Alpha 1抗胰蛋白酶核基质附着区增强RNA聚合酶Ⅱ依赖的转录

核基质附着区是一种真核生物的DNA元件,能调控染色体结构和活性。为研究体内MAR相关的染色质准入和转录调控,我们从人基因组中克隆了Alpha 1抗胰蛋白酶核基质附着区（α1-AT MAR）并将其连入pEGFP-C1载体。分别以空载体和携带MAR的载体通过脂质体法转染人胚肾293细胞系。经G418筛选20天的细胞池用作实验分析。半定量RT-PCR及荧光显微镜观察显示此MAR能够增强临近基因的表达。进一步用染色质免疫共沉淀（ChIP）共定位CMV启动子和RNA聚合酶Ⅱ（RNAPⅡ）,PCR结果显示存在MAR元件时,更多的RNA聚合酶Ⅱ被富集到启动子区。ChIP方法可用于证实MAR介导的转录激活,在实时检测方面比RT-PCR提供了更多动力学信息。这项技术为我们进一步研究基因表达调控提供了技术平台。     

Shear stress modulates macrophage-induced urokinase plasminogen activator expression in human chondrocytes

Introduction

Synovial macrophages, which can release proinflammatory factors, are responsible for the upregulation of cartilage-breakdown proteases and play critical roles in cartilage degradation during the progression of osteoarthritis (OA). In addition, shear stress exerts multifunctional effects on chondrocytes by inducing the synthesis of catabolic or anabolic genes. However, the interplay of macrophages, chondrocytes, and shear stress during the regulation of cartilage function remains poorly understood. We investigated the mechanisms underlying the modulation of human chondrocyte urokinase plasminogen activator (uPA) expression by macrophages and shear stress.

Methods

Human chondrocytes were stimulated by peripheral blood-macrophage- conditioned medium (PB-MCM), or exposure of chondrocytes cultured in PB-MCM to different levels of shear stress (2 to 20 dyn/cm²). Real-time polymerase chain reaction was used to analyze uPA gene expression. Inhibitors and small interfering RNA were used to investigate the mechanism for the effects of PB-MCM and shear stress in chondrocytes.

Results

Stimulation of human chondrocytes with PB-MCM was found to induce uPA expression. We demonstrated that activation of the JNK and Akt pathways and NF-κB are critical for PB-MCM-induced uPA expression. Blocking assays by using IL-1ra further demonstrated that IL-1β in PB-MCM is the major mediator of uPA expression in chondrocytes. PB-MCM-treated chondrocytes subjected to a lower level of shear stress showed inhibition of MCM-induced JNK and Akt phosphorylation, NF-κB activation, and uPA expression. The PB-MCM-induced uPA expression was suppressed by AMP-activated protein kinase (AMPK) agonist. The inhibitor or siRNA for AMPK abolished the shear-mediated inhibition of uPA expression.

Conclusions

These data support the hypothesis that uPA upregulation stimulated by macrophages may play an active role in the onset of OA and in the shear-stress protection against this induction.     

Signaling mechanisms responsible for lysophosphatidic acid-induced urokinase plasminogen activator expression in ovarian cancer cells

Lysophosphatidic acid (LPA) enhances urokinase plasminogen activator (uPA) expression in ovarian cancer cells; however, the molecular mechanisms responsible for this event have not been investigated. In this study, we used the invasive ovarian cancer SK-OV-3 cell line to explore the signaling molecules and pathways essential for LPA-induced uPA up-regulation. With the aid of specific inhibitors and dominant negative forms of signaling molecules, we determined that the G(i)-associated pathway mediates this LPA-induced event. Moreover, constitutively active H-Ras and Raf-1-activating H-Ras mutant enhance uPA expression, whereas dominant negative H-Ras and Raf-1 block LPA-induced uPA up-regulation, suggesting that the Ras-Raf pathway works downstream of G(i) to mediate this LPA-induced process. Surprisingly, dominant negative MEK1 or Erk2 displays only marginal inhibitory effect on LPA-induced uPA up-regulation, suggesting that a signaling pathway distinct from Raf-MEK1/2-Erk is the prominent pathway responsible for this process. In this report, we demonstrate that LPA activates NF-kappaB in a Ras-Raf-dependent manner and that blocking NF-kappaB activation with either non-phosphorylable IkappaB or dominant negative IkappaB kinase abolished LPA-induced uPA up-regulation and uPA promoter activation. Furthermore, introducing mutations to knock out the NF-kappaB binding site of the uPA promoter results in over 80% reduction in LPA-induced uPA promoter activation, whereas this activity is largely intact with the promoter containing mutations in the AP1 binding sites. Thus these results suggest that the G(i)-Ras-Raf-NF-kappaB signaling cascade is responsible for LPA-induced uPA up-regulation in ovarian cancer cells.     

小鼠白蛋白启动子的组织特异性功能研究

以绿色荧光蛋白(GFP)基因作为报告基因,通过对比小鼠白蛋白启动子在不同来源细胞系中启动HGFP基因的转录活性,对小鼠白蛋白启动子的组织特异性进行了研究。结果发现,小鼠白蛋白启动子在小鼠肝癌细胞系Hepa 1—6和人肝癌细胞系：HepG2均有很强的转录起始功能,荧光显微镜下可以观察到IGFP表达。Hepa 1—6细胞在转染早期的48h内,CMV的启动子和增强子序列是小鼠白蛋白启动子转录活性的4倍。G418加压筛选2周后,CMV的启动子的转录活性下降到只有小鼠白蛋白启动子活性的1／2。转染人肝癌细胞系HepG2 2周后,荧光显微镜下可以观察到GFP表达。其他的细胞如中华仓鼠卵巢细胞系CHO和人肺癌细胞系PLA 801中转染的小鼠白蛋白启动子不能启动GFP的表达,而对照CMV启动子控制下的GFP基因可在CHO和PLA 801中表达。以上结果说明,小鼠白蛋白启动子仅在肝脏来源的细胞中可以起始下游基因的转录,在其他组织来源的细胞中不能起始转录,这表明小鼠白蛋白启动子具有肝脏组织特异的转录活性,但没有种属特异性。     

A porcine gene, PBK, differentially expressed in the longissimus muscle from Meishan and Large White pig

An investigation of differences in gene expression in the longissimus muscle of Meishan and Large White pigs was undertaken, using the mRNA display technique. A fragment of one differentially expressed gene was isolated and sequenced, whereupon the complete cDNA sequence was then obtained by using the rapid amplification of cDNA ends (RACE). The nucleotide sequence of the gene is not related to any known porcine gene. Sequence analysis revealed that the open reading frame of this gene encodes a protein with 322 amino acids, thus displaying high sequence identity with the PDZ binding kinase (PBK) of eleven other animal species - dog, horse, cattle, human, chimpanzee, crab-eating macaque, rhesus monkey, rat, mouse, gray short-tailed opossum and platypus, so it can be defined as the porcine PBK gene. This gene was finally assigned GeneID:100141310. Phylogenetic tree analysis revealed that the swine PBK gene has a closer genetic relationship with the PBK gene of platypus. Gene expression analysis of eight tissues of a Meishan x Large White cross showed that the porcine PBK gene is differentially expressed in various tissues. Our experiment established the primary foundation for further research on this gene.     

Multiple instability-regulating sites in the 3'' untranslated region of the urokinase-type plasminogen activator mRNA.

In LLC-PK1 cells urokinase-type plasminogen activator (uPA) mRNA has a short half-life. It is stabilized by inhibition of protein synthesis and by downregulation of protein kinase C (PKC). In the present study on uPA mRNA metabolism, we focused our attention on the 3' untranslated region (3'UTR) of the uPA mRNA, as this region is long and highly conserved among several mammalian species, including mice and humans. To investigate the possible role of the 3'UTR of uPA mRNA in mRNA metabolism, we inserted this region into the 3'UTR of the rabbit beta-globin gene that is linked to the cytomegalovirus promoter and stably transfected it into LLC-PK1 cells. While the parental globin mRNA was stable, the chimeric mRNA was degraded as rapidly as endogenous uPA mRNA, suggesting that the 3'UTR of uPA mRNA contains most of the information required for its rapid turnover. Further analysis showed that there are at least three independent determinants of instability in the 3'UTR; one is an AU-rich sequence located immediately 3' of the poly(A) addition signal, and one is a sequence containing a stem structure. One determinant seems to require ongoing RNA synthesis for its activity. All chimeric unstable globin mRNAs became stable in the presence of cycloheximide, a protein synthesis inhibitor, suggesting that the stabilization of mRNA by protein synthesis inhibition is not through a specific sequence in the mRNA. In PKC-downregulated cells, globin mRNAs with the complete 3'UTR or the AU-rich sequence were stabilized, suggesting that PKC downregulation stabilizes uPA mRNA through the AU-rich sequence. Here we discuss the significance of multiple, independently acting instability determinants in the regulation of uPA mRNA metabolism.     

Fisetin Inhibits Migration and Invasion of Human Cervical Cancer Cells by Down-Regulating Urokinase Plasminogen Activator Expression through Suppressing the p38 MAPK-Dependent NF-κB Signaling Pathway

Fisetin (3,3’,4’,7-tetrahydroxyflavone), a naturally occurring flavonoid, has been reported to inhibit proliferation and induce apoptosis in several cancer types. However, its effect on the anti-metastatic potential of cervical cancer cells remains unclear. In the present study, we found that fisetin inhibits the invasion and migration of cervical cancer cells. The expression and activity of urokinase plasminogen activator (uPA) was significantly suppressed by fisetin in a dose-dependent manner. We also demonstrated that fisetin reduces the phosphorylation of p38 MAPK, but not that of ERK1/2, JNK1/2, or AKT. Addition of a p38 MAPK inhibitor, SB203580, further enhanced the inhibitory effect of fisetin on the expression and activity of uPA and the invasion and motility in cervical cancer cells. Fisetin suppressed the TPA (tetradecanoylphorbol-13-acetate)-induced activation of p38 MAPK and uPA, and inhibited the TPA-enhanced migratory and invasive abilities. Furthermore, the promoter activity of the uPA gene was dramatically repressed by fisetin, which disrupted the nuclear translocation of NF-κB and its binding amount on the promoter of the uPA gene, and these suppressive effects could be further enhanced by SB203580. This study provides strong evidence for the molecular mechanism of fisetin in inhibiting the aggressive phenotypes by repression of uPA via interruption of p38 MAPK-dependent NF-κB signaling pathway in cervical cancer cells and thus contributes insight to the potential of using fisetin as a therapeutic strategy against cervical cancer by inhibiting migration and invasion.     

Autocrine and paracrine regulation of tissue inhibitor of metalloproteinases, transin, and urokinase gene expression in metastatic and nonmetastatic mammary carcinoma cells.

Acquisition of metastatic competence by tumor cells is frequently accompanied by increased expression of extracellular proteases capable of degrading basement membrane and extracellular matrix. However, very little is known about how the genes encoding these enzymes and their inhibitor proteins are regulated in metastatic versus nonmetastatic cells. In this report, we have compared autocrine and paracrine regulation of tissue inhibitor of metalloproteinases (TIMP), transin, and urokinase plasminogen activator (uPA) genes in genetically related nonmetastatic SP1 and metastatic A3a cell lines. Compared to SP1 cells, metastatic A3a cells showed 15-20-fold higher transin, 3-5-fold less TIMP mRNA, and comparable levels of uPA mRNA. A qualitatively similar shift in expression of these genes was rapidly (i.e., 4-8 h) induced in nonmetastatic SP1 cells following the addition of conditioned medium from A3a cells. The gene-regulating activity present in A3a conditioned medium was heat-labile, suggesting that it was protein in nature. The responsiveness of SP1 cells to the factor(s) secreted by A3a conditioned medium was inhibited by cycloheximide. Basic fibroblast growth factor mimicked the effect of the A3a conditioned medium as an inducer of transin expression in the tumor cells. Although medium conditioned by the tumor cells did not affect uPA expression, addition of epidermal growth factor to the tumor cells transiently induced expression of uPA with a biphasic response that differed in SP1 and A3a cells. Initial induction of uPA at 2-4 h was similar for both cell lines, but after 24 h of exposure to epidermal growth factor, SP1 cells showed a net reduction in uPA, whereas metastatic cells returned to the unstimulated levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Arsenic trioxide (As2O3) inhibits invasion of HT1080 human fibrosarcoma cells: role of nuclear factor-kappaB and reactive oxygen species

In order to define the role of As2O3 in regulating the tumor cell invasiveness, the effects of As2O3 on secretion of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA), and in vitro invasion of HT1080 human fibrosarcoma cells were examined. As2O3 inhibited cell adhesion to the collagen matrix in a concentration dependent manner, whereas the same treatment enhanced cell to cell interaction. In addition, As2O3 inhibited migration and invasion of HT1080 cells stimulated with phorbol 12-myristate 13-aceate (PMA), and suppressed the expression of MMP-2, -9, membrane type-1 MMP, uPA, and uPA receptor (uPAR). In contrast, As2O3 increased the expression of tissue inhibitor of metalloproteinase (TIMP)-1 and PA inhibitor (PAI)-1, and reduced the MMP-2, -9, and uPA promoter activity in the presence and absence of PMA. Furthermore, the promoter stimulating and DNA binding activity of nuclear factor-kappaB (NF-kappaB) was blocked by As2O3, whereas the activator protein-1 activity was unchanged. Pretreatment of the cells with N-acetyl-L-cysteine (NAC) significantly prevented suppression of MMPs and uPA secretion, DNA binding activity of NF-kappaB, and in vitro invasion of HT1080 cells by As2O3, suggesting a role of reactive oxygen species (ROS) in this process. These results suggest that As2O3 inhibits tumor cell invasion by modulating the MMPs/TIMPs and uPA/uPAR/PAI systems of extracellular matrix (ECM) degradation. In addition, the generation of ROS and subsequent suppression of NF-kappaB activity by As2O3 might partly be responsible for the phenomena. Overall, As2O3 shows potent activity controlling tumor cell invasiveness in vitro.