Dominant-negative STAT3 Mutant Research Articles

Ciliary Neurotrophic Factor Activated Signaling Pathways In Retinal Müller Cells

Abstract Ciliary neurotrophic factor (CNTF) is a well-tested, neuroprotective agent that has been shown to retard photoreceptor degeneration in several animal models of retinitis pigmentosa. The molecular mechanisms underlying CNTF-mediated neuroprotection are currently not understood. CNTF could act directly on photoreceptors or it could act indirectly by stimulating Müller glial cells to produce photoreceptor neuroprotective agents. To better characterize CNTF action on Müller cells, we have studied signaling pathways activated by CNTF using an established retinal Müller cell line, rMC-1. RNA was isolated from CNTF-treated cultures, and suppressor of signal transducer and activator of transcription (SOCS3) and Glial fibrillary acidic protein (GFAP) transcript levels were assessed by quantitative real-time PCR. Immunoblotting was used to examine activation ofmitogen activated protein kinase (ERK1/2/MAPK) and phosphoinositide 3-kinase (PI3-K)/Aktpathways in response to CNTF. Additionally, the level of5' AMP-activated protein kinase (AMPK), an enzyme that plays a key role in cellular energy homeostasis levels, was determined by immunoblotting. CNTF treatment resulted strong upregulation of SOCS3 and GFAP transcripts that were blocked by expression of a dominant-negative STAT3 mutant. CNTF treatment also resulted in transient activation of ERK1/2/MAPK but not PI3K/Akt signaling pathway. There was no change in activation of AMPK. We conclude that CNTF treatment leads to stimulation of JAK-STAT and MAPK signaling pathways but not the PI3K/AKT pathway, associated with cell death, in Müller cells.

A Novel Small Molecule STAT Inhibitor, BP-4-018, Demonstrates Significant Anti-Tumour Activity and Synergism With Bortezomib In Pre-Clinical In Vivo Models Of Multiple Myeloma

Despite significant advances in the treatment of Multiple Myeloma (MM), it remains incurable and thus, novel therapeutic strategies are critically needed. Prior work has demonstrated that the transcription factor STAT3 is aberrantly activated in a number of human tumors and drives hallmark oncogenic processes such as proliferation, survival, angiogenesis and drug resistance. As such, this master regulator of malignant processes has become the focus of therapeutic targeting in human tumors that harbor constitutive STAT3 activation, including MM. In the present studies, we characterize a newly developed small molecule, BP-4-018. The inhibitory mechanism of BP-4-018 is based on its ability to function as a phosphotyrosine mimetic, and thus bind the STAT3 SH2 domain to inhibit STAT3 phosphorylation (pSTAT3), STAT3 dimerization and DNA binding. Based on the 3D crystal structure of STAT3, SH2 docking simulations and fluorescence polarization assays, BP-4-018 demonstrates high affinity binding to the SH2 domain of STAT3. Using a panel of molecularly heterogeneous human myeloma cell lines (HMCLs), BP-4-018 demonstrated potent and broad anti-tumor activity in MTT assays, with IC50 values in the low μM range (1.91 μM - 6.48 μM). These anti-MM effects are mediated through the induction of the apoptosis as evidence by Annexin V+ staining, PARP cleavage and activation of caspases 3 and 9. Using patient derived bone marrow (BM) cells, BP-4-018 was shown to retain its anti-MM activity against HMCLs cultured in the presence of a BM stroma, induce apoptosis in primary MM tumor cells, and importantly, displayed little to no toxicity against non-malignant BM cells. Using cell-based assays, we show that BP-4-018 inhibits basal and cytokine-induced pSTAT3, as well as STAT3 transcriptional activity in HMCLs that stably express a STAT3-driven luciferase reporter construct. Consistent with these effects, western blot analyses of BP-4-018-treated cells reveal decreased expression of STAT3 target genes including c-Myc, Mcl-1 and Bcl-xL. Interestingly, BP-4-018 did not demonstrate preferential inhibition of cytokine induced STAT3 phosphorylation over that of STATs 1 or 5 suggesting that this compound may in fact be a pan-STAT inhibitor. BP-4-018 does not however inhibit the phosphorylation of other proteins whose regulation is SH2-dependent including AKT, ERK, SRC and LYN. In vitro combinatorial studies revealed that BP-4-018 is synergistic in combination with dexamethasone (CI=0.62), melphalan (CI=0.94), lenalidomide (CI=0.82) and is highly synergistic with bortezomib (BTZ) (CI=0.37). To examine the potential mechanisms of combinatorial synergy with BTZ, we evaluated the effects of BTZ on STAT3 signaling in HMCLs. BTZ treatment alone augmented STAT3 transcriptional activity and led to the accumulation of Mcl-1 protein, which was reversed when cells were co-treated with BP-4-018. Furthermore, HMCLs expressing a dominant negative STAT3 mutant display increased sensitivity to BTZ. Collectively these data suggest that the combinatorial synergism with BTZ is at least in part related to BP-4-018-mediated inhibition of STAT3. Finally, we used a novel STAT3-driven luciferase xenograft model of MM to show that BP-4-018 (15 mg/kg/day, PO) reduces STAT3-driven luciferase activity in vivo, significantly delays tumor growth as a single agent, and in combination with BTZ, induces marked and in some animals complete tumor regression. Neither BP-4-018 alone or in combination elicited significant toxicities as assessed by weight loss, and more extensive pharmacodynamic, pharmacokinetic and toxicology studies of BP-4-018 are currently underway. Taken together, our results highlight the promising therapeutic potential of BP-4-018 and support the continued development of targeted therapies for MM with a focus on aberrant STAT signaling. Disclosures: No relevant conflicts of interest to declare.

Dominant-negative activity of the STAT3-Y705F mutant depends on the N-terminal domain

BackgroundSTAT3 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.ResultsExpression 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.ConclusionOur 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.

Abstract A260: BP-4-018, a novel inhibitor of STATs demonstrates anti-myeloma activity in preclinical models: Therapeutic implications of targeting aberrant STAT3 signaling in multiple myeloma.

Abstract As a master regulator of the malignant process, the oncogenic transcription factor STAT3 has become the focus of therapeutic targeting in human tumors that harbor constitutive STAT3 activity, including Multiple Myeloma (MM). In the present studies, we employ genetic and chemical tools to further validate STAT3 as a therapeutic target in MM and investigate a novel small molecule STAT3 inhibitor, BP-4-018, with promising in vitro and in vivo anti-MM activity. Using three molecularly heterogeneous human myeloma cell lines (HMCLs), we show that shRNA-mediated knockdown (KD) of STAT3 induces apoptosis, as evidenced by increased Annexin V+ staining and PARP cleavage. Consistent with this biological readout, we observed a decrease in protein expression of STAT3-regulated survival genes including c-Myc, Mcl-1, Bcl-2 and Bcl-xL in STAT3 KD cells. These findings are therapeutically relevant as treatment of HMCLs with BP-4-018 displays broad anti-myeloma activity with IC50 values in the low μM range (1.91-6.48 μM). This rationally designed STAT3 SH2 domain-binding compound induces apoptotic responses in HMCLs and primary MM cells. Mechanistic studies reveal that BP-4-018 dose-dependently inhibits constitutive STAT3 phosphorylation (pSTAT3). Moreover, in selected HMCLs the dose of BP-4-018 required to inhibit pSTAT3 correlated precisely with their respective sensitivity to BP-4-018. HMCLs expressing a STAT3-driven luciferase reporter construct confirmed inhibition of STAT3 transcriptional activity following BP-4-018 treatment, which was associated with a decrease in protein expression of STAT3 target genes. In cell-based assays, BP-4-018 did not demonstrate selectivity for inhibiting pSTAT3 over that of STATs 1 or 5. Importantly however, BP-4-018 did not inhibit phosphorylation of other SH2-dependent proteins including AKT, ERK, SRC and LYN. In vitro combinatorial studies reveal that BP-4-018 is highly synergistic with bortezomib (Btz; CI=0.37). Preliminary data suggests that this interaction may be related to Btz-induced STAT3 activation and induction of Mcl-1 accumulation as concomitant treatment with BP-4-018 inhibited Btz-induced Mcl-1 accumulation. Supporting these findings, cells expressing a dominant negative STAT3 mutant demonstrate enhanced sensitivity to Btz. Finally, we report findings from in vivo efficacy studies using a novel STAT3-driven luciferase xenograft model of MM. We show that BP-4-018 (15 mg/kg/day, PO) reduces STAT3-driven luciferase expression in vivo, significantly delays tumor growth when administered as a single agent, and in combination with Btz, promotes tumor regression without significant toxicities. Taken together, our results highlight the validity of STAT3 as a therapeutic target in MM and support continued study of BP-4-018 and its analogs for the treatment of MM. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A260. Citation Format: Danielle C. Croucher, Zhi Hua Li, Brent DG Page, Ellen Wei, Patrick T. Gunning, Suzanne Trudel. BP-4-018, a novel inhibitor of STATs demonstrates anti-myeloma activity in preclinical models: Therapeutic implications of targeting aberrant STAT3 signaling in multiple myeloma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A260.

STAT3 mediates TGF-β1-induced TWIST1 expression and prostate cancer invasion

TGF-β1 induces epithelial-mesenchymal transition (EMT) to stimulate cancer cell progression, and TWIST1 is a critical regulator of EMT. In the present study, we determined the underlying mechanisms of TGF-β1-induced TWIST1 expression and its effect on prostate cancer cell invasion. TGF-β1 stimulated STAT3 phosphorylation and HIF-1α expression. Silencing either STAT3 or HIF-1α efficiently attenuated TGF-β1-induced TWIST1 expression. Further ectopic expression of a dominant negative mutant of STAT3 reduced TGF-β1-induced TWIST1 expression. In addition, STAT3 and HIF-1α up-regulated TWIST1 expression by direct binding to a TWIST1 promoter. Strikingly, STAT3 also enhanced TGF-β1-induced TWIST1 expression through HIF-1α stabilization. Collectively, we demonstrate a mechanistic cascade of TGF-β1 up-regulating STAT3 activation and HIF-1α stabilization and subsequent TWIST1 expression, leading to prostate cancer invasion.

STAT3 suppresses transcription of proapoptotic genes in cancer cells with the involvement of its N-terminal domain

Activation of STAT3 in cancers leads to gene expression promoting cell proliferation and resistance to apoptosis, as well as tumor angiogenesis, invasion, and migration. In the characterization of effects of ST3-H2A2, a selective inhibitor of the STAT3 N-terminal domain (ND), we observed that the compound induced apoptotic death in cancer cells associated with robust activation of proapoptotic genes. Using ChIP and tiling human promoter arrays, we found that activation of gene expression in response to ST3-H2A2 is accompanied by altered STAT3 chromatin binding. Using inhibitors of STAT3 phosphorylation and a dominant-negative STAT3 mutant, we found that the unphosphorylated form of STAT3 binds to regulatory regions of proapoptotic genes and prevents their expression in tumor cells but not normal cells. siRNA knockdown confirmed the effects of ST3-HA2A on gene expression and chromatin binding to be STAT3 dependent. The STAT3-binding region of the C/EBP-homologous protein (CHOP) promoter was found to be localized in DNaseI hypersensitive site of chromatin in cancer cells but not in nontransformed cells, suggesting that STAT3 binding and suppressive action can be chromatin structure dependent. These data demonstrate a suppressive role for the STAT3 ND in the regulation of proapoptotic gene expression in cancer cells, providing further support for targeting STAT3 ND for cancer therapy.

Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube length by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.

Essential role of Stat3 in PI3K-induced oncogenic transformation

Cells transformed by the p110α-H1047R mutant of PI3K show increased tyrosine phosphorylation of Stat3. This activation of Stat3 is important for the transformation process, because a dominant-negative mutant of Stat3 interferes with PI3K-induced oncogenesis. GDC-0941, a specific inhibitor of PI3K reduces the level of Stat3 phosphorylation. The effect of PI3K on Stat3 appears to be mediated by a member of the Tec kinase family. The Tec kinase inhibitor LFM-A13 blocks Stat3 phosphorylation in H1047R-transformed cells. The Janus kinase inhibitor AG490 and the Src kinase inhibitor Src-1, as well as rapamycin, have no effect on Stat3 phosphorylation in H1047R-transformed cells. The H1047R-transformed cells also release a factor that induces Stat3 phosphorylation in normal cells with possible effects on the cellular microenvironment. In some human tumor cell lines, the enhanced phosphorylation of Stat3 is inhibited by both PI3K and by Tec kinase inhibitors, suggesting that the link between PI3K and Stat3 is significant in human cancer.

Tristetraprolin Mediates Anti-inflammatory Effects of Nicotine in Lipopolysaccharide-stimulated Macrophages

Nicotine inhibits the release of TNF-α from macrophage through activation of STAT3. Tristetraprolin (TTP) is known to destabilize pro-inflammatory transcripts containing AU-rich elements (ARE) in 3'-untranslated region (3'-UTR). Here we show that in LPS-stimulated human macrophages the anti-inflammatory action of nicotine is mediated by TTP. Nicotine induced activation of STAT3 enhanced STAT3 binding to the TTP promoter, increased TTP promoter activity, and increased TTP expression resulting in the suppression of LPS-stimulated TNF-α production. Overexpression of a dominant negative mutant of STAT3 (R382W) or down-regulation of STAT3 by siRNA abolished nicotine-induced TTP expression and suppression of LPS-stimulated TNF-α production. Nicotine enhanced the decay of TNF-α mRNA and decreased luciferase expression of a TNF-α 3'-UTR reporter plasmid in U937 cells. However, siRNA to TTP abrogated these effects of nicotine. In this experiment, we are reporting for the first time the involvement of TTP in the cholinergic anti-inflammatory cascade consisting of nicotine-STAT3-TTP-dampening inflammation.

Activation of the Signal Transducer and Activator of Transcription 3 Pathway Up-Regulates Estrogen Receptor-β Expression in Lung Adenocarcinoma Cells

Estrogens contribute to the pathogenesis of female lung cancer and function mainly through estrogen receptor-β (ERβ). However, the way in which ERβ expression is regulated in lung cancer cells remains to be explored. We have found that signal transducer and activator of transcription 3 (Stat3) activation up-regulates ERβ expression in PC14PE6/AS2 lung cancer cells in a preliminary Affymetrix oligonucleotide array study, and we sought to confirm the findings. In this study, we show that IL-6 induced ERβ mRNA and protein expression in lung cancer cells. The induction of ERβ in response to IL-6 was abolished by Janus kinase 2 inhibitor-AG490, dominant-negative mutant of Stat3, and Stat3-targeting short interfering RNA. The luciferase reporter assay and chromatin immunoprecipitation assay confirmed that IL-6-activated Stat3 binds to the ERβ promoter. Besides the Janus kinase 2/Stat3 pathway, the MEK/Erk pathway contributes to ERβ up-regulation induced by IL-6; however, the phosphoinositide 3'-kinase/Akt pathway does not. We also found that epidermal growth factor (EGF) stimulation or L858R mutation in EGF receptor (EGFR) induced Stat3 activation as well as ERβ expression in lung cancer cells. Inhibiting Stat3 activity by pharmacological or genetic approaches reduced EGF- and L858R mutant EGFR-induced ERβ expression, indicating that Stat3 activation is required for EGFR signaling-mediated ERβ up-regulation. Silencing ERβ decreased cell proliferation in lung cancer cells that overexpress L858R mutant EGFR. In conclusion, we have identified that Stat3 activation is essential for ERβ induction by IL-6, EGF, and the presence of EGFR mutation. The findings shed light on new therapeutic targets for female lung cancer, especially for those with EGFR mutations.

Abstract 2888: SOCS3-deficient cells are defective in the DNA damage response

Abstract The SOCS family of proteins are negative feedback inhibitors of cytokine and growth factor signal transduction, targeting primarily the STAT family of transcription factors. Several SOCS genes, including SOCS1 and SOCS3 are hypermethylated and silenced in solid tumors, which is believed to impart a growth advantage via hyperactivation of the STAT pathway. Despite the growing role of SOCS in the pathogenesis of cancer, very little is known of their role in modulating tumor cell responses to therapy. We recently identified an unexpected function for SOCS3 in the context of the DNA damage response. We found that cells lacking SOCS3 are sensitive to ionizing radiation (IR) in a clonogenic survival assay due to impaired p21 (Cip1/Waf-1) upregulation, which results in failure of the radiation-induced G1-S checkpoint. Normal cell cycle arrest and p21 upregulation could be restored in SOCS3-/- MEFs by overexpression of a phosphorylation-deficient, dominant-negative (DN) mutant of STAT3. Two STAT3-dependent genes known to suppress p21 expression, c-Myc and BCL6 are overrepresented in SOCS3-deficient MEFs. Likewise, human tumor cells that repress SOCS3 expression by epigenetic silencing are also more radiosensitive than their SOCS3+ counterparts. In agreemwnt with our data in MEFs, c-Myc and BCL6 are overexpressed in human tumor cells that silence SOCS3 expression by promoter hypermethulation, suggesting that tumors lacking SOCS3 may be more vulnerable to IR-based therapies due to impaired cell cycle arrest and DNA repair. Our data suggest that therapeutic targeting of SOCS3 in tumor cells could sensitize cells to IR and improve the effectiveness of radiotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2888. doi:10.1158/1538-7445.AM2011-2888

The β2-adrenergic receptor and Her2 comprise a positive feedback loop in human breast cancer cells

In this study, β2-AR level was found to be up-regulated in MCF-7 cells overexpressing Her2 (MCF-7/Her2). Correlation of β2-AR level with Her2 status was demonstrated in breast cancer tissue samples. Constitutive phosphorylation of ERK, mRNA expression up-regulation of catecholamine-synthesis enzymes, and increased epinephrine release were detected in MCF-7/Her2 cells. β2-AR expression induced by epinephrine and involvement of ERK signaling were validated. The data indicate that Her2 overexpression and excessive phosphorylation of ERK cause epinephrine autocrine release from breast cancer cells, resulting in up-regulation of β2-AR expression. The data also showed that catecholamine prominently stimulated Her2 mRNA expression and promoter activity. The activation and nuclear translocation of STAT3 triggered by isoproterenol were observed. Enhanced binding activities of STAT3 to the Her2 promoter after isoproterenol stimulation were verified. Using STAT3 shRNA and dominant negative STAT3 mutant, the role of STAT3 in isoproterenol-induced Her2 expression was further confirmed. The data support a model where β2-AR and Her2 comprise a positive feedback loop in human breast cancer cells.

Diesel Particle–Induced Transcriptional Expression of p21 Involves Activation of EGFR, Src, and Stat3

Exposure to diesel exhaust particles (DEP) has been associated with adverse health outcomes such as inflammation, adjuvancy, and mutagenesis. However, the molecular mechanisms by which DEP inhalation exerts these effects are still largely unknown. We previously reported that exposure to DEP activates the transcription factor Stat3 in airway epithelial cells, a primary target cell of inhaled DEP. To elucidate the functional role of Stat3 activation in these cells, we investigated the function of Stat3 in DEP-induced expression of the p21 gene in the human bronchial epithelial cell line BEAS-2B. We report that DEP exposure induces increased levels of p21 mRNA and protein in a manner that is independent of p53 and Sp1 expression or DNA binding to the p21 gene. Using chromatin immunoprecipitation assays and expression of a dominant-negative Stat3 mutant, we show that activation of Stat3 and its binding to the p21 promoter are required for DEP-induced expression of p21, suggesting that Stat3 plays an essential role in the induction of p21 by DEP. Additional experiments demonstrated that activation of p21 gene expression is dependent on the activation of epidermal growth factor receptor and Src kinase activities. Finally, we provide evidence suggesting that DEP exposure can inhibit the proliferation of human bronchial epithelial cells, suggesting a functional role of p21 activation airway epithelial cells exposed to DEP.

Modulation of PKCδ signaling alters the shear stress-mediated increases in endothelial nitric oxide synthase transcription: role of STAT3

We have previously shown that the regulation of endothelial nitric oxide synthase (eNOS) in endothelial cells isolated from fetal lamb under static conditions is positively regulated by PKCdelta. In this study, we explore the role of PKCdelta in regulating shear-induced upregulation of eNOS. We found that shear caused a decrease in PKCdelta activation. Modulation of PKCdelta before shear with a dominant negative mutant of PKCdelta (DN PKCdelta) or bryostatin (a known PKCdelta activator) demonstrated that PKCdelta inhibition potentiates the shear-mediated increases in eNOS expression and activity, while PKCdelta activation inhibited these events. To gain insight into the mechanism by which PKCdelta inhibits shear-induced eNOS expression, we examined activation of STAT3, a known target for PKCdelta phosphorylation. We found that shear decreased the phosphorylation of STAT3. Further the transfection of cells with DN PKCdelta reduced, while PKCdelta activation enhanced, STAT3 phosphorylation in the presence of shear. Transfection of cells with a dominant negative mutant of STAT3 enhanced eNOS promoter activity and nitric oxide production in response to shear. Finally, we found that mutating the STAT3 binding site sequence within the eNOS promoter increased promoter activity in response to shear and that this was no longer inhibited by bryostatin. In conclusion, shear decreases PKCdelta activity and, subsequently, reduces STAT3 binding to the eNOS promoter. This signaling pathway plays a previously unidentified role in the regulation of eNOS expression by shear stress.

SOCS3 regulates p21 expression and cell cycle arrest in response to DNA damage

Genotoxic agents such as ionizing radiation trigger cell cycle arrest at the G1/S and G2/M checkpoints, allowing cells to repair damaged DNA before entry into mitosis. DNA damage-induced G1 arrest involves p53-dependent expression of p21 (Cip1/Waf-1), which inhibits cyclin-dependent kinases and blocks S phase entry. While much of the core DNA damage response has been well-studied, other signaling proteins that intersect with and modulate this response remain uncharacterized. In this study, we identify Suppressor of Cytokine Signaling (SOCS)-3 as an important regulator of radiation-induced G1 arrest. SOCS3-deficient fibroblasts fail to undergo G1 arrest and accumulate in the G2/M phase of the cell cycle. SOCS3 knockout cells phosphorylate p53 and H2AX normally in response to radiation, but fail to upregulate p21 expression. In addition, STAT3 phosphorylation is elevated in SOCS3-deficient cells compared to WT cells. Normal G1 arrest can be restored in SOCS3 KO cells by retroviral transduction of WT SOCS3 or a dominant-negative mutant of STAT3. Our results suggest a novel function for SOCS3 in the control of genome stability by negatively regulating STAT3-dependent radioresistant DNA synthesis, and promoting p53-dependent p21 expression.

STAT3 and Oct-3/4 Control Histone Modification through Induction of Eed in Embryonic Stem Cells

Mouse embryonic stem (ES) cells can self-renew in the presence of leukemia inhibitory factor (LIF). Several essential transcription factors have been identified for the self-renewal of mouse ES cells, including STAT3, Oct-3/4, and Nanog. The molecular mechanism of ES cell self-renewal, however, is not fully understood. In the present study, we identified Eed, a core component of Polycomb repressive complex 2, as a downstream molecule of STAT3 and Oct-3/4. Artificial activation of STAT3 resulted in increased expression of Eed, whereas expression of a dominant negative mutant of STAT3 or suppression of Oct-3/4 expression led to down-regulation of Eed. Reporter, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that STAT3 and Oct-3/4 directly bind to the promoter region of Eed, suggesting that Eed is a common target molecule of STAT3 and Oct-3/4. We also found that suppression of STAT3, Oct-3/4, or Eed causes induction of differentiation-associated genes as well as loss of Lys(27)-trimethylated histone H3 at the promoter regions of the differentiation-associated genes. Suppression of STAT3 and Oct-3/4 also resulted in the absence of Eed at the promoter regions. These results suggest that STAT3 and Oct-3/4 maintain silencing of differentiation-associated genes through up-regulation of Eed in self-renewing ES cells.

PSM/SH2B1 splice variants: Critical role in src catalytic activation and the resulting STAT3s‐mediated mitogenic response

A role of PSM/SH2B1 had been shown in mitogenesis and extending to phenotypic cell transformation, however, the underlying molecular mechanism remained to be established. Here, four alternative PSM splice variants and individual functional protein domains were compared for their role in the regulation of Src activity. We found that elevated cellular levels of PSM variants resulted in phenotypic cell transformation and potentiated cell proliferation and survival in response to serum withdrawal. PSM variant activity presented a consistent signature pattern for any tested response of highest activity observed for gamma, followed by delta, alpha, and beta with decreasing activity. PSM-potentiated cell proliferation was sensitive to Src inhibitor herbimycin and PSM and Src were found in the same immune complex. PSM variants were substrates of the Src Tyr kinase and potentiated Src catalytic activity by increasing the V(max) and decreasing the K(m) for ATP with the signature pattern of variant activity. Dominant-negative PSM peptide mimetics including the SH2 or PH domains inhibited Src catalytic activity as well as Src-mediated phenotypic cell transformation. Activation of major Src substrate STAT3 was similarly potentiated by the PSM variants in a Src-dependent fashion or inhibited by PSM domain-specific peptide mimetics. Expression of a dominant-negative STAT3 mutant blocked PSM variant-mediated phenotypic cell transformation. Our results implicate an essential role of the PSM variants in the activation of the Src kinase and the resulting mitogenic response--extending to phenotypic cell transformation and involving the established Src substrate STAT3.

Leptin Increases the Expression of the Iron Regulatory Hormone Hepcidin in HuH7 Human Hepatoma Cells

Obesity is a major global health problem and is associated with low-grade inflammation and, in a number of cases, poor iron status. We speculated that the adipokine leptin might play a role in regulating iron metabolism in the overweight population because it shares a number of common biological features with IL-6, a major factor in the development of the anemia of chronic disease via its stimulatory actions on the production and release of the iron regulatory hormone hepcidin. To test this hypothesis, we exposed HuH7 human hepatoma cells to leptin and measured hepcidin mRNA expression by quantitative PCR. HuH7 cells were also transfected with a hepcidin promoter–luciferase reporter gene construct to investigate transcriptional regulation of hepcidin. In leptin-treated cells, hepcidin mRNA expression was enhanced significantly. Preincubation with a Janus kinase (JAK) 2 inhibitor significantly diminished this response. Hepcidin promoter activity was also increased in the presence of leptin. This effect was decreased either by mutation of the signal transducer and activator of transcription (STAT) 3 binding motif in the hepcidin promoter or by coexpressing a dominant-negative STAT3 mutant. These data suggest that leptin upregulates hepatic hepcidin expression through the JAK2/STAT3 signaling pathway. As a consequence, the increased production of leptin in overweight individuals might be a major contributor to the aberrant iron status observed in these population groups.

LIGHT, a member of the TNF superfamily, activates Stat3 mediated by NIK pathway

Stat3, a member of the signal transducers and activators of transcription (STAT) family, is a key signal transduction protein activated by numerous cytokines, growth factors, and oncoproteins that controls cell proliferation, differentiation, development, survival, and inflammation. Constitutive activation of Stat3 has been found frequently in a wide variety of human tumors and induces cellular transformation and tumor formation. In this study, we demonstrated that LIGHT, a member of tumor necrosis factor superfamily, activates Stat3 in cancer cells. LIGHT induces dose-dependent activation of Stat3 by phosphorylation at both the tyrosine 705 and serine 727 residues. The activation of Stat3 by LIGHT appears to be mediated by NIK phosphorylation. Expression of a kinase-inactive NIK mutant abolished LIGHT induced Stat3 activation. Overexpression of an active NIK induces Stat3 activation by phosphorylation at the both tyrosine 705 and serine 727 residues. Activation of Stat3 by NIK requires NIK kinase activity as showed by kinase assays. In addition, LIGHT increases the expression of Stat3 target genes including cyclin D1, survivin, and Bcl-xL, and stimulates human LNCaP prostate cancer cell growth in vitro which can be blocked by expression of a dominant-negative Stat3 mutant. Taken together, these results indicate that in addition to activating NF-κB/p52, LIGHT also activates Stat3. Activation of Stat3 together with activating non-canonical NF-κB/p52 signaling by LIGHT may maximize its effects on cellular proliferation, survival, and inflammation.

TIMP-1 mediates the inhibitory effect of interleukin-6 on the proliferation of a hepatocarcinoma cell line in a STAT3-dependent manner

The tissue inhibitor of metalloproteinases (TIMP)-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs) but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line), L-02 (a normal liver cell line) and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma) using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60% and reduced the migration to about 30%. Meanwhile, our data showed that interleukin-6 (IL-6) (100 ng/mL) could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.