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)

Topical tretinoin therapy produces clinical improvements in the fine wrinkling of photodamaged skin, possibly by enhancement of collagen synthesis. A major biochemically and histologically detectable change in photodamaged skin is the accumulation of abnormal elastic fibers (elastotic material). However, little is known about the effects of retinoic acid and ultraviolet B (UVB) on elastin gene expression. Consequently, we examined the effects of all-trans-retinoic acid (t-RA) and UVB on elastin gene expression in cultured human skin fibroblasts in vitro. Elastin mRNA gene expression was up-regulated in response to UVB by approximately equal to 3-fold, in a dose dependent manner, between 3 and 10 mJ/cm2 doses. Similar results were obtained by chloramphenicol acetyltransferase assay, in which a maximal promoter activation more than 5.4-fold that in nonirradiated controls occurred after a single dose of 20 mJ/cm2. Also t-RA inhibited the increase in elastin mRNA level following a single exposure to UVB by approximately 16%, and the increase in promotor activity by about 65%. The inhibitory effect of t-RA on elastin induced by UVB was also demonstrated by indirect immunofluorescence studies. Taken together, t-RA down-regulated human elastin gene expression elevated by a single exposure to UVB at transcriptional and possibly protein levels. These results suggest that the anti-photoaging effect of t-RA may be related, at least in part, to down-regulation of elastin gene expression elevated by UVB.
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PMID:All-trans-retinoic acid down-regulates elastin promoter activity elevated by ultraviolet B irradiation in cultured skin fibroblasts. 969 46

Hepatic stellate cells (HSCs) become activated into myofibroblast-like cells during the early stages of hepatic injury associated with fibrogenesis. The subsequent dysregulation of alphaI(I) collagen gene expression is a central pathogenetic step during the development of cirrhosis. Our recent study in rat HSCs (Davis, B. H., Chen, A., and Beno, D. (1996) J. Biol. Chem. 271, 11039-11042) found that ERK1,2 activation might be required for maximal alphaI(I) collagen gene expression. However, the role of the parallel JNK cascade in regulating alphaI(I) collagen gene expression was unknown. In this study, we initially found that UV irradiation of HSCs activated JNK but not ERK1,2. Furthermore, UV irradiation increased endogenous alpha I(I) collagen mRNA abundance and stimulated alpha I(I) collagen gene transcription in HSCs. The effect of the activation of JNK and Jun on alpha I(I) collagen gene expression was further evaluated via transfection of chloramphenicol acetyltransferase reporter plasmids with various sizes of truncated 5' upstream promoter sequence (UPS) of the alphaI(I) collagen gene. This revealed that dominant negative transcription factor JUN suppressed alpha I(I) collagen gene transcription in HSCs maintained in media with 20% serum and constitutively activated JUN increased alphaI(I) collagen gene transcription in HSCs cultured in media with 0.4% serum. UV activated JNK utilized a distal GC box in the 5'-UPS of the collagen gene to regulate gene transcription. This observation was confirmed by site-directed mutagenesis. In co-transfection experiments, the col-chloramphenicol acetyltransferase reporter with a mutagenized GC box was not suppressed by dn-JUN and was not stimulated by activated JUN or by UV irradiation. Southwestern blotting analyses and gel shift assays with basic transcription element-binding protein antiserum suggested that the GC box was bound by basic transcription element-binding protein, a recently described DNA-binding protein. In conclusion, the current study combined with our previous report suggests that ERK1,2 and JNK cascades regulate alphaI(I) collagen expression in HSCs through different regions of the 5'-UPS of the gene. The distal GC box in the 5'-UPS of the alphaI(I) collagen gene may play a central role in receiving extracellular signals through the JNK pathway.
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PMID:UV irradiation activates JNK and increases alphaI(I) collagen gene expression in rat hepatic stellate cells. 986 24

Among its plethora of activities as an inflammatory mediator, TNF-alpha has potent regulatory control on extracellular matrix production and degradation. Earlier studies have documented that TNF-alpha inhibits type I collagen gene (COL1A2) expression at the transcriptional level, but the characterization of the transcription factors involved has been elusive. In the present study, using transient cell transfection of human dermal fibroblasts with a battery of 5' end deletion/chloramphenicol acetyltransferase (CAT) reporter gene constructs, we have characterized the TNF-alpha response element of the COL1A2 promoter. The TNF-alpha response element was attributed to a specific region that comprises noncanonical activator protein-1 (AP-1) (CGAGTCA) and NF-kappa B (AGAGTTTCCC) binding sites. TNF-alpha effect was eliminated by a 2-bp substitution mutation in the NF-kappa B1 binding half site of the NF-kappa B cis element. Electrophoretic mobility shift assays (EMSA) showed that recombinant human NF-kappa B heterodimers as well as NF-kappa B1 and RelA homodimers, but not AP-1, were capable of binding this element. Further, EMSA with human fibroblast nuclear extracts demonstrated enhanced binding of a single, specific complex within 5 min of TNF-alpha stimulation, which reached a plateau by 1 h and was not affected by preincubation of cells with cycloheximide. Gel supershift assays identified the complex as the NF-kappa B (p50/p65) heterodimer, whereas Abs to nuclear factor of activated T cells (NF-AT) and Jun family members failed to recognize the complex. These data suggest that in fibroblasts TNF-alpha activates and initiates the nuclear translocation of NF-kappa B that binds a divergent NF-kappa B element and plays a critical role in the observed inhibition of alpha 2(I) collagen gene transcription.
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PMID:Nuclear factor-kappa B mediates TNF-alpha inhibitory effect on alpha 2(I) collagen (COL1A2) gene transcription in human dermal fibroblasts. 1020 51

Matrix metalloproteinase-2 (MMP-2) plays a critical role in tumor cell invasion and metastasis. Inhibitors of this enzyme effectively suppress tumor metastasis in experimental animals and are currently being tested in clinical trials. MMP-2 transcriptional regulation is a part of a delicate balance between the expression of various extracellular matrix (ECM) constituents and ECM degrading enzymes. Halofuginone, a low-molecular-weight quinazolinone alkaloid, is a potent inhibitor of collagen type alpha1 (I) gene expression and ECM deposition. We now report that expression of the MMP-2 gene by murine (MBT2-t50) and human (5637) bladder carcinoma cells is highly susceptible to inhibition by halofuginone. Fifty percent inhibition was obtained in the presence of as little as 50 ng/ml halofuginone. This inhibition is due to an effect of halofuginone on the activity of the MMP-2 promoter, as indicated by a pronounced suppression of chloramphenicol acetyltransferase activity driven by the MMP-2 promoter in transfected MBT2 cells. There was no effect on chloramphenicol acetyltransferase activity driven by SV40 promoter in these cells. Halofuginone-treated cells failed to invade through reconstituted basement-membrane (Matrigel) coated filters, in accordance with the inhibition of MMP-2 gene expression. A marked reduction (80-90%) in the lung colonization of MBT2 bladder carcinoma cells was obtained after the i.v. inoculation of halofuginone-treated cells as compared with the high metastatic activity exhibited by control untreated cells. Under the same conditions, there was almost no effect of halofuginone on the rate of MBT2 cell proliferation. These results indicate that the potent antimetastatic activity of halofuginone is due primarily to a transcriptional suppression of the MMP-2 gene, which results in a decreased enzymatic activity, matrix degradation, and tumor cell extravasation. This is the first description, to our knowledge, of a drug that inhibits experimental metastasis through the inhibition of MMP-2 at the transcriptional level. Combined with its known inhibitory effect on collagen synthesis and ECM deposition, halofuginone is expected to exert a profound anticancerous effect by inhibiting both the primary tumor stromal support and metastatic spread.
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PMID:Inhibition of matrix metalloproteinase-2 expression and bladder carcinoma metastasis by halofuginone. 1047 75

A single-stranded 27-mer phosphorothioate oligodeoxynucleotide (ssPT) containing the transforming growth factor-beta (TGF-beta) response element was synthesized. Rat fetal lung fibroblasts were stably transfected with the ColCat 3.6 plasmid, which contains a portion of the 5'-flanking region of the proalpha1(I) collagen gene linked to the chloramphenicol acetyltransferase (CAT) gene. The cells were transiently transfected with the modified oligodeoxynucleotides in both the presence and absence of bleomycin, a fibrogenic antineoplastic agent. At 50 microg ssPT, the bleomycin-induced increase in CAT activity was abrogated. The ability of ssPT to inhibit collagen synthesis in rat fetal lung fibroblasts was determined. Single-stranded PTs inhibited both collagen synthesis and noncollagen protein synthesis induced by TGF-beta1, the mediator of the bleomycin fibrogenic effect. Inflamed granulation tissue fibroblasts were prepared from polyvinyl alcohol sponges implanted in the backs of rats. These fibroblasts were treated with various doses of ssPTs in the presence and absence of TGF-beta1. Single-stranded PTs also blocked both the TGF-beta1-induced increase in collagen synthesis and noncollagen synthesis in these fibroblasts. However, the TGF-beta1-induced increase in collagen and noncollagen protein synthesis was not blocked by ssPTs containing a mutated TGF-beta response element. In addition, ssPT did not significantly alter the basal levels of collagen and noncollagen protein synthesis in rat lung fibroblasts or in granuloma derived fibroblasts. Since dexamethasone was also able to block the TGF-beta1-induced increase in collagen and noncollagen protein synthesis (Meisler et al., [1997] J. Invest. Dermatol. 108:285-289), these data indicate that phosphorothioate oligodeoxynucleotide antifibrotic agents mimic the inhibitory effect of glucocorticoids on collagen synthesis without the untoward side effects of these steroids.
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PMID:Promoter competitors as novel antifibrotics that inhibit transforming growth factor-beta induction of collagen and noncollagen protein synthesis in fibroblasts. 1050 92

We examined the effect of parathyroid hormone and various signaling molecules on collagen synthesis and chloramphenicol acetyltransferase activity in cultured transgenic mouse calvariae carrying fusion genes of the rat Col1a1 promoter and the chloramphenicol acetyltransferase reporter. After 48 h of culture, parathyroid hormone, forskolin, dibutyryl cAMP, 8-bromo cAMP, and phorbol myristate acetate inhibited transgene activity, while the calcium ionophore ionomycin had no effect. Pretreatment of calvariae with the phosphodiesterase inhibitor isobutylmethylxanthine potentiated the inhibitory effect of 1 nM parathyroid hormone on transgene activity and collagen synthesis. Parathyroid hormone further inhibited transgene activity and collagen synthesis in the presence of phorbol myristate acetate. Parathyroid hormone inhibition of transgene activity and collagen synthesis was not affected by indomethacin or interleukin-6. After 48 h of culture, parathyroid hormone inhibited chloramphenicol acetyltransferase activity by 50-85% in cultured calvariae carrying transgenes having progressive 5' upstream deletions of promoter DNA down to -1683 bp. These data show that the inhibitory effect of parathyroid hormone on Col1a1 expression in mouse calvariae is mediated mainly by the cAMP signaling pathway. Prostaglandins and IL-6 are not local mediators of the parathyroid hormone response in this model. Finally, regions of the Col1a1 promoter downstream of -1683 bp are sufficient for parathyroid hormone inhibition of the Col1a1 promoter.
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PMID:Parathyroid hormone inhibits collagen synthesis and the activity of rat col1a1 transgenes mainly by a cAMP-mediated pathway in mouse calvariae. 1067 25

Dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) are the most abundant steroids in humans whose low levels are related to aging, greater incidence of various cancers, immune dysfunction, atherosclerosis, and osteoporosis. It has been shown that collagen and collagenase gene expression decreases in fibroblasts taken from more aged donors. In this paper, to investigate the relationship between DHEA and skin aging, we examined the effects of DHEA on the regulation of collagen, collegians and stromelysin-1 genes in cultured human skin fibroblasts. In collagen assay, DHEA slightly increased collagen production in a dose-related fashion, its maximal effect occurred at 10(-5) M DHEA (P>0.05). In the presence of DHEA, steady-state levels of alpha1 (I) procollagen mRNA increased to 1. 6-fold of the non-treated group, while those of fibronectin were not. Interestingly, DHEA differently regulated collagenase and stromelysin-1 gene expression. The steady-state levels of collagenase mRNA decreased in response to DHEA by 40%, whereas those of stromelysin-1 mRNA increased up to 2.4-fold, compared to controls. Similar results were obtained for chloramphenicol acetyltransferase assay (CAT); maximal promoter activation of stromelysin-1 gene occurred at 10(-6) M DHEA, 4.5-fold higher than control. CAT assay revealed that treatment with 10(-5) M DHEA resulted in a strong ( approximately 70%) inhibition of the collagenase promoter activity. In our experiments, the effects of DHEA on these gene expressions were higher at pharmacologic concentration (>/=10(-5) M) than those at physiologic concentration (10(-8)-10(-6) M). This study suggests that the level of DHEA may be related to the process of skin aging through the regulation of production and degradation in extracellular matrix.
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PMID:Effects of dehydroepiandrosterone on collagen and collagenase gene expression by skin fibroblasts in culture. 1080 27

Hepatocyte growth factor (HGF) is thought to play a role in cell motility and invasion. Matrix metalloproteinases (MMPs) have been implicated in invasion and metastasis of tumor cells. We have previously reported that the Ets-oncogene family transcription factor E1AF positively regulates transcription of MMP genes in transient expression assays and that overexpression of the E1AF gene confers an invasive phenotype on breast cancer cells. Here we examined the effect of HGF on E1AF and MMP gene expression in terms of the invasive potential of the oral squamous cell carcinoma cell line HSC3. HGF stimulated expression of the E1AF gene. The levels of MMP-1, -3 and -9 mRNAs increased in cells treated with HGF and correlated with E1AF upregulation. In contrast, no obvious upregulation of MMP-1 and -9 mRNA was observed in ASE1AFHSC3 cells transfected with the antisense E1AF expression vector into parental HSC3 cells. The wild-type MMP-9 gene promoter was activated by endogenous E1AF in HSC3 cells, and chloramphenicol acetyltransferase (CAT) activities increased when HGF was added to transfected cells. On the other hand, CAT activity was reduced to almost two-thirds of the wild-type activity when HSC3 cells were transfected with a CAT reporter plasmid driven by a mutant MMP-9 promoter lacking the Ets-binding site, and induction of CAT activity was not observed upon addition of HGF. Analysis of organotypic raft cultures revealed that HSC3 cells invaded and degraded collagen gel actively upon addition of HGF. These results suggest that HGF induces expression of the Ets-related E1AF transcription factor gene whose product in turn activates MMP genes and leads to oral cancer cell invasion.
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PMID:Hepatocyte growth factor upregulates E1AF that induces oral squamous cell carcinoma cell invasion by activating matrix metalloproteinase genes. 1083 94

The purpose of this study was to gain insights into the underlying mechanism of myocardial fibrosis during hypothyroidism. Treatment of cardiac fibroblasts with a medium lacking thyroid hormone led to a 47% increase in [3H]thymidine incorporation into the cell nuclei compared with that in untreated cells. Northern blot analysis of RNA from cardiac fibroblasts grown in a thyroid hormone depleted medium resulted in a 38% increase in the abundance of mRNA for pro-alpha1(I) collagen. At the protein level, the amount of type I collagen, as determined by immunoprecipitation, was increased either in the cell lysate (46%) of cardiac fibroblasts grown in a thyroid hormone depleted medium or in the medium (44%). The chimeric plasmid, ColCAT 3.6, contains the 5'-flanking region of the rat pro-alphal(I) collagen gene (from bases -3520 to +115) fused to the chloramphenicol acetyltransferase (CAT) gene. The plasmid was cotransfected with thyroid hormone receptor (TR) expression plasmid into rat cardiac fibroblasts and COS-l cells (monkey mesangial cells). Cells transfected with the ColCAT plasmid in the presence of thyroid hormone (100 nM T(3)) had a significant decrease (39% in fibroblasts, P<0.01; 52% in COS-1 cells, P<0.001) in CAT activity when compared to cells not exposed to thyroid hormone. Transient co-transfection of TR with various pro-alphal(I) collagen/CAT deletion constructs showed that T(3)-dependent repression was preserved with the deletion from 3520 bp of the flanking sequence to a 5' end point at position -224, indicating that a thyroid hormone-response element (TRE) was localized at the region -224 to +115. The TR-DNA binding assays demonstrated binding of the human TRbeta1 to a fragment containing a proposed TRE located between position -35 and +115 in the 5'-flanking region of the rat pro-alphal(I) collagen gene.
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PMID:Molecular characterization of myocardial fibrosis during hypothyroidism: evidence for negative regulation of the pro-alpha1(I) collagen gene expression by thyroid hormone receptor. 1085 97

The consensus TGF-beta element (TGCCCACGGCCAG) located at approximately -161Obp from the start site of transcription of the rat pro alpha1(I) collagen gene has recently been shown to be required for the basal promoter activity of this gene (Meisler et al., J. Cell Biochem. 75: 196, 1999). Site directed mutation of this TGF-beta element resulted in almost complete abolishment of the basal promoter activity of the fibroblasts transfected with the 3.6 ColCat plasmid which contains a 3.6 kb portion of the 5' flanking region of the rat pro alpha1(I) collagen gene linked to the reporter gene, chloramphenicol acetyltransferase (CAT). Southwestern analysis of the nuclear protein binding to the TGF-beta element revealed a 34,000 Da complex while after UV-crosslinking, studies revealed a TGF-beta element nuclear protein complex of 82,000 Da (Ritzenthaler et al., J. Biol. Chem. 268: 13625, 1993). Thus, a multiple protein TGF-beta DNA element complex may exist which may promote the transcription of the rat pro alpha1(I) collagen gene. Since literature findings indicate that a nuclear factor interacts with an SP1-like binding site of the human pro alpha1(I) collagen promoter and an AP-1 binding sequence has been shown to be involved in the regulation of the human pro alpha2(I) collagen gene and both these binding sequences are TGF-beta1 responsive, we determined whether the TGF-beta element located in the 5' flanking region of the rat pro alpha1(I) collagen gene formed complexes with either of these nuclear factors or both.
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PMID:Human SP1 but not human AP1 binding to the TGF-beta element in the 5' flanking region of the rat PROalpha1(I) collagen gene. 1125 9


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