Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The promoter regions of the three mammalian transforming growth factor-beta genes (TGF-beta s 1, 2, and 3) have been recently cloned and characterized. The sequences show little similarity, suggesting different mechanisms of transcriptional control of these genes. To study differences in transcriptional regulation of mammalian and avian TGF-beta, we have cloned and sequenced the 5'-flanking region of chicken TGF-beta 3. Characterization of this region showed a TATA box and cAMP-responsive element (CRE) and AP-2 binding site consensus sequences starting at 12 and 28 base pairs, respectively, upstream from the TATA box. Moreover, four additional AP-2-like sites, 10 binding sites for the transcription factor Sp1, as well as two AP-1-like sites were also identified. Except for 32 base pairs of identity centered around the TATA box and CRE site and four other relatively small regions of identity, the chicken TGF-beta 3 promoter was found to be structurally very different from the human TGF-beta 3 promoter. Promoter fragments were cloned into a chloramphenicol acetyltransferase reporter plasmid to study functional activity. Basal transcriptional activity of the promoter was regulated in quail fibrosarcoma QM7 cells and in human adenocarcinoma A375 cells by multiple upstream elements including the TATA, CRE, and AP-2 sites. As in the human TGF-beta 3 promoter, the CRE site showed activation by forskolin, an effect which could be shown by expression of TGF-beta 3 mRNA in cultured chicken and quail cells as well. Our results indicate a complex pattern of transcriptional regulation of the chicken TGF-beta 3 gene and suggest that differences in the regulation of expression of the genes for mammalian and avian TGF-beta 3 may result in part from the unique structure of their 5'-flanking regions.
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PMID:Identification and characterization of the chicken transforming growth factor-beta 3 promoter. 140 6

Using human heart fibroblasts (HHF), we studied the effect of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) on the gene expression of type I collagen, collagenase and tissue inhibitor of metalloproteinases (TIMP). Initially, treatment of HHF with bFGF alone (10 ng/ml) resulted in elevated secretion of collagenase into the culture medium. Subsequent treatment of HHF with TGF-beta in combination with bFGF suppressed collagenase secretion. Northern blot analysis reinforced this observation by revealing an enhancement of the steady-state mRNA level of collagenase in response to bFGF. In order to examine if the collagenase gene was affected by bFGF at the transcriptional level, transfection experiments were carried out with a plasmid containing collagenase promoter linked to chloramphenicol acetyltransferase gene (CAT). Basic FGF stimulated CAT activity by four-fold, indicating increased promoter activity whereas the combination of TGF-beta and bFGF resulted in decreased CAT activity. TGF-beta was shown to increase type I collagen and TIMP mRNA levels by 2.5- and 2.1-fold, respectively. These results suggest that TGF-beta and bFGF may play a pivotal role in regulating collagen metabolism in HHF.
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PMID:Effect of growth factors on collagen metabolism in cultured human heart fibroblasts. 166 Aug 2

Decorin, a leucine-rich proteoglycan with ubiquitous tissue distribution, may play essential biological roles during inflammation and cancer growth through its ability to bind extracellular matrix constituents and growth factors. In this study, we demonstrate that decorin gene expression is greatly enhanced after normal diploid fibroblasts reach confluency and cease to proliferate. Elevation of decorin mRNA steady state levels was maintained for up to 16 days postconfluency. In vitro transcription analyses indicated enhanced transcriptional activity in quiescent fibroblasts when compared to cells harvested in their logarithmic phase of growth. This phenotypic trait was reversed by the exogenous addition of tumor necrosis factor-alpha (TNF-alpha). Furthermore, transforming growth factor-beta (TGF-beta) down-regulated decorin gene expression in an additive manner with TNF-alpha. Transient cell transfection assays using plasmid constructs harboring the decorin promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated a dose-dependent transcriptional repression by TNF-alpha. These findings were further corroborated by in vitro transcription experiments using nuclear extracts from control and TNF-alpha-treated quiescent fibroblasts. In contrast, the decorin promoter constructs failed to respond to TGF-beta, thus suggesting either post-transcriptional regulation by this growth factor or lack of TGF-beta-responsive elements. Further experiments with 5' deletion constructs showed two TNF-alpha response elements, one residing within the 5'-untranslated region (exon Ib), the other one between residues -188 and -140 of the decorin promoter. Collectively, our results indicate that TNF-alpha, through its ability to transcriptionally inhibit decorin gene expression in growth-arrested cells, may be a key modulator of the biological functions of this proteoglycan.
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PMID:Transcriptional regulation of decorin gene expression. Induction by quiescence and repression by tumor necrosis factor-alpha. 774 9

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.
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PMID:Structural and functional characterization of the human decorin gene promoter. A homopurine-homopyrimidine S1 nuclease-sensitive region is involved in transcriptional control. 827 54

Previously, we demonstrated that the progestin components (19-nortestosterone derivatives) in oral contraceptives are able to stimulate human breast cancer cell proliferation via an estrogen receptor (ER)-mediated mechanism. We now examine RU486, an antiprogestin, to determine whether it has estrogenic properties because it is also a 19-nortestosterone derivative. We found that RU486 stimulated the growth of MCF-7 human breast cancer cells at a concentration of 10(-6) M, which is similar to the pharmacological concentration (micromolar range) found in women taking RU486. The antiestrogens 4-hydroxytamoxifen and ICI 164,384 blocked RU486-induced cell proliferation. The estrogenic activity of RU486 is not due to impurities or aromatization to estrogenic metabolites. To determine whether the proliferative action of RU486 was mediated through the ER, cells were transfected with a chloramphenicol acetyltransferase reporter gene under the control of an estrogen response element derived from the Xenopus laevis vitellogenin 2A gene. We found that RU486 was able to induce chloramphenicol acetyltransferase activity at the concentrations that stimulated cell proliferation, and this induction was blocked by the addition of 4-hydroxytamoxifen and ICI 164,384. The estrogenic potential of RU486 to regulate ER target gene expression was also investigated. We found that, like 17 beta-estradiol (E2), RU486 was able to alter the expression and synthesis of progesterone receptor. The level of progesterone receptor (145 and 186 fmol/mg cytosol protein, respectively) was increased significantly compared to the control value (3 fmol/mg cytosol protein) with the addition of 10(-6) M RU486 or 10(-10) M E2, as determined by an enzyme immunoassay. The levels of transforming growth factor-beta 2 (TGF beta 2) and TGF beta 3 mRNA, but not TGF beta 1 mRNA, were decreased dramatically with the addition of 10(-6) M RU486. This is consistent with the effects of E2 on TGF beta expression. Therefore, RU486 has estrogen-like activities in its regulation of ER target gene expression. These results demonstrate that RU486 is a weak estrogen in human breast cancer cells and suggest that the RU486-induced cell proliferation is mediated via ER. The novel finding that RU486 exhibits some estrogen-like activity may be important for the interpretation of its action at high dosages as an abortifacient and also if RU486 is going to be evaluated clinically, again at high doses, for the treatment of breast cancer.
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PMID:Estrogenic actions of RU486 in hormone-responsive MCF-7 human breast cancer cells. 850 63

A transforming growth factor-beta (TGF-beta) activating element (TAE), with a nuclear factor-1 (NF-1)-like sequence, was previously located 1.6 kilobases upstream from the transcription start site in the alpha 1(I) collagen promoter (Ritzenthaler, J. D., Goldstein, R. H., Fine, A., Lichtler, A., Rowe, D. W., and Smith, B. D. (1991) Biochem. J. 280, 157-162). Double-stranded TAE, but not NF-1 consensus sequences, abrogated TGF-beta stimulation of co-transfected collagen promoter-chloramphenicol acetyltransferase constructs. Mutations in non-NF-1 binding sites, located by methylation interference, eliminated activity of the TAE oligonucleotide. However, TAE sequences failed to bind in vitro expressed NF-1 protein, to compete for NF-1-binding proteins, and to bind with protein which reacts with antibodies to NF-1 family of proteins. Within the TAE there was an activator protein 2 (AP-2) binding site. Although AP-2 protein bound to TAE, antibodies to AP-2 did not react with nuclear protein-TAE complexes. TAE bound to a 34,000-Da protein on Southwestern analysis. However, the UV-cross-linked TAE-nuclear protein complex was 82,000 Da. Finally, a dose-response study demonstrated that TGF-beta increased TAE nuclear binding proteins at lower doses with a different response curve than NF-1 nuclear binding proteins. Taken together these data demonstrated that TGF-beta functions in human lung fibroblasts to activate collagen transcription through TAE sites by protein complexes independent of NF-1 or AP-2 protein.
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PMID:Regulation of the alpha 1(I) collagen promoter via a transforming growth factor-beta activation element. 851 94

Glucocorticoids have previously have shown to decrease Type I collagen synthesis in vivo and in fibroblast cell culture. Several studies have demonstrated that glucocorticoids decrease Type I procollagen gene expression. These latter studies have included uridine incorporation into pro alpha 1 (I) and pro alpha 2 (I) mRNAs and nuclear run-off experiments. Using the ColCat 3.6 plasmid, which contains part of the 5' flanking region of the pro alpha 1 (I) collagen gene and the reporter gene, chloramphenicol acetyltransferase, the present studies demonstrate by stable transfection of fetal rat skin fibroblasts that dexamethasone down regulates the promoter activity of the pro alpha 1 (I) collagen gene. The glucocorticoid-mediated down-regulation of procollagen gene expression was demonstrated using the ColCat 3.6, 2.4, 1.7, or 0.9 plasmid. In addition, competitive oligonucleotide transfection experiments and site specific mutation of the glucocorticoid response element (GRE) in the whole ColCat 3.6 plasmid did not eliminate the effect. The possibility existed that another cis-element in the 5' flanking region of the pro alpha 1 (I) collagen gene was also required for the collagen glucocorticoid-mediated down-regulation of procollagen gene expression, since TGF-beta has been shown to stimulate in a decrease of transforming growth factor-beta (TGF-beta) secretion into the media. Gel mobility studies demonstrated that glucocorticoid treatment of rat skin fibroblasts decreased glucocorticoid receptor binding to the GRE and TGF-beta activator protein to the TGF-beta element which were brought back to control values by coordinate exogenous TGF-beta treatment. Thus the interaction of these TGF-beta molecules with cellular membrane receptors and subsequent transduction is dramatically decreased resulting in less signals to regulate collagen gene expression. These data indicate that glucocorticoids coordinately regulate procollagen gene expression through both the GRE and TGF-beta elements. Depression of procollagen gene expression by glucocorticoids through the TGF-beta element is mediated by decreased TGF-beta secretion, possibly involving a secondary effect on regulatory protein(s) encoded by noncollagenous protein gene(s). The present studies provide the basis for a novel mechanism of glucocorticoid-mediator regulation of eukaryotic genes containing the TGF-beta element.
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PMID:Glucocorticoids coordinately regulate type I collagen pro alpha 1 promoter activity through both the glucocorticoid and transforming growth factor beta response elements: a novel mechanism of glucocorticoid regulation of eukaryotic genes. 856 55

Previous studies have shown that transforming growth factor-beta (TGF-be ta) and tumor necrosis factor-alpha (TNF-alpha) modulate type I collagen gene expression in fibroblasts. To fine-map the corresponding response elements in the human alpha2(I) collagen (COL1A2) promoter, we have generated a series of 5' deletion promoter/chloramphenicol acetyltransferase (CAT) reporter gene constructs. Transient cell transfection assays using human dermal fibroblasts and stable transfection experiments using NIH 3T3 fibroblasts identified the region located between residues -265 and -241, as critical for TGF-beta response. Specifically, we demonstrate that this 25-base pair region mediates the up-regulatory effect of TGF-beta on COL1A2 promoter activity and allows antagonistic activity of TNF-alpha on the TGF-beta effect. Gel mobility shift assays indicate that nuclear factor binding to this 25-base pair region of COL1A2 promoter is competed by AP-1, but not NF-1 or NF-kappaB, oligonucleotides. Transient cell transfection experiments with plasmid constructs in which the potential AP-1-binding site located within this short region of promoter was modified by site-directed mutagenesis indicated that this element plays a significant role in the basal activity of the promoter. Furthermore, this sequence is essential for TGF-beta response and does not require the presence of the three Sp-1-binding sites located further upstream, between nucleotides -273 and -304. In addition, overexpression of c-jun in co-transfection experiments with COL1A2 promoter/CAT constructs blocks the TGF- beta response, further implicating AP-1 in the regulation of COL1A2 gene expression. Our results clarify the molecular mechanisms involved in the regulation of type I collagen gene expression and further emphasize the importance of AP-1 in mediating some of the TGF-beta effects on gene transcription.
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PMID:An AP-1 binding sequence is essential for regulation of the human alpha2(I) collagen (COL1A2) promoter activity by transforming growth factor-beta. 862 30

Type I collagen is the most abundant component of the extracellular matrix of human connective tissues. We have examined the effect of okadaic acid (OA), an inhibitor of phosphoserine- and-phosphothreonine-specific protein phosphatases 1 and 2A, on type I collagen gene expression by fibroblasts in culture. Treatment of human skin fibroblasts with OA potently reduced type I and type III collagen mRNA levels, maximally by over 90%. The inhibitory effect of OA on type I and III collagen mRNA abundance was not prevented by cycloheximide, and was not affected by simultaneous treatment with dexamethasone or retinoic acid. OA also abrogated the enhancing effect of transforming growth factor-beta (TGF-beta) on type I and III collagen mRNA levels. Treatment of transiently transfected NIH-3T3 fibroblasts with OA suppressed the activity of a 3.5 kb human pro alpha 2(I) collagen promoter/chloramphenicol acetyltransferase construct maximally, by 70%. In addition, OA treatment of NIH-3T3 cells abrogated enhancement of pro alpha 2(I) collagen promoter activity by TGF-beta. These results indicate that protein phosphatases 1 and 2A have an important role as positive regulators of type I and III collagen gene expression. The results also suggest that selective inhibition of activity of protein phosphatases 1 and 2A may offer a novel approach for preventing excessive collagen accumulation in fibrotic disorders.
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PMID:The protein phosphatase inhibitor okadaic acid suppresses type I collagen gene expression in cultured fibroblasts at the transcriptional level. 894 61

Perlecan, a modular heparan sulfate proteoglycan of basement membranes and cell surfaces, plays a crucial role in regulating the assembly of extracellular matrices and the binding of nutrients and growth factors to target cells. To achieve a molecular understanding of perlecan gene regulation, we isolated the 5'-flanking region and investigated its functional promoter activity and its response to cytokines. Transient cell transfection assays, using plasmid constructs harboring the perlecan promoter linked to the chloramphenicol acetyltransferase reporter gene, demonstrated that the largest approximately 2.5-kilobase construct contained maximal promoter activity. This promoter region was functionally active in a variety of cells of diverse histogenetic origin, thus corroborating the widespread expression of this gene product. Stepwise 5' deletion analyses demonstrated that the -461-base pair (bp) proximal promoter retained approximately 90% of the total activity, and internal deletions confirmed that the most proximal sequence was essential for proper promoter activity. Nanomolar amounts of transforming growth factor-beta induced 2-3-fold perlecan mRNA and protein core levels in normal human skin fibroblasts, and this induction was transcriptionally regulated; in contrast, tumor necrosis factor-alpha had no effect and was incapable of counteracting the effects of TGF-beta. Using additional 5' deletions and DNase footprinting analyses, we mapped the TGF-beta responsive region to a sequence of 177 bp contained between -461 and -285. This region harbored a 14-bp element similar to a TGF-beta-responsive element present in the promoters of collagen alpha1(I), alpha2(I), elastin, and growth hormone. Electrophoretic mobility shift assays and mutational analyses demonstrated that the perlecan TGF-beta-responsive element bound specifically to TGF-beta-inducible nuclear proteins with high affinity for NF-1 member(s) of transcription factors.
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PMID:Structural and functional characterization of the human perlecan gene promoter. Transcriptional activation by transforming growth factor-beta via a nuclear factor 1-binding element. 903 May 92


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