examined by using cultured cells expressing Stat1 protein and its variants. In the absence of Stat1, no antiviral state is established. Full complementation of the antiviral state requires full-length Stat1 which is phosphorylated on both tyrosine 701 and serine 727. The closely related signal transducer and activator of transcription protein Stat3 cannot substitute for the antiviral properties of Stat1.
Diffuse large B-cell lymphoma (DLBCL) with an activated B-cell (ABC) gene-expression profile has been shown to have a poorer prognosis compared with tumors with a germinal center B-cell type. ABC cell lines have constitutive activation of STAT3; however, the mechanisms regulating STAT3 signaling in lymphoma are unknown. In studies of class-I histone deacetylase (HDAC) expression, we found overexpression of HDAC3 in phospho STAT3-positive DLBCL and the HDAC3 was found to be complexed with STAT3. Inhibition of HDAC activity by panobinostat (LBH589) increased p300-mediated STAT3Lys685 acetylation with increased nuclear export of STAT3 to the cytoplasm. HDAC inhibition abolished STAT3Tyr705 phosphorylation with minimal effect on STAT3Ser727 and JAK2 tyrosine activity. pSTAT3Tyr705-positive DLBCLs were more sensitive to HDAC inhibition with LBH589 compared with pSTAT3Tyr705-negative DLBCLs. This cytotoxicity was associated with downregulation of the direct STAT3 target Mcl-1. HDAC3 knockdown upregulated STAT3Lys685 acetylation but prevented STAT3Tyr705 phosphorylation and inhibited survival of pSTAT3-positive DLBCL cells. These studies provide the rationale for targeting STAT3-positive DLBCL tumors with HDAC inhibitors.
lymphoma; HDAC3; LBH589; STAT3; Mcl-1
Inactivation of p27Kip1 is implicated in tumorigenesis and has both prognostic and treatment-predictive values for many types of human cancer. The transcription factor Stat1 is essential for innate immunity and tumor immunosurveillance through its ability to act downstream of interferons. Herein, we demonstrate that Stat1 functions as a suppressor of Ras transformation independently of an interferon response. Inhibition of Ras transformation and tumorigenesis requires the phosphorylation of Stat1 at tyrosine 701 but is independent of Stat1 phosphorylation at serine 727. Stat1 induces p27Kip1 expression in Ras transformed cells at the transcriptional level through mechanisms that depend on Stat1 phosphorylation at tyrosine 701 and activation of Stat3. The tumor suppressor properties of Stat1 in Ras transformation are reversed by the inactivation of p27Kip1. Our work reveals a novel functional link between Stat1 and p27Kip1, which act in coordination to suppress the oncogenic properties of activated Ras. It also supports the notion that evaluation of Stat1 phosphorylation in human tumors may prove a reliable prognostic factor for patient outcome and a predictor of treatment response to anticancer therapies aimed at activating Stat1 and its downstream effectors.
Gene regulation by cytokine-activated transcription factors of the signal transducer and activator of transcription (STAT) family requires serine phosphorylation within the transactivation domain (TAD). STAT1 and STAT3 TAD phosphorylation occurs upon promoter binding by an unknown kinase. Here, we show that the cyclin-dependent kinase 8 (CDK8) module of the Mediator complex phosphorylated regulatory sites within the TADs of STAT1, STAT3, and STAT5, including S727 within the STAT1 TAD in the interferon (IFN) signaling pathway. We also observed a CDK8 requirement for IFN-γ-inducible antiviral responses. Microarray analyses revealed that CDK8-mediated STAT1 phosphorylation positively or negatively regulated over 40% of IFN-γ-responsive genes, and RNA polymerase II occupancy correlated with gene expression changes. This divergent regulation occurred despite similar CDK8 occupancy at both S727 phosphorylation-dependent and -independent genes. These data identify CDK8 as a key regulator of STAT1 and antiviral responses and suggest a general role for CDK8 in STAT-mediated transcription. As such, CDK8 represents a promising target for therapeutic manipulation of cytokine responses.
► CDK8 phosphorylates STAT transactivation domains in cytokine responses ► The findings implicate CDK8 as a broad regulator of STATs and cytokine responses ► CDK8 selectively regulates IFN-γ target-gene expression ► The CDK8 module is required for IFN-inducible antiviral responses
Stat (signal transducers and activators of transcription) and Jak (Janus kinases) proteins are central components in the signal transduction events in hematopoietic and epithelial cells. They are rapidly activated by various cytokines, hormones, and growth factors. Upon ligand binding and cytokine receptor dimerization, Stat proteins are phosphorylated on tyrosine residues by Jak kinases. Activated Stat proteins form homo- or heterodimers, translocate to the nucleus, and induce transcription from responsive genes. Stat5 and Stat6 are transcription factors active in mammary epithelial cells and immune cells. Prolactin activates Stat5, and interleukin-4 (IL-4) activates Stat6. Both cytokines are able to stimulate cell proliferation, differentiation, and survival. We investigated the transactivation potential of Stat6 and found that it is not restricted to lymphocytes. IL-4-dependent activation of Stat6 was also observed in HC11 mammary epithelial cells. In these cells, Stat6 activation led to the induction of the beta-casein gene promoter. The induction of this promoter was confirmed in COS7 cells. The glucocorticoid receptor was able to further enhance IL-4-induced gene transcription through the action of Stat6. Deletion analysis of the carboxyl-terminal region of Stat6 and recombination of this region with a heterologous DNA binding domain allowed the delimitation and characterization of the transactivation domain of Stat6. The potencies of the transactivation domains of Stat5, Stat6, and viral protein VP16 were compared. Stat6 had a transactivation domain which was about 10-fold stronger than that of Stat5. In pre-B cells (Ba/F3), the transactivation domain of Stat6 was IL-4 regulated, independently from its DNA binding function.
The Stat (signal transducer and activator of transcription) factors transmit cytokine, growth factor, and hormone responses. Seven members of the Stat gene family are known. MGF-Stat5a has been discovered as a mediator of the prolactin response in mammary epithelial cells. Two closely related variants of Stat5, Stat5a and Stat5b, are encoded by distinct genes. We examined the functional properties of the carboxyl termini of these molecules. Wild-type Stat5a (794 amino acids) and the carboxyl-terminal deletion mutant Stat5a delta 772 supported prolactin-induced transcription of a beta-casein promoter-reporter construct in COS7 cells; Stat5a delta 750 did not. Upon prolactin activation, tyrosine phosphorylation and the specificity of DNA binding were indistinguishable among the three Stat5a variants. Tyrosine dephosphorylation and the downregulation of the DNA-binding activity were delayed in the Stat5a delta 750 mutant. The carboxyl-terminal transactivation domain of Stat5a, amino acids 722 to 794, can be conferred to the DNA-binding domain of the yeast transcription factor GAL4. Coexpression of Stat5a or Stat5b and of the carboxyl-terminal deletion mutants resulted in the suppression of transcriptional induction in COS or Ba/F3 cells. We propose that Stat5a delta 750 and Stat5b delta 754 are lacking functional transactivation domains and exert their dominant negative effects by blocking the DNA-binding site in Stat5-responsive gene promoters.
Src tyrosine kinase family was recently identified as a novel upstream modulator of MAP kinase subfamily, p42/p44, whose activation is required for urocortin (Ucn)-mediated cardioprotection. Src kinase was also shown to reduce apoptosis in different cancer cell lines, enhancing phosphorylation and DNA binding affinity of signal transducer and activator of transcription (STAT)3. In order to evaluate the effects of Ucn on the activation status of different STAT family members, HL-1 cardiac cells were incubated with Ucn (10 nM) for increasing periods of time. STAT3 was rapidly phosphorylated at Tyr705, while neither phosphorylation at Ser727 nor induction of total STAT3 was observed. Pretreatment with PP2, a selective inhibitor of Src tyrosine kinase, reduced the pSTAT−T705 phosphorylation and transcriptional activity induced by Ucn in a dose-dependent manner. Overexpression of STAT3 in HL-1 cardiac myocytes pretreated with Ucn reduced the magnitude of cell death as compared with Ucn treatment alone, while transfection of HL-1 cells with a STAT3 mutant functionally inactive, acting as a dominant negative (DN-STAT3), enhanced the extent of cell death in a dose-dependent manner. In line with this finding, in HL-1 cardiac myocytes overexpressing STAT3 treated with Ucn, addition of the Src kinase inhibitor PP2 reversed the cytoprotective effects of Ucn, proving that the cytoprotective effects of Ucn are also mediated via the Src-pSTAT−T705 phosphorylation pathway. By immunocytochemistry, Ucn induced nuclear translocation of pST3-T705, which was inhibited by pretreatment with PP2. Together, these data strongly suggest that Ucn can mediate cardioprotection by activating the Src-pSTAT-T705 phosphorylation pathway.
STAT3; cardioprotection; ischemia/reperfusion injury; signal transduction; urocortin
The signal transducer and activator of transcription-3 (Stat3) is a member of the STAT family of cytoplasmic transcription factors. Overactivation of Stat3 is detected with high frequency in human cancer and is considered a molecular abnormality that supports the tumor phenotype. Despite concerted investigative efforts, the molecular mechanisms leading to the aberrant Stat3 activation and Stat3-mediated transformation and tumorigenesis are still not clearly defined. Recent evidence reveals a crosstalk close relationship between Stat3 signaling and members of the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Specifically, Rac1, acting in a complex with the MgcRacGAP (male germ cell RacGAP), promotes tyrosine phosphorylation of Stat3 by the IL6-receptor family/Jak kinase complex, as well as its translocation to the nucleus. Studies have further revealed that the mutational activation of Rac1 and Cdc42 results in Stat3 activation, which occurs in part through the upregulation of IL6 family cytokines that in turn stimulates Stat3 through the Jak kinases. Interestingly, evidence also shows that the engagement of cadherins, cell to cell adhesion molecules, specifically induces a striking increase in Rac1 and Cdc42 protein levels and activity, which in turn results in Stat3 activation. In this review we integrate recent findings clarifying the role of the Rho family GTPases in Stat3 activation in the context of malignant progression.
Rho GTPases; MgcRacGAP; Stat3
Signal transducers and activators of transcription (STATs) are mediators of cytokine and growth factor signaling. In recent years, STAT5b has emerged as a key regulator of tumorigenesis. STAT5b phosphorylation and activation is mediated by several kinases known to be overexpressed in breast cancer, such as epidermal growth factor receptor, HER2, and c-Src. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6, is a nonreceptor tyrosine kinase expressed in more than 60% of breast cancers. Only a few substrates of the Brk tyrosine kinase have been identified, the most recent being STAT3. In the present article we investigate the potential role of Brk in the phosphorylation and activation STAT5b.
To determine whether Brk can phosphorylate STAT5b, transient transfection and in vitro kinase assays were performed. Luciferase reporter assays were used to measure Brk-induced STAT5b transcriptional activity. siRNA technology was utilized to investigate the biological significance of Brk-induced activation of STAT5b in breast cancer cell models.
Phosphospecific antibodies, mutational analysis, and in vitro kinase assays demonstrated that Brk specifically mediated STAT5b phosphorylation at the activating tyrosine, Y699. Transient transfection of Brk into the Brk-negative BT-549 breast cancer cell line enhanced STAT5b transcriptional activity, as measured by a STAT5-specific luciferase reporter. Furthermore, overexpression of kinase active c-Src enhanced Brk-induced STAT5b transcriptional activity. In Brk-positive breast cancer cell lines BT-20 and SKBr3, knockdown of Brk protein or of STAT5b protein using siRNA methodology resulted in a decrease in DNA synthesis. Knockdown of Brk and STAT5b together did not further decrease DNA synthesis compared with each alone, suggesting that Brk and STAT5b converge on the same pathway, ultimately leading to cellular proliferation.
Our studies demonstrate that Brk phosphorylates STAT5b on Y699, leading to increased STAT5b transcriptional activity. Furthermore, analysis of DNA synthesis suggests that STAT5b and Brk are converging upon the same proproliferative signaling pathway in breast cancer cells. We propose that Brk, like other tyrosine kinases, signals downstream to STAT5b to mediate proliferation of breast cancer cells. These results further establish STAT5b as well as Brk as potential targets for breast cancer therapy.
The pathogenic mechanism by which parvovirus B19 may induce inflammatory cardiomyopathy (iCMP) is complex but is known to involve inflammatory processes, possibly including activation of JAK/STAT signaling. The nonstructural B19 protein NS1 acts as a transactivator triggering signaling cascades that eventually lead to activation of interleukin 6 (IL-6). We examined the impact of NS1 on modulation of STAT signaling in human endothelial cells (HMEC-1). The NS1 sequences were identified from B19 DNA isolated from the myocardia of patients with fatal iCMP. B19 infection as well as NS1 overexpression in HMEC-1 cells produced a significant upregulation in the phosphorylation of both tyrosine705 and serine727 STAT3 (P < 0.05). The increased STAT3 phosphorylation was accompanied by dimerization, nuclear translocation, and DNA binding of pSTAT3. In contrast, NS1 expression did not result in increased STAT1 activation. Notably, the expression levels of the negative regulators of STAT activation, SOCS1 and SOCS3, were not altered by NS1. However, the level of PIAS3 was upregulated in NS1-expressing HMEC-1 cells. Analysis of the transcriptional activation of target genes revealed that NS1-induced STAT3 signaling was associated with upregulation of genes involved in immune response (e.g., the IFNAR1 and IL-2 genes) and downregulation of genes associated with viral defense (e.g., the OAS1 and TYK2 genes). Our results demonstrate that B19 NS1 modulates the STAT/PIAS pathway. The NS1-induced upregulation of STAT3/PIAS3 in the absence of STAT1 phosphorylation and the lack of SOCS1/SOCS3 activation may contribute to the mechanisms by which B19 evades the immune response and establishes persistent infection in human endothelial cells. Thus, NS1 may play a critical role in the mechanism of viral pathogenesis in B19-associated iCMP.
STAT3 is a transcription factor of central importance in chronic inflammation and cancer. In response to cytokine stimulation STAT3 is phosphorylated on a single tyrosine residue at position 705, dimerizes and accumulates in the nucleus to induce target gene expression. The substitution of tyrosine 705 to phenylalanine leads to a dominant-negative STAT3 mutant (STAT3-YF) which influences the activation of WT-STAT3 in stimulated cells through a mechanism that is not completely understood. In this study we analyzed the molecular mechanism of STAT3-YF dominant-negative activity in IL-6-induced STAT3 signaling and the relevance of the N-terminal domain.
Expression of STAT3-YF-YFP impairs tyrosine phosphorylation, nuclear translocation and the transcriptional activity of WT-STAT3 in IL-6-stimulated cells. The fluorescently labelled STAT3-YF mutant binds to a phosphorylated gp130 receptor-peptide comparable to WT-STAT3-YFP. STAT3-YF-YFP forms homodimers as well as heterodimers with WT-STAT3 in the presence and absence of IL-6. The preformed heterodimers in unstimulated cells are detectable by colocalization of STAT3-CFP with STAT3-YF-YFP fused to a nuclear localization signal. STAT3/STAT3-YF heterodimers are not able to bind to DNA in stimulated cells, but the presence of the mutant reduces DNA-binding of WT-STAT3 homodimers. STAT3-YF-ΔN-YFP lacking the N-terminal domain forms no dimers and only marginally affects the activity of WT-STAT3.
Our findings demonstrate that dominant-negative STAT3-YF affects the activation of WT-STAT3 at multiple levels. Unexpectedly, the N-terminal domain of STAT3-YF plays an important role for the dominant-negative effect. We show that (i) STAT3-YF competes with WT-STAT3 in binding to activated gp130-receptors, (ii) the formation of WT-STAT3/STAT3-YF heterodimers in IL-6-stimulated cells results in inactive, semiphosphorylated dimers which do not bind to DNA and thus fail to induce target gene expression, (iii) the N-terminal domain-mediated formation of preformed STAT3/STAT3-YF heterodimers in unstimulated cells which affects the IL-6-induced homodimerization of WT-STAT3 contributes to the dominant-negative effect of STAT3-YF. These findings will contribute to our understanding of naturally occuring dominant-negative STAT3 mutants that cause the hyper-IgE syndrome.
JAK-STAT signaling; STAT3; IL-6; STAT3-YF mutant; Dominant-negative; Hyper-IgE syndrome (HIES)
The fibroblast growth factors (FGFs) play diverse roles in development, wound healing and angiogenesis. The intracellular signal transduction pathways which mediate these pleiotropic activities remain incompletely understood. We show here that the proangiogenic factors FGF2 and FGF8b can activate signal transducers and activators of transcription (STATs) in mouse microvascular endothelial cells. Both FGF2 and FGF8b activate STAT5 and to a lesser extent STAT1, but not STAT3. The FGF2-dependent activation of endothelial STAT5 was confirmed in vivo with the matrigel plug angiogenesis assay. In tissue samples of human gliomas, a tumor type where FGF-induced angiogenesis is important, STAT5 is detected in tumor vessel endothelial cell nuclei, consistent with STAT5 activation. By forced expression of constitutively active or dominant-negative mutant STAT5A in mouse brain endothelial cells, we further show that STAT5 activation is both necessary and sufficient for FGF-induced cell migration, invasion and tube formation, which are key events in vascular endothelial morphogenesis and angiogenesis. In contrast, STAT5 is not required for brain endothelial cell mitogenesis. The cytoplasmic tyrosine kinases Src and Janus kinase 2 (Jak2) both appear to be involved in the activation of STAT5, as their inhibition reduces FGF2 and FGF8b induced STAT5 phosphorylation and endothelial cell tube formation. Constitutively active STAT5A partially restores tube formation in the presence of Src or Jak2 inhibitors. These observations demonstrate that FGFs utilize distinct signaling pathways to induce angiogenic phenotypes. Together, our findings implicate the FGF-Jak2/Src-STAT5 cascade as a critical angiogenic FGF signaling pathway.
FGF; STAT; Src; Jak2; angiogenesis; endothelial cells
Both nuclear factor (NF)-κB–inducing kinase (NIK) and inhibitor of κB (IκB) kinase (IKK) have been implicated as essential components for NF-κB activation in response to many external stimuli. However, the exact roles of NIK and IKKα in cytokine signaling still remain controversial. With the use of in vivo mouse models, rather than with enforced gene-expression systems, we have investigated the role of NIK and IKKα in signaling through the type I tumor necrosis factor (TNF) receptor (TNFR-I) and the lymphotoxin β receptor (LTβR), a receptor essential for lymphoid organogenesis. TNF stimulation induced similar levels of phosphorylation and degradation of IκBα in embryonic fibroblasts from either wild-type or NIK-mutant mice. In contrast, LTβR stimulation induced NF-κB activation in wild-type mice, but the response was impaired in embryonic fibroblasts from NIK-mutant and IKKα-deficient mice. Consistent with the essential role of IKKα in LTβR signaling, we found that development of Peyer's patches was defective in IKKα-deficient mice. These results demonstrate that both NIK and IKKα are essential for the induction of NF-κB through LTβR, whereas the NIK–IKKα pathway is dispensable in TNFR-I signaling.
alymphoplasia; cytokine signaling; IκB; Akt kinase; Peyer's patch
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, we examined the growth factor-dependent serine phosphorylation of STAT1 and STAT3. We provide in vitro and in vivo evidence that the ERK family of mitogen-activated protein (MAP) kinases, but not JNK or p38, specifically phosphorylate 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.
Activation of early response genes by interferons (IFNs) and other cytokines requires tyrosine phosphorylation of a family of transcription factors termed signal transducers and activators of transcription (Stats). The Janus family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2) is required for cytokine-induced tyrosine phosphorylation and dimerization of the Stat proteins. In order for IFNs to stimulate maximal expression of Stat1alpha-regulated genes, phosphorylation of a serine residue in the carboxy terminus by mitogen-activated protein kinase (MAPK) is also required. In HeLa cells, both IFN-beta and oncostatin M (OSM) stimulated MAPK and Raf-1 enzyme activity, in addition to Stat1 and Stat3 tyrosine phosphorylation. OSM stimulation of Raf-1 correlated with GTP loading of Ras, whereas IFN-beta activation of Raf-1 was Ras independent. IFN-beta- and OSM-induced Raf-1 activity could be coimmunoprecipitated with either Jak1 or Tyk2. Furthermore, HeLa cells lacking Jak1 displayed no activation of STAT1alpha, STAT3, and Raf-1 by IFN-beta or OSM and also demonstrated no increase in the relative level of GTP-bound p21ras in response to OSM. The requirement for Jak1 for IFN-beta- and OSM-induced activation of Raf-1 was also seen in Jak1-deficient U4A fibrosarcoma cells. Interestingly, basal MAPK, but not Raf-1, activity was constitutively enhanced in Jak1-deficient HeLa cells. Transient expression of Jak1 in both Jak-deficient HeLa cells and U4A cells reconstituted the ability of IFN-beta and OSM to activate Raf-1 and decreased the basal activity of MAPK, while expression of a kinase-inactive form of the protein showed no effect. Moreover, U4A cells selected for stable expression of Jak1, or COS cells transiently expressing Jak1 or Tyk2 but not Jak3, exhibited enhanced Raf-1 activity. Therefore, it appears that Jak1 is required for Raf-1 activation by both IFN-beta and OSM. These results provide evidence for a link between the Jaks and the Raf/MAPK signaling pathways.
STAT proteins are a group of latent cytoplasmic transcription factors which function as signal transducers and activators of transcription. Stat1 and -2 were originally identified to function in interferon signaling, and Stat1 was also found to be activated by epidermal growth factor (EGF) and other cytokines. New members of the STAT gene family are identified. Among them, Stat3 has 52.5% amino acid sequence homology with Stat1 and is activated by platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), EGF, interleukin-6, and other cytokines. Treatment of cells with EGF activates Stat1 and Stat3, which become phosphorylated on tyrosine residues to form homo - or heterodimers and translocate into the nucleus, binding to the sis-inducible element (SIE) in the c-fos promoter. Somatic cell genetic analyses demonstrated that Jaks, a family of nontransmembrane protein tyrosine kinases, are required for the activation of Stat1 and Stat2 in interferon-treated cells. However, little is known about the activation of Stat3 by growth factors. Here we report that in all v-Src-transformed cell lines examined, Stat3 is constitutively activated to bind to DNA and the phosphorylation of tyrosine on Stat3 is enhanced by the induction of v-Src expression. We also report that Src is shown to be associated with Stat3 in vivo, as well as in vitro, and phosphorylates Stat3 in vitro. Stat3 is also activated by CSF-1, possibly through CSF-1 receptor-c Src association in NIH 3T3 cells overexpressing CSF-1 receptors. Together, the data suggest that Src is involved in activation of Stat3 in growth factor signal transduction.