Doxycycline-inducible Promoter Research Articles

Phospholipase C-independent Activation of Glycogen Synthase Kinase-3β and C-terminal Src Kinase by Gαq

It is generally thought that activation of phospholipase Cbeta (PLCbeta) by Galphaq accounts for most of the effects of Gq-coupled receptors. Here we describe a novel effect of Galphaq that is independent of the PLCbeta pathway. Expression of the constitutively active Galphaq mutant Galphaq(Q209L) promoted an increase in glycogen synthase kinase-3beta (GSK-3beta) activity that was associated with increased phosphorylation of Tyr216 on GSK-3beta. Galphaq(Q209L)-AA, a mutant that cannot activate PLCbeta, also induced GSK-3beta activation and phosphorylation of Tyr216. We speculate that the protein-tyrosine kinase Csk (C-terminal Src kinase), which is also activated by Galphaq(Q209L) and Galphaq(Q209L)-AA, acts upstream of GSK-3beta. Expression of Csk accentuated the activation of GSK-3beta by Galphaq(Q209L), whereas catalytically inactive Csk blocked GSK-3beta activation by Galphaq(Q209L). Recombinant Csk phosphorylated and activated GSK-3beta in vitro, and GSK-3beta coprecipitated with Csk from cell lysates. These results suggest that activation of Csk and GSK-3beta by Galphaq may contribute to the physiological and pathological effects of Gq-coupled receptors.

Lymphoid tissue and emphysema in the lungs of transgenic mice inducibly expressing tumor necrosis factor-alpha.

To develop a model in which the pathogenic effects of the proinflammatory cytokine tumor necrosis factor-alpha (TNF) could be investigated, transgenic mice that express TNF in the lung under the control of a doxycycline-inducible promoter were generated. TNF transgene message was expressed at a low level in the absence of doxycycline treatment and was induced in the lung by administration of the drug. Analysis of lung lavage fluid indicated increases in neutrophils and lymphocytes in doxycycline-treated transgenic mice. Histologic analysis of lungs from adult transgenic mice treated with doxycycline revealed prominent development of lymphoid tissue and increases in airspace size. Genes upregulated in TNF transgenic mice, as identified by oligonucleotide microarray analysis, included a variety of transcripts expressed in lymphoid tissues. Immunohistochemical analysis demonstrated the presence of B lymphocytes and, to a lesser extent, T lymphocytes within lymphoid aggregates in TNF transgenic mice. CD8-positive T cells were absent from lymphocytic nodules, but in the lung parenchyma were more abundant in transgenic than in nontransgenic mice. These results indicate that induction of TNF in adult lung promotes the formation of lymphoid tissue and emphysema, and provides a model in which the pathogenic effects of TNF on the lung can be investigated.

Signal transducer and activator of transcription 5 activation is sufficient to drive transcriptional induction of cyclin D2 gene and proliferation of rat pancreatic beta-cells.

Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic beta-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the beta-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.

A search for doxycycline-dependent mutations that increase Drosophila melanogaster life span identifies the VhaSFD, Sugar baby, filamin, fwd and Cctl genes

Screening for conditional mutations that increase Drosophila life span has identified genes implicated in membrane transport, phospholipid metabolism and signaling, and actin cytoskeleton organization.

Hepatitis B virus X protein induced expression of interleukin 18 (IL-18): a potential mechanism for liver injury caused by hepatitis B virus (HBV) infection.

The hepatitis B virus X protein (HBx), a major viral transactivator, is implicated in hepatic inflammation, since it induces many pro-inflammatory cytokines at transcriptional level. The aim of this study was to investigate role of HBx in expression of interleukin 18 (IL-18), a newly identified cytokine that up-regulates Fas ligand (FasL) expression. Chang X-34 that expressing HBx under the control of a doxycycline-inducible promoter, and hepatitis B virus (HBV)-integrated hepatoma cell lines were examined for IL-18 expression by Northern and Western blotting analysis. To test the role of IL-18 produced by hepatoma cells, FasL expression was examined by flow cytometry after treatment with neutralizing anti-IL-18 antibodies. Further, IL-18 expression was examined in the liver tissues of HBx-transgenic mice. Induction of IL-18 following HBx expression in Chang X-34 and the pattern of IL-18 expression in HBV-integrated cell lines, implicated that HBx transcriptionally induces IL-18 expression. Neutralizing anti-IL-18 antibodies blocked the expression of FasL, suggesting that IL-18 plays a critical role in FasL expression. Further, IL-18 expression in the HBx-transgenic liver, was correlated with the degree of hepatitis. Our results demonstrated that HBx induces IL-18 expression in liver, which may be associated with hepatic injury by amplifying FasL expression during HBV infection.

Hepatitis B Virus X Protein Induced Expression of the Nur77 Gene

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in development of HBV-associated hepatocellular carcinoma (HCC). Recently, we reported that HBx induces Fas Ligand (FasL) expression, which may help HCC cells to evade host-immune surveillance. The aim of this study was to investigate the role of HBx in expression of Nur77, an orphan nuclear receptor implicated in the upregulation of FasL. When Chang X-34 expressing HBx under the control of a doxycycline-inducible promoter was examined, induction of Nur77 was observed following HBx expression. Blocking of Nur77 function by introduction of an antisense or a dominant negative mutant Nur77 significantly inhibited the induction of FasL, indicating that Nur77 plays critical roles in FasL expression. Further, a high-level expression of transcripts and DNA binding of Nur77 were observed in the HBV-integrated cell lines established from HCC patients that express HBx. These results suggested that Nur77 may contribute to leading the HBx-induced Fas/FasL signaling pathway which eliminates invading Fas-expressing lymphocytes.

High-frequency generation of conditional mutations affecting Drosophila melanogaster development and life span.

Genome sequencing reveals that a large percentage of Drosophila genes have homologs in humans, including many human disease genes. The goal of this research was to develop methods to efficiently test Drosophila genes for functions in vivo. An important challenge is the fact that many genes function at more than one point during development and during the life cycle. Conditional expression systems such as promoters regulated by tetracycline (or its derivative doxycycline) are often ideal for testing gene functions. However, generation of transgenic animals for each gene of interest is impractical. Placing the doxycycline-inducible ("tet-on") promoter directed out of the end of the P transposable element produced a mobile, doxycycline-inducible promoter element, named PdL. PdL was mobilized to 228 locations in the genome and was found to generate conditional (doxycycline-dependent), dominant mutations at high frequency. The temporal control of gene overexpression allowed generation of mutant phenotypes specific to different stages of the life cycle, including metamorphosis and aging. Mutations characterized included inserts in the alpha-mannosidase II (dGMII), ash1, and pumilio genes. Novel phenotypes were identified for each gene, including specific developmental defects and increased or decreased life span. The PdL system should facilitate testing of a large fraction of Drosophila genes for overexpression and misexpression phenotypes at specific developmental and life cycle stages.

Regulation of MCP-3 and BRCA2 mRNA Expression Levels by β1 Integrins

The integrin cytoplasmic domain has been shown to modulate several cellular functions, including cell proliferation, adhesion, migration, and intracellular signaling. The β1 integrin subunits β1C and β1A, which contain variant cytoplasmic domains, differentially affect cancer and normal cell functions. To identify target genes selectively regulated by these β1 cytoplasmic variants, stable cell transfectants expressing either β1A or β1C under the control of a doxycycline-inducible promoter were obtained using murine β1-deficient GD25 cells. Screening of 1176 murine cDNAs using first-strand cDNA of mRNA isolated from either β1C- or β1A-expressing cells showed a striking differential expression of few genes. The differential expression of two genes, MCP-3 and BRCA2 (monocyte chemoattractant protein-3 and breast cancer susceptibility gene 2, respectively), whose products are involved, respectively, in chemotaxis and embryonic proliferation, was confirmed by Northern blot analysis. Increased MCP-3 and decreased BRCA2 mRNA levels in cells expressing β1C compared to those in cells expressing β1A were observed. Since β1C and β1A stable cell transfectants showed comparable adhesion to fibronectin, upregulation of MCP-3 and downregulation of BRCA2 mRNA levels did not appear to be due to a differential ability of the β1C cells to adhere to the β1 ligand fibronectin. Overall, our data show that β1 integrin cytoplasmic domain variants control expression of downstream target genes in a differential manner without affecting cell adhesion.

AP-1 blockade inhibits the growth of normal and malignant breast cells.

We have previously demonstrated that basal AP-1 transcriptional activity is high in normal human mammary epithelial cells, intermediate in immortal breast cells, and relatively low in breast cancer cells. In this study we investigated whether differences in AP-1 transcriptional activity reflect differences in breast cells' dependence on AP-1 for proliferation. The cJun dominant negative, TAM-67, was used to determine the effect of AP-1 blockade on the growth of normal, immortal and malignant breast cells. We first showed that TAM-67 inhibits AP-1 activity in normal and malignant breast cells. We then determined whether this AP-1 inhibitor affected colony forming efficiency of the immortalized and malignant breast cells. The AP-1 inhibitor reduced colony formation of immortal breast cells by over 50% (by 58% in 184B5 cells and 62% in MCF10A cells), and reduced colony formation in the breast cancer cell line MCF7 by 43%, but did not reduce colony formation in the other breast cancer cell lines (T47D, MDA MB231 and MDA MB 435). We also determined the effect of AP-1 blockade on the growth of normal breast cells using a single cell proliferation assay. Using this assay, the growth of normal breast cells was extremely sensitive to AP-1 blockade, while immortal breast cells were moderately sensitive. We next directly tested the effect of TAM-67 expression on the growth of MCF7 breast cancer cells, using cells stably transfected with TAM-67 under the control of a doxycycline-inducible promoter. Upon induction, TAM-67 was expressed and AP-1 activity was inhibited in these cells. We then measured the growth of these cells in the presence or absence of TAM-67. The results of these studies show that the growth of MCF7 cells was suppressed by the AP-1 inhibitor, TAM-67. These results demonstrate that normal and immortalized breast cells, and some breast cancer cells (such as MCF7), require AP-1 to transduce proliferative signals, while other breast cancer cells (such as T47D, MDA MB 231 and MDA MB 435) do not. These studies suggest that the AP-1 transcription factor is a potential target for future agents for the prevention or treatment of breast cancer.