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ABSTRACT: 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.
Cancer Research 10/2010; 70(20):8222-32. · 7.86 Impact Factor
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ABSTRACT: ETS transcription factors are implicated in gene regulation during cell proliferation and in the development of the haematopoietic cell lineage. Characteristically, ETS proteins act in concert with other transcription factors and are regulated by post-translational modifications, most frequently phosphorylation. These events have been shown to modulate the DNA binding affinity and interactions of ETS transcription factors with co-activators, events that can ultimately determine the formation of productive transcription complexes on target gene promoters. However, direct implication of a transcription factor or one of its post-translational modifications in the regulation of a given gene requires detection of the modified factor at the target gene promoter. Chromatin immunoprecipitation assays were originally adopted to probe modifications to histone proteins associated with transcriptionally active genes in yeast. They have since been used to confirm the presence of numerous proteins at diverse gene promoters including, for example, recruitment of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 to the promoters of mitogen-responsive genes. Here chromatin immunoprecipitation is used to demonstrate the inducible appearance of phosphorylated Elk-1 at the human c-fos promoter.
Methods in molecular biology (Clifton, N.J.) 01/2010; 647:279-89.
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ABSTRACT: Many eukaryotic genes are acutely regulated by extra-cellular signals. The c-fos serum response element (SRE) mediates transcriptional activation in response to mitogens through serum response factor (SRF)-dependent recruitment of Elk-1, a mitogen-activated protein kinase (MAPK)-responsive transcription factor. How subsequent events at SRE promoters stimulate initiation of transcription has yet to be fully resolved. Here we show that extra-cellular signal-regulated kinase (ERK) and mitogen and stress-activated kinase (MSK) are recruited to SRE promoter complexes in vitro and in vivo. Their recruitment in vitro correlates with Elk-1 binding and for ERK the D domain/KIM of Elk-1 is specifically involved. In vivo, recruitment of ERK and MSK is stimulated by mitogens, correlates with histone H3 phosphorylation and is impaired by Elk-1 knockdown. Immunocytochemistry and confocal microscopy reveal that ERK appears to associate to some extent with initiating rather than elongating RNA polymerase II. Taken together, our data add to the body of evidence implying that ERK and related MAPKs may fulfil a generic role at the promoters of acutely regulated genes.
Nucleic Acids Research 06/2008; 36(8):2594-607. · 8.03 Impact Factor
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ABSTRACT: The Signal Transducer and Activator of Transcription 3 (STAT3) is necessary for ES cell renewal, plays critical roles during vertebrate development, and has oncogenic potential. STAT3 also mediates cytokine responses notably in the induction of acute phase response genes in the liver. Thus STAT3 is a pleiotropic regulator during cell proliferation and a cell-specific mediator of pro-inflammatory responses. How STAT3 fulfils both roles is unclear. To address this question we attempted to characterise pre-initiation complexes (PICs) on STAT3-responsive promoters containing the c-myc P2 promoter element (P2E) or c-fos Serum-Inducible Element (SIE). Although both promoters mediated cytokine responses in HepG2 cells, poor binding of STAT1 and STAT3 in vitro precluded isolation of active promoter complexes on the P2E. The inability of STAT3 to bind the P2E in vitro correlated with failure of the P2E to mediate cytokine-responsive gene expression in several other cell types. Thus the c-myc P2E behaves as a dual-purpose STAT3 element with anomalous characteristics in HepG2 cells.
Biochemical and Biophysical Research Communications 01/2008; 364(3):627-32. · 2.48 Impact Factor
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ABSTRACT: Signal transducer and activator of transcription (STAT) proteins are involved in cell proliferation and survival, aspects of tissue differentiation and immune function. STAT3 appears to be fundamentally important for vertebrate organisms, being required for the self-renewal of embryonal stem cells in response to leukemia inhibitory factor signaling and for proliferation of some somatic cell types. Moreover, STAT3 is up-regulated in a range of tumors, and a modified version of STAT3 (STAT3C) has been shown to function as an oncogene, whereas inhibition of STAT3 can suppress tumor cell growth. The constitutive activity of oncogenic STAT3C was reported to depend on spontaneous dimerization directed by disulfide bonds in the absence of tyrosine phosphorylation. In fact, tyrosine phosphorylation consequent upon cytokine or mitogen-induced signaling events remains obligatory for STAT3C activation. Instead, the DNA-binding affinity of phospho-STAT3C is elevated resulting in a faster on-rate and slower off-rate. The faster on-rate sensitizes STAT3C to cytokine stimulation, and the slower off-rate protects it from inactivation by nuclear phosphatases. These changes account for the ability of STAT3C to up-regulate persistently the expression of STAT3 target genes and promote cell cycle progression.
Journal of Biological Chemistry 12/2006; 281(44):33172-81. · 4.77 Impact Factor
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ABSTRACT: Signal transducer and activator of transcription (STAT) proteins are activated by cytokines and growth factors to play distinct roles in immune responses and developmental processes. STATs were thought to exist as latent, cytoplasmic monomers and activation to require dimer formation was mediated exclusively by reciprocal phospho-tyrosine/SH2-domain interactions, but recent evidence of cytoplasmic STAT complexes, including dimers, and unphosphorylated STATs in the nucleus has challenged these notions. STAT complexes detected by conventional SDS-PAGE, including a STAT3 dimer, have been reported. We show that such complexes can form during cell lysis and be disrupted with DTT, suggesting inter-chain disulphide bridging. STAT3 also forms a related complex in cells upon oxidative stress. We map the interaction to the amino-terminal domain of STAT3 and use mass spectrometry to implicate cysteine 259 as the reactive residue. The redox sensitivity of STAT3 may be significant, given its activation in cells in response to reactive oxygen species.
Biochemical and Biophysical Research Communications 10/2004; 322(3):1005-11. · 2.48 Impact Factor
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ABSTRACT: Cell growth is promoted by mitogens and survival factors, which activate intracellular signalling pathways to control cell cycle progression and cellular integrity. Proliferation signals are transmitted through Ras and Rho family small G-proteins coupled to mitogen-activated protein kinase (MAPK) cascades, while survival signals are propagated by lipid-dependent kinases such as phosphatidylinositide 3-kinases (PI3Ks) and protein kinase B (Akt/PKB). Recently, signal transducer and activator of transcription (STAT) proteins were identified as positive regulators of proliferation in a variety of cell types. Persistent activation of these pathways is associated with tumour cell growth, whereas their inhibition can halt proliferation and precipitate apoptotic cell death. The human pathogen Pseudomonas aeruginosa uses quorum-sensing signal molecules (QSSMs) to regulate virulence gene expression. QSSMs also suppress host immune responses although the mechanism of suppression is unknown. Here, we demonstrate that the QSSM N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) from P. aeruginosa blocks proliferation and induces apoptosis in human BC cell lines. Analyses of signalling events reveal that OdDHL has little or no effect on MAPK cascades, partially inhibits the Akt/PKB pathway and ablates STAT3 activity. Pharmacological inhibition of each pathway independently indicates that STAT3 activity is critical for BC cell proliferation and survival, while a constitutively active STAT3 confers resistance to OdDHL. These results support the notion of OdDHL as a bioactive molecule in eukaryotic systems and a paradigm for a novel class of antiproliferative compounds.
Oncogene 07/2004; 23(28):4894-902. · 6.37 Impact Factor
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ABSTRACT: The intracellular signals driving the proliferation of breast carcinoma (BC) cells have been widely studied. Both the mitotic and metastatic potential of BC cells have been linked to the frequent overexpression of ErbB family members. Other signaling molecules, including the estrogen receptor, the tyrosine kinases c-Src and Syk, and STAT proteins, especially STAT3, have also been implicated in BC tumor growth. Here we have examined ErbB and STAT protein expression and activation in six BC-derived cell lines. ErbB expression and tyrosine phosphorylation varied considerably among the six cell lines. However, STAT protein expression and activation were more consistent. Two levels of STAT3 activation were distinguished in DNA-binding assays: an epidermal growth factor-inducible, high level that requires both ErbB1 and Janus kinase (JAK) activity and an elevated serum-dependent level that is maintained by autocrine/paracrine signaling and requires JAK activity but is independent of ErbB1 kinase activity. BC cell growth could be inhibited by dominant-negative versions of STAT3 and the JAK inhibitor AG490 but not by PD153035 or PD168393, inhibitors of ErbB1 kinase activity. This indicates that BC cell proliferation may be a consequence of STAT3 activation by autocrine/paracrine signals.
Journal of Biological Chemistry 06/2002; 277(20):17397-405. · 4.77 Impact Factor
