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)

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

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

The gene encoding PTH-related peptide (PTHrP), a protein that plays a primary role in the development of humoral hypercalcemia of malignancy, is widely expressed in normal and neoplastic tissues. This study demonstrates that expression of the PTHrP gene has features of early response genes, including up-regulation after serum repletion of serum-starved ROS 17/2.8 (rat osteosarcoma) cells. The PTHrP messenger RNA (mRNA) levels were induced within 30 min and peaked at 4 h. Elevated mRNA levels were accompanied by an increase in secreted PTHrP. The serum effects on PTHrP mRNA levels were blocked by actinomycin D, suggesting a requirement for gene transcription. Nuclear run-on assays revealed a 3-fold increase in PTHrP gene transcription 4 h after exposure to serum. Deletions of the 5' flanking sequence of the rat PTHrP gene fused to the chloramphenicol acetyltransferase gene and transfected into ROS 17/2.8 cells showed that the serum-responsive region is located between -1.05 kb and -0.3 kb upstream of the transcription start site. PTHrP mRNA levels were also induced by cycloheximide, another feature common to early response genes. The PTHrP mRNA half-life in serum-starved cells was 56 min. Serum treatment prolonged the half-life 2.7-fold, suggesting serum-induced stabilization of the mRNA. Insulin and epidermal growth factor also induced PTHrP mRNA expression in a time-dependent manner analogous to serum, indicating that the effects of serum may be mediated, at least partially, through these agents. In summary, serum up-regulated PTHrP mRNA expression through both transcriptional and posttranscriptional mechanisms. This rapid stimulation by growth factors suggests that PTHrP may contribute to the early cellular response after growth factor stimulation.
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PMID:Serum stimulation of parathyroid hormone-related peptide gene expression in ROS 17/2.8 osteosarcoma cells through transcriptional and posttranscriptional mechanisms. 875 33

Parathyroid hormone (PTH) activates both adenylate cyclase and phospholipase C in target cells, and cloned PTH/PTH-related protein (PTHrP) receptor can mediate both responses when expressed in host cells such as LLC-PK1 renal epithelial cells. Because calcitonin (CT) is known to augment 70-kDa heat shock protein (HSP70) mRNA by an adenosine 3',5'-cyclic monophosphate (cAMP)-independent mechanism in LLC-PK1 cells, we examined regulation of HSP70 transcription by PTH in these cells. Like CT, human PTH-(1-34) [hPTH-(1-34); 10(-10) to 10(-7) M)] increased porcine HSP70 mRNA and human HSP70 promoter-chloramphenicol acetyltransferase (CAT) expression within 4 h in LLC-PK1 cells that stably express > or = 100,000 PTH/PTHrP receptors per cell. The effect of PTH on HSP70 mRNA was not mimicked by cAMP analogues, forskolin, phorbol esters, Ca2+ ionophores, or alpha-thrombin; was insensitive to pertussis toxin; and was not due to increased mRNA stability. The upregulation of HSP70 gene transcription by hPTH (and CT) was clearly observed even after deletion of the functional heat shock consensus element in the promoter region of the human HSP70/CAT reporter. Upregulation of HSP70 transcription via endogenous PTH receptors also was observed in the osteoblastic cell lines SaOS-2 and ROS 17/2.8. Regulation of HSP70 gene transcription by PTH may be a common cellular response to the hormone, which, in some cells, may not be mediated by activation of adenylate cyclase or protein kinase C.
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PMID:Regulation of HSP70 by PTH: a model of gene regulation not mediated by changes in cAMP levels. 876 37

Developmental studies of oncogene expression implicate the Fos and Jun family of transcription factors in the regulation of bone growth and differentiation. Promoters of many developmentally regulated genes, including osteocalcin, a marker of osteoblast differentiation, contain AP-1 sites that bind Fos/Jun dimers. Here, we demonstrate that the selective expression of fos- and jun-related genes is functionally related to the stage of osteoblast growth and differentiation in vitro. During osteoblast proliferation, nuclear protein levels of all seven activating protein-1 (AP-1) members are maximal. Subsequently, during the period of extracellular matrix maturation, levels decline. In fully differentiated osteoblasts, Fra-2 and (to a lesser extent) Jun D are the principal AP-1 members detectable by Western blot analysis. AP-1 complex composition and binding activity also exhibit developmental changes. All Fos and Jun family members are involved in AP-1 complex formation in proliferating cells, whereas Fra-2 and Jun D predominate in AP-1 complexes in differentiated osteoblasts. Overexpression of Fos and Jun family members in ROS 17/2.8 cells markedly affects the expression of an osteocalcin promoter-chloramphenicol acetyltransferase construct. Coexpression of only one AP-1 pair, Fra-2 and Jun D, stimulated reporter expression, whereas coexpression of other AP-1 pairs down-regulated expression (i.e. c-jun and any Fos family member) or had no effect (i.e. Fra-1 and Jun B). Promoter deletion analyses indicate that these effects are site specific. Consequential effects of Fra-2 on osteoblast differentiation are further demonstrated by antisense studies in which osteoblast differentiation and the development of a bone tissue-like organization were suppressed. Consistent with recent findings suggesting that AP-1 complex composition can selectively regulate gene transcription, our findings demonstrate that differential expression of Fos and Jun family members could play a role in the developmental regulation of bone-specific gene expression and, as a result, may be functionally significant for osteoblast differentiation.
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PMID:Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation. 882 1

Mothers against decapentaplegic-related proteins (Smads) are essential intracellular components for the signal transduction of transforming growth factor-beta (TGF-beta) family members. Smad1 mediates bone morphogenetic protein (BMP) signals, whereas Smad2 functions downstream of TGF-beta. TGF-beta is expressed in osteoblastic cells and acts as an autocrine and/or paracrine factor in regulation of osteoblastic functions. In this study, we examined the levels and functions of Smad2 in osteoblastic cells. Smad2 mRNA expression was hardly detectable by Northern blot analysis in an osteoblast-like cell line, ROS17/2.8, as well as in primary rat calvaria (PRC) cells. Overexpression of Smad2 gene enhanced endogenous Smad4 gene expression in both ROS17/2.8 and PRC cells, while Smad3 levels were not altered. Smad2 overexpression suppressed osteocalcin mRNA expression in ROS17/2.8 cells. Furthermore, Smad2 overexpression also suppressed transcriptional activity of the 1-kilobase pair osteocalcin gene promoter, which was linked to chloramphenicol acetyltransferase reporter gene in both ROS and PRC cells. Since core binding factor A1 (CBFA1) is involved in osteocalcin gene expression, we further examined CBFA1 expression in the Smad2-overexpressing ROS17/2.8 and PRC cells. The levels of CBFA1 mRNA were suppressed by the overexpression of Smad2 by about 50% in both ROS17/2.8 and PRC cells. TGF-beta treatment enhanced Smad4 expression in PRC cells, and this TGF-beta effect was blocked by the cotreatment with BMP, indicating that TGF-beta signaling pathway is interfered by BMP. These data indicate that Smad2 regulates Smad4 specifically and that CBFA1 gene is one of the downstream targets of Smad2.
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PMID:Smad2 overexpression enhances Smad4 gene expression and suppresses CBFA1 gene expression in osteoblastic osteosarcoma ROS17/2.8 cells and primary rat calvaria cells. 981 98

Three Cbfa motifs are strategically positioned in the bone-specific rat osteocalcin (rOC) promoter. Sites A and B flank the vitamin D response element in the distal promoter and sites B and C flank a positioned nucleosome in the proximal promoter. The functional significance of each Cbfa element was addressed by mutating individual or multiple Cbfa sites within the context of the -1.1-kb rOC promoter fused to a chloramphenicol acetyltransferase reporter gene. Promoter activity was assayed following transient transfection and after stable genomic integration in ROS 17/2.8 osteoblastic cell lines. We show that all three Cbfa sites are required for maximal basal expression of the rOC promoter. However, the distal sites A and B each contribute significantly more (P < 0.001) to promoter activity than site C. In a genomic context, sites A and B can largely compensate for a mutation at the proximal site C, and paired mutations involving site A (mAB or mAC) result in a far greater loss of activity than the mBC mutation. Strikingly, mutation of the three Cbfa sites leads to abrogation of responsiveness to vitamin D. Vitamin D-enhanced activity is also not observed when sites A and B are mutated. Significantly, related to these losses in transcriptional activity, mutation of the three Cbfa sites results in altered chromatin structure as reflected by loss of DNase I-hypersensitive sites at the vitamin D response element and over the proximal tissue-specific basal promoter. These findings strongly support a multifunctional role for Cbfa factors in regulating gene expression, not only as simple transcriptional transactivators but also by facilitating modifications in promoter architecture and chromatin organization.
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PMID:Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization. 1052 37


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