Signal Transducer And Activator Of Transcription 3 Mutant Research Articles

Modulation of Gene Expression and Tumor Cell Growth by Redox Modification of STAT3

Reactive oxygen species (ROS) promote tumor cell proliferation and survival by directly modulating growth-regulatory molecules and key transcription factors. The signal transducer and activator of transcription 3 (STAT3) is constitutively active in a variety of tumor cell types, where the effect of ROS on the Janus kinase/STAT pathway has been examined. We report here that STAT3 is directly sensitive to intracellular oxidants. Oxidation of conserved cysteines by peroxide decreased STAT3 binding to consensus serum-inducible elements (SIE) in vitro and in vivo and diminished interleukin (IL)-6-mediated reporter expression. Inhibitory effects produced by cysteine oxidation in STAT3 were negated in redox-insensitive STAT3 mutants. In contrast, ROS had no effect on IL-6-induced STAT3 recruitment to the c-myc P2 promoter. Expression of a redox-insensitive STAT3 in breast carcinoma cells accelerated their proliferation while reducing resistance to oxidative stress. Our results implicate STAT3 in coupling intracellular redox homeostasis to cell proliferation and survival.

Sunitinib Induces Apoptosis and Growth Arrest of Medulloblastoma Tumor Cells by Inhibiting STAT3 and AKT Signaling Pathways

Medulloblastomas are the most frequent malignant brain tumors in children. Sunitinib is an oral multitargeted tyrosine kinase inhibitor used in clinical trials as an antiangiogenic agent for cancer therapy. In this report, we show that sunitinib induced apoptosis and inhibited cell proliferation of both a short-term primary culture (VC312) and an established cell line (Daoy) of human medulloblastomas. Sunitinib treatment resulted in the activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase and upregulation of proapoptotic genes, Bak and Bim, and inhibited the expression of survivin, an antiapoptotic protein. Sunitinib treatment also downregulated cyclin E, cyclin D2, and cyclin D3 and upregulated p21Cip1, all of which are involved in regulating cell cycle. In addition, it inhibited phosphorylation of signal transducer and activator of transcription 3 (STAT3) and AKT (protein kinase B) in the tumor cells. Dephosphorylation of STAT3 (Tyr(705)) induced by sunitinib was helped by a reduction in activities of Janus-activated kinase 2 and Src. Additionally, sodium vanadate, an inhibitor of protein tyrosine phosphatases, partially blocked the inhibition of phosphorylated STAT3 by sunitinib. Loss of phosphorylated AKT after sunitinib treatment was accompanied by decreased phosphorylation of downstream proteins glycogen synthase kinase-3beta and mammalian target of rapamycin. Expression of a constitutively activated STAT3 mutant or myristoylated AKT partially blocked the effects of sunitinib in these tumor cells. Sunitinib also inhibited the migration of medulloblastoma tumor cells in vitro. These findings suggest the potential use of sunitinib for the treatment of pediatric medulloblastomas.

Signal Transducer and Activator of Transcription 3 (STAT3) Mediates Amino Acid Inhibition of Insulin Signaling through Serine 727 Phosphorylation

Nutrient overload is associated with the development of obesity, insulin resistance, and type II diabetes. High plasma concentrations of amino acids have been found to correlate with insulin resistance. At the cellular level, excess amino acids impair insulin signaling, the mechanisms of which are not fully understood. Here, we report that STAT3 plays a key role in amino acid dampening of insulin signaling in hepatic cells. Excess amino acids inhibited insulin-stimulated Akt phosphorylation and glycogen synthesis in mouse primary hepatocytes as well as in human hepatocarcinoma HepG2 cells. STAT3 knockdown protected insulin sensitivity from inhibition by amino acids. Amino acids stimulated the phosphorylation of STAT3 at Ser(727), but not Tyr(705). Replacement of the endogenous STAT3 with wild-type, but not S727A, recombinant STAT3 restored the ability of amino acids to inhibit insulin signaling, suggesting that Ser(727) phosphorylation was critical for STAT3-mediated amino acid effect. Furthermore, overexpression of STAT3-S727D was sufficient to inhibit insulin signaling in the absence of excess amino acids. Our results also indicated that mammalian target of rapamycin was likely responsible for the phosphorylation of STAT3 at Ser(727) in response to excess amino acids. Finally, we found that STAT3 activity and the expression of its target gene socs3, known to be involved in insulin resistance, were both stimulated by excess amino acids and inhibited by rapamycin. In conclusion, our study reveals STAT3 as a novel mediator of nutrient signals and identifies a Ser(727) phosphorylation-dependent and Tyr(705) phosphorylation-independent STAT3 activation mechanism in the modulation of insulin signaling.

The STAT3 NH2-terminal Domain Stabilizes Enhanceosome Assembly by Interacting with the p300 Bromodomain

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. Here we 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. We previously reported that the STAT3 NH(2)-terminal domain is acetylated by p300 at Lys-49 and Lys-87. By introducing K49R/K87R mutations, here we 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, our 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.

Suppressor of cytokine signaling 3 negative regulation of signal transducer and activator of transcription 3 in platelet‐derived growth factor‐induced fibroblast migration

Platelet-derived growth factor (PDGF) is involved in wound healing, but PDGF-induced fibroblast migration and the intracellular signaling mechanisms of fibroblast migration are poorly understood. Signal transducer and activator of transcription 3 (STAT3) is involved in migration and is negatively regulated by the suppressor of cytokine signaling 3 (SOCS3). We studied the PDGF induction of fibroblast migration in vitro and the involvement of STAT3 and SOCS3. We found that PDGF activated STAT3 and strongly induced fibroblast migration. Transfection with a dominant-negative mutant of STAT3 almost completely abolished PDGF-induced fibroblast migration and STAT3 phosphorylation. Next, we studied the mechanisms that regulate fibroblast migration. PDGF enhanced the expression of SOCS3 by 2.8-fold at 1 h. Transfection with SOCS3 almost completely abolished PDGF-induced STAT3 phosphorylation and reduced fibroblast migration to 47% of control, indicating that SOCS3 acts as a negative regulator of PDGF-induced fibroblast migration. In conclusion, PDGF induces fibroblast migration under the control of STAT3-SOCS3.

LIF- and IL-6-Induced Acetylation of STAT3 at Lys-685 through PI3K/Akt Activation

Signal transducer and activator of transcription 3 (STAT3), which mediates biological actions in many physiological processes, is activated by cytokines and growth factors via specific tyrosine or serine phosphorylation, dimerization and nuclear translocation. A recent study has demonstrated, by using antibody to acetylated lysine, and a STAT3 mutant with Lys-685-to-Arg substitution, that STAT3 is acetylated at Lys-685 by histone acetyltransferase p300, and that acetylation at Lys-685 is critical for STAT3 activation. In the present study, we created an acetyl-specific antibody against STAT3 acetylated at Lys-685, and found that leukemia inhibitory factor (LIF) or interleukin (IL)-6 induced acetylation of STAT3 at Lys-685 in 293T and Hep3B cells. Moreover, acetylation of STAT3 at Lys-685 was suppressed by PI3K inhibitor LY294002, or a dominant negative Akt. Taken together, our findings demonstrate that endogenous STAT3 is acetylated at Lys-685 by LIF or IL-6 through PI3K/Akt activation.

Phosphorylation of STAT3 Serine-727 by Cyclin-Dependent Kinase 1 Is Critical for Nocodazole-Induced Mitotic Arrest

Signal transducer and activator of transcription 3 (STAT3) mediates cellular responses to diverse cytokines and growth factors by modulating the expression of specific target genes. While phosphorylation of STAT3 at Tyr-705 has been demonstrated to be a prerequisite for STAT3 dimerization, nuclear translocation, and activation of gene transcription, the role of Ser-727 in regulation of STAT3 activity is controversial. Kinetworks KPSS-1.1 phospho-site screening of nocodazole-treated HeLa cells revealed that STAT3 Ser-727 phosphorylation was enhanced during mitosis, and this correlated with a reduction of Tyr-705 phosphorylation. Overexpression of STAT3 mutants in which these phosphorylation sites were separately abolished revealed that phosphorylation at these sites appeared to be mutually antagonistic. The nocodazole-induced STAT3 Ser-727 phosphorylation was reduced by selective inhibition of CDK1 phosphotransferase activity, and CDK1 could directly phosphorylate GST-STAT3 Ser-727 in vitro and co-immunoprecipitate with STAT3 in vivo. Blocking Ser-727 phosphorylation enhanced STAT3 DNA-binding activity toward its target gene promoters, implying a negative effect of Ser-727 phosphorylation on its transcriptional activity. Interference of Ser-727 phosphorylation resulted in an exit from mitotic arrest induced by nocodazole treatment and a cell cycle arrest at the G1 phase, as indicated by the accumulation of 2N cell population and enhanced expression of G1 cell cycle regulators including p21(CIP1/WAF1), p27(Kip1), and cyclin E. Taken together, our observations point to a novel role of STAT3 Ser-727 phosphorylation in control of the onset and maintenance of the M phase during the cell cycle through downregulation of CDK inhibitors.

Neoplastic transformation by the gep oncogene, Gα12, involves signaling by STAT3

Galpha(12), the alpha-subunit of G12, which has been referred to as the gep oncogene, stimulates mitogenic pathways in different cell types and readily induces neoplastic transformation of fibroblast cell lines. Recently, we have shown that the oncogenic pathway activated by Galpha(12) involves the receptor tyrosine kinase platelet derived growth factor receptor-alpha (PDGFRalpha) and JAK3. In the present study, we demonstrate that the GTPase-deficient activated mutant of Galpha(12) activates signal transducer and activator of transcription 3 (STAT3) via PDGFRalpha as well as JAK3. Here we show that Galpha(12) stimulates the phosphorylation of STAT3 at both Tyrosine-705 and Serine-727 residues. Studies to delineate the mechanism by which Galpha(12) stimulates STAT3 have indicated that the Tyrosine-705-phosphorylation of STAT3 involves the tyrosine kinases, Janus Kinase-3 as well as Src kinase, whereas the Serine-727 phosphorylation of STAT3 occurs via the receptor tyrosine kinase, PDGFRalpha and phosphatidylinositol 3-OH kinase pathway. Our results also indicate that the coexpression of the dominant negative, DNA binding mutant of STAT3 (STAT3DB) inhibits the foci formation as well as anchorage-independent growth of Galpha(12)QL-transfectants, thereby establishing the critical role of STAT3 in Galpha(12)QL-mediated neoplastic cell growth. The results presented here demonstrate, for the first time, the ability of Galpha(12) to recruit multiple receptor-, nonreceptor-, and Ser/Thr kinases to stimulate STAT3-signaling to promote neoplastic transformation.

ERK1/2 contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells.

Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growth-promoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream Ras-MAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3 pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 mM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely. Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059. Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2 negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.

Ral GDP Dissociation Stimulator and Ral GTPase Are Involved in Myocardial Hypertrophy

Ras-related GTPase (Ral) is converted to the GTP-bound form by Ral GDP dissociation stimulator (Ral-GDS), a putative effector protein of Ras. Although a number of studies indicate that Ras induces cardiac hypertrophy, the functional role of Ral-GDS/Ral signaling pathway is as yet unknown in cardiac myocytes. We investigated the role of the Ral-GDS/Ral pathway in cardiac hypertrophy. Transfection of Ral-GDS and constitutively active mutant of Ral (RalG23V) in cultured rat neonatal myocytes stimulated promoter activity of c-fos (5.4-fold and 2.6-fold, P<0.01), alpha-skeletal actin (2.7-fold and 2.1-fold, P<0.01), and beta-myosin heavy chain-luciferase (2.8-fold and 2.3-fold, P<0.01). Ral-GDS-induced or RalG23V-induced promoter activation was increased synergistically with activated Ras (RasG12V). Dominant-negative mutant of Ral (RalS28N) partially inhibited RasG12V induced promoter activation. Cardiac myocytes transfected with RalG23V showed increased cell size compared with nontransfected or vector-transfected cells (2.1-fold, P<0.01). Cardiotrophin-1 (CT-1) upregulated Ral-GDS mRNA expression and induced Ral activation. CT-1-induced Ral-GDS mRNA expression was inhibited by overexpression of the dominant-negative mutant of STAT3. Moreover, Ral activity was elevated in hypertrophied hearts (2.1-fold, P<0.01) by mechanical stress in association with increased CT-1 expression and signal transducer and activator of transcription 3 (STAT3) phosphorylation in the rat aortic banding model. Ral-GDS/Ral pathway is involved in a wide range of gene expressions and is activated by hypertrophic stimuli in vitro and in vivo. SATA3 may play a key role in Ral-GDS expression and Ral activation. Our data provide evidence that the Ral-GDS/Ral signaling pathway is a link to the process of cardiac hypertrophy.

The Role of Phosphatidylinositol 3-Kinase, Rho Family GTPases, and STAT3 in Ros-induced Cell Transformation

Using loss-of-function mutants of Ros and inducible epidermal growth factor receptor-Ros chimeras we investigated the role of various signaling pathways in Ros-induced cell transformation. Inhibition of the mitogen-activated protein kinase (MAPK) pathway with the MEK (MAP/extracellular signal-regulated kinase kinase) inhibitor PD98059 had little effect on the Ros-induced monolayer and anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells even though more than 70% of the MAPK was inhibited. In contrast, inhibiting the phosphatidylinositol 3-kinase (PI3K) pathway with the drug LY294002, a dominant negative mutant of PI3K, Deltap85, or the phosphatidylinositol phosphatase PTEN (phosphatase and tensin homologue deleted in chromosome ten) resulted in a dramatic reduction of v-Ros- and epidermal growth factor receptor-Ros-promoted anchorage-independent growth of chicken embryo fibroblasts and NIH3T3 cells, respectively. Parallel and downstream components of PI3K signaling such as the Rho family GTPases (Rac, Rho, Cdc42) and the survival factor Akt were all shown to contribute to Ros-induced anchorage-independent growth, although Rac appeared to be less important for Ros-induced colony formation in NIH3T3 cells. Furthermore, the transformation-attenuated v-Ros mutants F419 and DI could be complemented by constitutively active mutants of PI3K and Akt. Finally, we found that overexpressing a constitutively active mutant of STAT3 (STAT3C) conferred a resistance to the inhibition of Ros-induced anchorage-independent growth by LY294002, suggesting a possible overlap of functions between PI3K and STAT3 signaling in mediating Ros-induced anchorage-independent growth.