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)

A proinflammatory cytokine cascade, including IL-1 alpha, IL-1 beta, TNF-alpha, IL-6, and IL-8, is activated in response to infection or immunologic insult. Besides their immunologic effects, several of these mediators stimulate bone resorption and inhibit bone formation. Osteocalcin, the most abundant noncollagenous protein present in bone, is an osteoblast-specific product whose production closely correlates with bone formation, and which has also been implicated in control of bone resorption. IL-1 and TNF have previously been shown to down-regulate osteocalcin production in vitro and in vivo, although the mechanism of this inhibition is unknown. In the present studies, IL-1 beta and TNF-alpha both inhibited 1,25-dihydroxyvitamin D3-stimulated production of osteocalcin protein and mRNA by ROS 17/2.8 osteosarcoma cells, whereas IL-6 had no effect on protein and only weakly inhibited mRNA. To determine if down-regulation was exerted at the transcriptional level, an osteocalcin promoter-chloramphenicol acetyltransferase (CAT) fusion gene was constructed (PHOC-CAT). After transient transfection of PHOC-CAT into ROS 17/2.8 osteosarcoma cells, reporter CAT activity was up-regulated by vitamin D at concentrations above 10(-12) M. In screening studies, TNF-alpha (-57%) and IL-6 (-37%) inhibited vitamin D-stimulated osteocalcin transcription, whereas IL-1 alpha, IL-1 beta, and IL-8 had no effect. Other immune cytokines and growth factors, including IL-2, IL-3, IL-7, and M-CSF, also failed to regulate osteocalcin transcription. Despite their lack of promoter regulation, IL-1 alpha and IL-1 beta also stimulated PGE2 production by ROS 17/2.8, further confirming the ability of the host cell to respond to these mediators. In dose-response experiments, down-regulation by TNF-alpha was significant at concentrations as low as 0.14 pM (0.1 U/ml), whereas approximately 10(4)-fold higher concentration of IL-6 was required to exert a similar effect. TNF-alpha-mediated down-regulation was unaffected by indomethacin. These data demonstrate that of these cytokines, TNF-alpha alone potently down-regulates osteocalcin promoter function, whereas IL-1 acts post-transcriptionally, possibly by reducing mRNA stability. Heterogeneity therefore exists among the proinflammatory cytokines with respect to the level at which control of osteocalcin expression is exerted.
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PMID:Proinflammatory cytokines tumor necrosis factor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin gene promoter. 130 41

Several endocrine hormones which influence liver metabolism are known to increase in activity during the acute phase of injury or inflammation. We determined whether these hormones have the potential to influence acute-phase protein production in human and rat hepatoma cells. Catecholamines, glucagon, growth hormone, triiodothyronine, and cyclic nucleotides individually or in combination did not modulate the basal or the interleukin-1 (IL-1)-, IL-6-, and dexamethasone-stimulated levels of acute-phase plasma proteins. Insulin, however, was found to be a rapid, nonspecific, and dose-dependent inhibitor of the cytokine and glucocorticoid stimulation of acute-phase protein gene expression and to exert its effect at the transcriptional level. The insulin inhibition applied to all cytokines tested but to various degrees, depending upon the particular acute-phase gene. Insulin resulted in an early and prominent increase in the transcription of genes encoding the AP-1 components of JunA, JunB, and c-Fos, as has been observed for other growth factors. However, the effect of insulin on C/EBP beta was unexpected and paradoxical: while insulin completely inhibited the transcriptional activation of the C/EBP beta gene in cytokine- and dexamethasone-treated cells, the level of cytoplasmic C/EBP beta RNA was elevated. Quantitation of C/EBP beta mRNA by Northern (RNA) blot analysis and of C/EBP beta DNA binding activity by Southwestern (DNA-protein) blot analysis showed that insulin, when combined with cytokines and dexamethasone, stimulated both the mRNA and DNA binding activity by a factor of 1.6 compared with that of cells treated with cytokines and dexamethasone alone. Transient transfection of H-35 and HepG2 cells with a chloramphenicol acetyltransferase (CAT) gene expression vector containing the C/EBP beta response element also resulted in a 1.5-fold increase of C/EBP beta-mediated transcription in insulin-treated cells. Transfection of CAT gene constructs containing increasing lengths of heptaglobin gene 5' flanking sequences indicated that insulin inhibition of IL-6 stimulation required the presence of the region from -4100 to -1030. These results suggest that insulin has the potential to control the transcription of acute-phase genes by at least two separate mechanisms.
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PMID:Insulin is a prominent modulator of the cytokine-stimulated expression of acute-phase plasma protein genes. 137 89

The induction of human immunodeficiency virus type 1 (HIV-1) gene expression by cytokines was investigated in cells of central nervous system origin. These were human neuroblastoma, glioblastoma, and astrocytoma cell lines, a murine oligodendroglioma and primary murine astrocyte cultures. The cytokines used were tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), IL-6, and interferons alpha and gamma (IFN alpha, gamma). Transient transfection of cells with a chloramphenicol acetyltransferase (CAT) reporter gene under the control of the HIV-1 long terminal repeat (LTR) showed significant augmentation following treatment by particular cytokines. TNF alpha was found to augment HIV LTR-directed CAT activity in all cell types. IL-1 beta also activated the HIV LTR reporter gene in glioblastoma, astrocytoma, and astrocyte cells. IL-6 enhanced HIV gene expression in one example only, the primary astrocyte cultures. The interferons generally suppressed expression from the LTR except IFN gamma which produced a twofold rise in the murine glial cells and IFN alpha augmenting expression in one neuroblastoma cell line. No synergy was observed between pairs of activating cytokines tested. The HIV tat gene product was found to be functional in all cells, cotransfection of a tat expression vector transactivating expression from the LTR, with varying degrees of efficiency. In some cell lines the combination of an activating cytokine and tat resulted in an enhancement above that obtained by cotransfection of tat alone. In others, the level of CAT activity did not significantly change. Analysis of nuclear extracts from cytokine-treated cells further implicated the involvement of NFKB in the induction of HIV-1 gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cytokine augmentation of HIV-1 LTR-driven gene expression in neural cells. 159 55

The transcription rate of the haptoglobin (Hp) gene is stimulated by interleukin-1 (IL-1), IL-6, and dexamethasone in rat hepatoma (H-35) cells. To identify the cis-acting regulatory elements responsive to these hormones, various lengths of 5' Hp gene-flanking regions, including the promoter, were inserted into chloramphenicol acetyltransferase gene expression vectors and transiently introduced into H-35 cells. The first 4 kb of 5' region mediated a severalfold increase in expression after treatment with IL-6 and dexamethasone. No response to IL-1 was detectable. When, however, upstream sequences were deleted to position -165 relative to the transcription start site, a significant stimulation by IL-1 was gained without appreciably affecting the IL-6 response. With the apparent removal of an inhibitory sequence, the promoter-proximal 165-bp region also displayed a severalfold enhanced response to the combination of dexamethasone, IL-1, and IL-6. The sequence from -165 to -147, termed the A-element, was found to be crucial for all hormone regulatory functions. Two copies of the A-element linked to a heterologous promoter responded to the three hormones, but to a lesser degree than in the Hp gene promoter context. The regulatory elements of the rat Hp gene were similarly active in human hepatoma cells. Optimal regulation by IL-6 in HepG2 cells was, however, independent of the A-element. The A-element functioned in these cells exclusively as an IL-1 response sequence. The results suggest that genomic sequences upstream of the rat Hp gene suppress the regulation by specific cytokines more prominently in transient expression assays than in the normal chromosomal context. Moreover, the functional comparison indicated that specific regulatory regions of the rat Hp gene do not function identically in different hepatic cell types.
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PMID:Distinct regulation of the interleukin-1 and interleukin-6 response elements of the rat haptoglobin gene in rat and human hepatoma cells. 217 89

The 5'-flanking region of the gene coding for the gamma chain of human fibrinogen was characterized for its promoter activity. Reporter gene studies using chloramphenicol acetyltransferase as the indicator along with mutations in the DNA showed that a TATA-like sequence (-20 to -23 base pairs (bp)), a CAAT-like sequence (-54 to -57 bp), and an upstream stimulatory factor (USF) binding site (-66 to -77 bp) constitute a minimal promoter that mediates liver-specific expression of the gene. Electrophoretic gel mobility assays and antibody binding studies confirmed the interaction of USF with the binding site. An IL-6 responsive element with a sequence of CTGGAA located at -301 to -306 bp was shown to be a functional element in the IL-6 response. In contrast to the IL-6 responsive elements in the human alpha- and beta-fibrinogen genes, the element in the gene for the gamma chain of human fibrinogen was unaffected by the presence or elevated levels of the beta or delta isoforms of the CCAAT/enhancer binding proteins. A negative element with sequence homology to several silencer elements was also identified in the region of -348 to -390 bp of the gene for the gamma chain of human fibrinogen. A comparison of the regulatory elements in the genes coding for all three chains in fibrinogen is also presented.
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PMID:Characterization of the 5'-flanking region of the gene for the gamma chain of human fibrinogen. 749 36

The receptor for granulocyte colony stimulating factor (G-CSFR) and chimeric receptors consisting of the extracellular domain of G-CSFR and the transmembrane and cytoplasmic domain of the leukemia inhibitory factor receptor, gp130, or c-mpl function as homodimeric complexes. These receptors mediate a similar stimulation of gene transcription via separate regulatory elements of acute phase plasma protein genes. To identify the receptor regions within the cytoplasmic domains necessary for transcriptional regulation, the receptors were transiently expressed in rat hepatoma cells. Each receptor form reconstituted G-CSF-induced expression of a chloramphenicol acetyltransferase gene construct containing the cytokine response element of the rat alpha 1-acid glycoprotein gene. This regulation required the presence of two conserved sequence motifs (referred to as box 1 and box 2) in the cytoplasmic domains of each receptor. With the exception of G-CSFR-MPL chimera, the receptors also mediated a similarly high stimulation via the IL-6 response element of the rat beta-fibrinogen and hemopexin genes. Regulation of the IL-6 response element required, however, in addition to boxes 1 and 2, a third sequence motif (box 3). This motif is absent in the cytoplasmic domain of c-mpl, possibly explaining its inability to activate the IL-6 response element. When cells which express receptor forms with prominent box 3 function were treated with suramin, a ligand-independent gene stimulation via the IL-6 response element was observed. The suramin effect probably involves a receptor dimerization mediated by the extracellular G-CSFR domain and by the intracellular regions that include box 3.
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PMID:Signaling by the cytoplasmic domain of hematopoietin receptors involves two distinguishable mechanisms in hepatic cells. 751 79

Hepatic expression of various members of the cytochrome P-450 (CYP) superfamily is suppressed during inflammatory responses. We have shown that the specific expression of P-450 2C11 in male rat liver is suppressed transcriptionally by endotoxin treatment. To investigate the molecular mechanisms underlying this phenomenon, we studied the effects of the inflammatory cytokines interleukin (IL)-1, IL-6, tumor necrosis factor-alpha (TNF), interferon (IFN)-alpha, and IFN-gamma on the expression of P-450 2C11 and the mRNAs of two typical acute-phase protein genes, alpha 1-acid glycoprotein (AGP) and fibrinogen, in primary hepatocyte cultures. IL-1, IL-6, TNF, and IFN-alpha all suppressed P-450 2C11 mRNA, whereas IFN-gamma had no effect. IL-1 and TNF were more effective than IL-6 in the suppression of P-450 2C11 mRNA. Whereas IL-1 and IL-6 effects on P-450 2C11 were accompanied by induction of AGP and fibrinogen mRNAs, IFN-alpha and TNF treatments had no effects on AGP. The suppression of P-450 2C11 and the induction of AGP by IL-1 showed similar time courses. The combination of IL-1 and IL-6 showed additivity in suppression of P-450 2C11, at maximally effective concentrations of cytokines. The effects of IL-1 on P-450 2C11 and AGP expression were blocked by IL-1 receptor antagonist protein. We also studied the effects of IL-1 and IL-6 on the transient expression of chloramphenicol acetyl-transferase reporter gene constructs containing 200 or 1287 base pairs of the 5' flanking region of the CYP2C11 gene, transfected into primary hepatocytes. The chloramphenicol acetyltransferase activities in cells transfected with the 200-base pair construct were reduced to about 33% and 58% of control levels by treatment with IL-1 or IL-6, respectively, suggesting that sequences important for cytokine down-regulation lie within the proximal promoter region of the CYP2C11 gene.
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PMID:Suppression of the constitutive expression of cytochrome P-450 2C11 by cytokines and interferons in primary cultures of rat hepatocytes: comparison with induction of acute-phase genes and demonstration that CYP2C11 promoter sequences are involved in the suppressive response to interleukins 1 and 6. 753 97

Expression of the pancreatitis-associated protein I (PAP I), an exocrine pancreatic protein, increases rapidly and strongly in acinar cells during the acute phase of pancreatitis. This is reminiscent of the response to stress of acute phase proteins. We have previously demonstrated that serum factors from rats with acute pancreatitis, but not from healthy rats, could induce endogenous PAP I gene expression in the acinar cell line AR-42J (Dusetti, N., Mallo, G., Dagorn, J.-C., Iovanna, J. L. (1994) Biochem. Biophys. Res. Commun. 204, 238-243). In the present work, we have evaluated the influence of several mediators of inflammation on rat PAP I gene transcription in these cells. Tumor necrosis factor alpha induced an increase in PAP I mRNA expression, and interferon gamma caused an even greater increase in PAP I mRNA level. These stimulations were antagonized by dexamethasone. Interleukin (IL)-1, IL-6, or dexamethasone alone were ineffective. Combinations of IL-1 with IL-6 or dexamethasone were also ineffective. IL-6 and dexamethasone together induced a marked stimulation of PAP I gene transcription, and this effect was slightly attenuated by IL-1. To analyze the cis-regulatory elements responsible for the induction of transcription, we fused a 1.2-kilobase segment of the rat PAP I promoter to the chloramphenicol acetyltransferase (CAT) gene as reporter. The resultant chimeric DNA was transfected into AR-42J cells. Addition of IL-6 or dexamethasone was ineffective, whereas their mixture increased the CAT activity 12 times. Progressive deletions of the PAP I promoter were then fused to the CAT gene, and the constructs were transfected to AR-42J cells. A 12-fold increase in CAT activity was seen upon IL-6/dexamethasone treatment with constructs containing more than 274 base pairs upstream from the cap site. In that region, two sequences are similar to the canonical IL-6 response element. Site-directed mutagenesis of these regions strongly decreased induction, showing that they were functional. PAP I should therefore be classified among acute phase proteins of class 2, whose expression is increased by IL-6 acting in combination with glucocorticoids.
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PMID:Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identification of two functional interleukin-6 response elements in the rat PAP I promoter region. 754 77

In the present report we describe a heretofore unrecognized role for a Jak/STAT signaling pathway, namely the stimulation of expression of the aromatase P450 (CYP19) gene, and hence of estrogen biosynthesis, in human adipose tissue. Expression of this gene in adipose tissue as well as in adipose stromal cells maintained in the presence of serum and glucocorticoids is regulated by a distal TATA-less promoter, I.4, which contains a glucocorticoid response element, an Sp1 binding site, and an interferon-gamma activation site (GAS) element. The stimulatory action of serum (in the presence of dexamethasone) can be replaced by interleukin (IL)-11, leukemia inhibitory factor, and oncostatin-M, as well as by IL-6, providing the IL-6 soluble receptor is also present. Stimulation of the cells by these factors led to rapid phosphorylation of Jak1, but not Jak2 or Jak3, on tyrosine residues. STAT3 but not STAT1 was also phosphorylated and bound to the GAS element in the I.4 promoter region. When regions of this promoter were fused upstream of the chloramphenicol acetyltransferase reporter gene and transfected into the cells, mutagenesis or deletion of the GAS element led to complete loss of reporter gene expression. Since adipose tissue is the major site of estrogen biosynthesis in men and in postmenopausal women, this pathway involving a Jak/STAT signaling mechanism acting together with glucocorticoids and Sp1 appears to be the principal means whereby estrogen biosynthesis is regulated in the elderly.
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PMID:Aromatase P450 gene expression in human adipose tissue. Role of a Jak/STAT pathway in regulation of the adipose-specific promoter. 760 17

Although 17 beta-estradiol (E2) replacement therapy has been shown to be effective in treating postmenopausal osteoporosis, the underlying mechanism remains unclear. The presence of low levels of functional endogenous estrogen receptor (ER) in some osteoblastic cells has been demonstrated, and the suggestion that the abundance of ER may be rate-limiting in the action of E2 on these cells has been made. To study the mechanism of ER in regard to E2-mediated effects, we stably transfected a human osteosarcoma cell line, SaOS-2, with an expression vector, pMV-7-ER, containing the human ER gene. We characterized six of the stably transfected clones. One of the stable clones, SaOS-2-ER, expressed extra copies of ER genes integrated into the genome as detected by Southern blot analysis, showed a significantly increased level of ER mRNA by RT-PCR, and contained an increased level of ER cytosolic protein as detected by an ER-specific EIA. The overexpressed ER was functional and sensitive to E2 in a dose-dependent fashion after transient transfection with a vector containing an estrogen response element (ERE) linked to a chloramphenicol acetyltransferase (CAT) reporter gene. Scatchard analysis revealed a single high-affinity binding site with a Kd similar to values obtained for the ER in MCF-7 breast cancer cells. These SaOS-2-ER cells had altered osteoblast phenotypic features including growth inhibition, decreased basal alkaline phosphatase activity, and decreased IL-6 expression and secretion. In response to E2, a greater than 2-fold increase in TGF-beta 1 mRNA was quantitatively measured in these ER-overexpressing osteoblasts. These cells may provide a sensitive and unique model for understanding the mechanism of E2 and ER in overall bone metabolism.
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PMID:Generation and characterization of a human osteosarcoma cell line stably transfected with the human estrogen receptor gene. 763 12


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