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)

Type I collagen is expressed in a variety of connective tissue cells and its transcriptional regulation is highly complex because of the influence of numerous developmental, environmental, and hormonal factors. To investigate the molecular basis for one aspect of this complex regulation, the expression of alpha 1(I) collagen (COL1A1) gene in osseous tissues, we fused a 3.6-kb DNA fragment between bases -3,521 and +115 of the rat COL1A1 promoter, and three deletion mutants, to the chloramphenicol acetyltransferase (CAT) marker gene. The expression of these ColCAT transgenes was measured in stably transfected osteoblastic cell lines ROS 17/2.8, Py-la, and MC3T3-E1 and three fibroblastic lines NIH-3T3, Rat-1, and EL2. Deletion of the distal 1.2-kb fragment of the full-length ColCAT 3.6 construct reduced the promoter activity 7- to 30-fold in the osteoblastic cell lines, twofold in EL2 and had no effect in NIH-3T3 and Rat-1 cells. To begin to assess the function of COL1A1 upstream regulatory elements in intact animals, we established transgenic mouse lines and examined the activity of the ColCAT3.6 construct in various tissues of newborn animals. The expression of this construct followed the expected distribution between the high and low collagen-producing tissues: high levels of CAT activity in calvarial bone, tooth, and tendon, a low level in skin, and no detectable activity in liver and brain. Furthermore, CAT activity in calvarial bone was three- to fourfold higher than that in the adjacent periosteal layer. Immunostaining for CAT protein in calvaria and developing tooth germ of ColCAT3.6 mice also confirmed the preferred expression of the transgene in differentiated osteoblasts and odontoblasts compared to fibroblast-like cells of periosteum and dental papilla. This study suggests that the 3.6-kb DNA fragment confers the strong expression of COL1A1 gene in high collagen producing tissues of intact animals and that the 5' flanking promoter sequence between -3,521 and -2,295 bp contains one or more stimulatory elements which are preferentially active in osteoblastic cells.
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PMID:Differential utilization of regulatory domains within the alpha 1(I) collagen promoter in osseous and fibroblastic cells. 173 Jul 46

We have located a cis-acting element (alpha 1-TAE) within the promoter sequences of the rat collagen alpha 1(I) gene (COL1A1) 1600 bases upstream of the transcription start site which mediates transcriptional activation by transforming growth factor beta (TGF-beta). The functional significance of this region was established by (1) deletion analysis of the alpha 1(I) promoter cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene and (2) by co-transfection of promoter constructs with double-stranded oligonucleotides. DNA-mobility-shift assays with radiolabelled alpha 1-TAE demonstrated increased nuclear binding activity after TGF-beta stimulation. Oligonucleotides encoding the alpha 1-TAE, additional upstream regions within the alpha 1(I) promoter, as well as consensus nuclear-factor-1 (NF-1) sequences, competed with the alpha 1-TAE sequence. The two collagen type I genes are stimulated by TGF-beta through different regions of their promoters.
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PMID:Transforming-growth-factor-beta activation elements in the distal promoter regions of the rat alpha 1 type I collagen gene. 174 43

The synthesis of type I collagen in bone cells is inhibited by the calcium-regulating hormone 1,25-dihydroxyvitamin D3. Earlier work from our laboratories has indicated that vitamin D regulation is at the level of transcription, based on results from both nuclear run-off assays and functional promoter analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloramphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D-mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the HindIII-Pstl restriction fragment between nucleotides -2295 and -1670) which is necessary for 1,25-dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone-mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides -2256 and -2216) contains a sequence which is very similar to vitamin D-responsive elements identified in the osteocalcin gene. Extracts from cultured cells which express a high level of vitamin D receptor contain a hormone:receptor complex that binds specifically to this 41 bp fragment, as demonstrated by bandshift analysis. However, deletion of this vitamin D receptor binding region from either a -3.5 kb or a -2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other vitamin D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25-dihydroxyvitamin D3.
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PMID:Analysis of regulatory regions in the COL1A1 gene responsible for 1,25-dihydroxyvitamin D3-mediated transcriptional repression in osteoblastic cells. 789 Aug 7

The activity of fusion genes containing fragments of the COL1A1 promoter was measured in tissues from 6- to 8-day-old transgenic mice. ColCAT3.6 contains approximately 3.6 kb (-3521 to 115 bp) of the rat COL1A1 gene, the chloramphenicol acetyltransferase (CAT) reporter gene, and the SV40 splice and polyadenylation sequences. ColCAT2.3 and ColCAT1.7 are deletion constructs that contain 2296 and 1667 bp of COL1A1 upstream from the RNA start site, respectively. For each transgene, up to six lines of mice were characterized. Both ColCAT3.6 and ColCAT2.3 had similar activity in bone and tooth; ColCAT1.7 was inactive. In transgenic calvariae, levels of transgene mRNA paralleled levels of CAT activity. In tendon, the activity of ColCAT2.3 was 3- to 4-fold lower than that of ColCAT3.6, and the activity ColCAT1.7 was 16-fold lower than that of ColCAT2.3. There was little activity of the ColCAT constructs in liver and brain. These data show that DNA sequences between -2.3 and -1.7 kb are required for COL1A1 promoter expression in bone and tooth; sequences that control expression in tendon are distributed between -3.5 and -1.7 kb of the promoter, with sequences downstream of -1.7 kb still capable of directing expression to this tissue. The cis elements that govern basal expression of COL1A1 in transgenic calvariae appear to be different from those required for optimal expression of the COL1A1 promoter in stably transfected osteoblastic cells.
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PMID:Upstream regulatory elements necessary for expression of the rat COL1A1 promoter in transgenic mice. 802 54

We have previously reported that the expression of the ColCAT3.6 transgene containing 3.5 kilobases (kb) of alpha 1(I) collagen (COL1A1) promoter sequence fused to the chloramphenicol acetyltransferase (CAT) reporter gene paralleled the expression of the endogenous gene in several connective tissues. We report here that the activity of the reporter gene in aorta from 7-day-old transgenic mice is 10-64-fold lower than in tendon or bone, whereas the endogenous gene is highly expressed in all three tissues. In contrast, the COL1A1 minigene containing 2.3 kb of upstream sequence, the first five exon/intron units, the last six exon/intron units, and 2 kb of 3'-flanking sequence showed high CAT activity in aorta. These results suggest that cis sequences found in ColCAT3.6 mediate high levels of COL1A1 expression in bone and tendon, but not in vascular smooth muscle cells (VSMC), whereas sequences located within the minigene, but not found in ColCAT3.6, mediate VSMC-specific expression. Analysis of promoter activity in cultured cells derived from transgenic tissues further suggests the presence of VSMC-specific regulatory domains. Transient transfection studies, however, failed to shows differential regulation. These differences stress the importance of not relying exclusively on transient transfection data when mapping tissue-specific regulatory domains.
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PMID:Regulation of the alpha 1(I) collagen promoter in vascular smooth muscle cells. Comparison with other alpha 1(I) collagen-producing cells in transgenic animals and cultured cells. 810 63

To directly compare the patterns of collagen promoter expression in cells and tissues, the activity of COL1A1 fusion genes in calvariae of neonatal transgenic mice and in primary bone cell cultures derived by sequential digestion of transgenic calvariae was measured. ColCAT3.6 contains 3.6 kb (positions -3521 to +115) of the rat COL1A1 gene ligated to the chloramphenicol acetyltransferase (CAT) reporter gene. ColCAT2.3 and ColCAT1.7 are 5' deletion mutants which contain 2,296 and 1,672 bp, respectively, of COL1A1 DNA upstream from the transcription start site. ColCAT3.6 activity was 4- to 6-fold lower in primary bone cell cultures than in intact calvariae, while ColCAT2.3 activity was at least 100-fold lower in primary bone cells than in calvariae. These changes were accompanied by a threefold decrease in collagen synthesis and COL1A1 mRNA levels in primary bone cells compared with collagen synthesis and COL1A1 mRNA levels in freshly isolated calvariae. ColCAT3.6 and ColCAT2.3 activity was maintained in calvariae cultured in the presence or absence of serum for 4 to 7 days. Thus, when bone cells are removed from their normal microenvironment, there is parallel downregulation of collagen synthesis, collagen mRNA levels, and ColCAT3.6 activity, with a much greater decrease in ColCAT2.3. These data suggest that a 624-bp region of the COL1A1 promoter between positions -2296 and -1672 is active in intact and cultured bone but inactive in cultured cells derived from the bone. We suggest that the downregulation of COL1A1 activity in primary bone cells may be due to the loss of cell shape or to alterations in cell-cell and/or cell-matrix interactions that normally occur in intact bone.
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PMID:Transgenic expression of COL1A1-chloramphenicol acetyltransferase fusion genes in bone: differential utilization of promoter elements in vivo and in cultured cells. 835 76

Our previous studies have shown that the 49-base pair region of promoter DNA between -1719 and -1670 base pairs is necessary for transcription of the rat COL1A1 gene in transgenic mouse calvariae. In this study, we further define this element to the 13-base pair region between -1683 and -1670. This element contains a TAAT motif that binds homeodomain-containing proteins. Site-directed mutagenesis of this element in the context of a COL1A1-chloramphenicol acetyltransferase construct extending to -3518 base pairs decreased the ratio of reporter gene activity in calvariae to tendon from 3:1 to 1:1, suggesting a preferential effect on activity in calvariae. Moreover, chloramphenicol acetyltransferase-specific immunofluorescence microscopy of transgenic calvariae showed that the mutation preferentially reduced levels of chloramphenicol acetyltransferase protein in differentiated osteoblasts. Gel mobility shift assays demonstrate that differentiated osteoblasts contain a nuclear factor that binds to this site. This binding activity is not present in undifferentiated osteoblasts. We show that Msx2, a homeodomain protein, binds to this motif; however, Northern blot analysis revealed that Msx2 mRNA is present in undifferentiated bone cells but not in fully differentiated osteoblasts. In addition, cotransfection studies in ROS 17/2.8 osteosarcoma cells using an Msx2 expression vector showed that Msx2 inhibits a COL1A1 promoter-chloramphenicol acetyltransferase construct. Our results suggest that high COL1A1 expression in bone is mediated by a protein that is induced during osteoblast differentiation. This protein may contain a homeodomain; however, it is distinct from homeodomain proteins reported previously to be present in bone.
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PMID:Identification of a TAAT-containing motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice. 866 11

Recent studies have demonstrated that tumour necrosis factor-alpha (TNF-alpha) decreases alpha 1(I) procollagen gene (COL1A1) expression in cultured human dermal fibroblasts. The purpose of this study was to analyse the transcriptional control of COL1A1 by TNF-alpha. Cultured human dermal fibroblasts were transiently transfected with plasmids containing 5' flanking sequences of COL1A1 fused to the chloramphenicol acetyltransferase (CAT) gene, and were incubated for 48 h in medium with or without TNF-alpha. TNF-alpha inhibited the CAT activity of fibroblasts transfected with plasmids containing 2.3 kb of 5' flanking sequences of COL1A1, whereas the activity of control plasmids containing the herpes simplex thymidine kinase promoter gene (pBLCAT) was unaltered. A series of deletion constructs of various small substitution mutations of the COL1A1 5' flanking region fused to the CAT gene were also transfected, and CAT activity was measured after incubation with TNF-alpha. TNF-alpha suppressed COL1A1 promoter activity through proximal short promoter elements containing only 107 bp. Short substitution mutations between -101 and -97 bp or between -46 and -38 bp abolished TNF-alpha suppression of COL1A1 promoter activity. DNA-protein complex formation was observed involving both sites in gel retardation assays. These results suggest that TNF-alpha suppressed COL1A1 promoter activity through elements located between -101 and -97 bp and between -46 and -38 bp of the COL1A1 promoter, and that the suppression involved DNA-protein interactions.
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PMID:The transcription of human alpha 1(I) procollagen gene (COL1A1) is suppressed by tumour necrosis factor-alpha through proximal short promoter elements: evidence for suppression mechanisms mediated by two nuclear-factorbinding sites. 892 Sep 84

Interferon-gamma (IFN-gamma), a multifunctional cytokine produced by activated Th1 lymphocytes, exerts potent effects on the extracellular matrix by regulating fibroblast function. In this study, we examined the modulation of alpha1(I) procollagen gene (COL1A1) expression by recombinant IFN-gamma. The results showed that IFN-gamma stimulated the rapid accumulation of interferon regulated factor (IRF)-1 mRNA, followed by a delayed and dose-dependent inhibition of alpha1(I) procollagen mRNA expression in skin fibroblasts from several different donors. The inhibitory response was abrogated in fibroblasts stably expressing IRF-1 in the antisense orientation. A marked decrease in the amount of heterogeneous nuclear pre-mRNA preceded the inhibition of COL1A1 mRNA expression. In fibroblasts transiently transfected with COL1A1 promoter-chloramphenicol acetyltransferase reporter gene plasmids, IFN-gamma selectively inhibited promoter activity and abrogated its stimulation induced by TGF-beta. The inhibition by IFN-gamma was not due to downregulation of TGF-beta receptor mRNA expression in the fibroblasts or decreased ligand binding to the receptor. IFN-alpha and IFN-beta by themselves had little effect on promoter activity, but IFN-alpha augmented the inhibitory effect of IFN-gamma. Using a series of 5' deletion constructs, a proximal region of the COL1A1 promoter was shown to function as an IFN-gamma response element. This region of the gene harbors overlapping binding sites for transcription factors Sp1, Sp3, and NF-1 but no homologs of previously characterized IFN-gamma response elements. The putative IFN-gamma response region was sufficient to confer inhibition of reporter gene expression by treatment with IFN-gamma. Gel mobility shift analysis showed that two distinct and specific DNA-protein complexes were formed when fibroblast nuclear extracts were incubated with oligonucleotides spanning the IFN-gamma response region. IFN-gamma did not modify the ability of nuclear proteins to bind to this region. The results indicate that IFN-gamma inhibits COL1A1 expression in fibroblasts principally at the level of gene transcription. Inhibition involves IRF-1 and is mediated through a short proximal promoter segment but without an apparent change in promoter occupancy. The findings provide novel insight into the mechanism of IFN-gamma regulation of fibroblast function.
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PMID:Negative modulation of alpha1(I) procollagen gene expression in human skin fibroblasts: transcriptional inhibition by interferon-gamma. 1008 37

Hepatic stellate cells (HSC) differ in their phenotype depending on the initiation and progression of their activation. Our hypothesis was that different mechanisms govern type I collagen synthesis depending on stage of HSC activation. We investigated the role of alpha(5)beta(1)-integrin as a regulator of type I collagen gene COL1A1 expression in primary and passaged HSC cultures using transgenic mouse containing type I collagen gene COL1A1 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The alpha(5)beta(1) protein levels increased during the activation and were highest in day 6 primary cultures but decreased in passaged HSC. CAT activity, reflecting COL1A1 expression, was upregulated by alpha(5)beta(1)-integrin. Inhibition of alpha(5)beta(1)-integrin by echistatin and blocking antibody resulted in reduced transgene activity only in early primary cultures (compared with the control, 53.3 +/- 12% echistatin and 58.8 +/- 7% blocking antibody, respectively, P < 0.05). Treatment of passaged HSC with either echistatin or blocking antibody had no effect. Fibronectin, an alpha(5)beta(1)-integrin ligand, increased transgene activity in primary (210 +/- 33%, P < 0.05) but not in passaged HSC cultures (119 +/- 8%). This alpha(5)beta(1)-integrin effect appears to be at least in part mediated by CCAAT enhancer binding protein-beta (C/EBPbeta), because fibronectin increased and alpha(5)-gene silencing by small interfering RNA decreased C/EBPbeta levels. In addition, C/EBPbeta knockout mice showed reduced type I collagen synthesis compared with wild-type littermates. Therefore alpha(5)beta(1)-integrin is an important regulator of type I collagen production in early primary HSC cultures but appears to have no direct role once the HSC are fully activated.
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PMID:Differences in regulation of type I collagen synthesis in primary and passaged hepatic stellate cell cultures: the role of alpha5beta1-integrin. 1751 Jan 95


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