过量表达Stat3诱导COS7细胞显著的形态变化

信号转导和转录激活蛋白Stat3在细胞的生长、分化、存活和运动过程中扮演重要角色。通过免疫细胞化学染色 ,免疫沉淀结合免疫蛋白质印迹法 ,以及瞬时转染由Stat3特异识别DNA元件所指导的报道基因等技术 ,证明COS7细胞中存在活性Stat3蛋白的表达。Stat3cDNA和报道基因在COS7细胞中的共转染实验表明 ,外源的Stat3蛋白具有DNA结合活性 ,并可增强报道基因的表达。同时 ,过量表达Stat3的COS7细胞呈现显著的细胞形态变化 ,如细胞体积增大、胞体伸出突起 ,有些突起有分枝和伪足。这些结果提示 ,Stat3蛋白在细胞的粘附和迁移 ,以及细胞骨架的重建过程中可能具有重要功能。     

Stat3 Activates the Receptor Tyrosine Kinase Like Orphan Receptor-1 Gene in Chronic Lymphocytic Leukemia Cells

Background

The receptor tyrosine kinase like orphan receptor (ROR)-1 gene is overexpressed in chronic lymphocytic leukemia (CLL). Because Stat3 is constitutively activated in CLL and sequence analysis revealed that the ROR1 promoter harbors γ-interferon activation sequence-like elements typically activated by Stat3, we hypothesized that Stat3 activates ROR1.

Methodology/Principal Findings

Because IL-6 induced Stat3 phosphorylation and upregulated Ror1 protein levels in MM1 cells, we used these cells as a model. We transfected MM1 cells with truncated ROR1 promoter luciferase reporter constructs and found that IL-6 induced luciferase activity of ROR1-195 and upstream constructs. Co-transfection with Stat3 siRNA reduced the IL-6-induced luciferase activity, suggesting that IL-6 induced luciferase activity by activating Stat3. EMSA and the ChIP assay confirmed that Stat3 binds ROR1, and EMSA studies identified two Stat3 binding sites. In CLL cells, EMSA and ChIP studies determined that phosphorylated Stat3 bound to the ROR1 promoter at those two ROR1 promoter sites, and ChIP analysis showed that Stat3 co-immunoprecipitated DNA of STAT3, ROR1, and several Stat3-regulated genes. Finally, like STAT3-siRNA in MM1 cells, STAT3-shRNA downregulated STAT3, ROR1, and STAT3-regulated genes and Stat3 and Ror1 protein levels in CLL cells.

Conclusion/Significance

Our data suggest that constitutively activated Stat3 binds to the ROR1 promoter and activates ROR1 in CLL cells.     

Regulation of matrilysin expression in endothelium by fibroblast growth factor-2

Matrilysin (MMP7) is a secreted matrix metalloproteinase, which contributes to angiogenesis by breaking down basement membranes. We show that the angiogenic factor FGF-2 induces MMP7 expression in human endothelial cells. The promoter contains a Lef/Tcf consensus sequence, but using wildtype or Lef/Tcf-mutated promoter constructs, FGF-2-induced MMP7 reporter activity is independent from Lef/Tcf sites. Instead, we show that overexpression of a dominant negative Stat3 mutant reduces FGF-2-mediated MMP7 promoter activity. However, Stat3 does not bind to the MMP7 promoter, but activates MMP7 gene expression indirectly via AP-1. This is confirmed by MMP7 promoter constructs with mutated AP-1 sites which did not respond to FGF-2 and by siRNAs against Stat1 and Stat3, which repressed FGF-2-induced MMP7 protein expression. In conclusion, we show that FGF-2-induced MMP7 expression in endothelium depends on AP-1 and FGF-2 signaling to AP-1 involves a Stat1/3-dependent pathway.     

The JAK2/STAT3 and mitochondrial pathways are essential for quercetin nanoliposome-induced C6 glioma cell death

The formulation of quercetin nanoliposomes (QUE-NLs) has been shown to enhance QUE antitumor activity in C6 glioma cells. At high concentrations, QUE-NLs induce necrotic cell death. In this study, we probed the molecular mechanisms of QUE-NL-induced C6 glioma cell death and examined whether QUE-NL-induced programmed cell death involved Bcl-2 family and mitochondrial pathway through STAT3 signal transduction pathway. Downregulation of Bcl-2 and the overexpression of Bax by QUE-NL supported the involvement of Bcl-2 family proteins upstream of C6 glioma cell death. In addition, the activation of JAK2 and STAT3 were altered following exposure to QUE-NLs in C6 glioma cells, suggesting that QUE-NLs downregulated Bcl-2 mRNAs expression and enhanced the expression of mitochondrial mRNAs through STAT3-mediated signaling pathways either via direct or indirect mechanisms. There are several components such as ROS, mitochondrial, and Bcl-2 family shared by the necrotic and apoptotic pathways. Our studies indicate that the signaling cross point of the mitochondrial pathway and the JAK2/STAT3 signaling pathway in C6 glioma cell death is modulated by QUE-NLs. In conclusion, regulation of JAK2/STAT3 and ROS-mediated mitochondrial pathway agonists alone or in combination with treatment by QUE-NLs could be a more effective method of treating chemical-resistant glioma.     

A novel cis-acting element in Her2 promoter regulated by Stat3 in mammary cancer cells

Stat3 plays important roles in the development of breast malignancies and oncogenesis. In the present study, a palindromic cis-acting element displaying repression activity in breast cancer cells expressing low level of Her2 was found in Her2 promoter. Deletion analysis showed that the novel element was located within Pal2 region spanning nucleotides -529 to -505. The sequence analysis of Pal2 region revealed a DNA sequence (TTAAGATAA) homologous to the binding site of Stat3, starting from position -529 to -521bp. By reporter assay, Pal2 was found to be regulated by constitutive activated Stat3C. A stimulatory effect both on Her2 mRNA and protein expressions was observed in MCF-7 cells stably expressing Stat3C, suggesting that Stat3 regulated Her2 expression. Using ChIP assays the binding of Stat3 to Her2 promoter was confirmed. The data obtained in this study indicate constitutive activated Stat3 regulates Her2 expression. Further investigation of differential effects of Stat3 exerting on breast cancer cells expressing Her2 at different levels will provide more insights into the roles of Stat3 in Her2 expression as well as the regulation of diverse biological activities.