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

Osteocalcin is an abundant noncollagenous protein in bone, and its synthesis is stimulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In this study, the rat osteocalcin gene was isolated, sequenced, and found to be a single-copy gene that is highly conserved between human and rat. Northern blot analysis of RNAs from a number of rat tissues revealed osteocalcin mRNA only in calvariae, consistent with bone-specific expression of osteocalcin. In order to investigate promoter activity and its modulation by 1,25(OH)2D3, plasmids containing the osteocalcin promoter region linked to the reporter enzyme bacterial chloramphenicol acetyltransferase (CAT) were used to transfect rat osteosarcoma ROS 17/2.8 cells, which express osteocalcin endogenously, and UMR 106 cells, which lack osteocalcin expression. Transfected ROS 17/2.8 cells exhibited a higher basal CAT activity than UMR 106 cells. Moreover, 1,25(OH)2D3 stimulated the CAT expression 5-10-fold only in ROS 17/2.8 cells and not in UMR 106 cells. By use of unidirectional deletion analysis, a domain strongly responsive to 1,25(OH)2D3 was identified between bases -1035 and -871 upstream from the site of transcription initiation, while a weakly responsive region was found further downstream.
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PMID:Characterization of the rat osteocalcin gene: stimulation of promoter activity by 1,25-dihydroxyvitamin D3. 326 36

Osteocalcin is a major noncollagenous protein of bone regulated by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] and is believed to be expressed only by differentiated osteoblasts. We introduced a 3.9-kilobase human osteocalcin gene promoter (hOCP)-chloramphenicol acetyltransferase (CAT) fusion gene into the germ line of mice. Examination of tissue extracts from these transgenic mice demonstrated that the expression of CAT was restricted to bone-associated tissues and the brain. Immunohistochemical staining of femur tissue sections using CAT antibodies localized the production of CAT protein to osteoblasts and maturing chondrocytes. Previous studies via transient transfection into osteoblast-like cells have identified a vitamin D response element approximately 500 basepairs up-stream of the hOCP capable of mediating 1,25-(OH)2D3 induction. As a consequence, regulation of the transgene was examined in homozygous transgenic lines for sensitivity to 1,25-(OH)2D3. Hormonal deficiency was created using a low calcium diet supplemented with 0.8% SrCl2 for 7 days and was restored in experimental mice by injection of 25 ng 1,25-(OH)2D3/day, ip, for 3 days. The low vitamin D3 diet decreased CAT activity several-fold in extracts from calvaria, femur, and brain compared to that in mice maintained on a normal diet, while 1,25-(OH)2D3 supplementation restored and enhanced CAT activity over control values. These data demonstrate that hOCP is sufficient to direct osteoblast-specific 1,25-(OH)2D3-sensitive gene expression in mice in addition to the unexpected regulatable expression in brain tissue.
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PMID:The human osteocalcin promoter directs bone-specific vitamin D-regulatable gene expression in transgenic mice. 848 81

Osteocalcin (OC), a bone specific protein expressed during differentiation and mineralization of the bone extracellular matrix, is down-regulated upon treatment with transforming growth factor (TGF)-beta 1. To address the potential role of OC gene expression in relation to TGF-beta 1 regulation of bone formation and resorption, we examined the transcriptional activity of the rat OC promoter after TGF-beta 1 treatment. 5' deletion analysis of rat OC promoter-chloramphenicol acetyltransferase constructs demonstrated that TGF-beta 1 treatment repressed chloramphenicol acetyltransferase activity by 2.4-fold in transient transfections of ROS 17/2.8 cells. A 29-bp region between -162 and -134 identified as the TGF-beta 1 response domain, conferred TGF-beta 1 responsiveness to the -108 to +24 rat OC basal promoter in an orientation dependent manner. Mutation of an activator protein-1/cAMP-response element-like motif (- 146 to -139) abolished TGF-beta 1 responsiveness of the construct. In vitro gel-mobility shift and competition assays using wild-type and mutated oligonucleotides and antibodies indicate that Fra-2, a Fos related transcription factor, binds to this motif. We show that Fra-2 is an activator of the OC promoter, and TGF-beta 1 inhibits this activation. Our results demonstrate that Fra-2 is hyperphosphorylated upon TGF-beta 1 treatment of ROS 17/2.8 cells. Additionally, treatment of cells with a staurosporine protein kinase C inhibitor abrogates TGF-beta 1 mediated down-regulation of the OC promoter activity. Together, these results demonstrate that TGF-beta 1 responsiveness of the rat osteocalcin gene in ROS 17/2.8 cells is mediated through an activator protein-1 like cis-acting element that interacts with Fra-2. Furthermore, our results are consistent with a critical role for TGF-beta 1 induced phosphorylation of Fra-2 in the repression of OC gene transcription.
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PMID:Transforming growth factor-beta 1 responsiveness of the rat osteocalcin gene is mediated by an activator protein-1 binding site. 861 40