Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Several treatments which regulate tyrosine hydroxylase (TH) transcription, such as stress in vivo, or 12-O-tetradecanoylphorbol-13-acetate (TPA) in cell culture, induce both Egr1 and AP1 factors. Previously, we identified a functional Egr1 motif overlapping with Sp1 site in the rat TH promoter. Its response to Egr1 also required the presence of an AP1/Ebox motif. Here, we further examined the cross-talk between these sites. Insertion of 10- or 20-bp between the Sp1/Egr1 and AP1/Ebox elements, reduced the ability of Egr1 to upregulate luciferase reporter activity controlled by the proximal 272 nucleotides of the rat TH promoter in PC12 cells. Electrophoretic mobility shift assays with nuclear extracts from TPA treated cells were used to identify the composition of the factors which bound the AP1/Ebox motif and whether there is competition with factors which bind the Sp1/Egr1 motif. The complexes formed with labeled AP1/E box oligonucleotide were reduced or supershifted with antisera to Fos family, c-Fos, Fra-2, and Jun D. Excess Sp1/Egr1 oligonucleotide or anti Egr1 antisera did not compete. Fra-2 was a major component of the complex after 2-4 h TPA. Transfection of PC12 cells with Fra-2 induced reporter activity requiring the AP1, but not the Egr1 motif. However, when cotransfected with Fra-2, Egr1 expression plasmids elicited lower induction of luciferase activity than observed with Egr1 alone. Our results suggest that although it does not compete for binding to the promoter, Egr1 can modulate the regulation of TH transcription by AP1 factors.
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PMID:Interactions between Egr1 and AP1 factors in regulation of tyrosine hydroxylase transcription. 1267 Jul 3

We have isolated a novel gene, LDOC1, which encodes for a leucine zipper protein that was downregulated in a series of human pancreatic cancer cell lines but was expressed in corresponding normal tissues. We report the initial characterization of LDOC1 as a novel regulator of the transcriptional response mediated by the nuclear factor kappa B (NF-kappaB). Transient expression of LDOC1 significantly inhibited the luciferase activity in LDOC1-negative BxPC-3 pancreatic cancer cell line transfected with the NF-kappaB reporter plasmid, activated with mitogen-activated protein kinase/ERK kinase kinase-1 (MEEK). LDOC1, however, does not affect p53, AP1 and CRE-dependent reporter gene expression. The activation of NF-kappaB through ligand-induced stimulation by tumor necrosis factor-alpha (TNF-alpha) or phorbol 12-myristate 13-acetate (PMA) was also inhibited by transient expression of LDOC1 in a dose dependent manner. To determine the growth effect of LDOC1 expression on cancer cells, BxPC-3 cells were stably transfected with LDOC1 cDNA. Viability studies demonstrated that TNF-alpha or PMA-induced antiproliferative effects were significantly enhanced by stable transfection of cells with LDOC1. These observations suggest that LDOC1 is a novel regulator of NF-kappaB that can affect the PMA or TNF-alpha-mediated pathway to apoptosis through inhibition of NF-kappaB activation in BxPC3 pancreatic cancer cells.
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PMID:Leucine-zipper protein, LDOC1, inhibits NF-kappaB activation and sensitizes pancreatic cancer cells to apoptosis. 1271 34

Altered transcriptional control is likely to contribute to the down-regulation of connexin 43 (Cx43) expression observed in many forms of heart disease. However, little is known about the factors regulating Cx43 transcription in the heart under (patho)physiological conditions. Therefore, a systematic study of rat Cx43 (rCx43) proximal promoter regulation in rat primary neonatal ventricular cardiomyocytes (NCM) and, for comparison, different cell types was initiated. Luciferase assays revealed that, in NCM, the proximal promoter is preserved in a conserved region extending from 148 nucleotides upstream towards 281 nucleotides downstream relative to the transcription initiation site (TIS). Further deletional analysis suggested the involvement of four putative Sp- and two AP1-binding sites. The binding of both Sp1 and Sp3 to the Sp-binding elements and AP1 to the AP1-binding elements was demonstrated by electrophoretic mobility shift assays (EMSA). Promoter-luciferase assays using the natural rCx43 proximal promoter and mutated derivatives in NCM, HL-1 and A7r5 cells revealed that all sites contribute to basal promoter activity. Trans-activation of the Cx43 proximal promoter with Sp1 and Sp3 in Drosophila Schneider line 2 (SL2) cells demonstrated that Sp1 and, to a lesser extent, Sp3 determine rCx43 promoter activation. Thus Sp1, Sp3 and AP1 determine basal Cx43 expression. In addition, we studied the effect of the cardiac transcription factor Nkx2.5 on Cx43 regulation. NCM were infected with adenovirus encoding either beta-galactosidase (control) or Nkx2.5. Cx43 protein and mRNA were significantly decreased after Nkx2.5 infection as shown by Western and Northern blot analyses. Promoter-reporter assays demonstrated that the rCx43 promoter was down-regulated approximately twofold upon Nkx2.5 overexpression. Therefore, in NCM, Nkx2.5 appears to play a role in the regulation of Cx43 expression.
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PMID:Analysis of the rat connexin 43 proximal promoter in neonatal cardiomyocytes. 1464 4

Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV8), has significant sequence homology to Epstein-Barr virus (EBV). In cell culture, HHV8 is primarily latent, and viral genes associated with lytic replication are not expressed. Two lytic origins of DNA replication (oriLyt) are present within the HHV8 genome and are composed of an AT-rich region adjacent to GC-rich DNA sequences. We have now identified essential cis- and trans-acting elements required for oriLyt-dependent DNA replication. The transient replication assay was used to show that two AT-rich elements, three consensus AP1 transcription factor-binding sites, an ORF50 response element (RE), and a consensus TATA box motif are essential for efficient origin-dependent DNA replication. Transient transfection of luciferase reporter constructs indicated that the downstream region of the HHV8 oriLyt responds to ORF50 and suggests that part of the oriLyt may be an enhancer/promoter. In addition, a transient cotransfection-replication assay elucidated the set of trans-acting factors required for lytic DNA replication. These factors consist of homologues to the core replication proteins: ORF6 (ssDNA binding protein), ORF9 (DNA polymerase), ORF40-41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), and ORF59 (polymerase processivity factor) common to all herpesviruses along with ORF50 (K-Rta) and K8 (K-bZIP).
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PMID:Amplification of the Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 lytic origin of DNA replication is dependent upon a cis-acting AT-rich region and an ORF50 response element and the trans-acting factors ORF50 (K-Rta) and K8 (K-bZIP). 1497 23

The breast cancer resistance protein (BCRP) is an ATP-binding cassette half transporter that confers resistance to anticancer drugs such as mitoxantrone, anthracyclines, topotecan, and SN-38. Initial characterization of the BCRP promoter revealed that it is TATA-less with 5 putative Sp1 sites downstream from a putative CpG island and several AP1 sites (K. J. Bailey-Dell et al., Biochim. Biophys. Acta, 1520: 234-241, 2001). Here, we examined the sequence of the 5'-flanking region of the BCRP gene and found a putative estrogen response element (ERE). We showed that estrogen enhanced the expression of BCRP mRNA in the estrogen receptor (ER)-positive T47D:A18 cells and PA-1 cells stably expressing ERalpha. In BCRP promoter-luciferase assays, sequential deletions of the BCRP promoter showed that the region between -243 and -115 is essential for the ER effect. Mutation of the ERE found within this region attenuated the estrogen response, whereas deletion of the site completely abrogated the estrogen effect. Furthermore, electrophoretic mobility shift assays revealed specific binding of ERalpha to the BCRP promoter through the identified ERE. Taken together, we provide evidence herein for a novel ERE in the BCRP promoter.
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PMID:Identification of a novel estrogen response element in the breast cancer resistance protein (ABCG2) gene. 1497 83

In this study, we investigated osteoblastic differentiation by trichostatin A (TSA), a histone deacetylase inhibitor in mouse undifferentiated mesenchymal cell line. TSA increased the osteopontin (OPN) mRNA level and OPN protein. Deletion analysis of the promoter region revealed TSA-induced luciferase response was regulated by -75 to -65 of the OPN promoter. There was an AP1-binding sequence at the site of the OPN promoter. In an electrophoretic mobility shift assay, bands of the complexes were supershifted by addition of antibody to c-fos and phosphorylated c-jun. These data suggested that AP1 plays a crucial role in the TSA-induced OPN expression.
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PMID:Trichostatin A activates the osteopontin gene promoter through AP1 site. 1498 5

Interleukin-1beta (IL-1beta) induces degradation via hyperexpression of an array of genes, including metalloproteinases (MMP), in cartilage cells during articular degenerative diseases. In contrast, natural ligands for peroxisome proliferator-activated receptors (PPARs) display protective anti-cytokine effects in these cells. We used the PPAR agonist rosiglitazone (Rtz) to investigate PPAR-gamma isotype on IL-1beta-target genes. Immunocytochemistry, electrophoretic mobility shift, and transient transfection assays revealed a functional PPAR-gamma in chondrocytes in vitro. Rtz displayed significant inhibition of IL-1beta effects in chondrocytes. Low Rtz concentrations (close to K(d) values for PPAR-gamma, 0.1 to 1 microm) inhibited the effects of IL-1beta on (35)S-sulfated proteoglycan production and gelatinolytic activities and downregulated MMP1 expression at mRNA and protein levels. We have investigated the mechanism of action of Rtz against IL-1beta-mediated MMP1 gene hyperexpression. Rtz effect occurs at the transcriptional level of the MMP1 promoter, as observed in transiently transfected cells with pMMP1-luciferase vector. Transient expression of wild type PPAR-gamma enhanced Rtz inhibitory effect in chondrocytes, whereas a mutated dominant negative PPAR-gamma abolished it, supporting the role of PPAR-gamma in this effect. MMP1 gene promoter analysis revealed the involvement of a cis-acting element located at -83 to -77, shown to be a composite PPRE/AP1 site. Gel mobility and supershift assays demonstrated that PPAR-gamma and c-Fos/c-Jun proteins bind this cis-acting element in a mutually exclusive way. Our data highlight a new PPAR-gamma-dependent inhibitory mechanism on IL-1beta-mediated cartilage degradation occurring through DNA binding competition on the composite PPRE/AP1 site in the MMP1 promoter.
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PMID:Peroxisome proliferator-activated receptor-gamma down-regulates chondrocyte matrix metalloproteinase-1 via a novel composite element. 1509 May 44

In our continued studies on corticotropin releasing factor receptor (CRFR1) signaling in the skin, we tested functional activity of CRFR1alpha, e, f, g and h isoforms after transfection to COS cells. Both membrane-bound and soluble variants are translated in vivo into final protein products that undergo further post-translational modifications. CRFR1alpha was the only isoform coupled directly to adenylate cyclase with the exception of an artificial isoform (CRFR1h2) with the insertion of 37 amino acids between the ligand binding domain and the first extracellular loop that was capable of producing detectable levels of cyclic AMP (cAMP). Soluble isoforms could modulate cell response with CRFR1e attenuating and CRFR1h amplifying CRFR1alpha-coupled cAMP production stimulated by urocortin. Testing with plasmids containing the luciferase reporter gene, and inducible cis-elements (CRE, CaRE, SRE, AP1 or NF-kappaB) demonstrated that only CRFR1alpha was involved directly in the transcriptional regulation, while CRFR1g inhibited CRE activity. Significantly higher reporter gene expression by CRF was observed than that mediated by 4beta-phorbol 12-myristate 13-acetate and forskolin alone, being compatible with the concomitant treatment by phorbol 12-myristate 13-acetate and forskolin. This suggests that both protein kinase A and C can be involved in CRF-dependent signal transduction.
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PMID:Molecular and functional characterization of novel CRFR1 isoforms from the skin. 1520 47

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found associated with tumors that appear and regress on a seasonal basis. There are quantitative and qualitative differences in the amount of virus expression between developing and regressing tumors. To understand the role of host cell factors in WDSV expression, DNase I footprint analysis, electrophoretic mobility shift assays (EMSA), and reporter gene assays were employed. DNase I footprint analysis of the U3 region of the WDSV long terminal repeat with nuclear extract prepared from a walleye cell line revealed protection of an Oct1, AP1, Whn, and two E4BP4 sites. Additionally, three regions that contained no putative transcription factor binding sites were protected. EMSA confirmed the specific binding of the protected sites and revealed three additional sites, NF1, AP3, and LVa, not protected in DNase I footprint analysis. Site-directed mutagenesis of the individual sites, in the context of a luciferase reporter plasmid, revealed that the NF1, Oct1, AP1, E4BP4#2, AP3, and LVa sites contributed to transcription activation driven by the WDSV U3 region. Mutation of Novel#2 resulted in an increase in luciferase activity, suggesting the Novel#2 site may function to bind a negative regulator of transcription. Anti-Jun and anti-Fos antiserum specifically inhibited protein-DNA complex formation, indicating the presence of c-Jun and c-Fos in the walleye cell nuclear extracts and their participation in binding to the AP1 site. Interestingly, degenerative 15-bp repeats found in the U3 region are differentially protected in DNase I footprint analysis by the walleye cell line nuclear extract and regressing-tumor nuclear extract. EMSA utilizing the 15-bp repeat probe revealed that there are similarities of binding with W12 cell and developing-tumor nuclear extracts and that the binding differs from that observed with regressing-tumor nuclear extract.
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PMID:Identification and characterization of cis-acting elements residing in the walleye dermal sarcoma virus promoter. 1522 Apr 34

Testes determining factor Sry is encoded by the Sry locus on the Y chromosome and may be involved in the regulation of blood pressure. Here we tested the hypothesis that Sry regulates transcription of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of catecholamines. Sry was found to be expressed in catecholaminergic regions, in male but not female rats. Co-transfection of PC12 cells with expression vector for Sry and the reporter construct [p5'TH(-773/+27)/Luc], containing 773 of the proximal nucleotides of the TH promoter directing luciferase reporter activity, led to elevation of reporter activity. The reporter activity of a shorter construct [p5'TH(-272/+27)/Luc] lacking putative Sry sites also responded to Sry. However, mutation of the AP1 site in the TH promoter greatly reduced induction by Sry, indicating that the regulation is primarily at this motif. The remaining, significantly increased expression with the mutated TH promoter construct may reflect Sry function at other sites in addition to the AP1 motif. These results reveal that Sry can regulate TH transcription and suggest that this may be one of the mechanisms of Sry mediated regulation of catecholamine biosynthesis in catecholaminergic neurons in males.
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PMID:Regulation of tyrosine hydroxylase gene transcription by Sry. 1546 65


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