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

To identify cis-acting sequences transcriptionally regulating the human calcitonin receptor (hCTR) gene, hCTR promoter/luciferase gene constructs were transiently or stably transfected into hCTR-positive and -negative cell lines. Luciferase assays demonstrated that the proximal hCTR promoter (hCTRP1) was transcriptionally active in all cell lines tested. High-level hCTRP1 activity depended on an 11 bp Sp1/Sp3 binding site. Electrophoretic mobility shift assay showed that this region bound the transcription factors Sp1 and Sp3. We further showed that hCTRP1 was strongly activated by the 11 bp Sp1/Sp3 binding site in hCTRP1/luciferase-, Sp1-transfected Drosophila S2 cells. Bisulphite-mediated sequencing of genomic DNA from hCTR-expressing and -non-expressing cell lines demonstrated that the endogenous hCTRP1 was hypomethylated in all cell lines tested. These results suggest that the hCTRP1 is activated by the tissue-ubiquitous transcription factor Sp1 and that an epigenetic process unrelated to CpG methylation represses its activity in hCTR-negative tissues.
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PMID:Tissue-specific activity of the proximal human calcitonin receptor promoter is mediated by Sp1 and an epigenetic phenomenon. 1462 7

We examined the effect of suboptimal concentrations of cyclin-dependent kinase inhibitors, which do not interfere with cell proliferation, on retinoblastoma expression in hamster (Chinese hamster ovary K1) and human (K562 and HeLa) cells. To achieve this, we used the chemical inhibitors roscovitine and olomoucine (which inhibit CDK2 preferentially), UCN-01 (which also inhibits CDK4/6) and p21 (as an intrinsic inhibitor). All chemical inhibitors and overexpression of p21 strongly induced retinoblastoma protein expression. UCN-01-mediated retinoblastoma expression was caused by an increase in both the levels of retinoblastoma mRNA and the stability of the protein. The expression of the transcription factor Sp1, a retinoblastoma-interacting protein, was also enhanced by all the cyclin-dependent kinase inhibitors tested. However, Sp1 expression was caused by an increase in the levels of Sp1 mRNA without modification in the stability of the protein. By using luciferase experiments, the transcriptional activation of both retinoblastoma and Sp1 promoters by UCN-01 was confirmed. Bisindolylmaleimide I, at concentrations causing a similar or higher inhibition of protein kinase C than UCN-01, provoked a lower activation of retinoblastoma and Sp1 expression. Finally, the effects of cyclin-dependent kinase inhibitors on dihydrofolate reductase gene expression were evaluated. Treatment with UCN-01 increased cellular dihydrofolate reductase mRNA levels, and dihydrofolate reductase enzymatic activity was enhanced by UCN-01, roscovitine, olomoucine and p21, in transient transfection experiments. These results support a mechanism for the self-regulation of retinoblastoma expression, and point to the need to establish the appropriate dose of cyclin-dependent kinase inhibitors as antiproliferative agents in anticancer treatments.
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PMID:The expression of retinoblastoma and Sp1 is increased by low concentrations of cyclin-dependent kinase inhibitors. 1465 8

In this report, we examined the role of activin in the regulation of cell growth inhibition of human hepatocarcinoma cells. Using RNase protection assay for various cell cycle regulators and Western blotting experiments, we show that activin treatment of HepG2 cells leads to increased gene expression of the cyclin-dependent kinase inhibitor (CDKI) p15INK4B. Furthermore, transient co-transfection studies of the p15INK4B promoter/luciferase construct performed in HepG2 cells demonstrates that activin induction of the p15INK4B promoter is mediated through the Smad pathway. p15INK4B gene promoter mapping analysis revealed a 66-bp region within the proximal domain of the promoter, which contains a consensus site for the transcription factor Sp1, as critical for mediating the activin effect on p15INK4B gene expression. Finally, gel mobility shift experiments, using the Sp1 consensus site, revealed increased DNA binding of Sp1 in response to activin treatment of HepG2 cells, further confirming the involvement of Sp1 in activin-mediated p15INK4B gene promoter activation. Together, our data indicates an important role for the cyclin-dependent kinase inhibitor p15INK4B in activin-induced cell cycle arrest in liver cells.
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PMID:Activin induces hepatocyte cell growth arrest through induction of the cyclin-dependent kinase inhibitor p15INK4B and Sp1. 1509 10

Coumarins and structurally related compounds have been recently shown to present anti-human immunodeficiency virus, type 1 (HIV-1) activity. Among them, the dietary furanocoumarin imperatorin is present in citrus fruits, in culinary herbs, and in some medicinal plants. In this study we report that imperatorin inhibits either vesicular stomatitis virus-pseudotyped or gp160-enveloped recombinant HIV-1 infection in several T cell lines and in HeLa cells. These recombinant viruses express luciferase as a marker of viral replication. Imperatorin did not inhibit the reverse transcription nor the integration steps in the viral cell cycle. Using several 5' long terminal repeat-HIV-1 constructs where critical response elements were either deleted or mutated, we found that the transcription factor Sp1 is critical for the inhibitory activity of imperatorin induced by both phorbol 12-myristate 13-acetate and HIV-1 Tat. Moreover in transient transfections imperatorin specifically inhibited phorbol 12-myristate 13-acetate-induced transcriptional activity of the Gal4-Sp1 fusion protein. Since Sp1 is also implicated in cell cycle progression we further studied the effect of imperatorin on cyclin D1 gene transcription and protein expression and in HeLa cell cycle progression. We found that imperatorin strongly inhibited cyclin D1 expression and arrested the cells at the G(1) phase of the cell cycle. These results highlight the potential of Sp1 transcription factor as a target for natural anti-HIV-1 compounds such as furanocoumarins that might have a potential therapeutic role in the management of AIDS.
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PMID:Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway. 1521 31

The expression of human Parvovirus B19 nonstructural protein 1 (NS1) induces cell cycle arrest at the G1 phase and is accompanied by increased expression of the cyclin-dependent kinase inhibitor, p21/WAF1. Here, we provide direct evidence that NS1 mediates the transactivation of p21/WAF1. Up-regulation of p21/WAF1 by wild-type NS1 but not an NS1 mutant deleted of its NTP binding motif was observed. We also demonstrated that the wild-type NS1 is unable to induce G1 arrest in p21-deficient cells. Using reporter plasmids containing various mutants of the p21/WAF1 promoter, luciferase assay further revealed that the binding sites of the promoter to the transcription factor Sp1 are critical for NS1-mediated transactivation. Indeed Sp1 interacts only with the wild-type NS1 but not the NS1 mutant. These results indicate a cooperative contribution of NS1 and Sp1 to the transactivation of p21/WAF1, which leads to G1 arrest.
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PMID:Human Parvovirus B19 nonstructural protein transactivates the p21/WAF1 through Sp1. 1551 26

Early T lymphocyte activator 1 (Eta-1), also known as Osteopontin, is a cytokine produced by macrophages and T lymphocytes. It is involved in the regulation of IL-12 and IL-10 expression in macrophages and stimulates the polarization of T cells to the Th1 subset. Three promoter polymorphisms of the human Eta-1 gene, -443T/C, -156delG/G, -66T/G, were investigated for possible influence on gene expression. Electrophoretic mobility shift assays (EMSA) with nuclear extract from the human myeloid leukaemia premonocyte cell line, THP-1, revealed sequence specific binding of the transcription factor Sp1 to the -66T allele but not the -66G allele, and haplotype -443C/-156G/-66T showed a marked increase in promoter activity of a luciferase reporter gene. Thus, a substitution of the T-base with G at position -66 in the Eta-1 promoter modulates the promoter activity of the Eta-1 gene, which might influence the Th1 versus Th2 balance. These observations are discussed in relation to a recently reported related observation on the same gene, and it is argued that discrepancies between reporter gene assays in the two studies may be due to the use of different cell lines and may reflect requirements for different transcription factors in cells involved in immune responses compared with other cells.
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PMID:A functional polymorphism in the Eta-1 promoter is associated with allele specific binding to the transcription factor Sp1 and elevated gene expression. 1600 26

Previous studies have demonstrated that the mechanical compressive loading affects the biosynthesis of chondrocytes seeded in three dimensional scaffolds. In this study, the level of type II collagen mRNA expression was increased by a continuous dynamic compression at 10% compressive strain and 0.1 Hz in chondrocytes seeded in a biodegradable, elastomeric scaffold, poly(L-lactide-co-epsilon-caprolactone) (PLCL). To further examine this molecular mechanism, the promoter region of COL2A1 gene, which is encoding type II collagen, was analyzed using rabbit chondrocytes transfected with luciferase reporter vectors containing the 5'-flanking regions of human COL2A1 gene. A deletion mutant analysis revealed that the most active short promoter in response to continuous dynamic compression is in the region between -509 and -109 base pairs, where the transcription factor Sp1 is located. Additionally, an mRNA decay experiment using transcription inhibitor actinomycin D demonstrated that dynamic compression do not stabilize type II collagen mRNA. Our results indicate that mechanical compression increases the level of type II mRNA expression by transcriptional activation possibly through the Sp1 binding sites residing in the proximal region of the COL2A1 gene promoter.
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PMID:Mechanical compressive loading stimulates the activity of proximal region of human COL2A1 gene promoter in transfected chondrocytes. 1665 Mar 79

Multidrug resistance-associated protein MRP3/Mrp3 (ABCC3) is upregulated in cholestasis, an adaptive response that may protect the liver from accumulation of toxic compounds, such as bile salts and bilirubin conjugates. However, the mechanism of this upregulation is poorly understood. We and others have previously reported that fetoprotein transcription factor/liver receptor homolog-1 is an activator of MRP3/Mrp3 expression. In searching for additional regulatory elements in the human MRP3 promoter, we have now identified nuclear receptor retinoic X receptor-alpha:retinoic acid receptor-alpha (RXRalpha:RARalpha) as a repressor of MRP3 activation by transcription factor Sp1. A luciferase reporter assay demonstrated that cotransfection of transcription factor Sp1 stimulates the MRP3 promoter activity and that additions of RXRalpha:RARalpha abrogated this activation in a dose-dependent manner. Site mutations and gel shift assays have identified a Sp1 binding GC box motif at -113 to -108 nts upstream from the MRP3 translation start site, where RXRalpha:RARalpha specifically reduced Sp1 binding to this site. Mutation of the GC box also reduced MRP3 promoter activity. The functional role of RXRalpha:RARalpha as a repressor of MRP3 expression was further confirmed by RARalpha small-interfering RNA knockdown in HepG2 cells, which upregulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXRalpha:RARalpha act in concert to regulate MRP3 expression. Since RXRalpha:RARalpha expression is diminished by cholestatic liver injury, loss of RXRalpha:RARalpha may lead to upregulation of MRP3/Mrp3 expression in these disorders.
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PMID:Nuclear receptors RXRalpha:RARalpha are repressors for human MRP3 expression. 1727 13

Ataxin 1 (Atxn1) is a protein of unknown function associated with spinocerebellar ataxia type 1 (SCA1), a neurodegenerative disease of late onset with variable degrees of cerebellar ataxia, ophthalmoplegia and neuropathy. SCA1 is caused by the toxic effects triggered by an expanded polyglutamine (polyQ) within Atxn1 resulting in neurodegeneration in the cerebellum, brain stem and spinocerebellar tracts. To gain insights into Atxn1 function, we have analysed the cerebellar gene expression profiles by microarray analysis in Atxn1-null mice, and identified alterations in expression of genes regulated by Sp1-dependent transcription, including the dopamine receptor D2 (Drd2), retinoic acid/thyroid hormone and Wnt-signalling. Interestingly, Drd2 expression levels are reduced in both Atxn1-null and transgenic mice expressing a pathogenic human Atxn1 with an expanded polyglutamine in cerebellar Purkinje cells. Our co-transfection experiments in human neuroblastoma SH-SY5Y cells and luciferase assays provide evidence for transcriptional regulation of Drd2 by Atxn1 and its AXH module. We show that Atxn1 occupies at the Drd2 promoter in vivo, and interacts and functions synergistically with the zinc-finger transcription factor Sp1 to co-regulate Drd2 expression. The interaction and transcriptional effects are mediated by the AXH domain within Atxn1 and are abrogated by the expanded polyQ within Atxn1. Therefore, this study identifies novel molecular targets that are regulated by Atxn1 which might contribute to the motor deficits in SCA1, and provides new insights into the mechanisms by which Atxn1 co-regulates transcription.
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PMID:Down-regulation of the dopamine receptor D2 in mice lacking ataxin 1. 1759 52

While studying differentially expressed genes between sensitive and 10(-5)M Methotrexate (MTX) resistant HT29 human colon cancer cells, we identified some members of the aldo-keto reductase (AKR) superfamily. The study was followed with the member AKR1C1 (EC 1.1.1.213), validating its increase in mRNA and protein levels in MTX resistant cells. The genomic content for AKR1C1 remained unchanged between sensitive and resistant cells, thereby excluding a mechanism of AKR1C1 gene amplification. Thus, we cloned the AKR1C1 human promoter and performed luciferase experiments that revealed a transcriptional regulation of the gene in the resistant cells. Computational studies showed a putative binding site for the transcription factor Sp1. The co-transfection of Sp1 or Sp3 with different constructs of AKR1C1 promoter deletions, including and excluding the proximal GC-box, demonstrated a key role for these factors in regulating AKR1C1 transcriptional activity. Gel-shift assays revealed an increase in Sp1 and Sp3 binding in resistant compared to sensitive cells, without differences in Sp1 protein levels. Dephosphorylation of the extracts coincided with a decrease in Sp1 binding, which is consistent with a process of regulation of Sp1 by phosphorylation. We also investigated the possible relationship between AKR1C1 expression and MTX action. Overexpression of AKR1C1 counteracted the S-phase accumulation of cells and apoptosis caused by MTX treatment. This suggests a role of AKR1C1 in cell proliferation. Finally, overexpression of AKR1C1 in MTX sensitive HT29 cells conferred resistance to the chemotherapeutic agent and silencing of AKR1C1 by means of iRNA technology sensitized the cells to MTX.
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PMID:Transcriptional regulation of aldo-keto reductase 1C1 in HT29 human colon cancer cells resistant to methotrexate: role in the cell cycle and apoptosis. 1794 94


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